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Novel rapid nongenomic and slow genomic mechanisms of ovarian steroid modulation of the hypothalamic-pituitary-adrenal axis and the cardiovascular system during ovine pregnancy

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Novel rapid nongenomic and slow genomic mechanisms of ovarian steroid modulation of the hypothalamic-pituitary-adrenal axis and the cardiovascular system during ovine pregnancy
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Roesch, Darren Michael, 1971-
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English

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Subjects / Keywords:
Baroreflexes ( jstor )
Corticosteroids ( jstor )
Ewes ( jstor )
Hippocampus ( jstor )
Plasmas ( jstor )
Pregnancy ( jstor )
Rats ( jstor )
Receptors ( jstor )
Sheep ( jstor )
Steroids ( jstor )

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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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NOVEL RAPID NONGENOMIC AND SLOW GENOMIC MECHANISMS OF
OVARIAN STEROID MODULATION OF THE HYPOTHALAMIC-PITUITARY-
ADRENAL AXIS AND THE CARDIOVASCULAR SYSTEM DURING OVINE
PREGNANCY














By

DARREN MICHAEL ROESCH













A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

1998































my quest to understand: to my mother
this volume: to my sister
completely: to GNV
















ACKNOWLEDGMENTS

This collection of data and hypotheses represents a much larger accumulation of

personal intellectual and emotional enlightenment. I will forever be grateful to those who

have contributed to my advancement: most of whom I will fail to mention in these pages.

First, I must thank my mentor, Dr. Maureen Keller-Wood, for providing the

intellectual and material fuel for this achievement. Her unselfish dedication to my

education has earned her a special place in my heart: I will always think of her as a

surrogate mother. I also am indebted to Dr. Charles E. Wood, the other role model in the

Wood lab family, for continuous sage and patient instruction.

I also thank the additional members of my supervisory committee for encouraging

and assuring a high standard of academic accomplishment: Dr. Pushpa S. Kalra, Dr.

Michael J. Katovich, Dr. William J. Millard, and Dr. Donna Wielbo.

I thank my family, especially my father, Mr. Daniel P. Roesch, for providing the

resources to make my education possible. In many ways, this dissertation also is their

achievement.

The Health Science Center has truly become an extended family, and many people

have contributed to my education and this dissertation. I will not attempt to thank

everyone because I will undoubtedly omit a dear friend. However, I must thank the

members of the Wood lab family for rolling up their sleeves to assist with the "dirty

work": Ms. Sara Caldwell, Ms. Deanna Deauseault, Mr. David Husted, Dr. Eun-Kyung


iii









Kim, Ms. Ellen Manlove, Mr. Pini Orbach, Ms. Melanie Pockey, Mr. Scott Purinton, Dr.

Christine Saoud, and Dr. Hiayan Tong.

I thank Dr. Joanna Peris and the rest of the Department of Pharmacodynamics

faculty for believing in me when even I did not believe in myself. I thank Dr. Janice

Wachtel Walton for helping me learn to live, and I acknowledge Mr. Gilbert N. Vansoi,

my living "candle on the water."






































iv
















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

NOVEL RAPID NONGENOMIC AND SLOW GENOMIC MECHANISMS OF
OVARIAN STEROID MODULATION OF THE HYPOTHALAMIC-PITUITARY-
ADRENAL AXIS AND THE CARDIOVASCULAR SYSTEM DURING OVINE
PREGNANCY

By

Darren Michael Roesch

May 1998

Chair: Maureen Keller-Wood, Ph.D.
Major Department: Pharmacodynamics

The setpoint and response of both the cardiovascular system and the hypothalamic-

pituitary-adrenal (HPA) axis are altered during pregnancy. Evidence strongly

demonstrates estrogens regulate these systems, and the role of progesterone is becoming

better appreciated. However, multiple receptor types may contribute to progesterone's

effects. In addition to the well-described progesterone receptor, progesterone interacts

with two other intracellular steroid-binding transcription factors: the mineralocorticoid

(MR) and glucocorticoid (GR) receptors. The 5cc-reduced metabolite of progesterone,

tetrahydroprogesterone (THP), also interacts with GABAA receptors to mediate rapid

physiological effects. The dissertation examines the possibility that progesterone acts

through these novel receptors to affect the cardiovascular system and the HPA axis.





v











Plasma progesterone levels characteristic of ovine pregnancy rapidly reduced mean

arterial pressure (MAP) in ovary-intact ewes. THP could induce these rapid progesterone

effects. However, since supraphysiological levels of progesterone did not alter MAP,

multiple receptor types appear to contribute to the rapid cardiovascular effects of

progesterone.

Physiological levels of progesterone also rapidly reduced arterial pressure in

ovariectomized ewes when starting baseline pressures were slightly elevated; suggesting

baseline arterial pressure and/or reproductive state influence the rapid effects of

progesterone. Decreased 5a-reduction of progesterone was confirmed in the livers of

ovariectomized ewes, suggesting ovarian steroids modulate progesterone metabolism.

MR and GR availability and immunoreactivity are differentially regulated in the

ovine hippocampus during pregnancy. Progesterone could contribute to these observed

changes. However, chronic treatment of ovary-intact ewes with estrone, but not chronic

treatment of ovariectomized ewes with progesterone, induced changes in these receptors

characteristic of ovine pregnancy. This finding suggests estrogens modulate these

receptors, but does not eliminate the possibility the progesterone also regulates these

receptors. Differential regulation of hippocampal MR and GR could contribute to altered

activity and responsiveness of the HPA axis during pregnancy, and altered HPA axis

activity could contribute to cardiovascular adaptation to pregnancy since steroid and

peptide components of the axis modulate fluid homeostasis, vascular responsiveness, and

neural blood pressure control.






vi











These studies support the hypothesis that ovarian steroids contribute to adaptation

of the HPA axis and the cardiovascular system to pregnancy via novel rapid nongenomic

and slow genomic mechanisms.













































vii


















TABLE OF CONTENTS
page


ACKNOWLEDGMENTS ............................................... iii

ABSTRACT................................................................................................................... v


CHAPTERS

1 INTRODUCTION .............................................................................................. 1

2 REVIEW OF THE LITERATURE ........................................................................... 4
2.1 Cardiovascular Adaptation to Pregnancy.......................................... ............. 4
2.1.1 Plasma Volume ...................................................................................... 5
2.1.1.1 M ineralocorticoids....................................................................... 5
2.1.1.2 Glucocorticoids .................................................................. ........... 6
2.1.1.3 AVP ......................... ........ .............. ........................................... 7
2.1.1.4 ANP .......................................................................................... 7
2.1.1.5 Estradiol...................................................................... ....... .... 8
2.1.1.6 Progesterone ............................................................. ......................... 9
2.1.2 Cardiac Output................................................................................. ....... 10
2.1.3 M ean Arterial Pressure (M AP) ................................................................ 11
2.1.4 Reflex Control.......................................................................................... 14
2.5 The HPA Axis.................................................................................................... 18
2.5.1 Introduction ............................................................................................. 18
2.5.2 Corticosteroid Receptors......................................................................... 19
2.5.2.1 Ligand-binding characteristics....................................................... 19
2.5.2.2 Receptor activation................................................................... .. 20
2.5.2.3 Genetic characteristics ................................................................... 21
2.5.2.4 Intracellular location.................................................................. ..... 22
2.5.2.5 Differentiation between mineralocorticoid and glucocorticoid
signals ................................................................................................. 22
2.5.2.6 Receptor auto-regulation ................................................................. 24
2.5.3 Feedback Inhibition ............................................................................... 24
2.5.3.1 Intermediate and slow feedback inhibition........................................25
2.5.3.2 Fast feedback inhibition .................................................................. 30
2.5.3.3 HPA axis during pregnancy. ................... ...................................31



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2.6 Role of Altered HPA Axis Function in Cardiovascular Adaptation to
Pregnancy ........................................................................................... ............ 35
2.7 Objectives ................................................................................... ................ 41

3 GENERAL MATERIALS AND METHODS ......................................................44
3.1 Animal Care........... ................................................... .... 44
3.2 General Surgical Procedures ...........................................................................45
3.2.1 O variectom y.......... ............................................................... .......... .. 45
3.2.2 Catheterization.................................................................... ........... ..... 45
3.3 Chronic Steroid Implantation .................................. .......................................46
3.4 Acute Progesterone Infusions...................................................................... 46
3.5 Handling and Analysis of Blood Samples........................................ ............ 47
3.5.1 Sampling and Storage.......................................................... .............. 47
3.5.2 Plasm a A nalysis.............................................. ...................................... 47
3.6 Cardiovascular Measurements........................................................................ 48
3.6.1 Mean Arterial Pressure..................... .................... ............................ 48
3.6.2 Plasm a V olum e .......................... ................... ................................... 48
3.6.3 B aroreflex ....................... ............... .................................................49
3.6.3.1 Bolus injection method .................................................................49
3.6.3.2 Steady-state method ......................................................................49
3.6.3.3 Expression of data ................................................... ............ ... 50
3.7 Tissue Collection.......................................................................................... 51
3.8 5a-Reductase / 3a-Hydroxysteroid Dehydrogenase Activity Assay ................ 52
3.8.1 Tissue Preparation............................................................................... 52
3.8.2 A ctivity A ssay....... ..... ............... ......................................... .......... ... 52
3.9 Radioligand Binding Assays ............................................................................. 53
3.9.1 Tissue Preparation................................................................ ........... 53
3.9.2 Incubations................................................... ..................................... 54
3.9.3 Separation of bound from free ............................................. ........... 54
3.10 W estern B lots ............................................................................................ 55
3.10.1 Tissue Preparation............................................................ ........... ... 55
3.10.2 Electrophoresis and Transfer ............................................ ........... .... 56
3.10.3 Densitometry................................................................................... 56
3.11 Statistical Analysis ................................................................................... 57

4 ACUTE EFFECT OF PROGESTERONE ON CARDIOVASCULAR
FUNCTION IN THE OVARY-INTACT EWE................................. ............. 58
4.1 Introduction ...................... ........................ ................................................ 58
4.2 M ethods...... ............................................... ...... ............................................ 59
4.2.1 Progesterone Infusion and MAP............................................ .......... .. 60
4.2.2 B aroreflex Test.................................................................... ............ 60
4.2.3 Statistical Analysis............................................................................. 60
4.3 R esults ....... ............................................. ..................................................... 61
4.3.1 Plasma Progesterone Levels ..................................................................61
4.3.2 A rterial Pressure..................................................................................... 61
4.3.3 B aroreflex ............................................................................................ 62


ix











4.3.4 Plasma Na' and AVP............................................................................... 63
4.3.5 Plasma Protein......................................................................................... 63
4.4 D iscussion............................................................................................. ............. 63

5 ACUTE EFFECT OF PROGESTERONE ON BLOOD PRESSURE, BLOOD
VOLUME, AND BAROREFLEX FUNCTION IN THE
OVARIECTOMIZED EWE .......................................... .......................................73
5.1 Introduction ........... .............................................................................. 73
5.2 Methods........................................................ .................................................. 75
5.2.1 30-Minute Infusion Protocol................................................................... 76
5.2.2 4-Hour Infusion Protocol ................................................ ..............77
5.2.3 Statistical Analysis................................................................................... 77
5.3 R esults............................................................................................................... 78
5.3.1 30-Minute Infusion Protocol................................................................... 78
5.3.1.1 Plasma progesterone levels.......................................... .............. 78
5.3.1.2 Arterial pressure ................................................... ................. 78
5.3.1.3 Plasma volume............................................................................... 78
5.3.2 4-Hour Infusion Protocol ..................................................... ........... 79
5.3.2.1 Plasma progesterone levels............................................... .... 79
5.3.2.4 Baroreflex..................................................................................... 80
5.4 D iscussion.......................................................................................................... 80

6 CHARACTERIZATION OF Sax-REDUCTASE ACTIVITY IN THE LIVER
AND BRAINSTEM OF THE NON-PREGNANT, PREGNANT, AND
OVARIECTOMIZED EWE ............................................................................92
6.1 Introduction ................ ......... .................................... .................................... 92
6.2 Methods.................................................................................................... 93
6.3 Results......... ................... ....................................................... ..................... 93
6.4 Discussion......................................................................................................... 95

7 CHARACTERIZATION OF BRAIN MINERALOCORTICOID AND
GLUCOCORTICOID RECEPTOR AVAILABILITY IN THE NON-
PREGNANT AND PREGNANT EWE ................................................................ 101
7.1 Introduction................................................................................................... 101
7.2 M ethods........................................................................................................... 102
7.3 Results ............................................... .. .................................................. 103
7.4 D iscussion........................................... ............... ........................................ 104

8 EFFECT OF PREGNANCY ON THE APPARENT BINDNG AFFINITY OF
THE MINERALOCORTICOID AND GLUCOCORTICOID RECEPTOR........... 111
8.1 Introduction ........................ ........................................................................ 111
8.2 M ethods........................................................................................................... 112
8 .3 R esu lts ................................ ................ .. ....................................................... 113
8.4 D iscussion...................... ...... ........................................................................ 114




x











9 CHARACTERIZATION OF CYTOSOLIC IMMUNOREACTIVE
MINERALOCORTIOID AND GLUCOCORTICOID RECEPTORS IN THE
NONPREGNANT AND PREGNANT EWE ....................................................... 121
9.1 Introduction................................ ..... ............................................................... 121
9.2 M ethods..................................................................................................... 122
9.3 R esults.................................... ................... ............................ .................... 122
9.3.1 M R Im m unoreactivity........................ .................. ........................... 122
9.3.2 GR Immunoreactivity ........... .. ............................................................... 123
9.4 D iscussion........ ........................ .. ....................................................... 124

10 EFFECT OF CHRONIC PROGESTERONE TREATMENT ON
HIPPOCAMPAL MINERALOCORTICOID AND GLUCOCORTICOID
RECEPTOR AVAILABILITY AND CYTOSOLIC IMMUNOREACTIVITY
IN THE OVARIECTOMIZED EWE................................................................ 134
10.1 Introduction ........... ...... ................. ................................................................ 134
10.2 M ethods................................................................................................... 134
10.3 R esults .............. ......... ..................................................................... ......... 135
10.3.1 Plasm a Steroid Levels....................................................................... 135
10.3.2 MR and GR Availability ........................................................................ 135
10.3.3 MR and GR Immunoreactivity............................................................... 136
10.4 D iscussion................................................................................................ 136

11 EFFECT OF CHRONIC ESTRONE TREATMENT ON HIPPOCAMPAL
MINERALOCORTICOID AND GLUCOCORTICOID RECEPTOR
AVAILABILITY AND CYTOSOLIC IMMUNOREACTIVITY IN THE
OVARY-INTACT EW E........................... ................................................... 143
11.1 Introduction ............................................................................................... 143
11.2 M ethods...................... ..................... .. .... .................................... 143
11.3 R esults ............................................................................................... 144
11.3.1 M R and GR Availability ..................................................................... 144
11.3.2 MR and GR Total Immunoreactivity...................................................... 144
11.4 D iscussion............................. .. ...................... ........................... ......... 144

12 SU M M ARY .................................................................................................... 149
12.1 O verview ....................................................................................................... 149
12.2 Specific Hypotheses Tested:...................................................................... 149
12.2.1 Hypothesis 1: Progesterone Rapidly Alters Arterial Pressure, Blood
Volume and Baroreflex Sensitivity............................................................. 150
2.2.2 Hypothesis 2: MR and GR Availability, Immunoreactivity, and
Apparent Affinity are Altered During Pregnancy....................................... 154
12.2.3 Hypothesis 3: Ovarian Steroids Alter MR and GR Availability and
Im m unoreactivity ........................................................................... 158

REFEREN CES...................................................................................................... 159

BIOGRAPHICAL SKETCH ... ............. ......................................................... 196


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CHAPTER 1
INTRODUCTION

Maternal cardiovascular homeostasis is drastically adjusted during pregnancy,

presumably for the benefit of fetal development. Many investigators have hypothesized

endocrinologic mechanisms contribute to cardiovascular adaptation to pregnancy since the

plasma concentrations of numerous reproductive hormones rise as gestation progresses.

However, the precise mechanisms leading to the observed increase in blood volume, and

decrease in MAP, vascular tone, and baroreflex responsiveness are not understood.

Remarkably, circulating estrogens and progestins increase as much as 100-fold

over non-pregnant levels during pregnancy (8). Evidence suggests progesterone is the

steroid responsible for the maintenance of pregnancy since progesterone maintains the

myometrium of the uterus in a quiescent state (8). In women, progesterone derived from

the corpus luteum of the ovary briefly supports early gestation, but progesterone is

derived primarily from the placenta for the duration of human pregnancy (8).

Progesterone levels rise to about 100 ng/ml about 45 days into a human pregnancy, fall to

about 60 ng/ml by about the 70'h day of gestation and then rise steadily to peak at about

200 ng/ml at parturition (about 289 days). In the sheep, progesterone levels are about

one-tenth the levels observed in human pregnancy (8). As in the human, progesterone

derived from the placenta can maintain ovine pregnancy in the absence of the ovaries, but

this independence of the placenta occurs at a later stage of pregnancy (8). During ovine


1








2

pregnancy, progesterone levels rise steadily and peak at about 10 ng/ml after about 130

days of gestation. At this point, plasma progesterone levels rapidly decline toward and

reach non-pregnant levels when parturition occurs after about 145 days of gestation (8).

In contrast to progesterone, estrogens enhance rhythmic contraction of the uterus

(8). Therefore, it has been suggested that progesterone dominates and maintains

pregnancy throughout the course of gestation and an increased ratio of

estrogens/progestins at the termination of gestation allows the initiation of parturition (8).

In women, estrogen biosynthesis occurs in both the fetus and placenta (8). Plasma

estrogen levels begin to rise steadily beginning about 100 days into human gestation and

peak at about 200 ng/ml at parturition. In sheep, estrogen concentrations are much lower

than they are in women and estrogens are primarily synthesized by the placenta in sheep

(8). In this species, plasma estrogen levels remain below 30 pg/ml until about the 140&

day of gestation then rise rapidly to about 200 pg/ml at parturition (8).

Over the years, numerous studies have revealed elevated estrogens contribute to

cardiovascular adaptation to pregnancy. In contrast, the importance ofprogestins has

long been debated since studies using supra-physiological levels ofprogestins actually

suggest progestins decrease MAP by reducing blood volume. More recent studies

demonstrate chronic treatment with physiological levels of progesterone results in

expanded plasma volume and reduced MAP, suggesting physiological levels of

progesterone contribute to maternal cardiovascular homeostasis.

Hormones mediate specific effects by interacting with specific receptors in target

tissues, and progesterone certainly produces many of its effects by interacting with the

isoforms of the intracellular steroid-binding transcription factor commonly known as the








3


progesterone receptor. Evidence also suggests progesterone and its metabolites interact

with intracellular and extracellular receptors not commonly considered progesterone

receptors. A metabolite of progesterone, tetrahydroprogesterone (THP), is now known to

potentiate the effects of the neurotransmitter y-aminobutyric acid (GABA) at the GABAA

chloride channel and accumulating evidence suggests this progesterone metabolite rapidly

modulates cardiovascular function by interacting with this receptor type.

Progesterone also interacts, with varying affinity and efficacy, with two

intracellular steroid-binding transcription factors known as the corticosteroid

(mineralocorticoid (MR) and glucocorticoid (GR)) receptors. These two novel

progesterone receptors modulate the responsiveness and mediate the effects of the

hypothalamic-pituitary-adrenal (HPA) axis, an endocrine axis known to respond to

physiological and psychological stress, cardiovascular disturbances, and to contribute to

cardiovascular homeostasis. Therefore, interaction of progesterone with the

corticosteroid receptor system may be an important genomic mechanism of cardiovascular

adaptation to pregnancy.

The availability of these numerous progesterone receptors probably contributes to

a multitude of time- and concentration-dependent progesterone effects. The studies

presented in this dissertation were designed to determine if physiological levels of

progesterone could contribute to cardiovascular adaptation to pregnancy by interacting

with these novel progesterone receptors.


















CHAPTER 2
REVIEW OF THE LITERATURE

The setpoint and the response of both the cardiovascular system and the

hypothalamic-pituitary-adrenal (HPA) axis are altered during pregnancy. This dissertation

is based on the hypothesis that elevated concentrations of ovarian steroids contribute to

adaptation of these two functionally interdependent systems to pregnancy. This chapter

reviews current knowledge of cardiovascular and HPA axis function during pregnancy,

outlines the functional integration of the two systems, and considers potential mechanisms

of adaptation to pregnancy.

2.1 Cardiovascular Adaptation to Pregnancy


Studies consistently describe decreased mean arterial pressure (MAP) and

increased plasma volume, heart rate, and cardiac output in pregnant women (68), rabbits

(110), dogs (27), rats (51), and ruminants (186). These changes are essential for normal

fetal development since the incidence ofgestational complications increases when these

variables fail to adjust (92, 206). The correlation of altered cardiovascular function with

hormonal rhythms both during pregnancy and during the menstrual cycle led to the

hypothesis that ovarian steroids contribute to adaptation of the cardiovascular system

(169). As will be discussed below, numerous studies demonstrate estradiol induces many

cardiovascular changes characteristic of pregnancy, and recent evidence suggests



4








5


physiological concentrations of progesterone also contribute to cardiovascular adaptation

to pregnancy.

2.1.1 Plasma Volume

Plasma volume expands by 40-50% in gravid humans and rats, but in species such

as the sheep plasma volume increases only about 10% during pregnancy (187).

Differential modulation of plasma mineralocorticoids, glucocorticoids, arginine

vasopressin (AVP), and atrial natriuretic peptide (ANP) contribute to volume expansion

during pregnancy.

2.1.1.1 Mineralocorticoids

The renin-angiotensin system regulates adrenocortical secretion of the

mineralocorticoid aldosterone. The smooth muscle cells in the afferent and efferent

arterioles of the juxtaglomerular apparatus release renin, the rate-limiting enzyme of the

renin-angiotensin system, in response to decreased perfusion pressure, increased renal

sympathetic nerve activity, and decreased sodium delivery to the macula densa. Renin

converts liver-derived angiotensinogen to angiotensin I, and angiotensin I is subsequently

converted to angiotensin II by the ubiquitously distributed endothelial angiotensin

converting-enzymes. Angiotensin II stimulates aldosterone secretion from the zona

glomerulosa of the adrenal cortex by increasing activity of steroid synthesis enzymes.

Adrenocorticotropin (ACTH), a peptide released from the anterior pituitary when the

HPA axis is stimulated, also contributes to mineralocorticoid synthesis by increasing

enzyme activity. Both angiotensin II and aldosterone are important modulators of plasma

volume. Angiotensin II stimulates vasoconstriction of arterioles, AVP secretion from the

posterior pituitary, sodium (Na') reabsorption in the proximal tubule, and acts in the








6

hypothalamus to stimulate drinking behavior. Aldosterone also stimulates Na' and

subsequent water reabsorption in the late portion of the distal tubule and the collecting

duct (152). During pregnancy plasma angiotensinogen, renin activity, angiotensin II,

angiotensin converting-enzymes, and aldosterone increase and may contribute to volume

expansion (173).

2.1.1.2 Glucocorticoids

The doubling of maternal plasma glucocorticoid (cortisol is the primary

glucocorticoid in the human and the sheep) concentrations during pregnancy (65, 132,

199) may contribute to the shift of fluid into the vascular space. Like mineralocorticoids,

glucocorticoids shift Na+ and subsequently fluid out of cells (162). However,

mineralocorticoids alone are not sufficient to replenish plasma volume in adrenalectomized

dogs (263). Cortisol also increases hepatic protein production and accelerates movement

of interstitial protein into the vascular system (16, 266). As a result, plasma proteins

increase and enhance oncotic pull of fluid into the vascular system. Gann and Pirkle (83,

218-220) propose cortisol first shifts electrolytes and fluids into the interstitial space. As a

result of the increased interstitial pressure, transcapillary filling and lymphatic flow

increase, and the increased lymphatic flow increases circulating protein and oncotic pull of

fluid into the vascular system. Although electrolyte and protein shifts induced by

mineralocorticoids and glucocorticoids could contribute to volume expansion during

pregnancy, plasma oncotic pressure and osmolality would be expected to remain

unchanged following equilibration (257). Since plasma oncotic pressure and osmolality

actually fall during pregnancy (64), additional mechanisms probably contribute to volume

expansion and hemodilution during pregnancy.








7


2.1.1.3 AVP

AVP release from the posterior pituitary also is regulated differently during

pregnancy. Hypothalamic osmoreceptors and, to a lesser extent, the baroreceptors

mediate increased AVP release in response to hyperosmolality or hypotension,

respectively (152). AVP increases aquaporin expression and water reabsorption in the

collecting duct (149). Basal concentrations of plasma AVP are not changed in pregnant

humans, rats, or sheep, but the setpoint for stimulation of AVP does reset during

pregnancy (134, 165). The osmotic threshold for stimulation of AVP and thirst is reduced

during all stages of human pregnancy and the slope of the linear relationship between

plasma osmolality and AVP is reduced in late pregnancy (165). In humans, increased

placental production ofAVP-degrading aminopeptidases contributes to the apparent reset

in AVP regulation (165), but these enzymes are not increased in the sheep and other

species during pregnancy (230). In pregnant ewes, the relationship between plasma

osmolality and AVP is not altered, but the relationship between MAP and AVP is shifted

to the left in this species (134) and in dogs (27). These findings suggest altered AVP

responsiveness contributes to decreased arterial pressure in the presence of expanded

volume, but it is unclear if AVP modulates volume expansion during pregnancy.

2.1.1.4 ANP

Differential regulation of plasma ANP levels and target tissue responsiveness to

ANP during pregnancy contributes to cardiovascular adaptation to pregnancy. Normally,

volume-induced atrial stretch stimulates atrial myocytes to release ANP and increase water

and Na' excretion. ANP is a potent vasodilator, increases glomerular filtration rate and

filtered Na' load and inhibits renin, aldosterone, and AVP release and Na' reabsorption by








8


the collecting duct (152). Basal circulating ANP either increases or remains the same

during pregnancy (127) and pregnancy attenuates both atrial ANP release in response to

atrial distension and renal responsiveness to ANP (126, 203, 204). Thus, differential

attenuation of the ANP system contributes to volume expansion and hemodilution during

pregnancy.

2.1.1.5 Estradiol

Chronic treatment of ewes with estradiol (174, 277) and women with estrogen-

containing oral contraceptives (3, 14) increases blood volume. Estrogens do not appear to

increase volume by altering endocrine control of fluid homeostasis.

Although some evidence suggests estrogens increase plasma volume by

augmenting activity of the renin-angiotensin system (128), a recent study by Magness and

colleagues suggests the estrogen-induced increase in renin-angiotensin system activity is a

temporary baroreflex-mediated response to the estrogen-induced decrease in MAP (174).

These investigators demonstrate renin activity and angiotensin II concentrations normalize

as volume shifts into the dilated vascular beds (174).

AVP content of the hypothalamic paraventricular nucleus and the posterior

pituitary peaks in the rat at proestrus when plasma estrogen concentrations are the highest

(53, 94). However, administration of estrogens to rats does not alter the threshold for

osmotic stimulation of AVP and actually increases the slope of the relationship between

plasma osmolality and AVP (59). Investigators have found the effect of estrogen

replacement on plasma AVP depends on the route and duration of administration (215).

As a result, further study is required to establish the role of estrogens in adaptation of the

AVP response during pregnancy.








9


Similarly, estrogen treatment does not mimic the effects of pregnancy on ANP

release (126). As will be discussed below, estrogens probably contribute to volume

expansion by decreasing vascular resistance and allowing volume to shift into the vascular

space.

2.1.1.6 Progesterone

Recent evidence suggests progesterone also may contribute to volume expansion

during pregnancy. Early studies demonstrated supraphysiological concentrations of

progesterone antagonize the salt and fluid retaining actions ofmineralocorticoids (254,

287). However, studies demonstrate plasma renin activity, aldosterone, and volume are

increased in women during the progesterone-dominant luteal phase of the menstrual cycle

(97, 247) and in men after administration of progesterone (187). Chronic treatment with

physiological concentrations of progesterone over 10-14 days also increases plasma

volume in ovariectomized ewes, but without increasing activity of the renin-angiotensin

system (210). These findings are contradictory to the anti-mineralocorticoid hypothesis of

progesterone action and suggest progesterone enhances volume expansion during

pregnancy.

Progesterone does not appear to expand plasma volume by increasing activity of

the renin-angiotensin system (210) or decreasing release of ANP in response to atrial

stretch (296). However, progesterone treatment does diminish the natriuretic effect of

ANP in the kidney (203). Progesterone also may contribute to plasma volume expansion

in the ewe by increasing AVP (210), although evidence suggests progesterone actually

decreases plasma AVP in ovariectomized rats (52). Progesterone may indirectly increase

AVP by increasing sodium appetite (50, 178, 188) and plasma Na concentration (210) or








10


progesterone may modulate regulation of AVP by corticosteroid receptors in the

hypothalamus.

2.1.2 Cardiac Output

Increased heart rate and stroke volume augment cardiac output during pregnancy

(173). Magness and colleagues (173) have demonstrated estradiol increases cardiac

output. In the acute phase of estradiol treatment (approximately 3 days), estradiol does

not alter stroke volume and increases cardiac output by increasing heart rate (174). Blood

volume expands after about 7 days of estradiol treatment and increases stroke volume and

further enhances cardiac output (174). This finding suggests increased blood volume and

end-diastolic filling volume augment stroke volume. Some evidence suggests estrogens

enlarge the left ventricular chamber and allow increased end-diastolic volume without

increased end-diastolic filling pressure (86). Although evidence suggests estrogens

increase cardiac output, the effect of progesterone on cardiac output has not been

reported.

A recent study by Brooks and colleagues (26) suggests altered parasympathetic

and sympathetic neural control allows heart rate to increase during pregnancy. Basal

sympathetic output to the heart increases, and the observed decrease in gain of reflex

control of heart rate is due to decreased sympathetic response reserve. Furthermore,

reduced cardiac sensitivity to cholinergic agents decreases the ability of the

parasympathetic system to maximally reduce heart rate.

Currently, the effect of ovarian steroids on control of the heart rate is not

completely understood. As discussed above, estradiol increases heart rate (174, 277), but

physiological concentrations of progesterone may actually decrease heart rate (210).








11

Further study is required to determine the effects of estradiol and progesterone on

sympathetic and parasympathetic output to the heart and on cardiac responsiveness to

adrenergic and cholinergic neurotransmitters.

2.1.3 Mean Arterial Pressure (MAP)

A decrease in total peripheral resistance reduces MAP by 5-10 mm Hg during

pregnancy (67). An early hypothesis argued that the addition of a low-resistance

uteroplacental circulation contributes to decrease in total peripheral resistance (169).

However, a recent study of the pregnant guinea pig demonstrates the increase in total

vascular conductance is due largely to an increase in non-uteroplacental conductance (54).

Evidence suggests ovarian steroids decrease resistance in non-uteroplacental vascular

beds.

Chronic estradiol treatment decreases MAP and vascular tone (174, 277) and since

the progesterone-induced decrease in MAP is accompanied by an expansion of plasma

volume, progesterone also appears to decrease vascular tone (210). Chronic progesterone

treatment also reduces blood pressure in humans, dogs, and rats under conditions in which

mineralocorticoid levels are not elevated (5, 31, 63, 84, 260). The contribution of ovarian

steroids to decreased vascular tone and arterial pressure is incompletely understood but

may include direct regulation of the vascular system or indirect regulation of endocrine

and neural modulators of vascular reactivity and tone.

Estrogen and progesterone receptors are located in vascular endothelial nuclei

(107, 114, 121, 148, 160, 164, 205, 214). Estrogen and progesterone may bind these

vascular steroid receptors and decrease peripheral vascular resistance via direct or indirect

mechanisms.








12

Evidence demonstrates progesterone has direct and indirect effects on vascular

tone. Using isolated placental arteries and veins, Omar and colleagues have recently

demonstrated progesterone directly elicits an acute reduction in vascular tone via a direct

mechanism in the vascular endothelium that is mediated by both intracellular progesterone

receptors and cyclic adenosine monophosphate (202). It is unclear whether this

progesterone effect on placental vascular tone also occurs in the maternal peripheral

vascular system. Evidence also suggests a 5c-reduced metabolite of progesterone,

dihydroprogesterone (DHP), contributes to decreased vascular tone and reactivity via an

indirect mechanism. DHP attenuates the pressor response to angiotensin II by increasing

circulating prostaglandins through an unknown receptor mechanism (71). Vascular

refractoriness to angiotensin II can be abolished in normal gravid women by administering

the prostaglandin synthesis inhibitor, indomethacin. Dihydroprogesterone restores

vascular refractoriness to angiotensin II in normal pregnancy women treated with

indomethacin and in women with pregnancy-induced hypertension who do not normally

exhibit refractoriness to angiotensin II (71). Since infusions of DHP were used in these

studies, it is not known if the GABAA-active 3a-dehyrogenated metabolite,

tetrahydroprogesterone (THP), contributes to the effects of DHP.

Estrogens appear to have an indirect effect on both vascular tone and vascular

reactivity. Estrogens contribute to decreased vascular tone by increasing production of

the endothelium derived relaxing factor, nitric oxide (NO), and vasodilatory

prostaglandins (173, 248). As described above, prostaglandins also contribute to

decreased vascular reactivity. Molnar and Hertelendy (191) have reported that in vivo

inhibition of nitric oxide synthase in pregnant rats increases vascular responsiveness to








13


angiotensin II, norepinephrine and AVP so that the responsiveness of the pregnant rats is

indistinguishable from the responsiveness of postpartum control rats.

Ovarian steroids could modulate sympathetic output to the vascular system.

However, decreased sympathetic output probably does not contribute to the observed

decrease in peripheral resistance during pregnancy since evidence suggests sympathetic

vascular tone increases during pregnancy (264). Ovarian steroid receptors are located in

brain regions associated with cardiovascular control (259). Ovarian steroids could have

currently uncharacterized direct effects in these brain regions or ovarian steroids could

modulate other central components involved in sympathetic control of the vascular

system. For example, ovarian steroids do regulate brain corticosteroid receptors (271)

known to modulate neural control of blood pressure.

As will be discussed below, there are two types of corticosteroid receptors:

mineralocorticoid (MR) and glucocorticoid (GR) receptors. Brain MR and GR appear to

regulate neural control of blood pressure. Central infusion of aldosterone increases blood

pressure by increasing vascular resistance in the rat and dog without increasing plasma

sodium concentration (119, 216), and the MR antagonist RU 28318 blocks the central

effect ofaldosterone (90). These finding suggest brain MR modulate increases in

sympathetic outflow (279-282). Some evidence suggests that the brain renin-angiotensin

system mediates increases in sympathetic outflow induced by centrally administered

corticosteroids (265). The effects of centrally administered hydrocortisone on blood

pressure, heart rate, and sympathetic nerve activity can be abolished by

intracerebroventricular pretreatment with an angiotensin II receptor antagonist or an

angiotensin I converting-enzyme inhibitor (265). Although Gomez-Sanchez and








14

colleagues did not observe a change in blood pressure after central administration of the

GR-selective agonist RU 26988 (91), van den Berg and colleagues suggest activation of

central GR actually decreases arterial pressure (282). As a result, currently available data

suggest activation of central MR increases sympathetic outflow and central GR decreases

or does not control sympathetic outflow to the vascular system.

As will be discussed below, the sheep brain does not express significant MR-

protecting 1 Ip-hydroxysteroid dehydrogenate activity (145). Despite this fact,

intracerebroventricular infusion of aldosterone (269) or cortisol (144) does not change

MAP in this species. The failure of central corticosteroids to increase arterial pressure in

the sheep may be due to decreased brain MR availability in this species. The failure of

central glucocorticoids to decrease arterial pressure may be due to the lack of the

opportunity of infused glucocorticoids to inhibit sympathetic outflow in the resting,

unstressed animal. It is currently unknown how central MR and GR regulate arterial

pressure in any species during pregnancy when circulating corticosteroids, ovarian

steroids, and other endocrine factors that may modify MR and GR are elevated.

2.1.4 Reflex Control

The evidence presented above details potential mechanisms of adaptation of

resting cardiovascular function to pregnancy. Studies also demonstrate reflex control of

arterial pressure also is altered during pregnancy (29, 98, 133). Neural stretch receptors

located in the wall of internal carotid artery and in the wall of the aortic arch respond to

perturbations in arterial pressure (234). Signals from the baroreceptors are integrated

with other pressure-regulating inputs in the nucleus of the tractus solitarius and other brain

regions (4, 41, 283) and neural output to the periphery is modified accordingly (234).








15


Heart rate and renal sympathetic nerve responses to perturbations in arterial pressure are

commonly used to index reflex control of arterial pressure (69), and numerous studies of

reflex control of arterial pressure during pregnancy have been conducted (27, 28, 47, 51,

109-113, 133, 159, 179, 223). The results of these studies have led investigators to

conclude the gain of the baroreflex may be augmented, attenuated, or not changed.

Brooks and colleagues (29) argue these studies draw conflicting conclusions because they

look at different portions of the baroreflex curve. While some studies examine the entire

baroreflex curve, most studies focus only on the response to an increase or decrease in

blood pressure. A careful analysis of the entire baroreflex curve suggests an attenuated

response to hypotension accounts for a decrease in overall baroreflex sensitivity during

pregnancy (29). Elevated ovarian steroids could modify reflex control of arterial pressure

during pregnancy by modifying responsiveness of the pressure sensitive stretch receptors,

signal processing in the brain integration centers, or sensitivity of targets controlled by

neural outflow.

Estrogens do not appear to reduce the gain of the baroreflex (29), but evidence

does suggest progesterone metabolites have rapid, nongenomic actions on the baroreflex.

Progesterone can be sequentially metabolized (see Figure 6.1) by 5oc-reductase and 3oc-

hydroxysteroid dehydrogenase in the brain and periphery to form tetrahydroprogesterone

(THP). THP, which circulates at elevated levels during pregnancy, enhances activation of

the GABAA receptor by GABA (176). Rogers and Heesch (98) recently demonstrated

that THP rapidly causes a leftward shift in setpoint and at the same time decreases the

overall sensitivity of renal sympathetic nerve responses to changes in arterial pressure.

These investigators propose that THP acts in the rostral ventral lateral medulla (RVLM)








16

to enhance GABAergic activity and attenuate sympathetic outflow (98). Baroreceptor

neurons that monitor pressure in the internal carotid and aortic arch send information to

the nucleus of the tractus solitarius in the caudal dorsomedial medulla oblongata. This

information is integrated and relayed to GABA-containing cell bodies in the caudal ventral

lateral medulla via at least one intermediate synapse. These GABA-containing cell bodies

project to the RVLM where GABA inhibits excitatory amino acid projections to the

intermediolateral cell column. The intermediolateral cell column is the source of

preganglionic sympathetic cell bodies and all sympathetic outflow (4, 41, 262, 283).

Therefore, by enhancing GABA inhibition of excitatory projections to the

intermediolateral cell column, the elevated concentrations of THP present during

pregnancy may attenuate sympathetic outflow.

A more recent study by Masilamini and Heesch (179) suggests pregnancy and THP

selectively alter the renal sympathetic nerve response and not the heart rate response to

changes in arterial pressure. However, resting heart rate was elevated in this study and the

majority of the response curve was obtained from the bradycardic response to pressure

elevation. Accumulated evidence suggests sympathetic outflow to both the heart and the

vascular system increases during pregnancy (26, 264), and reduced sympathetic reserve

may account for the observed decrease in reflex gain. As a result, future investigations of

the effect of ovarian steroids on components of sympathetic outflow and reflex gain must

carefully ensure experiments commence at resting conditions. In conclusion, these studies

suggest progesterone metabolites may modify central processing of pressure-regulating

signals. The effects of ovarian steroids on the responsiveness of pressure-sensing neurons








17


and on target responsiveness to the outflow arm of the reflex also should be examined as

potential targets for ovarian steroid modulation of reflex control.

The pregnancy-induced plasma volume expansion also could contribute to a

decrease in baroreflex sensitivity since other investigators have reported an attenuation of

the baroreflex in the volume-loaded state (49). Expanded plasma volume is sensed by the

low-pressure cardiopulmonary stretch receptors (13). These receptors sense volume

expansion and increase heart rate (285), decrease renal sympathetic nerve activity (239),

and attenuate reflex control of the heart and peripheral resistance (12). Increased

stimulation of the cardiac mechanoreceptors by increased volume may contribute to many

of the observed changes in reflex control of arterial pressure during pregnancy.

The accumulated evidence demonstrates cardiovascular function is altered during

pregnancy. Cardiovascular adaptation to pregnancy appears to commence as increased

concentrations of circulating vasodilators (nitric oxide and prostaglandins) reduce vascular

resistance and reactivity and allow volume to expand and other variables to reset

reflexively. Evidence strongly suggests ovarian steroids contribute to the initiation and

maintenance of this adaptation. The importance of estrogens in cardiovascular adaptation

to pregnancy is fairly well established, and the importance of progesterone appears to have

been previously underestimated. Further study is needed to define the role of

progesterone in cardiovascular adaptation to pregnancy and to define which of the many

potential receptor mechanisms contribute to progesterone's effects on the cardiovascular

system.








18

2.5 The HPA Axis


2.5.1 Introduction

While the adrenal medulla functions primarily as an epinephrine-secreting

sympathetic ganglion, the adrenal cortex secretes corticosteroids (85). Steroid

synthesizing enzymes are differentially distributed in the cortex (85). As a result, the zona

glomerulosa, the outer portion of the cortex, primarily secretes the mineralocorticoid

aldosterone (85). The inner cortex, the zonas fasciculata and reticularis, primarily secrete

glucocorticoids (cortisol in the human, dog, and sheep and corticosterone in the rat) and

lower concentrations of androgens (85). Aldosterone and cortisol (or corticosterone in

the rat) were originally labeled the primary mineralocorticoid and glucocorticoid

hormones, respectively, based on their relative effects on electrolyte and glucose

homeostasis (245).

While, as discussed above, the renin-angiotensin system and plasma potassium

levels regulate synthesis of aldosterone by the zona glomerulosa, anterior pituitary-derived

adrenocorticotropin (ACTH) controls adrenocortical secretion of cortisol (246). ACTH

secretion is controlled by release of corticotropin releasing-factor (CRF) and arginine

vasopressin (AVP) from parvocellular neurons of the hypothalamic paraventricular

nucleus (100). This system, commonly referred to as the hypothalamic-pituitary-adrenal

(HPA) axis, has become known as a stress-responsive system that initiates mechanisms

essential for immediate survival and/or contributes to recovery from a homeostatic

imbalance. However, the role of adrenal glucocorticoids in basal and stimulated

homeostasis is still under debate (196).








19

The parvocellular neurons of the paraventricular nucleus receive neural signals

from multiple brain regions (100). Except in the dog (142) and sheep (9) there is a

circadian rhythmicity in HPA axis activity in all species studied (142). Neurally integrated

light input from the retina entrains the suprachiasmatic nucleus of the hypothalamus (193),

and neural projections from the suprachiasmatic nucleus control paraventricular activity in

these species (10, 194).

There are numerous forms of stress (82) and multiple neural pathways impinge on

the paraventricular nucleus (100). While information about systemic or physiological

stress is relayed to the paraventricular nucleus through brainstem aminergic/peptidergic

pathways or from projections originating in blood-brain-barrier-deficient subfornical

organs, information about psychological or experiential stress is relayed indirectly from the

prefrontal cortex, hippocampus, or amygdala (100). Since multiple pathways mediate

stress-induced activation of the HPA axis, each pathway could be modulated by several

factors.

2.5.2 Corticosteroid Receptors

2.5.2.1 Ligand-binding characteristics

Corticosteroids modulate target tissues through two intracellular receptors (182).

The two corticosteroid receptors co-localize in most tissues (182). One of these cytosolic

receptors is known as the mineralocorticoid receptor (MR) based on its role in classic

mineralocorticoid target tissues (81). The MR can be described by its rank order of

affinity for the major circulating corticosteroids: corticosterone, cortisol, and aldosterone.

In the rat, the MR has a high affinity for corticosterone and aldosterone (-0.5 nM) and a

lower affinity for cortisol (226). In a species in which cortisol is the major glucocorticoid








20

(the dog), the MR also has a very high affinity for corticosterone (- 0.05 nM) and a lower

affinity (-0.2 nM) for cortisol and aldosterone (227). The other cytosolic corticosteroid

receptor is known as the glucocorticoid receptor (GR). In the rat, the GR also has a high

affinity (but lower than the MR) for corticosterone (-5 nM) and an even lower affinity

(-20 nM) for cortisol and aldosterone (226). In the dog, the GR has an equal but lower

affinity than the MR for corticosterone and cortisol (-5 nM) and a two-fold lower affinity

(-10 nM) for aldosterone (227). As a result of these binding studies, the MR is frequently

described as the high affinity, low capacity receptor, and the GR is described as the lower

affinity, high capacity receptor (61). These binding parameters demonstrate these two

corticosteroid receptors cross-reactively bind the major circulating corticosteroids. Of

course, binding studies do not reveal the efficacy of a given ligand-receptor complex.

However Rupprecht and colleagues (233) have demonstrated, using human

neuroblastoma cell cultures co-transfected with vectors containing human MR or GR and

a mouse mammary tumor virus-luciferase reporter gene, that each of the major circulating

corticosteroids is efficacious at both receptors. The human MR (6) and GR (104) have

been cloned. Both receptors are intracellular, steroid-binding transcription factors and

members of the steroid and thyroid hormone superfamily (37, 70).

2.5.2.2 Receptor activation

In the absence of hormone, steroid receptors form a complex with heat-shock

proteins 90, 70, and 56 (270). Receptor-associated heat shock proteins appear to

maintain the receptor in a state capable of binding ligand and incapable of mediating

transcription (270). However, once steroids bind with the receptor, the steroid-receptor

complex dissociates from the heat shock proteins, dimerizes with another steroid-receptor








21

complex, and mediates gene silencing and activation (270). Once the steroid-receptor

complex obtains this activated state, the ligand does not freely dissociate (42). Evidence

suggests phosphorylation regulates steroid receptor activity and ligand-independent events

also can activate steroid receptors (270).

2.5.2.3 Genetic characteristics

The amino acid sequences of the two proteins are highly homologous and their

structural and functional domains are similar (6). The amino terminal region varies in size

and bares no structural homology among the human MR and GR, but the carboxy-terminal

ligand-binding domain (57% identity) and the cysteine-rich zinc-finger DNA-binding

domain (94% identity) are highly homologous (6). These structural similarities allow the

two receptors to have similar steroid binding and activation profiles (233) and to bind

closely related DNA target sequences (6).

There are two splice variants of the human and rat MR and each isoform is

regulated by a separate promoter (15, 39, 155, 295), but the functional role of each

isoform has not been described. There also are two isoforms of the human GR (104), and

GRJ3 inhibits GRa activity (200). The ovine MR has not been cloned, but 942 base pair

segment of the ovine GR corresponding to residues 143-453 of the human GR (80%

identity) has been cloned (294). The availability of multiple receptor forms suggests

steroid receptor physiology is immensely complex. Each receptor isoform would be

expected to have a different rank order of affinity for the endogenous ligands and each

ligand-receptor complex would be expected to bind various genetic response elements

with varying affinity and efficacy.








22

2.5.2.4 Intracellular location

Considerable debate has focused on the intracellular location of steroid receptors.

Since early studies detected radiolabeled steroid binding in the cytosoluble fraction of

tissue homogenates, a two-step model of steroid receptor activation was proposed (117).

This model proposed steroids bind their receptor in the cytosol and the activated steroid-

receptor complex translocates to the nucleus and regulates transcription (117). The

cytosolic location of functional MR and GR has been largely discounted by

immunocytochemical evidence demonstrating nuclear localization (185). Although some

immunocytochemical studies demonstrate cytosolic location, tissue fixation technique can

distort the apparent intracellular location of the steroid receptors (185). Studies clearly

demonstrating an absence of cytosolic steroid receptor binding in enucleated cells suggest

detectable "cytosolic" binding represents extraction of nuclear proteins into the

cytosoluble homogenate fraction (289). Of course, steroid receptors must be translated in

the cytosol, and an energy-requiring nuclear shuttling mechanism for these proteins does

exist (95). The evidence strongly suggests the functional steroid receptors are located in

the nucleus and can be easily extracted into the cytosol during tissue homogenization, but

the debate continues.

2.5.2.5 Differentiation between mineralocorticoid and glucocorticoid signals

The microsomal enzyme 11P -hydroxysteroid dehydrogenase (11-PHSD) plays a

role in determining when MR is exposed to circulating glucocorticoids. There are at least

two isoforms of this enzyme (244). One isoform (110-HSD2) reacts primarily in one

direction and converts cortisol to cortisone or corticosterone to 11-dehydrocorticosterone

(244). Funder and colleagues have demonstrated 11 3-HSD2 colocalizes with the MR and








23

protects the MR from activation by circulating glucocorticoids in the distal nephron of

most species and in the rat brain (80). However, significant conversion ofcortisol to

cortisone does not occur in the ovine brain (145). Another isoform of the enzyme (110-

HSD1) appears to be the predominate isoform in the liver of most species and the brain of

species including the sheep and human (244). This isoform of 110I-HSD is bi-directional

(192). This isoform (111-HSD1) has a relatively low affinity (in the pM range) for

cortisol and corticosterone and appears to proceed in the direction favoring conversion of

cortisone to cortisol or 11-dehydrocorticosterone to corticosterone at physiological

glucocorticoid concentrations (244). As a result, 11 -HSD appears to inactivate

glucocorticoids only when circulating concentrations are high (244). In summary, there

appear to be species and tissue differences in the protection of MR from circulating

glucocorticoids by 11-PHSD, and the MR in the human and ovine brain appears to be

particularly vulnerable to activation by circulating glucocorticoids.

Some evidence suggests intrinsic receptor properties also contribute to the ability

of MR to differentiate between aldosterone and glucocorticoids. Although the MR binds

corticosterone and cortisol with a high affinity, the aldosterone-MR complex is more

potent than the cortisol/corticosterone-MR complex since the half maximal effective dose

is approximately 100-fold lower (168, 233). However, aldosterone and cortisol both

promote transcription in cells transfected with human MR, and the cortisol-MR complex is

actually more efficacious than the aldosterone-MR complex since cortisol induces almost

2-fold more reporter gene transcription than aldosterone (233). This suggests that all of

the major corticosteroid ligands exert physiological effects through the MR. Evidence

collected by Pearce and colleagues demonstrates one level at which MR and GR obtain








24


selectivity: the MR and GR interact differently with non-receptor transcription factors

(209). However, the mechanisms by which the MR and GR obtain selectivity are still

poorly understood, and corticosteroid physiology will probably be most accurately

portrayed when described in terms of cross-reactivity.

2.5.2.6 Receptor auto-regulation

The MR and GR respond to circulating corticosteroids in a manner that has been

termed auto-regulation (34, 231, 288). When plasma corticosteroid concentrations

increase, transcription of MR and GR decreases. In contrast, when plasma corticosteroid

concentrations decrease, transcription of MR and GR increases. These findings

demonstrate MR and GR are auto-regulated by corticosteroids, suggesting that the

expression of both MR and GR should be decreased during pregnancy when circulating

corticosteroids increase.

2.5.3 Feedback Inhibition

Negative feedback inhibition of the HPA axis occurs in three time domains (141):

fast (seconds to minutes), intermediate (hours), and slow (hours to days). MR and GR

mediate intermediate and slow feedback inhibition of the axis in the hippocampus (115),

hypothalamus, pituitary, and perhaps even the adrenal itself (141). In addition to these

feedback sites located at central sites in the HPA axis, MR and GR may mediate feedback

inhibition at diffuse relay stations in the afferent nerve pathways (141). As will be

discussed below, the receptors involved in fast feedback inhibition are unknown.








25

2.5.3.1 Intermediate and slow feedback inhibition

Intermediate and slow feedback inhibition requires genomic events and protein

synthesis (141). While intermediate feedback inhibits the release of ACTH and the

synthesis and release of CRF, slow feedback inhibits both the synthesis and release of CRF

and ACTH (141).


2.5.3.1.1 Role ofMR and GR
Recent studies suggest distinct roles for MR and GR in feedback inhibition. Reul

and de Kloet observed the high affinity MR is almost completely activated and the lower

affinity GR is mostly unoccupied at the trough of the HPA axis circadian rhythm in the

unstressed animal (226). The lower affinity GR becomes activated at the peak of the

rhythm and in the stressed animal (226). This evidence led to a binary theory of

corticosteroid receptor action: basal concentrations ofcorticosteroids exert their feedback

effects through the MR and high concentrations (at the peak of the rhythm and during

stress) exert their effects through the GR (55, 226, 228, 229). A study by Dallman and

colleagues suggests the GR alone is not able to suppress activity of the HPA axis;

activation of both the MR and GR is required for feedback inhibition at the peak of the

rhythm (22). This finding suggests that although the MR may function as the high affinity

receptor and the GR may function as the lower affinity receptor, the two receptors

probably function in concert. Indeed, accumulating evidence suggests the two

components of this binary receptor system may interact. Steroid receptors are known to

form dimers before binding to the genetic response element (270), and recent evidence

suggests the MR and GR can form heterodimers (166, 267). The precise role of

corticosteroid receptor heterodimers in target tissue activity and feedback control of the








26


axis has not been studied. Genetic studies may eventually differentiate between MR and

GR homodimer and heterodimer responsive elements, and future physiological studies

should consider the relative availability of MR and GR in the target tissue of interest, the

concentration of corticosteroid obtained, and the resulting relative amounts of activated

MR and GR homodimers and heterodimers.


2.5.3.1.2 Role of the Hippocampus
The hippocampus is a functionally unique site that may be involved in basal activity

of the axis and feedback inhibition and facilitation. The hippocampus was first suspected

as a regulatory site after McEwen and colleagues demonstrated the hippocampus

concentrates more radiolabeled corticosterone than any other portion of the rat brain

(184). The hippocampus integrates afferent exteroceptive and interoceptive sensory data

for efferent projection to the prefrontal cortex, autonomic centers (including

cardiovascular centers), and motor nuclei, and plays a primary role in cognition and

emotions (19, 235). Therefore, it seems likely the hippocampus regulates the stress-

activated HPA axis (115). In fact, the hippocampus appears to be the primary site for

MR-mediated effects on HPA axis activity in the rat since MR is nearly undetectable in the

rodent pituitary and hypothalamus (226). Since the hippocampus lacks direct projections

to the paraventricular nucleus of the hypothalamus, the hippocampus probably regulates

the paraventricular nucleus via at least one intermediary synapse in the bed nucleus of the

stria terminalis (100). As a result of integration ofhippocampal control of the axis with

other neural inputs at relay synapses, the effect of the hippocampus on axis activity is

probably extremely sensitive to alteration by other neural inputs.








27


2.5.3.1.2.1 Basal Activity
The role of the hippocampus in regulation of the HPA axis has been thoroughly

reviewed by Jacobson and Sapolsky (115) and more recently by Feldman and Weidenfeld

(73) and Herman and colleagues (100). A majority of studies suggest excitatory output

from the hippocampus inhibits activity of the axis (115). The hippocampus inhibits basal

activity of the axis: electrical stimulation of the hippocampus decreases circulating

corticosteroids in the pigeon (17), human (177, 232) and cat (249) and ablative

hippocampal or fornix lesions also increase plasma corticosteroids, ACTH or P-endorphin

levels in several species (17, 76, 101, 150, 161, 172, 190, 198, 237, 292).


2.5.3.1.2.2 Circadian Rhythms
The hippocampus also regulates the circadian rhythm of the axis (115). The

hippocampus may inhibit activity of the axis at the nadir of the rhythm since trough

corticosteroid levels tend to increase after hippocampal damage (17, 76, 161, 190, 198).

However, corticosteroid levels at the nadir (5 jgg/dl) tended to be higher than normal

(-~l.g/dl) even in the control animals used in these studies (115). Since basal

corticosteroid levels were elevated, these studies most likely reveal a hypersensitivity to

"background" stress at the trough of the rhythm in hippocampal lesioned animals.

As Dallman and colleagues (56) have outlined, autonomous output of the pituitary

and adrenal accounts for output of the axis at the trough of the rhythm in the rat. Basal

hypothalamus (120), paraventricular nucleus (57), and suprachiasmatic nucleus (38)

lesions do not decrease plasma ACTH levels below normal at the nadir of the rhythm in

the rat. CRF antibodies do not decrease ACTH and corticosterone levels at the trough of

the rhythm in the rat (7), and trough corticosterone levels in adrenal- intact rats are








28

indistinguishable from corticosterone concentrations measured in adrenalectomized rats

(55). Therefore, the hippocampus most likely contributes to regulation of the axis during

the peak of the rhythm and during stress when neural components activate the axis.

Moreover, although neural inputs into the paraventricular nucleus increase in certain

instances, these neural inputs are not necessarily routed through the hippocampus. Studies

have observed corticosteroid output at the peak of the rhythm (when neural inputs

probably increase activity of the axis) is reduced in the male rat (161, 190) and increased

in the female rat (292). However, a recent, carefully designed, study by Dallman and

colleagues failed to demonstrate an effect of fimbria fornix-lesions on diurnal HPA axis

activity in adrenal-intact rats (23).


2.5.3.1.2.3 Stress-Induced Activity
The role of the hippocampus in stress-induced activity of the axis is not clear

(115). Jacobson and Sapolsky (115) have offered possible interpretations of numerous

hippocampal lesion studies which suggest the hippocampus does not regulate stress-

induced HPA axis activity. For example, individual study results could be influenced by

the precise subfield location of the hippocampal lesion, the period of time elapsed between

induction of the lesion and the study, and the timing of blood sampling (115). The

simplest and most attractive interpretation is that the hippocampus exerts different effects

on different stresses. Although many sensory and emotional stresses are processed

through the hippocampus, cognitive stress is probably most completely processed through

the hippocampus. Indeed, Coover, Goldman, and Levine (48) demonstrated the response

ofhippocampectomized rats subjected to ether stress or the "frustrative-emotional" stress

of introduction to a novel environment does not differ from the response of control rats.







29

In contrast, hippocampectomy did attenuate the corticosteroid response to extinction of a

learned lever-press reward system (48). Similarly, although non-emotional and non-

cognitive cues control the diurnal HPA axis rhythm, it is conceivable that under certain

experimental conditions neural input into the paraventricular nucleus may receive stronger

influence from the sensory integrating hippocampus and the diurnal rhythm may be more

strongly influenced by the hippocampus in those circumstances.


2.5.3.1.2.4 Facilitation
Keller-Wood and Dallman (141) noted that non-systemic stresses (skin incision,

laparotomy, restraint stress, and electric shock), which would be expected to influence

paraventricular nucleus activity through more complicated (or even multiple) neural

pathways than systemic stresses (hypotension or hypoglycemia), are uniquely able to

induce facilitation of axis response to subsequent or repeated stress. A recent study by de

Kloet and colleagues (284) suggests the hippocampus could modulate axis facilitation; as

expected, hippocampal MR blockade enhances but hippocampal GR antagonism

suppresses basal HPA axis activity at the peak of the rhythm. This finding suggests the

hippocampal MR inhibits activity of the axis and the hippocampal GR enhances activity of

the axis. A study by Bradbury and Dallman (20), conducted in adrenalectomized rats

replaced with corticosterone at concentrations well below peak levels (3-6 ,tg/dl),

confirms blockade of hippocampal MR with a MR-specific antagonist increases ACTH at

the peak of the rhythm. However, blockade of hippocampal GR with a GR-specific

antagonist actually increases ACTH when corticosterone levels are maintained at this fairly

low concentration. Together these findings suggest low activated concentrations of

hippocampal GR contribute to MR inhibition of the axis, but higher concentrations of






30

activated GR actually enhance output of the axis. These combined pharmacological data

demonstrates MR and GR actions may be regulated by the relative activation of MR and

GR and resulting amounts of MR and GR homo- and heterodimers.

In summary, the available evidence does not clearly demonstrate the precise role of

the hippocampus in regulating the diurnal rhythm of the axis, but suggests the

hippocampal MR is capable of inhibiting activity of the axis and high numbers of activated

hippocampal GR are capable of facilitating activity the axis. Moreover, these studies

suggest hippocampal regulation ofHPA axis activity is extremely sensitive to influence

from other neural inputs and the degree to which all neural input into the paraventricular

nucleus is routed through the hippocampus at the time of observation.

2.5.3.2 Fast feedback inhibition

Fast feedback inhibition appears to require a membrane-mediated event to inhibit

release of CRF and ACTH and decrease pituitary sensitivity to CRF (141), but the

mechanism of fast feedback inhibition is currently unknown. Corticosteroid-binding

membrane receptors have only recently been described (181, 208), and have yet to be

cloned. Recent evidence demonstrates the GABAA receptor is an important modulator of

rapid steroid effects; metabolites of cortisol, corticosterone, and progesterone are potent

benzodiazepine-like modulators of the GABAA receptor (176). Since benzodiazepines are

known to inhibit activity of the HPA axis (222), and excitatory hippocampal outputs are

thought to act in the bed nucleus of the stria terminalis to stimulate GABA inhibition of

CRF neurons in the paraventricular nucleus, the GABAA-active neurosteroids also may

mediate fast feedback inhibition. Moreover, GABAA receptors are known to play a

significant role in the circadian time-keeping system (189, 195), so the steroid metabolites







31

may regulate the time-keeping system. Much work is needed to characterize and

determine the mechanisms of rapid steroid effects.

2.5.3.3 HPA axis during pregnancy


2.5.3.3.1 Basal Activity
Resting plasma cortisol is increased in pregnant sheep and women, suggesting that

the setpoint of the HPA axis increases during pregnancy (133). Although elevated

concentrations of plasma corticosteroid binding globulin (CBG) may contribute to the

elevation in plasma cortisol in women, CBG concentration does not increase in the

pregnant ewe and the concentration of free plasma cortisol is increased in both species

during pregnancy (132). ACTH probably stimulates the increase in plasma cortisol since

plasma ACTH concentrations increase during the course of gestation and adrenal

responsiveness to ACTH increases during pregnancy (132). The mechanism leading to

increased ACTH concentrations during pregnancy is unknown, but in the sheep, plasma

ACTH is not increased by CRF or ACTH derived from the placenta (137, 138).


2.5.3.3.2 Feedback Sensitivity
These observations suggest feedback regulation of the axis may be attenuated

during gestation. Indeed, evidence does suggest the ability of dexamethasone to reduce

free cortisol concentrations is attenuated in the pregnant women (199, 201). However,

pregnancy does not reduce the absolute suppression of ACTH by a two-hour infusion (the

intermediate feedback time domain) of cortisol in the sheep (132). Interestingly, the initial

rate of feedback inhibition during the first two-hour infusion (in the rapid feedback time

domain) is reduced in the pregnant ewe (132). This suggests genomic feedback

mechanisms are not altered in pregnant ewes. A decrease in hypothalamic or pituitary






32

GABAA receptor expression or sensitivity or in metabolism of cortisol to the GABAA-

active metabolite, tetrahydrocortisol, may decrease the rate of feedback sensitivity in the

rapid feedback time domain.

Although absolute suppression of ACTH by cortisol is not reduced in the pregnant

ewe, plasma ACTH is significantly increased during pregnancy when cortisol

concentration is maintained below 5 ng/ml, according to a study of non-pregnant and

pregnant adrenalectomized ewes replaced with varying concentrations of cortisol (131).

Collectively these findings suggest that although the genomic feedback mechanism is not

altered during ovine pregnancy, plasma ACTH at basal, but not stimulated, cortisol

concentrations is increased in the pregnant ewe. Differential modulation of hippocampal,

hypothalamic, or pituitary MR and GR and/or neurotransmitter systems by ovarian

steroids during pregnancy may attenuate the ability of low concentrations of cortisol to

reduce plasma ACTH. Since the MR is the high affinity receptor that is activated by basal

concentrations of cortisol, the inability of low concentrations of cortisol to decrease

ACTH suggests that the MR, in particular, may be less responsive during pregnancy.


2.5.3.3.3 Responsiveness
Responsiveness of the HPA axis also is altered during ovine pregnancy in a

stimulus-specific manner; the ACTH response to hypotension is reduced but the ACTH

response to hypoglycemia is augmented during gestation (130). Since each stimulus

reaches the paraventricular nucleus through a distinct neural pathway (82), pregnancy

must selectively modify input into the axis. Ovarian steroids may modulate the processing

of input into the axis and the effects of ovarian steroids on MR and GR may play a

primary role in adapting the responsiveness of the axis to pregnancy.






33

2.5.3.3.4 Role of Ovarian Steroids
Currently available evidence suggests ovarian steroids contribute to differential

regulation of HPA axis setpoint and responsiveness (96). Female rats tend to have higher

circulating levels of both ACTH and corticosterone than male rats. This sex difference

appears to be due to effects of ovarian steroids on both the steroid synthesizing enzymes

in the adrenal gland and on neuroendocrine control of the axis (96). In cycling female

rats, the HPA axis is significantly more responsive during proestrus (when estradiol levels

are highest) than during estrous or diestrous (286). Treatment ofovariectomized rats with

estradiol mimics the effects of proestrus on the responsiveness of the axis (286), and co-

administration of progesterone attenuates the effects of estradiol on the axis (225, 286).

However, a more recent, and carefully designed study by Carey and colleagues (36)

confirmed estradiol increases activity of the axis and did not find an inhibitory effect of

progesterone. Estrogens appear to increase activity of the axis by increasing hypothalamic

CRF expression (102), adrenal responsiveness to ACTH (147), and attenuating the

glucocorticoid feedback signal (32, 225). In summary, these studies suggest estrogens

increase activity of the axis by increasing the activating components of the axis and by

decreasing feedback signal effectiveness. However, these studies do not clearly define the

effect of progesterone on HPA axis activity. The observed activating effects of estrogens

may play a role in the adaptation of the axis to pregnancy, but since absolute feedback

sensitivity is not reduced during ovine pregnancy, the effects of estrogens on feedback do

not appear to be physiologically relevant to pregnancy.

Evidence suggests progesterone could antagonize feedback regulation of the HPA

axis. High concentrations of progesterone antagonize glucocorticoid inhibition of P-







34

endorphin (a protein processed from the same precursor molecule as ACTH) secretion in

pituitary cultures (1), of corticotropin-releasing activity from isolated hypothalami (118),

and of ACTH in vivo (66). Similarly, an acute infusion of physiological concentrations of

progesterone antagonizes the feedback effects of cortisol in the non-pregnant ewe (136).

In vitro evidence demonstrates progesterone has a high affinity but very low efficacy at

both the MR and GR (233). This finding suggests progesterone could act as an

endogenous antagonist of cortisol actions at the hippocampus, hypothalamus, and

pituitary, inhibit the feedback control of the axis, and contribute to the increase in setpoint

observed during pregnancy. Absolute suppression of ACTH by increased concentrations

ofcortisol is not reduced during ovine pregnancy (132), suggesting progesterone does not

alter GR-mediated feedback to contribute to resetting the HPA axis setpoint. However,

the rate of feedback inhibition by increased cortisol is reduced during ovine pregnancy

(132). This effect could be due to GR antagonism by high circulating concentrations of

progesterone. However, if this effect is due to progesterone, progesterone only appears

to slow the rate of the feedback signal under these circumstances. Since plasma ACTH is

increased in pregnant ewes compared to non-pregnant ewes maintained at plasma cortisol

concentrations below 5 ng/ml (131), the setpoint for ACTH at this level of cortisol does

appear to be altered. This effect could be due to progesterone antagonism of the MR

component of the feedback signal that is only unmasked at low levels ofcortisol.

However, since treatment with progesterone alone does not increase the ratio of ACTH to

cortisol (129), progesterone alone probably does not induce the change in setpoint by

altering the sensitivity of the feedback system. Other signals are probably required to







35

increase neural output to the paraventricular nucleus and increase the setpoint of the HPA

axis.

2.6 Role of Altered HPA Axis Function in Cardiovascular Adaptation to Pregnancy


The studies presented above clearly demonstrate both the cardiovascular system

and the HPA axis are altered during pregnancy and strongly suggest ovarian steroids

contribute to the adaptation of each system to pregnancy. As discussed above, adaptation

of the HPA axis to pregnancy may further contribute to cardiovascular adaptation to

pregnancy since corticosteroids modulate fluid homeostasis, vascular responsiveness (58),

and central blood pressure control. In addition to these corticosteroid-mediated effects on

the cardiovascular system, several of the peptide components of the HPA axis (CRF,

AVP, and ACTH) modulate cardiovascular function. Therefore, ovarian steroids may

contribute to cardiovascular adaptation to pregnancy by directly modifying expression of

these peptides and their subsequent effects on downstream effectors; the downstream

effectors of CRF, AVP, and ACTH include the adrenal and neural outflow to the

cardiovascular system. In addition, ovarian steroids may contribute to differential

regulation of CRF, AVP, and ACTH by modifying the way in which MR and/or GR

respond to circulating corticosteroids. Ovarian steroids could modify MR and/or GR

regulation of CRF, AVP, and ACTH by altering expression of MR and/or GR and/or

modifying the ability of circulating corticosteroids to bind and activate MR and/or GR.

The effect of AVP, CRF, and ACTH on cardiovascular function are discussed below.

Studies demonstrate ACTH has effects on cardiovascular function that may be

independent of corticosteroid secretion. Therefore, the increased circulating







36

concentrations of ACTH found during pregnancy may directly contribute to

cardiovascular adaptation to pregnancy. However, most of the described effects of ACTH

on cardiovascular function appear to contradict a role for elevated concentrations of

circulating ACTH in cardiovascular adaptation to pregnancy.

In the sheep (241), rat (290), and human (291), supraphysiological concentrations

of peripherally administered ACTH increase blood pressure. Sheep treated chronically

with ACTH also have attenuated baroreceptor-heart rate reflexes (258), and exhibit

increases in cardiac output and no change in total peripheral resistance (93). A study of

the time course of development of ACTH-induced hypertension reveals that, in the first

few hours after initiation of ACTH treatment, MAP does not increase since an early

increase in cardiac output is offset by a decrease in total peripheral resistance (250). After

several hours, total peripheral resistance returns toward normal and MAP begins to

increase in the ACTH-treated sheep (250). If the ACTH-induced increase in heart rate

and cardiac output is prevented pharmacologically with a 0-adrenergic receptor

antagonist, total peripheral resistance increases and ACTH-induced hypertension is

maintained (93). However, if vasoconstriction is prevented with a calcium channel

antagonist or vascular smooth muscle relaxants, ACTH administration does not increase

arterial pressure (250). Therefore, ACTH increases blood pressure by increasing cardiac

output and an ACTH-induced change in the regulation of peripheral resistance is an

essential component in the development of ACTH-induced cardiovascular changes.

The central nervous system appears to play a selective role in ACTH-induced

hypertension. Although P-adrenergic receptor (propanolol), autonomic ganglion

(phentolamine) and sympathetic nervous system (methyldopa and clonidine) antagonists







37


prevent increases in heart rate and cardiac output by peripherally administered ACTH,

these drugs fail to prevent ACTH-induced hypertension (251, 253). In fact, autonomic

ganglion blockade with phentolamine actually enhances the pressor response to

peripherally administered ACTH. Together, these findings demonstrate peripherally

administered ACTH modifies neural control of the heart, but during peripheral ACTH

treatment neural control of the vascular system appears to attempt to maintain pressure at

the normal setpoint. Therefore, peripherally administered ACTH appears to alter vascular

tone and increase blood pressure through non-neural mechanisms. However, the precise

mechanism by which peripherally administered ACTH increases blood pressure is still

unknown. Intracerobroventricular administration of ACTH also increases blood pressure

in sheep (243), suggesting central ACTH-containing neurons stimulate increases in blood

pressure. However, centrally administered ACTH may not modulate blood pressure by

the same mechanisms as peripherally administered ACTH, and the mechanisms behind

centrally administered ACTH-induced hypertension have been less thoroughly studied.

Peripherally administered ACTH could increase blood pressure by increasing

concentrations of circulating corticosteroids. However, the role of corticosteroids in

development of ACTH-induced hypertension has been debated. Scoggins and colleagues

(72) have found cocktails containing various combinations of intravenously administered

corticosteroids (including cortisol, corticosterone, deoxycorticosterone, 11-deoxycortisol,

and 18-hydroxydeoxycorticosterone), designed to mimic the adrenal response to ACTH

administration, fail to elevate blood pressure in the sheep. Despite this finding, adrenal

steroids do appear to be an essential component of ACTH-induced hypertension. The

effects of ACTH on blood pressure are abolished in the adrenalectomized rat (290) and







38

other investigators have demonstrated intravenous infusions of physiological

concentrations ofcortisol are sufficient to increase blood pressure in the ewe (144).

Scoggins and colleagues have suggested steroid infusions can only mimic the effects of

ACTH on blood pressure in the sheep when 17a,20a-hydroxyprogesterone and 17ct-

hydroxyprogesterone are included with both aldosterone and cortisol in the steroid

infusion cocktail (252). However, it is unclear why two steroids with minimal

mineralocorticoid and glucocorticoid activity would be required to reveal the pressure

increasing effects of aldosterone and cortisol, and this finding is disputed by the

observation that intravenously-administered physiological concentrations of cortisol

increase blood pressure (144).

If ACTH increases cardiac output and blood pressure by first stimulating

corticosteroid secretion, corticosteroids do not appear to act centrally to increase blood

pressure in the sheep. Intracerebroventricular infusions of aldosterone (269), cortisol

(144), and cocktails containing 17a,20a-hydroxyprogesterone and 17a-

hydroxyprogesterone (268) all fail to increase blood pressure in this species. These

findings are consistent with the observation that centrally acting pharmacological agents

do not prevent ACTH-induced hypertension (251, 253). However, the role of

corticosteroids in ACTH-induced hypertension requires further investigation.

Together, these observations suggest elevated concentrations of circulating ACTH

could contribute to pregnancy-associated increases in cardiac output and baroreflex

attenuation. However, additional factors must prevent increased concentrations of ACTH

from changing the regulation of peripheral resistance and elevating MAP during

pregnancy. Circulating progesterone may allow elevated concentrations of ACTH to







39

contribute to increasing cardiac output without increasing MAP during pregnancy.

Indeed, Scoggins and colleagues (254) have demonstrated pre-administration of

progesterone prevents ACTH from increasing blood pressure, but progesterone

administration does not reverse hypertension established by previous ACTH

administration.

Further study is needed to determine if ACTH has direct effects on the vascular

system. The studies discussed above suggest ACTH may be a vasoconstrictor. More than

likely, the effects of ACTH on cardiovascular function observed by Scoggins and

colleagues are not applicable to pregnancy since these investigators consistently used

extremely high infusion rates (20 pg/kg/day) in their studies. Moreover, these

investigators demonstrated that the hypertensive effects of ACTH can only be obtained at

infusion rates above 1 mg/kg/day (240). This infusion rate elevates plasma ACTH beyond

the normal increase (about 25 pg/ml) observed during pregnancy.

One study demonstrates ACTH actually vasodilates the human placental

circulation (44). It is unclear if ACTH has the same effect on the peripheral vascular

system and to what degree the effects of ACTH on the vascular tone are altered during

pregnancy. Collectively, these studies demonstrate ACTH modulates cardiovascular

function. However, the mechanisms by which ACTH modulates cardiovascular function

are still unclear, and the role of ACTH in cardiovascular adaptation to pregnancy requires

further investigation.

CRF is another peptide that appears to have effects on cardiovascular function.

Since circulating concentrations of CRF tend to be minimal (-12 pg/ml), the effects of

CRF on cardiovascular function are probably mostly mediated via central neuronal







40

projections that release CRF. In rats (79), sheep (242), and dogs (30),

intracerebroventricular infusions of CRF increase MAP and heart rate, and this effect

appears to be mediated by CRF-induced increases in sympathetic outflow (30, 78).

Moreover, when CRF is administered centrally, the baroreflex maintains pressure and

heart rate about the new setpoint with reduced gain and range of responsiveness (77).

The effect CRF on the heart rate response to baroreceptor loading and unloading appears

to be mediated via CRF effects on parasympathetic outflow to the heart since

parasympathetic but not sympathetic antagonists prevent the effects of CRF on the

baroreflex (77). The role of central CRF in regulating cardiovascular function during

pregnancy has not been investigated, but ovarian steroids may have direct or indirect

effects on this cardiovascular-regulatory peptide during pregnancy.

AVP is another peptide that may play a role in cardiovascular adaptation to

pregnancy. As discussed above, AVP is released directly into the circulation from the

posterior pituitary and circulating AVP plays an important role in regulating fluid

homeostasis, vascular tone, and baroreflex sensitivity in non-pregnant animals, and the role

of altered AVP control in gestational cardiovascular function is still under investigation.

AVP also has central cardiovascular effects on cardiovascular function, but these effects

are somewhat controversial. While some studies suggest central AVP release decreases

sensitivity of the baroreflex and increases blood pressure and heart rate by stimulating

sympathetic outflow (221), other studies suggest central AVP actually increases sensitivity

of the baroreflex and decreases blood pressure and heart rate (25). These seemingly

contradictory findings may be due to differences in the dose of AVP administered or the

site of administration in the respective studies. Additional studies are required to clarify







41

the role of central AVP in regulation of cardiovascular function and to determine how

central AVP may contribute to cardiovascular adaptation to pregnancy.

2.7 Objectives


The literature demonstrates ovarian steroids contribute to cardiovascular

adaptation to pregnancy through direct and indirect mechanisms. Some of the indirect

effects of ovarian steroids on cardiovascular function appear to be mediated through the

effects of ovarian steroids on the HPA axis. However, further study is needed to expand

our understanding of the effects of ovarian steroids on both systems and to further clarify

the role of altered HPA axis function in cardiovascular regulation during pregnancy.

To date, the effects of estrogens on both the HPA axis and the cardiovascular

system have been examined more extensively than have the effects of progesterone on

both systems. Therefore, the studies presented in this dissertation were designed to

enhance understanding of the effect of progesterone on both the HPA axis and the

cardiovascular system. In particular, these studies were designed to determine the validity

of the general hypothesis that progesterone modulates the cardiovascular system and the

HPA axis via three novel progesterone receptor types: the GABAA receptor and the MR

and GR. In order to determine the validity of this general hypothesis three specific

hypotheses were tested:

1. Progesterone rapidly alters blood pressure, baroreflex sensitivity, and blood

volume.

Since 5a-reduced GABAA-active progesterone metabolites are elevated during

pregnancy and GABAA receptors are located in the paraventricular nucleus and







42


cardiovascular-regulatory nuclei in the brainstem, progesterone may have acute effects on

the cardiovascular system and the HPA axis that are mediated via Sa-reduced metabolites.

Chapters 4, 5, and 6 test the hypothesis that progesterone rapidly modulates

cardiovascular function

2. Pregnancy is associated with changes in MR and GR availability,

immunoreactivity, and apparent affinity.

Since progesterone also interacts with MR and GR, progesterone could more

slowly modulate the HPA axis and the cardiovascular system by interacting with these two

receptors. Chapters 7, 8, and 9 compare the availability, affinity, and immunoreactivity of

the MR and GR in non-pregnant and pregnant ewes and reveal these receptors are

differentially regulated during pregnancy when circulating progesterone concentrations are

high.

3. Ovarian steroids alter MR and GR availability and immunoreactivity during

pregnancy.

Chapter 10 tests the hypothesis that progesterone contributes to differential

regulation of MR and GR during pregnancy but does not establish or eliminate a role for

progesterone in differential modulation of MR and GR. The study presented in Chapter

11 reveals estrogen contributes to differential modulation of MR and GR during

pregnancy.







43


In summary, the studies presented in this dissertation reveal progesterone may

contribute to adaptation of the HPA axis and the cardiovascular system to pregnancy via

three novel progesterone receptors: the GABAA, and the MR and GR. As discussed in

Chapter 12, future study will be required to confirm and clarify the role of these novel

receptors in progesterone-mediated effects on the HPA axis and the cardiovascular

system.















CHAPTER 3
GENERAL MATERIALS AND METHODS


3.1 Animal Care


This dissertation represents a collection of in vivo and in vitro studies of the ewe

(female Ovis aries). Ewes were cared for according to the guidelines established by the

American Association for Accreditation of Laboratory Care and all experimental

procedures were approved by the University of Florida's Institutional Animal Care and

Use Committee.

During the acute steroid infusion studies, ewes were housed in the animal

resources facility at the University of Florida Health Sciences Center, maintained in a

controlled environment (12-hour light/dark cycle and a constant 19-21 C temperature),

and allowed access to food and water ad libitum.

Ewes treated chronically (60 days) with steroid implants were allowed to recover

from the surgical procedure for approximately two weeks in an enclosed animal resources

facility. The animals then spent the duration of the treatment period in an open pasture

and were moved to the animal resources facility at the Health Sciences Center

approximately 48 hours prior to sacrifice.

Body temperature was measured at the end of each experiment and twice daily for

five days after a surgical procedure to assess health. Ewes also were treated with 750-





44







45


1000 mg ampicillin (Polyflex, Aveco, Fort Dodge, IA) after each infusion experiment and

twice daily for five days after a surgical procedure.

3.2 General Surgical Procedures


Ewes were fasted for 24 hours prior to a surgical procedure. Anesthesia was

induced with ketamine (20 mg/kg i.m.) and maintained with halothane in -3% oxygen. All

surgical procedures were performed using aseptic techniques.

3.2.1 Ovariectomy

Bilateral ovariectomies were performed as previously described (9). Briefly,

ovaries were isolated through a midline abdominal incision, the vascular supply was

ligated, and the ovary was removed.

3.2.2 Catheterization

In some studies, catheters were introduced bilaterally into the femoral artery and

vein for steroid delivery, blood sampling, and arterial pressure measurement. This method

has been previously described in detail (9). Briefly, an incision was made in the femoral

triangle, and the artery and vein were exposed. Each vessel was ligated distally, and a

0.05 mm catheter was advanced approximately 10 inches into the descending aorta or

inferior vena cava. Each catheter was secured with surgical ties, filled with heparinized

saline, plugged with a sterile nail, and routed subcutaneously with a trocar to exit the

flank. When not in use, the exteriorized catheters were stored in a paper pocket and

protected by a spandage wrap.







46


3.3 Chronic Steroid Implantation


Subcutaneous implants were used to chronically administer steroids. Estrone and

placebo implants were purchased from Innovative Research of America (Toledo, OH).

Estradiol and progesterone (Sigma, St. Louis, MO) implants were prepared using aseptic

techniques by filling shells with crystalline steroid (123, 124). Estradiol was packed to a

height of 3 cm in 4 cm long pieces of Silastic tubing (0.132 in. inner diameter, 0.183 in.

outer diameter, Dow-Coming, Midland, MI). Progesterone was packed into PhamElastM

silicon polymer (SF Medical, Hudson, MA) implants measuring 75 X 55 mm. These

implants were sealed with Silastic adhesive (Dow-Coring). To eliminate a surge in

plasma steroid concentrations immediately after implantation, implants were placed in

sterile, isotonic saline and maintained at 37C for 24 hours prior to implantation (123).

All implants were placed into the dorsal scapular region of anesthetized ewes using aseptic

technique.

3.4 Acute Progesterone Infusions


For acute progesterone infusion studies, progesterone (Sigma) was prepared and

stored in 100% ethanol. Prior to an experiment, the progesterone stock was prepared in

10 or 20% ethanol in 0.9% saline in a syringe. Steroid solutions were infused into venous

catheters through 0.2 pm sterile Acrodisc filters to prevent the introduction of

microbacterial contaminants into the ewes.







47

3.5 Handling and Analysis of Blood Samples


3.5.1 Sampling and Storage

Aliquots of blood were collected in tubes containing heparin (for analysis of blood

volume and plasma sodium) or sodium EDTA (0.015 M, for analysis of hormones). These

whole blood samples were placed on ice until the end of the experiment. Samples were

then centrifuged, and plasma aliquots were stored at -200C until analyzed.

3.5.2 Plasma Analysis

Plasma progesterone was measured by radioimmunoassay (RIA) using kits

purchased from Diagnostic Products, Inc. (Los Angeles, CA). The lower limit of

detection of the assay was 0.1 ng/ml. The antibody used in this kit displays about 0.03%

cross-reactivity with cortisol and 9.0% cross-reactivity with THP. Plasma AVP was

extracted with bentonite (Sigma) and measured by RIA as previously described (224).

The lower limit of detection for this assay was 0.39 pg/ml when 0.5 ml of plasma was

extracted. For both radioimmunoassays, samples measuring below the lower limit of

detection were assigned the value of the limit of detection for purposes of statistical

analysis.

Plasma Na and K* were analyzed using a Na'/K analyzer (NOVA, Waltham,

MA). Plasma protein concentration was measured with a refractometer (Fisher Scientific,

Inc.) in order to assess whether either steroid treatment altered fluid distribution.






48

3.6 Cardiovascular Measurements


3.6.1 Mean Arterial Pressure

An arterial catheter was connected to a Statham P23Db transducer (Gould,

Oxnard, CA) and Grass model 7 recorder (Quincey, MA) to continuously monitor blood

pressure during infusions. Arterial pressure was sampled at 1 or 20 Hz with a Keithley

system 570 analog-to-digital converter and a microcomputer using software from

AsystantTM (Asyst Software Technologies, Rochester, NY). Mean arterial pressure

(MAP) was calculated as the mean value for each one-minute interval.

3.6.2 Plasma Volume

Plasma volume was estimated using the Evans blue dye dilution technique (18).

Before beginning a volume test, a control plasma sample (30 ml) was withdrawn. This

sample was used to create the standard curve. A known volume (EI) of a 5 mg/ml

solution (calculated by weighing the injection syringe before and after injection) of Evans

blue (Sigma) was flushed into a venous catheter with excess saline (30 ml) at the start of

the volume test. Blood samples were then withdrawn from an arterial catheter at 10-

minute intervals. All blood volume samples were placed into tubes containing heparin and

centrifuged at 3,000 rpm to obtain a plasma sample. The resulting plasma sample was

again centrifuged at 12,000 rpm to remove all contaminating blood cells.

The Evans blue stock (5 mg/ml) was diluted into the control plasma sample to

create standards (0.0025- 0.03 mg/ml). The absorbances of the standard and unknown

samples were read at 620 nm on a diode array spectrophotometer (Beckman Instruments,

Inc., Fullerton, CA). The relationship between standard absorbance (Add) and







49


concentration (C,t) was fit to the equation of a line (Aid = mtd x Cd + bad) using

SigmaPlot (Jandel Scientific, San Rafael, CA). The relationship between sample

absorbance (A.m) and sampling time (turn) also was fit to the equation of a line (A. =

m.m x tn + b.m) using SigmaPlot(. The concentration of Evans blue at the instant of

injection (Co) was extrapolated (Co = (b.u bud)/mstd) and the volume of distribution of

the dye was calculated (Vd = EI/Co). Finally, the plasma volume (Vp) was expressed by

normalizing the Vd to the ewe's body weight (Bw, Vp = Vd / Bw).

3.6.3 Baroreflex

3.6.3.1 Bolus injection method

In some experiments, baroreceptor function was evaluated using bolus injections

ofphenylephrine and nitroprusside. Resting heart rate and resting MAP were sampled

over 15-second period immediately after steroid infusion and prior to the baroreflex test.

A 3 pg/kg bolus of phenylephrine (American Regent Laboratories, Shirley, NY) was

injected into a venous catheter and the resulting pressor response was recorded at 20 Hz

as described above. Blood pressure and heart rate were then allowed to return to baseline

levels (15-20 minutes). Next, a 50 pg/kg bolus of nitroprusside (Nitropress, Elkins-Sinn,

Cherry Hill, NJ) was injected into the venous catheter and the resulting depressor response

was recorded.

3.6.3.2 Steady-state method

In other experiments, baroreceptor function was evaluated by recording MAP and

heart rate at a plateau after graded continuous intravenous infusions of phenylephrine and

nitroprusside. Solutions of phenylephrine and nitroprusside (20 mg/kg/ml) were prepared






50

in sterile isotonic saline and were infused at rates of 0.1, 0.2, 0.4, and 0.8 ml/min. In order

to establish a plateau in pressure and heart rate, each infusion rate was continued for 3

minutes before stepping up to the next infusion rate. The phenylephrine was infused first,

blood pressure and heart rate were allowed to return to baseline (30-40 minutes), and the

test was concluded with the graded nitroprusside infusions.

3.6.3.3 Expression of data

Baroreflex function curves express the relation between MAP and heart period.

When the bolus injection method was used, the heart period was calculated as the time

interval (in msec) between peaks in the first derivative of the arterial pressure wave, and

MAP at each peak was referenced from a smoothed curve of the arterial pressure wave.

Only the portions of the arterial pressure curves during the period of increasing or

decreasing pressure were used in the analysis.

When the steady-state method was used, MAP and heart period were calculated

from a 30-second recording obtained at the plateau for each dose of phenylephrine or

nitroprusside.

For each experiment in each animal, the baroreflex function relation was fit to the

logistic equation y = [a/(l+eb()]+d using SigmaPlot (Jandel Scientific, San Rafael,

CA). In order to prevent distortion of fit parameters by data sets that did not fit well to a

logistic equation (3/21 curves), the parameters were constrained to values of a<1000,

70250. The parameters obtained from this logistic fit have physiological

meaning; a represents the maximum range of the heart period (ms); b represents the slope,

or sensitivity, of the reflex (Ams/AmmHg); c represents the MAP (mm Hg) at the

maximum rate of change (set-point or inflexion point); and d represents the minimum heart






51


period (ms) (143). The maximum gain of the reflex was calculated from the first

derivative of the logistic function (143). Threshold (MAP that produces the shortest heart

period) and saturation (MAP that produces longest heart period) values of MAP were

calculated from the third derivative of the equation (143). Mean baroreflex curves were

computer generated using the mean parameter values obtained in each treatment group.

When the bolus injection method was used, it also was desirable to determine if

treatment selectively altered the sensitivity of one portion of the baroreflex curve. The

slope of the rapid response to nitroprusside and phenylephrine was determined by linear

regression for each experiment in each animal.

3.7 Tissue Collection


All ewes were housed in the animal resources facility at the Health Sciences Center

for at least 24 hours prior to sacrifice. In instances when the ewe had not undergone

femoral catheterization, a jugular catheter was inserted approximately 24 hours prior to

sacrifice. These catheter lines were used to minimize stressful interaction between the ewe

and the investigator, to obtain a pre-sacrifice blood sample, and to administer an overdose

of sodium pentobarbital.

Immediately after sacrifice, the brain was perfused with a cold 10% solution of

dimethylsulfoxide in isotonic saline. Dimethylsulfoxide has been shown to cryoprotect

corticosteroid receptors (171). Unperfused tissues (kidney and liver) were washed and

stored in the cryoprotective solution. All tissue samples were rapidly excised, frozen in a

dry ice-acetone bath, and stored at -800C until assayed.






52


3.8 5ct-Reductase / 3a-Hydroxysteroid Dehydrogenase Activity Assay


The in vitro conversion of progesterone into the GABAA-active metabolites DHP

and THP was assayed according to a modification of the method previously described by

Krause and Karavolas (154).


3.8.1 Tissue Preparation

Tissues were obtained and stored as described above. At the time of assay, liver

and brainstem samples from non-pregnant, pregnant, and ovariectomized ewes were

homogenized using Krebs-Ringer bicarbonate (KRB) buffer (Sigma, pH=7.4, 40C, 5ml of

buffer per gram of tissue). Homogenized tissues were first centrifuged at 3,000 x g for 20

minutes at 40C to remove large particles from the homogenate. The supernatant was

retained and centrifuged at 100,000 x g for 60 minutes at 4C. The pellet was retained

and resuspended in an equal volume of KRB buffer to yield a microsomal fraction.

3.8.2 Activity Assay

The reaction was started by adding 500 pl of the microsomal fraction to conical-

bottom, screw-top, borosilicate centrifuge tubes (Kimble Glass Inc, Vineland, NJ)

containing approximately 20,000 dpm oftritiated (1,2,6,7-H) progesterone (H-P,

Amersham International, Buckinghamshire, England), 500 pM NADPH, and 5 mM of

dithiothreitol in KRB buffer (final reaction volume = 1 ml). The reaction was allowed to

run for 30 minutes at 370C, and the reaction was stopped with 5 ml of anhydrous ethyl

ether. The tubes were then immersed in an acetone/dry ice bath, and the organic phase

was transferred to new tubes spiked with steroid carriers (100 .Lg of progesterone and






53


DHP and 200 p.g of THP). To determine the amount of background radioactivity carried

by DHP and THP, blank tubes were prepared containing progesterone, DHP, THP, and

3H-P. All tubes were evaporated under a stream of air and the residual steroids were

reconstituted in 100 pl of warmed ethanol. The reconstituted samples were spotted on to

Thin Layer Chromatography (TLC) plates (silica gel 60 A, 0.25 mm thickness, Whatman,

Clifton, NJ) and developed in benzene: methanol (19:1 v/v, Sigma). Steroid bands were

visualized by exposure to ultraviolet light and iodine vapors, and the bands were scraped

into scintillation vials and counted.

The amount of radioactivity recovered in the DHP and THP bands was expressed

as the percentage of radioactivity recovered in all three bands for each lane, and the

average percentage of background activity carried in each band in the blank reaction tubes

was subtracted.

3.9 Radioligand Binding Assays


The binding oftritiated cortisol to available cytosolic corticosteroid receptors was

assayed according to a modification of a previously described method (226).

3.9.1 Tissue Preparation

Hippocampus, hypothalamus, pituitary, and kidney were homogenized using a

motor-driven Teflon pestle and glass tube (Wheaton Science Products) in a reducing

homogenization buffer (RHB): ice-cold Tris-HCI (10 mM, pH 7.4) containing 10%

glycerol, 10 mM sodium molybdate, 4 mM dithiothreitol, 2 mM EDTA, and 4 mM B-

mercaptoethanol (10 ml buffer per gram of tissue). The components of this buffer

(purchased from Sigma) have been shown to stabilize corticosteroid receptors (108). The






54

homogenate was centrifuged at 100,000xg for 60 minutes to obtain a cytosolic fraction

(supernatant). Aliquots of cytosol were stored at -800C until assayed.

3.9.2 Incubations

The radioligand-binding assay was conducted in borosilicate test tubes at 40C. For

determination of nonspecific and mineralocorticoid receptor binding, an appropriate

amount of stock steroid was added to each tube and the tubes were evaporated to dryness

in a centrifugal evaporator (Jouan, Inc., Winchester, VA). The residual steroids were

reconstituted with 100 pl of RHB containing the desired amount oftritiated (1,2,6,7-3-H)

cortisol (3H-F, Amersham, final concentrations ranging 0.1-25 nM). The binding reaction

was initiated by adding 100 pl ofcytosol (final incubation volume of 200 pl with 1-2 Ag of

protein/pl).

MR binding was determined in the presence of 1.25 pM RU 28362, a specific GR

agonist (Roussel-Uclaf, Romainville, France). Nonspecific binding was determined in the

presence of 12.5uM dexamethasone sodium phosphate (American Regent Laboratories,

Inc., Shirley, NY). Binding to GR was calculated from the difference between total

specific cortisol binding and specific cortisol binding in the presence of excess RU 28362.

Specific binding was expressed in fmol/mg protein, and protein concentration was

determined according to the method of Bradford (24) using a kit purchased from Bio-Rad

Laboratories (Hercules, CA).

3.9.3 Separation of bound from free

Steroid-bound receptors were separated from the unbound ligand by LH-20 gel

exclusion chromatography (170). Chromatography columns were prepared by plugging






55


5ml PYREXTM serological pipettes (Corning, Inc.) with 3 mm diameter borosilicate beads

(Kimble Glass Company). The columns were filled to a height of 8 cm with Sephadex

LH-20 (Pharmacia Biotech, Uppsala, Sweden) reconstituted in RHB. For separation of

bound from free, a 150 il aliquot of the incubate was loaded to the top of the column bed,

washed into the column bed with 100 ll buffer, and eluted with an additional 1 ml of

buffer. After each use, the columns were washed with 15 volumes ofmethanol and 15

volumes of RHB. During the buffer RHB re-equilibration portion of the wash, the bead

bed was agitated and allowed to resettle. Columns were maintained at 40C.

3.10 Western Blots


Total cytosolic and whole cell MR and GR were estimated using a semi-

quantitative Western blot procedure.

3.10.1 Tissue Preparation

Tissues were homogenized in RHB as described above. Whole cell samples were

obtained from the unprocessed homogenate, and cytosolic samples were obtained after

centrifugation at 100,000xg as described above. The concentration of protein in each

samples was determined according to the method of Bradford (24) using a kit purchased

from Bio-Rad. All samples were stored at -800C until further processed. Prior to gel

electrophoresis, samples were diluted in a Laemmli (156) reducing sampling buffer

(0.0625 M Tris-HCI, 10% glycerol (v/v), 2% SDS (w/v), 0.72 M B-mercaptoethanol, and

0.001% bromophenol blue) and heated at 950C for 5 minutes.






56


3.10.2 Electrophoresis and Transfer

Samples were loaded onto precast 10% polyacrylamide, Tris-HCl gels (Bio-Rad

Laboratories) and electrophoresed at approximately 100 V using a Mini-Protean II gel

electrophoresis system (Bio-Rad Laboratories). An equal volume of sample (30 1l) and

amount of protein (20 .g) was loaded into each lane. One lane of each gel was loaded

with a mixture of stained proteins of known molecular weights (Rainbow"T coloured

protein molecular weight markers, Amersham).

After electrophoresis, proteins were transferred overnight (at 22 V) onto a

nitrocellulose membrane using a Mini Trans-Blot cell (Bio-Rad Laboratories). The

membranes were blocked with 10% w/v nonfat powdered milk solution, and incubated

with an anti-MR or anti-GR antibody (both purchased from Affinity Bioreagents, Golden,

CO) and an appropriate horseradish-peroxidase conjugated IgG antibody (Sigma). The

membranes were then incubated with RenaissanceT Western Blot Chemiluminesence

Reagent Plus (NENT Life Science Products, Boston, MA) and exposed to ReflectionTM

Autoradiography Film (NENT Life Science Products) for approximately 30 seconds.

3.10.3 Densitometry

The resulting films were scanned with model GS-670 Imaging Densitometer (Bio-

Rad) and immunoreactive band density was analyzed using Molecular Analyst/PC

software. Only the densities of samples developed on the same Western Blot were

compared. The linear relationship between electrophoretic mobility and the logarithm of

the molecular weight of RainbowTM markers was used to estimate the molecular weight of

the resulting immunoreactive bands.






57

3.11 Statistical Analysis


Methods of statistical analysis were chosen based on experimental design and the

character of the obtained data set (87); these are detailed for each experiment in each

chapter. For all statistical tests, the null hypothesis was rejected when p < 0.05.














CHAPTER 4
ACUTE EFFECT OF PROGESTERONE ON CARDIOVASCULAR FUNCTION IN THE
OVARY-INTACT EWE



4.1 Introduction


Associated with the increase in progesterone and estrogen during pregnancy is a

substantial increase in heart rate, cardiac output, and plasma volume (169). A concurrent

decrease in total peripheral resistance results in a 5-10 mm Hg reduction in MAP (MAP) (169).

Several studies suggest the baroreflex maintains arterial pressure about this new setpoint with

reduced sensitivity when the baroreflex response to decreased pressure is included in the analysis

(29). The correlation of altered cardiovascular function during pregnancy and the menstrual cycle

with hormonal rhythms has led to the hypothesis that ovarian steroids are important regulators of

the cardiovascular system (169).

Estradiol has been shown to induce many cardiovascular changes characteristic of

pregnancy. Chronic estradiol treatment decreases vascular tone and MAP, and increases renin

activity, plasma volume, and cardiac output (174, 277). Recently collected evidence suggests that

progesterone also modulates these variables. In ovariectomized ewes, physiological levels of

progesterone, alone or in combination with estradiol, significantly reduce arterial pressure and

increase plasma volume and arginine vasopressin (AVP) without increasing activity of the renin-

angiotensin system (210). Therefore, progesterone also appears to be a regulator of

cardiovascular function.



58









59
Previous studies attributed progesterone's antihypertensive actions to its ability to

antagonize the renal salt and fluid retaining activity ofmineralocorticoids (254, 287). Evidence

now suggests progesterone also has rapid, nongenomic actions on the baroreflex. Progesterone

can be sequentially metabolized by 5oc-reductase and 3oc-hydroxy steroid dehydrogenase in the

brain and periphery to form tetrahydroprogesterone (THP). THP, which circulates at elevated

levels during pregnancy, enhances activation of the GABAA receptor by GABA (176). Heesch

and Rogers (98) recently demonstrated that THP rapidly causes a leftward shift in setpoint, and at

the same time, decreases the overall sensitivity of renal sympathetic nerve responses to changes in

arterial pressure. These investigators propose that THP acts in the rostral ventral lateral medulla

to enhance GABAergic activity, thereby increasing the GABAergic inhibition of excitatory amino

acid projections to the preganglionic sympathetic cell bodies of the intermediolateral cell column

(98).

This experiment was designed to test for a rapid effect of progesterone on blood pressure,

Na AVP, volume, and baroreflex sensitivity in non-pregnant ewes. Two doses of progesterone

are used to test for this effect: one within the physiological range for pregnant ewes and one

exceeding this range.

4.2 Methods


Seven adult, ovary-intact, anestrous ewes were studied during and immediately after 2-

hour venous infusions. Each ewe was subjected to three infusion protocols: vehicle (10% ethanol

in 0.9% saline), progesterone (Sigma, St. Louis, MO) at 3 pg/kg/min (3P4), and progesterone at 6

pg/kg/min (6P4) in a randomized, crossover design.









60
4.2.1 Progesterone Infusion and MAP

During a given experiment, a ewe was treated with a continuous 2-hour infusion of

control vehicle or steroid. In order to minimize the possibility of residual steroid effects, each

ewe was allowed at least a two-day recovery period between treatments. Arterial pressure was

continuously monitored during the infusions. Blood samples (7 ml each) were collected at 30-

minute intervals during the first two hours of the infusion for measurement of plasma Na+,

protein, AVP, and progesterone.

4.2.2 Baroreflex Test

Following a 2-hour infusion of vehicle, 3P4 or 6P4, baroreceptor function was evaluated

using bolus injections ofphenylephrine (3 gg/kg) and nitroprusside (50 gg/kg) while the infusion

continued.

4.2.3 Statistical Analysis

Plasma AVP, protein, sodium, and progesterone concentrations and MAP responses

measured over time were analyzed by two-way analysis of variance (ANOVA) corrected for

repeated measures. Logistic and linear curve fit parameters were compared by one-way ANOVA

corrected for repeated measures; when values were not normally distributed, a Friedman repeated

measures ANOVA on ranks was used. Individual means were compared with a Duncan's multiple

range test. For all statistical tests, the null hypothesis was rejected when p < 0.05. Data are

expressed as the mean + standard error of the mean (SEM).






61


4.3 Results


4.3.1 Plasma Progesterone Levels

When progesterone was infused at a rate of 3 jg/kg/min, plasma progesterone

concentration increased to 10.4 3.9 ng/ml after 30 minutes (Fig. 4.1C), and remained at

this level until 120 minutes when the plasma concentration averaged 8.4 2.9 ng/ml.

These concentrations are significantly greater than during the infusion of vehicle (mean

concentration 2.1 0.7 ng/ml at 120 minutes). When progesterone was infused at a

higher rate (6 pg/kg/min), progesterone increased to 37.8 14.3 ng/ml at 60 minutes into

the experiment (Fig. 4. 1E), and was 19.4 3.0 ng/ml by the end of the 6 gg/kg/min

treatment period. This plasma concentration was significantly greater than the level

obtained at the end of the infusion of 3 pg/kg/min or at the end of the infusion of vehicle.

The tendency for plasma progesterone concentration to decrease toward the end of the 6

pg/kg/min infusion is most likely attributable to the poor solubility of high concentrations

of progesterone in the 10% ethanol vehicle. Progesterone crystals tended to form in the

infusion lines of some of the larger animals as the experiment progressed, and in some

instances the infusion line had to be cleared with a bolus of saline.

4.3.2 Arterial Pressure

Mean arterial pressures were statistically similar at the beginning of each infusion

protocol. Arterial pressures averaged 95.9 7.9 mm Hg at the beginning of the 2-hour

control vehicle infusion (Fig. 4.1B), and was not significantly different at the end of the

experiment (94.0 7.5 mm Hg). However, infusion of progesterone at a rate of 3

pg/kg/min significantly reduced MAP over the two hours (Fig. 4. D). MAP was






62

significantly decreased by the 17th minute of 3P4 treatment (90.4 1.3 mm Hg), and

remained significantly reduced until the end of the experiment (mean value after 120

minutes of infusion of 3pg/kg/min: 86.2 1.7 mm Hg). In contrast, the higher rate of

infusion (6pg/kg/min) did not significantly change MAP (Fig. 4. IF).

4.3.3 Baroreflex

Representative logistic curve fits from an individual animal (W94) are presented in

Figs. 4.2A-C. Mean logistic curve fit parameter values are presented in Table 4.1, and

mean baroreflex curves are diagramed in Fig. 4.2D. When the entire baroreflex curve was

analyzed, there were no significant differences among treatment groups.

The slope of the phenylephrine-induced portion of the baroreflex curve was not

significantly altered by either treatment (Table 4.2). The linear response to phenylephrine

was highly variable in the control group (23.2 16.9 Ams/AmmHg), and this was due to a

steep response in one animal (G157, 124.0 Ams/AmmHg). When this animal was

eliminated from the analysis, the slope of the bradycardic response to phenylephrine after

vehicle infusion was 6.3 1.5 Ams/AmmHg, and there were no significant differences

among groups. The slope of the nitroprusside-induced portion of the baroreflex curve

was not significantly altered after infusion of progesterone at a rate of 3pg/kg/min (Table

4.2). Although infusion of progesterone at a rate of 6 pg/kg/min tended to attenuate the

slope of the response to nitroprusside injection compared to vehicle, this was not

significantly different. However, the slope of the response to nitroprusside after infusion

of progesterone at a rate of 6 pg/kg/min was significantly attenuated compared to the

slope after infusion of progesterone at a rate of 3 pg/kg/min.






63

4.3.4 Plasma Na and AVP

There were no significant effects of either the 3 pg/kg/min or the of 6 pg/kg/min

infusions on plasma Na (Figure 4.3A). Infusion of progesterone at rate of 3 pg/kg/min

significantly increased plasma AVP over time (2.1 0.9 to 3.2 1.0 pg/ml at 120 minutes;

Figure 4.3B). Plasma AVP concentration after 120 minutes of infusion of progesterone at

a rate of 3 ug/kg/min was significantly greater than after 120 minutes of vehicle infusion.

In contrast, plasma AVP was not significantly altered by infusion of progesterone at rate

of 6 ;g/kg/min.

4.3.5 Plasma Protein

Plasma protein concentration did not significantly change during infusion of

control vehicle (7.3 0.1 to 7.4 0.1 g/l00ml, Figure 4.3C) or progesterone at a rate of 3

pg/kg/min (7.5 0.2 to 7.6 0.2 g/100ml). Prior to infusion of progesterone at a rate of

pg/kg/min, plasma protein concentration was significantly lower (7.1 0.2 g/100ml) than

plasma protein concentration prior to the beginning of vehicle infusion (7.3 0.1

g/100ml). Although plasma protein concentration significantly increased over time during

the infusion of progesterone at a rate of pg/kg/min (7.1 0.2 to 7.6 0.1 g/100ml), it was

not significantly different from vehicle after 120 minutes of infusion.

4.4 Discussion


Plasma progesterone levels (10-12 ng/ml) characteristic of ovine pregnancy (9)

rapidly reduced MAP in ovary-intact, anestrous, adult ewes, suggesting progesterone

decreases blood pressure through nongenomic mechanisms in this animal model. In

contrast, supraphysiological levels of progesterone (25-40 ng/ml) did not alter MAP






64


within 2 hours. This finding indicates plasma progesterone concentration and MAP are

not linked through a simple, linear dose-response relationship. Consequently,

progesterone appears to regulate cardiovascular function through multiple mechanisms.

Initial interest in the antihypertensive actions of progesterone arose from the

observation that progesterone antagonizes the salt and fluid retaining actions of

mineralocorticoids (5). Subsequent studies have attributed the antihypertensive effect of

progesterone to its anti-mineralocorticoid properties (254, 287). However, chronic

progesterone treatment also reduces blood pressure in humans, dogs, and rats under

conditions in which mineralocorticoid levels are not elevated (5, 31, 63, 84, 260). Chronic

progesterone treatment over 10-14 days reduces arterial pressure in ewes, but expands

plasma volume (210), a finding that is contradictory to the anti-mineralocorticoid

hypothesis of progesterone action. In this study physiological levels of progesterone

decrease arterial pressure more rapidly than previously noted. This study reveals a

significant change in blood pressure within 17 minutes, but no evidence of changes in

plasma electrolytes or volume over the 120 minutes of study.

The mechanism by which progesterone decreases systemic pressure is currently

unknown. Progesterone may decrease arterial pressure by an extrarenal action at

progestin-binding sites. Homogenate and autoradiographic binding studies reveal

radiolabeled progestin binding in both the vascular system and in cardiovascular centers in

the hypothalamus and brain stem (114, 259). Using isolated placental arteries and veins,

Omar and colleagues have recently demonstrated progesterone elicits an acute reduction

in vascular tone via a mechanism mediated by intracellular progesterone receptors and

cyclic adenosine monophosphate (202). This mechanism may be specific to the placental






65


circulation and may not contribute significantly to systemic vascular tone. Similarly,

Magness and Rosenfeld (175) have shown that the effects ofestradiol on uterine blood

flow and vascular resistance are much more profound than the systemic effects.

Heesch and Rogers (98) have hypothesized progesterone metabolites alter blood

pressure and decrease baroreflex sensitivity by decreasing sympathetic outflow through an

action at GABAA receptors in brain stem cardiovascular-regulatory areas. An attenuation

of baroreflex sensitivity at physiological levels of progesterone using heart rate as the

index of sympathetic outflow was not observed in this study. It is possible that

sympathetic outflow to the vascular system is altered by this concentration of

progesterone. There was a tendency towards attenuation of the linear response to

nitroprusside after infusion of supraphysiological levels of progesterone, suggesting that

the tachycardic response to hypotension is reduced during infusion of progesterone at a

high rate. Glick and Braunwald have suggested that the rapid bradycardic response to

hypertension is primarily mediated via parasympathetic activity while the rapid tachycardic

response to hypotension is predominately mediated by sympathetic activity (88).

Therefore, the attenuated slope of the baroreflex response to nitroprusside observed after

infusion of 6 Pg P4 /kg/min may be due to compromised sympathetic control of the heart

rate. If this effect is mediated by progesterone metabolites, supraphysiological

progesterone levels appear to be required to achieve active metabolite concentrations.

Progesterone also could modulate blood pressure by acting as an antagonist or

weak agonist at mineralocorticoid (MR) or glucocorticoid (GR) receptors (233). The KD

for progesterone binding to each of these receptors is within the physiological range of

plasma progesterone, but progesterone has a much higher affinity for MR than for GR






66


(233). MR and GR have already been implicated in central blood pressure regulation in

the rat (89, 282); intracerebroventricular MR agonists increase, but GR agonists decrease,

blood pressure (282). Intracellular progesterone receptors may interact with this system

since one form (PR-A) has been shown to inhibit transcription of MR and GR in

transfected cell cultures (180). The interaction of progesterone with these numerous

potential receptor types could therefore contribute to the complexity of progesterone's

actions on the cardiovascular system by the second hour of infusion.

In these experiments there was no acute effect of progesterone infusion on plasma

sodium or plasma proteins, suggesting the infusion did not alter electrolyte or fluid

balance. The infusion of progesterone at 3 ig/kg/min increased plasma AVP, but this was

not significant until 120 minutes. The effect of low concentrations of progesterone on

plasma AVP can not be readily explained as an enhancement of GABAA activity by

progesterone metabolites. Current evidence suggests GABAA activation actually inhibits

AVP release (146). Instead, the increase in plasma AVP at 120 minutes may be mediated

by genomic mechanisms. Previous investigations have observed increases in both plasma

sodium and AVP concentrations after six days of progesterone treatment (unpublished

observations), suggesting an indirect effect of progesterone on AVP through osmolality.

This effect of progesterone could be mediated by renal actions of progesterone however;

further study of the acute and chronic effects of progesterone on plasma Na+, AVP, and

volume is required.

This study confirms physiological concentrations of progesterone significantly

diminish arterial pressure and increase plasma AVP. Furthermore, the change in arterial

pressure occurs within 17 minutes, consistent with a nongenomic mechanism. However,






67


there was not a significant effect of 2 hours of infusion of progesterone at this physiologic

dose on the baroreflex heart rate response to acute increases or decreases in blood

pressure. In contrast, supraphysiological progesterone levels did not alter arterial pressure

within 2 hours, but did tend to decrease the slope of the tachycardic response to acute

hypotension. The availability of numerous potential progesterone receptor sites may

contribute to progesterone's multiple actions.






68



Table 4.1. Logistic baroreflex curve parameters.

Experimental Group


Vehicle (n=7) 3P4 (n=7) 6P4 (n=7)

Resting MAP (mmHg) 97.3 7.8 94.0 6.7 97.7 6.9

Resting heart rate (beats/min) 71.4 5.4 69.1 3.7 73.7 3.0


(a) heart period range (ms) 647.0 105.4 564.2 78.1 488.4 97.1


(b) slope (Ams/AmmHg) -0.14 0.04 -0.11 0.04 -0.57 0.32


(c) MAP at inflexion point 93.2 8.8 91.0 6.8 93.9 5.7


(d) heart period minimum (ms) 435.2 35.4 487.6 19.6 475 47.0


Maximum gain (Ams/AmmHg) 19.3 4.2 14.0 3.8 48.0 23.5


Threshold pressure (mmHg) 80.1 9.4 65.5 13.2 69.4 17.1


Saturation pressure (mmHg) 106.2 8.9 116.4 7.2 118.5 19.1






69



Table 4.2. Linear baroreflex responses to nitroprusside and phenylephrine.

Experimental Group


Vehicle (n=7) 3P4 (n=7) 6P4 (n=7)


Nitroprusside (Ams/AmmHg) 10.2 2.1 11.8 2.3 5.7 1.3 *


Phenylephrine (Ams/AmmHg) 23.2 16.9 # 5.9 2.4 7.4 2.1



*significantly different from 3P4,
#n=6 after omitting outlying value of 124.0 Ams/AmmHg








70





50 -A 110 ,-
40 105
40 -
100
95
20 90
10 85
0 80
I I I II i
0 30 60 90 120 E 0 30 60 90 120
0 D
S50 -C 1105
C 40- 05
t 100 -
0 30- 0A
C 0 95
0 20- o
10- 1 85-
0 0 80 -
0 0 30 60 90 120 0 30 80 90 120
1.
IL
50 t110-
Ec
100
30 0
95
20 90

10 85 -
0 80
I I I I i I
0 30 60 90 120 0 30 60 90 120

Time (min)










Figure 4.1. Plasma progesterone (P4; A,C,E) and MAP (B,D,F) during 2-hour infusions
of vehicle (0; A,B), or P4 at rates of 3 pg/kg/min (M; C,D) and 6 pg/kg/min (A; E,F).
Data are means SEM (n=7).







71







1200 A 1200 B
00000

a pa

800 800



400 400 -
0 I I I I I I I I I I
o. 30 60 90 120 150 30 60 90 120 150
L

O 1200 C 1200 -

11

S800 800
MA A *


400 400
I I I I I I I I
30 60 90 120 150 30 60 90 120 150

Mean Arterial Pressure (mmHg)












Figure 4.2. Representative baroreflex curves from ewe W94 after infusion of vehicle (0;
A), 3 pg P4/kg/min (I; B), and 6 pg Pdkg/min('; C). Mean baroreflex curves were
computer generated using mean logistic curve fit parameter values (D); vehicle (solid line,
n=7), 6 pg P4/kg/min (dotted line, n=7), and 6 pg P4kg/min (dashed line, n=7).








72





2- A



5- 1
E





-2
I i I I I
0 30 60 90 120

5- B
4
3-




I I I I I



E 0.8 -

0.0
CL



-0.4-

S-0.8-
0 30 60 90 120

Time (min)









Figure 4.3. Effect of 2-hour infusions of vehicle (0, n=7) or P4 at rates of 3 pg/kg/min
(U, n=7) and 6 gg/kg/min (', n=7) P4 on plasma Na' (A), AVP (B), and protein (C).
Data are means SE.














CHAPTER 5
ACUTE EFFECT OF PROGESTERONE ON BLOOD PRESSURE, BLOOD VOLUME, AND
BAROREFLEX FUNCTION IN THE OVARIECTOMIZED EWE


5.1 Introduction


A recent study by Heesch and Rogers (98) demonstrated the progesterone metabolite

THP, which circulates at elevated levels during pregnancy (46), rapidly causes a leftward shift in

setpoint and decreases the sensitivity of renal sympathetic nerve responses to changes in arterial

pressure. These investigators propose THP modulates a GABAergic component of the baroreflex

in the rostral ventral lateral medulla (RVLM). In the RVLM, GABAergic neurons inhibit

excitatory amino acid projections to pre-ganglionic sympathetic cell bodies in the

intermediolateral cell column.

The results of the study presented in Chapter 4 suggest progesterone also has rapid effects

on arterial pressure, and the relationship between plasma progesterone concentration and MAP is

not a simple, linear dose-response relationship. Studies have not confirmed an effect of

progesterone alone on baroreflex sensitivity; progesterone did not decrease baroreflex sensitivity

after two hours (Chapter 4) or two weeks (210) of progesterone treatment despite a reduction in

MAP in both time intervals. However, these studies measured the rapid heart rate responses to

single doses ofphenylephrine and nitroprusside to evaluate baroreflex responsiveness. This

method may measure the rapid vagal response to pressure changes, but not the slower-responding

GABA-modulated sympathetic efferent response (45).




73









74
A study of the ovariectomized ewe demonstrated chronic treatment with progesterone at

concentrations typical of ovine pregnancy also expands plasma volume (210). The time-course

and mechanisms of progesterone-induced changes in plasma volume are unknown. The plasma

protein data presented in Chapter 4 did not suggest an acute effect of progesterone on plasma

volume, but a more complete analysis is required. Slower genomic action at receptors in the

vascular system, kidney, hypothalamus, and brain stem may be required for progesterone to

increase plasma volume and maintain decreased pressure in the presence of increased volume.

Progesterone could slowly increase plasma volume by acting directly in the kidney or by

increasing AVP synthesis in the hypothalamus and subsequent release from the posterior pituitary.

However, chronic volume expansion is generally associated with an increase in blood pressure

rather than the decrease in pressure observed with progesterone. This suggests progesterone also

must decrease vascular tone by acting directly in the vascular system (202) or by modifying

sympathetic outflow from the brainstem (98). A progesterone-induced decrease in vascular tone

also would contribute to the expansion of plasma volume by progesterone. Expanded plasma

volume also could contribute to a decrease in baroreflex sensitivity since other investigators have

reported an attenuation of the baroreflex in the volume-loaded state (49). A complete assessment

of the time course of progesterone-induced changes in MAP, plasma volume, and baroreflex

responsiveness would further clarify the role of progesterone in cardiovascular adaptation to

pregnancy.

Two studies were conducted to more completely evaluate the rapid nongenomic and early

genomic effects of progesterone infusion on arterial pressure, blood volume and baroreflex

sensitivity. The first study was a 30-minute infusion designed to determine if, in addition to an









75
alteration in arterial pressure, progesterone rapidly alters plasma volume and the slower-

responding sympathetic component of the heart rate response to perturbations in arterial pressure.

The second study consisted of a 4-hour infusion of control vehicle or progesterone at rates of 1.5,

3, and 6 pg/kg/min. This study was designed to further evaluate the relationship between

progesterone concentration and arterial pressure and to determine if progesterone has slower

effects on plasma volume and the baroreflex. Ovariectomized ewes were studied in order to

eliminate possible confounding changes in plasma steroid levels during the natural estrous cycle,

and estradiol was replaced to a basal level since many of progesterone's effects are known to

require estrogen priming (123, 124).

5.2 Methods


Eight adult ovariectomized-estradiol treated ewes were studied in a 30-minute protocol

and a 4-hour progesterone infusion protocol. Five of the eight ewes were studied beginning

approximately four months after ovariectomy and three of the ewes were studied beginning

approximately one week after ovariectomy. In the previous study progesterone was dissolved in a

10% ethanol vehicle, but in this study progesterone was administered in a 20% ethanol vehicle.

The concentration of vehicle ethanol was increased in this study to ensure the more highly

concentrated progesterone preparations would remain in solution throughout the experiment. In

addition, a single estradiol implant (3 cm) was placed in dorsal scapular region of each ewe since

many of progesterone's effects are known to require prior estrogen priming and at least one form

of the intracellular progesterone receptor is induced by estrogen (125). The implants were

implanted one week before beginning the experiments, and the implants have been shown to









76
maintain plasma estradiol at levels (-4 pg/ml) characteristic of the luteal phase of ovine menstrual

cycle (123, 124). In order to minimize the possibility of residual progesterone effects, each ewe

was allowed at least a 2-day recovery period between infusion treatments.

5.2.1 30-Minute Infusion Protocol

In the 30-minute protocol, each ewe was subjected to three infusions: vehicle (20%

ethanol in 0.9% saline, 0.7 ml/min), and progesterone at rates of 1.5 (1.5P4) and 3 (3P4)

pg/kg/min in a randomized, crossover design. Arterial pressure was recorded continuously for 45

minutes, and the 30-minute infusion began after the first 15 minutes of pressure recording. Blood

samples were collected before beginning (40 ml) and at 5-minute intervals (18 ml) after Evans

blue injection and during the infusion. These blood samples were used to determine plasma

volume and progesterone concentrations. The volume of blood (approximately 166 ml)

withdrawn during the course of the study was about 2% of the total blood volume (approximately

7.5 L). Blood volume was measured using an adaptation of the Evans blue dye dilution technique

(18) designed to detect rapid shifts of fluid into the vascular compartment. Evans blue was

injected after ten minutes of baseline pressure recording and five minutes prior to beginning the

infusion. Plasma dye concentration was measured in samples obtained at 5-minute intervals

thereafter. Normally the dye is eliminated from plasma linearly over time, but if progesterone

rapidly shifts fluid into the vascular compartment, the slope would increase over time and

relationship between time and dye concentration would become curve-linear. Following the 30-

minute infusion of vehicle or progesterone, baroreceptor function was evaluated using graded

continuous infusions of phenylephrine and nitroprusside while the infusion continued. This









77
method allows closer examination of the more slowly responding sympathetic component of heart

rate control.

5.2.2 4-Hour Infusion Protocol

In the 4-hour protocol, each ewe was subjected to four infusions: vehicle (20% ethanol in

0.9% saline, 0.7 ml/min), and progesterone at 1.5 (1.5P4), 3 (3P4), and 6 (6P4) pg/kg/min in a

randomized crossover design. Arterial pressure was monitored continuously for 250 minutes, and

the 4-hour infusions began after the first ten minutes of pressure recording. Blood samples were

collected before beginning the infusion (40 ml), at 10-minute intervals during the first and last 30

minutes (10 ml) of the infusion, and at hourly intervals (10 ml each) throughout the experiment.

These blood samples were used for measurement of plasma progesterone concentration and

determination of plasma volume. Plasma volume was measured using the Evans blue dye dilution

technique during the final thirty minutes of the infusion. Following the 4-hour infusion of vehicle

or progesterone, baroreceptor function was evaluated using graded continuous infusions of

phenylephrine and nitroprusside while the infusion continued.

5.2.3 Statistical Analysis

MAP responses measured over time were analyzed by two-way ANOVA corrected for

repeated measures. Plasma progesterone concentrations, logistic curve fit parameters, and plasma

volume concentrations were compared by one-way ANOVA corrected for repeated measures;

when values were not normally distributed, a Friedman repeated measures ANOVA on ranks was

used. Individual progesterone means were compared using a Student-Newman-Keuls post-hoc

test. For all statistical tests, the null hypothesis was rejected when p < 0.05. Data are expressed

as the mean standard error of the mean (SEM).






78


5.3 Results


5.3.1 30-Minute Infusion Protocol

5.3.1.1 Plasma progesterone levels

Plasma progesterone concentration was 0.8 0.3 ng/ml after thirty minutes of

vehicle infusion (Figure 5.1A) and was significantly increased to 7.2 1.6 ng/ml and 15.0

3.4 ng/ml after infusion of progesterone at rates of 1.5 Lg/kg/min and 3 gg/kg/min,

respectively.

5.3.1.2 Arterial pressure

MAP averaged 92.2 2.9 mmHg during the 15-minute baseline recording, (Figure

5.1B, C, and D) and did not change significantly after 30 minutes of control vehicle

infusion (91.2 2.3 mmHg, Figure 5.1B). Similarly, infusion of progesterone at a rate of

1.5 ig/kg/min did not significantly change arterial pressure after 30 minutes (95.0 2.9

vs. 95.0 2.0 mmHg, Figure 5.1C). The 30-minute infusion of progesterone at a rate of 3

ig/kg/min significantly reduced arterial pressure over time from 95.0 2.9 to 92.4 1.7

mmHg (Figure 5.1D). However, arterial pressure at the end of the 30-minute infusion was

not significantly different from arterial pressure at the end of the vehicle infusion.

5.3.1.3 Plasma volume

Representative Evans blue dye elimination curves from ewe Y254 are presented in

figure 5.2. Optical density of plasma decreased linearly over time during infusion of

vehicle, or progesterone at rates of 1.5 and 3 pg/kg/min; the r2 values of the linear

regressions were 0.79 0.09, 0.68 + 0.12, and 0.83 0.11, respectively. As a result,






79


there was no evidence of rapid movement of fluid into the vascular compartment. The

slopes of the lines were 3.0 0.4, 2.1 0.3, and 2.5 0.4 AO.D./Amin, respectively.

Since these values were not significantly different among treatment groups, there was no

evidence of differential dye elimination during any of the infusion treatments. Plasma

volume (Figure 5.3) was 53.44.1 ml/kg after thirty minutes of vehicle infusion, and was

not significantly different after infusion of progesterone at rates of 1.5 (53.46.3 ml/kg) or

3 pg/kg/min (53.24.3 ml/kg).

5.3.2 4-Hour Infusion Protocol

5.3.2.1 Plasma progesterone levels

Plasma progesterone concentration was 2.4 0.9 ng/ml after four hours of vehicle

infusion (Figure 5.4). When progesterone was infused at a rate of 1.5 gg/kg/min, plasma

concentration was significantly increased to 8.1 1.5 ng/ml after four hours. Infusion of

progesterone at a rate of 3 [tg/kg/min significantly elevated progesterone levels to 19.5

4.9 ng/ml. Plasma progesterone concentration was 25.9 7.4 ng/ml after four hours of

infusion at a rate of 6 gg/kg/min, but this concentration was not significantly different

from the concentration obtained after infusion of 3 lg/kg/min.

5.3.2.2 Arterial pressure.

MAP did not change over time in response to any dose of progesterone (Figure

5.5). MAP averaged 88.3 6.8 mmHg at the beginning of the 4-hour vehicle infusion,

and was not significantly different at the end of the experiment (86.7 6.2 mmHg).

Similarly, MAP was not significantly different from control values after 4-hour infusions of






80


progesterone at rates of 1.5 (85.9 5.0 vs. 86.5 3.9), 3 (92.0 5.0 vs. 90.6 4.9), and

6 (85.3 5.7 vs. 87.6 5.6) lg/kg/min.

5.3.2.3 Plasma volume.

Plasma volume was 37.6 6.5 ml/kg after four hours of vehicle infusion (Figure

5.6), and was not significantly different after infusion of progesterone at rates of 1.5 (39.3

5.4), 3 (39.2 4.5), or 6 (37.9 4.5) 6 gg/kg/min.

5.3.2.4 Baroreflex

Representative logistic curve fits from an individual animal (ewe Y258) are

presented in Figure 5.7. Mean logistic curve-fit parameter values are presented in Table

5.1. Baroreflex curves were not significantly different among treatment groups.

5.4 Discussion


The results of the 30-minute progesterone infusion confirm a rapid reduction in

arterial pressure when progesterone is infused at a rate of 3 lg/kg/min, but a lower

infusion rate of 1.5 tg/kg/min did not decrease arterial pressure. The change in arterial

pressure during the infusion of progesterone at a rate of 3 ug/kg/min was not

accompanied by an increase in plasma volume, suggesting progesterone decreases arterial

pressure before expanding plasma volume. Progesterone may decrease arterial pressure

by directly reducing vascular tone (202) or by increasing the concentration of GABAA-

active progesterone metabolites (98). A decrease in vascular tone could contribute to a

subsequent expansion of plasma volume that also may require progesterone actions in the

hypothalamus and/or kidney.






81

The results of the 4-hour infusion do not confirm a rapid effect of progesterone on

MAP in the ovariectomized ewe, and demonstrate 4-hour infusions of physiological and

supraphysiological concentrations of progesterone do not alter plasma volume or the heart

rate response to perturbations in arterial pressure.

Baseline arterial pressure was significantly lower at the beginning of the 4-hour

(87.9 2.6 mmHg) infusion protocol than at the beginning of the 30-minute infusion

protocol (94.4 2.9 mmHg) and the study presented in Chapter 4 (94.9 2.1 mmHg).

This finding suggests progesterone rapidly reduces arterial pressure when baseline

pressures are slightly elevated. In all three studies care was taken to minimize stress to the

animals and to allow time for the animals to recover from the stress of interaction with the

investigator. However, the protocols of the two rapid infusion studies were probably

more stressful to the ewes than the 4-hour protocol. In the study presented in Chapter 4,

ewes were removed from their home cages and taken to a nearby laboratory for study. In

both the 30-minute and 4-hour protocols presented in this chapter the ewes were studied

in their home pens. However, the design of the 30-minute experiment could have been

more stressful to the ewes since the protocol required increased investigator activity

outside of the animal pen immediately before and during the study.

If progesterone rapidly reduces arterial pressure via GABAA active metabolites,

these metabolites may be most effective when sympathetic activity is increased. The

sympathetic nervous system contributes minimally to resting vascular tone, but increased

sympathetic tone may contribute to the observed elevations in baseline pressure in the two

rapid infusion experiments. This may explain why an infusion of progesterone at a rate of

3 Ig/kg/min rapidly reduces arterial pressure when baseline arterial pressures are slightly






82

elevated. Studies suggest sympathetic tone actually increases during pregnancy (264) and

sympathetic response gain is reduced due to reduced sympathetic reserve (29). Since

sympathetic outflow appears to be increased during pregnancy, the true role of elevated

progesterone metabolites in cardiovascular adaptation to pregnancy may be limited to their

effects on sympathetic gain (98) and the gain of components of sympathetic output also

may be selectively regulated (179). Progesterone metabolites may rapidly reduce arterial

pressure in the ewe by transiently reducing sympathetic outflow and may contribute to a

selective long-term reduction in sympathetic gain, but additional mechanisms may

contribute to the chronic effects of progesterone on cardiovascular function.

Although a steady-state baroreflex test was used to reveal potential changes in the

slow-responding sympathetic component of heart rate control, the 4-hour progesterone

infusions did not alter the heart rate response to perturbations in arterial pressure. This

finding is consistent with a recent report from Masilamani and Heesch which demonstrates

THP alters renal sympathetic nerve responses but not heart rate responses to changes in

arterial pressure obtained using graded continuous infusions of phenylephrine and

nitroprusside (179).

In the previous experiment the ewes were ovary-intact, anestrous, adults, but in

the current study chronically ovariectomized ewes were used. Evidence collected by other

investigators suggests the rapid pressure response to progesterone may be attenuated in

the ovariectomized animal. A recent study by Heesch et al. (157) demonstrated the effect

of THP on the baroreflex is abolished in the ovariectomized rat. The response to THP

may be altered in the ovariectomized rat since estrogens regulate GABAA receptor subunit

composition (99). To determine if the time response to progesterone decreases with time






83

after ovariectomy, the responses of the animals studied beginning one week after

ovariectomy (acutely ovariectomized ewes) were analyzed separately from the responses

of the animals studied beginning four months after ovariectomy (chronically

ovariectomized ewes). The findings of the study were not changed when the acutely and

chronically ovariectomized ewes were analyzed. This suggests that if ovariectomy

modifies the ability of progesterone to rapidly reduce arterial pressure, the response is

modified approximately one week after ovariectomy. Previous studies also have

demonstrated a reduction in 5a-reductase activity in the ovariectomized rat (153).

Therefore, if 5c-reductase activity also is reduced in the ovariectomized ewe, the ability of

progesterone to rapidly reduce arterial pressure may be compromised in this model.

However, chronic progesterone treatment (2 weeks) decreases blood pressure in

ovariectomized ewes, suggesting progesterone does not only modify blood pressure

through GABAA receptor mechanisms.

In summary, infusion of progesterone at a rate of 3 pg/kg/min rapidly reduces

arterial pressure in ovariectomized, estradiol-treated ewes when baseline pressure is

slightly elevated. Progesterone does not alter blood volume or the heart rate response to

perturbations in arterial pressure after thirty minutes or four hours. Further study is

needed to determine the effect of progesterone on arterial pressure when infusion is begun

at different starting pressures.







84








25 105 -
A B
E A
'" 20 T 100 -
.5 I
S15 E
2 E 95
10 a-
o 90
2 5
0 85
I I I I
0 15 30 45
Time (min)


105 C 105- D

S100- 100 -
I I
E 95- E 95
0- -
90- 2 90

85 85 -
I I I I I I I
0 15 30 45 0 15 30 45
Time (min) Time (min)





Figure 5.1. Effect of 30-minute progesterone infusions on plasma progesterone
concentration and MAP. A) Plasma progesterone concentration after 30-minute infusions
of vehicle (open bar, n=8), 1.5P4 (horizontal stripe bar, n=8), and 3P4 (diagonal stripe bar,
n=8). B) MAP during 30-minute infusions of vehicle (B, n=8), 1.5P4 (C, n=8), and 3P4
(D, n=8). Data are means SE.






85








S0.8- 0.6
S A B
d d
0.7 -

0.5 -
S0.6 -

W 0.5- 0.4
0 35 70 0 35 70
Time (min) Time (min)

0.6 -
O C
0.5 -

o 0.4

w 0.3
0 35 70
Time (min)


Figure 5.2. Representative Evans blue dye elimination curves from ewe Y254. Plasma
optical density decreases linearly over time during 30-minute infusions of vehicle (A) or
progesterone at rates of 1.5P4 (B) and 3P4 (C).






86





80



= 60
E T
E
-= 40-


E
S20



0



Figure 5.3. Plasma volume after 30-minute infusions of vehicle (open bar, n=8), 1.5P4
(horizontal stripe bar, n=8), and 3P4 (diagonal stripe bar, n=8). Data are means SE.







87


Table 5.1. Logistic baroreflex curve parameters obtained after 4-hour infusions of vehicle
or progesterone.



Experimental Group

Vehicle (n=8) 1.5P4 (n=8) 3P4 (n=8) 6P4 (n=8)

Resting MAP,
79.6 6.1 77.2 6.3 81.3 8.1 82.3 5.0
mmHg

Resting heart period,
804.5 71.2 701.7 80.8 788.1 95.7 795.0 110.8
ms

Heart period range,
683.6 108.4 755.7 + 115.7 646.9 110.2 645.2 107.9
ms

Slope, Ams/AmmHg -0.92 0.80 -1.14 0.69 -0.63 0.37 -0.40 0.16

MAP at inflexion
89.2 5.7 91.6 4.6 92.7 + 3.3 90.0 5.5
point, mmHg

Heart period
506.4 32.2 561.0 50.5 501.3 45.1 484.6 48.2
minimum, ms

Maximum gain,
85.5 65.3 265.5 174.0 77.8 45.7 51.9 19.2
Ams/AmmHg

Threshold pressure,
67.1 16.6 81.8 5.7 82.3 5.9 80.6 4.3
mmHg

Saturation pressure,
111.3 12.0 101.45.9 103.1 4.2 99.48.1
mmHg

Data are means SE.







88








40

35

30
E
'd 25 -
C
a)
5 20




















Figure 5.4. Plasma progesterone concentration after 4-hour infusions of vehicle (open
bar, n=8), 1.5P4 (horizontal stripe bar, n=8), 3P4 (diagonal stripe bar, n=8), and 6P4
(lattice bar, n=8). Data are means SE.
10




0-






89




110- 110 -
A B
0-100 100

E go 90 -
E 80 80 -

3 70 70 -
0I I I I I I
(0 0 50 100 150 200 250 0 50 100 150 200 250
0.
15

E 110-C 110
0 C
100- 100

90 90 -

80 80 -

70 70 -
I I I I I I I I I I I
0 50 100 150 200 250 0 50 100 150 200 250

Time (min)






Figure 5.5. MAP during 4-hour infusions of vehicle (A, n=8), 1.5P4 (B, n=8), 3P4 (C,
n=8) and 6P4 (D, n=8). Data are means SE.




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NOVEL RAPID NONGENOMIC AND SLOW GENOMIC MECHANISMS OF
OVARIAN STEROID MODULATION OF THE HYPOTHALAMIC-PITUITARY-
ADRENAL AXIS AND THE CARDIOVASCULAR SYSTEM DURING OVINE
PREGNANCY
By
DARREN MICHAEL ROESCH
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1998

my quest to understand: to my mother
this volume: to my sister
completely: to GNV

ACKNOWLEDGMENTS
This collection of data and hypotheses represents a much larger accumulation of
personal intellectual and emotional enlightenment. I will forever be grateful to those who
have contributed to my advancement: most of whom I will fail to mention in these pages.
First, I must thank my mentor, Dr. Maureen Keller-Wood, for providing the
intellectual and material fuel for this achievement. Her unselfish dedication to my
education has earned her a special place in my heart: I will always think of her as a
surrogate mother. I also am indebted to Dr. Charles E. Wood, the other role model in the
Wood lab family, for continuous sage and patient instruction.
I also thank the additional members of my supervisory committee for encouraging
and assuring a high standard of academic accomplishment: Dr. Pushpa S. Kalra, Dr.
Michael J. Katovich, Dr. William J. Millard, and Dr. Donna Wielbo.
I thank my family, especially my father, Mr. Daniel P. Roesch, for providing the
resources to make my education possible. In many ways, this dissertation also is their
achievement.
The Health Science Center has truly become an extended family, and many people
have contributed to my education and this dissertation. I will not attempt to thank
everyone because I will undoubtedly omit a dear friend. However, I must thank the
members of the Wood lab family for rolling up their sleeves to assist with the “dirty
work”: Ms. Sara Caldwell, Ms. Deanna Deauseault, Mr. David Husted, Dr. Eun-Kyung
iii

Kim, Ms. Ellen Manlove, Mr. Pini Orbach, Ms. Melanie Pockey, Mr. Scott Purinton, Dr.
Christine Saoud, and Dr. Hiayan Tong.
I thank Dr. Joanna Peris and the rest of the Department of Pharmacodynamics
faculty for believing in me when even I did not believe in myself. I thank Dr. Janice
Wachtel Walton for helping me learn to live, and I acknowledge Mr. Gilbert N. Vansoi,
my living “candle on the water.”
IV

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
NOVEL RAPID NONGENOMIC AND SLOW GENOMIC MECHANISMS OF
OVARIAN STEROID MODULATION OF THE HYPOTHALAMIC-PITUITARY-
ADRENAL AXIS AND THE CARDIOVASCULAR SYSTEM DURING OVINE
PREGNANCY
By
Darren Michael Roesch
May 1998
Chair: Maureen Keller-Wood, Ph.D.
Major Department: Pharmacodynamics
The setpoint and response of both the cardiovascular system and the hypothalamic-
pituitary-adrenal (HPA) axis are altered during pregnancy. Evidence strongly
demonstrates estrogens regulate these systems, and the role of progesterone is becoming
better appreciated. However, multiple receptor types may contribute to progesterone’s
effects. In addition to the well-described progesterone receptor, progesterone interacts
with two other intracellular steroid-binding transcription factors: the mineralocorticoid
(MR) and glucocorticoid (GR) receptors. The 5a-reduced metabolite of progesterone,
tetrahydroprogesterone (THP), also interacts with GABAa receptors to mediate rapid
physiological effects. The dissertation examines the possibility that progesterone acts
through these novel receptors to affect the cardiovascular system and the HPA axis.
v

Plasma progesterone levels characteristic of ovine pregnancy rapidly reduced mean
arterial pressure (MAP) in ovary-intact ewes. THP could induce these rapid progesterone
effects. However, since supraphysiological levels of progesterone did not alter MAP,
multiple receptor types appear to contribute to the rapid cardiovascular effects of
progesterone.
Physiological levels of progesterone also rapidly reduced arterial pressure in
ovariectomized ewes when starting baseline pressures were slightly elevated; suggesting
baseline arterial pressure and/or reproductive state influence the rapid effects of
progesterone. Decreased 5a-reduction of progesterone was confirmed in the livers of
ovariectomized ewes, suggesting ovarian steroids modulate progesterone metabolism.
MR and GR availability and immunoreactivity are differentially regulated in the
ovine hippocampus during pregnancy. Progesterone could contribute to these observed
changes. However, chronic treatment of ovary-intact ewes with estrone, but not chronic
treatment of ovariectomized ewes with progesterone, induced changes in these receptors
characteristic of ovine pregnancy. This finding suggests estrogens modulate these
receptors, but does not eliminate the possibility the progesterone also regulates these
receptors. Differential regulation of hippocampal MR and GR could contribute to altered
activity and responsiveness of the HPA axis during pregnancy, and altered HPA axis
activity could contribute to cardiovascular adaptation to pregnancy since steroid and
peptide components of the axis modulate fluid homeostasis, vascular responsiveness, and
neural blood pressure control.
vi

These studies support the hypothesis that ovarian steroids contribute to adaptation
of the HPA axis and the cardiovascular system to pregnancy via novel rapid nongenomic
and slow genomic mechanisms.
Vll

TABLE OF CONTENTS
page
ACKNOWLEDGMENTS iii
ABSTRACT v
CHAPTERS
1 INTRODUCTION 1
2 REVIEW OF THE LITERATURE 4
2.1Cardiovascular Adaptation to Pregnancy 4
2.1.1 Plasma Volume 5
2.1.1.1 Mineralocorticoids 5
2.1.1.2 Glucocorticoids 6
2.1.1.3 A VP 7
2.1.1.4 ANP 7
2.1.1.5 Estradiol 8
2.1.1.6 Progesterone 9
2.1.2 Cardiac Output 10
2.1.3 Mean Arterial Pressure (MAP) 11
2.1.4 Reflex Control 14
2.5 The HPA Axis 18
2.5.1 Introduction 18
2.5.2 Corticosteroid Receptors 19
2.5.2.1 Ligand-binding characteristics 19
2.5.2.2 Receptor activation 20
2.5.2.3 Genetic characteristics 21
2.5.2.4 Intracellular location 22
2.5.2.5 Differentiation between mineralocorticoid and glucocorticoid
signals 22
2.5.2.6 Receptor auto-regulation 24
2.5.3 Feedback Inhibition 24
2.5.3.1 Intermediate and slow feedback inhibition 25
2.5.3.2 Fast feedback inhibition 30
2.5.3.3 HPA axis during pregnancy 31
viii

2.6 Role of Altered HPA Axis Function in Cardiovascular Adaptation to
Pregnancy 35
2.7 Objectives 41
3 GENERAL MATERIALS AND METHODS 44
3.1 Animal Care 44
3.2 General Surgical Procedures 45
3.2.1 Ovariectomy 45
3.2.2 Catheterization 45
3.3 Chronic Steroid Implantation 46
3.4 Acute Progesterone Infusions 46
3.5 Handling and Analysis of Blood Samples 47
3.5.1 Sampling and Storage 47
3.5.2 Plasma Analysis 47
3.6 Cardiovascular Measurements 48
3.6.1 Mean Arterial Pressure 48
3.6.2 Plasma Volume 48
3.6.3 Baroreflex 49
3.6.3.1 Bolus injection method 49
3.6.3.2 Steady-state method 49
3.6.3.3 Expression of data 50
3.7 Tissue Collection 51
3.8 5a-Reductase / 3a-Hydroxysteroid Dehydrogenase Activity Assay 52
3.8.1 Tissue Preparation 52
3.8.2 Activity Assay 52
3.9 Radioligand Binding Assays 53
3.9.1 Tissue Preparation 53
3.9.2 Incubations 54
3.9.3 Separation of bound from free 54
3.10 Western Blots 55
3.10.1 Tissue Preparation 55
3.10.2 Electrophoresis and Transfer 56
3.10.3 Densitometry 56
3.11 Statistical Analysis 57
4 ACUTE EFFECT OF PROGESTERONE ON CARDIOVASCULAR
FUNCTION IN THE OVARY-INTACT EWE 58
4.1 Introduction 58
4.2 Methods 59
4.2.1 Progesterone Infusion and MAP 60
4.2.2 Baroreflex Test 60
4.2.3 Statistical Analysis 60
4.3 Results 61
4.3.1 Plasma Progesterone Levels 61
4.3.2 Arterial Pressure 61
4.3.3 Baroreflex 62
IX

4.3.4 PlasmaNa+ and AVP 63
4.3.5 Plasma Protein 63
4.4Discussion 63
5 ACUTE EFFECT OF PROGESTERONE ON BLOOD PRESSURE, BLOOD
VOLUME, AND BAROREFLEX FUNCTION IN THE
OVARIECTOMIZED EWE 73
5.1 Introduction 73
5.2 Methods 75
5.2.1 30-Minute Infusion Protocol 76
5.2.2 4-Hour Infusion Protocol 77
5.2.3 Statistical Analysis 77
5.3 Results 78
5.3.1 30-Minute Infusion Protocol 78
5.3.1.1 Plasma progesterone levels 78
5.3.1.2 Arterial pressure 78
5.3.1.3 Plasma volume 78
5.3.2 4-Hour Infusion Protocol 79
5.3.2.1Plasma progesterone levels 79
5.3.2.4 Baroreflex 80
5.4 Discussion 80
6 CHARACTERIZATION OF 5a-REDUCTASE ACTIVITY IN THE LIVER
AND BRAINSTEM OF THE NON-PREGNANT, PREGNANT, AND
OVARIECTOMIZED EWE 92
6.1 Introduction 92
6.2 Methods 93
6.3 Results 93
6.4 Discussion 95
7 CHARACTERIZATION OF BRAIN MINERALOCORTICOID AND
GLUCOCORTICOID RECEPTOR AVAILABILITY IN THE NON¬
PREGNANT AND PREGNANT EWE 101
7.1 Introduction 101
7.2 Methods 102
7.3 Results 103
7.4 Discussion 104
8 EFFECT OF PREGNANCY ON THE APPARENT BINDNG AFFINITY OF
THE MINERALOCORTICOID AND GLUCOCORTICOID RECEPTOR Ill
8.1 Introduction Ill
8.2 Methods 112
8.3 Results 113
8.4 Discussion 114
x

9 CHARACTERIZATION OF CYTOSOLIC IMMUNOREACTIVE
MINERALOCORTIOID AND GLUCOCORTICOID RECEPTORS IN THE
NONPREGNANT AND PREGNANT EWE 121
9.1 Introduction 121
9.2 Methods 122
9.3 Results 122
9.3.1 MR Immunoreactivity 122
9.3.2 GR Immunoreactivity 123
9.4 Discussion 124
10 EFFECT OF CHRONIC PROGESTERONE TREATMENT ON
HIPPOCAMPAL MINERALOCORTICOID AND GLUCOCORTICOID
RECEPTOR AVAILABILITY AND CYTOSOLIC IMMUNOREACTIVITY
IN THE OVARIECTOMIZED EWE 134
10.1 Introduction 134
10.2 Methods 134
10.3 Results 135
10.3.1 Plasma Steroid Levels 135
10.3.2 MR and GR Availability 135
10.3.3 MR and GR Immunoreactivity 136
10.4 Discussion 136
11 EFFECT OF CHRONIC ESTRONE TREATMENT ON HIPPOCAMPAL
MINERALOCORTICOID AND GLUCOCORTICOID RECEPTOR
AVAILABILITY AND CYTOSOLIC IMMUNOREACTIVITY IN THE
OVARY-INTACT EWE 143
11.1 Introduction 143
11.2 Methods 143
11.3 Results 144
11.3.1 MR and GR Availability 144
11.3.2 MR and GR Total Immunoreactivity 144
11.4 Discussion 144
12 SUMMARY 149
12.1 Overview 149
12.2 Specific Hypotheses Tested: 149
12.2.1Hypothesis 1: Progesterone Rapidly Alters Arterial Pressure, Blood
Volume and Baroreflex Sensitivity 150
2.2.2 Hypothesis 2: MR and GR Availability, Immunoreactivity, and
Apparent Affinity are Altered During Pregnancy 154
12.2.3Hypothesis 3: Ovarian Steroids Alter MR and GR Availability and
Immunoreactivity 158
REFERENCES 159
BIOGRAPHICAL SKETCH 196
xi

CHAPTER 1
INTRODUCTION
Maternal cardiovascular homeostasis is drastically adjusted during pregnancy,
presumably for the benefit of fetal development. Many investigators have hypothesized
endocrinologic mechanisms contribute to cardiovascular adaptation to pregnancy since the
plasma concentrations of numerous reproductive hormones rise as gestation progresses.
However, the precise mechanisms leading to the observed increase in blood volume, and
decrease in MAP, vascular tone, and baroreflex responsiveness are not understood.
Remarkably, circulating estrogens and progestins increase as much as 100-fold
over non-pregnant levels during pregnancy (8). Evidence suggests progesterone is the
steroid responsible for the maintenance of pregnancy since progesterone maintains the
myometrium of the uterus in a quiescent state (8). In women, progesterone derived from
the corpus luteum of the ovary briefly supports early gestation, but progesterone is
derived primarily from the placenta for the duration of human pregnancy (8).
Progesterone levels rise to about 100 ng/ml about 45 days into a human pregnancy, fall to
about 60 ng/ml by about the TO^day of gestation and then rise steadily to peak at about
200 ng/ml at parturition (about 289 days). In the sheep, progesterone levels are about
one-tenth the levels observed in human pregnancy (8). As in the human, progesterone
derived from the placenta can maintain ovine pregnancy in the absence of the ovaries, but
this independence of the placenta occurs at a later stage of pregnancy (8). During ovine
1

2
pregnancy, progesterone levels rise steadily and peak at about 10 ng/ml after about 130
days of gestation. At this point, plasma progesterone levels rapidly decline toward and
reach non-pregnant levels when parturition occurs after about 145 days of gestation (8).
In contrast to progesterone, estrogens enhance rhythmic contraction of the uterus
(8). Therefore, it has been suggested that progesterone dominates and maintains
pregnancy throughout the course of gestation and an increased ratio of
estrogens/progestins at the termination of gestation allows the initiation of parturition (8).
In women, estrogen biosynthesis occurs in both the fetus and placenta (8). Plasma
estrogen levels begin to rise steadily beginning about 100 days into human gestation and
peak at about 200 ng/ml at parturition. In sheep, estrogen concentrations are much lower
than they are in women and estrogens are primarily synthesized by the placenta in sheep
(8). In this species, plasma estrogen levels remain below 30 pg/ml until about the 140th
day of gestation then rise rapidly to about 200 pg/ml at parturition (8).
Over the years, numerous studies have revealed elevated estrogens contribute to
cardiovascular adaptation to pregnancy. In contrast, the importance of progestins has
long been debated since studies using supra-physiological levels of progestins actually
suggest progestins decrease MAP by reducing blood volume. More recent studies
demonstrate chronic treatment with physiological levels of progesterone results in
expanded plasma volume and reduced MAP, suggesting physiological levels of
progesterone contribute to maternal cardiovascular homeostasis.
Hormones mediate specific effects by interacting with specific receptors in target
tissues, and progesterone certainly produces many of its effects by interacting with the
isoforms of the intracellular steroid-binding transcription factor commonly known as the

3
progesterone receptor. Evidence also suggests progesterone and its metabolites interact
with intracellular and extracellular receptors not commonly considered progesterone
receptors. A metabolite of progesterone, tetrahydroprogesterone (THP), is now known to
potentiate the effects of the neurotransmitter y-aminobutyric acid (GABA) at the GABAa
chloride channel and accumulating evidence suggests this progesterone metabolite rapidly
modulates cardiovascular function by interacting with this receptor type.
Progesterone also interacts, with varying affinity and efficacy, with two
intracellular steroid-binding transcription factors known as the corticosteroid
(mineralocorticoid (MR) and glucocorticoid (GR)) receptors. These two novel
progesterone receptors modulate the responsiveness and mediate the effects of the
hypothalamic-pituitary-adrenal (HPA) axis, an endocrine axis known to respond to
physiological and psychological stress, cardiovascular disturbances, and to contribute to
cardiovascular homeostasis. Therefore, interaction of progesterone with the
corticosteroid receptor system may be an important genomic mechanism of cardiovascular
adaptation to pregnancy.
The availability of these numerous progesterone receptors probably contributes to
a multitude of time- and concentration-dependent progesterone effects. The studies
presented in this dissertation were designed to determine if physiological levels of
progesterone could contribute to cardiovascular adaptation to pregnancy by interacting
with these novel progesterone receptors.

CHAPTER 2
REVIEW OF THE LITERATURE
The setpoint and the response of both the cardiovascular system and the
hypothalamic-pituitary-adrenal (HPA) axis are altered during pregnancy. This dissertation
is based on the hypothesis that elevated concentrations of ovarian steroids contribute to
adaptation of these two functionally interdependent systems to pregnancy. This chapter
reviews current knowledge of cardiovascular and HPA axis function during pregnancy,
outlines the functional integration of the two systems, and considers potential mechanisms
of adaptation to pregnancy.
2,1 Cardiovascular Adaptation to Pregnancy
Studies consistently describe decreased mean arterial pressure (MAP) and
increased plasma volume, heart rate, and cardiac output in pregnant women (68), rabbits
(110), dogs (27), rats (51), and ruminants (186). These changes are essential for normal
fetal development since the incidence of gestational complications increases when these
variables fail to adjust (92, 206). The correlation of altered cardiovascular function with
hormonal rhythms both during pregnancy and during the menstrual cycle led to the
hypothesis that ovarian steroids contribute to adaptation of the cardiovascular system
(169). As will be discussed below, numerous studies demonstrate estradiol induces many
cardiovascular changes characteristic of pregnancy, and recent evidence suggests
4

5
physiological concentrations of progesterone also contribute to cardiovascular adaptation
to pregnancy.
2.1.1 Plasma Volume
Plasma volume expands by 40-50% in gravid humans and rats, but in species such
as the sheep plasma volume increases only about 10% during pregnancy (187).
Differential modulation of plasma mineralocorticoids, glucocorticoids, arginine
vasopressin (AVP), and atrial natriuretic peptide (ANP) contribute to volume expansion
during pregnancy.
2.1.1.1 Mineralocorticoids
The renin-angiotensin system regulates adrenocortical secretion of the
mineralocorticoid aldosterone. The smooth muscle cells in the afferent and efferent
arterioles of the juxtaglomerular apparatus release renin, the rate-limiting enzyme of the
renin-angiotensin system, in response to decreased perfusion pressure, increased renal
sympathetic nerve activity, and decreased sodium delivery to the macula densa. Renin
converts liver-derived angiotensinogen to angiotensin I, and angiotensin I is subsequently
converted to angiotensin II by the ubiquitously distributed endothelial angiotensin
converting-enzymes. Angiotensin II stimulates aldosterone secretion from the zona
glomerulosa of the adrenal cortex by increasing activity of steroid synthesis enzymes.
Adrenocorticotropin (ACTH), a peptide released from the anterior pituitary when the
HPA axis is stimulated, also contributes to mineralocorticoid synthesis by increasing
enzyme activity. Both angiotensin II and aldosterone are important modulators of plasma
volume. Angiotensin II stimulates vasoconstriction of arterioles, AVP secretion from the
posterior pituitary, sodium (Na+) reabsorption in the proximal tubule, and acts in the

6
hypothalamus to stimulate drinking behavior. Aldosterone also stimulates Na+ and
subsequent water reabsorption in the late portion of the distal tubule and the collecting
duct (152). During pregnancy plasma angiotensinogen, renin activity, angiotensin II,
angiotensin converting-enzymes, and aldosterone increase and may contribute to volume
expansion (173).
2,1,1,2 Glucocorticoids
The doubling of maternal plasma glucocorticoid (cortisol is the primary
glucocorticoid in the human and the sheep) concentrations during pregnancy (65, 132,
199) may contribute to the shift of fluid into the vascular space. Like mineralocorticoids,
glucocorticoids shift Na+ and subsequently fluid out of cells (162). However,
mineralocorticoids alone are not sufficient to replenish plasma volume in adrenalectomized
dogs (263). Cortisol also increases hepatic protein production and accelerates movement
of interstitial protein into the vascular system (16, 266). As a result, plasma proteins
increase and enhance oncotic pull of fluid into the vascular system. Gann and Pirkle (83,
218-220) propose cortisol first shifts electrolytes and fluids into the interstitial space. As a
result of the increased interstitial pressure, transcapillary filling and lymphatic flow
increase, and the increased lymphatic flow increases circulating protein and oncotic pull of
fluid into the vascular system. Although electrolyte and protein shifts induced by
mineralocorticoids and glucocorticoids could contribute to volume expansion during
pregnancy, plasma oncotic pressure and osmolality would be expected to remain
unchanged following equilibration (257). Since plasma oncotic pressure and osmolality
actually fall during pregnancy (64), additional mechanisms probably contribute to volume
expansion and hemodilution during pregnancy.

7
2.1.1.3 A VP
AVP release from the posterior pituitary also is regulated differently during
pregnancy. Hypothalamic osmoreceptors and, to a lesser extent, the baroreceptors
mediate increased AVP release in response to hyperosmolality or hypotension,
respectively (152). AVP increases aquaporin expression and water reabsorption in the
collecting duct (149). Basal concentrations of plasma AVP are not changed in pregnant
humans, rats, or sheep, but the setpoint for stimulation of AVP does reset during
pregnancy (134, 165). The osmotic threshold for stimulation of AVP and thirst is reduced
during all stages of human pregnancy and the slope of the linear relationship between
plasma osmolality and AVP is reduced in late pregnancy (165). In humans, increased
placental production of AVP-degrading aminopeptidases contributes to the apparent reset
in AVP regulation (165), but these enzymes are not increased in the sheep and other
species during pregnancy (230). In pregnant ewes, the relationship between plasma
osmolality and AVP is not altered, but the relationship between MAP and AVP is shifted
to the left in this species (134) and in dogs (27). These findings suggest altered AVP
responsiveness contributes to decreased arterial pressure in the presence of expanded
volume, but it is unclear if AVP modulates volume expansion during pregnancy.
21,1.4 ANP
Differential regulation of plasma ANP levels and target tissue responsiveness to
ANP during pregnancy contributes to cardiovascular adaptation to pregnancy. Normally,
volume-induced atrial stretch stimulates atrial myocytes to release ANP and increase water
and Na+ excretion. ANP is a potent vasodilator, increases glomerular filtration rate and
filtered Na+ load and inhibits renin, aldosterone, and AVP release and Na+ reabsorption by

8
the collecting duct (152). Basal circulating ANP either increases or remains the same
during pregnancy (127) and pregnancy attenuates both atrial ANP release in response to
atrial distension and renal responsiveness to ANP (126, 203, 204). Thus, differential
attenuation of the ANP system contributes to volume expansion and hemodilution during
pregnancy.
2.1.1.5 Estradiol
Chronic treatment of ewes with estradiol (174, 277) and women with estrogen-
containing oral contraceptives (3, 14) increases blood volume. Estrogens do not appear to
increase volume by altering endocrine control of fluid homeostasis.
Although some evidence suggests estrogens increase plasma volume by
augmenting activity of the renin-angiotensin system (128), a recent study by Magness and
colleagues suggests the estrogen-induced increase in renin-angiotensin system activity is a
temporary baroreflex-mediated response to the estrogen-induced decrease in MAP (174).
These investigators demonstrate renin activity and angiotensin II concentrations normalize
as volume shifts into the dilated vascular beds (174).
AVP content of the hypothalamic paraventricular nucleus and the posterior
pituitary peaks in the rat at proestrus when plasma estrogen concentrations are the highest
(53, 94). However, administration of estrogens to rats does not alter the threshold for
osmotic stimulation of AVP and actually increases the slope of the relationship between
plasma osmolality and AVP (59). Investigators have found the effect of estrogen
replacement on plasma AVP depends on the route and duration of administration (215).
As a result, further study is required to establish the role of estrogens in adaptation of the
AVP response during pregnancy.

9
Similarly, estrogen treatment does not mimic the effects of pregnancy on ANP
release (126). As will be discussed below, estrogens probably contribute to volume
expansion by decreasing vascular resistance and allowing volume to shift into the vascular
space.
2,1,1,6 Progesterone
Recent evidence suggests progesterone also may contribute to volume expansion
during pregnancy. Early studies demonstrated supraphysiological concentrations of
progesterone antagonize the salt and fluid retaining actions of mineralocorticoids (254,
287). However, studies demonstrate plasma renin activity, aldosterone, and volume are
increased in women during the progesterone-dominant luteal phase of the menstrual cycle
(97, 247) and in men after administration of progesterone (187). Chronic treatment with
physiological concentrations of progesterone over 10-14 days also increases plasma
volume in ovariectomized ewes, but without increasing activity of the renin-angiotensin
system (210). These findings are contradictory to the anti-mineralocorticoid hypothesis of
progesterone action and suggest progesterone enhances volume expansion during
pregnancy.
Progesterone does not appear to expand plasma volume by increasing activity of
the renin-angiotensin system (210) or decreasing release of ANP in response to atrial
stretch (296). However, progesterone treatment does diminish the natriuretic effect of
ANP in the kidney (203). Progesterone also may contribute to plasma volume expansion
in the ewe by increasing AVP (210), although evidence suggests progesterone actually
decreases plasma AVP in ovariectomized rats (52). Progesterone may indirectly increase
AVP by increasing sodium appetite (50, 178, 188) and plasma Na+ concentration (210) or

10
progesterone may modulate regulation of AVP by corticosteroid receptors in the
hypothalamus.
2,1.2 Cardiac Output
Increased heart rate and stroke volume augment cardiac output during pregnancy
(173). Magness and colleagues (173) have demonstrated estradiol increases cardiac
output. In the acute phase of estradiol treatment (approximately 3 days), estradiol does
not alter stroke volume and increases cardiac output by increasing heart rate (174). Blood
volume expands after about 7 days of estradiol treatment and increases stroke volume and
further enhances cardiac output (174). This finding suggests increased blood volume and
end-diastolic filling volume augment stroke volume. Some evidence suggests estrogens
enlarge the left ventricular chamber and allow increased end-diastolic volume without
increased end-diastolic filling pressure (86). Although evidence suggests estrogens
increase cardiac output, the effect of progesterone on cardiac output has not been
reported.
A recent study by Brooks and colleagues (26) suggests altered parasympathetic
and sympathetic neural control allows heart rate to increase during pregnancy. Basal
sympathetic output to the heart increases, and the observed decrease in gain of reflex
control of heart rate is due to decreased sympathetic response reserve. Furthermore,
reduced cardiac sensitivity to cholinergic agents decreases the ability of the
parasympathetic system to maximally reduce heart rate.
Currently, the effect of ovarian steroids on control of the heart rate is not
completely understood. As discussed above, estradiol increases heart rate (174, 277), but
physiological concentrations of progesterone may actually decrease heart rate (210).

11
Further study is required to determine the effects of estradiol and progesterone on
sympathetic and parasympathetic output to the heart and on cardiac responsiveness to
adrenergic and cholinergic neurotransmitters.
2,1,3 Mean Arterial Pressure (MAPI
A decrease in total peripheral resistance reduces MAP by 5-10 mm Hg during
pregnancy (67). An early hypothesis argued that the addition of a low-resistance
uteroplacental circulation contributes to decrease in total peripheral resistance (169).
However, a recent study of the pregnant guinea pig demonstrates the increase in total
vascular conductance is due largely to an increase in non-uteroplacental conductance (54).
Evidence suggests ovarian steroids decrease resistance in non-uteroplacental vascular
beds.
Chronic estradiol treatment decreases MAP and vascular tone (174, 277) and since
the progesterone-induced decrease in MAP is accompanied by an expansion of plasma
volume, progesterone also appears to decrease vascular tone (210). Chronic progesterone
treatment also reduces blood pressure in humans, dogs, and rats under conditions in which
mineralocorticoid levels are not elevated (5, 31, 63, 84, 260). The contribution of ovarian
steroids to decreased vascular tone and arterial pressure is incompletely understood but
may include direct regulation of the vascular system or indirect regulation of endocrine
and neural modulators of vascular reactivity and tone.
Estrogen and progesterone receptors are located in vascular endothelial nuclei
(107, 114, 121, 148, 160, 164,205,214). Estrogen and progesterone may bind these
vascular steroid receptors and decrease peripheral vascular resistance via direct or indirect
mechanisms.

12
Evidence demonstrates progesterone has direct and indirect effects on vascular
tone. Using isolated placental arteries and veins, Omar and colleagues have recently
demonstrated progesterone directly elicits an acute reduction in vascular tone via a direct
mechanism in the vascular endothelium that is mediated by both intracellular progesterone
receptors and cyclic adenosine monophosphate (202). It is unclear whether this
progesterone effect on placental vascular tone also occurs in the maternal peripheral
vascular system. Evidence also suggests a 5a-reduced metabolite of progesterone,
dihydroprogesterone (DHP), contributes to decreased vascular tone and reactivity via an
indirect mechanism. DHP attenuates the pressor response to angiotensin II by increasing
circulating prostaglandins through an unknown receptor mechanism (71). Vascular
refractoriness to angiotensin II can be abolished in normal gravid women by administering
the prostaglandin synthesis inhibitor, indomethacin. Dihydroprogesterone restores
vascular refractoriness to angiotensin II in normal pregnancy women treated with
indomethacin and in women with pregnancy-induced hypertension who do not normally
exhibit refractoriness to angiotensin II (71). Since infusions of DHP were used in these
studies, it is not known if the GABAA-active 3a-dehyrogenated metabolite,
tetrahydroprogesterone (THP), contributes to the effects of DHP.
Estrogens appear to have an indirect effect on both vascular tone and vascular
reactivity. Estrogens contribute to decreased vascular tone by increasing production of
the endothelium derived relaxing factor, nitric oxide (NO), and vasodilatory
prostaglandins (173, 248). As described above, prostaglandins also contribute to
decreased vascular reactivity. Molnar and Hertelendy (191) have reported that in vivo
inhibition of nitric oxide synthase in pregnant rats increases vascular responsiveness to

13
angiotensin II, norepinephrine and AVP so that the responsiveness of the pregnant rats is
indistinguishable from the responsiveness of postpartum control rats.
Ovarian steroids could modulate sympathetic output to the vascular system.
However, decreased sympathetic output probably does not contribute to the observed
decrease in peripheral resistance during pregnancy since evidence suggests sympathetic
vascular tone increases during pregnancy (264). Ovarian steroid receptors are located in
brain regions associated with cardiovascular control (259). Ovarian steroids could have
currently uncharacterized direct effects in these brain regions or ovarian steroids could
modulate other central components involved in sympathetic control of the vascular
system. For example, ovarian steroids do regulate brain corticosteroid receptors (271)
known to modulate neural control of blood pressure.
As will be discussed below, there are two types of corticosteroid receptors:
mineralocorticoid (MR) and glucocorticoid (GR) receptors. Brain MR and GR appear to
regulate neural control of blood pressure. Central infusion of aldosterone increases blood
pressure by increasing vascular resistance in the rat and dog without increasing plasma
sodium concentration (119, 216), and the MR antagonist RU 28318 blocks the central
effect of aldosterone (90). These finding suggest brain MR modulate increases in
sympathetic outflow (279-282). Some evidence suggests that the brain renin-angiotensin
system mediates increases in sympathetic outflow induced by centrally administered
corticosteroids (265). The effects of centrally administered hydrocortisone on blood
pressure, heart rate, and sympathetic nerve activity can be abolished by
intracerebroventricular pretreatment with an angiotensin II receptor antagonist or an
angiotensin I converting-enzyme inhibitor (265). Although Gomez-Sanchez and

14
colleagues did not observe a change in blood pressure after central administration of the
GR-selective agonist RU 26988 (91), van den Berg and colleagues suggest activation of
central GR actually decreases arterial pressure (282). As a result, currently available data
suggest activation of central MR increases sympathetic outflow and central GR decreases
or does not control sympathetic outflow to the vascular system.
As will be discussed below, the sheep brain does not express significant MR-
protecting 1 l|3-hydroxysteroid dehydrogenate activity (145). Despite this fact,
intracerebroventricular infusion of aldosterone (269) or cortisol (144) does not change
MAP in this species. The failure of central corticosteroids to increase arterial pressure in
the sheep may be due to decreased brain MR availability in this species. The failure of
central glucocorticoids to decrease arterial pressure may be due to the lack of the
opportunity of infused glucocorticoids to inhibit sympathetic outflow in the resting,
unstressed animal. It is currently unknown how central MR and GR regulate arterial
pressure in any species during pregnancy when circulating corticosteroids, ovarian
steroids, and other endocrine factors that may modify MR and GR are elevated.
2,1,4 Reflex Control
The evidence presented above details potential mechanisms of adaptation of
resting cardiovascular function to pregnancy. Studies also demonstrate reflex control of
arterial pressure also is altered during pregnancy (29, 98, 133). Neural stretch receptors
located in the wall of internal carotid artery and in the wall of the aortic arch respond to
perturbations in arterial pressure (234). Signals from the baroreceptors are integrated
with other pressure-regulating inputs in the nucleus of the tractus solitarius and other brain
regions (4, 41, 283) and neural output to the periphery is modified accordingly (234).

15
Heart rate and renal sympathetic nerve responses to perturbations in arterial pressure are
commonly used to index reflex control of arterial pressure (69), and numerous studies of
reflex control of arterial pressure during pregnancy have been conducted (27, 28, 47, 51,
109-113, 133, 159, 179, 223). The results of these studies have led investigators to
conclude the gain of the baroreflex may be augmented, attenuated, or not changed.
Brooks and colleagues (29) argue these studies draw conflicting conclusions because they
look at different portions of the baroreflex curve. While some studies examine the entire
baroreflex curve, most studies focus only on the response to an increase or decrease in
blood pressure. A careful analysis of the entire baroreflex curve suggests an attenuated
response to hypotension accounts for a decrease in overall baroreflex sensitivity during
pregnancy (29). Elevated ovarian steroids could modify reflex control of arterial pressure
during pregnancy by modifying responsiveness of the pressure sensitive stretch receptors,
signal processing in the brain integration centers, or sensitivity of targets controlled by
neural outflow.
Estrogens do not appear to reduce the gain of the baroreflex (29), but evidence
does suggest progesterone metabolites have rapid, nongenomic actions on the baroreflex.
Progesterone can be sequentially metabolized (see Figure 6.1) by 5oc-reductase and 3oc-
hydroxysteroid dehydrogenase in the brain and periphery to form tetrahydroprogesterone
(THP). THP, which circulates at elevated levels during pregnancy, enhances activation of
the GABAa receptor by GABA (176). Rogers and Heesch (98) recently demonstrated
that THP rapidly causes a leftward shift in setpoint and at the same time decreases the
overall sensitivity of renal sympathetic nerve responses to changes in arterial pressure.
These investigators propose that THP acts in the rostral ventral lateral medulla (RVLM)

16
to enhance GABAergic activity and attenuate sympathetic outflow (98). Baroreceptor
neurons that monitor pressure in the internal carotid and aortic arch send information to
the nucleus of the tractus solitarius in the caudal dorsomedial medulla oblongata. This
information is integrated and relayed to GABA-containing cell bodies in the caudal ventral
lateral medulla via at least one intermediate synapse. These GABA-containing cell bodies
project to the RVLM where GABA inhibits excitatory amino acid projections to the
intermediolateral cell column. The intermediolateral cell column is the source of
preganglionic sympathetic cell bodies and all sympathetic outflow (4, 41, 262, 283).
Therefore, by enhancing GABA inhibition of excitatory projections to the
intermediolateral cell column, the elevated concentrations of THP present during
pregnancy may attenuate sympathetic outflow.
A more recent study by Masilamini and Heesch (179) suggests pregnancy and THP
selectively alter the renal sympathetic nerve response and not the heart rate response to
changes in arterial pressure. However, resting heart rate was elevated in this study and the
majority of the response curve was obtained from the bradycardic response to pressure
elevation. Accumulated evidence suggests sympathetic outflow to both the heart and the
vascular system increases during pregnancy (26, 264), and reduced sympathetic reserve
may account for the observed decrease in reflex gain. As a result, future investigations of
the effect of ovarian steroids on components of sympathetic outflow and reflex gain must
carefully ensure experiments commence at resting conditions. In conclusion, these studies
suggest progesterone metabolites may modify central processing of pressure-regulating
signals. The effects of ovarian steroids on the responsiveness of pressure-sensing neurons

17
and on target responsiveness to the outflow arm of the reflex also should be examined as
potential targets for ovarian steroid modulation of reflex control.
The pregnancy-induced plasma volume expansion also could contribute to a
decrease in baroreflex sensitivity since other investigators have reported an attenuation of
the baroreflex in the volume-loaded state (49). Expanded plasma volume is sensed by the
low-pressure cardiopulmonary stretch receptors (13). These receptors sense volume
expansion and increase heart rate (285), decrease renal sympathetic nerve activity (239),
and attenuate reflex control of the heart and peripheral resistance (12). Increased
stimulation of the cardiac mechanoreceptors by increased volume may contribute to many
of the observed changes in reflex control of arterial pressure during pregnancy.
The accumulated evidence demonstrates cardiovascular function is altered during
pregnancy. Cardiovascular adaptation to pregnancy appears to commence as increased
concentrations of circulating vasodilators (nitric oxide and prostaglandins) reduce vascular
resistance and reactivity and allow volume to expand and other variables to reset
reflexively. Evidence strongly suggests ovarian steroids contribute to the initiation and
maintenance of this adaptation. The importance of estrogens in cardiovascular adaptation
to pregnancy is fairly well established, and the importance of progesterone appears to have
been previously underestimated. Further study is needed to define the role of
progesterone in cardiovascular adaptation to pregnancy and to define which of the many
potential receptor mechanisms contribute to progesterone’s effects on the cardiovascular
system.

18
2.5 The HPA Axis
2,5,1 Introduction
While the adrenal medulla functions primarily as an epinephrine-secreting
sympathetic ganglion, the adrenal cortex secretes corticosteroids (85). Steroid
synthesizing enzymes are differentially distributed in the cortex (85). As a result, the zona
glomerulosa, the outer portion of the cortex, primarily secretes the mineralocorticoid
aldosterone (85). The inner cortex, the zonas fasciculata and reticularis, primarily secrete
glucocorticoids (cortisol in the human, dog, and sheep and corticosterone in the rat) and
lower concentrations of androgens (85). Aldosterone and cortisol (or corticosterone in
the rat) were originally labeled the primary mineralocorticoid and glucocorticoid
hormones, respectively, based on their relative effects on electrolyte and glucose
homeostasis (245).
While, as discussed above, the renin-angiotensin system and plasma potassium
levels regulate synthesis of aldosterone by the zona glomerulosa, anterior pituitary-derived
adrenocorticotropin (ACTH) controls adrenocortical secretion of cortisol (246). ACTH
secretion is controlled by release of corticotropin releasing-factor (CRF) and arginine
vasopressin (AVP) from parvocellular neurons of the hypothalamic paraventricular
nucleus (100). This system, commonly referred to as the hypothalamic-pituitary-adrenal
(HPA) axis, has become known as a stress-responsive system that initiates mechanisms
essential for immediate survival and/or contributes to recovery from a homeostatic
imbalance. However, the role of adrenal glucocorticoids in basal and stimulated
homeostasis is still under debate (196).

19
The parvocellular neurons of the paraventricular nucleus receive neural signals
from multiple brain regions (100). Except in the dog (142) and sheep (9) there is a
circadian rhythmicity in HP A axis activity in all species studied (142). Neurally integrated
light input from the retina entrains the suprachiasmatic nucleus of the hypothalamus (193),
and neural projections from the suprachiasmatic nucleus control paraventricular activity in
these species (10, 194).
There are numerous forms of stress (82) and multiple neural pathways impinge on
the paraventricular nucleus (100). While information about systemic or physiological
stress is relayed to the paraventricular nucleus through brainstem aminergic/peptidergic
pathways or from projections originating in blood-brain-barrier-deficient subfornical
organs, information about psychological or experiential stress is relayed indirectly from the
prefrontal cortex, hippocampus, or amygdala (100). Since multiple pathways mediate
stress-induced activation of the HPA axis, each pathway could be modulated by several
factors.
2,5.2 Corticosteroid Receptors
2.5.2.1 Ligand-binding characteristics
Corticosteroids modulate target tissues through two intracellular receptors (182).
The two corticosteroid receptors co-localize in most tissues (182). One of these cytosolic
receptors is known as the mineralocorticoid receptor (MR) based on its role in classic
mineralocorticoid target tissues (81). The MR can be described by its rank order of
affinity for the major circulating corticosteroids: corticosterone, cortisol, and aldosterone.
In the rat, the MR has a high affinity for corticosterone and aldosterone (-0.5 nM) and a
lower affinity for cortisol (226). In a species in which cortisol is the major glucocorticoid

20
(the dog), the MR also has a very high affinity for corticosterone (~ 0.05 nM) and a lower
affinity (~0.2 nM) for cortisol and aldosterone (227). The other cytosolic corticosteroid
receptor is known as the glucocorticoid receptor (GR). In the rat, the GR also has a high
affinity (but lower than the MR) for corticosterone (-5 nM) and an even lower affinity
(-20 nM) for cortisol and aldosterone (226). In the dog, the GR has an equal but lower
affinity than the MR for corticosterone and cortisol (-5 nM) and a two-fold lower affinity
(-10 nM) for aldosterone (227). As a result of these binding studies, the MR is frequently
described as the high affinity, low capacity receptor, and the GR is described as the lower
affinity, high capacity receptor (61). These binding parameters demonstrate these two
corticosteroid receptors cross-reactively bind the major circulating corticosteroids. Of
course, binding studies do not reveal the efficacy of a given ligand-receptor complex.
However Rupprecht and colleagues (233) have demonstrated, using human
neuroblastoma cell cultures co-transfected with vectors containing human MR or GR and
a mouse mammary tumor virus-luciferase reporter gene, that each of the major circulating
corticosteroids is efficacious at both receptors. The human MR (6) and GR (104) have
been cloned. Both receptors are intracellular, steroid-binding transcription factors and
members of the steroid and thyroid hormone superfamily (37, 70).
2.5,2.2 Receptor activation
In the absence of hormone, steroid receptors form a complex with heat-shock
proteins 90, 70, and 56 (270). Receptor-associated heat shock proteins appear to
maintain the receptor in a state capable of binding ligand and incapable of mediating
transcription (270). However, once steroids bind with the receptor, the steroid-receptor
complex dissociates from the heat shock proteins, dimerizes with another steroid-receptor

21
complex, and mediates gene silencing and activation (270). Once the steroid-receptor
complex obtains this activated state, the ligand does not freely dissociate (42). Evidence
suggests phosphorylation regulates steroid receptor activity and ligand-independent events
also can activate steroid receptors (270).
2,5,2,3 Genetic characteristics
The amino acid sequences of the two proteins are highly homologous and their
structural and functional domains are similar (6). The amino terminal region varies in size
and bares no structural homology among the human MR and GR, but the carboxy-terminal
ligand-binding domain (57% identity) and the cysteine-rich zinc-finger DNA-binding
domain (94% identity) are highly homologous (6). These structural similarities allow the
two receptors to have similar steroid binding and activation profiles (233) and to bind
closely related DNA target sequences (6).
There are two splice variants of the human and rat MR and each isoform is
regulated by a separate promoter (15, 39, 155, 295), but the functional role of each
isoform has not been described. There also are two isoforms of the human GR (104), and
GR3 inhibits GRa activity (200). The ovine MR has not been cloned, but 942 base pair
segment of the ovine GR corresponding to residues 143-453 of the human GR (80%
identity) has been cloned (294). The availability of multiple receptor forms suggests
steroid receptor physiology is immensely complex. Each receptor isoform would be
expected to have a different rank order of affinity for the endogenous ligands and each
ligand-receptor complex would be expected to bind various genetic response elements
with varying affinity and efficacy.

22
2.5.2.4 Intracellular location
Considerable debate has focused on the intracellular location of steroid receptors.
Since early studies detected radiolabeled steroid binding in the cytosoluble fraction of
tissue homogenates, a two-step model of steroid receptor activation was proposed (117).
This model proposed steroids bind their receptor in the cytosol and the activated steroid-
receptor complex translocates to the nucleus and regulates transcription (117). The
cytosolic location of functional MR and GR has been largely discounted by
immunocytochemical evidence demonstrating nuclear localization (185). Although some
immunocytochemical studies demonstrate cytosolic location, tissue fixation technique can
distort the apparent intracellular location of the steroid receptors (185). Studies clearly
demonstrating an absence of cytosolic steroid receptor binding in enucleated cells suggest
detectable “cytosolic” binding represents extraction of nuclear proteins into the
cytosoluble homogenate fraction (289). Of course, steroid receptors must be translated in
the cytosol, and an energy-requiring nuclear shuttling mechanism for these proteins does
exist (95). The evidence strongly suggests the functional steroid receptors are located in
the nucleus and can be easily extracted into the cytosol during tissue homogenization, but
the debate continues.
2.5.2.5 Differentiation between mineralocorticoid and glucocorticoid signals
The microsomal enzyme 11 P-hydroxysteroid dehydrogenase (11-PHSD) plays a
role in determining when MR is exposed to circulating glucocorticoids. There are at least
two isoforms of this enzyme (244). One isoform (11P-HSD2) reacts primarily in one
direction and converts cortisol to cortisone or corticosterone to 11-dehydrocorticosterone
(244). Funder and colleagues have demonstrated 11P-HSD2 colocalizes with the MR and

23
protects the MR from activation by circulating glucocorticoids in the distal nephron of
most species and in the rat brain (80). However, significant conversion of cortisol to
cortisone does not occur in the ovine brain (145). Another isoform of the enzyme (11(3-
HSD1) appears to be the predominate isoform in the liver of most species and the brain of
species including the sheep and human (244). This isoform of 1 1J3-HSD is bi-directional
(192). This isoform (11J3-HSD1) has a relatively low affinity (in the jaM range) for
cortisol and corticosterone and appears to proceed in the direction favoring conversion of
cortisone to cortisol or 11-dehydrocorticosterone to corticosterone at physiological
glucocorticoid concentrations (244). As a result, 11(3-HSD1 appears to inactivate
glucocorticoids only when circulating concentrations are high (244). In summary, there
appear to be species and tissue differences in the protection of MR from circulating
glucocorticoids by 11-0HSD, and the MR in the human and ovine brain appears to be
particularly vulnerable to activation by circulating glucocorticoids.
Some evidence suggests intrinsic receptor properties also contribute to the ability
of MR to differentiate between aldosterone and glucocorticoids. Although the MR binds
corticosterone and cortisol with a high affinity, the aldosterone-MR complex is more
potent than the cortisol/corticosterone-MR complex since the half maximal effective dose
is approximately 100-fold lower (168, 233). However, aldosterone and cortisol both
promote transcription in cells transfected with human MR, and the cortisol-MR complex is
actually more efficacious than the aldosterone-MR complex since cortisol induces almost
2-fold more reporter gene transcription than aldosterone (233). This suggests that all of
the major corticosteroid ligands exert physiological effects through the MR. Evidence
collected by Pearce and colleagues demonstrates one level at which MR and GR obtain

24
selectivity: the MR and GR interact differently with non-receptor transcription factors
(209). However, the mechanisms by which the MR and GR obtain selectivity are still
poorly understood, and corticosteroid physiology will probably be most accurately
portrayed when described in terms of cross-reactivity.
2,5.2,6 Receptor auto-regulation
The MR and GR respond to circulating corticosteroids in a manner that has been
termed auto-regulation (34, 231, 288). When plasma corticosteroid concentrations
increase, transcription of MR and GR decreases. In contrast, when plasma corticosteroid
concentrations decrease, transcription of MR and GR increases. These findings
demonstrate MR and GR are auto-regulated by corticosteroids, suggesting that the
expression of both MR and GR should be decreased during pregnancy when circulating
corticosteroids increase.
2.5.3 Feedback Inhibition
Negative feedback inhibition of the HPA axis occurs in three time domains (141):
fast (seconds to minutes), intermediate (hours), and slow (hours to days). MR and GR
mediate intermediate and slow feedback inhibition of the axis in the hippocampus (115),
hypothalamus, pituitary, and perhaps even the adrenal itself (141). In addition to these
feedback sites located at central sites in the HPA axis, MR and GR may mediate feedback
inhibition at diffuse relay stations in the afferent nerve pathways (141). As will be
discussed below, the receptors involved in fast feedback inhibition are unknown.

25
2.5.3.1 Intermediate and slow feedback inhibition
Intermediate and slow feedback inhibition requires genomic events and protein
synthesis (141). While intermediate feedback inhibits the release of ACTH and the
synthesis and release of CRF, slow feedback inhibits both the synthesis and release of CRF
and ACTH (141).
2.5.3.1.1 Role of MR and GR
Recent studies suggest distinct roles for MR and GR in feedback inhibition. Reul
and de Kloet observed the high affinity MR is almost completely activated and the lower
affinity GR is mostly unoccupied at the trough of the HPA axis circadian rhythm in the
unstressed animal (226). The lower affinity GR becomes activated at the peak of the
rhythm and in the stressed animal (226). This evidence led to a binary theory of
corticosteroid receptor action: basal concentrations of corticosteroids exert their feedback
effects through the MR and high concentrations (at the peak of the rhythm and during
stress) exert their effects through the GR (55, 226, 228, 229). A study by Dallman and
colleagues suggests the GR alone is not able to suppress activity of the HPA axis;
activation of both the MR and GR is required for feedback inhibition at the peak of the
rhythm (22). This finding suggests that although the MR may function as the high affinity
receptor and the GR may function as the lower affinity receptor, the two receptors
probably function in concert. Indeed, accumulating evidence suggests the two
components of this binary receptor system may interact. Steroid receptors are known to
form dimers before binding to the genetic response element (270), and recent evidence
suggests the MR and GR can form heterodimers (166, 267). The precise role of
corticosteroid receptor heterodimers in target tissue activity and feedback control of the

26
axis has not been studied. Genetic studies may eventually differentiate between MR and
GR homodimer and heterodimer responsive elements, and future physiological studies
should consider the relative availability of MR and GR in the target tissue of interest, the
concentration of corticosteroid obtained, and the resulting relative amounts of activated
MR and GR homodimers and heterodimers.
2.5.3.1.2 Role of the Hippocampus
The hippocampus is a functionally unique site that may be involved in basal activity
of the axis and feedback inhibition and facilitation. The hippocampus was first suspected
as a regulatory site after McEwen and colleagues demonstrated the hippocampus
concentrates more radiolabeled corticosterone than any other portion of the rat brain
(184). The hippocampus integrates afferent exteroceptive and interoceptive sensory data
for efferent projection to the prefrontal cortex, autonomic centers (including
cardiovascular centers), and motor nuclei, and plays a primary role in cognition and
emotions (19, 235). Therefore, it seems likely the hippocampus regulates the stress-
activated HPA axis (115). In fact, the hippocampus appears to be the primary site for
MR-mediated effects on HPA axis activity in the rat since MR is nearly undetectable in the
rodent pituitary and hypothalamus (226). Since the hippocampus lacks direct projections
to the paraventricular nucleus of the hypothalamus, the hippocampus probably regulates
the paraventricular nucleus via at least one intermediary synapse in the bed nucleus of the
stria terminalis (100). As a result of integration of hippocampal control of the axis with
other neural inputs at relay synapses, the effect of the hippocampus on axis activity is
probably extremely sensitive to alteration by other neural inputs.

27
2.5.3.1.2.1 Basal Activity
The role of the hippocampus in regulation of the HPA axis has been thoroughly
reviewed by Jacobson and Sapolsky (115) and more recently by Feldman and Weidenfeld
(73) and Herman and colleagues (100). A majority of studies suggest excitatory output
from the hippocampus inhibits activity of the axis (115). The hippocampus inhibits basal
activity of the axis: electrical stimulation of the hippocampus decreases circulating
corticosteroids in the pigeon (17), human (177, 232) and cat (249) and ablative
hippocampal or fornix lesions also increase plasma corticosteroids, ACTH or P-endorphin
levels in several species (17, 76, 101, 150, 161, 172, 190, 198, 237, 292).
2.5.3.1.2.2 Circadian Rhythms
The hippocampus also regulates the circadian rhythm of the axis (115). The
hippocampus may inhibit activity of the axis at the nadir of the rhythm since trough
corticosteroid levels tend to increase after hippocampal damage (17, 76, 161, 190, 198).
However, corticosteroid levels at the nadir (5 pg/dl) tended to be higher than normal
(~l|ug/dl) even in the control animals used in these studies (115). Since basal
corticosteroid levels were elevated, these studies most likely reveal a hypersensitivity to
“background” stress at the trough of the rhythm in hippocampal lesioned animals.
As Dallman and colleagues (56) have outlined, autonomous output of the pituitary
and adrenal accounts for output of the axis at the trough of the rhythm in the rat. Basal
hypothalamus (120), paraventricular nucleus (57), and suprachiasmatic nucleus (38)
lesions do not decrease plasma ACTH levels below normal at the nadir of the rhythm in
the rat. CRF antibodies do not decrease ACTH and corticosterone levels at the trough of
the rhythm in the rat (7), and trough corticosterone levels in adrenal- intact rats are

28
indistinguishable from corticosterone concentrations measured in adrenalectomized rats
(55). Therefore, the hippocampus most likely contributes to regulation of the axis during
the peak of the rhythm and during stress when neural components activate the axis.
Moreover, although neural inputs into the paraventricular nucleus increase in certain
instances, these neural inputs are not necessarily routed through the hippocampus. Studies
have observed corticosteroid output at the peak of the rhythm (when neural inputs
probably increase activity of the axis) is reduced in the male rat (161, 190) and increased
in the female rat (292). However, a recent, carefully designed, study by Dallman and
colleagues failed to demonstrate an effect of fimbria fornix-lesions on diurnal HPA axis
activity in adrenal-intact rats (23).
2.5.3.1.2.3 Stress-Induced Activity
The role of the hippocampus in stress-induced activity of the axis is not clear
(115). Jacobson and Sapolsky (115) have offered possible interpretations of numerous
hippocampal lesion studies which suggest the hippocampus does not regulate stress-
induced HPA axis activity. For example, individual study results could be influenced by
the precise subfield location of the hippocampal lesion, the period of time elapsed between
induction of the lesion and the study, and the timing of blood sampling (115). The
simplest and most attractive interpretation is that the hippocampus exerts different effects
on different stresses. Although many sensory and emotional stresses are processed
through the hippocampus, cognitive stress is probably most completely processed through
the hippocampus. Indeed, Coover, Goldman, and Levine (48) demonstrated the response
of hippocampectomized rats subjected to ether stress or the “frustrative-emotional” stress
of introduction to a novel environment does not differ from the response of control rats.

29
In contrast, hippocampectomy did attenuate the corticosteroid response to extinction of a
learned lever-press reward system (48). Similarly, although non-emotional and non-
cognitive cues control the diurnal HPA axis rhythm, it is conceivable that under certain
experimental conditions neural input into the paraventricular nucleus may receive stronger
influence from the sensory integrating hippocampus and the diurnal rhythm may be more
strongly influenced by the hippocampus in those circumstances.
2.5.3.1.2.4 Facilitation
Keller-Wood and Dallman (141) noted that non-systemic stresses (skin incision,
laparotomy, restraint stress, and electric shock), which would be expected to influence
paraventricular nucleus activity through more complicated (or even multiple) neural
pathways than systemic stresses (hypotension or hypoglycemia), are uniquely able to
induce facilitation of axis response to subsequent or repeated stress. A recent study by de
Kloet and colleagues (284) suggests the hippocampus could modulate axis facilitation; as
expected, hippocampal MR blockade enhances but hippocampal GR antagonism
suppresses basal HPA axis activity at the peak of the rhythm. This finding suggests the
hippocampal MR inhibits activity of the axis and the hippocampal GR enhances activity of
the axis. A study by Bradbury and Dallman (20), conducted in adrenalectomized rats
replaced with corticosterone at concentrations well below peak levels (3-6 pg/dl),
confirms blockade of hippocampal MR with a MR-specific antagonist increases ACTH at
the peak of the rhythm. However, blockade of hippocampal GR with a GR-specific
antagonist actually increases ACTH when corticosterone levels are maintained at this fairly
low concentration. Together these findings suggest low activated concentrations of
hippocampal GR contribute to MR inhibition of the axis, but higher concentrations of

30
activated GR actually enhance output of the axis. These combined pharmacological data
demonstrates MR and GR actions may be regulated by the relative activation of MR and
GR and resulting amounts of MR and GR homo- and heterodimers.
In summary, the available evidence does not clearly demonstrate the precise role of
the hippocampus in regulating the diurnal rhythm of the axis, but suggests the
hippocampal MR is capable of inhibiting activity of the axis and high numbers of activated
hippocampal GR are capable of facilitating activity the axis. Moreover, these studies
suggest hippocampal regulation of HP A axis activity is extremely sensitive to influence
from other neural inputs and the degree to which all neural input into the paraventricular
nucleus is routed through the hippocampus at the time of observation.
2,5.3,2 Fast feedback inhibition
Fast feedback inhibition appears to require a membrane-mediated event to inhibit
release of CRF and ACTH and decrease pituitary sensitivity to CRF (141), but the
mechanism of fast feedback inhibition is currently unknown. Corticosteroid-binding
membrane receptors have only recently been described (181, 208), and have yet to be
cloned. Recent evidence demonstrates the GABAa receptor is an important modulator of
rapid steroid effects; metabolites of cortisol, corticosterone, and progesterone are potent
benzodiazepine-like modulators of the GABAa receptor (176). Since benzodiazepines are
known to inhibit activity of the HPA axis (222), and excitatory hippocampal outputs are
thought to act in the bed nucleus of the stria terminalis to stimulate GABA inhibition of
CRF neurons in the paraventricular nucleus, the GABAA-active neurosteroids also may
mediate fast feedback inhibition. Moreover, GABAa receptors are known to play a
significant role in the circadian time-keeping system (189, 195), so the steroid metabolites

31
may regulate the time-keeping system. Much work is needed to characterize and
determine the mechanisms of rapid steroid effects.
2,5,3.3 HP A axis during pregnancy
2.5.3.3.1 Basal Activity
Resting plasma cortisol is increased in pregnant sheep and women, suggesting that
the setpoint of the HPA axis increases during pregnancy (133). Although elevated
concentrations of plasma corticosteroid binding globulin (CBG) may contribute to the
elevation in plasma cortisol in women, CBG concentration does not increase in the
pregnant ewe and the concentration of free plasma cortisol is increased in both species
during pregnancy (132). ACTH probably stimulates the increase in plasma cortisol since
plasma ACTH concentrations increase during the course of gestation and adrenal
responsiveness to ACTH increases during pregnancy (132). The mechanism leading to
increased ACTH concentrations during pregnancy is unknown, but in the sheep, plasma
ACTH is not increased by CRF or ACTH derived from the placenta (137, 138).
2.5.3.3.2 Feedback Sensitivity
These observations suggest feedback regulation of the axis may be attenuated
during gestation. Indeed, evidence does suggest the ability of dexamethasone to reduce
free cortisol concentrations is attenuated in the pregnant women (199, 201). However,
pregnancy does not reduce the absolute suppression of ACTH by a two-hour infusion (the
intermediate feedback time domain) of cortisol in the sheep (132). Interestingly, the initial
rate of feedback inhibition during the first two-hour infusion (in the rapid feedback time
domain) is reduced in the pregnant ewe (132). This suggests genomic feedback
mechanisms are not altered in pregnant ewes. A decrease in hypothalamic or pituitary

32
GABAa receptor expression or sensitivity or in metabolism of cortisol to the GABAa-
active metabolite, tetrahydrocortisol, may decrease the rate of feedback sensitivity in the
rapid feedback time domain.
Although absolute suppression of ACTH by cortisol is not reduced in the pregnant
ewe, plasma ACTH is significantly increased during pregnancy when cortisol
concentration is maintained below 5 ng/ml, according to a study of non-pregnant and
pregnant adrenalectomized ewes replaced with varying concentrations of cortisol (131).
Collectively these findings suggest that although the genomic feedback mechanism is not
altered during ovine pregnancy, plasma ACTH at basal, but not stimulated, cortisol
concentrations is increased in the pregnant ewe. Differential modulation of hippocampal,
hypothalamic, or pituitary MR and GR and/or neurotransmitter systems by ovarian
steroids during pregnancy may attenuate the ability of low concentrations of cortisol to
reduce plasma ACTH. Since the MR is the high affinity receptor that is activated by basal
concentrations of cortisol, the inability of low concentrations of cortisol to decrease
ACTH suggests that the MR, in particular, may be less responsive during pregnancy.
2.5.3.3.3 Responsiveness
Responsiveness of the HPA axis also is altered during ovine pregnancy in a
stimulus-specific manner; the ACTH response to hypotension is reduced but the ACTH
response to hypoglycemia is augmented during gestation (130). Since each stimulus
reaches the paraventricular nucleus through a distinct neural pathway (82), pregnancy
must selectively modify input into the axis. Ovarian steroids may modulate the processing
of input into the axis and the effects of ovarian steroids on MR and GR may play a
primary role in adapting the responsiveness of the axis to pregnancy.

33
2.5.3.3.4 Role of Ovarian Steroids
Currently available evidence suggests ovarian steroids contribute to differential
regulation of HP A axis setpoint and responsiveness (96). Female rats tend to have higher
circulating levels of both ACTH and corticosterone than male rats. This sex difference
appears to be due to effects of ovarian steroids on both the steroid synthesizing enzymes
in the adrenal gland and on neuroendocrine control of the axis (96). In cycling female
rats, the HPA axis is significantly more responsive during proestrus (when estradiol levels
are highest) than during estrous or diestrous (286). Treatment of ovariectomized rats with
estradiol mimics the effects of proestrus on the responsiveness of the axis (286), and co¬
administration of progesterone attenuates the effects of estradiol on the axis (225, 286).
However, a more recent, and carefully designed study by Carey and colleagues (36)
confirmed estradiol increases activity of the axis and did not find an inhibitory effect of
progesterone. Estrogens appear to increase activity of the axis by increasing hypothalamic
CRF expression (102), adrenal responsiveness to ACTH (147), and attenuating the
glucocorticoid feedback signal (32, 225). In summary, these studies suggest estrogens
increase activity of the axis by increasing the activating components of the axis and by
decreasing feedback signal effectiveness. However, these studies do not clearly define the
effect of progesterone on HPA axis activity. The observed activating effects of estrogens
may play a role in the adaptation of the axis to pregnancy, but since absolute feedback
sensitivity is not reduced during ovine pregnancy, the effects of estrogens on feedback do
not appear to be physiologically relevant to pregnancy.
Evidence suggests progesterone could antagonize feedback regulation of the HPA
axis. High concentrations of progesterone antagonize glucocorticoid inhibition of 0-

34
endorphin (a protein processed from the same precursor molecule as ACTH) secretion in
pituitary cultures (1), of corticotropin-releasing activity from isolated hypothalami (118),
and of ACTH in vivo (66). Similarly, an acute infusion of physiological concentrations of
progesterone antagonizes the feedback effects of cortisol in the non-pregnant ewe (136).
In vitro evidence demonstrates progesterone has a high affinity but very low efficacy at
both the MR and GR (233). This finding suggests progesterone could act as an
endogenous antagonist of cortisol actions at the hippocampus, hypothalamus, and
pituitary, inhibit the feedback control of the axis, and contribute to the increase in setpoint
observed during pregnancy. Absolute suppression of ACTH by increased concentrations
of cortisol is not reduced during ovine pregnancy (132), suggesting progesterone does not
alter GR-mediated feedback to contribute to resetting the HPA axis setpoint. However,
the rate of feedback inhibition by increased cortisol is reduced during ovine pregnancy
(132). This effect could be due to GR antagonism by high circulating concentrations of
progesterone. However, if this effect is due to progesterone, progesterone only appears
to slow the rate of the feedback signal under these circumstances. Since plasma ACTH is
increased in pregnant ewes compared to non-pregnant ewes maintained at plasma cortisol
concentrations below 5 ng/ml (131), the setpoint for ACTH at this level of cortisol does
appear to be altered. This effect could be due to progesterone antagonism of the MR
component of the feedback signal that is only unmasked at low levels of cortisol.
However, since treatment with progesterone alone does not increase the ratio of ACTH to
cortisol (129), progesterone alone probably does not induce the change in setpoint by
altering the sensitivity of the feedback system. Other signals are probably required to

35
increase neural output to the paraventricular nucleus and increase the setpoint of the HPA
axis.
2.6 Role of Altered HPA Axis Function in Cardiovascular Adaptation to Pregnancy
The studies presented above clearly demonstrate both the cardiovascular system
and the HPA axis are altered during pregnancy and strongly suggest ovarian steroids
contribute to the adaptation of each system to pregnancy. As discussed above, adaptation
of the HPA axis to pregnancy may further contribute to cardiovascular adaptation to
pregnancy since corticosteroids modulate fluid homeostasis, vascular responsiveness (58),
and central blood pressure control. In addition to these corticosteroid-mediated effects on
the cardiovascular system, several of the peptide components of the HPA axis (CRF,
AVP, and ACTH) modulate cardiovascular function. Therefore, ovarian steroids may
contribute to cardiovascular adaptation to pregnancy by directly modifying expression of
these peptides and their subsequent effects on downstream effectors; the downstream
effectors of CRF, AVP, and ACTH include the adrenal and neural outflow to the
cardiovascular system. In addition, ovarian steroids may contribute to differential
regulation of CRF, AVP, and ACTH by modifying the way in which MR and/or GR
respond to circulating corticosteroids. Ovarian steroids could modify MR and/or GR
regulation of CRF, AVP, and ACTH by altering expression of MR and/or GR and/or
modifying the ability of circulating corticosteroids to bind and activate MR and/or GR.
The effect of AVP, CRF, and ACTH on cardiovascular function are discussed below.
Studies demonstrate ACTH has effects on cardiovascular function that may be
independent of corticosteroid secretion. Therefore, the increased circulating

36
concentrations of ACTH found during pregnancy may directly contribute to
cardiovascular adaptation to pregnancy. However, most of the described effects of ACTH
on cardiovascular function appear to contradict a role for elevated concentrations of
circulating ACTH in cardiovascular adaptation to pregnancy.
In the sheep (241), rat (290), and human (291), supraphysiological concentrations
of peripherally administered ACTH increase blood pressure. Sheep treated chronically
with ACTH also have attenuated baroreceptor-heart rate reflexes (258), and exhibit
increases in cardiac output and no change in total peripheral resistance (93). A study of
the time course of development of ACTH-induced hypertension reveals that, in the first
few hours after initiation of ACTH treatment, MAP does not increase since an early
increase in cardiac output is offset by a decrease in total peripheral resistance (250). After
several hours, total peripheral resistance returns toward normal and MAP begins to
increase in the ACTH-treated sheep (250). If the ACTH-induced increase in heart rate
and cardiac output is prevented pharmacologically with a P-adrenergic receptor
antagonist, total peripheral resistance increases and ACTH-induced hypertension is
maintained (93). However, if vasoconstriction is prevented with a calcium channel
antagonist or vascular smooth muscle relaxants, ACTH administration does not increase
arterial pressure (250). Therefore, ACTH increases blood pressure by increasing cardiac
output and an ACTH-induced change in the regulation of peripheral resistance is an
essential component in the development of ACTH-induced cardiovascular changes.
The central nervous system appears to play a selective role in ACTH-induced
hypertension. Although P-adrenergic receptor (propanolol), autonomic ganglion
(phentolamine) and sympathetic nervous system (methyldopa and clonidine) antagonists

37
prevent increases in heart rate and cardiac output by peripherally administered ACTH,
these drugs fail to prevent ACTH-induced hypertension (251, 253). In fact, autonomic
ganglion blockade with phentolamine actually enhances the pressor response to
peripherally administered ACTH. Together, these findings demonstrate peripherally
administered ACTH modifies neural control of the heart, but during peripheral ACTH
treatment neural control of the vascular system appears to attempt to maintain pressure at
the normal setpoint. Therefore, peripherally administered ACTH appears to alter vascular
tone and increase blood pressure through non-neural mechanisms. However, the precise
mechanism by which peripherally administered ACTH increases blood pressure is still
«
unknown. Intracerobroventricular administration of ACTH also increases blood pressure
in sheep (243), suggesting central ACTH-containing neurons stimulate increases in blood
pressure. However, centrally administered ACTH may not modulate blood pressure by
the same mechanisms as peripherally administered ACTH, and the mechanisms behind
centrally administered ACTH-induced hypertension have been less thoroughly studied.
Peripherally administered ACTH could increase blood pressure by increasing
concentrations of circulating corticosteroids. However, the role of corticosteroids in
development of ACTH-induced hypertension has been debated. Scoggins and colleagues
(72) have found cocktails containing various combinations of intravenously administered
corticosteroids (including cortisol, corticosterone, deoxycorticosterone, 11-deoxycortisol,
and 18-hydroxydeoxycorticosterone), designed to mimic the adrenal response to ACTH
administration, fail to elevate blood pressure in the sheep. Despite this finding, adrenal
steroids do appear to be an essential component of ACTH-induced hypertension. The
effects of ACTH on blood pressure are abolished in the adrenalectomized rat (290) and

38
other investigators have demonstrated intravenous infusions of physiological
concentrations of cortisol are sufficient to increase blood pressure in the ewe (144).
Scoggins and colleagues have suggested steroid infusions can only mimic the effects of
ACTH on blood pressure in the sheep when 17a,20a-hydroxyprogesterone and 17a-
hydroxyprogesterone are included with both aldosterone and cortisol in the steroid
infusion cocktail (252). However, it is unclear why two steroids with minimal
mineralocorticoid and glucocorticoid activity would be required to reveal the pressure
increasing effects of aldosterone and cortisol, and this finding is disputed by the
observation that intravenously-administered physiological concentrations of cortisol
increase blood pressure (144).
If ACTH increases cardiac output and blood pressure by first stimulating
corticosteroid secretion, corticosteroids do not appear to act centrally to increase blood
pressure in the sheep. Intracerebroventricular infusions of aldosterone (269), cortisol
(144), and cocktails containing 17a,20a-hydroxyprogesterone and 17a-
hydroxyprogesterone (268) all fail to increase blood pressure in this species. These
findings are consistent with the observation that centrally acting pharmacological agents
do not prevent ACTH-induced hypertension (251, 253). However, the role of
corticosteroids in ACTH-induced hypertension requires further investigation.
Together, these observations suggest elevated concentrations of circulating ACTH
could contribute to pregnancy-associated increases in cardiac output and baroreflex
attenuation. However, additional factors must prevent increased concentrations of ACTH
from changing the regulation of peripheral resistance and elevating MAP during
pregnancy. Circulating progesterone may allow elevated concentrations of ACTH to

39
contribute to increasing cardiac output without increasing MAP during pregnancy.
Indeed, Scoggins and colleagues (254) have demonstrated pre-administration of
progesterone prevents ACTH from increasing blood pressure, but progesterone
administration does not reverse hypertension established by previous ACTH
administration.
Further study is needed to determine if ACTH has direct effects on the vascular
system. The studies discussed above suggest ACTH may be a vasoconstrictor. More than
likely, the effects of ACTH on cardiovascular function observed by Scoggins and
colleagues are not applicable to pregnancy since these investigators consistently used
extremely high infusion rates (20 pg/kg/day) in their studies. Moreover, these
investigators demonstrated that the hypertensive effects of ACTH can only be obtained at
infusion rates above 1 mg/kg/day (240). This infusion rate elevates plasma ACTH beyond
the normal increase (about 25 pg/ml) observed during pregnancy.
One study demonstrates ACTH actually vasodilates the human placental
circulation (44). It is unclear if ACTH has the same effect on the peripheral vascular
system and to what degree the effects of ACTH on the vascular tone are altered during
pregnancy. Collectively, these studies demonstrate ACTH modulates cardiovascular
function. However, the mechanisms by which ACTH modulates cardiovascular function
are still unclear, and the role of ACTH in cardiovascular adaptation to pregnancy requires
further investigation.
CRF is another peptide that appears to have effects on cardiovascular function.
Since circulating concentrations of CRF tend to be minimal (~12 pg/ml), the effects of
CRF on cardiovascular function are probably mostly mediated via central neuronal

40
projections that release CRF. In rats (79), sheep (242), and dogs (30),
intracerebroventricular infusions of CRF increase MAP and heart rate, and this effect
appears to be mediated by CRF-induced increases in sympathetic outflow (30, 78).
Moreover, when CRF is administered centrally, the baroreflex maintains pressure and
heart rate about the new setpoint with reduced gain and range of responsiveness (77).
The effect CRF on the heart rate response to baroreceptor loading and unloading appears
to be mediated via CRF effects on parasympathetic outflow to the heart since
parasympathetic but not sympathetic antagonists prevent the effects of CRF on the
baroreflex (77). The role of central CRF in regulating cardiovascular function during
pregnancy has not been investigated, but ovarian steroids may have direct or indirect
effects on this cardiovascular-regulatory peptide during pregnancy.
AVP is another peptide that may play a role in cardiovascular adaptation to
pregnancy. As discussed above, AVP is released directly into the circulation from the
posterior pituitary and circulating AVP plays an important role in regulating fluid
homeostasis, vascular tone, and baroreflex sensitivity in non-pregnant animals, and the role
of altered AVP control in gestational cardiovascular function is still under investigation.
AVP also has central cardiovascular effects on cardiovascular function, but these effects
are somewhat controversial. While some studies suggest central AVP release decreases
sensitivity of the baroreflex and increases blood pressure and heart rate by stimulating
sympathetic outflow (221), other studies suggest central AVP actually increases sensitivity
of the baroreflex and decreases blood pressure and heart rate (25). These seemingly
contradictory findings may be due to differences in the dose of AVP administered or the
site of administration in the respective studies. Additional studies are required to clarify

41
the role of central AVP in regulation of cardiovascular function and to determine how
central AVP may contribute to cardiovascular adaptation to pregnancy.
2.7 Objectives
The literature demonstrates ovarian steroids contribute to cardiovascular
adaptation to pregnancy through direct and indirect mechanisms. Some of the indirect
effects of ovarian steroids on cardiovascular function appear to be mediated through the
effects of ovarian steroids on the HPA axis. However, further study is needed to expand
our understanding of the effects of ovarian steroids on both systems and to further clarify
the role of altered HPA axis function in cardiovascular regulation during pregnancy.
To date, the effects of estrogens on both the HPA axis and the cardiovascular
system have been examined more extensively than have the effects of progesterone on
both systems. Therefore, the studies presented in this dissertation were designed to
enhance understanding of the effect of progesterone on both the HPA axis and the
cardiovascular system. In particular, these studies were designed to determine the validity
of the general hypothesis that progesterone modulates the cardiovascular system and the
HPA axis via three novel progesterone receptor types: the GABAa receptor and the MR
and GR. In order to determine the validity of this general hypothesis three specific
hypotheses were tested:
1. Progesterone rapidly alters blood pressure, baroreflex sensitivity, and blood
volume.
Since 5a-reduced GABAA-active progesterone metabolites are elevated during
pregnancy and GABAa receptors are located in the paraventricular nucleus and

42
cardiovascular-regulatory nuclei in the brainstem, progesterone may have acute effects on
the cardiovascular system and the HPA axis that are mediated via 5a-reduced metabolites.
Chapters 4, 5, and 6 test the hypothesis that progesterone rapidly modulates
cardiovascular function
2. Pregnancy is associated with changes in MR and GR availability,
immunoreactivity, and apparent affinity.
Since progesterone also interacts with MR and GR, progesterone could more
slowly modulate the HPA axis and the cardiovascular system by interacting with these two
receptors. Chapters 7, 8, and 9 compare the availability, affinity, and immunoreactivity of
the MR and GR in non-pregnant and pregnant ewes and reveal these receptors are
differentially regulated during pregnancy when circulating progesterone concentrations are
high.
3. Ovarian steroids alter MR and GR availability and immunoreactivity during
pregnancy.
Chapter 10 tests the hypothesis that progesterone contributes to differential
regulation of MR and GR during pregnancy but does not establish or eliminate a role for
progesterone in differential modulation of MR and GR. The study presented in Chapter
11 reveals estrogen contributes to differential modulation of MR and GR during
pregnancy.

43
In summary, the studies presented in this dissertation reveal progesterone may
contribute to adaptation of the HPA axis and the cardiovascular system to pregnancy via
three novel progesterone receptors: the GABAa, and the MR and GR. As discussed in
Chapter 12, future study will be required to confirm and clarify the role of these novel
receptors in progesterone-mediated effects on the HPA axis and the cardiovascular
system.

CHAPTER 3
GENERAL MATERIALS AND METHODS
3.1 Animal Care
This dissertation represents a collection of in vivo and in vitro studies of the ewe
(female Ovis dries). Ewes were cared for according to the guidelines established by the
American Association for Accreditation of Laboratory Care and all experimental
procedures were approved by the University of Florida’s Institutional Animal Care and
Use Committee.
During the acute steroid infusion studies, ewes were housed in the animal
resources facility at the University of Florida Health Sciences Center, maintained in a
controlled environment (12-hour light/dark cycle and a constant 19-21°C temperature),
and allowed access to food and water ad libitum.
Ewes treated chronically (60 days) with steroid implants were allowed to recover
from the surgical procedure for approximately two weeks in an enclosed animal resources
facility. The animals then spent the duration of the treatment period in an open pasture
and were moved to the animal resources facility at the Health Sciences Center
approximately 48 hours prior to sacrifice.
Body temperature was measured at the end of each experiment and twice daily for
five days after a surgical procedure to assess health. Ewes also were treated with 750-
44

45
1000 mg ampicillin (Polyflex, Aveco, Fort Dodge, IA) after each infusion experiment and
twice daily for five days after a surgical procedure.
3.2 General Surgical Procedures
Ewes were fasted for 24 hours prior to a surgical procedure. Anesthesia was
induced with ketamine (20 mg/kg i.m.) and maintained with halothane in ~3% oxygen. All
surgical procedures were performed using aseptic techniques.
3.2.1 Ovariectomy
Bilateral ovariectomies were performed as previously described (9). Briefly,
ovaries were isolated through a midline abdominal incision, the vascular supply was
ligated, and the ovary was removed.
3.2.2 Catheterization
In some studies, catheters were introduced bilaterally into the femoral artery and
vein for steroid delivery, blood sampling, and arterial pressure measurement. This method
has been previously described in detail (9). Briefly, an incision was made in the femoral
triangle, and the artery and vein were exposed. Each vessel was ligated distally, and a
0.05 mm catheter was advanced approximately 10 inches into the descending aorta or
inferior vena cava. Each catheter was secured with surgical ties, filled with heparinized
saline, plugged with a sterile nail, and routed subcutaneously with a trocar to exit the
flank. When not in use, the exteriorized catheters were stored in a paper pocket and
protected by a spandage wrap.

46
3.3 Chronic Steroid Implantation
Subcutaneous implants were used to chronically administer steroids. Estrone and
placebo implants were purchased from Innovative Research of America (Toledo, OH).
Estradiol and progesterone (Sigma, St. Louis, MO) implants were prepared using aseptic
techniques by filling shells with crystalline steroid (123, 124). Estradiol was packed to a
height of 3 cm in 4 cm long pieces of Silastic® tubing (0.132 in. inner diameter, 0.183 in.
outer diameter, Dow-Coming, Midland, MI). Progesterone was packed into PhamElastâ„¢
silicon polymer (SF Medical, Hudson, MA) implants measuring 75 X 55 mm. These
implants were sealed with Silastic® adhesive (Dow-Corning). To eliminate a surge in
plasma steroid concentrations immediately after implantation, implants were placed in
sterile, isotonic saline and maintained at 37°C for 24 hours prior to implantation (123).
All implants were placed into the dorsal scapular region of anesthetized ewes using aseptic
technique.
3.4 Acute Progesterone Infusions
For acute progesterone infusion studies, progesterone (Sigma) was prepared and
stored in 100% ethanol. Prior to an experiment, the progesterone stock was prepared in
10 or 20% ethanol in 0.9% saline in a syringe. Steroid solutions were infused into venous
catheters through 0.2 |im sterile Acrodisc® filters to prevent the introduction of
microbacterial contaminants into the ewes.

47
3.5 Handling and Analysis of Blood Samples
3.5.1 Sampling and Storage
Aliquots of blood were collected in tubes containing heparin (for analysis of blood
volume and plasma sodium) or sodium EDTA (0.015 M, for analysis of hormones). These
whole blood samples were placed on ice until the end of the experiment. Samples were
then centrifuged, and plasma aliquots were stored at -20°C until analyzed.
3.5.2 Plasma Analysis
Plasma progesterone was measured by radioimmunoassay (RIA) using kits
purchased from Diagnostic Products, Inc. (Los Angeles, CA). The lower limit of
detection of the assay was 0.1 ng/ml. The antibody used in this kit displays about 0.03%
cross-reactivity with cortisol and 9.0% cross-reactivity with THP. Plasma AVP was
extracted with bentonite (Sigma) and measured by RIA as previously described (224).
The lower limit of detection for this assay was 0.39 pg/ml when 0.5 ml of plasma was
extracted. For both radioimmunoassays, samples measuring below the lower limit of
detection were assigned the value of the limit of detection for purposes of statistical
analysis.
Plasma Na+ and K+ were analyzed using a Na7K+ analyzer (NOVA, Waltham,
MA). Plasma protein concentration was measured with a refractometer (Fisher Scientific,
Inc.) in order to assess whether either steroid treatment altered fluid distribution.

48
3.6 Cardiovascular Measurements
3.6.1 Mean Arterial Pressure
An arterial catheter was connected to a Statham P23Db transducer (Gould,
Oxnard, CA) and Grass model 7 recorder (Quincey, MA) to continuously monitor blood
pressure during infusions. Arterial pressure was sampled at 1 or 20 Hz with a Keithley
system 570 analog-to-digital converter and a microcomputer using software from
Asystantâ„¢ (Asyst Software Technologies, Rochester, NY). Mean arterial pressure
(MAP) was calculated as the mean value for each one-minute interval.
3.6.2 Plasma Volume
Plasma volume was estimated using the Evans blue dye dilution technique (18).
Before beginning a volume test, a control plasma sample (30 ml) was withdrawn. This
sample was used to create the standard curve. A known volume (E¡) of a 5 mg/ml
solution (calculated by weighing the injection syringe before and after injection) of Evans
blue (Sigma) was flushed into a venous catheter with excess saline (30 ml) at the start of
the volume test. Blood samples were then withdrawn from an arterial catheter at 10-
minute intervals. All blood volume samples were placed into tubes containing heparin and
centrifuged at 3,000 rpm to obtain a plasma sample. The resulting plasma sample was
again centrifuged at 12,000 rpm to remove all contaminating blood cells.
The Evans blue stock (5 mg/ml) was diluted into the control plasma sample to
create standards (0.0025- 0.03 mg/ml). The absorbances of the standard and unknown
samples were read at 620 nm on a diode array spectrophotometer (Beckman Instruments,
Inc., Fullerton, CA). The relationship between standard absorbance (A,td) and

49
concentration (Cstd) was fit to the equation of a line (A^d = mstd x Cstd + bstd) using
SigmaPlot® (Jandel Scientific, San Rafael, CA). The relationship between sample
absorbance (A„m) and sampling time (t^m) also was fit to the equation of a line (A*,,, =
m8am x tsam + bsam) using SigmaPlot®. The concentration of Evans blue at the instant of
injection (C0) was extrapolated (C0 = (bsam - bstd)/mstd) and the volume of distribution of
the dye was calculated (Vd = Ei/C0). Finally, the plasma volume (Vp) was expressed by
normalizing the Vd to the ewe’s body weight (Bw, Vp = Vd / Bw).
3.6.3 Baroreflex
3.6.3.1 Bolus injection method
In some experiments, baroreceptor function was evaluated using bolus injections
of phenylephrine and nitroprusside. Resting heart rate and resting MAP were sampled
over 15-second period immediately after steroid infusion and prior to the baroreflex test.
A 3pg/kg bolus of phenylephrine (American Regent Laboratories, Shirley, NY) was
injected into a venous catheter and the resulting pressor response was recorded at 20 Hz
as described above. Blood pressure and heart rate were then allowed to return to baseline
levels (15-20 minutes). Next, a 50 pg/kg bolus of nitroprusside (Nitropress, Elkins-Sinn,
Cherry Hill, NJ) was injected into the venous catheter and the resulting depressor response
was recorded.
3.6.3.2 Steady-state method
In other experiments, baroreceptor function was evaluated by recording MAP and
heart rate at a plateau after graded continuous intravenous infusions of phenylephrine and
nitroprusside. Solutions of phenylephrine and nitroprusside (20 mg/kg/ml) were prepared

50
in sterile isotonic saline and were infused at rates of 0.1, 0.2, 0.4, and 0.8 ml/min. In order
to establish a plateau in pressure and heart rate, each infusion rate was continued for 3
minutes before stepping up to the next infusion rate. The phenylephrine was infused first,
blood pressure and heart rate were allowed to return to baseline (30-40 minutes), and the
test was concluded with the graded nitroprusside infusions.
3,6.3.3 Expression of data
Baroreflex function curves express the relation between MAP and heart period.
When the bolus injection method was used, the heart period was calculated as the time
interval (in msec) between peaks in the first derivative of the arterial pressure wave, and
MAP at each peak was referenced from a smoothed curve of the arterial pressure wave.
Only the portions of the arterial pressure curves during the period of increasing or
decreasing pressure were used in the analysis.
When the steady-state method was used, MAP and heart period were calculated
from a 30-second recording obtained at the plateau for each dose of phenylephrine or
nitroprusside.
For each experiment in each animal, the baroreflex function relation was fit to the
logistic equation^ = [al{\+eb(x'c))]+dusing SigmaPlot® (Jandel Scientific, San Rafael,
CA). In order to prevent distortion of fit parameters by data sets that did not fit well to a
logistic equation (3/21 curves), the parameters were constrained to values of a< 1000,
70250. The parameters obtained from this logistic fit have physiological
meaning; a represents the maximum range of the heart period (ms); b represents the slope,
or sensitivity, of the reflex (Ams/AmmHg); c represents the MAP (mm Hg) at the
maximum rate of change (set-point or inflexion point); and d represents the minimum heart

51
period (ms) (143). The maximum gain of the reflex was calculated from the first
derivative of the logistic function (143). Threshold (MAP that produces the shortest heart
period) and saturation (MAP that produces longest heart period) values of MAP were
calculated from the third derivative of the equation (143). Mean baroreflex curves were
computer generated using the mean parameter values obtained in each treatment group.
When the bolus injection method was used, it also was desirable to determine if
treatment selectively altered the sensitivity of one portion of the baroreflex curve. The
slope of the rapid response to nitroprusside and phenylephrine was determined by linear
regression for each experiment in each animal.
3,7 Tissue Collection
All ewes were housed in the animal resources facility at the Health Sciences Center
for at least 24 hours prior to sacrifice. In instances when the ewe had not undergone
femoral catheterization, a jugular catheter was inserted approximately 24 hours prior to
sacrifice. These catheter lines were used to minimize stressful interaction between the ewe
and the investigator, to obtain a pre-sacrifice blood sample, and to administer an overdose
of sodium pentobarbital.
Immediately after sacrifice, the brain was perfused with a cold 10% solution of
dimethylsulfoxide in isotonic saline. Dimethylsulfoxide has been shown to cryoprotect
corticosteroid receptors (171). Unperfused tissues (kidney and liver) were washed and
stored in the cryoprotective solution. All tissue samples were rapidly excised, frozen in a
dry ice-acetone bath, and stored at -80°C until assayed.

52
3.8 5a-Reductase / 3a-Hvdroxvsteroid Dehydrogenase Activity Assay
The in vitro conversion of progesterone into the GABAA-active metabolites DHP
and THP was assayed according to a modification of the method previously described by
Krause and Karavolas (154).
3.8.1 Tissue Preparation
Tissues were obtained and stored as described above. At the time of assay, liver
and brainstem samples from non-pregnant, pregnant, and ovariectomized ewes were
homogenized using Krebs-Ringer bicarbonate (KRB) buffer (Sigma, pH=7.4, 4°C, 5ml of
buffer per gram of tissue). Homogenized tissues were first centrifuged at 3,000 x g for 20
minutes at 4°C to remove large particles from the homogenate. The supernatant was
retained and centrifuged at 100,000 x g for 60 minutes at 4°C. The pellet was retained
and resuspended in an equal volume of KRB buffer to yield a microsomal fraction.
3.8.2 Activity Assay
The reaction was started by adding 500 pi of the microsomal fraction to conical-
bottom, screw-top, borosilicate centrifuge tubes (Kimble Glass Inc, Vineland, NJ)
containing approximately 20,000 dpm of tritiated (1,2,6,7-3H) progesterone (3H-P,
Amersham International, Buckinghamshire, England), 500 pM NADPH, and 5 mM of
dithiothreitoi in KRB buffer (final reaction volume = 1 ml). The reaction was allowed to
run for 30 minutes at 37°C, and the reaction was stopped with 5 ml of anhydrous ethyl
ether. The tubes were then immersed in an acetone/dry ice bath, and the organic phase
was transferred to new tubes spiked with steroid carriers (100 pg of progesterone and

53
DHP and 200 pg of THP). To determine the amount of background radioactivity carried
by DHP and THP, blank tubes were prepared containing progesterone, DHP, THP, and
3H-P. All tubes were evaporated under a stream of air and the residual steroids were
reconstituted in 100 pi of warmed ethanol. The reconstituted samples were spotted on to
Thin Layer Chromatography (TLC) plates (silica gel 60 A, 0.25 mm thickness, Whatman,
Clifton, NJ) and developed in benzene: methanol (19:1 v/v, Sigma). Steroid bands were
visualized by exposure to ultraviolet light and iodine vapors, and the bands were scraped
into scintillation vials and counted.
The amount of radioactivity recovered in the DHP and THP bands was expressed
as the percentage of radioactivity recovered in all three bands for each lane, and the
average percentage of background activity carried in each band in the blank reaction tubes
was subtracted.
3.9 Radioligand Binding Assays
The binding of tritiated cortisol to available cytosolic corticosteroid receptors was
assayed according to a modification of a previously described method (226).
3,9,1 Tissue Preparation
Hippocampus, hypothalamus, pituitary, and kidney were homogenized using a
motor-driven Teflon pestle and glass tube (Wheaton Science Products) in a reducing
homogenization buffer (RHB): ice-cold Tris-HCl (10 mM, pH 7.4) containing 10%
glycerol, 10 mM sodium molybdate, 4 mM dithiothreitol, 2 mM EDTA, and 4 mM B-
mercaptoethanol (10 ml buffer per gram of tissue). The components of this buffer
(purchased from Sigma) have been shown to stabilize corticosteroid receptors (108). The

54
homogenate was centrifuged at 100,000xg for 60 minutes to obtain a cytosolic fraction
(supernatant). Aliquots of cytosol were stored at -80°C until assayed.
3.9.2 Incubations
The radioligand-binding assay was conducted in borosilicate test tubes at 4°C. For
determination of nonspecific and mineralocorticoid receptor binding, an appropriate
amount of stock steroid was added to each tube and the tubes were evaporated to dryness
in a centrifugal evaporator (Jouan, Inc., Winchester, VA). The residual steroids were
reconstituted with 100 pi of RHB containing the desired amount of tritiated (1,2,6,7-3H)
cortisol (3H-F, Amersham, final concentrations ranging 0.1-25 nM). The binding reaction
was initiated by adding 100 pi of cytosol (final incubation volume of 200 pi with 1-2 pg of
protein/pl).
MR binding was determined in the presence of 1.25 pM RU 28362, a specific GR
agonist (Roussel-Uclaf, Romainville, France). Nonspecific binding was determined in the
presence of 12.5pM dexamethasone sodium phosphate (American Regent Laboratories,
Inc., Shirley, NY). Binding to GR was calculated from the difference between total
specific cortisol binding and specific cortisol binding in the presence of excess RU 28362.
Specific binding was expressed in fmol/mg protein, and protein concentration was
determined according to the method of Bradford (24) using a kit purchased from Bio-Rad
Laboratories (Hercules, CA).
3.9.3 Separation of bound from free
Steroid-bound receptors were separated from the unbound ligand by LH-20 gel
exclusion chromatography (170). Chromatography columns were prepared by plugging

55
5ml PYREXâ„¢ serological pipettes (Corning, Inc.) with 3 mm diameter borosilicate beads
(Kimble Glass Company). The columns were filled to a height of 8 cm with Sephadex®
LH-20 (Pharmacia Biotech, Uppsala, Sweden) reconstituted in RHB. For separation of
bound from free, a 150 til aliquot of the incubate was loaded to the top of the column bed,
washed into the column bed with 100 pi buffer, and eluted with an additional 1 ml of
buffer. After each use, the columns were washed with 15 volumes of methanol and 15
volumes of RHB. During the buffer RHB re-equilibration portion of the wash, the bead
bed was agitated and allowed to resettle. Columns were maintained at 4°C.
310 Western Blots
Total cytosolic and whole cell MR and GR were estimated using a semi-
quantitative Western blot procedure.
3,10,1 Tissue Preparation
Tissues were homogenized in RHB as described above. Whole cell samples were
obtained from the unprocessed homogenate, and cytosolic samples were obtained after
centrifugation at 100,000xg as described above. The concentration of protein in each
samples was determined according to the method of Bradford (24) using a kit purchased
from Bio-Rad. All samples were stored at -80°C until further processed. Prior to gel
electrophoresis, samples were diluted in a Laemmli (156) reducing sampling buffer
(0.0625 M Tris-HCl, 10% glycerol (v/v), 2% SDS (w/v), 0.72 M B-mercaptoethanol, and
0.001% bromophenol blue) and heated at 95°C for 5 minutes.

56
3.10.2 Electrophoresis and Transfer
Samples were loaded onto precast 10% polyacrylamide, Tris-HCl gels (Bio-Rad
Laboratories) and electrophoresed at approximately 100 V using a Mini-Protean® II gel
electrophoresis system (Bio-Rad Laboratories). An equal volume of sample (30 pi) and
amount of protein (20 pg) was loaded into each lane. One lane of each gel was loaded
with a mixture of stained proteins of known molecular weights (Rainbowâ„¢ coloured
protein molecular weight markers, Amersham).
After electrophoresis, proteins were transferred overnight (at 22 V) onto a
nitrocellulose membrane using a Mini Trans-Blot® cell (Bio-Rad Laboratories). The
membranes were blocked with 10% w/v nonfat powdered milk solution, and incubated
with an anti-MR or anti-GR antibody (both purchased from Affinity Bioreagents, Golden,
CO) and an appropriate horseradish-peroxidase conjugated IgG antibody (Sigma). The
membranes were then incubated with Renaissanceâ„¢ Western Blot Chemiluminesence
Reagent Plus (NENâ„¢ Life Science Products, Boston, MA) and exposed to Reflectionâ„¢
Autoradiography Film (NENâ„¢ Life Science Products) for approximately 30 seconds.
3.10.3 Densitometry
The resulting films were scanned with model GS-670 Imaging Densitometer (Bio-
Rad) and immunoreactive band density was analyzed using Molecular Analyst®/PC
software. Only the densities of samples developed on the same Western Blot were
compared. The linear relationship between electrophoretic mobility and the logarithm of
the molecular weight of Rainbowâ„¢ markers was used to estimate the molecular weight of
the resulting immunoreactive bands.

57
3.11 Statistical Analysis
Methods of statistical analysis were chosen based on experimental design and the
character of the obtained data set (87); these are detailed for each experiment in each
chapter. For all statistical tests, the null hypothesis was rejected when p < 0.05.

CHAPTER 4
ACUTE EFFECT OF PROGESTERONE ON CARDIOVASCULAR FUNCTION IN THE
OVARY-INTACT EWE
4.1 Introduction
Associated with the increase in progesterone and estrogen during pregnancy is a
substantial increase in heart rate, cardiac output, and plasma volume (169). A concurrent
decrease in total peripheral resistance results in a 5-10 mm Hg reduction in MAP (MAP) (169).
Several studies suggest the baroreflex maintains arterial pressure about this new setpoint with
reduced sensitivity when the baroreflex response to decreased pressure is included in the analysis
(29). The correlation of altered cardiovascular function during pregnancy and the menstrual cycle
with hormonal rhythms has led to the hypothesis that ovarian steroids are important regulators of
the cardiovascular system (169).
Estradiol has been shown to induce many cardiovascular changes characteristic of
pregnancy. Chronic estradiol treatment decreases vascular tone and MAP, and increases renin
activity, plasma volume, and cardiac output (174, 277). Recently collected evidence suggests that
progesterone also modulates these variables. In ovariectomized ewes, physiological levels of
progesterone, alone or in combination with estradiol, significantly reduce arterial pressure and
increase plasma volume and arginine vasopressin (AVP) without increasing activity of the renin-
angiotensin system (210). Therefore, progesterone also appears to be a regulator of
cardiovascular function.
58

59
Previous studies attributed progesterone's antihypertensive actions to its ability to
antagonize the renal salt and fluid retaining activity of mineralocorticoids (254, 287). Evidence
now suggests progesterone also has rapid, nongenomic actions on the baroreflex. Progesterone
can be sequentially metabolized by 5oc-reductase and 3oc-hydroxy steroid dehydrogenase in the
brain and periphery to form tetrahydroprogesterone (THP). THP, which circulates at elevated
levels during pregnancy, enhances activation of the GABAa receptor by GABA (176). Heesch
and Rogers (98) recently demonstrated that THP rapidly causes a leftward shift in setpoint, and at
the same time, decreases the overall sensitivity of renal sympathetic nerve responses to changes in
arterial pressure. These investigators propose that THP acts in the rostral ventral lateral medulla
to enhance GABAergic activity, thereby increasing the GABAergic inhibition of excitatory amino
acid projections to the preganglionic sympathetic cell bodies of the intermediolateral cell column
(98).
This experiment was designed to test for a rapid effect of progesterone on blood pressure,
Na+, AVP, volume, and baroreflex sensitivity in non-pregnant ewes. Two doses of progesterone
are used to test for this effect: one within the physiological range for pregnant ewes and one
exceeding this range.
4,2 Methods
Seven adult, ovary-intact, anestrous ewes were studied during and immediately after 2-
hour venous infusions. Each ewe was subjected to three infusion protocols: vehicle (10% ethanol
in 0.9% saline), progesterone (Sigma, St. Louis, MO) at 3 pg/kg/min (3P4), and progesterone at 6
pg/kg/min (6P4) in a randomized, crossover design.

4.2.1Progesterone Infusion and MAP
60
During a given experiment, a ewe was treated with a continuous 2-hour infusion of
control vehicle or steroid. In order to minimize the possibility of residual steroid effects, each
ewe was allowed at least a two-day recovery period between treatments. Arterial pressure was
continuously monitored during the infusions. Blood samples (7 ml each) were collected at 30-
minute intervals during the first two hours of the infusion for measurement of plasma Na+,
protein, AVP, and progesterone.
4.2.2 Baroreflex Test
Following a 2-hour infusion of vehicle, 3P4 or 6P4, baroreceptor function was evaluated
using bolus injections of phenylephrine (3 pig/kg) and nitroprusside (50 pg/kg) while the infusion
continued.
4.2.3 Statistical Analysis
Plasma AVP, protein, sodium, and progesterone concentrations and MAP responses
measured over time were analyzed by two-way analysis of variance (ANOVA) corrected for
repeated measures. Logistic and linear curve fit parameters were compared by one-way ANOVA
corrected for repeated measures; when values were not normally distributed, a Friedman repeated
measures ANOVA on ranks was used. Individual means were compared with a Duncan's multiple
range test. For all statistical tests, the null hypothesis was rejected when p < 0.05. Data are
expressed as the mean ± standard error of the mean (SEM).

61
4 3 Results
4.3.1 Plasma Progesterone Levels
When progesterone was infused at a rate of 3 pg/kg/min, plasma progesterone
concentration increased to 10.4 ± 3.9 ng/ml after 30 minutes (Fig. 4.1C), and remained at
this level until 120 minutes when the plasma concentration averaged 8.4 ± 2.9 ng/ml.
These concentrations are significantly greater than during the infusion of vehicle (mean
concentration 2.1 ± 0.7 ng/ml at 120 minutes). When progesterone was infused at a
higher rate (6 pg/kg/min), progesterone increased to 37.8 ± 14.3 ng/ml at 60 minutes into
the experiment (Fig. 4. IE), and was 19.4 ±3.0 ng/ml by the end of the 6 pg/kg/min
treatment period. This plasma concentration was significantly greater than the level
obtained at the end of the infusion of 3 pg/kg/min or at the end of the infusion of vehicle.
The tendency for plasma progesterone concentration to decrease toward the end of the 6
pg/kg/min infusion is most likely attributable to the poor solubility of high concentrations
of progesterone in the 10% ethanol vehicle. Progesterone crystals tended to form in the
infusion lines of some of the larger animals as the experiment progressed, and in some
instances the infusion line had to be cleared with a bolus of saline.
4.3.2 Arterial Pressure
Mean arterial pressures were statistically similar at the beginning of each infusion
protocol. Arterial pressures averaged 95.9 ± 7.9 mm Hg at the beginning of the 2-hour
control vehicle infusion (Fig. 4. IB), and was not significantly different at the end of the
experiment (94.0 ± 7.5 mm Hg). However, infusion of progesterone at a rate of 3
pg/kg/min significantly reduced MAP over the two hours (Fig. 4. ID). MAP was

62
significantly decreased by the 17th minute of 3P4 treatment (90.4 ± 1.3 mm Hg), and
remained significantly reduced until the end of the experiment (mean value after 120
minutes of infusion of 3pg/kg/min: 86.2 ± 1.7 mm Hg). In contrast, the higher rate of
infusion (6pg/kg/min) did not significantly change MAP (Fig. 4. IF).
4,3,3 Baroreflex
Representative logistic curve fits from an individual animal (W94) are presented in
Figs. 4.2A-C. Mean logistic curve fit parameter values are presented in Table 4.1, and
mean baroreflex curves are diagramed in Fig. 4.2D. When the entire baroreflex curve was
analyzed, there were no significant differences among treatment groups.
The slope of the phenylephrine-induced portion of the baroreflex curve was not
significantly altered by either treatment (Table 4.2). The linear response to phenylephrine
was highly variable in the control group (23.2 ± 16.9 Ams/AmmHg), and this was due to a
steep response in one animal (G157, 124.0 Ams/AmmHg). When this animal was
eliminated from the analysis, the slope of the bradycardic response to phenylephrine after
vehicle infusion was 6.3 ± 1.5 Ams/AmmHg, and there were no significant differences
among groups. The slope of the nitroprusside-induced portion of the baroreflex curve
was not significantly altered after infusion of progesterone at a rate of 3 pg/kg/min (Table
4.2). Although infusion of progesterone at a rate of 6 pg/kg/min tended to attenuate the
slope of the response to nitroprusside injection compared to vehicle, this was not
significantly different. However, the slope of the response to nitroprusside after infusion
of progesterone at a rate of 6 pg/kg/min was significantly attenuated compared to the
slope after infusion of progesterone at a rate of 3 pg/kg/min.

63
4.3.4 Plasma Na* and AVP
There were no significant effects of either the 3 pg/kg/min or the of 6 pg/kg/min
infusions on plasma Na+ (Figure 4.3 A). Infusion of progesterone at rate of 3 pg/kg/min
significantly increased plasma AVP over time (2.1 ± 0.9 to 3.2 ± 1.0 pg/ml at 120 minutes;
Figure 4.3B). Plasma AVP concentration after 120 minutes of infusion of progesterone at
a rate of 3 pg/kg/min was significantly greater than after 120 minutes of vehicle infusion.
In contrast, plasma AVP was not significantly altered by infusion of progesterone at rate
of 6 pg/kg/min.
4.3.5 Plasma Protein
Plasma protein concentration did not significantly change during infusion of
control vehicle (7.3 ± 0.1 to 7.4 ± 0.1 g/100ml, Figure 4.3C) or progesterone at a rate of 3
pg/kg/min (7.5 ± 0.2 to 7.6 ± 0.2 g/100ml). Prior to infusion of progesterone at a rate of
pg/kg/min, plasma protein concentration was significantly lower (7.1 ± 0.2 g/100ml) than
plasma protein concentration prior to the beginning of vehicle infusion (7.3 ± 0.1
g/100ml). Although plasma protein concentration significantly increased over time during
the infusion of progesterone at a rate of pg/kg/min (7.1 ± 0.2 to 7.6 ±0.1 g/100ml), it was
not significantly different from vehicle after 120 minutes of infusion.
4,4 Discussion
Plasma progesterone levels (10-12 ng/ml) characteristic of ovine pregnancy (9)
rapidly reduced MAP in ovary-intact, anestrous, adult ewes, suggesting progesterone
decreases blood pressure through nongenomic mechanisms in this animal model. In
contrast, supraphysiological levels of progesterone (25-40 ng/ml) did not alter MAP

64
within 2 hours. This finding indicates plasma progesterone concentration and MAP are
not linked through a simple, linear dose-response relationship. Consequently,
progesterone appears to regulate cardiovascular function through multiple mechanisms.
Initial interest in the antihypertensive actions of progesterone arose from the
observation that progesterone antagonizes the salt and fluid retaining actions of
mineralocorticoids (5). Subsequent studies have attributed the antihypertensive effect of
progesterone to its anti-mineralocorticoid properties (254, 287). However, chronic
progesterone treatment also reduces blood pressure in humans, dogs, and rats under
conditions in which mineralocorticoid levels are not elevated (5, 31, 63, 84, 260). Chronic
progesterone treatment over 10-14 days reduces arterial pressure in ewes, but expands
plasma volume (210), a finding that is contradictory to the anti-mineralocorticoid
hypothesis of progesterone action. In this study physiological levels of progesterone
decrease arterial pressure more rapidly than previously noted. This study reveals a
significant change in blood pressure within 17 minutes, but no evidence of changes in
plasma electrolytes or volume over the 120 minutes of study.
The mechanism by which progesterone decreases systemic pressure is currently
unknown. Progesterone may decrease arterial pressure by an extrarenal action at
progestin-binding sites. Homogenate and autoradiographic binding studies reveal
radiolabeled progestin binding in both the vascular system and in cardiovascular centers in
the hypothalamus and brain stem (114, 259). Using isolated placental arteries and veins,
Omar and colleagues have recently demonstrated progesterone elicits an acute reduction
in vascular tone via a mechanism mediated by intracellular progesterone receptors and
cyclic adenosine monophosphate (202). This mechanism may be specific to the placental

65
circulation and may not contribute significantly to systemic vascular tone. Similarly,
Magness and Rosenfeld (175) have shown that the effects of estradiol on uterine blood
flow and vascular resistance are much more profound than the systemic effects.
Heesch and Rogers (98) have hypothesized progesterone metabolites alter blood
pressure and decrease baroreflex sensitivity by decreasing sympathetic outflow through an
action at GABAa receptors in brain stem cardiovascular-regulatory areas. An attenuation
of baroreflex sensitivity at physiological levels of progesterone using heart rate as the
index of sympathetic outflow was not observed in this study. It is possible that
sympathetic outflow to the vascular system is altered by this concentration of
progesterone. There was a tendency towards attenuation of the linear response to
nitroprusside after infusion of supraphysiological levels of progesterone, suggesting that
the tachycardic response to hypotension is reduced during infusion of progesterone at a
high rate. Glick and Braunwald have suggested that the rapid bradycardic response to
hypertension is primarily mediated via parasympathetic activity while the rapid tachycardic
response to hypotension is predominately mediated by sympathetic activity (88).
Therefore, the attenuated slope of the baroreflex response to nitroprusside observed after
infusion of 6 pg P4 /kg/min may be due to compromised sympathetic control of the heart
rate. If this effect is mediated by progesterone metabolites, supraphysiological
progesterone levels appear to be required to achieve active metabolite concentrations.
Progesterone also could modulate blood pressure by acting as an antagonist or
weak agonist at mineralocorticoid (MR) or glucocorticoid (GR) receptors (233). The KD
for progesterone binding to each of these receptors is within the physiological range of
plasma progesterone, but progesterone has a much higher affinity for MR than for GR

66
(233). MR and GR have already been implicated in central blood pressure regulation in
the rat (89, 282); intracerebroventricular MR agonists increase, but GR agonists decrease,
blood pressure (282). Intracellular progesterone receptors may interact with this system
since one form (PR-A) has been shown to inhibit transcription of MR and GR in
transfected cell cultures (180). The interaction of progesterone with these numerous
potential receptor types could therefore contribute to the complexity of progesterone's
actions on the cardiovascular system by the second hour of infusion.
In these experiments there was no acute effect of progesterone infusion on plasma
sodium or plasma proteins, suggesting the infusion did not alter electrolyte or fluid
balance. The infusion of progesterone at 3 pg/kg/min increased plasma AVP, but this was
not significant until 120 minutes. The effect of low concentrations of progesterone on
plasma AVP can not be readily explained as an enhancement of GABAa activity by
progesterone metabolites. Current evidence suggests GABAa activation actually inhibits
AVP release (146). Instead, the increase in plasma AVP at 120 minutes may be mediated
by genomic mechanisms. Previous investigations have observed increases in both plasma
sodium and AVP concentrations after six days of progesterone treatment (unpublished
observations), suggesting an indirect effect of progesterone on AVP through osmolality.
This effect of progesterone could be mediated by renal actions of progesterone however;
further study of the acute and chronic effects of progesterone on plasma Na+, AVP, and
volume is required.
This study confirms physiological concentrations of progesterone significantly
diminish arterial pressure and increase plasma AVP. Furthermore, the change in arterial
pressure occurs within 17 minutes, consistent with a nongenomic mechanism. However,

67
there was not a significant effect of 2 hours of infusion of progesterone at this physiologic
dose on the baroreflex heart rate response to acute increases or decreases in blood
pressure. In contrast, supraphysiological progesterone levels did not alter arterial pressure
within 2 hours, but did tend to decrease the slope of the tachycardic response to acute
hypotension. The availability of numerous potential progesterone receptor sites may
contribute to progesterone's multiple actions.

68
Table 4.1. Logistic baroreflex curve parameters.
Experimental Group
Vehicle (n=7)
3P4 (n=7)
6P4 (n=7)
Resting MAP (mmHg)
97.3 ±7.8
94.0 ±6.7
97.7 ±6.9
Resting heart rate (beats/min)
71.4 ±5.4
69.1 ±3.7
73.7 ±3.0
(a) heart period range (ms)
647.0 ± 105.4
564.2 ±78.1
488.4 ±97.1
(A) slope (Ams/AmmHg)
-0.14 ±0.04
-0.11 ±0.04
-0.57 ±0.32
(c) MAP at inflexion point
93.2 ±8.8
91.0 ± 6.8
93.9 ±5.7
(d) heart period minimum (ms)
435.2 ±35.4
487.6 ± 19.6
475 ± 47.0
Maximum gain (Ams/AmmHg)
19.3 ±4.2
14.0 ±3.8
48.0 ±23.5
Threshold pressure (mmHg)
80.1 ±9.4
65.5 ± 13.2
69.4 ± 17.1
Saturation pressure (mmHg)
106.2 ±8.9
116.4 ±7.2
118.5 ± 19.1

69
Table 4.2. Linear baroreflex responses to nitroprusside and phenylephrine.
Experimental Group
Vehicle (n=7) 3P4 (n=7) 6P4(n=7)
Nitroprusside (Ams/AmmHg) 10.2 ±2.1 11.8 ±2.3 5.7 ±1.3*
Phenylephrine (Ams/AmmHg) 23.2 ±16.9# 5.9 ±2.4 7.4 ±2.1
* significantly different from 3P4,
#n=6 after omitting outlying value of 124.0 Ams/AmmHg

70
30 60 90 120
O
CL
30 60 90 120
E
E
30 60 90 120
<
C
co
o
30 60 90 120
110 n
105
100
95
90
85 -
80 -
i i l i
30 60 90 120
Time (min)
Figure 4.1. Plasma progesterone (P4; A,C,E) and MAP (B,D,F) during 2-hour infusions
of vehicle (O; A,B), or P4 at rates of 3 pg/kg/min (â– ; C,D) and 6 pg/kg/min (â– *; E,F).
Data are means ± SEM (n=7).

Heart Period (ms)
71
1200 -i
800 -
400 J
~ l 1 1 1
30 60 90 120 150
1200 -i
800 -
400 J
A A A
30 60 90 120 150
30 60 90 120 150
Mean Arterial Pressure (mmHg)
Figure 4.2. Representative baroreflex curves from ewe W94 after infusion of vehicle (O;
A), 3 pg PvTcg/min (â– ; B), and 6 pg P4/kg/min(A; C). Mean baroreflex curves were
computer generated using mean logistic curve fit parameter values (D); vehicle (solid line,
n=7), 6 pg P4/kg/min (dotted line, n=7), and 6 pg P4/kg/min (dashed line, n=7).

72
Time (min)
Figure 4.3. Effect of 2-hour infusions of vehicle (O, n=7) or P4 at rates of 3 gg/kg/min
(â– , n=7) and 6 gg/kg/min (A, n=7) P4 on plasma Na+ (A), AVP (B), and protein (C).
Data are means ± SE.

CHAPTER 5
ACUTE EFFECT OF PROGESTERONE ON BLOOD PRESSURE, BLOOD VOLUME, AND
BAROREFLEX FUNCTION IN THE OVARIECTOMIZED EWE
5.1 Introduction
A recent study by Heesch and Rogers (98) demonstrated the progesterone metabolite
THP, which circulates at elevated levels during pregnancy (46), rapidly causes a leftward shift in
setpoint and decreases the sensitivity of renal sympathetic nerve responses to changes in arterial
pressure. These investigators propose THP modulates a GABAergic component of the baroreflex
in the rostral ventral lateral medulla (RVLM). In the RVLM, GABAergic neurons inhibit
excitatory amino acid projections to pre-ganglionic sympathetic cell bodies in the
intermediolateral cell column.
The results of the study presented in Chapter 4 suggest progesterone also has rapid effects
on arterial pressure, and the relationship between plasma progesterone concentration and MAP is
not a simple, linear dose-response relationship. Studies have not confirmed an effect of
progesterone alone on baroreflex sensitivity; progesterone did not decrease baroreflex sensitivity
after two hours (Chapter 4) or two weeks (210) of progesterone treatment despite a reduction in
MAP in both time intervals. However, these studies measured the rapid heart rate responses to
single doses of phenylephrine and nitroprusside to evaluate baroreflex responsiveness. This
method may measure the rapid vagal response to pressure changes, but not the slower-responding
GABA-modulated sympathetic efferent response (45).
73

74
A study of the ovariectomized ewe demonstrated chronic treatment with progesterone at
concentrations typical of ovine pregnancy also expands plasma volume (210). The time-course
and mechanisms of progesterone-induced changes in plasma volume are unknown. The plasma
protein data presented in Chapter 4 did not suggest an acute effect of progesterone on plasma
volume, but a more complete analysis is required. Slower genomic action at receptors in the
vascular system, kidney, hypothalamus, and brain stem may be required for progesterone to
increase plasma volume and maintain decreased pressure in the presence of increased volume.
Progesterone could slowly increase plasma volume by acting directly in the kidney or by
increasing AVP synthesis in the hypothalamus and subsequent release from the posterior pituitary.
However, chronic volume expansion is generally associated with an increase in blood pressure
rather than the decrease in pressure observed with progesterone. This suggests progesterone also
must decrease vascular tone by acting directly in the vascular system (202) or by modifying
sympathetic outflow from the brainstem (98). A progesterone-induced decrease in vascular tone
also would contribute to the expansion of plasma volume by progesterone. Expanded plasma
volume also could contribute to a decrease in baroreflex sensitivity since other investigators have
reported an attenuation of the baroreflex in the volume-loaded state (49). A complete assessment
of the time course of progesterone-induced changes in MAP, plasma volume, and baroreflex
responsiveness would further clarify the role of progesterone in cardiovascular adaptation to
pregnancy.
Two studies were conducted to more completely evaluate the rapid nongenomic and early
genomic effects of progesterone infusion on arterial pressure, blood volume and baroreflex
sensitivity. The first study was a 30-minute infusion designed to determine if, in addition to an

75
alteration in arterial pressure, progesterone rapidly alters plasma volume and the slower-
responding sympathetic component of the heart rate response to perturbations in arterial pressure.
The second study consisted of a 4-hour infusion of control vehicle or progesterone at rates of 1.5,
3, and 6 pg/kg/min. This study was designed to further evaluate the relationship between
progesterone concentration and arterial pressure and to determine if progesterone has slower
effects on plasma volume and the baroreflex. Ovariectomized ewes were studied in order to
eliminate possible confounding changes in plasma steroid levels during the natural estrous cycle,
and estradiol was replaced to a basal level since many of progesterone’s effects are known to
require estrogen priming (123, 124).
5.2 Methods
Eight adult ovariectomized-estradiol treated ewes were studied in a 30-minute protocol
and a 4-hour progesterone infusion protocol. Five of the eight ewes were studied beginning
approximately four months after ovariectomy and three of the ewes were studied beginning
approximately one week after ovariectomy. In the previous study progesterone was dissolved in a
10% ethanol vehicle, but in this study progesterone was administered in a 20% ethanol vehicle.
The concentration of vehicle ethanol was increased in this study to ensure the more highly
concentrated progesterone preparations would remain in solution throughout the experiment. In
addition, a single estradiol implant (3 cm) was placed in dorsal scapular region of each ewe since
many of progesterone’s effects are known to require prior estrogen priming and at least one form
of the intracellular progesterone receptor is induced by estrogen (125). The implants were
implanted one week before beginning the experiments, and the implants have been shown to

76
maintain plasma estradiol at levels (~4 pg/ml) characteristic of the luteal phase of ovine menstrual
cycle (123, 124). In order to minimize the possibility of residual progesterone effects, each ewe
was allowed at least a 2-day recovery period between infusion treatments.
5.2,1 30-Minute Infusion Protocol
In the 30-minute protocol, each ewe was subjected to three infusions: vehicle (20%
ethanol in 0.9% saline, 0.7 ml/min), and progesterone at rates of 1.5 (I.5P4) and 3 (3P4)
pg/kg/min in a randomized, crossover design. Arterial pressure was recorded continuously for 45
minutes, and the 30-minute infusion began after the first 15 minutes of pressure recording. Blood
samples were collected before beginning (40 ml) and at 5-minute intervals (18 ml) after Evans
blue injection and during the infusion. These blood samples were used to determine plasma
volume and progesterone concentrations. The volume of blood (approximately 166 ml)
withdrawn during the course of the study was about 2% of the total blood volume (approximately
7.5 L). Blood volume was measured using an adaptation of the Evans blue dye dilution technique
(18) designed to detect rapid shifts of fluid into the vascular compartment. Evans blue was
injected after ten minutes of baseline pressure recording and five minutes prior to beginning the
infusion. Plasma dye concentration was measured in samples obtained at 5-minute intervals
thereafter. Normally the dye is eliminated from plasma linearly over time, but if progesterone
rapidly shifts fluid into the vascular compartment, the slope would increase over time and
relationship between time and dye concentration would become curve-linear. Following the 30-
minute infusion of vehicle or progesterone, baroreceptor function was evaluated using graded
continuous infusions of phenylephrine and nitroprusside while the infusion continued. This

77
method allows closer examination of the more slowly responding sympathetic component of heart
rate control.
5.2.2 4-Hour Infusion Protocol
In the 4-hour protocol, each ewe was subjected to four infusions: vehicle (20% ethanol in
0.9% saline, 0.7 ml/min), and progesterone at 1.5 (1.5P4), 3 (3P4), and 6 (6P4) pg/kg/min in a
randomized crossover design. Arterial pressure was monitored continuously for 250 minutes, and
the 4-hour infusions began after the first ten minutes of pressure recording. Blood samples were
collected before beginning the infusion (40 ml), at 10-minute intervals during the first and last 30
minutes (10 ml) of the infusion, and at hourly intervals (10 ml each) throughout the experiment.
These blood samples were used for measurement of plasma progesterone concentration and
determination of plasma volume. Plasma volume was measured using the Evans blue dye dilution
technique during the final thirty minutes of the infusion. Following the 4-hour infusion of vehicle
or progesterone, baroreceptor function was evaluated using graded continuous infusions of
phenylephrine and nitroprusside while the infusion continued.
5.2.3 Statistical Analysis
MAP responses measured over time were analyzed by two-way ANOVA corrected for
repeated measures. Plasma progesterone concentrations, logistic curve fit parameters, and plasma
volume concentrations were compared by one-way ANOVA corrected for repeated measures;
when values were not normally distributed, a Friedman repeated measures ANOVA on ranks was
used. Individual progesterone means were compared using a Student-Newman-Keuls post-hoc
test. For all statistical tests, the null hypothesis was rejected when p < 0.05. Data are expressed
as the mean ± standard error of the mean (SEM).

78
5.3 Results
5.3.1 30-Minute Infusion Protocol
5.3.1.1 Plasma progesterone levels
Plasma progesterone concentration was 0.8 + 0.3 ng/ml after thirty minutes of
vehicle infusion (Figure 5.1 A) and was significantly increased to 7.2 ± 1.6 ng/ml and 15.0
± 3.4 ng/ml after infusion of progesterone at rates of 1.5 pg/kg/min and 3 pg/kg/min,
respectively.
5.3.1.2 Arterial pressure
MAP averaged 92.2 ± 2.9 mmHg during the 15-minute baseline recording, (Figure
5. IB, C, and D) and did not change significantly after 30 minutes of control vehicle
infusion (91.2 ± 2.3 mmHg, Figure 5. IB). Similarly, infusion of progesterone at a rate of
1.5 pg/kg/min did not significantly change arterial pressure after 30 minutes (95.0 ± 2.9
vs. 95.0 ± 2.0 mmHg, Figure 5.1C). The 30-minute infusion of progesterone at a rate of 3
pg/kg/min significantly reduced arterial pressure over time from 95.0 ± 2.9 to 92.4 ±1.7
mmHg (Figure 5. ID). However, arterial pressure at the end of the 30-minute infusion was
not significantly different from arterial pressure at the end of the vehicle infusion.
5.3.1.3 Plasma volume
Representative Evans blue dye elimination curves from ewe Y254 are presented in
figure 5.2. Optical density of plasma decreased linearly over time during infusion of
vehicle, or progesterone at rates of 1.5 and 3 pg/kg/min; the r2 values of the linear
regressions were 0.79 ± 0.09, 0.68 ± 0.12, and 0.83 ±0.11, respectively. As a result,

79
there was no evidence of rapid movement of fluid into the vascular compartment. The
slopes of the lines were 3.0 ± 0.4, 2.1 ± 0.3, and 2.5 ± 0.4 AO. D./Amin, respectively.
Since these values were not significantly different among treatment groups, there was no
evidence of differential dye elimination during any of the infusion treatments. Plasma
volume (Figure 5.3) was 5 3.4±4.1 ml/kg after thirty minutes of vehicle infusion, and was
not significantly different after infusion of progesterone at rates of 1.5 (53.4±6.3 ml/kg) or
3 pg/kg/min (53.2±4.3 ml/kg).
5,3,2 4-Hour Infusion Protocol
5.3.2.1 Plasma progesterone levels
Plasma progesterone concentration was 2.4 ± 0.9 ng/ml after four hours of vehicle
infusion (Figure 5.4). When progesterone was infused at a rate of 1.5 pg/kg/min, plasma
concentration was significantly increased to 8.1 ± 1.5 ng/ml after four hours. Infusion of
progesterone at a rate of 3 pg/kg/min significantly elevated progesterone levels to 19.5 ±
4.9 ng/ml. Plasma progesterone concentration was 25.9 ± 7.4 ng/ml after four hours of
infusion at a rate of 6 pg/kg/min, but this concentration was not significantly different
from the concentration obtained after infusion of 3 pg/kg/min.
5.3.2.2 Arterial pressure.
MAP did not change over time in response to any dose of progesterone (Figure
5.5). MAP averaged 88.3 ±6.8 mmHg at the beginning of the 4-hour vehicle infusion,
and was not significantly different at the end of the experiment (86.7 ± 6.2 mmHg).
Similarly, MAP was not significantly different from control values after 4-hour infusions of

80
progesterone at rates of 1.5 (85.9 ± 5.0 vs. 86.5 ± 3.9), 3 (92.0 ± 5.0 vs. 90.6 ±4.9), and
6 (85.3 ± 5.7 vs. 87.6 ± 5.6) pg/kg/min.
5.3.2.3 Plasma volume.
Plasma volume was 37.6 ± 6.5 ml/kg after four hours of vehicle infusion (Figure
5.6), and was not significantly different after infusion of progesterone at rates of 1.5 (39.3
± 5.4), 3 (39.2 ± 4.5), or 6 (37.9 ± 4.5) 6 pg/kg/min.
5.3.2.4 Baroreflex
Representative logistic curve fits from an individual animal (ewe Y258) are
presented in Figure 5.7. Mean logistic curve-fit parameter values are presented in Table
5.1. Baroreflex curves were not significantly different among treatment groups.
5.4Discussion
The results of the 30-minute progesterone infusion confirm a rapid reduction in
arterial pressure when progesterone is infused at a rate of 3 pg/kg/min, but a lower
infusion rate of 1.5 pg/kg/min did not decrease arterial pressure. The change in arterial
pressure during the infusion of progesterone at a rate of 3 pg/kg/min was not
accompanied by an increase in plasma volume, suggesting progesterone decreases arterial
pressure before expanding plasma volume. Progesterone may decrease arterial pressure
by directly reducing vascular tone (202) or by increasing the concentration of GABAa-
active progesterone metabolites (98). A decrease in vascular tone could contribute to a
subsequent expansion of plasma volume that also may require progesterone actions in the
hypothalamus and/or kidney.

81
The results of the 4-hour infusion do not confirm a rapid effect of progesterone on
MAP in the ovariectomized ewe, and demonstrate 4-hour infusions of physiological and
supraphysiological concentrations of progesterone do not alter plasma volume or the heart
rate response to perturbations in arterial pressure.
Baseline arterial pressure was significantly lower at the beginning of the 4-hour
(87.9 ± 2.6 mmHg) infusion protocol than at the beginning of the 30-minute infusion
protocol (94.4 ± 2.9 mmHg) and the study presented in Chapter 4 (94.9 ±2.1 mmHg).
This finding suggests progesterone rapidly reduces arterial pressure when baseline
pressures are slightly elevated. In all three studies care was taken to minimize stress to the
animals and to allow time for the animals to recover from the stress of interaction with the
investigator. However, the protocols of the two rapid infusion studies were probably
more stressful to the ewes than the 4-hour protocol. In the study presented in Chapter 4,
ewes were removed from their home cages and taken to a nearby laboratory for study. In
both the 30-minute and 4-hour protocols presented in this chapter the ewes were studied
in their home pens. However, the design of the 30-minute experiment could have been
more stressful to the ewes since the protocol required increased investigator activity
outside of the animal pen immediately before and during the study.
If progesterone rapidly reduces arterial pressure via GABAa active metabolites,
these metabolites may be most effective when sympathetic activity is increased. The
sympathetic nervous system contributes minimally to resting vascular tone, but increased
sympathetic tone may contribute to the observed elevations in baseline pressure in the two
rapid infusion experiments. This may explain why an infusion of progesterone at a rate of
3 4g/kg/min rapidly reduces arterial pressure when baseline arterial pressures are slightly

82
elevated. Studies suggest sympathetic tone actually increases during pregnancy (264) and
sympathetic response gain is reduced due to reduced sympathetic reserve (29). Since
sympathetic outflow appears to be increased during pregnancy, the true role of elevated
progesterone metabolites in cardiovascular adaptation to pregnancy may be limited to their
effects on sympathetic gain (98) and the gain of components of sympathetic output also
may be selectively regulated (179). Progesterone metabolites may rapidly reduce arterial
pressure in the ewe by transiently reducing sympathetic outflow and may contribute to a
selective long-term reduction in sympathetic gain, but additional mechanisms may
contribute to the chronic effects of progesterone on cardiovascular function.
Although a steady-state baroreflex test was used to reveal potential changes in the
slow-responding sympathetic component of heart rate control, the 4-hour progesterone
infusions did not alter the heart rate response to perturbations in arterial pressure. This
finding is consistent with a recent report from Masilamani and Heesch which demonstrates
THP alters renal sympathetic nerve responses but not heart rate responses to changes in
arterial pressure obtained using graded continuous infusions of phenylephrine and
nitroprusside (179).
In the previous experiment the ewes were ovary-intact, anestrous, adults, but in
the current study chronically ovariectomized ewes were used. Evidence collected by other
investigators suggests the rapid pressure response to progesterone may be attenuated in
the ovariectomized animal. A recent study by Heesch et al. (157) demonstrated the effect
of THP on the baroreflex is abolished in the ovariectomized rat. The response to THP
may be altered in the ovariectomized rat since estrogens regulate GABAa receptor subunit
composition (99). To determine if the time response to progesterone decreases with time

83
after ovariectomy, the responses of the animals studied beginning one week after
ovariectomy (acutely ovariectomized ewes) were analyzed separately from the responses
of the animals studied beginning four months after ovariectomy (chronically
ovariectomized ewes). The findings of the study were not changed when the acutely and
chronically ovariectomized ewes were analyzed. This suggests that if ovariectomy
modifies the ability of progesterone to rapidly reduce arterial pressure, the response is
modified approximately one week after ovariectomy. Previous studies also have
demonstrated a reduction in 5a-reductase activity in the ovariectomized rat (153).
Therefore, if 5a-reductase activity also is reduced in the ovariectomized ewe, the ability of
progesterone to rapidly reduce arterial pressure may be compromised in this model.
However, chronic progesterone treatment (2 weeks) decreases blood pressure in
ovariectomized ewes, suggesting progesterone does not only modify blood pressure
through GABAa receptor mechanisms.
In summary, infusion of progesterone at a rate of 3 pg/kg/min rapidly reduces
arterial pressure in ovariectomized, estradiol-treated ewes when baseline pressure is
slightly elevated. Progesterone does not alter blood volume or the heart rate response to
perturbations in arterial pressure after thirty minutes or four hours. Further study is
needed to determine the effect of progesterone on arterial pressure when infusion is begun
at different starting pressures.

84
O 15 30 45
Time (min)
105 -i
C
o>
X
E
E
CL
<
100 -
95 -
90 -
85 J
0 15 30 45
Time (min)
105
o>
x
E
E
CL
<
100
95
90
85
Time (min)
Figure 5.1. Effect of 30-minute progesterone infusions on plasma progesterone
concentration and MAP. A) Plasma progesterone concentration after 30-minute infusions
of vehicle (open bar, n=8), 1.5P4 (horizontal stripe bar, n=8), and 3P4 (diagonal stripe bar,
n=8). B) MAP during 30-minute infusions of vehicle (B, n=8), 1.5P4 (C, n=8), and 3P4
(D, n=8). Data are means ± SE.

Evans Blue (O.D.) Evans Blue (O.D.)
85
Figure 5.2. Representative Evans blue dye elimination curves from ewe Y254. Plasma
optical density decreases linearly over time during 30-minute infusions of vehicle (A) or
progesterone at rates of 1.5P4 (B) and 3P4(C).

86
Figure 5.3. Plasma volume after 30-minute infusions of vehicle (open bar, n=8), 1.5P4
(horizontal stripe bar, n=8), and 3P4 (diagonal stripe bar, n=8). Data are means ± SE.

87
Table 5.1. Logistic baroreflex curve parameters obtained after 4-hour infusions of vehicle
or progesterone.
Experimental Group
Vehicle (n=8)
1.5P4 (n=8)
3P4 (n=8)
6P4 (n=8)
Resting MAP,
mmHg
79.6 ±6.1
77.2 ±6.3
81.3 ±8.1
82.3 ±5.0
Resting heart period,
ms
804.5 ±71.2
701.7 ±80.8
788.1 ±95.7
795.0 ± 110.8
Heart period range,
ms
683.6 ± 108.4
755.7 ± 115.7
646.9 ± 110.2
645.2 ± 107.9
Slope, Ams/AmmHg
-0.92 ±0.80
-1.14 ± 0.69
-0.63 ±0.37
-0.40 ±0.16
MAP at inflexion
point, mmHg
89.2 ±5.7
91.6 ± 4.6
92.7 ±3.3
90.0 ±5.5
Heart period
minimum, ms
506.4 ±32.2
561.0 ±50.5
501.3 ±45.1
484.6 ±48.2
Maximum gain,
Ams/AmmHg
85.5 ±65.3
265.5 ± 174.0
77.8 ±45.7
51.9 ± 19.2
Threshold pressure,
mmHg
67.1 ± 16.6
81.8 ± 5.7
82.3 ±5.9
80.6 ±4.3
Saturation pressure,
mmHg
111.3 ± 12.0
101.4 ±5.9
103.1 ±4.2
99.4 ±8.1
Data are means ± SE.

88
40 -T
35 -
30 -
I
Figure 5.4. Plasma progesterone concentration after 4-hour infusions of vehicle (open
bar, n=8), 1.5P4 (horizontal stripe bar, n=8), 3P4 (diagonal stripe bar, n=8), and 6P4
(lattice bar, n=8). Data are means ± SE.

Mean Arterial Pressure (mmHg)
89
Time (min)
Figure 5.5. MAP during 4-hour infusions of vehicle (A, n=8), 1.5P4 (B, n=8), 3P4 (C,
n=8) and 6P4 (D, n=8). Data are means ± SE.

90
Figure 5.6. Plasma volume after 4-hour infusions of vehicle (open bar, n=8), 1.5P4
(horizontal stripe bar, n=8), 3P4 (diagonal stripe bar, n=8), and 6P4 (lattice bar, n=8).
Data are means ± SE.

Heart Period (ms)
91
Mean Arterial Pressure (mmHg)
Figure 5.7. Representative baroreflex curves from ewe Y258 after infusions of vehicle
(A), 1.5 P4 (B), 3P4 (C), and 6P4 (D).

CHAPTER 6
CHARACTERIZATION OF 5a-REDUCTASE ACTIVITY IN THE LIVER AND
BRAINSTEM OF THE NON-PREGNANT, PREGNANT, AND OVARIECTOMIZED
EWE
6.1 Introduction
Progesterone can be sequentially metabolized (Figure 6.1) by 5a-reductase and
3a-hydroxysteroid dehydrogenase in the brain and periphery to sequentially form
dihydroprogesterone (DHP) and tetrahydroprogesterone (THP). As discussed in Chapters
2,4, and 5, THP is a GABAA-active metabolite that has been proposed to contribute to
progesterone’s ability to rapidly reduce arterial pressure in the ovary-intact, adult ewe.
Karavolas and colleagues have previously reported a tissue specific regulation of 5a-
reductase and 3a-hydroxysteroid dehydrogenase activities by ovarian steroids in rats (11,
153, 163). This study was designed to compare activity of the enzymes responsible for
formation of GABAA-active progesterone metabolites in non-pregnant, pregnant
(approximately 120 days gestation), and ovariectomized (65 days) ewes. Changes in the
activity of the 5a-reduction pathway during pregnancy or in the absence of the ovaries
may alter the ability of progesterone to induce rapid physiological effects. In this study,
the liver was assayed since this is the primary peripheral site of steroid metabolism and the
brainstem was assayed since this is the site at which GABAA-active neurosteroids are
hypothesized to modify cardiovascular function.
92

93
6 2 Methods
Liver and brainstem microsomal preparations were prepared from tissues obtained
from 5 non-pregnant (ovary-intact, anestrous), 5 pregnant (approximately 120 days of
gestation), and 5 ovariectomized (65 days) ewes. The microsomal preparations were
incubated with tritiated progesterone (3HP4) for thirty minutes. The resulting steroid
metabolites were extracted from the preparation using anhydrous ethyl ether and were
resolved on thin layer chromatography. Steroid bands corresponding to progesterone,
dihydroprogesterone (DHP), and tetrahydroprogesterone (THP) were scraped from the
chromatography plates and the radioactivity of each band was counted in a scintillation
counter. Conversion of 3HP4 to DHP and THP was calculated as the percentage of total
radioactivity recovered from the three steroid bands in each lane.
The effect of reproductive state on enzyme activity was analyzed Kruskal-Wallis
one way analysis of variance on ranks since the data sets were not normally distributed.
Individual means were compared by a Student-Newman-Keuls post-hoc test. The null
hypothesis was rejected when p < 0.05. Data are expressed as the mean ± standard error
of the mean (SEM).
6.3 Results
In the blank incubations, THP and DHP carried 22.1± 6.2 % and 0.89+ 0.31 % of
total counts respectively. These values represent background activity and were subtracted
from all calculated conversion rates.

94
3a-hydroxysteroid dehydrogenase activity was observed in microsomal
preparations prepared from the livers of pregnant, non-pregnant, and ovariectomized ewes
as exhibited by the conversion of 3HP4 to THP (Figure 6.2A). There was a significant
effect of reproductive state on conversion to THP, and all groups were significantly
different from each other. The mean percentage of P4 conversion to THP was highest in
the non-pregnant ewe (56.8 ±1.1) and lowest in the ovariectomized ewe (23.8± 3.8).
Conversion to THP was intermediate in the pregnant ewe (35.5 ± 1.6) and was
significantly less than in the non-pregnant ewe
There also was a significant effect of reproductive state in the conversion of P4 to
DHP in the liver (Figure 6.2B). The mean percentage of conversion of P4 into DHP in the
pregnant, non-pregnant and ovariectomized ewe was 23.9 ± 0.7, 7.2 + 0.8, and 15.2 ± 1.7,
respectively. All groups were significantly different from each other.
There was no detectable conversion of P4 to THP in the brainstem, suggesting an
absence of 3a-hydroxysteroid dehydrogenase activity. However, there was a significant
effect of reproductive state on conversion of P4 to DHP by 5a-reductase in the brainstem
(Figure 6.3). The mean percentage of conversion to DHP in the pregnant, non-pregnant,
and ovariectomized ewe was 21.0 ± 2.6, 35.3 ± 1.4, and 41.7 ± 3.7, respectively. The
pregnant ewe converted significantly less P4 to DHP than the non-pregnant and
ovariectomized ewe. In contrast to the liver, the ovariectomized ewe demonstrated the
highest level of 5a-reductase activity in the brainstem.

95
6.4 Discussion
The results of this study suggest reproductive state influences metabolism of
progesterone into DHP and the GABAA-active metabolite, THP. In the rostral ventral-
lateral medullary portion of the brainstem, GABAergic neurons inhibit excitatory amino
acid projections to pre-ganglionic sympathetic cell bodies in the intermediolateral cell
column and these sympathetic cell bodies are the source of sympathetic outflow to the
heart and vascular system. Therefore, regulation of the activity of the 5a-reductase, 3a-
hydroxysteroid dehydrogenase pathway may be an important mechanism contributing to
modulation of sympathetic outflow to the cardiovascular system.
In the brainstem, conversion to DHP was approximately equal in the non-pregnant
and ovariectomized ovine brainstem and was significantly lower in the pregnant brainstem.
This finding suggests activity of the rate-limiting enzyme (5a-reductase) in this metabolic
pathway decreases in the brainstem of pregnant ewes. Conversion of 3HP4to THP was
undetectable in microsomal preparations supplemented with NADPH. Studies of the rat
5a-reductase, 3a-hydroxysteroid dehydrogenase pathway (see figure 6.1) demonstrate
there is a particulate isoform of 3a-hydroxysteroid dehydrogenase that can be isolated in
microsomal preparations (122). However, this enzyme actually reacts predominately in
the oxidative direction (converts THP to DHP) and requires NAD+ as a cofactor (122).
The isoform of 5a-reductase which favors reduction is actually located in the cytosol and
is NADPH-dependent (122). These observations suggest 3a-hydroxysteroid
dehydrogenase activity may be detectable in ovine brainstem cytosols supplemented with
tritiated DHP and NADPH. Further study is needed to accurately characterize the effect if

96
reproductive state on the ovine brainstem 5oc-reductase, 3a-hydroxysteroid
dehydrogenase pathway.
Both 5a-reductase and 3a-hydroxysteroid dehydrogenase activity was detected in
ovine liver microsomes supplemented with NADPH. Since studies in the rat demonstrate
the reductive form of 3a-hydroxysteroid dehydrogenase is located in the cytosol (122), it
is unclear why conversion to THP was observed. The liver may contain more
concentrated enzymatic activity and, as a result, the cytosolic enzymes may be more easily
extracted intro the microsomal preparations. Conversion to THP was highest in livers
obtained from non-pregnant ewes, intermediate in livers obtained from pregnant ewes, and
lowest in livers obtained from ovariectomized ewes. This suggests that for a given
concentration of progesterone, significantly more progesterone would be metabolized to
THP in the non-pregnant ewe than in the ovariectomized ewe. If reproductive state
influences brainstem enzyme activity in the same manner, these results suggest the absence
of a rapid pressure response to progesterone in the 4-hour protocol presented in Chapter 5
may be partially attributable to reduced metabolism to GABAA-active THP in
cardiovascular-regulating brain areas in the ovariectomized ewe.
Although activity of the 5a-reductase, 3a-hydroxysteroid dehydrogenase pathway
appears to be decreased in the both the brainstem and liver of the pregnant ewe, the
pregnant ewe would still be expected to generate more GABAA-active progesterone
metabolites since starting substrate (progesterone) increases during pregnancy. In fact,
the tendency towards decreased enzymatic activity during pregnancy may be an adaptation
to prevent excessive formation of GABAA-active progesterone metabolites in the presence
of increased concentrations of precursor progesterone.

97
It is interesting to speculate peripheral steroid metabolism contributes to the
formation of centrally active GABAA-modulating neurosteroids since steroids are highly
lipophilic and can easily cross the blood brain barrier. However, neurosteroids are
typically found in the brain at concentrations many times higher than those found in the
plasma (176), suggesting that centrally active neurosteroids are predominately synthesized
in the brain. It is unclear what role, if any, peripherally derived neurosteroids play in the
formation of centrally active metabolites. Many studies have demonstrated central effects
of peripherally administered neurosteroids and neurosteroid precursors (176). Future
studies which investigate the ability of peripherally administered neurosteroid precursors
to induce central effects in the presence of centrally (intracerebroventricular) administered
metabolism inhibitors may provide insight into the physiological role of peripheral
metabolism.
In summary, the results of this study suggest ovarian steroids regulate metabolism
of progesterone into GABAa active metabolites. Differential regulation of this enzyme
system may be one mechanism by which the physiological effects of progesterone are
modulated. However, further study is needed to accurately characterize the ovine 5a-
reductase, 3a-hydroxysteroid dehydrogenase pathway and the regulation of each enzyme
by ovarian steroids. A better understanding of this enzyme system may provide insight
into the mechanisms by which progesterone induces its physiological effects.

98
5a-reductase
3a-hydroxysteroid
dehydrogenase
Figure 6.1. Progesterone (P4) is metabolized by 5a -reductase to form
dihydroprogesterone (DHP) and 3a-hydroxysteroid dehydrogenase to form
tetrahydroprogesterone (THP), the GABAA-active metabolite. 5a -reductase is the rate-
limiting enzyme for formation of THP. 5a -reductase only exhibits reductive activity and
uses NADPH as a cofactor. There are multiple isoforms of 5a -reductase, but all isoforms
are located in microsomes. The also are multiple isoforms of 3a -hydroxysteroid
dehydrogenase. One isoform is located in the cytosol, reacts predominately in the
reductive direction and uses NADPH as a cofactor. Another isoform is particulate and
can be isolated in the microsomal fraction but reacts predominately in the oxidative
direction and uses NADH as a cofactor. This metabolic pathway is described in a review
prepared by Karavolas and Hodges (122).

99
Figure 6.2. Conversion of 3HP4 into GABAa active metabolites in the liver microsomes
obtained from non-pregnant (diagonal stripe bar, n=5), pregnant (diagonal crosshatch bar,
n=5), and ovariectomized (open bar, n=5) ewes. A) Percent conversion to THP. B)
Percent conversion to DHP. Data are means ± SE. *Significantly different from values
obtained in non-pregnant and ovariectomized ewes. *#Significantly different from values
obtained in non-pregnant and pregnant ewes.

100
Figure 6.3. Conversion of 3HP4 into DHP in brainstem microsomes obtained from non¬
pregnant (diagonal stripe bar, n=5), pregnant (diagonal crosshatch bar, n=5), and
ovariectomized (open bar, n=5) ewes. * + Significantly different from values obtained in
non-pregnant and ovariectomized ewes (p<0.05).

CHAPTER 7
CHARACTERIZATION OF BRAIN MINERALOCORTICOID AND
GLUCOCORTICOID RECEPTOR AVAILABILITY IN THE NON-PREGNANT AND
PREGNANT EWE
7.1 Introduction
The setpoint and response of the HPA axis is altered during ovine pregnancy
(133). Glucocorticoids regulate activity of the HPA axis via a binary gene-regulating
corticosteroid receptor system, and this system consists of a high affinity, low capacity
MR and a lower affinity, higher capacity GR (21, 62). Associated heat-shock proteins
maintain intracellular steroid-binding transcription factors in a steroid-binding competent
state (270). When a steroid binds its receptor, the heat shock proteins dissociate and the
steroid bound to the activated steroid-receptor complex no longer freely dissociates. As a
result of this property, steroid receptor radioligand-binding assays only estimate the
amount of unactivated receptor present in an assayed tissue. Reul and de Kloet (226)
used this characteristic of MR and GR to determine the role of the binary corticosteroid
receptor system in responding to circadian- and stress-induced activity of the axis. They
measured available MR or GR in the hippocampus of adrenal-intact and adrenalectomized
rats at different points in the circadian rhythm and after stress and demonstrated the MR is
almost fully occupied even when plasma glucocorticoids are lowest (at the trough of the
HPA axis rhythm). Higher plasma glucocorticoid levels (the peak of the rhythm and after
stress) fully saturate the MR and activate the GR. This finding suggests corticosteroids
101

102
exert their effects through the MR when circulating concentrations are low, but when
corticosteroid secretion is increased during stress and at the peak of the rhythm, GR is
activated.
The setpoint and response of the cardiovascular system also is altered during ovine
pregnancy (133). Since MR and GR also modulate cardiovascular function (89, 282),
these receptors may be involved in adaptation of both the HP A axis and the cardiovascular
system to gestation.
This study was designed to compare saturation binding (Bmx) of MR and GR in
the non-pregnant and pregnant ewe. The kidney, hypothalamus, pituitary, and
hippocampus were evaluated. The hypothalamus, pituitary, and hippocampus were
analyzed since these are the major HPA axis feedback sites. Changes in MR and/or GR
regulation at these sites during pregnancy could contribute to differential regulation of the
HPA axis activity during pregnancy. The kidney was analyzed as a control peripheral
tissue known to include both MR and GR. Since circulating concentrations of cortisol and
aldosterone are elevated during ovine pregnancy (9), availability of both the MR and GR
should decrease in the pregnant ewe.
7,2 Methods
Tissues (kidney, hypothalamus, pituitary, and hippocampus) were obtained from
five ovary-intact, anestrous, non-pregnant and five pregnant ewes (average gestational age
= 120/145 days) and were prepared and assayed as described in Chapter 3. Briefly,
cytosolic aliquots were incubated in the presence of a saturating (25 nM) concentration of
radiolabeled cortisol to determine total corticosteroid binding. MR availability was

103
determined by incubating radiolabeled cortisol in the presence of 50-fold excess of the
specific GR agonist RU 28362. Nonspecific binding was determined in the presence of a
500-fold excess of unlabeled dexamethasone (a MR and GR ligand). Samples were eluted
through Sephadex LH-20 chromatography columns to separate bound from free. Binding
to GR was calculated from the difference between total specific cortisol binding and
specific cortisol binding in the presence of excess RU 28362. Specific binding was
expressed as fmol/mg protein.
Since data sets were not normally distributed, saturation binding was compared
using a Mann-Whitney rank sum test. The null hypothesis was rejected when p < 0.05.
Data are expressed as the mean ± standard error of the mean (SEM).
7,3 Results
Pregnancy did not significantly alter total corticosteroid binding in any of the
tissues studied. GR binding was measured in the hippocampus, kidney, hypothalamus, and
pituitary of non-pregnant and pregnant ewes (Figure 7.1 A). There was a significant
decrease in hippocampal GR binding in the pregnant ewe (n=5, 14.8 ± 8.7 fmol/mg
protein) compared to the non-pregnant ewe (n=5, 46.3 ± 17.4 fmol/mg protein). GR
binding averaged 39.9 + 7.9 fmol/mg protein in kidneys obtained from non-pregnant ewes
(n=4) and was not significantly different in kidneys obtained from pregnant ewes (n=5,
28.4 ± 6.4 fmol/mg protein). GR binding averaged 43.1 ±7.1 fmol/mg protein in
hypothalamic cytosols obtained from non-pregnant ewes (n=5) and was not significantly
different in hypothalamic cytosols obtained from pregnant ewes (n=5, 25.7 ± 8.9 fmol/mg
protein). One outlying hypothalamic GR binding value was eliminated from the data set

104
obtained from pregnant ewes (656.2 fmol/mg protein) after applying a test for an extreme
mean. In pituitary cytosols obtained from non-pregnant ewes (n=4) GR binding averaged
60.2 ± 10.2 fmol/mg protein (after omitting an outlying value of 176.6 fmol/mg protein).
GR binding was not significantly different in pituitary cytosols obtained from pregnant
ewes (n=5, 87.9 ± 14.4 fmol/mg protein after omitting an outlying value of 650.7 fmol/mg
protein).
MR binding was measured in cytosols prepared from the hippocampus, kidney,
hypothalamus, and pituitary of non-pregnant ewes (Figure 7. IB). MR binding capacity
significantly increased from undetectable levels in the hippocampus of non-pregnant ewes
(n=5) to 2.8 ±1.6 fmol/mg protein in pregnant ewes (n=5). There was a tendency
towards increased MR availability in all other tissues assayed. MR binding averaged 5.5 ±
0.5 fmol/mg protein in kidney cytosols obtained from non-pregnant ewes (n=4) and was
8.2 ± 3.7 fmol/mg protein in kidney cytosols obtained from pregnant ewes (n=5). In
hypothalamic cytosols obtained from non-pregnant ewes (n=4), MR binding averaged 4.0
± 2.3 fmol/mg protein and was not significantly different in hypothalamic cytosols
obtained from pregnant ewes (n=7, 5.7 ±3.2 fmol/mg protein). MR binding averaged 4.3
±1.5 fmol/mg protein in pituitary cytosols obtained from non-pregnant ewes (n=6) and
was not significantly different in pituitary cytosols obtained from pregnant ewes (n=4, 4.9
± 2.9 fmol/mg protein after omitting an outlying value of 43.4 fmol/mg protein).
7,4 Discussion
This study demonstrates MR binding capacity increases and GR binding capacity
decreases in the hippocampus of the pregnant ewe. Steroid receptors exist in two forms:

105
an unactivated form available for steroid activation and a bound, activated form that is
incapable of exchanging steroid in a radioligand binding assay. As a result, saturation
point radioligand binding assays such as this one provide a measure of unactivated or
"available" receptors and do not reveal information about receptors already activated by
endogenous steroid.
Binding curves demonstrate the available MR becomes about 50% saturated at
0.5nM cortisol, a typical concentration of free plasma cortisol in the non-pregnant ewe.
During pregnancy, free plasma cortisol would be expected to approximately double (~1
nM) and contribute to decreased MR availability, but this increase in plasma cortisol
would still not approach the 50% saturation point for GR (~5 nM). Therefore, one would
predict decreased radioligand binding to the MR and perhaps decreased binding to the GR
in the presence of the higher levels of circulating corticosteroids found during pregnancy.
The decrease in GR availability observed during ovine pregnancy is consistent with
increased receptor activation due to elevated circulating cortisol. However, the tendency
towards increased MR availability is contradictory to what would be expected in the
presence of elevated corticosteroids. These results suggest there is a shift from complete
MR activation at physiological corticosteroid levels in non-pregnant ewes to increased GR
activation and decreased MR activation in the pregnant ewe. Differential modulation of
corticosteroid receptors may be one mechanism contributing to adaptation of the
cardiovascular system and the HPA axis to pregnancy. However, the precise contributing
mechanisms and the physiological relevance of these receptor changes remain unclear.
This study demonstrates the hypothalamus and pituitary of the ewe actually
contain more available MR than the hippocampus. In contrast, MR is concentrated in the

106
rodent hippocampus and nearly undetectable in the hypothalamus and pituitary (226), but
MR binding is essential in feedback regulation of the HPA axis (22). These findings have
lead some investigators to propose MR-mediated feedback inhibition of the rat HPA axis
requires corticosteroid binding in the hippocampus or other brain regions outside of the
hypothalamus and pituitary. Since the ovine hypothalamus and pituitary contain both
available MR and GR, these sites may respond to both small and large increases in
corticosteroid concentrations while the hippocampus may only respond when
corticosteroid concentrations are increased to levels high enough to activate the GR.
The dog hypothalamus and pituitary also contain high concentrations of MR (227).
Like the sheep, the dog also secretes cortisol as the primary glucocorticoid and both
species lack a circadian rhythm in HPA axis activity (9, 141). Since the sheep and dog
lack an HPA axis circadian rhythm and basal pituitary and adrenal output without
hypothalamic drive accounts for basal activity of the HPA axis (56), neural drive (and
hippocampal influence) may be reserved for stress-induced activation in these species.
Indeed, a study by Dallman and colleagues suggests the pituitary is the primary site for
feedback in the dog since cortisol and dexamethasone inhibit the response both
hypoglycemia and ovine CRF (135). The pituitary may also be the primary feedback site
in the sheep. A recent study by de Kloet and colleagues suggests hippocampal GR
actually increase activity of the axis presumably by “disinhibiting” the axis (284).
Together, these findings make it interesting to speculate that the hippocampus plays a
more important role in regulating the axis when activity of the axis is increased (the peak
of the rhythm in the rat and perhaps during pregnancy in the sheep and other species).

107
At this point, it can not be concluded whether the observed changes in MR and GR
availability in the hippocampus of the pregnant ewe reflect a response to changes in
circulating glucocorticoid levels, an adaptation which drives differential activity of the
axis, or a combination of the two possibilities. Based on what we know about the affinity
of the MR and GR and circulating glucocorticoid concentrations during pregnancy, it
seems likely that the increase in MR availability reflects a modification of receptor
expression. In contrast, the decrease in GR availability could be either a response to
increased circulating glucocorticoids or a modification of receptor expression. Further
study is needed to determine the role of the hippocampus in basal, stimulated and
pregnancy-associated HPA axis activity in the sheep. If the ovine hippocampus proves to
regulate HPA axis activity further study will be required to determine the role of
hippocampal MR and GR and the mechanisms contributing to the observed changes in
receptor availability.
The cell exerts multiple levels of control on corticosteroid receptors, and altered
modulation at any of these levels may contribute to the observed changes in hippocampal
MR and GR availability. Corticosteroid receptors may be transcribed, translated, spliced,
activated, or degraded differently in the pregnant state. Further study is needed to clarify
which levels of corticosteroid receptor regulation contribute to the observed changes in
MR and GR and availability. The increased levels of ovarian steroids present during
pregnancy may alter MR and GR binding capacity by modulating any of these levels of
control. In fact, the effect of ovarian steroids on corticosteroid receptor binding has been
previously examined by several investigators (271).

108
Koch and colleagues (151) first demonstrated a potential effect of ovarian steroids
on corticosteroid receptor binding capacity when they observed increased MR binding in
the hippocampus of female compared to male Wistar-Kyoto normotensive rats. However,
the results of this study left it difficult to conclude a gender difference in MR binding since
only one Scatchard plot was generated for each experimental group and the relationship
between male and female binding capacity was reversed in spontaneously hypertensive
rats. In more extensive studies, Turner and colleagues demonstrated total corticosteroid
binding differs between genders in a region specific fashion: capacity was increased in the
hippocampus (276) and decreased in the hypothalamus and pituitary of female compared
to male rats (272, 276). Region specific differences also were demonstrated when MR
and GR binding were distinguished: there were not any gender differences in binding
capacity in the hippocampus (273), but MR binding was increased in the pituitary (274) of
female rats. In those studies, gender differences in MR or GR binding capacity in the
hippocampus may have been masked by failure to synchronize the estrous cycles and/or
the time of day of sacrifice (105, 106). Overall, these comparisons between genders of
corticosteroid receptor binding capacity support the possibility that ovarian steroids
contribute to the increased hippocampal MR capacity observed in this study.
Several studies in ovariectomized-adrenalectomized rats have demonstrated
chronic subcutaneous estrogen replacement (4-10 days) has region specific effects on
corticosteroid binding. Estrogens appear to decrease total corticosteroid binding in both
the pituitary and hippocampus (272, 273). In the pituitary, this decrease appears to be
attributable to a decrease in MR capacity (74, 75, 272). In the hippocampus, the decrease
in total binding capacity may be partially attributable to a decrease in GR binding (273).

109
However, it is unclear whether estradiol increases (75) or decreases (36) MR binding.
These findings suggest the high levels of circulating estrogens present during pregnancy
may contribute to the observed changes in hippocampal MR and GR availability.
Chronic treatment with progesterone alone decreases total corticosteroid receptor
binding in the hippocampus (238), and this effect appears to be selective for the MR (36).
However, when given in combination with estradiol, in a very carefully designed study by
Carey and colleagues (35), progesterone opposed the tendency of estradiol to decrease
MR binding. Therefore, these studies suggest that, in the presence of high circulating
estrogens, progesterone may contribute to the increase in MR binding capacity observed
during pregnancy, but progesterone probably does not modulate GR.
In summary, binding capacity of MR and GR are differentially regulated in the
hippocampus during ovine pregnancy. These changes may contribute to adaptation of the
HPA axis and the cardiovascular system to pregnancy. Currently available evidence
suggests ovarian steroids may contribute to these changes; estrogen may decrease GR
availability and the combination of estrogen and progesterone may increase MR binding
capacity.

110
MR Availability
GR Availability
Figure 7.1. Maximal corticosteroid receptor binding capacity in non-pregnant (open bars)
and pregnant (solid bars) ewes. A) MR availability. B) GR availability. Hipp =
hippocampus, Kid = Kidney, Hyp= hypothalamus, Pit = pituitary. Data are meansiSE.
* p<0.05.

CHAPTER 8
EFFECT OF PREGNANCY ON THE APPARENT BINDNG AFFINITY OF THE
MINERALOCORTICOID AND GLUCOCORTICOID RECEPTOR
8.1 Introduction
Progesterone binds the MR with an extremely high affinity (233) and is a potent
anti-mineralocorticoid (158, 254, 287). These findings suggest the high circulating levels
of progesterone present during pregnancy may act as a competitive antagonist, antagonize
in vivo activation of the MR by circulating corticosteroids and contribute to the observed
increase in hippocampal MR availability. Indeed, Carey and colleagues (36) have
demonstrated a decrease in the apparent binding affinity of MR in hippocampal cytosols
obtained from rats four hours after injection with progesterone.
Although most evidence characterizes progesterone as an anti-mineralocorticoid,
progesterone exhibits partial agonist activity in cell cultures transfected with the human
MR and a MR-responsive reporter gene (233). This finding suggests that if circulating
progesterone concentrations are high enough, progesterone may actually activate the
receptor and activated receptors would not be detectable in an in vitro ligand-binding
assay.
Progesterone also interacts with the GR with a lower affinity (42, 183, 233), and
appears to have partial agonistic activity at this receptor (233). This suggests
progesterone may activate GR and contribute to the decreased GR availability observed in
the hippocampus during ovine pregnancy.
Ill

112
There is also in vivo evidence that progesterone acts as an endogenous antagonist
of cortisol actions in the hippocampus, hypothalamus, and pituitary to inhibit the feedback
control of the axis, and contribute to the increase in setpoint observed during pregnancy.
High concentrations of progesterone antagonize glucocorticoid inhibition of (3-endorphin
(a protein processed from the same precursor molecule as ACTH) secretion in pituitary
cultures (1), of corticotropin-releasing activity from isolated hypothalami (118), and of
ACTH in vivo (66). Similarly, an acute infusion of physiological concentrations of
progesterone antagonizes the feedback effects of cortisol in the non-pregnant ewe (136).
This study was specifically designed to test for evidence of the presence of an
endogenous competitive MR antagonist during pregnancy. If an endogenous competitive
MR antagonist is present in cytosols obtained from pregnant ewes, the apparent binding
affinity of the MR should be reduced in these tissues. To test this hypothesis, the
dissociation constant (KD) for 3H-cortisol binding to both the MR and GR was determined
in pituitary cytosols obtained from non-pregnant and pregnant ewes. The pituitary was
assayed since, in the previous study, the pituitary exhibited the highest level of 3H-cortisol
binding per mg of tissue and provides the strongest signal for measurement of binding
over a wide concentration range.
8,2 Methods
Pituitary samples from non-pregnant and pregnant ewes were prepared and
assayed as detailed in Chapter 3. Cytosols from 1-3 animals were pooled to obtain
enough tissue for each assay. Binding curves were created from four different pools of

113
pituitary cytosols obtained from non-pregnant and four different pools of pituitary cytosols
obtained from pregnant ewes (approximately 120 days of gestation).
To determine total corticosteroid binding, cytosols were incubated in the presence
of a range of 3H-cortisol concentrations (0. l-25nM). At each concentration, MR
availability was determined by incubating 3H-cortisol in the presence of 50-fold excess of
the specific GR agonist RU 28362 and nonspecific binding was determined in the presence
of a 500-fold excess of unlabeled dexamethasone (a MR and GR ligand). Each incubation
point was performed in duplicate. GR binding was calculated and bound was separated
from free as detailed in Chapter 3.
The results of each binding curve were fit to a 3 parameter Hill formulation (B =
FNxBmax/(KD+FN)) using SigmaPlot® (Jandel Scientific). In this equation, B represents
specific binding at a given concentration of free ligand (F). B^x represents the maximal
binding capacity, Kd represents the dissociation constant and N is the Hill coefficient. The
fit parameters were compared by a Mann-Whitney rank sum test since the data sets were
not normally distributed. Data are expressed as the mean ± standard error of the mean
(SEM). The null hypothesis was rejected when p < 0.05. The computer program
SigmaPlot® was used to generate average binding curves from the average fit parameters.
8,3 Results
Average MR binding parameters are presented in Table 8.1, and average GR
binding parameters are presented in Table 8.2. When compared to non-pregnant ewes,
pregnant ewes did not exhibit any significant differences in any of the binding parameters.

114
Average MR binding curves are illustrated in Fig 8.1. There was a tendency
towards a decrease in MR binding affinity in pituitary cytosols obtained from pregnant
ewes, but this was not significant. Average GR binding curves are illustrated in Fig 8.2.
Maximal GR binding tended to decrease in pituitary cytosols obtained from pregnant ewes
as would be expected to occur in the presence of high concentrations of circulating
corticosteroids, but there were no significant differences between the average non¬
pregnant and pregnant GR binding curves.
8.4 Discussion
The results of this study fail to demonstrate a significant change in the KD of the
pituitary MR or GR during pregnancy, but suggest a tendency towards decreased affinity
of the MR. This tendency towards a decreased MR affinity could be due to the presence
of an endogenous competitive MR antagonist such as progesterone. Since a pool of
tissues obtained from one to three animals was used in each determination of KD, plasma
progesterone concentration and binding affinity were not obtained from individual animals
and the two parameters could not be correlated. If each binding assay were conducted in
an individual animal, a correlation of apparent affinity with in vivo progesterone level may
reveal an effect of progesterone on the apparent binding affinity of the MR.
Previous studies have attempted to determine the effect of in vivo progesterone
treatment on MR binding affinity in rats (197, 271). One study failed to demonstrate an
effect of progesterone on the apparent affinity of the receptor (275) while another study
suggests in vivo progesterone does indeed reduce the KD of the MR in rats (35, 36). The
contradictory findings of these two studies may be explained by the degree of

115
progesterone metabolism that occurs between the last progesterone injection and time of
sacrifice of the animal (271). In the study by Carey and colleagues (36), progesterone
levels were not measured, but rats were sacrificed only four hours after the last
progesterone injection when plasma levels should have been very high and the brains were
not perfused prior to sacrifice. The current study failed to demonstrate a significant
reduction in MR affinity in pituitary cytosols obtained from pregnant ewes with
continuously elevated progesterone levels. A correlation between pre-sacrifice
progesterone level and MR binding affinity may provide a better method for evaluating the
effect of in vivo progesterone on MR binding affinity. Moreover, it may be necessary to
more closely reproduce the methods of Carey and colleagues (36) and conduct binding
studies in cytosols obtained from unperfused brains.
Progesterone also may modify MR affinity in a region specific manner. For
example, McEwen and colleagues have observed MR and GR in the hippocampus are
much more sensitive to activation by low basal corticosteroid levels than the MR and GR
in the pituitary (255). Similarly, the hippocampus may be more sensitive to physiological
concentrations of progesterone, and apparent MR affinity may actually be reduced during
ovine pregnancy in the hippocampus, a tissue associated with decreased MR availability.
However, the low total MR availability in the hippocampus of the non-adrenalectomized
ewe makes it impossible to measure the affinity of the receptor in this brain region. Future
studies in adrenalectomized, pregnant ewes could begin to address the effect of pregnancy
and progesterone on MR affinity since MR availability should increase in the hippocampus
of the adrenalectomized ewe replaced with low levels of aldosterone and cortisol.

116
Studies also have demonstrated progesterone interacts with the GR via a unique
binding domain (261), increases the rate of dissociation of glucocorticoids from the
receptor (42), and exhibits anti-glucocorticoid effects (293). These findings suggest the
elevated progesterone levels found during ovine pregnancy also may decrease the apparent
affinity of the GR. However, this study failed to demonstrate an effect of progesterone on
the affinity of the GR. Some evidence suggests progesterone is actually a fairly effective
agonist at the GR (233). Therefore, progesterone may actually activate the GR in vivo
and contribute to the observed decrease in GR availability in the hippocampus during
ovine pregnancy.
Interestingly, the results of this study suggest there is little difference between the
affinity of the MR and the GR for 3H-cortisol in both the non-pregnant and pregnant ewe.
Turner and Ansari (274) have previously reported the affinity of the MR and GR for 3H-
dexamethasone is different in the male rat hypothalamus but the Kd of the MR is reduced
in the female rat hypothalamus and more closely resembles the KD of the GR. The study
by Carey et al. (36) suggests this decrease in MR affinity may be due to the presence of
higher circulating progesterone levels in the female. Since progesterone has an
exceptionally high affinity for the MR (233), it is possible that the progesterone levels
found in the non-pregnant, luteal-phase female are sufficient to maximally reduce the KD
of the MR. Indeed, plasma progesterone levels approach 10 nM in the cycling ewe (9),
and this concentration may be as much as 1000 times the K¡ of progesterone for the MR
(233). Since the tissues used in this study were pooled and the estrous cycles of the non¬
pregnant ewes were not synchronized, it is difficult to determine the precise relationship
between MR affinity and progesterone level. The very high levels of progesterone found

117
during ovine pregnancy (~60 nM) may not antagonize the MR any more than luteal-phase
progesterone levels.
In summary, this study suggests there is a tendency towards decreased MR affinity
in the ovine pituitary during pregnancy, but this difference is not significant. Further study
is needed to investigate potential regional differences in MR affinity during pregnancy and
to correlate actual plasma progesterone levels with apparent MR binding affinity.

118
Table 8-1. Average MR Binding Parameters.
MR Binding Parameters
Kd (n=4)
Bmax (n=4)
N(n=4)
Non-pregnant
1.5 ±0.4
2.2 ± 1.2
5.4 ±4.1
Pregnant
2.2 ±0.5
2.1 ± 1.2
6.7 ± 1.7
Bmax, maximal binding capacity; Ki
3, the dissociation constant; N, Hill coefficient.
Table 8-2. Average GR Binding Parameters.
GR Binding Parameters
Kd (n=4)
Bmax (n=4)
N(n=4)
Non-pregnant
4.0 ± 1.3
129.6 ±56.4
1.3 ±0.2
Pregnant
7.6 ±2.6
40.8 ± 16.6
1.3 ±0.2
Bmax, maximal binding capacity; KD) the dissociation constant; N, Hill coefficient.

119
Figure 8.1. 3H-Cortisol binding to the MR in the non-pregnant (solid line) and pregnant
(dotted line) ewe. Curves were generated from the average fit parameters described in
Table 8.1.

120
Figure 8.2. 3H- cortisol binding to the GR in the non-pregnant (solid line) and pregnant
(dotted line) ewe. Curves were generated from the average fit parameters described in
Table 8.2.

CHAPTER 9
CHARACTERIZATION OF CYTOSOLIC IMMUNOREACTIVE
MINERALOCORTIOID AND GLUCOCORTICOID RECEPTORS IN THE
NONPREGNANT AND PREGNANT EWE
9.1 Introduction
MR availability is increased and GR availability is decreased in the ovine
hippocampus during pregnancy. However, since radioligand-binding studies only measure
unactivated, available steroid receptors, it is not possible to extrapolate total receptor
number from the results of a radioligand-binding assay. Since apparent changes in
receptor availability may actually be due to changes in the level of MR and GR protein
expression, it is necessary to estimate the total amount of MR and GR protein expressed
in the non-pregnant and pregnant ewe.
This study uses a semi-quantitative Western blot analysis to estimate total
immunoreactive MR and GR protein in the cytosols of tissues obtained from non-pregnant
and pregnant ewes. Previous studies have demonstrated that although unoccupied steroid
receptors are normally localized in the cell nucleus, these receptors are extracted into
“cytosolic” preparations (289). Prior to gel electrophoresis the proteins are denatured and
this allows the antibodies to interact with antigenic epitopes regardless of the original in
vivo steroid binding state (116). In this study immunoreactive MR (/MR) was estimated
using an anti-idiotypic antibody designed to fit into the steroid binding domain of the MR
(167). The anti-MR antibody recognizes a 116 KD protein representing the MR in the rat.
121

122
Immunoreactive GR (/GR) was estimated using an antibody designed to recognize a
sequence in the amino terminus of the human GR (43). The anti-GR antibody recognizes
a 96 KD protein representing the GR.
9.2 Methods
Tissue samples (hippocampus, kidney, hypothalamus, and pituitary) from four non¬
pregnant and four pregnant ewes were prepared and analyzed by Western blot as
described in Chapter 3. For each tissue evaluated, two gels consisting of samples from
four non-pregnant and four pregnant ewes were run. The proteins on each gel were
transferred onto a nitrocellulose membrane and one membrane was blotted with the anti-
MR antibody and the other membrane was blotted with the anti-GR antibody. The
immunoreactivity present on each membrane was quantified by chemiluminescent
densitometry. Only the optical densities of samples run on the same Western blot were
compared. Optical densities were compared by a Mann-Whitney rank sum test because the
data sets were not normally distributed. The null hypothesis was rejected when p < 0.05.
Data are expressed as the mean ± standard error of the mean (SEM).
9.3 Results
9,3,1 MR Immunoreactivity
The anti-MR antibody reacted at several molecular weights in all of the tissues
studied. All of these bands appeared to be specific since the optical density of the bands
was dependent on primary antibody concentration.

123
In the kidney, the anti-MR antibody reacted with proteins at five molecular
weights (131, 111, 54, 35, and 28 KD) and each band represented approximately 13, 20,
23, 22, and 22 % of the total immunoreactivity, respectively (Table 9.1). In the
hippocampus, the anti-MR antibody reacted with proteins at 138, 119, 64, and 37 KD
representing approximately 17, 15, 32, and 36% of the total immunoreactivity (Table 9.1).
In the hypothalamus, the anti-MR antibody reacted with proteins at 130, 66, and 48 KD
representing approximately 14, 46, and 40 % of the total immunoreactivity (Table 9.1).
In the pituitary, the anti-MR antibody stained proteins at 165, 128, 98, and 49 KD
representing approximately 22, 21, 25, and 32 % of the total immunoreactivity (Table
9.1). The antibody used in these assays recognizes a full length MR with a weight of 116
KD in the rat. In these assays, the antibody consistently reacted with a species at
approximately 132 KD (131, 138, 130, and 128 KD in the kidney, hippocampus,
hypothalamus, and pituitary, respectively). This 132 KD immunoreactive protein appears
to represent the full-length ovine MR. Total MR immunoreactivity (Figures 9.1 A, 9.2A,
9.3A, and 9.4A), and the reactivity of each band (Table 9.1) was significantly increased
during pregnancy in all tissues studied.
9,3,2 GR Immunoreactivity
In these assays, the anti-GR antibody consistently reacted with proteins
corresponding to a 111 KD full- length receptor (106, 113, 112, and 111 KD in the
kidney, hippocampus, hypothalamus, and pituitary, respectively). Although human full-
length receptor is 96KD (43), this 111 KD species appears to represent the ovine full-
length receptor under these assay conditions. An immunoreactive species corresponding
to a 57 KD (57, 54, 57, and 60 KD in the kidney, hippocampus, hypothalamus, and

124
pituitary, respectively) half-length receptor was also observed in all tissues studied.
Another study has also demonstrated a half-length receptor in the sheep (236). In the
hippocampus, kidney, and hypothalamus, the anti-GR antibody also reacted with a smaller
molecular weight (36, 40, and 40 KD, respectively). The half-length receptor accounted
for a majority of the total immunoreactivity (50-60%), and the remainder of the
immunoreactivity was equally distributed among the other two molecular weight bands.
Total GR immunoreactivity significantly increased during ovine pregnancy in the
pituitary (Figure 9.4B), but did not significantly change in any of the other tissues studied
(Figures 9. IB, 9.2B, 9.3B). In the hippocampus, the 36 KD band was significantly
increased during pregnancy and the 57 KD band was significantly decreased during
pregnancy in the hypothalamus, but there were no significant changes in the optical density
of any other bands during pregnancy (Table 9.2).
9,4 Discussion
Both the anti-MR and anti-GR antibodies used in this study reacted specifically
with proteins below the molecular weights of the full-length MR and GR proteins. These
proteins could be degradation products of the full-length receptor that result from
turnover of activated receptors. MR immunoreactivity has not been studied very
extensively with the antibody used in these assays, but GR immunoreactivity has
previously been studied with this antibody in a study of the sheep fetal hypothalamus and
pituitary (236). A half-length immunoreactive GR was also observed by another set of
investigators when a different antibody was used to screen cellular extracts (256). In both
instances, the investigators proposed the half-length immunoreactive species were

125
proteolytic fragments of the full-length receptor. In the study of the sheep fetal
hypothalamus and pituitary, the ratio of the optical density of the half-length species to the
full-length species significantly increased with developmental age as plasma cortisol
concentrations increase (236). This finding strongly supports the hypothesis that the half-
length immunoreactive GR, and the other smaller immunoreactive species represent
turnover of the full-length receptor after activation.
The current study demonstrates immunoreactive MR increases in the
hippocampus, kidney, hypothalamus, and pituitary during ovine pregnancy.
Immunoreactive GR also significantly increases in the pituitary, but does not significantly
change in the hippocampus, kidney, or hypothalamus. These findings suggest differential
expression of MR during ovine pregnancy may contribute to the observed changes in
hippocampal MR availability.
The observed increase in MR immunoreactivity in the hippocampus suggests the
increase in hippocampal MR availability during ovine pregnancy is due to increased
expression of MR protein. However, the significant 3-fold increase in immunoreactive
MR in the kidney, hypothalamus, and pituitary was not accompanied by a significant
increase in MR availability in these tissues; suggesting another mechanism contributes to
the selective increase in hippocampal MR availability. A study by Carey et al. (36)
suggests high endogenous levels of progesterone may antagonize activation of the
hippocampal MR by circulating corticosteroids, but the pituitary data presented in the
previous chapter does not support this hypothesis. Progesterone may be a more effective
MR antagonist in the hippocampus than in other tissues and further contribute to the
observed increase in MR availability.

126
Ovarian steroids could contribute to the increase in immunoreactive MR. There
are no known studies of the effect of ovarian steroids on MR immunoreactivity, but the
effect of ovarian steroids on MR mRNA levels has been previously investigated (271).
Several studies of have demonstrated progesterone alone does not change MR mRNA
levels in the adrenalectomized-ovariectomized rat but progesterone does attenuate the
tendency of estradiol to decrease MR mRNA levels in the adrenalectomized-
ovariectomized rat model (36, 207). These findings suggest ovarian steroids may
contribute to increased MR mRNA and immunoreactivity during pregnancy when
estrogens are high and progestins are dominant. Indeed, Castrón et al. have recently
demonstrated progesterone increases MR mRNA levels in primary hippocampal neuron
cultures and this effect can be reproduced in vivo in the adrenalectomized-ovariectomized
rat after chronic estrogen priming (40). However, the effect of progesterone MR
expression may depend on the isoform of the progesterone receptor expressed in the
target tissue since one form of the progesterone receptor (PR-A) has been shown to
inhibit transcription of MR in transfected cell cultures (180). In summary, the results of
these studies suggest estrogens alone do not contribute to the observed increase in
hippocampal MR immunoreactivity but progesterone, in combination with estrogens, may
increase the expression of the MR.
Ovarian steroids also have been shown to modulate GR immunoreactivity (2) and
mRNA levels (33, 35, 36, 207, 212, 213). These studies suggest ovariectomy increases
expression of the GR in the rat hippocampus and pituitary (212, 213, 217), and this
increase is reversed by estrogens (33, 212, 217). However, estrogen (32, 33) and
progesterone (207) actually appear to reverse glucocorticoid-induced GR down-regulation

127
in adrenalectomized-ovariectomized rat. These findings suggest estrogen and
progesterone would attenuate down-regulation of GR by the elevated circulating
glucocorticoids present during pregnancy. Indeed, GR immunoreactivity significantly
increased in the pituitary, tended to increase in the hippocampus and kidney, but did not
significantly change in the hypothalamus during ovine pregnancy. Therefore, the observed
decrease in hippocampal GR availability during ovine pregnancy appears to reflect
increased GR activation in the presence of increased circulating glucocorticoids.
The combined results of these studies suggest the hippocampal GR responds to
increased HPA axis activity (hippocampal GR availability decreases) but is not
differentially regulated (GR immunoreactivity does not increase in all tissues) in all tissues
during ovine pregnancy. In contrast, the MR, particularly in the hippocampus, appears to
be differentially regulated during pregnancy. MR expression appears to increase in all
tissues and increased expression appears to contribute to increased hippocampal MR
availability during pregnancy. However, the results of these studies do not indicate if the
levels of activated MR increase, decrease, or remain the same during ovine pregnancy.
Further study is needed to determine the mechanisms contributing to the increase in MR
expression observed during ovine pregnancy and the contribution of these changes to
adaptation of the HPA axis and the cardiovascular system to pregnancy.

Total Immunoreactivity (O.D.)
128
Figure 9.1. Total corticosteroid receptor immunoreactivity in the kidney of the non¬
pregnant (open bar, n=4) and pregnant (solid bar, n=4) ewe. A) MR immunoreactivity; B)
GR immunoreactivity. *p<0.05.

Total Immunoreactivity (O.D.)
129
Figure 9.2. Total corticosteroid receptor immunoreactivity in the hippocampus of the
non-pregnant (open bar, n=4) and pregnant (solid bar, n=4) ewe. A) MR
immunoreactivity; B) GR immunoreactivity. *p<0.05.

Total Immunoreactivity (O.D.)
130
Figure 9.3. Total corticosteroid receptor immunoreactivity in the hypothalamus of the
non-pregnant (open bar, n=4) and pregnant (solid bar, n=4) ewe. A) MR
immunoreactivity; B) GR immunoreactivity. *p<0.05.

Total Immunoreactivity (O.D.)
131
Figure 9.4. Total corticosteroid receptor immunoreactivity in the pituitary of the non¬
pregnant (open bar, n=4) and pregnant (solid bar, n=4) ewe. A) MR immunoreactivity; B)
GR immunoreactivity. *p<0.05.

132
Table 9.1. Optical densities of immunoreactive mineralocorticoid receptor protein bands
in the non-pregnant and pregnant ewe.
MR Immunoreactivity (O.D.)
Non-Pregnant (n=4)
Pregnant (n=4)
# 138 KD
1.8 ±0.6
5.6 ±0.9*
Hippocampus
119 KD
1.3 ±0.6
6.0 ±0.6*
64 KD
3.5 ±0.6
11.1 ±0.3*
37 KD
4.6 ± 1.0
10.2 ±0.2*
# 131 KD
1.6 ±0.2
4.8 ± 1.1*
111 KD
2.7 ±0.4
6.3 ±0.2*
Kidney
54 KD
2.8 ±0.5
9.1 ±0.8*
35 KD
2.4 ±0.4
8.6 ±0.2*
28 KD
2.7 ±0.8
8.4 ±0.5*
# 130 KD
0.9 ±0.3
2.9 ±0.6*
Hypothalamus
66 KD
2.9 ±0.9
10.5 ±1.1*
48 KD
2.6 ±0.7
8.6 ± 1.3*
165 KD
2.1 ±0.7
6.6 ±0.8*
# 128 KD
2.1 ±0.7
6.6 ±0.8*
Pituitary
98 KD
2.4 ±0.6
7.8 ± 1.3*
49 KD
3.4 ±0.9
8.8 ± 1.4*
*P<0.05, # full-length receptor

133
Table 9.2. Optical densities of immunoreactive glucocorticoid receptor protein bands in
the non-pregnant and pregnant ewe.
GR Immunoreactivity (O.D.)
# 106 KD
Non-Pregnant (n=4)
1.1 ±0.1
Pregnant(n=4)
3.9 ±2.1
Hippocampus
t 57 KD
6.1 ±0.5
11.7 ±2.1
36 KD
1.9 ± 0.1
5.6 ±1.5*
# 113 KD
0.14 ±0.05
1.8 ±0.7
Kidney
154KD
2.9 ±0.5
8.2 ±2.8
# 112 KD
4.0 ±0.6
2.8 ±0.6
Hypothalamus
t 57 KD
11.9 ± 0.6
8.2 ± 1.0*
40 KD
6.2 ±0.4
5.1 ±0.8
# 111 KD
1.0 ± 0.1
3.2 ±0.3*
Pituitary
160 KD
6.1 ±0.4
12.0 ± 1.3*
*P<0.05, # full-length receptor, t half-length receptor

CHAPTER 10
EFFECT OF CHRONIC PROGESTERONE TREATMENT ON HIPPOCAMPAL
MINERALOCORTICOID AND GLUCOCORTICOID RECEPTOR AVAILABILITY
AND CYTOSOLIC IMMUNOREACTIVITY IN THE OVARIECTOMIZED EWE
10.1 Introduction
The results of the previous chapters demonstrate hippocampal MR availability and
immunoreactivity increases and GR availability decreases during ovine pregnancy. As
discussed in Chapters 7,8, and 9, the increased concentrations of ovarian steroids found
during ovine pregnancy may contribute to the observed changes in MR and GR availability
and immunoreactivity. This study was designed to determine the effects of chronic
treatment with concentrations of progesterone typical of ovine pregnancy on hippocampal
MR and GR availability and immunoreactivity.
10,2 Methods
Ovariectomized ewes were studied in order to eliminate the possible confounding
effects of changes in plasma expected to occur during the natural estrous cycle in ovary-
intact ewes. Ten ovariectomized ewes were studied. Five of the ovariectomized ewes
were studied as controls and five of the ewes were implanted with 3 progesterone implants
and a 3 cm estradiol implant for 65 days. A previous study has shown that the
progesterone implants increase plasma progesterone concentration to levels characteristic
of ovine pregnancy (210). The estradiol implant was used to replace plasma estradiol to a
134

135
level characteristic of the luteal phase of the ovine estrus cycle since many of
progesterone’s effects are known to require estrogen priming (123, 124).
A blood sample was taken prior to sacrifice for determination of plasma
progesterone levels. Cytosolic samples were prepared and hippocampal MR and GR
availability and immunoreactivity was evaluated as described in Chapters 3, 7, and 9.
Groups were compared by a Mann-Whitney rank sum test since the data sets were not
normally distributed. The null hypothesis was rejected when p < 0.05. Data are expressed
as the mean ± standard error of the mean (SEM).
10.3 Results
10.3.1 Plasma Steroid Levels
Plasma progesterone concentration was 0.3 ± 0.2 ng/ml in the ovariectomized ewe
(Figure 10.1). Plasma progesterone concentration was significantly increased (2.2 ± 0.3
ng/ml) in ovariectomized ewes treated with progesterone implants for 65 days, but this
progesterone concentration was not as high as the plasma progesterone concentrations
observed in the pregnant ewes used in previous studies (9.1 ± 1.7 ng/ml).
10.3.2 MR and GR Availability
MR availability was 3.8 ± 1.6 fmol/mg protein in the hippocampus of
ovariectomized ewes (Figure 10.2A), and was not significantly different in progesterone-
treated ewes (2.6 ± 1.2 fmol/mg protein).

136
GR availability was 26.5 + 0.9 fmol/mg protein in the hippocampus of
ovariectomized ewes (Figure 10.2B), and was not significantly different in progesterone
treated ewes (28.3 ± 2.9 fmol/mg protein).
10.3,3 MR and GR Immunoreactivitv
Total MR immunoreactivity (Figure 10.3 A) was not significantly altered by 65
days of progesterone treatment. In contrast, progesterone significantly decreased total
GR immunoreactivity (Figure 10.3B).
10,4 Discussion
The results of this study demonstrate chronic progesterone treatment does not
significantly alter hippocampal MR availability or immunoreactivity in ovariectomized
ewes. Moreover, chronic progesterone treatment does not change hippocampal GR
availability but appears to decrease total hippocampal GR immunoreactivity in
ovariectomized ewes. These findings do not support the hypothesis that progesterone
contributes to the changes in hippocampal MR and GR availability and immunoreactivity
observed during ovine pregnancy.
Some available evidence suggests elevated concentrations of progesterone could
act as an in vivo competitive antagonist and increase MR availability during pregnancy.
Progesterone has potent anti-mineralocorticoid properties (158, 254, 287) and
progesterone injection reduces the apparent binding affinity of MR in hippocampal
cytosols obtained from rats four hours after injection, presumably by competitive inhibition
(35). However, this study did not reveal an effect on MR availability after chronic
treatment of the ovariectomized ewe with progesterone. Although progesterone

137
concentration was significantly increased in the progesterone-treated ewes after 65 days of
treatment, progesterone levels were not as high at the end of the study as the
concentrations typically observed during ovine pregnancy (~10 ng/ml). Therefore, it is
possible that the effects of progesterone on MR availability may be more evident when
progesterone concentrations are maintained at higher levels. Although this progesterone-
treatment protocol elevated progesterone concentrations into the range observed during
pregnancy in a two-week study (210) and a 60-80 day study (129), the implants may not
have contained enough crystalline progesterone to maintain adequate release for 65 days
or scar tissue formation may have hindered the release rate in this 65-day protocol.
Evidence suggests progesterone may modulate MR expression alone or by
antagonizing the effects of estradiol on MR expression. One form of the progesterone
receptor (PR-A, the isoform which appears to not be regulated by estradiol (125)) has
been shown to inhibit transcription of MR in transfected cell cultures (180) and
progesterone attenuates estradiol-induced decreases in MR mRNA levels in the
adrenalectomized-ovariectomized rat model (36, 40, 207). Since chronic progesterone
treatment did not change MR immunoreactivity in ovariectomized ewes, these mechanisms
do not appear to have been induced by the steroid treatment protocol used in this study.
Burgess and Handa (33) have previously observed a significant increase in MR
mRNA expression in the ovariectomized rat. Indeed, MR availability in the hippocampus
of the chronically ovariectomized ewe was significantly higher than in the non-pregnant
ewe and was not significantly different from the pregnant ewe. Therefore, the ability to
observe progesterone-induced effects on MR availability and immunoreactivity in this
study may have been obscured by changes in MR availability and/or expression induced by

138
ovariectomy. However, it is unclear if, in addition to availability, hippocampal MR
immunoreactivity and/or expression in the ovariectomized ewe is increased over levels
found in the non-pregnant ewe. Further study is needed to determine the effect of
ovariectomy on MR expression; a western blot study that allows comparison of MR
immunoreactivity in the non-pregnant, ovariectomized, and pregnant ewe should be
conducted.
The results of this study also did not demonstrate an effect of progesterone on GR
availability in the hippocampus of the ovariectomized ewe. However, GR
immunoreactivity was decreased in the progesterone- and estradiol-treated ovariectomized
ewe. Ovariectomy increases expression of the GR in the rat hippocampus and pituitary
(212, 213, 217), and this increase is reversed by physiological concentrations of estradiol
(33, 212, 217). These finding suggest the estradiol component of the replacement regimen
could contribute to the observed decrease in GR immunoreactivity.
It also is possible progesterone alone could decrease GR expression and/or
immunoreactivity. Physiological concentrations of progesterone induce reporter gene
expression in cell cultures transfected with the human GR (233), suggesting that
progesterone is a partial GR agonist. Studies consistently demonstrate glucocorticoids
auto-regulate their own receptors: GR mRNA expression decreases after exposure to
glucocorticoids (34, 231, 288). This auto-regulatory mechanism may be induced after
progesterone treatment in the ovariectomized ewe, but the observed decrease in GR
immunoreactivity should be correlated with a decrease in mRNA levels to confirm
decreased expression.

139
The possibility that physiological concentrations of progesterone act as a weak
glucocorticoid agonist in vivo has not been thoroughly explored. However, the results of
other studies suggest progesterone is a glucocorticoid agonist: (1) After two weeks of
treatment with physiological concentrations of progesterone, plasma volume increases in
the ovariectomized ewe (210). As discussed in Chapter 2, glucocorticoids may contribute
to volume expansion by shifting Na+ and subsequently fluid out of cells (162). (2) 60-80
days of progesterone treatment augments the ACTH and AVP response to hypotension
and enhances the inhibition of ACTH by cortisol (210). These findings could be explained
as glucocorticoid effects of progesterone on hippocampal GR. Although hypothalamic
and pituitary GR enhance feedback (60), hippocampal GR activation enhances feedback at
the neural output to the axis (284).
Changes in the regulation of MR and GR availability and expression after
ovariectomy also may contribute to observed changes in HPA axis responsiveness.
Previous studies have demonstrated the response to hypotension is altered in the
ovariectomized ewe: the ACTH and plasma renin response is attenuated (139, 211) and
the AVP response to hypotension is augmented (139). Ovariectomy appears to alter
responsiveness of the axis by modifying a component in the brain since pituitary
responsiveness to AVP and CRF is not altered after ovariectomy (211). In summary, this
study does not demonstrate a direct role for progesterone in modulating MR and GR
during pregnancy. The results do suggest MR and GR availability and expression may
change in response to ovariectomy. However, direct comparisons of MR and GR
availability and immunoreactivity in tissues obtained from non-pregnant and pregnant ewes
are needed.

140
Figure 10.1. Plasma progesterone levels in chronically ovariectomized ewes (open bar,
n=5) and chronically ovariectomized ewes implanted with progesterone and estradiol
(solid bar, n=5). Data are means ± SE. *p<0.05.

141
Figure 10.2. MR (A) and GR (B) availability in the hippocampus of chronically
ovariectomized, estradiol-treated ewes (open bar, n=5) and chronically ovariectomized
ewes implanted with progesterone and estradiol (solid bar, n=5).

142
Figure 10.3. MR (A) and GR (B) total immunoreactivity in the hippocampus of
chronically ovariectomized, estradiol-treated ewes (open bar, n=4) and chronically
ovariectomized ewes treated with progesterone and estradiol (solid bar, n=4)

CHAPTER 11
EFFECT OF CHRONIC ESTRONE TREATMENT ON HIPPOCAMPAL
MINERALOCORTICOID AND GLUCOCORTICOID RECEPTOR AVAILABILITY
AND CYTOSOLIC IMMUNOREACTIVITY IN THE OVARY-INTACT EWE
11.1 Introduction
The results of Chapters 7 and 9 demonstrate differential regulation of hippocampal
MR and GR receptor availability and immunoreactivity in pregnant ewes. However, as
detailed in Chapter 10, chronic progesterone treatment in the ovariectomized ewe did not
induce changes in MR and GR characteristic of ovine pregnancy.
Evidence suggest estrogens are important modulators of MR and GR (271). This
study was designed to determine if estrone, the major circulating estrogen of ovine
pregnancy, induces changes in MR and GR availability and immunoreactivity characteristic
of ovine pregnancy.
11.2 Methods
Hippocampus samples were collected from ovary-intact, ewes sacrificed 21 days
after subcutaneous implantation with placebo (n=5) or Ei (n=5) pellets. The tissues used
in this study were not processed at the time of sacrifice exactly as described in Chapter 3.
Although the brains were rapidly removed, dissected and frozen, the brains used in this
study were not perfused with the cryoprotective 10% dimethylsulfoxide solution and the
tissue samples were stored at -20°C for approximately 3 years prior to assay.
143

144
Hippocampal cytosols were prepared exactly as described in Chapter 3. MR and GR
availability was measured using radioligand-binding as described in Chapters 3 and 7.
Quantitative Western blots were used to estimate total immunoreactive MR and GR
protein as described in Chapters 3 and 9. Groups were compared by a Mann-Whitney
rank sum test since the data sets were not normally distributed. The null hypothesis was
rejected when p < 0.05. Data are expressed as the mean ± SEM.
11.3 Results
11.3.1 MR and GR Availability
MR availability tended to be greater in the estrone-treated ewe, but was not
significantly different from control (Figure 11.1 A). In contrast, GR availability was
significantly decreased in the estrone-treated ewe (Figure 11. IB).
11.3.2 MR and GR Total Immunoreactivitv
Estrone treatment significantly increased total immunoreactive MR (Figure 11.2A)
and immunoreactive GR (Figure 11.2B).
11.4 Discussion
The results of this study demonstrate chronic estrone treatment induces changes in
hippocampal corticosteroid receptor availability and immunoreactivity characteristic of
ovine pregnancy. As observed during ovine pregnancy, estrone-treatment increased
hippocampal MR immunoreactivity and decreased hippocampal GR availability.

145
Other studies suggest estrogens modulate MR and GR (271). Although it is
unclear whether estradiol increases (75) or decreases (36) hippocampal MR availability,
two studies suggest estrogens decrease MR mRNA levels in the adrenalectomized-
ovariectomized rat model (36, 207). In contrast, the results of this study suggest chronic
estrone treatment actually increased MR immunoreactivity in the ewe. Whereas those
studies were conducted in tissues obtained from adrenalectomized-ovariectomized rats,
the current study was conducted in tissues obtained from ovary-intact ewes; suggesting
circulating corticosteroids may modulate the estrogen response. Although estrone
increased MR immunoreactivity and there was a tendency towards increased MR
availability, estrone-treatment did not significantly increase MR availability. This finding
suggests that elevated circulating progesterone may be necessary to increase MR
availability even when circulating estrogens increase MR expression.
Estrogens have been shown to reverse glucocorticoid-induced GR down-
regulation in adrenalectomized-ovariectomized rat (32, 33), suggesting estrone increases
GR expression by opposing auto-regulation of the GR by circulating glucocorticoids.
Moreover, a recent study by Kushner (278) and colleagues demonstrates the GR and the
estrogen receptor (ER) oppose each other via interactions at regulated response elements.
Therefore, the observed increase in MR and GR immunoreactivity may be the result of the
ability of the estrone-ER complex to oppose MR and GR auto-regulation by endogenous
corticosteroids.
Estrone treatment increased GR immunoreactivity and decreased GR availability.
Although not significantly different, basal ACTH and cortisol levels also tended to be
higher in the estrone-treated ewes (140), suggesting basal activity of the HP A axis was

146
elevated by estrone treatment. Other investigators also have found estrogens increase
basal activity of the axis (96). Together these findings suggest estrone increases both
circulating cortisol and hippocampal GR expression and the increased concentrations of
circulating cortisol increase GR activation and decrease GR availability.
In summary, the results of this study suggest estrogens modulate MR and GR and
contribute to the changes in MR and GR availability and immunoreactivity observed
during ovine pregnancy

147
Figure 11.1. MR (A) and GR (B) availability in the hippocampus of the ovary-intact ewe
(open bar, n=5) and the ovary-intact ewe chronically treated with estrone (solid bar, n=5).
Data are means ± SE.
‘ if

148
Figure 11.2. MR (A) and GR (B) total immunoreactivity in the hippocampus of the
ovary-intact ewes (open bar, n=4) and ovary-intact ewes chronically treated with estrone
(solid bar, n=4). Data are means ± SE.

CHAPTER 12
SUMMARY
12.1 Overview
The studies presented in this dissertation were designed to test the validity of three
very general hypotheses. These general hypotheses are:
1. Progesterone induces physiological effects on the HP A axis and the
cardiovascular system by binding multiple receptor types. Among the
physiologically relevant progesterone receptor types are the MR, GR and the
GAB Aa receptor.
2. Elevated concentrations of progesterone modulate both the HPA axis and the
cardiovascular system during pregnancy.
3. Adapted HPA axis activity contributes to cardiovascular adaptation to
pregnancy.
12,2 Specific Hypotheses Tested:
In order to determine the validity of the general hypotheses outlined above, the
following specific hypotheses were tested in this dissertation:
1. Progesterone rapidly alters blood pressure, baroreflex sensitivity, and blood
volume.
149

150
2. Pregnancy is associated with changes in MR and GR availability,
immunoreactivity, and apparent affinity.
3. Ovarian steroids contribute to the changes in MR and GR availability and
immunoreactivity observed during ovine pregnancy.
The results of the studies designed to test these specific hypotheses are discussed
below:
12.2.1 Hypothesis 1: Progesterone Rapidly Alters Arterial Pressure. Blood Volume and
Baroreflex Sensitivity
The study presented in Chapter 4 demonstrates physiological concentrations of
progesterone rapidly reduce arterial pressure in ovary-intact, adult ewes. This study is the
first study to suggest physiological concentrations of progesterone decrease blood
pressure through an extracellular and nongenomic mechanism. It is interesting to
speculate that progesterone is 5a-reduced to the GABAA-active metabolite THP and that
this metabolite and the GABAa receptor contribute to the rapid effects of progesterone.
However, further study is required to verify that the GABAa receptor is involved in the
rapid, apparently nongenomic, effects of progesterone on cardiovascular function.
Unfortunately, the only currently available GABAa receptor antagonist is the highly toxic
tetrodotoxin. Therefore, these studies can not be repeated in the presence of a GABAa
receptor antagonist. However, it would be interesting to repeat the study in the presence
of a 5a-reductase inhibitor such as finasteride. If the rapid pressure response to
progesterone were abolished in the presence of a 5a-reductase inhibitor, this would
strongly suggest that 5a-reduced, GABAA-active progesterone metabolites are responsible
for the rapid cardiovascular effects of progesterone.

151
The results of the study presented in chapter 4 suggest arterial pressure and plasma
progesterone concentration are not linked through a simple, linear dose-response
relationship since supraphysiological levels of progesterone did not alter arterial pressure
within 2 hours. This finding suggests that multiple receptors may be involved in the
effects of progesterone on cardiovascular function, and the concentration of circulating
progesterone appears to determine which receptors are activated. Additional studies
designed to clarify the dose-response relationship between progesterone concentration and
its effects on arterial pressure and blood volume would provide additional means to assess
the receptor types involved in the cardiovascular responses to progesterone. Once the
relationship between progesterone concentration and progesterone’s various effects on
cardiovascular function is established, it will possible to use the known affinities of the
potential progesterone receptors to correlate the observed responses with each of the
potential progesterone receptor types.
These studies did not demonstrate a rapid effect of progesterone on the heart-rate
responses to perturbations in arterial pressure even when graded continuous infusions of
phenylephrine and nitroprusside were used to uncover potential changes in the more
slowly adapting sympathetic component of the baroreflex. Therefore, none of the studies
conducted to date have demonstrated an effect of progesterone on baroreflex function in
the ewe. However, the effect of acute progesterone infusion in the intact ewe and the
effect of chronic progesterone treatment in the ovariectomized ewe on baroreflex function
should still be assessed using the graded continuous infusion method; important effects of
progesterone on the baroreflex may be uncovered in these circumstances.

152
The studies presented in Chapter 5 were designed to determine if, in addition to
rapidly reducing MAP, progesterone also rapidly expands plasma volume. These studies
did not reveal an effect of progesterone on plasma volume after 30 minutes or 4 hours,
suggesting that longer periods of time (and genomic events) are required for physiological
concentrations of progesterone to expand plasma volume. It is possible that chronic
progesterone treatment has effects on baroreflex sensitivity that follow its effects on
plasma volume since cardiopulmonary receptor stimulation is known to induce changes in
the baroreflex characteristic of pregnancy.
The studies presented in Chapter 5 also revealed that the rapid effects of
physiological levels of progesterone also appear to depend on starting baseline pressure;
progesterone appears to have its most profound effects on arterial pressure when starting
pressures (and presumably sympathetic outflow to the vascular system) are slightly
elevated. It has been proposed that metabolites of progesterone decrease blood pressure
by interacting with GABAa receptors in the RVLM and GABAa receptors in the RVLM
are known to inhibit excitatory outflow to the sympathetic nervous system. Therefore, it
makes sense that progesterone metabolites would only be effective when this system is
activated.
Since the studies presented in Chapter 5 were also conducted in ovariectomized
ewes (but the studies presented in Chapter 4 were conducted in ovary-intact, anestrous
ewes), it is also possible that progesterone’s rapid effects are altered in ovariectomized
animals. The study presented in Chapter 6 was tests the hypothesis that metabolism of
progesterone is reduced in ovariectomized ewes. The results of Chapter 6, although
preliminary, do indeed suggest that 5a-reduction of progesterone to the GABAA-active

153
metabolite, THP, is decreased in ovariectomized ewes. This reduction in progesterone
metabolism could contribute to the abolishment of the rapid pressure response to
progesterone during the 4-hour protocol. Further study is needed to fully characterize the
5a-reduction of progesterone in physiologically relevant tissues in ovary-intact,
ovariectomized, and pregnant ewes. Moreover, studies will be required to fully establish
whether there is a true relationship between starting arterial pressure (and sympathetic
tone) and the ability of progesterone to rapidly reduce arterial pressure.
These studies of the rapid cardiovascular effects of progesterone also contribute to
the general aims of this area of research by supporting the hypothesis that progesterone
contributes to cardiovascular adaptation to pregnancy. Furthermore, since these studies
demonstrated progesterone has effects on cardiovascular function that occur more quickly
than would be expected to occur via genomic mechanisms, these studies also support the
hypothesis that novel progesterone types are involved in the physiological effects of
progesterone.
These studies have not explored the possibility that progesterone also rapidly alters
function of the HPA axis. It is interesting to speculate that progesterone metabolites
modulate the HPA axis since GABAa receptors are concentrated in the paraventricular
nucleus and the axis is known to be responsive to benzodiazepines. Progesterone
metabolites may blunt activation of the axis and/or enhance corticosteroid feedback signals
to the hypothalamus. These areas are worthy of further study since some of
progesterone’s effects on cardiovascular function may also be mediated via rapid (and
slow) effects on the HPA axis.

154
The most important effects of progesterone are probably not mediated via these
rapid, apparently nongenomic, mechanisms. Instead, these rapid effects of progesterone
are probably fairly transient and serve more to amplify more prolonged genomic effects of
progesterone.
2.2.2 Hypothesis 2: MR and GR Availability. Immunoreactivitv. and Apparent Affinity are
Altered During Pregnancy
Overwhelming pharmacological evidence suggests progesterone mediates
physiological effects through the MR and GR. The studies presented in Chapter 7 were
designed to test the hypothesis that the amount of available (unactivated) MR and GR
changes in HPA axis feedback sites in the brain in pituitary during pregnancy when
circulating progesterone and corticosteroids are elevated. The results of this study reveal
that the hippocampus is an important site for differential regulation of MR and GR
availability. Hippocampal GR availability tended to decrease during pregnancy, a finding
that is consistent with increased receptor activation in the presence of elevated
concentrations of circulating corticosteroids. In contrast, MR availability significantly
increased in pregnant ewes. This finding suggests an endogenous receptor antagonist such
as progesterone could be protecting the MR from activation in the presence of high
concentrations of circulating corticosteroids.
If an endogenous MR antagonist were present in cytosols obtained from pregnant
ewes, the apparent binding affinity of the receptor would be reduced in cytosols obtained
from pregnant ewes. Chapter 8 did reveal a strong tendency towards reduced MR affinity
in cytosols obtained from pregnant ewes, but this change did not reach statistical
significance. In vitro studies conducted by other investigators have shown progesterone

155
can increase the rate of corticosteroid dissociation from the GR, and this effect may also
be true for the MR. However, the results of the study presented in Chapter 8 suggest this
antagonistic effect of progesterone on the corticosteroid receptors may not be the most
physiologically relevant effect of progesterone on these receptor types. Indeed, studies of
transfected cell cultures have shown that progesterone is a partial agonist at both the MR
and GR. This finding suggests that progesterone causes heat shock proteins to dissociate
from the receptor so that the receptor can assume an activated, unavailable conformation
that is capable of mediating transcription. Therefore, the tendency of progesterone to
increase the rate of corticosteroid dissociation from intracellular receptors may enhance
the apparent corticosteroid antagonist activity of progesterone, but the activated
progesterone-MR/GR complex also appears to be physiologically relevant.
Although it was speculated that the significant increase in MR availability in
hippocampal cytosols was due to protection from an endogenous corticosteroid
antagonist, the study conducted in Chapter 7 did not test the possibility that increased
expression of MR contributes to the increase in hippocampal MR availability during
pregnancy. Therefore, the study presented in Chapter 9 was conducted to determine if
immunoreactivity of MR and GR changes during pregnancy. This study demonstrated that
MR immunoreactivity does indeed increase during pregnancy, suggesting that increased
MR expression is the primary mechanism leading to increased hippocampal MR
availability during pregnancy.
These studies are the first to demonstrate that corticosteroid receptors are
differentially regulated during pregnancy. The change in MR and GR availability and
immunoreactivity could represent a response of the receptor system to increased HPA axis

156
activity and/or MR and GR availability and expression could be changed during pregnancy
in order allow axis activity to be regulated during pregnancy. The changes in MR
availability and immunoreactivity in particular suggest that the latter hypothesis may be
true. These studies demonstrate that MR and GR are differentially regulated during
pregnancy when progesterone, corticosteroids, and other circulating factors are increased.
These changes could contribute to differential regulation of the HPA axis, and as
discussed above, differential regulation of the HPA axis could contribute to cardiovascular
adaptation to pregnancy.
One interesting finding of these studies is that the hippocampus is the primary site
for differential regulation of the MR and GR during ovine pregnancy. The hippocampus
has long been suspected as an HPA feedback site, but studies fail to consistently
demonstrate a role for the hippocampus in feedback regulation of the HPA axis. As
discussed in Chapter 2, it is possible that the role of the hippocampus in regulating the
HPA axis depends on both the time of day and the type of stress applied to the axis.
Moreover, it is interesting to speculate that the role of the hippocampus in regulating the
axis is increased during pregnancy when neural and hypothalamic output to the pituitary
appears to be increased.
Novel approaches are required to elucidate the role of the hippocampus in control
of the HPA axis during the normal circadian rhythm, stress, and pregnancy. Hippocampal
lesion studies have been used extensively to assess the role of the hippocampus in
regulation of the axis, but these studies have been inconclusive. It would be interesting to
determine which stresses actually route their signal through the hippocampus before
impacting the paraventricular nucleus. One way to determine this would be to look for

157
increases in early intermediate gene immunoreactivity in the hippocampus in response to
various stresses. Early intermediate genes are transcription factors that tend to be
expressed rapidly in response to cellular activation (103). By looking at the pattern of
early intermediate gene activation in regions of the hippocampus in response to various
stresses, it may become apparent that only certain stresses route their signals through the
hippocampus. In contrast, it may become evident that all stresses that increase activity of
the HPA axis share a common pattern of hippocampal cell type activation. By combining
these studies with other immunoreactive, retrograde tracing, and more specific lesion
studies it may be possible to determine the precise role of the hippocampus in regulation
of the HPA axis.
It also would be interesting to look at the pattern of hippocampal activation under
various circumstances. For example, hippocampal (or downstream in the bed nucleus of
the stria terminalis or the paraventricular nucleus) activation may be different if a stress is
applied after application of a feedback signal (infusion of corticosteroid). Moreover, the
pattern of cellular activation may be different if a stress is applied during instances when
basal neural output to the axis is increased (the peak of the circadian rhythm or perhaps
during pregnancy).
Further study is required to determine the mechanisms leading to differential
regulation of hippocampal MR and GR during pregnancy (Chapters 10 and 11 represent
one attempt at answering this question) and the contribution of these receptors changes to
differential regulation of the HPA axis. One way to determine the role of the changes in
hippocampal MR and GR activation would be to infuse MR and GR antagonists and/or
agonists directly into the hippocampus. By monitoring basal activity and responsiveness

158
of the axis in the presence of these drugs, it may be possible to begin to elucidate the role
of these receptors in adapting the HPA axis to pregnancy.
In summary, these studies demonstrate hippocampal corticosteroid receptors are
differentially regulated during pregnancy. Progesterone or other endocrine factors could
induce these receptor changes. These changes may contribute to adaptation of the HPA
axis and subsequently cardiovascular function during pregnancy.
12.2.3 Hypothesis 3: Ovarian Steroids Alter MR and GR Availability and
Immunoreactivitv
The studies conducted in Chapter 10 and Chapter 11 were conducted to determine
if ovarian steroids could induce changes in MR availability and immunoreactivity. Chronic
treatment of ovary-intact ewes with estrone, but not chronic treatment of the
ovariectomized ewe with progesterone, induced changes in these receptors characteristic
of ovine pregnancy. This finding suggests estrogens contribute to adaptation of the
cardiovascular system and the hypothalamic-pituitary-adrenal axis to pregnancy by
modulating these receptors. Since hippocampal MR availability also significantly increases
in ovariectomized ewes (the control animal used in the chronic progesterone study), the
possibility that progesterone also regulates MR and GR can not be eliminated.
Together these studies support the hypothesis that ovarian steroids contribute to
adaptation of the hypothalamic-pituitary-adrenal axis and the cardiovascular system during
pregnancy via novel rapid nongenomic and slow genomic mechanisms.

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BIOGRAPHICAL SKETCH
Darren Roesch was bom in Fort Wayne, Indiana, in June of 1971. During the first
year of his life he completed a conquest to the top of his grandparents’ kitchen table in his
hometown of Warsaw, Indiana. However, Darren quickly grew bored with the small mid-
western town lifestyle and moved his family to sunny New Smyrna Beach, Florida, where
he and his sister Amanda supervised their parents’ management of their businesses.
When Darren was ten years old, his mother died of complications of insulin-
dependent diabetes. The loss left him deeply saddened and a little neurotic, but not as
cute, rich, or famous as Prince William.
Darren was required by law to attend public school for so long that he acquired a
diploma from New Smyrna Beach High School. Darren was even named an outstanding
senior, but one of his school science teachers told Darren he did not deserve it because he
was too much of a slacker. The science teacher was right, of course. Since his high
school guidance counselors did not seem to have any better ideas, Darren decided to
attend the University of Florida and become a medical doctor like everyone else. Darren
did not like college too much because there were too many students and none of them
seemed to notice Darren. That is probably because Darren is only 5 feet and 5 inches tall.
As Darren neared completion of his B .S. in microbiology, he looked at his
transcripts and realized no medical school outside of the Caribbean would admit him, so
196

197
he decided to go to graduate school in the Department of Pharmacodynamics at the
University of Florida.
In graduate school, Darren finally narrowed his life goals to curing diabetes so his
mother would be proud or pursuing his life-long fetish with sheep. The latter option
seemed like a lot more fun, so Darren began his study in Dr. Maureen Keller-Wood’s lab.
Graduate school was a lot of fun for Darren, but eventually Dr. Keller-Wood exhausted all
of her resources, Darren’s now ex-wife became jealous of the sheep, and Darren’s
therapist finally wore out his accurately active imagination. As a result, Darren decided it
was time to write the masterpiece you are now holding in your hands.
Rumor says Darren will soon move to Washington, D.C., to begin a postdoctoral
fellowship at Georgetown University. In truth, nobody really knows what Darren will do
next. He just might return to that kitchen table top in Warsaw, Indiana, and shout “Oh,
no! Darren fall! Darren fall!” but then again, he is probably too big now for his Grammy
to rescue him from dangerous summits.

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
jájlÉLt - //A
M&ureen Keller-Wood, Chair
Associate Professor of
Pharmacodynamics
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
4^oJLd—
Pushpa S: Kalra
Professor of Physiology
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Micháel J. Kato\
Professor of Pharmacodynamics
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is/fully adequate, in scope
as a dissertation for the degree of Doctor of Philosophy
William J.
Professo
acodynamics
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Ltek&O*. LaI^OQ
EL
Donna Wielbo
Assistant Professor of Medicinal
Chemistry

This dissertation was submitted to the Graduate Faculty of the College of
Pharmacy and to the Graduate School and was accepted as partial fulfillment of
requirements for the degree of Doctor of Philosophy
May 1998
Dean, Graduate School




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