Novel rapid nongenomic and slow genomic mechanisms of ovarian steroid modulation of the hypothalamic-pituitary-adrenal a...

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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














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.





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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


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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




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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.