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Pulmonary vascular responses of perinatal and adult sheep to arachidonic acid during normoxia and hypoxia

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Title:
Pulmonary vascular responses of perinatal and adult sheep to arachidonic acid during normoxia and hypoxia
Alternate title:
Arachidonic acid during normoxia and hypoxia
Creator:
Tod, Mary Lee, 1953-
Publication Date:
Language:
English
Physical Description:
vii, 114 leaves : illustrations, graphs ; 29 cm.

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Subjects / Keywords:
Arachidonic Acids ( mesh )
Vascular Resistance ( mesh )
Vasoconstrictor Agents ( mesh )
Physiology thesis M.S ( mesh )
Dissertations, Academic -- Physiology -- UF ( mesh )
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bibliography ( marcgt )
non-fiction ( marcgt )
Academic theses. ( lcgft )
Academic theses ( fast )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida.
Bibliography:
Includes bibliographical references (leaves 104-113).
General Note:
Photocopy of typescript.
General Note:
Vita.
Statement of Responsibility:
Mary Lee Tod.

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University of Florida
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University of Florida
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Copyright Mary Lee Tod. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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10180827 ( OCLC )

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Full Text
PULMONARY VASCULAR RESPONSES OF PERINATAL AND ADULT SHEEP
TO ARACHIDONIC ACID DURING NORMOXIA AND HYPOXIA
By
MARY LEE TOD
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
1984


I dedicate this dissertation to my father, for his example of
determination in overcoming obstacles, and to my mother, for teaching
me the patience I needed to complete a difficult task.


ACKNOWLEDGEMENTS
The author wishes to express her appreciation for the comments,
criticisms and guidance of her supervisory committee, Dr. S. Cassin,
Dr. A. B. Otis, Dr. W. N. Stainsby and Dr. A. H. Neims. In addition,
thanks are due to the members of the laboratory who aided at various
stages of this research project: H. Kuck, T. Cupp, R. Shoup, E.
McJett, R. Su and R. Cooper.
Special appreciation is extended the chairman of the supervisory
committee, Dr. Sidney Cassin, for the provision of laboratory facili
ties and animals, support and encouragement during the completion of
this manuscript.
Special thanks are due Calvin Timmerman for his constant
encouragement and support during this project, and for his endless
understanding and patience.


TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS iii
ABSTRACT vi
INTRODUCTION 1
Historical Overview 1
Prostaglandin Biochemistry 2
Prostaglandins and the Pulmonary Circulation 8
Statement of Purpose 15
PERINATAL PULMONARY RESPONSES TO ARACHIDONIC ACID
DURING NORMOXIA AND HYPOXIA 17
Introduction 17
Materials and Methods 19
Results 22
Discussion 33
THROMBOXANE SYNTHETASE INHIBITION AND PERINATAL
PULMONARY RESPONSE TO ARACHIDONIC ACID 38
Introduction 38
Materials and Methods 39
Results 41
Discussion 51
EFFECTS OF PROSTAGLANDIN ON PERINATAL
PULMONARY CIRCULATION.. 57
Introduction 57
Materials and Methods 58
Results 61
Discussion 69
ARACHIDONIC ACID AND ADULT PULMONARY CIRCULATION 74
Introduction 74
Materials and Methods 75
Results 79
Discussion 94
CONCLUSIONS 99
i v


REFERENCES 104
BIOGRAPHICAL SKETCH 114
v


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
PULMONARY VASCULAR RESPONSES OF PERINATAL AND ADULT SHEEP
TO ARACHIDONIC ACID DURING NORMOXIA AND HYPOXIA
By
Mary Lee Tod
April 1984
Chairman: Dr. Sidney Cassin
Major Department: Physiology
The actions of exogenous arachidonic acid during normoxia and
hypoxia were studied in perinatal lambs using an in situ pump-perfused
lung preparation. Ventilated fetal and neonatal lambs responded to
arachidonate infusions with pulmonary vasoconstriction and systemic
hypotension. In ventilated fetuses the effect of arachidonic acid was
additive to the hypoxic pressor response. The pulmonary vascular
response in newborns to arachidonic acid was unaltered by hypoxia, pos
sibly because of the severity of the vasoconstriction to arachidonate
alone. Thus, hypoxia does not alter the response of the perinatal pul
monary circulation to exogenous arachidonic acid.
Inhibition of thromboxane synthetase with OKY-1581 depressed ara-
chidonate-induced increases in pulmonary vascular resistance. Pulmo
nary vasoconstriction still occurred in response to arachidonic acid
VI


after OKY-1581, but the peak increase in pulmonary vascular resistance
was 50 85 % less than the response before the inhibitor. The remain
ing response was attributed to formation of endoperoxides. Inhibition
of thromboxane synthetase resulted in a significant enhancement of sys
temic hypotension in response to arachidonic acid.
Injections of PGH2 into the pulmonary circulation of ventilated
fetuses caused increases in pulmonary vascular resistance. The pulmo
nary response to PGH2 was reduced following OKY-1581. The response
of unventilated fetuses to PGH2 was pulmonary vasodilatation. The
differences in response to PGH2 may be due in part to differences in
pulmonary vascular tone before and after ventilation. Another possible
explanation is that ventilation of fetal lungs alters the ratios of the
end products of PGH2 metabolism, so that after ventilation more
thromboxanes are formed in response to PGH2*
In contrast to studies in other species, low-dose arachidonate
infusions always resulted in pulmonary vasoconstriction in adult sheep.
The pulmonary circulation of hypoxic adults constricted even further
upon addition of arachidonic acid. The pulmonary pressor response to
arachidonate was not affected by prior elevation of pulmonary vascular
resistance with PGF2a. In both perinatal and adult sheep, eleva
tion of pulmonary vascular resistance by hypoxia or PGF2a does not
alter the pressor response to arachidonic acid. Pulmonary responses to
arachidonic acid appear to be species dependent.
vi i


CHAPTER ONE
INTRODUCTION
Historical Overview
Prostaglandin research had its beginnings over 50 years ago with a
report of contractile and relaxant activity of isolated uterine strips
in response to seminal fluid (1). Reports of vasodepressor activity of
human seminal fluid and an alcoholic watery extract from prostate
glands came from two independent groups (2, 3). This vasodepressor
substance from prostate glands was named "prostaglandin" by von Euler
(4), and its biological activity in lowering blood pressure was
established in several species.
Attempts at isolation and purification of "prostaglandin" were
successful with the separation of a prostaglandin factor, PGF, in 1957
(5). The compound was determined to be an unsaturated hydroxyacid.
Further purification and separation of extracts from sheep prostate
glands resulted in identification of two compounds, PGE and PGF (6, 7),
which retained the biological activity of the crude extracts when
tested on smooth muscle and for effects on blood pressure (8).
Prostaglandins were found to be derived from essential fatty acids (9,
10), with the most common precursor in mammals being arachidonic acid
(11). Prostaglandin biosynthesis has been detected in most tissues of
the body, including lung (12, 13).
1


2
In the past 10 years, many advances have been made in prostaglan
din research. Products of arachidonic acid metabolism other than the
original PG's, E and F, were isolated which also exhibited biological
activity. These unstable products include the endoperoxide intermedi
ates, PGG2 and PGH2 (14-17), prostacyclin (18-20), the thromboxanes
(TX's) (21) and the leukotrienes (22). In recognition of their vast
contributions to a field of research only 50 years old, the Nobel Prize
in Medicine was awarded in 1982 to Sune Bergstrom, Bengt Samuelsson and
John R. Vane.
Prostaglandin Biochemistry
Monoenoic, bisenoic and trienoic PG's are derived from the essen
tial fatty acids, 8,11,14-eicosatrienoic acid (dihomo-y-1inolenic
acid), 5,8,11,14-eicosatetraenoic acid (arachidonic acid) and
5,8,11,14,17-eicosapentaenoic acid, respectively (9, 10). Products of
these fatty acids are denoted by a subscript to indicate the degree of
unsaturation in the molecule; e.g., PGH2 contains two double bonds.
Arachidonic acid is the predominant PG precursor in mammals, and
it is esterified to phospholipid membranes (11). Arachidonic acid must
be liberated from membranes before entering into the biosynthesis of
prostaglandins; this liberation results from hydrolysis by phospholi
pases (11). Phospholipases can be activated by a number of stimuli; a
common factor is probably distortion of the cell membrane (13).
Release of free arachidonic acid begins the biosynthetic cascade (Fig
1); availability of free arachidonic acid is recognized as the rate-
limiting step in the formation of prostaglandins (11).


Figure 1: Biosynthetic scheme of the products of arachidonic acid is
presented. Points of inhibition are included, with the
inhibitors shown in parentheses.


(HMWOLSf A?
LEUKOTRIENE
PROSTAGLANDIN
PROSTACYCLIN
THROMBOXANE


5
Arachidonic acid enters one of two enzymatic pathways following
its release: (1) the cyclo-oxygenase system, which leads to formation
of PG's and TX's, or (2) the lipoxygenase pathway, which contributes
leukotrienes (LT's) and other oxygenated, non-cyclic fatty acids (15,
16, 22). The lipoxygenase pathway has generated much interest recent
ly; leukotrienes are involved in immune responses to inflammation,
asthma, and other immediate hyper-sensitivity reactions (22). Leuko
trienes C4, D4 and E4 have been identified as major components of
slow-reacting substance of anaphylaxis (SRS-A) (22). Another leukotri-
ene, LTB4, is involved in chemotactic responses leading to migration
of neutrophils and eosinophils (22). The prostaglandin synthetase com
plex is a group of membrane-bound enzymes which requires oxygen and
other cofactors (15). The initial step in the formation of PG's from
arachidonic acid appears to be a peroxidative reaction that allows the
fatty acid to be attacked by oxygen (23). This is quickly followed by
cyclization of the hydroperoxide into a cyclic endoperoxide intermedi
ate, PGG2 (15, 16). This endoperoxide intermediate can be reduced to
form PGH2, a second intermediate in the pathway (15, 16), or it may
also be converted enzymatically to PGE2, PGI2 or TXA2 (23). The
endoperoxides are highly unstable in aqueous solution, and rapidly form
other prostaglandins (24). The enzymes which convert the endoperoxides
to the other PG's and TXA2 are present in many tissues of most spe
cies, but they exhibit varying activities and concentrations in differ
ent organs, contributing to the wide range of responses of individual
systems to release of PG's (Table 1) (16, 18, 25-28).
The primary PG's, PGE2 and PGF2a, were the earlier ones
studied (12). A third primary PG is PGD2* The primary PG's are


6
Table 1. Major sites of formation and inactivation of prostaglandins
Formation
Inactivation
P6E2
seminal vesicles,
kidney
lung
PGF2a
connective tissue,
reproductive tissue
lung
pgd2
lung, brain, leukocytes,
gastrointestinal tissue
enzymatically in
blood
pgi2
vascular endothelium
spontaneous hydrolysis
in blood
txa2
platelets, lung,
spleen
spontaneous hydrolysis
in blood
Sources for material presented in this table are ref 16, 18, 25-28.


7
relatively stable in aqueous solution, and each is derived from PGH2
by different routes. PGE2 and PGF2a are produced by isomerization
and reduction, respectively, of the endoperoxide intermediate (15, 16).
The enzymatic formation of PGD2 can occur by one of two methods. A
soluble PGH-PGD isomerase is present in various organs, and in the
presence of glutathione it converts PGH2 solely to PGD2 without
formation of PGF2a (16). An additional enzyme isolated from sheep
lung is capable of producing both PGD2 and PGF2a in the presence
of glutathione (29). There is considerable interconversion of the pri
mary PG's, in particular the formation of PGF2a from PGD2 and PGE2
which occurs in sheep blood (28). Secondary prostaglandins, PGA2,
PGB2 and PGC2, are derived from PGE2 by dehydrogenation and ring
isomerization.
The primary and secondary PG's were relatively easily extracted
and purified, and thus have been studied extensively (12, 30-32).
Recently, new products of arachidonic acid were identified which are
highly potent but extremely unstable (14, 18, 19, 21). Prostacyclin
synthetase is an enzyme which is found in vascular endothelia; it con
verts PGH2 to prostacyclin, or PGI2 (18, 19). Prostacyclin hydrolyzes
spontaneously to the relatively inactive 6-keto-PGFia in blood;
the half-life for this reaction is 3 min (34). Prostacyclin has been
found to have powerful anti-aggregatory action, is a potent vasodilator
of both the pulmonary and systemic circulations, and relaxes arterial
smooth muscle strips (35).
Thromboxane synthetase is the enzyme which forms TXA2 from the
endoperoxides (21), and the primary site of TX formation is in plate
lets. The half-life of TXA2 in blood is approximately 30 sec, with


8
the stable and relatively inactive degradation product being TXB2
(21, 25, 36). Thromboxane A2 exhibits potent platelet aggregating
ability, and it is a component of rabbit aorta contracting substance,
RCS (21, 37). Normally, formation of TXA2 by platelets is counter
balanced by synthesis of PGI2 by the vascular endothelium, so that
platelet aggregation does not occur. However, in cases of injured ves
sels, there is little release of PGI2 and aggregation of platelets is
possible (18, 19, 35). Thus, the interaction of PGI2 and TXA2 is
important in platelet homeostasis and regulation of blood flow in small
vessels.
Prostaglandins and the Pulmonary Circulation
The lungs of many mammals are able to synthesize and release PG's
as a result of many stimuli, including air embolization (13), infusion
of particles (13, 38-40), anaphylaxis (41) and mechanical distension
(42, 43). Piper and Vane (13) suggested that the feature that is com
mon to these various stimuli is that of mechanical distortion or
stretching of cellular membranes. A possible role for dilator PG's,
released as a result of ventilation, could be the matching of ventila
tion with perfusion (44). This would result in increased blood flow to
areas of the lung that are more stretched, or better ventilated.
Release of primarily dilator PG's has been postulated as an important
part of the large decrease in pulmonary vascular resistance which oc
curs at birth (45), as demonstrated by the increase of PGI-1 ike com
pounds in the pulmonary venous blood of fetal lambs following initia
tion of ventilation.


9
The lungs also serve an important role in the metabolism of many
of the prostaglandins (26, 46, 47), inactivating up to 95 % of the
E-series prostaglandins and 75 % of the F-series prostaglandins. In
the adult the inactivation of PG's which occurs in the pulmonary
circulation "protects" the systemic circulation from the effects of
these vasoactive compounds. However, in the fetus there is a large
proportion of the cardiac output which is shunted through the foramen
ovale and the ductus arteriosus, bypassing the pulmonary circulation,
and metabolism of PG's in the fetus must therefore occur at alternate
sites, such as the liver, kidneys, brain and placenta (48, 49). The
enzyme responsible for the majority of inactivation of PG's, 15-hydroxy
PG dehydrogenase, is present in most tissues, and has been isolated
from the cytosol in the non-membrane bound fraction (48, 50). The
isolated enzyme exhibits substrate specificity for PGE2, PGA2 z2 PGH=^
and PGF2a, producing 15-keto PG's; it has no affinity for PGB2, PGD2 or
PGG2 as substrates (50, 51). Inactivation of PG's in vivo occurs
inside cells. Thus, Bito et^ al_. (52) have shown that a pulmonary PG
transport system exists which facilitates entry of some of the PG's
into the cell. Eling et aj[. (53) demonstrated that PGEj, PGE2 PGF^a
and PGF2a are readily transported into rat lung tissue, and PGD2 is
also thought to be a substrate for the transport system since it com
petitively inhibits uptake of PGE^. Pulmonary tissue (lung homoge
nates) of the rat is capable of inactivating PGI2 enzymatically, but
PGI2 is not transported into the cell (54). However, spontaneous
hydration of PGI2 in the blood shortens its actions (33, 34).
Administration of exogenous PG's intravenously or via the pulmo
nary artery results in dose-related changes in pulmonary vascular


10
resistance (Table 2). Prostaglandin F2a causes a marked pulmonary
vasoconstriction to occur in most adult animals, including dogs (55),
swine (56), sheep (57) and cats (58). The calf (59), fetal goat (60,
61) and newborn lamb (62) also respond to PGF2a by increasing pulmonary
vascular resistance. Similar effects are observed with PGF^ in both
adult and fetal animals (60, 63), and F-series prostaglandins cause
systemic hypertension when administered in doses that exceed the capac
ity of the lungs for inactivation.
Prostaglandins of the E-series are generally vasodilators in most
species and vascular beds studied. In the pulmonary circulations of
swine (56), sheep (57), dogs (64) and rabbits (65), PGE^ causes
decreases in pulmonary vascular resistance, while PGE2 produces pul
monary vasoconstriction. However, both PGE^ and PGE2 cause a drop
in systemic blood pressure in the above studies on adults. The action
of PGE1 in the pulmonary circulation of perinatal goats and newborn
lambs is similar to the response seen in adults, but the effects of
PGE2 are opposite (59, 61, 62, 66, 67). Pulmonary vascular resis
tance in calves (59), perinatal goats (61, 66, 67) and newborn lambs
(62) was decreased when PGE2 was administered. This observed differ
ence between fetus and adult in response of the pulmonary circulation
to PGE2 may be due in part to increased vascular tone of the fetal
lung (68), or to differences in P2* The vascular tone of the neona
tal lung remains elevated slightly over adult conditions, and may thus
explain the decrease in pulmonary vascular resistance with PGE2 seen
in newborn goats and lambs (61, 62, 66, 67).
Although initially described as having no biological activity
(16), PGD2 was found to increase tracheal insufflation pressure


11
Table 2. Summary of actions of prostaglandins on the pulmonary and
systemic circulations
PVR
Fetus
SVR
Newborn
PVR SVR
Adult
PVR SVR
PGF2a
4
4
4
4
+
4
pge2
4-
4
4-
4
4
4
pgd2
4-
-(f)
4,4*
-(f)
i
-(f)
pgi2
4-
4-
4-
4
4
4
txb2
?
?
?
?
4
4
pgh2
?
?
?
?
+
4
The effects of exogenous prostaglandins on pulmonary vascular resis
tance (PVR) and systemic vascular resistance (SVR) of fetal, neonatal
and adult animals are presented. Prostaglandins were administered
either directly into the pulmonary circulation or intravenously.
+ indicates vasoconstriction
4. indicates vasodilatation
- indicates no change
? response is not known
* Pulmonary response in newborn lambs was dependent on dose. Dilata
tion occurred with doses less than 2.5 yg/kg.min; constriction occurred
with doses greater than 8.0 yg/kg.min.


12
and systemic blood pressure in guinea pigs (17). Pulmonary vascular
resistance in the dog has been observed to increase with PGD2, while
systemic blood pressure was either unaffected or was decreased (69-71).
Infusions of PGD2 into the pulmonary circulation of fetal goats
caused dose-dependent decreases in pulmonary vascular resistance, with
either no effect on systemic pressure or a slight increase (72). New
born lambs showed a biphasic response to PGD2> with doses less than
2.5 yg/kg.min causing decreases in pulmonary tone, and doses greater
than 8.0 yg/kg.min producing pulmonary vasoconstriction (72). In
another study using conscious newborn lambs, PGD2 always caused vaso
constriction (62). Adult goats responded to PGD2 with increases in
pulmonary vascular resistance (72). The reason for differences between
fetus, newborn and adult is not clear, but it was postulated that
maturation of receptor population with differing affinities for PGD2
may occur (72).
Prostacyclin has been observed to be a very powerful pulmonary and
systemic vasodilator in both adult and perinatal animals (62, 73-80).
Using similar doses of PGI2 and PGE¡, it was found that PGI2 produced
a 4- to 10-fold greater decrease in systemic and pulmonary arterial
pressures (78). The use of prostacyclin to reduce elevated pulmonary
vascular resistance in the treatment of the clinical syndrome, persis
tent fetal circulation, has been suggested (81), but a caveat has been
issued against this practice due to the severe systemic hypotension
which accompanies infusion of PGI2 (82).
Thromboxanes have been shown to be vasoconstrictors in all vascu
lar beds studied (83, 84). There is greater potency of TXA2 than
TXB2 in stimulating platelet aggregation or smooth muscle contraction


13
(21, 37). Administration of TXB2 to dogs (85, 86) and cats (86)
caused pulmonary and systemic vasoconstriction. The actions of throm
boxanes have not been determined for the perinatal circulation.
Prostaglandin endoperoxides cause smooth muscle to contract (17),
and they have also been observed to increase pulmonary vascular resis
tance in adult monkeys, cats, dogs (87, 88) and sheep (89). Because
the half-life of PGH2 is extremely short, the responses observed could
be due to metabolites. However, use of an endoperoxide analog which is
not subject to further enzymatic action indicates that the vasocon
strictor activity of PGH2 is significant and is not merely due to the
action of a metabolite (69, 88, 90). Tyler et aK (61, 91) observed
pulmonary vasoconstriction in perinatal goats upon administration of
endoperoxide analogs. Systemic hypertension also occurred in these
studies, but the authentic endoperoxide intermediate, PGH2, does not
necessarily yield the same response when infused intravenously (87,
88). Systemic vasodilatation noted following infusion of PGH2 is most
probably the result of dilator prostaglandins such as PGI2 and/or PGE2
being produced from the endoperoxide during passage through the lung.
Arachidonic acid, the fatty acid prostaglandin precursor, produces
pulmonary and systemic vascular effects when injected into dogs (92).
These effects could be blocked by aspirin (92). Kadowitz et aK (69)
showed a dose-response relationship for bolus injections of arachidonic
acid in closed-chest dogs. Increases in pulmonary vascular resistance
were accompanied by decreases in systemic arterial pressure, and both
were eliminated following indomethacin (69). An additional study in
intact dogs demonstrated the ability of arachidonic acid to cause pul
monary vasoconstriction in the absence of formed elements in the blood


14
was diminished during hypoxia (93). Perinatal goats respond to infu
sions of arachidonic acid with pulmonary vasoconstriction and systemic
hypotension, and indomethacin blocked these responses (91). A report
by Mullane et aK (94) was the first to suggest that low dose infu
sions of arachidonic acid caused falls in pulmonary arterial pressure
as well as systemic arterial pressure. Bioassay of arterial blood con
firmed the production of PGI2 (94). A study in cats reported diver
gent responses to arachidonic acid (95). In this report, when pulmo
nary tone was elevated with infusion of PGH2 analog or PGF2a, low dose
infusions of arachidonic acid elicited pulmonary vasodilatation (95).
Thus, response of the pulmonary circulation to arachidonic acid appears
to be dependent in part on existing pulmonary tone.
The release of prostaglandin-like compounds from isolated cat
lungs exposed to alveolar hypoxia was reported in 1974 (96). There was
also a reduction in the hypoxic pressor response in cats which had been
pre-treated with aspirin (96), leading to the proposal that prostaglan
dins were responsible for the pulmonary pressor response to hypoxia.
Several reports followed which argued against a role for prostaglandins
as the mediators of the hypoxic pressor response; instead, they sugges
ted that dilator prostaglandins were released in response to hypoxia to
counter-balance the pulmonary vasoconstriction (97-101). Using isola
ted rat lungs (97, 100), anesthetized dogs (98-100), calves (100) and
perinatal goats (101), these reports demonstrated potentiation of the
hypoxic pressor response following inhibition of prostaglandin synthe
sis with indomethacin (97-101), meclofenamate (97, 100) or aspirin (97-
99). From these studies a moderating role for prostaglandins in
response to hypoxia was proposed.


15
Recently, studies in anesthetized dogs suggest that arachidonic
acid can reduce the hypoxic pressor response (102). In these experi
ments, infusion of arachidonic acid during a period of hypoxia caused
reduction in pulmonary vascular resistance to pre-hypoxic levels (102).
Thin layer chromatography of a sample of systemic arterial blood showed
a major peak corresponding to 6-keto-PGF^a, the metabolite of PGI2
(102). Additional studies demonstrated the release of vasodilator PG's
from isolated rat lung during pulmonary vasoconstriction caused by hy
poxia or angiotensin II (103). 6-Keto-PGFla was measured in the efflu
ent from lungs which were constricted with angiotensin II or hypoxia,
suggesting that pulmonary vasoconstriction causes release of PGI2
(103).
Statement of Purpose
There are conflicting observations on the effects of arachidonic
acid in the pulmonary circulation (69, 91-95). Some studies have
reported only pulmonary vasoconstriction in response to arachidonic
acid (69, 91-93). Others have indicated that low doses of arachidonic
acid cause pulmonary vasodilatation, while higher doses produce pulmo
nary vasoconstriction (94, 95). Hyman et al. (95) have suggested the
difference in response may be due in part to existing level of pulmo
nary vascular resistance. In cats with elevated pulmonary tone, infu
sion of arachidonic acid in low doses caused pulmonary vasodilatation
(95). A report on the actions of arachidonic acid in dogs demonstrated
that infusion of arachidonic acid could reduce the pulmonary vasocon
striction seen in response to hypoxia (102). The response of normoxic


16
perinatal goats to infusions of arachidonic acid was always pulmonary
vasoconstriction, accompanied by systemic hypotension (91).
Based on these observations, the effects of arachidonic acid dur
ing normoxia and hypoxia were evaluated in ventilated fetal and neona
tal lambs using an in situ pump-perfused lower left lung preparation.
The results of these studies indicated that pulmonary vasoconstrictors
were produced from arachidonic acid during both normoxia and hypoxia;
thus, an inhibitor of thromboxane synthetase was tested in this prepa
ration to determine the role of TX's in the pulmonary pressor response
observed with arachidonic acid. Additionally, PG^j an important
intermediate in the prostaglandin biosynthetic pathway, was injected in
fetal lambs before and after ventilation to measure the pulmonary
response. The response of the pulmonary circulation of adult sheep to
arachidonic acid was evaluated under basal conditions and when pulmo
nary tone was enhanced by hypoxia or by infusion of PGF2a. The results
from this last experiment extend the observations made in perinatal
lambs and suggest that the divergent response of the pulmonary circula
tion to arachidonic acid is partly due to species differences.


CHAPTER TWO
PERINATAL PULMONARY RESPONSES TO ARACHIDONIC ACID
DURING NORMOXIA AND HYPOXIA
Introduction
Establishment of normal pulmonary vascular tone in newly venti
lated mammals is due in part to release of dilator prostaglandins
(PG's) (104). Treatment of fetal goats with indomethacin, a PG synthe
sis inhibitor, prior to ventilation does not change the initial rapid
decrease in pulmonary vascular resistance (PVR) which occurs with the
onset of ventilation; however, the slower, prolonged decrease in PVR is
markedly attenuated by indomethacin (104). This attenuation of the
ventilation-induced fall in PVR indicates that under normal conditions
(without PG synthesis inhibition) there is formation of a PG which acts
on the pulmonary vasculature in a vasodilatory manner. Indomethacin
also increases baseline PVR in premature and mature newborn goats
(101), indicating the removal of a dilator influence on the pulmonary
circulation. Recently, Leffler and others (45) measured PG's in pulmo
nary arterial and pulmonary venous blood samples drawn before and after
ventilation of fetal goats and lambs. Ventilation results in a net
increase in PGI-like material produced by the lung. Prostacyclin
(PGI2) is the most potent vasodilator PG of the fetal pulmonary cir
culation (78). Prostacyclin is also the predominant product of PG syn
thesis in perinatal vasculature (105, 106) and is continuously released
17


18
into the systemic circulation from the lungs (107). These findings
suggest an important role of vasodilatory PG's in establishing and
maintaining the low PVR of the newly-ventilated lung.
Dilator PG's are involved in modulating the pulmonary pressor
response to alveolar hypoxia in newborn goats, as evidenced by the
potentiation of the hypoxic pressor response in the presence of a PG
synthesis inhibitor (101). The pressor response to hypoxia is not due
to PG's, as it occurs even when production of PG's is blocked; it
appears that dilator PG's are released during hypoxia and act to
counteract hypoxic pulmonary vasoconstriction (100, 101).
Although perinatal vessels form mostly PGl£, infusions of the
bisenoic PG precursor, arachidonic acid, directly into the pulmonary
circulation of fetal and neonatal goats are associated with dose-
related increases in PVR (91). The increases in PVR are accompanied by
decreases in systemic arterial pressure, and these responses are inhib
ited completely by administration of indomethacin. Thus, the effects
of exogenous arachidonic acid are due to synthesis of PG's, but the
combined pulmonary and systemic effects are unlike those produced by
any single PG infused in a similar manner (108).
The reason for the differences in response of the pulmonary circu
lation to exogenous and endogenous arachidonic acid is not understood.
The following studies are designed to evaluate the pulmonary vascular
responses of ventilated fetal and neonatal lambs to infusions of ara
chidonic acid during normoxia and hypoxia.


19
Materials and Methods
Surgical preparation. Thirteen newborn lambs (2-9 days of age,
4.6 0.2 kg) and mothers of 18 fetal lambs (0.91-0.97 gestation,
3.5 0.2 kg) were anesthetized with chloralose (50 mg/kg, iv) and
tracheotomies were performed. Chloralose (10 mg/kg) was administered
hourly to maintain anesthesia. Fetuses were delivered by cesarean sec
tion and placed on a warmed (40C) table adjacent to the ewe with
umbilical circulation undisturbed. A saline-filled rubber bag was
placed over the fetal head to prevent breathing, and the fetal abdomen
was sutured to the maternal skin to minimize exposure of the umbilical
cord. A tracheal cannula filled with warm (40C) saline was tied in
the fetal trachea and the head cover was removed. In both fetal and
neonatal lambs colonic temperature was monitored (Yellow Springs
Instrument) and maintained between 38 and 40C by use of an infra-red
lamp and placement of a heating pad beneath the animal. The femoral
artery was cannulated for monitoring systemic arterial pressure (SAP)
(Statham P23DC transducer), heart rate and arterial blood gases
(Instrumentation Laboratory pH/blood gas analyzer 213). Newborns were
paralyzed with d-tubocurare (0.2 mg/kg, iv), and controlled ventilation
with positive end-expiratory pressure (PEEP) of 2-4 cm H2O was initi
ated using either a Harvard respirator or a Healthdyne infant ventila
tor. A left thoracotomy was performed with removal of the fourth rib
to permit access to the left lung. Following isolation and dissection
from surrounding tissue of the left pulmonary artery, the lamb was
heparinized (2000 U/kg, iv). A catheter was placed in the femoral vein
and advanced into the inferior vena cava to a position above the level


20
of the liver. Blood was withdrawn from the inferior vena cava and
pumped (Cole-Parmer Masterflex pump no 7016) into the left pulmonary
artery. Between 35 and 40 % of the total lung tissue, as determined by
weight, was perfused in this manner. Flow through the ductus arterio
sus (in fetuses) and to the right lung was not disturbed. Left pulmo
nary blood flow (Q, Statham flowmeter M2202 with In Vivo Metric 3.0 or
4.0 mm cannulating electromagnetic flow probe) was maintained constant.
In fetal lambs, flow was set at a level at which pulmonary arterial
pressure (PAP) was equal to or slightly greater than mean SAP. In new
borns, flow was set to a minimal rate of 10 ml/kg body wt.min, and it
was increased to achieve a maximal flow at which PAP was not greater
than 30 mmHg (ave PAP = 22.4 1.0 mmHg). Flow was normalized for body
weight in order to account for variability in flow rates which were
related to the size of the animal. A catheter was placed in the left
atrium for measurement of left atrial pressure (LAP), and pulmonary
vascular resistance (PVR) was calculated as (PAP LAP) body weight

/ Q. Pressures, flow and heart rate were recorded continuously on a
Gould Brush 480 8-channel polygraph. An Apple 11+ computer was connec
ted to the polygraph with an analog-to-digital interface (AI13, Inter
active Structures, Inc.) for on-line data sampling and calculation of
PVR.
Experimental procedure. The sodium salt of arachidonic acid
(Sigma or NuChek Prep) was stored in dry form at -16C. Fresh solu
tions were prepared daily with saline (final cone = 1 mg/ml) and pro
tected from light prior to being infused (Harvard pump) at varying
rates (0.051 2.06 ml/min) into the pulmonary arterial circuit.


21
Following establishment of baseline values for pressures, flow and
normal arterial blood gases, fluid was aspirated from the fetal tra
chea, the umbilical cord was occluded, and ventilation was started.
Due to the ventilation-induced decrease in PAP, flow was increased to
reflect the normal increase in pulmonary perfusion occurring at birth.
Ventilated fetuses were paralyzed with d-tubocurare (0.2 mg/kg, iv) as
necessary for control of ventilation.
The following treatments were applied in random order to each
animal group after establishing baseline values for pressures and flow
and obtaining normal arterial blood gases and pH. (Criteria for rejec
tion of hemodynamic data were arterial blood samples with pH < 7.30,
PO2 < 75 mmHg, or PCO2 > 50 mmHg.) 1) Arachidonic acid was infused
directly into the left pulmonary arterial perfusion circuit for 2 min
at varying rates. Doses of arachidonic acid were expressed as amount/
kg body wt.min. 2) Hypoxia was produced by lowering the inspired O2
to 6 % for 3 min. 3) A combination of the two treatments was given,
with hypoxia started for 1 min, and arachidonic acid infused during the
second and third minutes of hypoxia. A recovery period of at least 10
min was allowed between each experimental period in order to return to
control values. Due to deterioration of the animal preparations after
several treatments, each animal did not receive every treatment. The
following variables were sampled at intervals of 1 min, with the first
minute of the treatment sampled at 20-sec intervals: PAP, LAP, Q, mean
SAP and heart rate. A reading was taken 1 min before the start of the
treatment, and this value and the value at the beginning of the treat
ment were averaged to give a control value for the variables PVR and
mean SAP. The control values were set to 100 %, and the values at the


22
remaining time intervals were expressed as a percentage of the control
value. Each of the treatments within each animal group was analyzed
using a one-way analysis of variance with repeated measures, and dif
ferences between means were tested with the Newman-Keuls test (109).
Differences between groups or between treatments within the same group
were tested for significance using the unpaired Student's t-test. The
level of significance for statistical differences was P < 0.05.
Results
The average responses to 2-min infusions of arachidonic acid are
shown in Fig 2. The average pulmonary vascular response of 8 venti
lated fetuses to 9 infusions of arachidonic acid (133.7 11.7
yg/kg.min) was a 26 % increase in PVR, at 2 min, over the control PVR.
The mean SAP at the same point was 96 % of the control value; this was
not significantly different from the control SAP. After the infusion
was turned off, PVR returned to baseline within 60 sec, but at the same
time, mean SAP significantly (P < 0.01) decreased to 91 % of the con
trol value and remained close to this level for the next 2 min. A
higher dose range (ave dose = 284.9 12.8 ug/kg.min) was administered
to 10 ventilated fetal lambs, and it resulted in PVR at 40 sec of 156 %
of the control PVR (P < 0.01) (Table 3). Mean SAP at that time was
99 % of control SAP, but it dropped to a low value of 81 % at the fifth
minute. The significant decrease in mean SAP and a longer time for
recovery to baseline at this dose of arachidonic acid were reasons for
not attempting even higher doses.


Figure 2: Average pulmonary and systemic responses to 2-min infusions
of arachidonic acid are shown for 8 ventilated fetuses (9
infusions, ave dose = 133.7 11.7 yg/kg.min) and 6 newborns
(7 infusions, ave dose = 164.0 15.9 yg/kg.min). Data are
expressed as mean SEM. Bars indicate the duration of the
treatment. Points marked with asterisks are significantly
different from the value at 0 min; P < 0.05, ** P < 0.01.


VENTILATED FETAL LAMBS
ARACH ACID-2 min
1 33.7 ^g/kg.hr
TIME (min)
% SAP % PVR
NEWBORN LAMBS
ARACH ACID-2 min
164.0 pg/kg.min
TIME (min)


25
Table 3. Maximal Change in PVR and SAP for all treatments in each group
Ventilated Fetuses
% PVR % SAP
Newborn Lambs
% PVR % SAP
1. Arachidonic Acid
(low dose)-2 min
(inc)
126.33
9.72
191.15
11.87
(dec)

91.23 2.92
--
93.15 3.29
(dose)
133.7
11.7 pg/kg.min
52.8
5.6 pg/kg.min
(n)
9
13
2. Arachidonic Acid
(high dose)-2 min
(inc)
155.71
15.39
301.37
20.24
(dec)

80.51 4.08

82.90 2.93
(dose)
284.9
12.8 pg/kg.min
164.0
15.9 pg/kg.min
(n)
15
7
3. Hypoxia
(6 %)-3 min
(inc)
160.25
13.36 120.47 7.22
143.76
4.51 136.48 5.99
(dec)
--

--
--
(n)
12
13
4. Combination of 1
+ 3
(inc)
192.33
14.96 136.38 12.39
221.26
9.51 121.84 2.08
(dec)

80.78 9.37

82.22 6.65
(dose)
155.2
14.8 pg/kg.min
39.5
4.7 pg/kg.min
(n)
4
5
5. Combination of 2
+ 3
(inc)
234.38
24.71 109.18 15.60
292.04
11.23 126.98 10.65
(dec)

72.43 6.21
--
72.74 4.63
(dose)
315.9
44.6 pg/kg.min
156.0
21.7 pg/kg.min
(n)
4
5
Values shown are maximal increases (inc) or decreases (dec) in PVR or
SAP during the treatment indicated. Data are expressed as mean SEM.
The number of treatments in each group is indicated by n. Treatments 4
and 5 are combinations of 3 min hypoxia and 2 min arachidonic acid infu
sion at dose ranges as in 1 (for treatment 4) or 2 (for treatment 5).
Locations with a dash indicate the response was in only one direction;
i.e., only vasoconstriction or vasodilatation. Note that the doses of
arachidonic acid which are compared in Fig 2 are from treatment 1 (ven
tilated fetuses) and treatment 2 (newborn lambs). Also, Fig 4 shows
doses from treatment 4 for ventilated fetuses and treatment 5 for new
born lambs.


26
In 6 newborn lambs, 7 infusions of arachidonic acid (ave dose =
164.0 15.9 pg/kg.min) caused PVR to increase to 301 % of control PVR
(P < 0.01) (Fig 2). Althoug the dose used was comparable to the lower
dose used in the ventilated fetal lambs, the pulmonary response was
markedly greater in the neonate (P < 0.001). The mean SAP at the end
of the 2-min infusion was 84 % of control SAP, and the combination of
the systemic hypotension and severe pulmonary vasoconstriction preven
ted further increasing the infusion rate to the same levels used in the
ventilated fetal lambs. Instead, the dose of arachidonic acid infused
was lowered (Table 3), so that 10 lambs received 13 infusions at an
average rate of 52.8 5.6 ug/kg.min. This lower dose caused a signif
icant (P < 0.01) increase in PVR to 191 % of baseline PVR, without
causing any significant reductions in mean SAP.
The responses to arachidonic acid observed in ventilated fetal and
neonatal lambs were due to production of PG's, as administration of
indomethacin (2 mg/kg, iv, 1 ventilated fetus) or meclofenamate (2
mg/kg, iv, 1 lamb) prevented any response when arachidonic acid was
infused after 15 min. Also, infusion of a fatty acid, 11,14,17-eicosa-
trienoic acid, which does not undergo conversion to PG's, did not cause
increases in PVR or systemic hypotension in 1 newborn and 1 ventilated
fetus.
The effects of 3 min of alveolar hypoxia (inspired O2 = 6 %) are
illustrated in Fig 3. The pulmonary vascular responses in neonates and
ventilated fetuses were similar with a slightly greater increase in PVR
in the ventilated fetal lambs (160 % of control) than in the newborns
(144 % of control) (Table 3). The increase in mean SAP was also simi
lar (ventilated fetuses, 120 %; newborn lambs, 134 %). This period of


Figure 3: Average pulmonary and systemic responses to 3-min periods of
hypoxia (FIq2 = 0*06) are shown for 10 ventilated fetuses
(12 cases) and 12 newborn lambs (13 cases). Symbols are as
in Fig 2.


VENTILATED FETAL LAMBS
HYPOXIA-6%-3 min
TIME (min)
UAd % dVS %
NEWBORN LAMBS
HYPOXIA-6%-3 min
r\s
Co
TIME (min)


29
hypoxic exposure produced a consistent response in each group of ani
mals and was accompanied by a severe decrease in arterial O2 tension
(Table 4), with recovery to baseline values occurring within 10 min in
most cases.
The results of the combination of 3 min of hypoxia with a 2-min
infusion of arachidonic acid are shown in Fig 4. In 5 newborn lambs,
PVR increased to 292 % of control PVR at the end of the combination,
but this response was not significantly different (P > 0.5) from the
response to a similar dose of arachidonic acid during normoxia (Table
3). In ventilated fetal lambs receiving the lower dose of arachidonic
acid, PVR increased to 192 % of control PVR during alveolar hypoxia and
arachidonic acid infusion. This increase was significantly greater
(P < 0.01) than the response to arachidonic acid alone, but appeared to
be an additive effect to the hypoxic pressor response. Thus, the pul
monary response to arachidonic acid was not affected by exposure to
alveolar hypoxia in newborn lambs, but was increased significantly in
ventilated fetal lambs during hypoxia. However, mean SAP was reduced
following the termination of the hypoxia and infusion in both groups of
animals (Fig 4). The fall in SAP to 81 % of control values at 3 min
after the end of the treatment in the ventilated fetuses receiving the
low dose of arachidonic acid was significantly different from the
increase in mean SAP to 136 % of control at the end of the treatment
(P < 0.01). In neonatal lambs, the increases in mean SAP during the
combined hypoxia and arachidonic acid (values at 40 sec, 1, 2 and 3
min) were significantly different from mean SAP following this treat
ment (values at 4, 5 and 6 min, P < 0.01). The time course and extent
of the systemic hypotension in the two animal groups during the


Table 4.
Arterial blood gases and pH before, during
and following hypoxia
pH
P02
PC02
(mmHg)
(mmHg)
1. Ventilated Fetal
Lambs
(n = 8)
Before Hypoxia
7.46
+
0.03
123.8 10.0
33.0 1.8
During Hypoxia
7.45
+
0.03
18.6 2.1
34.3 2.1
After Hypoxia
7.42
+
0.02
102.2 13.5
35.2 1.3
2. Newborn Lambs
(n = 12)
Before Hypoxia
7.43
+
0.02
127.7 8.6
33.5 2.1
During Hypoxia
7.43
+
0.02
20.9 0.9
31.8 1.8
After Hypoxia
7.41
+
0.02
112.8 8.5
34.3 1.8
Data are expressed as mean SEM.


Figure 4: Average pulmonary and systemic responses to a combination of
3-min periods of hypoxia (FIq2 = 0.06) and 2-min infusions
of arachidonic acid are shown in 2 ventilated fetuses (4
cases, ave dose = 155.2 14.8 wg/kg.min) and 5 newborns (5
cases, ave dose = 156.0 21.7 yg/kg.min). Symbols are as
in Fig 2.


VENTILATED FETAL LAMBS
HYPOXIA-6%-3 min
ARACH ACID-2 min
155.2 yg/kg.min
TIME (min)
* SAP % PVR
NEWBORN LAMBS
HYPOXIA-6%-3 min
ARACH ACID-2 min
1 56.0 *ig/kg.min
TIME (min)


33
combination of hypoxia and arachidonic acid were markedly different
from the responses to arachidonic acid alone (Fig 2 and 4).
Discussion
Intra-pulmonary infusions of arachidonic acid in 3 dose ranges
caused significant increases in PVR in ventilated fetal and neonatal
lambs. At comparable doses (100-200 yg/kg.min), newborns showed sig
nificantly greater increases in PVR than ventilated fetal lambs. This
finding is similar to an earlier report on arachidonic acid in fetal
and newborn goats (91). A possible explanation for the greater respon
siveness of the neonate to arachidonic acid is the increase in lung
microsome cyclo-oxygenase activity which occurs after birth (110).
This increase in cyclo-oxygenase activity results in higher concentra
tions of PGH2 available for further metabolism in newborns. Addi
tionally, PGH2 formed in the lung may exert a local effect on pulmo
nary vessels before undergoing metabolism to other PG's. Studies in
perinatal goats indicate that a stable endoperoxide analog caused pul
monary vasoconstriction (91). Thus, if similar amounts of arachidonic
acid were infused in newborns and ventilated fetuses, the ability of
newborns to form more PGH2 could result in more severe pulmonary
vasoconstriction (Fig 2).
Pulmonary metabolism of arachidonic acid is dependent on substrate
availability, relative activities of various PG enzymes, and cofactor
levels. Arachidonic acid is converted rapidly to PGH2 by cyclo-oxy
genase (15), and the endoperoxide intermediate either is released into
the circulation or is metabolized further. The quantity and nature of


34
PG's formed from PGH2 depend on the PG enzyme activities and the
amount of PGH2 present. In homogenates from adult cat lungs, the
production rates of 6-keto-PGF^a and TXB=2 (metabolites of PGI2 and
TXA2, respectively) were equal at low levels of PGH2 (less than 10 yM)
(111). However, as the concentration of PGH2 was increased, there
was up to 7 times more TXB2 than 6-keto-PGF^a produced. Studies in
fetal and newborn sheep lung homogenates indicate the activity of pros
tacyclin synthetase was relatively low and constant throughout gesta
tion (110). The enzyme also exhibited saturation at low substrate
levels. However, thromboxane synthetase activity increased during ges
tation and continued to rise post-natally, so that at higher concentra
tions of PGH2, neonatal lung homogenates formed up to 5 times as much
TXB2 as 6-keto-PGFja.
A problem with studies using isolated homogenates is a lack of
demonstrated biological activity of the products. Thus, the above stu
dies which measure amounts of PG products formed by lungs do not indi
cate any vasoactive properties of the compounds produced. The short
half-life of TXA2 makes it difficult to obtain a dose-response curve
in vivo, so that relative potencies for TXA2 and PGI2 cannot be
assessed. Based on the present study, the net effect of arachidonic
acid infusion was pulmonary vasoconstriction, indicating either activi
ty due to PGH2, greater formation of TXA2, greater potency of TXA2 on
pulmonary vascular smooth muscle, or a combination of the above. Also,
there could be an interaction with the primary PG's, such as PGF2a
or PGD2, which are pulmonary vasoconstrictors in newborn goats and
lambs (60, 72).


35
The pulmonary vascular effects of exogenous arachidonic acid
infused directly into the pulmonary circulation of neonatal lambs were
not altered by concomitant exposure to alveolar hypoxia. Arachidonic
acid increased PVR over the hypoxic pressor response, but the extent of
pulmonary vasoconstriction was so severe with infusions alone that it
appears the pulmonary vessels may have been unable to increase tone any
further in response to hypoxia. Although ventilated fetuses exhibited
a significantly greater response to arachidonic acid infused during
hypoxia than during normoxia, the increase in PVR was merely additive
to the hypoxic pressor response. Thus hypoxia does not appear to
affect the actions of exogenous arachidonic acid in the perinatal pul
monary circulation.
In contrast to these studies, recent reports using adult dogs
demonstrated a reduction in hypoxic pulmonary vasoconstriction during
infusion of arachidonic acid (102, 112). The dose administered in the
study by Gerber et al_. (102) was similar to the lower doses infused in
newborns in the present study; however, the only response seen in lambs
was pulmonary vasoconstriction, whether arachidonic acid was infused
during normoxia or hypoxia (Table 3). A recent study in conscious
adult sheep demonstrated that infusions of arachidonic acid at doses of
25, 50 and 100 pg/kg.min caused pulmonary vasoconstriction (113).
Thus, differences in responses may be due to species differences (27).
The hypotensive effects of arachidonic acid on the systemic circu
lation were insignificant at the low doses infused into newborns, but
with higher doses mean SAP decreased significantly in both newborn and
ventilated fetal lambs. This is consistent with reports of PGI2
being the predominant PG released by the lung (20, 45, 107). The


36
systemic response to hypoxia was an increase in mean SAP, with a rapid
return to base!in. The addition of arachidonic acid during hypoxia
caused SAP to drpo below control levels at the end of the combined
treatment, and at the end of 5 min mean SAP was still significantly
depressed. These findings are in agreement with studies indicating an
increase in vasodilator PG's during hypoxia (100, 101) and preliminary
data which show an increased efflux of PG's several minutes after in
fusing arachidonic acid from lungs of newborn lambs which were perfused
with Krebs solution (Leffler, personal communication).
Although arachidonic acid forms a moderate amount of a systemic
vasodilator when infused during normoxia, the significantly greater
reduction in mean SAP and the prolonged time for recovery from systemic
hypotension during hypoxia and arachidonic acid indicate an increase in
the amount of vasodilator substance coming from the lung. Additional
ly, PGI2 may be produced by vascular endothelia in the systemic cir
culation. Infusions of arachidonic acid produce only vasoconstriction
in the perinatal pulmonary circulation, during both normoxia and hypox
ia. The most likely explanation for these results is that high concen
trations of PGH2 are formed in the lung with subsequent release of
TXA2 and/or PGH2. Both of these compounds are presumed to have
vasoconstrictor activity, and both have short half-lives. The predomi
nant effect is thus pulmonary vasoconstriction caused by one of these
products of arachidonic acid metabolism, with further metabolism of
PGH2 to PGI2 and degradation of TXA2 to a relatively weak metabolite.
This would result in systemic hypotension due to the predominance of
PGI2 at this time. The effect of hypoxia upon arachidonate metabo
lism seems to reflect no change in PG products acting in the lung, but


37
may indicate an enhanced release of PGI2 from the lungs into the
systemic circulation.


CHAPTER THREE
THROMBOXANE SYNTHETASE INHIBITION AND PERINATAL PULMONARY
RESPONSE TO ARACHIDONIC ACID
Introduction
Biological activity of the potent vasoconstrictor and proaggrega-
tory substance TXA2 can be blocked either by inhibition of TX synthe
tase or with a TXA2 receptor antagonist (114). Inhibitors of TX syn
thetase include imidazole, and its derivatives (114), and pyridine, and
its derivatives (115, 116); TXA2 receptor antagonists include analogs
of PGH2, such as 9,ll-epoxyimino-5,13-dienoic acid (114). Pyridine
and its derivatives have been described as being capable of preventing
release of TXB2 from platelets and from lung microsomes by inhibiting
TX synthetase (115, 116). The pyridine derivative found to be the most
potent inhibitor of TX synthetase inhibition was characterized as p-[4-
(2-carboxy-l-propenyl)benzyl] pyridine hydrochloride (OKY-1555) (115),
and its sodium salt is known as OKY-1581 (sodium (E)-3-[4-(3-pyridyl-
methyl)phenyl]-2-methyl acrylate) (117).
OKY-1581 inhibited platelet aggregation induced by arachidonic
acid in vitro (118), and reduced concentrations of TXB2 in blood of
monkeys (118), rabbits (119), and humans (120) when administered
orally. The incidence of arachidonic acid-induced cerebral infarction
38


39
in rabbits was reduced by pre-treatment with OKY-1581 (118). In dogs
with a partially obstructed coronary artery, intravenous administration
of OKY-1581 inhibited the platelet aggregation caused by the obstruc
tion (121). Inhibition of TX synthetase occurred without affecting
synthesis of PGI2 (121).
In fetal and neonatal lambs and goats, infusions of arachidonic
acid into the pulmonary circulation produced dose-related increases in
pulmonary vascular resistance (91, Ch 2). This pressor response may be
due to 1) increased production of the pressor agent TXA2, 2) forma
tion of PGH2 or 3) mechanical obstruction due to platelet aggregation.
Thus, inhibition of TX synthesis might prevent pulmonary vasoconstric
tion due to arachidonic acid. The purpose of this study was to test
the effect of a specific TX synthetase inhibitor, OKY-1581, on the
response of the perinatal pulmonary circulation to infusions of arachi
donic acid and hypoxia.
Materials and Methods
Surgical preparation. The surgical procedure for the in situ
pump-perfused lower left lung preparation in perinatal lambs has been
described in detail in the preceding chapter. Mothers of 12 fetal
lambs (0.91-0.97 gestation, wt = 3.7 0.1 kg) and 13 newborn lambs
(2-9 days of age, wt = 4.6 0.2 kg) were anesthetized with chloralose
and prepared so that left pulmonary arterial pressure (PAP), left
atrial pressure (LAP), systemic arterial pressure (SAP) and heart rate
were monitored continuously under conditions of constant left pulmonary

arterial blood flow (Q). By maintaining Q constant, changes in PAP


40
reflected changes in pulmonary vascular resistance (PVR), which was
calculated as (PAP LAP) body wt / Q. In these experiments fetal
lambs were ventilated and the umbilical cord was occluded.
Experimental procedure. The sodium salt of arachidonic acid
(Sigma or NuChek Prep) was stored in dry form at -16C. Fresh solu
tions were prepared daily with saline (final cone = 1 mg/ml) and pro
tected from light prior to being infused (Harvard pump) at varying
rates (0.051 2.06 ml/min) into the pulmonary arterial circuit.
OKY-1581 (Ono Pharmaceutical Co.) was received and stored in dry form
at 6C. On the day of the experiment, the compound was diluted with
0.9 % saline to a concentration of 50 mg/ml.
The following treatments were applied in random order to newborn
lambs after establishing baseline values for pressures and flow and
obtaining normal arterial blood gases and pH. (Criteria for rejection
of hemodynamic data were arterial blood samples with pH < 7.30, PO2 <
75 mmHg, or PCO2 > 50 mmHg.) 1) Arachidonic acid was infused direct
ly into the left pulmonary arterial circuit for 2 min at varying rates.
2) Alveolar hypoxia was produced by lowering the inspired O2 to 6 %
for 3 min. 3) A combination of the two treatments was given, with
hypoxia started for 1 min, and arachidonic acid infused during the
second and third minutes of hypoxia. A recovery period of at least 10
min was allowed between successive experimental periods in order to
return to control values. Lambs then received 50 mg of OKY-1581,
injected slowly into the pulmonary arterial circuit, and the treatments
were repeated. Thromboxane inhibition lasted at least 2 hours, and
lambs showed responses to arachidonic acid at 2 hours after OKY-1581
which were similar to responses at 15 min after inhibition. Lambs


41
which did not receive OKY-1581 (Ch 2) did not show attenuation of pul
monary vascular responses to arachidonic acid after repeated infusions.
Ventilated fetuses were tested for responses to arachidonic acid only,
both before and after TX synthesis inhibition. Due to deterioration of
the animal preparations after several treatments, some animals did not
receive every treatment. The following variables were measured at
intervals of 1 min, with the first minute of the treatment sampled at
*
20-sec intervals: PAP, LAP, Q, mean SAP and heart rate. A measurement
was made 1 min before the start of the treatment, and this value and
the value at the beginning of the treatment were averaged to give a
control value for the variables PVR and mean SAP. The control values
were set to 100 %, and the values at the remaining time intervals were
expressed as a percentage of the control value. The treatments within
each animal group were analyzed using one-way analysis of variance with
repeated measures, and differences between means were tested with the
Newman-Keuls test (109). Differences between treatments within the
same animal group were tested for significance using the unpaired
Student's t-test. The level of significance for statistical differ
ences was P < 0.05.
Results
The effects of treatment with the specific TX synthetase inhibitor
OKY-1581 on infusions of arachidonic acid in neonatal lambs are shown
in Fig 5 and 6. In 8 lambs receiving 2-min infusions of arachidonic
acid (ave dose = 52.4 6.8 pg/kg.min, Fig 5), pre-treatment with
OKY-1581 significantly (P < 0.01) reduced the increase in PVR at 40 sec


Figure 5: Average pulmonary and systemic responses to 2-min infusions
of arachidonic acid are shown for 10 newborn lambs (13
infusions, ave dose = 52.8 5.6 ug/kg.min) and 8 newborns
treated with 50 mg OKY-1581 (13 infusions, ave dose = 52.4
6.8 yg/kg.min). Symbols are as in Fig 2.


NEWBORN LAMBS
ARACH ACID-2 min
52.8 >jg/kg.min
160
140 -
QC
a 120 -
*
100 ih
80
a.
<
CO
140
120
100
80
80
- 1
TIME (min)
NEWBORN LAMBS
ARACH ACID-2 min
52.4 pg/kg.min
FOLLOWING OKY-1581
l
J L J I I 1IL.
lili
00
r
J L-
J I I I I I I I I I I I I I I L.
TIME (min)


Figure 6: Average pulmonary and systemic responses to 2-min infusions
of arachidonic acid are shown for 6 newborn lambs (7
infusions, ave dose = 164.0 15.9 yg/kg.min) and 6 lambs
treated with 50 mg 0KY-1581 (6 infusions, ave dose = 141.5
14.5 yg/kg.min). Symbols are as in Fig 2.


NEWBORN LAMBS
ARACH ACID-2 min
184.0 jjg/kg.min
TIME (min)
% SAP % PVR
NEWBORN LAMBS
ARACH ACID-2 min
141.5 jjg/kg.min
FOLLOWING OKY-1581
TIME (min)


46
as compared with lambs without the inhibitor, and the response remained
lower than the pulmonary response to arachidonic acid (ave dose = 52.8
5.6 yg/kg.min) without OKY-1581 until the fifth minute. The peak
increase in PVR due to arachidonic acid following OKY-1581 occurred at
1 min, and was only 17 % of the peak increase produced by arachidonic
acid alone, which occurred at 2 min. At this dose of arachidonic acid
there were no significant differences in the response of the systemic
circulation in the presence or absence of TX synthesis inhibition.
There is a suggestion of a greater fall in systemic pressure. Injec
tion of OKY-1581 produced transient (less than 60 sec) decreases in
PAP. Pressure returned to baseline before infusions or hypoxia were
tested.
Figure 6 shows average responses to a higher dose of arachidonic
acid in 6 newborns. Addition of OKY-1581 significantly (P < 0.005)
reduced the pulmonary response to arachidonic acid (ave dose = 141.5
14.5 yg/kg.min) at 40 sec, and PVR remained at significantly lower
values than observed in lambs without OKY-1581 until the sixth minute.
The maximal increase in PVR in lambs receiving OKY-1581 occurred at 1
min, and was 26 % of the peak increase in PVR at 2 min in lambs without
the blocker. Systemic blood pressure decreased significantly from con
trol values in both groups of lambs (with and without OKY-1581) at 2
and 3 min (Fig 6), but the fall in SAP in lambs receiving the inhibitor
was more than double the decrease in SAP in lambs without OKY-1581.
This significant (P < 0.025) increase in the systemic hypotensive ef
fect of arachidonic acid at 3 min was sustained for the remaining time
intervals, so that at 6 min, mean SAP was only 75 % of control SAP in
treated lambs.


47
The effects of 3 min of hypoxia (inspired O2 = 6 %) in the
presence and absence of TX synthetase inhibition are shown in Fig 7.
There were no significant differences in the responses of the pulmonary
circulation in lambs receiving OKY-1581 prior to hypoxia as compared
with responses in lambs without the inhibitor. The effect of hypoxia
on the systemic blood pressure was also similar in the two groups of
lambs, with a slight, although insignificant, depression of the
increase in SAP during hypoxia occurring in the OKY-treated lambs.
After the hypoxic period ended, SAP in lambs receiving OKY-1581 fell
below baseline values. The decrease observed in SAP at 4, 5 and 6 min
was not significantly different from the control SAP in the TX inhibit
ed group; however, the value at 5 min was significantly (P < 0.01) less
than the SAP at 5 min in the lambs without OKY-1581.
Newborn lambs which had received OKY-1581 responded in a similar
manner to combined hypoxia and arachidonic acid infusions as lambs
which were not treated with the blocker (Table 5). In both groups ara
chidonic acid caused a further increase in PVR over the hypoxic pressor
response alone, but the combined response was not greater than the ad
ditive response of arachidonic acid and hypoxia alone. The pulmonary
response to the lower dose of arachidonic acid during hypoxia in 3 OKY-
treated lambs was significantly (P < 0.05) less than the increase in
PVR in 5 untreated lambs at 2 and 3 min. The peak PVR in lambs with
OKY was 62 % of the peak PVR in lambs without the blocker. At the
higher dose of arachidonic acid infused during hypoxia, 3 lambs receiv
ing OKY-1581 showed significantly smaller increases in PVR at the
second and third minutes of the response (P < 0.01) than 5 lambs with
out the inhibitor. The difference between the two groups was still


Figure 7: Average pulmonary and systemic responses to 3-min periods of
hypoxia (FIq2 = 0.06) are shown for 12 newborn lambs (13
cases) and 6 lambs treated with 50 mg OKY-1581 (7 cases).
Symbols are as in Fig 2.


NEWBORN LAMBS
HYPOXIA-6%-3 min
TIME (min)
% SAP % PVR
NEWBORN LAMBS
HYPOXIA-6%-3 min
FOLLOWING OKY-158 1
TIME (min)


50
Table 5. Maximal Change in PVR and SAP for all treatments
without OKY-1581
with OKY-1581
% PVR
% SAP
% PVR
% SAP
Venti1ated
Fetuses
1. Arachidonic Acid
(high dose)-2 min
(inc)
155.71
15.39
125.93
5.21
(dec)
96.01
2.33 80.51 4.08
80.68
2.10 69.48 3.62
(dose)
284.9
12.8 yg/kg.min
296.9
13.1 yg/kg.min
(n)
15
11
Newborn Lambs
1. Arachidonic Acid
(low dose)-2 min
(inc)
191.15
11.87
115.45
4.13
(dec)
--
93.15 3.29
--
86.58 4.01
(dose)
52.8
5.6 yg/kg.min
52.4
6.8 yg/kg.min
(n)
13
13
2. Arachidonic Acid
(high dose)-2 min
(inc)
301.37
20.24
152.60
9.20
(dec)

82.90 2.93
91.92
2.59 62.02 8.00
(dose)
164.0
15.9 yg/kg.min
141.5
14.5 yg/kg.min
(n)
7
6
3. Hypoxia
(6 %)-3 min
(inc)
143.76
4.51 136.48 5.99
153.37
9.86 124.49 3.66
(dec)

--
--
86.77 4.24
(n)
13
7
4. Combination of 1
+ 3
(inc)
221.26
9.51 121.84 2.08
174.58
14.23 130.63 4.44
(dec)
--
82.22 6.65
--
86.88 6.24
(dose)
39.5
4.7 yg/kg.min
39.5
0.4 yg/kg.min
(n)
5
4
5. Combination of 2
+ 3
(inc)
292.04
11.23 126.98 10.65
189.90
32.69 140.40 26.59
(dec)

72.74 4.63
--
75.40 7.28
(dose)
156.0
21.7 yg/kg.min
145.8
22.9 yg/kg.min
(n)
5
3
Values shown are maximal increases (inc) or decreases (dec) in PVR or
SAP during the treatment indicated. Data are expressed as mean SEM.
The number of observations in each treatment is indicated by n. Treat
ments 4 and 5 under newborn lambs are combinations of 3 min hypoxia and
2 min arachidonic acid infusion at dose ranges as in 1 (for treatment 4)
or 2 (for treatment 5). Locations with a dash indicate the response was
in only one direction; i.e., only vasoconstriction or vasodilatation.


51
present at 4 and 5 min (P < 0.05). There was a 47 % reduction in peak
PVR in the OKY-treated lambs as compared with the peak PVR in the
untreated group. There were no significant differences between the two
groups in responses of the systemic circulation to combinations of
hypoxia and arachidonic acid at either dose of arachidonate used (Table
5).
Pulmonary and systemic vascular responses to infusions of arachi
donic acid in ventilated fetal lambs are shown in Fig 8. The increase
in PVR at 40 sec in 6 fetuses receiving OKY-1581 was 54 % less than the
peak PVR, also at 40 sec, in 10 fetuses without the blocker. Although
the depressed PVR in treated fetal lambs was not significantly differ
ent from the peak PVR in the untreated group, the values at 2, 3, 4, 5
and 6 min in fetuses with OKY-1581 were significantly lower than cor
responding values in untreated ventilated fetal lambs (P < 0.025, 0.02,
0.001, 0.001, 0.001, respectively). Also, in the OKY-treated fetuses,
decreases in PVR at 3, 4, 5 and 6 min were significantly (P < 0.01)
less than control PVR (Fig 8). Systemic pressure was significantly (P
< 0.05) reduced from control SAP at 1 min in TX-inhibited fetuses, and
values for SAP at 2 and 3 min in the treated group were significantly
(P < 0.01, 0.025, respectively) lower than corresponding values in ven
tilated fetuses without OKY-1581.
Discussion
Inhibition of TX synthetase with the pyridine derivative OKY-1581
depressed the increases in PVR which occur with infusions of arachidon
ic acid in perinatal lambs. The dose of OKY-1581 used in this study


Figure 8: Average pulmonary and systemic responses to 2-min infusions
of arachidonic acid are shown for 10 ventilated fetal lambs
(13 infusions, ave dose = 284.9 12.8 yg/kg.min) and 6
ventilated fetuses treated with 50 mg OKY-1581 (11 infu
sions, ave dose = 296.9 13.1 yg/kg.min). Symbols are as
in Fig 2.


% SAP % PVR
VENTILATED FETAL LAMBS
ARACH ACID-2 min
284.9 pg/kg.min
120
100 Ih
0.
w 80 -
*
60 -
40
-1
TIME (min)
VENTILATED FETAL LAMBS
ARACH ACID-2 min
296.9 jjg/kg.min
FOLLOWING OKY- 158 1
l I i I il I l l I I l 1 A
TIME (min)


54
(50 mg, or 10 15 mg/kg) was at least 10 times the effective dose
administered to rabbits to prevent arachidonate-induced sudden death
(115). However, the effectiveness of the inhibition was not estab
lished in these studies. When animals were treated with OKY-1581, the
peak increase in PVR in response to infusions of arachidonic acid was
16 %, 26 % and 46 % of the peak pulmonary response without the inhibi
tor, depending on the dose of arachidonic acid infused (Table 5). The
hydrochloride compound OKY-1555 has been characterized as a noncompeti
tive inhibitor of TX synthetase (115). Presumably, the sodium salt
used in this study, OKY-1581, acts in the same way as the hydrochloride
salt. Therefore, one likely explanation for our findings is that the
increase in PVR seen in the presence of OKY-1581 is caused by PG endo-
peroxides. Alternatively, the enzyme may not have been inhibited com
pletely. The pharmacology of OKY-1581 has not been studied with res
pect to TX synthetase of developing sheep lung.
Treatment with OKY-1581 also significantly enhanced systemic hypo
tension caused by infusions of arachidonic acid in lambs. This could
result from increased production of vasodilator PG's in the lung due to
shunting of PGH2 away from TX synthetase. This is consistent with
studies showing increased formation of 6-keto-PGF^a with TX synthetase
inhibition (120, 121) and may contribute to the "steal" phenomenon pro
posed by Gryglewski and others (18). Thus, formation of PG endoperox-
ides could give rise to increased production of PGI2 which is a po
tent vasodilator of both systemic and pulmonary circulations in perina
tal goats and lambs (78). Additionally, any possible pressor action of
TXA2 itself on the systemic circulation would be missing.


55
Pulmonary vasoconstriction in response to hypoxia was unaffected
by inhibition of TX synthesis (Fig 7). Hypoxic pulmonary vasoconstric
tion is not believed to be produced by PG's (100, 101). Inhibition of
cyclo-oxygenase results in potentiation of the hypoxic pressor response
(100, 101), indicating removal of net PG dilator influence during
hypoxia. 0KY-1581 does not interfere with enzymes responsible for
producing dilator PG's (118, 120). PGI2 PGE2 and PGD2 are dilators in
the perinatal pulmonary circulation, and PGI2 and PGE2 are systemic
vasodilators as well (67, 72, 78). Thus, inhibition of TX synthetase
should not affect the production of these dilator PG's in response to
hypoxia. There was a greater reduction in SAP after hypoxia (at 5 min)
in lambs treated with 0KY-1581 as compared to untreated lambs (Fig 7),
perhaps due to increased formation of dilator PG's in the presence of
TX synthesis inhibition.
One of the problems not addressed in this study is the role of
platelets in the pulmonary response to arachidonic acid. It is con
ceivable that increases in PVR seen in response to infusions of arachi
donic acid are due in part to platelet aggregation leading to plugging
of the pulmonary microvasculature. Thromboxanes are potent platelet
aggregatory compounds, and they also stimulate vascular smooth muscle
contractions in vitro (21). Several authors (93, 122, Leffler, per
sonal communication) suggest that platelets are not necessary for pul
monary vasoconstriction in response to arachidonic acid. Hyman and
others (93) demonstrated that the pressor response to arachidonic acid
was similar in canine lungs perfused with either blood or platelet-poor
plasma. Leffler (personal communication) has found that neonatal lamb
lungs which were perfused with Krebs solution respond to exogenous


56
arachidonic acid with pulmonary vasoconstriction. Increases in PAP
occurred in isolated rabbit lungs perfused with Krebs-Henseleit albumin
buffer with addition of arachidonic acid to the perfusate (122). In
the above study, arachidonate-induced increases in PAP were substan
tially reduced by imidazole, a TX synthetase inhibitor (122). Thus, it
appears likely that platelets are not needed to demonstrate activity of
TX synthetase in producing pulmonary hypertension in response to ara
chidonic acid.
The inability of OKY-1581 to completely block the arachidonate-
induced increase in PVR was surprising. In adult sheep pulmonary
hypertension induced by endotoxin infusion was correlated with a sub
stantial increase in TXE$2 (123). If TXA2 were solely responsible
for the increase in PAP observed in adult sheep, it would appear that
prevention of TX formation should prevent pulmonary hypertension.
OKY-1581 was unable to completely prevent increases in PVR due to ara
chidonic acid, leading to the proposal that the remaining response is
due to formation of PGh^. Prostaglandin H2 is a potent pulmonary
vasoconstrictor in adult dogs (88) and in adult sheep (89). The endo-
peroxide formed in the lung may participate in a "steal" phenomenon,
resulting in increased formation of PGI2 Thromboxanes appear to be
responsible for the majority of the increase in PVR in response to ara
chidonic acid in perinatal lambs. Inhibition of TX synthetase with
OKY-1581 resulted in greater than 50 % reduction in pulmonary vasocon
striction caused by arachidonic acid, and there was significant en
hancement of systemic hypotension in the OKY-treated animals. The
increased systemic hypotension in OKY-treated lambs is probably a
result of increased formation of PGI2 from endoperoxides due to
prevention of formation of TXA2.


CHAPTER FOUR
EFFECTS OF PROSTAGLANDIN H? ON PERINATAL
PULMONARY CIRCULATION
Introduction
Prostaglandin H2 functions as the key intermediate in PG synthe
sis from which the stable products, PG2, PGE2 and PGF2a, as well as
the unstable compounds, PGI2 and TXA2, are derived. The terminal
enzymes which yield these various products are present in varying
amounts and activities in different organs, and this distribution
contributes to the wide range of responses of individual systems to
release of PG's (16, 18, 25-28 ). There is evidence that PGH2 is
capable of eliciting direct responses in biological systems without
undergoing conversion to other PG's (14, 17). Kadowitz et al_. (88)
demonstrated the ability of PGH2 to contract isolated segments of
canine intrapulmonary vein. Additionally, bolus injections of PGH2
caused increases in lobar arterial and small vein pressures in pump-
perfused canine lungs (88). Unanesthetized adult sheep responded to
infusions of PGH2 with dose-related increases of pulmonary arterial
pressure (89). In both of these studies, a stable analog of PGH2
also produced pulmonary vasoconstriction (88, 89). Endoperoxide
analogs have been shown to cause dose-dependent increases in pulmonary
vascular resistance (PVR) and systemic arterial pressure (SAP) in
57


58
perinatal goats (91). Recently it was demonstrated that increases in
PVR produced by infusions of arachidonic acid in perinatal lambs were
not abolished by TX synthetase inhibition (Ch 3). It was proposed that
a portion of the arachidonate-induced increase in PVR was due to PG
endoperoxides. The effects of PGH2 were studied in pump-perfused
lungs of perinatal lambs in the present study.
Materials and Methods
Surgical preparation. The details of the surgical procedure for
the in situ pump-perfused lower left lung preparation in perinatal
lambs have been described in Ch 2. Mothers of 7 fetal lambs (0.93 -
0.97 gestation, wt = 3.6 0.1 kg) were anesthetized with chloralose,
and fetuses were delivered by cesarean section. Fetuses were surgical
ly prepared so that left pulmonary arterial pressure (PAP), left atrial
pressure (LAP), SAP and heart rate were monitored continuously under
4
conditions of constant left pulmonary arterial blood flow (Q). Flow
was set at a level at which PAP was equal to or slightly greater than

mean SAP. By maintaining Q constant, changes in PAP reflected changes
in PVR, which was calculated as (PAP LAP) body wt / Q.
Experimental procedure. Prostaglandin H2 was prepared by Dr.
D.B. McNamara (Tulane Univ., New Orleans) using the method of Egan et
al. (124), as described by She et a]_. (111)* The endoperoxide was
shipped on dry ice and stored in a hexane:ether (4:6) solution at
-85C. Purity was estimated by chromatography of (1-^C) PGH2 on
silica gel plates and by radiochromatographic scan and scraping (111).
Purity of PGH2 was greater than 90 %. Immediately before injection


59
of PGH2, an aliquot of the stock solution was dried under a stream of
nitrogen, diluted with cold saline and drawn into a chilled Hamilton
syringe. An injection of 0.1 ml of various concentrations of PGH2
was performed within 15 sec of dilution with saline. The concentra
tions of PGH2 used were 1.0, 10.0, 20.0 and 40.0 yg/ml. OKY-1581
(Ono Pharmaceutical Co), a pyridine derivative, or UK 37,248-01 (Pfiser
Chemical Co), an imidazole derivative, was used to inhibit TX synthe
sis. OKY-1581 was diluted with saline to a concentration of 50 mg/ml
and 1.0 ml was injected into the pulmonary arterial circuit. UK
37,248-01 was diluted with saline to a concentration of 6.0 or 10.0
mg/ml, so that 1.0 ml of solution gave a dose of approximately 2 mg/kg.
These doses were greater than the effective doses in rabbits (115).
Following the surgical preparation, baseline values for pressures
and flow in fetuses were established, and arterial blood gases and pH
were analyzed. (Criteria for rejection of hemodynamic data in fetuses
were arterial blood samples with pH < 7.20, PO2 < 15 mmHg or PCO2 >
60 mmHg.) After baseline values were obtained (Table 6), bolus injec
tions of PGH2 were made into the pulmonary arterial circuit. Doses
were administered in random order. After several doses were given to
fetuses, ventilation was initiated with a Harvard respirator or a
Healthdyne infant ventilator, and the umbilical cord was occluded.
Following the ventilation-induced fall in PAP, Q was increased to
reflect the normal increase in pulmonary perfusion occurring at birth.
Control values were observed (Table 6), and bolus injections of PGH2
were administered to ventilated fetuses. (Criteria for rejection of
hemodynamic data in ventilated fetuses were arterial blood samples with
pH < 7.30, PO2 < 75 mmHg and PCO2 > 50 mmHg.) After several


Table 6. Control values in unventilated and ventilated fetal lambs
Hemodynamic Data
PAP
SAP
Q
PVR
(mmHg)
(mmHg)
(ml/kg.mi n)
(mmHg.kg.min/ml)
1. Unventilated
70.1
60.6
15.98
4.85
Fetuses
(n = 11)
2.0
1.7
1.85
0.47
2. Ventilated
27.4
42.3
24.00
1.12
Fetuses
1.4
3.0
1.95
0.12
(n = 10)
Arterial Blood Samples
pH
P02
(mmHg)
PC0¡
(mmHg1
1. Unventilated
7.31
25.7
52.7
Fetuses
(n = 11)
0.01
0.6
1.3
2. Ventilated
7.44
112.1
33.2
Fetuses
(n = 10)
0.02
7.6
2.0
Data are control values for pulmonary jirterial pressure (PAP), mean systemic arterial
pressure (SAP), pulmonary blood flow (Q), pulmonary vascular resistance (PVR), and
arterial blood gases and pH in 6 unventilated and 7 ventilated fetal lambs. Data are
expressed as mean SEM. The number of observations for the control values is indicated
by n.


61
injections, a TX synthetase inhibitor was injected into the pulmonary
arterial circuit, and doses of PGH2 were repeated. One animal
received 2 doses of OKY-1581 (separated by 40 min and 2 injections of
PGH2), 2 received only UK 37,248-01, and 1 received a dose of
OKY-1581, followed by an injection of PGF^, and then a dose of UK
37,248-01, followed by a second injection of PGH2 (separated by at
least 30 min). Due to deterioration of animal preparations after sev
eral injections, some animals did not receive all doses of PGH2 and
each of the ventilated fetuses did not receive a TX synthesis blocker.
The following variables were sampled 60 sec before injection, at

5-sec intervals for the first minute, and also at 120 sec: PAP, LAP, Q,
mean SAP and heart rate. The readings at 0 and -60 sec were averaged
to give a control value for the variables PVR and mean SAP. The con
trol values were set to 100 %, and the values at the remaining time
intervals were expressed as a percentage of the control value. Each of
the treatments within a group (fetus, ventilated fetus, and ventilated
fetus with TX synthetase inhibition) was analyzed using a one-way ana
lysis of variance with repeated measures, and differences between means
were tested with the Newman-Keuls test (109). Differences between ven
tilated fetuses with and without TX synthetase inhibition were tested
using the unpaired Student's t-test. Differences cited were statisti
cally significant at P < 0.05.
Results
The average response of the pulmonary circulation of 6 fetal lambs
to 11 bolus injections of PGH2 is shown in Fig 9. Doses of PGH2


Figure 9: Average pulmonary and systemic responses to bolus injec
tions of PGH2 are shown for 6 unventilated fetal lambs
(11 injections, ave dose = 0.50 0.09 yg/kg). Symbols are
as in Fig 2.


SAP % PVR
63
UNVENTILATED FETUSES
PGH2 BOLUSES
0.50 pg/kg
TIME (sec)


64
ranged from 0.24 1.18 yg/kg (ave dose = 0.50 0.09 yg/kg); however,
there was not a dose-dependent relationship over this range of doses.
Injections of PGH2 consistently produced decreases in PVR of 10 25 %
The fall in PVR was rapid in onset, reached a peak at 10 sec after
injection, and returned to baseline within 35 sec. Values at 5, 10,
15, 20 and 25 sec were significantly (P < 0.01) different from the
value at 0 sec. The peak response was only 79 % of the control PVR.
At the doses of PGH2 injected, there was no effect on SAP.
Figure 10 shows the results of 10 bolus injections of PGH2 in 7
ventilated fetal lambs. The range of doses was 0.24 0.61 yg/kg (ave
dose = 0.39 0.05 yg/kg). Within this dose range, injections of PGH2
caused significant pulmonary vasoconstriction, without producing a
systemic effect. Increases in PVR were significantly different from
control PVR at 10, 15, 20, 25, 30, 35, 40 (all P < 0.01), and 45 sec
(P < 0.05). The peak pressor response occurred at 15 sec, and it was
150 % of the baseline value. PVR remained elevated above control
values until 50 sec after injection.
Four ventilated fetal lambs were treated with either OKY-1581 (50
mg) or UK 37,248-01 (2-3 mg/kg) to inhibit TX synthesis. There was no
difference between the two blockers in the pulmonary response to PGH2;
therefore the data for the two drugs were combined (Fig 11). Injec
tions of PGH2 (ave dose = 0.31 0.03 yg/kg) following TX synthetase
inhibition were able to cause significant (P < 0.01) increases in PVR
at 10 and 15 sec. The maximal response at 10 sec was 128 % of control
PVR. This peak increase in PVR was 56 % of the maximal increase in PVR
in the lambs without the inhibitors. There were significant differ
ences in the increases in PVR in response to PGH2 in ventilated fetal


Figure 10: Average pulmonary and systemic responses to bolus injec
tions of PGH2 are shown for 7 ventilated fetal lambs (10
injections, ave dose = 0.39 0.05 yg/kg). Symbols are as
in Fig 2.


% SAP % PVR
66
VENTILATED FETUSES
PGH2 BOLUSES
0.39 jg/kg
TIME (sec)


Figure 11: Average pulmonary and systemic responses to bolus injec
tions of PGH2 following thromboxane synthetase inhibition
(TSI) are shown for 4 ventilated fetal lambs (7 injections,
ave dose = 0.31 0.03 yg/kg). Symbols are as in Fig 2.


SAP % PVR
68
VENTILATED FETUSES
PGH2 BOLUSES
0.31 pg/kg
FOLLOWING TSI
TIME (sec)


69
lambs with and without TX synthetase inhibition. In the group treated
with inhibitors, the PGh^-induced increases in PVR were significantly
depressed in comparison with the untreated group at 15, 20, 25, 30, 40,
45 and 55 sec (P < 0.05, 0.01, 0.05, 0.05, 0.05, 0.05 and 0.05, respec
tively). There were no differences between the treated and untreated
lambs in response of the systemic circulation to intra-pulmonary injec
tions of PGH2-
Control injections of ice-cold saline (0.1 ml) were given to 2
fetal lambs before ventilation and 2 different lambs after ventilation.
These injections did not elicit any changes in PVR or mean SAP. In 3
fetal lambs before and after ventilation injections were given of an
"inactivated" PGI^- The endoperoxide was prepared in the same manner
as for an injection, but the syringe containing the PGH2 in saline
was allowed to sit on a warmed surface under light for at least 15 min
before injection. There was no response of the pulmonary circulation
to "inactivated" PGH2 injections in ventilated fetal lambs. There
was a slight, but significant (P < 0.01), decrease in PVR in unventi
lated fetal lambs to 94 % of control PVR, perhaps due to degradation of
PGH2 to PGE2* Systemic pressure was not affected by intra-pulmonary
injections of "inactivated" PGH2*
Discussion
The finding that injections of PGH2 into the pulmonary circula
tion of ventilated fetal lambs produced increases in PVR confirms ear
lier reports on PGH2 and the adult lung (88, 89). Over the dose
range used in this study, PGH2 did not exhibit a dose-dependent


70
relationship with PVR. The range was small primarily due to limited
quantities of PGH2 available. The average dose of PGH2 in the present
study (0.39 yg/kg) was approximately 4 times the dose used in adult
dogs (88). The increase in PVR in ventilated fetal lambs was 50 %
above baseline, while the increase in lobar arterial pressure in adult
dogs was 24 % greater than control. Bowers et al. (89) infused PGH2
into the superior vena cava of unanesthetized adult sheep and measured
pulmonary arterial pressure and cardiac output. They reported a
tripling of PVR at the steady state during an infusion of 0.25
yg/kg.min PGH2 (89). The greater responsiveness of the pulmonary
circulation of adult sheep as compared with the ventilated fetal lambs
could be due to lack of anesthesia in the adults. Also, in the adults
the PGH2 was infused over a period of at least 15 min, and a steady
state increase in PVR was observed. In the present study, PGH2
injections lasted approximately 3 sec, so that comparisons with
responses obtained during infusions are difficult.
Intra-pulmonary injections of PGH2 produced decreases in PVR in
fetal lambs and increases in PVR in ventilated fetuses. The doses of
PGH2 administered to the two groups were similar; thus, there should
be no difference in response due to differing substrate concentrations.
Major differences which existed between the ventilated and unventilated
fetal lambs were decreased PVR, increased Q, increased PO2 and
decreased PCO2 after ventilation (Table 6). These changes reflect
the normal events which occur as a result of ventilation at birth
(68). The decrease in pulmonary vascular tone may be the most impor
tant factor in explaining the differing responses to PGH2* Studies
in adult cat lung demonstrated that infusion of the bisenoic PG


71
precursor, arachidonic acid, produced pulmonary vasodilatation when
pulmonary vascular tone was elevated (95). Hyman et al_. proposed that
the pulmonary vascular response to exogenous arachidonate infusions was
dependent in part on the pre-existing level of PVR. This agreed with
the finding by Gerber frt aK (102) that PVR in dogs undergoing a hypox
ic pressor response was returned toward pre-hypoxia baseline values by
infusion of arachidonic acid.
In contrast, an earlier study (91) reported only pulmonary vaso
constriction when arachidonic acid was infused into the pulmonary cir
culation of fetal and neonatal goats. These findings were confirmed in
ventilated fetal and neonatal lambs (Ch 2) which had low pulmonary vas
cular tone (comparable to that of the ventilated fetuses in the present
study). However, when PVR was increased above baseline by 6 % hypoxia,
infusion of arachidonic acid at all doses produced only further in
creases in PVR (Ch 2). The use of OKY-1581 to inhibit TX synthetase
prior to infusions of arachidonic acid did not prevent pulmonary vaso
constriction in ventilated fetal and neonatal lambs (Ch 3). Thus, the
remaining pulmonary pressor response was attributed to formation and
actions of PGH2* While the data in ventilated fetal lambs injected
with PGH2 agree with the above studies, there are differing results
from unventilated fetuses receiving PGH2 and arachidonic acid.
Histamine, a chemically different compound, also produces pulmo
nary vasoconstriction and vasodilatation in animals of differing ages
(68, 125). In fetal lambs prior to ventilation, injection of histamine
caused a very large increase in pulmonary blood flow; after ventila
tion, the same dose caused little or no increase in pulmonary blood
flow (68). In adult dogs, histamine actively constricted small lobar


72
veins (125). Another prostaglandin, PGD2> has similar actions on the
pulmonary circulation (72). When PGD2 was infused into the pulmonary
circulation of unventilated fetal goats, it caused dose-dependent
decreases in PVR. After ventilation of fetuses and in newborn lambs,
infusions of PGD2 at doses greater than 8.0 yg/kg.min resulted in
increases in PVR (72). It is conceivable that PGD2 could be formed
from the injections of PGH2 in the present study.
Development of PG metabolism by fetal and neonatal lamb lung
microsomes has been described by Friedman et aj_. (110). Fetal micro-
somes were capable of producing PGE2, PGI2 and TXA2 enzymatically from
PGH2- Prostacyclin synthetase exhibited enzyme saturation at low
levels of PGH2, and formation of PGI2 was low throughout gestation.
Thromboxane synthetase showed low activity when PGH2 concentrations
were low, but at high PGH2 concentrations (400 ng PGH2/25O yg lung
homogenate protein), TX's were a major product of PG synthesis in late-
term fetal lung. The product formed in greatest quantities by fetal
lamb lung homogenates was PGE2 (110), and formation of PGE2 by
fetal goat lung microsomes was enhanced by addition of reduced gluta
thione (GSH) (126). Thus, levels of endogenous GSH in fetal lung may
exert important control over products of PGH2 metabolism, with greater
amounts of TX's formed in the absence of GSH and more PGE2 formed in
the presence of GSH (126). Factors involved in the regulation of GSH
in fetal lung are unknown.
The above studies on PGH2 metabolism in fetal and neonatal lung
microsomes do not indicate whether PGD2 production was assayed. It
has been shown that there is a specific glutathione-S-transferase
present in adult sheep lung which causes large amounts of PGF2a and PGD2


73
to be produced (29). Incubation of the purified enzyme with PGH2
resulted in 3 times more PGD2 than PGE2 produced (29). Thus, if
this enzyme were present and active in fetal sheep lungs, formation of
PGD2 could be a significant factor in the response to PGH2-
Thromboxanes have a potent ability to contract vascular smooth
muscle in vitro (17); PGE2 and PGI2 are vasodilators of fetal and
neonatal pulmonary and systemic circulations (67, 82). In summary,
injections of PGH2 caused increases in PVR in ventilated fetuses.
This is consistent with 1) direct effect of PGH2 on vascular smooth
muscle (17) and 2) formation of products with vasoconstrictor activity,
such as TXA2, PGD2 and PGF2a. Inhibition of TX synthetase with either
OKY-1581 or UK 37,248-01 resulted in a reduction of pulmonary vasocon
striction, but did not abolish the response completely (Fig 10). In
contrast, unventilated fetal lambs always responded to injections of
PGH2 with decreases in PVR. These findings suggest metabolism of PGH2
to PGI2 PGD2 and/or PGE2, with little direct action of PGH2 on fetal
pulmonary vessels. It is possible that ventilation of fetal lungs al
ters the end products of PGH2 metabolism, perhaps by limiting availa
bility of GSH, so that after ventilation more TX's are formed in
response to a bolus of PGH2- Alternatively, the pulmonary vascular
response to PGH2 may depend in part on existing basal pulmonary vas
cular tone.


CHAPTER FIVE
ARACHIDONIC ACID AND ADULT PULMONARY CIRCULATION
Introduction
Initial observations by other authors on the effects of arachidon-
ic acid on the pulmonary circulation demonstrated that pulmonary vaso
constriction could be produced by infusion or injection of arachidonic
acid into the pulmonary circulation (69, 92, 93). Recent studies have
indicated that arachidonic acid can cause decreases in PVR when admin
istered by infusion at low doses (94, 95). Additional reports demon
strated the ability of infusions of arachidonic acid to reduce PVR
which had been elevated by hypoxia, PGF2a, or an analog of PGH2
(95, 102, 112). Assay of arterial blood during the infusion of arachi
donic acid in the above studies showed formation of 6-keto-PGFia
(95, 102, 112). These reports demonstrated that vasodilator PG's,
PGI2 in particular, were formed from exogenous arachidonic acid when
pulmonary tone was enhanced.
In contrast to these findings in adult animals, infusions of ara
chidonic acid into the pulmonary circulation of perinatal goats always
produced pulmonary vasoconstriction (91). The results presented in Ch
2 confirmed that arachidonic acid causes increases in PVR in ventilated
fetal and neonatal lambs. These results were not altered when the
74


75
inspired O2 was reduced to 6 %; instead, a hypoxic pressor response
was observed and the infusion of arachidonic acid caused further pulmo
nary vasoconstriction. Consequently, the following studies in adult
sheep were undertaken to determine if the pulmonary pressor actions of
arachidonic acid, when infused during hypoxia, were dependent upon
developmental state of the animal, or if the response was related to
oxygenation or elevated pulmonary vascular tone. Pulmonary vascular
resistance was elevated in two ways: 1) inspired oxygen was reduced to
10 %, or 2) PGF2a was infused while normoxic conditions were main
tained. The effects of infusion of arachidonic acid were tested under
both of these conditions.
Materials and Methods
Surgical preparation. The surgical procedure for the isolated
pump-perfused left lower lung preparation, described for perinatal
lambs in Ch 2, was modified slightly for use in adult sheep. Ten non
pregnant ewes (52.7 3.5 kg) were anesthetized with chloral ose (50
mg/kg, iv) and tracheotomies were performed. Anesthesia was maintained
with hourly supplements of chloralose (10 mg/kg). Ewes were paralyzed
with d-tubocurare (0.2 mg/kg, iv) and ventilation was initiated with a
Harvard respirator. Colonic temperature was monitored (Yellow Springs
Instrument) and maintained between 38 and 41C by placement of heating
pads beneath the animal. The femoral artery was cannulated for moni
toring SAP (Statham P23DC transducer), heart rate and arterial blood
gases and pH (Instrumentation Laboratory pH/blood gas analyzer 213).
An incision was made along the upper margin of the left fourth rib, and


76
the third and fourth ribs were retracted to permit access to the left
lung. Following isolation and dissection from surrounding tissue of
the left pulmonary artery, the ewe was heparinized (2000 U/kg, iv). A
catheter was placed in the femoral vein and advanced into the inferior
vena cava. Blood was withdrawn from the inferior vena cava and pumped
(Cole-Parmer Masterflex pump no. 7018) into the left pulmonary artery.
Between 35 and 40 % of the total lung tissue, as determined by weight,
was perfused in this manner. Flow to the right lung was not disturbed.
Left pulmonary blood flow (Q) (Statham flowmeter M4401 with Medicon
FloProbe Sensor A2208 electromagnetic flow probe) was maintained con
stant at a level that produced approximately 20 mmHg pressure in the
left pulmonary artery. A catheter was placed in the left atrium for
measurement of LAP, and PVR was calculated as (PAP LAP) body wt /
Q. Pressures, flow and heart rate were recorded continuously on a
Gould Brush 480 8-channel polygraph. An Apple 11+ computer was con
nected to the polygraph with an analog-to-digital interface (AI13,
Interactive Structures, Inc.) for on-line data sampling and calculation
of PVR.
Experimental procedure. The sodium salt of arachidonic acid
(Sigma or NuChek Prep) was stored in dry form at -16C. Fresh solu
tions were prepared daily with saline (final cone = 1 mg/ml) and pro
tected from light prior to being infused (Harvard pump) at varying
rates into the pulmonary arterial circulation. Prostaglandin ?2a
(tromethamine salt, Upjohn Co.) was stored in dry form at -16C, and an
ethanol solution (100 mg/ml) was diluted with saline (final cone = 10.8
mg/ml) and stored at 6C.


77
Following surgical preparation of the animals, baseline values for
pressures and flow were established and arterial blood gases and pH
were analyzed. (Criteria for rejection of hemodynamic data were arte
rial blood samples with pH < 7.30, PO2 < 75 mmHg or PCO2 > 50 mmHg.)
Control values were observed before each experimental period (Table 7),
and the following treatments were administered in random order. 1)
Arachidonic acid was infused into the pulmonary arterial circuit for 5
min at varying rates. Doses of arachidonic acid were expressed as
amount/kg body wt.min. 2) Alveolar hypoxia was produced by lowering
the inspired O2 to 10 % for 15 min. 3) A combination of treatments 1
and 2 was given, with hypoxia started for 5 min, arachidonic acid in
fused during the sixth through the tenth minutes, and hypoxia continued
for an additional 5 min after terminating the arachidonate infusion.
4) Prostaglandin F2a was infused into the pulmonary arterial circuit
for 15 min. 5) A combination of treatments 1 and 4 was given, with
PGF2a infused for a total of 15 min and arachidonic acid infused
during the sixth through the tenth minutes. 6) Hypoxia was produced by
lowering the inspired O2 to 10 % for 5 min. 7) Arachidonic acid was
infused directly into the perfusion circuit for 15 min. 8) A combina
tion of treatments 6 and 7 was given, with arachidonic acid infused for
15 min and the animal exposed to hypoxia during the sixth through the
tenth minutes. A recovery period of at least 5-10 min was allowed
between experimental periods in order to return to control values. The
minimal duration of an experiment in which an animal received every
treatment was 3 hours. However, due to deterioration of the animal
preparations after several experimental periods, some animals did not
receive every treatment. The following variables were sampled at


Table 7. Control values in adult sheep
A. Hemodynamic Data
PAP
SAP

Q
PVR
(mmHg)
(mmHg)
(ml/kg.min)
(mmHg.kg.min/ml)
(n = 55)
19.2
82.4
14.56
1.17
0.4
1.9
0.53
0.05
B. Arterial Blood Samples
pH
P02
PCOo
(mmHg)
(mmHg)
(n = 55)
7.47
108.4
37.4
0.01
3.1
0.7
Data are control values prior to each treatment for pulmonary.arterial pressure (PAP),
mean systemic arterial pressure (SAP), pulmonary blood flow (Q), pulmonary vascular
resistance (PVR), and arterial blood gases and pH in 10 adult sheep. Data are expressed
as mean SEM. The number of observations for each of the control values is given as n.


79
intervals of 1 min, with the first minute of the treatment sampled at
20-sec intervals: PAP, LAP, Q, mean SAP and heart rate. A reading was
taken 1 min before the start of the experimental period, and this value
and the value at the beginning of the treatment were averaged to give a
control value for the variables PVR and mean SAP. The control values
were set to 100 %, and the values at the remaining time intervals were
expressed as a percentage of the control value. Each of the treatments
was analyzed using a oneway analysis of variance with repeated mea
sures, and differences between means were tested with the Newman-Keuls
test (109). Differences between treatments were tested for signifi
cance using the unpaired Student's t-test. The level of significance
for statistical differences was P < 0.05.
Results
The average pulmonary and systemic responses of 6 adult sheep to
15 min of hypoxia (FIQ2 = 10 %) are shown in Fig 12. Hypoxia pro
duced significant (P < 0.01) increases in PVR from 2 min through 16
min. PVR returned to baseline within 3 min of terminating the hypoxic
exposure. Mean SAP was not significantly elevated from the control
value during hypoxia. After returning to normoxia, mean SAP signifi
cantly (P < 0.05) declined to 88 % of control SAP at 19 and 20 min.
Thirteen 5-min infusions of arachidonic acid (ave dose = 17.6
2.6 ug/kg.min) were given to 9 ewes. The average vascular responses to
these infusions are shown in Fig 13. Infusions of arachidonic acid in
low doses (as compared to doses used in neonatal lambs, Ch 2) for 5 mi n
caused significant (P < 0.01) increases in PVR at 1 through 6 min.


Figure 12: Average pulmonary and systemic responses to 15-min periods
of hypoxia (FIq2 = 10 %) are shown for 6 adult sheep (6
cases). Symbols are as in Fig 2.


% SAP
ADULT SHEEP
HYPOXIA- 1 0 %- 1 5 min
TIME (min)


Figure 13: Average pulmonary and systemic responses to 5-min infu
sions of arachidonic acid are shown for 9 adult sheep (13
infusions, ave dose = 17.6 + 2.6 pg/kg.min). Symbols are
as in Fig 2.


SAP % PVR
83
ADULT SHEEP
ARACHIDONIC ACID-5 min
17.6 jjg/kg.min
TIME (min)


84
Arachidonate-induced increases in PVR formed a plateau from 2 5 min;
PVR was 120 % of control at 4 min. A slight, but significant, reduc
tion in mean SAP occurred with the increases in pulmonary pressure.
Mean SAP was significantly decreased from control SAP at 4, 5, 6, 7 and
8 min (P < 0.05, 0.01, 0.05, 0.01 and 0.05, respectively). The maximal
fall in mean SAP was 96 % of control SAP at 5 min.
Arachidonic acid (ave dose = 16.4 2.8 yg/kg.min) was infused
during the sixth through the tenth minutes of hypoxia in 7 adult sheep.
Average pulmonary and systemic vascular responses to 7 combined treat
ments are shown in Fig 14. There were significant increases in PVR due
to hypoxia before and after infusion of arachidonic acid. Infusion of
arachidonic acid caused an additional increase in PVR during the sixth
through the tenth minutes. Increases in PVR during the combined treat
ments (from 7-10 min) were almost 150 % of control PVR. Values at 7
- 10 min were significantly (P < 0.01) greater than the values at 2, 3,
4 and 5 min, before arachidonic acid was infused. Increases in PVR
during the combined treatments were also significantly greater than the
increases in PVR after the infusion was terminated. Pulmonary vascular
resistance at 10 min was significantly greater than values at 11, 12,
13, 14 and 15 min, while hypoxia was still present (P < 0.05, 0.01,
0.01, 0.01 and 0.01, respectively). The response of the systemic cir
culation to the combination of 15 min of hypoxia and 5 min of arachi
donic acid progressed in stages (Fig 14). During the first 5 min of
hypoxia, mean SAP was significantly (P < 0.05) increased from control
SAP to 114 % at 5 min. During the sixth through the tenth minutes of
hypoxia, while arachidonic acid was infused, mean SAP returned toward
baseline. Following the termination of the infusion, mean SAP was not


Figure 14: Average pulmonary and systemic responses to a combination of
15-min periods of hypoxia (FIq2 = 10 %) and 5-min infusions
of arachidonic acid are shown for 7 adult sheep (7 cases,
ave dose = 16.4 2.8 yg/kg.min). Symbols are as in Fig 2.


% SAP % PVR
ADULT SHEEP
HYPOXIA- 1 0 %- 1 5 min
ARACHIDONIC ACID-5 min
16.4 jjg/kg.min
TIME (min)
t-4-i


87
significantly different from the value at 0 min. However, the values
at 13, 14, 15, 16, 17, 18, 19 and 20 min were significantly (P < 0.05,
0.05, 0.01, 0.01, 0.01, 0.01, 0.01 and 0.01, respectively) different
from the values at 4 and 5 min.
As an alternative to hypoxia as a means to elevate PVR, PGF2a
was infused for 15 min (ave dose = 4.4 1.3 yg/kg.min) in 5 ewes (Fig
15). The infusion rate used was chosen to produce approximately the
same increase in PVR as hypoxia caused; the two treatments were not
significantly different after the first 4 min. Infusion of PGF2a
produced a rapid increase in PVR; values at 40 sec and 1 min were sig
nificantly (P < 0.05) greater than control PVR. Values for PVR at the
remaining time intervals during the infusion were significantly greater
than control PVR at P < 0.01 (from 2 to 15 min). There were increases
in mean SAP during infusion of PGF2a, beginning at 1 min (P < 0.05)
and continuing from 2-18 min (P < 0.01). The value at 19 min was
still significantly (P < 0.05) elevated over control SAP, but mean SAP
had returned to baseline at 20 min.
The effects in 6 ewes of combining 15 min PGF2a (ave dose = 4.1
1.1 yg/kg.min) and 5 min of arachidonic acid (16.1 3.2 yg/kg.min) are
shown in Fig 16. There were significant increases in PVR due to infu
sion of PGF2a before and after infusion of arachidonic acid. Infusion
of arachidonic acid caused an increase in PVR during the sixth through
the tenth minutes. Increases in PVR during the combined treatments
(from 7-10 min) were 190 % of control PVR. The values at 7, 8, 9 and
10 min, during the arachidonate infusion, were significantly (P < 0.01)
greater than values during infusion of PGF2a (at 2, 3, 4, 5, 11, 12, 13,
14 and 15 min). The response of the systemic circulation to the


Figure 15: Average pulmonary and systemic responses to 15-min infusions
of PGF are shown for 5 adult sheep (5 infusions, ave
dose = 4.4 1.3 ug/kg.min). Symbols are as in Fig 2.


% SAP % PVR
ADULT SHEEP
PGF2alpha-15 min
4.4 jjg/kg.min
TIME (min)


Figure 16: Average pulmonary and systemic responses to a combination of
15-min infusions of PGF2a (ave dose = 4.1 1.1 ug/kg.min)
and 5-min infusions of arachidonic acid are shown for 6
adult sheep (6 infusions, ave dose = 16.1 3.2 pg/kg.min).
Symbols are as in Fig 2.


% PVR
ADULT SHEEP
PGF2alpha-15 min
4.1 pg/kg.min
ARACHIDONIC ACID-5 min
16.1 jjg/kg.min
TIME (min)


92
combined treatment of 15 min of PGF2a and 5 min of arachidonic
acid was not significantly different from the systemic response to
infusion of PGF2a alone. Mean SAP was increased significantly
from the value at 0 min at 2 18 min (P < 0.05 at 2, 17 and 18 min; P
< 0.01 at 3 16 min). The values for mean SAP during infusion of
arachidonic acid (6 10 min) were not significantly different from
values before and after arachidonic acid (2-5 and 11 15 min).
To study the effects of arachidonic acid infusion on the hypoxic
pressor response, arachidonic acid was infused for 15 min. After re
turning to baseline, responses to 5 min of hypoxia were measured. Then
a combination of the two treatments was given. Pulmonary and systemic
responses to these experimental treatments are summarized in Table 8.
Prolonged infusion of low-dose arachidonic acid (12.3 0.6 yg/kg.min)
caused significant (P < 0.05) increases in PVR at 3 15 min. The pul
monary hypertension was accompanied by a slight reduction in mean SAP
which was not significantly different from control SAP. The pulmonary
circulation responded to 5 min of reduced oxygen (FIQ2 = 10 %) with
significant (P < 0.01) increases in PVR at 2 6 min. There were sig
nificant (P < 0.01) increases in mean SAP at 3, 4 and 5 min during
hypoxia. Maximal increases in % PVR and % SAP occurred at 5 min (Table
8); however, the values from 2-5 min were not significantly different
from each other. The two treatments were combined in 5 sheep such that
arachidonic acid was infused for 15 min (ave dose = 12.3 0.6 yg/kg.
min) and inspired oxygen was reduced during the sixth through the tenth
minutes. Arachidonic acid caused significant (P < 0.05) increases in
PVR at 3, 4 and 5 min, and also at 11 15 min. Addition of hypoxia
produced further increases in PVR at 6 10 min, with values at these


93
Table 8. Maximal change in PVR and SAP for all treatments in adult
sheep
% PVR
% SAP
1. Arachidonic Acid (17.6 2.6 yg/kg.min)-5 min
9 Ewes (inc) 120.38 3.01
13 Treatments (dec)
2. Hypoxia (FIq2 = 10 %)-15 min
6 Ewes (inc) 135.00 7.71
6 Treatments (dec)
95.75 1.75
111.82 3.98
88.35 4.22
3. Hypoxia-15 min and Arachidonic
7 Ewes (inc)
7 Treatments (dec)
4. PGF2a (4.4 1.3 yg/kg.min)-15
5Ewes (inc)
5 Treatments (dec)
5. PGF2a (4.1 1.1 yg/kg.min)-15
(16.1 3.2 yg/kg.min)-
6 Ewes (inc)
6 Treatments (dec)
6. Hypoxia (FIq2 = 10 %)-5 min
7 Ewes (inc)
8 Treatments (dec)
7. Arachidonic Acid (12.3 0.6 yg/kg
5 Ewes (inc)
5 Treatments (dec)
Acid (16.4 2.8 yg/kg.min)-5 min
148.64 10.02 113.97 5.44
89.61 4.17
mi n
142.62 8.96 118.36 1.73
min and Arachidonic Acid
mi n
192.83 15.82 125.82 7.86
120.63 2.53 117.20 7.22
min)-15 min
124.76 12.24
88.16 8.58
8.Arachidonic Acid (12.3 0.6 yg/kg.min)-15 min and Hypoxia-5 min
5 Ewes (inc) 167.58 18.83 119.44 6.33
5 Treatments (dec) -- 91.64 7.39
Values shown are maximal increases (inc) or decreases (dec) in PVR or
SAP during the treatment indicated. Data are expressed as mean SEM.
Locations with a dash indicate the response was in only one direction;
i.e., only vasoconstriction or vasodilatation.


Full Text
PULMONARY VASCULAR RESPONSES OF PERINATAL AND ADULT SHEEP
TO ARACHIDONIC ACID DURING NORMOXIA AND HYPOXIA
By
MARY LEE TOD
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
1984

I dedicate this dissertation to my father, for his example of
determination in overcoming obstacles, and to my mother, for teaching
me the patience I needed to complete a difficult task.

ACKNOWLEDGEMENTS
The author wishes to express her appreciation for the comments,
criticisms and guidance of her supervisory committee, Dr. S. Cassin,
Dr. A. B. Otis, Dr. W. N. Stainsby and Dr. A. H. Neims. In addition,
thanks are due to the members of the laboratory who aided at various
stages of this research project: H. Kuck, T. Cupp, R. Shoup, E.
McJett, R. Su and R. Cooper.
Special appreciation is extended the chairman of the supervisory
committee, Dr. Sidney Cassin, for the provision of laboratory facili¬
ties and animals, support and encouragement during the completion of
this manuscript.
Special thanks are due Calvin Timmerman for his constant
encouragement and support during this project, and for his endless
understanding and patience.

TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS iii
ABSTRACT vi
INTRODUCTION 1
Historical Overview 1
Prostaglandin Biochemistry 2
Prostaglandins and the Pulmonary Circulation 8
Statement of Purpose 15
PERINATAL PULMONARY RESPONSES TO ARACHIDONIC ACID
DURING NORMOXIA AND HYPOXIA 17
Introduction 17
Materials and Methods 19
Results 22
Discussion 33
THROMBOXANE SYNTHETASE INHIBITION AND PERINATAL
PULMONARY RESPONSE TO ARACHIDONIC ACID 38
Introduction 38
Materials and Methods 39
Results 41
Discussion 51
EFFECTS OF PROSTAGLANDIN ON PERINATAL
PULMONARY CIRCULATION.. 57
Introduction 57
Materials and Methods 58
Results 61
Discussion 69
ARACHIDONIC ACID AND ADULT PULMONARY CIRCULATION 74
Introduction 74
Materials and Methods 75
Results 79
Discussion 94
CONCLUSIONS 99
TV

REFERENCES 104
BIOGRAPHICAL SKETCH 114
v

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
PULMONARY VASCULAR RESPONSES OF PERINATAL AND ADULT SHEEP
TO ARACHIDONIC ACID DURING NORMOXIA AND HYPOXIA
By
Mary Lee Tod
April 1984
Chairman: Dr. Sidney Cassin
Major Department: Physiology
The actions of exogenous arachidonic acid during normoxia and
hypoxia were studied in perinatal lambs using an in situ pump-perfused
lung preparation. Ventilated fetal and neonatal lambs responded to
arachidonate infusions with pulmonary vasoconstriction and systemic
hypotension. In ventilated fetuses the effect of arachidonic acid was
additive to the hypoxic pressor response. The pulmonary vascular
response in newborns to arachidonic acid was unaltered by hypoxia, pos
sibly because of the severity of the vasoconstriction to arachidonate
alone. Thus, hypoxia does not alter the response of the perinatal pul
monary circulation to exogenous arachidonic acid.
Inhibition of thromboxane synthetase with OKY-1581 depressed ara-
chidonate-induced increases in pulmonary vascular resistance. Pulmo¬
nary vasoconstriction still occurred in response to arachidonic acid
VI

after OKY-1581, but the peak increase in pulmonary vascular resistance
was 50 - 85 % less than the response before the inhibitor. The remain¬
ing response was attributed to formation of endoperoxides. Inhibition
of thromboxane synthetase resulted in a significant enhancement of sys¬
temic hypotension in response to arachidonic acid.
Injections of PGH2 into the pulmonary circulation of ventilated
fetuses caused increases in pulmonary vascular resistance. The pulmo¬
nary response to PGH2 was reduced following OKY-1581. The response
of unventilated fetuses to PGH2 was pulmonary vasodilatation. The
differences in response to PGH2 may be due in part to differences in
pulmonary vascular tone before and after ventilation. Another possible
explanation is that ventilation of fetal lungs alters the ratios of the
end products of PGH2 metabolism, so that after ventilation more
thromboxanes are formed in response to PGH2*
In contrast to studies in other species, low-dose arachidonate
infusions always resulted in pulmonary vasoconstriction in adult sheep.
The pulmonary circulation of hypoxic adults constricted even further
upon addition of arachidonic acid. The pulmonary pressor response to
arachidonate was not affected by prior elevation of pulmonary vascular
resistance with PGF2a. In both perinatal and adult sheep, eleva¬
tion of pulmonary vascular resistance by hypoxia or PGF2a does not
alter the pressor response to arachidonic acid. Pulmonary responses to
arachidonic acid appear to be species dependent.
vi i

CHAPTER ONE
INTRODUCTION
Historical Overview
Prostaglandin research had its beginnings over 50 years ago with a
report of contractile and relaxant activity of isolated uterine strips
in response to seminal fluid (1). Reports of vasodepressor activity of
human seminal fluid and an alcoholic watery extract from prostate
glands came from two independent groups (2, 3). This vasodepressor
substance from prostate glands was named "prostaglandin" by von Euler
(4), and its biological activity in lowering blood pressure was
established in several species.
Attempts at isolation and purification of "prostaglandin" were
successful with the separation of a prostaglandin factor, PGF, in 1957
(5). The compound was determined to be an unsaturated hydroxyacid.
Further purification and separation of extracts from sheep prostate
glands resulted in identification of two compounds, PGE and PGF (6, 7),
which retained the biological activity of the crude extracts when
tested on smooth muscle and for effects on blood pressure (8).
Prostaglandins were found to be derived from essential fatty acids (9,
10), with the most common precursor in mammals being arachidonic acid
(11). Prostaglandin biosynthesis has been detected in most tissues of
the body, including lung (12, 13).
1

2
In the past 10 years, many advances have been made in prostaglan¬
din research. Products of arachidonic acid metabolism other than the
original PG's, E and F, were isolated which also exhibited biological
activity. These unstable products include the endoperoxide intermedi¬
ates, PGG2 and PGH2 (14-17), prostacyclin (18-20), the thromboxanes
(TX's) (21) and the leukotrienes (22). In recognition of their vast
contributions to a field of research only 50 years old, the Nobel Prize
in Medicine was awarded in 1982 to Sune Bergstrom, Bengt Samuelsson and
John R. Vane.
Prostaglandin Biochemistry
Monoenoic, bisenoic and trienoic PG's are derived from the essen¬
tial fatty acids, 8,11,14-eicosatrienoic acid (dihomo-y-1inolenic
acid), 5,8,11,14-eicosatetraenoic acid (arachidonic acid) and
5,8,11,14,17-eicosapentaenoic acid, respectively (9, 10). Products of
these fatty acids are denoted by a subscript to indicate the degree of
unsaturation in the molecule; e.g., PGH2 contains two double bonds.
Arachidonic acid is the predominant PG precursor in mammals, and
it is esterified to phospholipid membranes (11). Arachidonic acid must
be liberated from membranes before entering into the biosynthesis of
prostaglandins; this liberation results from hydrolysis by phospholi¬
pases (11). Phospholipases can be activated by a number of stimuli; a
common factor is probably distortion of the cell membrane (13).
Release of free arachidonic acid begins the biosynthetic cascade (Fig
1); availability of free arachidonic acid is recognized as the rate-
limiting step in the formation of prostaglandins (11).

Figure 1: Biosynthetic scheme of the products of arachidonic acid is
presented. Points of inhibition are included, with the
inhibitors shown in parentheses.

fMMPHOLfPASÉ *2
LEUKQTRIENE
PROSTAGLANDIN
PROSTACYCLIN
THROMBOXANE

5
Arachidonic acid enters one of two enzymatic pathways following
its release: (1) the cyclo-oxygenase system, which leads to formation
of PG's and TX's, or (2) the lipoxygenase pathway, which contributes
leukotrienes (LT's) and other oxygenated, non-cyclic fatty acids (15,
16, 22). The lipoxygenase pathway has generated much interest recent¬
ly; leukotrienes are involved in immune responses to inflammation,
asthma, and other immediate hyper-sensitivity reactions (22). Leuko¬
trienes C4, D4 and E4 have been identified as major components of
slow-reacting substance of anaphylaxis (SRS-A) (22). Another leukotri-
ene, LTB4, is involved in chemotactic responses leading to migration
of neutrophils and eosinophils (22). The prostaglandin synthetase com¬
plex is a group of membrane-bound enzymes which requires oxygen and
other cofactors (15). The initial step in the formation of PG's from
arachidonic acid appears to be a peroxidative reaction that allows the
fatty acid to be attacked by oxygen (23). This is quickly followed by
cyclization of the hydroperoxide into a cyclic endoperoxide intermedi¬
ate, PGG2 (15, 16). This endoperoxide intermediate can be reduced to
form PGH2, a second intermediate in the pathway (15, 16), or it may
also be converted enzymatically to PGE2, PGI2 or TXA2 (23). The
endoperoxides are highly unstable in aqueous solution, and rapidly form
other prostaglandins (24). The enzymes which convert the endoperoxides
to the other PG's and TXA2 are present in many tissues of most spe¬
cies, but they exhibit varying activities and concentrations in differ¬
ent organs, contributing to the wide range of responses of individual
systems to release of PG's (Table 1) (16, 18, 25-28).
The primary PG's, PGE2 and PGF2a, were the earlier ones
studied (12). A third primary PG is PGÜ2* The primary PG's are

6
Table 1. Major sites of formation and inactivation of prostaglandins
Formation
Inactivation
pge2
seminal vesicles,
kidney
lung
PGF2a
connective tissue,
reproductive tissue
lung
pgd2
lung, brain, leukocytes,
gastrointestinal tissue
enzymatically in
blood
pgi2
vascular endothelium
spontaneous hydrolysis
in blood
txa2
platelets, lung,
spleen
spontaneous hydrolysis
in blood
Sources for material presented in this table are ref 16, 18, 25-28.

7
relatively stable in aqueous solution, and each is derived from PGH2
by different routes. PGE2 and PGF2a are produced by isomerization
and reduction, respectively, of the endoperoxide intermediate (15, 16).
The enzymatic formation of PGD2 can occur by one of two methods. A
soluble PGH-PGD isomerase is present in various organs, and in the
presence of glutathione it converts PGH2 solely to PGD2 without
formation of PGF2a (16). An additional enzyme isolated from sheep
lung is capable of producing both PGD2 and PGF2a in the presence
of glutathione (29). There is considerable interconversion of the pri¬
mary PG's, in particular the formation of PGF2a from PGD2 and PGE2
which occurs in sheep blood (28). Secondary prostaglandins, PGA2,
PGB2 and PGC2, are derived from PGE2 by dehydrogenation and ring
isomerization.
The primary and secondary PG's were relatively easily extracted
and purified, and thus have been studied extensively (12, 30-32).
Recently, new products of arachidonic acid were identified which are
highly potent but extremely unstable (14, 18, 19, 21). Prostacyclin
synthetase is an enzyme which is found in vascular endothelia; it con¬
verts PGH2 to prostacyclin, or PGI2 (18, 19). Prostacyclin hydrolyzes
spontaneously to the relatively inactive 6-keto-PGF^Q in blood;
the half-life for this reaction is 3 min (34). Prostacyclin has been
found to have powerful anti-aggregatory action, is a potent vasodilator
of both the pulmonary and systemic circulations, and relaxes arterial
smooth muscle strips (35).
Thromboxane synthetase is the enzyme which forms TXA2 from the
endoperoxides (21), and the primary site of TX formation is in plate¬
lets. The half-life of TXA2 in blood is approximately 30 sec, with

8
the stable and relatively inactive degradation product being TXB2
(21, 25, 36). Thromboxane A£ exhibits potent platelet aggregating
ability, and it is a component of rabbit aorta contracting substance,
RCS (21, 37). Normally, formation of TXA2 by platelets is counter¬
balanced by synthesis of PGI2 by the vascular endothelium, so that
platelet aggregation does not occur. However, in cases of injured ves¬
sels, there is little release of PGI2 and aggregation of platelets is
possible (18, 19, 35). Thus, the interaction of PGI2 and TXA2 is
important in platelet homeostasis and regulation of blood flow in small
vessels.
Prostaglandins and the Pulmonary Circulation
The lungs of many mammals are able to synthesize and release PG's
as a result of many stimuli, including air embolization (13), infusion
of particles (13, 38-40), anaphylaxis (41) and mechanical distension
(42, 43). Piper and Vane (13) suggested that the feature that is com¬
mon to these various stimuli is that of mechanical distortion or
stretching of cellular membranes. A possible role for dilator PG's,
released as a result of ventilation, could be the matching of ventila¬
tion with perfusion (44). This would result in increased blood flow to
areas of the lung that are more stretched, or better ventilated.
Release of primarily dilator PG's has been postulated as an important
part of the large decrease in pulmonary vascular resistance which oc¬
curs at birth (45), as demonstrated by the increase of PGI-1 ike com¬
pounds in the pulmonary venous blood of fetal lambs following initia¬
tion of ventilation.

9
The lungs also serve an important role in the metabolism of many
of the prostaglandins (26, 46, 47), inactivating up to 95 % of the
E-series prostaglandins and 75 % of the F-series prostaglandins. In
the adult the inactivation of PG's which occurs in the pulmonary
circulation "protects" the systemic circulation from the effects of
these vasoactive compounds. However, in the fetus there is a large
proportion of the cardiac output which is shunted through the foramen
ovale and the ductus arteriosus, bypassing the pulmonary circulation,
and metabolism of PG's in the fetus must therefore occur at alternate
sites, such as the liver, kidneys, brain and placenta (48, 49). The
enzyme responsible for the majority of inactivation of PG's, 15-hydroxy
PG dehydrogenase, is present in most tissues, and has been isolated
from the cytosol in the non-membrane bound fraction (48, 50). The
isolated enzyme exhibits substrate specificity for PGE2, PGA2 z2 , PGH=^
and PGF2a, producing 15-keto PG's; it has no affinity for PGB2, PGD2 or
PGG2 as substrates (50, 51). Inactivation of PG's in vivo occurs
inside cells. Thus, Bito et al_. (52) have shown that a pulmonary PG
transport system exists which facilitates entry of some of the PG's
into the cell. Eling et^ aJL (53) demonstrated that PGEj, PGE2> PGF^a
and PGF2a are readily transported into rat lung tissue, and PGD2 is
also thought to be a substrate for the transport system since it com¬
petitively inhibits uptake of PGE^. Pulmonary tissue (lung homoge¬
nates) of the rat is capable of inactivating PGI2 enzymatically, but
PGI2 is not transported into the cell (54). However, spontaneous
hydration of PGI2 in the blood shortens its actions (33, 34).
Administration of exogenous PG's intravenously or via the pulmo¬
nary artery results in dose-related changes in pulmonary vascular

10
resistance (Table 2). Prostaglandin F2a causes a marked pulmonary
vasoconstriction to occur in most adult animals, including dogs (55),
swine (56), sheep (57) and cats (58). The calf (59), fetal goat (60,
61) and newborn lamb (62) also respond to PGF2a by increasing pulmonary
vascular resistance. Similar effects are observed with PGF^ in both
adult and fetal animals (60, 63), and F-series prostaglandins cause
systemic hypertension when administered in doses that exceed the capac¬
ity of the lungs for inactivation.
Prostaglandins of the E-series are generally vasodilators in most
species and vascular beds studied. In the pulmonary circulations of
swine (56), sheep (57), dogs (64) and rabbits (65), PGE^ causes
decreases in pulmonary vascular resistance, while PGE2 produces pul¬
monary vasoconstriction. However, both PGE^ and PGE2 cause a drop
in systemic blood pressure in the above studies on adults. The action
of PGE1 in the pulmonary circulation of perinatal goats and newborn
lambs is similar to the response seen in adults, but the effects of
PGE2 are opposite (59, 61, 62, 66, 67). Pulmonary vascular resis¬
tance in calves (59), perinatal goats (61, 66, 67) and newborn lambs
(62) was decreased when PGE2 was administered. This observed differ¬
ence between fetus and adult in response of the pulmonary circulation
to PGE2 may be due in part to increased vascular tone of the fetal
lung (68), or to differences in PÜ2* The vascular tone of the neona¬
tal lung remains elevated slightly over adult conditions, and may thus
explain the decrease in pulmonary vascular resistance with PGE2 seen
in newborn goats and lambs (61, 62, 66, 67).
Although initially described as having no biological activity
(16), PGD2 was found to increase tracheal insufflation pressure

11
Table 2. Summary of actions of prostaglandins on the pulmonary and
systemic circulations
PVR
Fetus
SVR
Newborn
PVR SVR
Adult
PVR SVR
PGF2a
t
+
t
t
+
+
pge2
4-
4
4-
4-
t
4
pgd2
4
-(f)
4,4*
-(f)
f
-(4)
pgi2
4
4-
4-
4
4
4
txb2
?
?
?
?
+
4
pgh2
?
?
?
?
+
4
The effects of exogenous prostaglandins on pulmonary vascular resis¬
tance (PVR) and systemic vascular resistance (SVR) of fetal, neonatal
and adult animals are presented. Prostaglandins were administered
either directly into the pulmonary circulation or intravenously.
+ indicates vasoconstriction
4. indicates vasodilatation
- indicates no change
? response is not known
* Pulmonary response in newborn lambs was dependent on dose. Dilata¬
tion occurred with doses less than 2.5 ug/kg.min; constriction occurred
with doses greater than 8.0 yg/kg.min.

12
and systemic blood pressure in guinea pigs (17). Pulmonary vascular
resistance in the dog has been observed to increase with PGD2, while
systemic blood pressure was either unaffected or was decreased (69-71).
Infusions of PGD2 into the pulmonary circulation of fetal goats
caused dose-dependent decreases in pulmonary vascular resistance, with
either no effect on systemic pressure or a slight increase (72). New¬
born lambs showed a biphasic response to PGD25 with doses less than
2.5 yg/kg.min causing decreases in pulmonary tone, and doses greater
than 8.0 yg/kg.min producing pulmonary vasoconstriction (72). In
another study using conscious newborn lambs, PGD2 always caused vaso¬
constriction (62). Adult goats responded to PGD2 with increases in
pulmonary vascular resistance (72). The reason for differences between
fetus, newborn and adult is not clear, but it was postulated that
maturation of receptor population with differing affinities for PGD2
may occur (72).
Prostacyclin has been observed to be a very powerful pulmonary and
systemic vasodilator in both adult and perinatal animals (62, 73-80).
Using similar doses of PGI2 and PGE¡, it was found that PGI2 produced
a 4- to 10-fold greater decrease in systemic and pulmonary arterial
pressures (78). The use of prostacyclin to reduce elevated pulmonary
vascular resistance in the treatment of the clinical syndrome, persis¬
tent fetal circulation, has been suggested (81), but a caveat has been
issued against this practice due to the severe systemic hypotension
which accompanies infusion of PGI2 (82).
Thromboxanes have been shown to be vasoconstrictors in all vascu¬
lar beds studied (83, 84). There is greater potency of TXA2 than
TXB2 in stimulating platelet aggregation or smooth muscle contraction

13
(21, 37). Administration of TXB2 to dogs (85, 86) and cats (86)
caused pulmonary and systemic vasoconstriction. The actions of throm¬
boxanes have not been determined for the perinatal circulation.
Prostaglandin endoperoxides cause smooth muscle to contract (17),
and they have also been observed to increase pulmonary vascular resis¬
tance in adult monkeys, cats, dogs (87, 88) and sheep (89). Because
the half-life of PGH2 is extremely short, the responses observed could
be due to metabolites. However, use of an endoperoxide analog which is
not subject to further enzymatic action indicates that the vasocon¬
strictor activity of PGH2 is significant and is not merely due to the
action of a metabolite (69, 88, 90). Tyler et_ aK (61, 91) observed
pulmonary vasoconstriction in perinatal goats upon administration of
endoperoxide analogs. Systemic hypertension also occurred in these
studies, but the authentic endoperoxide intermediate, PGH2, does not
necessarily yield the same response when infused intravenously (87,
88). Systemic vasodilatation noted following infusion of PGH2 is most
probably the result of dilator prostaglandins such as PGI2 and/or PGE2
being produced from the endoperoxide during passage through the lung.
Arachidonic acid, the fatty acid prostaglandin precursor, produces
pulmonary and systemic vascular effects when injected into dogs (92).
These effects could be blocked by aspirin (92). Kadowitz et al_. (69)
showed a dose-response relationship for bolus injections of arachidonic
acid in closed-chest dogs. Increases in pulmonary vascular resistance
were accompanied by decreases in systemic arterial pressure, and both
were eliminated following indomethacin (69). An additional study in
intact dogs demonstrated the ability of arachidonic acid to cause pul¬
monary vasoconstriction in the absence of formed elements in the blood

14
was diminished during hypoxia (93). Perinatal goats respond to infu¬
sions of arachidonic acid with pulmonary vasoconstriction and systemic
hypotension, and indomethacin blocked these responses (91). A report
by Mullane et aK (94) was the first to suggest that low dose infu¬
sions of arachidonic acid caused falls in pulmonary arterial pressure
as well as systemic arterial pressure. Bioassay of arterial blood con¬
firmed the production of PGI2 (94). A study in cats reported diver¬
gent responses to arachidonic acid (95). In this report, when pulmo¬
nary tone was elevated with infusion of PGH2 analog or PGF2a, low dose
infusions of arachidonic acid elicited pulmonary vasodilatation (95).
Thus, response of the pulmonary circulation to arachidonic acid appears
to be dependent in part on existing pulmonary tone.
The release of prostaglandin-like compounds from isolated cat
lungs exposed to alveolar hypoxia was reported in 1974 (96). There was
also a reduction in the hypoxic pressor response in cats which had been
pre-treated with aspirin (96), leading to the proposal that prostaglan¬
dins were responsible for the pulmonary pressor response to hypoxia.
Several reports followed which argued against a role for prostaglandins
as the mediators of the hypoxic pressor response; instead, they sugges¬
ted that dilator prostaglandins were released in response to hypoxia to
counter-balance the pulmonary vasoconstriction (97-101). Using isola¬
ted rat lungs (97, 100), anesthetized dogs (98-100), calves (100) and
perinatal goats (101), these reports demonstrated potentiation of the
hypoxic pressor response following inhibition of prostaglandin synthe¬
sis with indomethacin (97-101), meclofenamate (97, 100) or aspirin (97-
99). From these studies a moderating role for prostaglandins in
response to hypoxia was proposed.

15
Recently, studies in anesthetized dogs suggest that arachidonic
acid can reduce the hypoxic pressor response (102). In these experi¬
ments, infusion of arachidonic acid during a period of hypoxia caused
reduction in pulmonary vascular resistance to pre-hypoxic levels (102).
Thin layer chromatography of a sample of systemic arterial blood showed
a major peak corresponding to 6-keto-PGF^a, the metabolite of PGI2
(102). Additional studies demonstrated the release of vasodilator PG's
from isolated rat lung during pulmonary vasoconstriction caused by hy¬
poxia or angiotensin II (103). 6-Keto-PGFla was measured in the efflu¬
ent from lungs which were constricted with angiotensin II or hypoxia,
suggesting that pulmonary vasoconstriction causes release of PGI2
(103).
Statement of Purpose
There are conflicting observations on the effects of arachidonic
acid in the pulmonary circulation (69, 91-95). Some studies have
reported only pulmonary vasoconstriction in response to arachidonic
acid (69, 91-93). Others have indicated that low doses of arachidonic
acid cause pulmonary vasodilatation, while higher doses produce pulmo¬
nary vasoconstriction (94, 95). Hyman et al. (95) have suggested the
difference in response may be due in part to existing level of pulmo¬
nary vascular resistance. In cats with elevated pulmonary tone, infu¬
sion of arachidonic acid in low doses caused pulmonary vasodilatation
(95). A report on the actions of arachidonic acid in dogs demonstrated
that infusion of arachidonic acid could reduce the pulmonary vasocon¬
striction seen in response to hypoxia (102). The response of normoxic

16
perinatal goats to infusions of arachidonic acid was always pulmonary
vasoconstriction, accompanied by systemic hypotension (91).
Based on these observations, the effects of arachidonic acid dur¬
ing normoxia and hypoxia were evaluated in ventilated fetal and neona¬
tal lambs using an in situ pump-perfused lower left lung preparation.
The results of these studies indicated that pulmonary vasoconstrictors
were produced from arachidonic acid during both normoxia and hypoxia;
thus, an inhibitor of thromboxane synthetase was tested in this prepa¬
ration to determine the role of TX's in the pulmonary pressor response
observed with arachidonic acid. Additionally, PG^j an important
intermediate in the prostaglandin biosynthetic pathway, was injected in
fetal lambs before and after ventilation to measure the pulmonary
response. The response of the pulmonary circulation of adult sheep to
arachidonic acid was evaluated under basal conditions and when pulmo¬
nary tone was enhanced by hypoxia or by infusion of PGF2a- The results
from this last experiment extend the observations made in perinatal
lambs and suggest that the divergent response of the pulmonary circula¬
tion to arachidonic acid is partly due to species differences.

CHAPTER TWO
PERINATAL PULMONARY RESPONSES TO ARACHIDONIC ACID
DURING NORMOXIA AND HYPOXIA
Introduction
Establishment of normal pulmonary vascular tone in newly venti¬
lated mammals is due in part to release of dilator prostaglandins
(PG's) (104). Treatment of fetal goats with indomethacin, a PG synthe¬
sis inhibitor, prior to ventilation does not change the initial rapid
decrease in pulmonary vascular resistance (PVR) which occurs with the
onset of ventilation; however, the slower, prolonged decrease in PVR is
markedly attenuated by indomethacin (104). This attenuation of the
ventilation-induced fall in PVR indicates that under normal conditions
(without PG synthesis inhibition) there is formation of a PG which acts
on the pulmonary vasculature in a vasodilatory manner. Indomethacin
also increases baseline PVR in premature and mature newborn goats
(101), indicating the removal of a dilator influence on the pulmonary
circulation. Recently, Leffler and others (45) measured PG's in pulmo¬
nary arterial and pulmonary venous blood samples drawn before and after
ventilation of fetal goats and lambs. Ventilation results in a net
increase in PGI-like material produced by the lung. Prostacyclin
(PGI2) is the most potent vasodilator PG of the fetal pulmonary cir¬
culation (78). Prostacyclin is also the predominant product of PG syn¬
thesis in perinatal vasculature (105, 106) and is continuously released
17

18
into the systemic circulation from the lungs (107). These findings
suggest an important role of vasodilatory PG's in establishing and
maintaining the low PVR of the newly-ventilated lung.
Dilator PG's are involved in modulating the pulmonary pressor
response to alveolar hypoxia in newborn goats, as evidenced by the
potentiation of the hypoxic pressor response in the presence of a PG
synthesis inhibitor (101). The pressor response to hypoxia is not due
to PG's, as it occurs even when production of PG's is blocked; it
appears that dilator PG's are released during hypoxia and act to
counteract hypoxic pulmonary vasoconstriction (100, 101).
Although perinatal vessels form mostly PGl£, infusions of the
bisenoic PG precursor, arachidonic acid, directly into the pulmonary
circulation of fetal and neonatal goats are associated with dose-
related increases in PVR (91). The increases in PVR are accompanied by
decreases in systemic arterial pressure, and these responses are inhib¬
ited completely by administration of indomethacin. Thus, the effects
of exogenous arachidonic acid are due to synthesis of PG's, but the
combined pulmonary and systemic effects are unlike those produced by
any single PG infused in a similar manner (108).
The reason for the differences in response of the pulmonary circu¬
lation to exogenous and endogenous arachidonic acid is not understood.
The following studies are designed to evaluate the pulmonary vascular
responses of ventilated fetal and neonatal lambs to infusions of ara¬
chidonic acid during normoxia and hypoxia.

19
Materials and Methods
Surgical preparation. Thirteen newborn lambs (2-9 days of age,
4.6 ± 0.2 kg) and mothers of 18 fetal lambs (0.91-0.97 gestation,
3.5 ± 0.2 kg) were anesthetized with chloralose (50 mg/kg, iv) and
tracheotomies were performed. Chloralose (10 mg/kg) was administered
hourly to maintain anesthesia. Fetuses were delivered by cesarean sec¬
tion and placed on a warmed (40°C) table adjacent to the ewe with
umbilical circulation undisturbed. A saline-filled rubber bag was
placed over the fetal head to prevent breathing, and the fetal abdomen
was sutured to the maternal skin to minimize exposure of the umbilical
cord. A tracheal cannula filled with warm (40°C) saline was tied in
the fetal trachea and the head cover was removed. In both fetal and
neonatal lambs colonic temperature was monitored (Yellow Springs
Instrument) and maintained between 38° and 40°C by use of an infra-red
lamp and placement of a heating pad beneath the animal. The femoral
artery was cannulated for monitoring systemic arterial pressure (SAP)
(Statham P23DC transducer), heart rate and arterial blood gases
(Instrumentation Laboratory pH/blood gas analyzer 213). Newborns were
paralyzed with d-tubocurare (0.2 mg/kg, iv), and controlled ventilation
with positive end-expiratory pressure (PEEP) of 2-4 cm H2O was initi¬
ated using either a Harvard respirator or a Healthdyne infant ventila¬
tor. A left thoracotomy was performed with removal of the fourth rib
to permit access to the left lung. Following isolation and dissection
from surrounding tissue of the left pulmonary artery, the lamb was
heparinized (2000 U/kg, iv). A catheter was placed in the femoral vein
and advanced into the inferior vena cava to a position above the level

20
of the liver. Blood was withdrawn from the inferior vena cava and
pumped (Cole-Parmer Masterflex pump no 7016) into the left pulmonary
artery. Between 35 and 40 % of the total lung tissue, as determined by
weight, was perfused in this manner. Flow through the ductus arterio¬
sus (in fetuses) and to the right lung was not disturbed. Left pulmo¬
nary blood flow (Q, Statham flowmeter M2202 with In Vivo Metric 3.0 or
4.0 mm cannulating electromagnetic flow probe) was maintained constant.
In fetal lambs, flow was set at a level at which pulmonary arterial
pressure (PAP) was equal to or slightly greater than mean SAP. In new¬
borns, flow was set to a minimal rate of 10 ml/kg body wt.min, and it
was increased to achieve a maximal flow at which PAP was not greater
than 30 mmHg (ave PAP = 22.4 ±1.0 mmHg). Flow was normalized for body
weight in order to account for variability in flow rates which were
related to the size of the animal. A catheter was placed in the left
atrium for measurement of left atrial pressure (LAP), and pulmonary
vascular resistance (PVR) was calculated as (PAP - LAP) • body weight
/ Q. Pressures, flow and heart rate were recorded continuously on a
Gould Brush 480 8-channel polygraph. An Apple 11+ computer was connec¬
ted to the polygraph with an analog-to-digital interface (AI13, Inter¬
active Structures, Inc.) for on-line data sampling and calculation of
PVR.
Experimental procedure. The sodium salt of arachidonic acid
(Sigma or NuChek Prep) was stored in dry form at -16°C. Fresh solu¬
tions were prepared daily with saline (final cone = 1 mg/ml) and pro¬
tected from light prior to being infused (Harvard pump) at varying
rates (0.051 - 2.06 ml/min) into the pulmonary arterial circuit.

21
Following establishment of baseline values for pressures, flow and
normal arterial blood gases, fluid was aspirated from the fetal tra¬
chea, the umbilical cord was occluded, and ventilation was started.
Due to the ventilation-induced decrease in PAP, flow was increased to
reflect the normal increase in pulmonary perfusion occurring at birth.
Ventilated fetuses were paralyzed with d-tubocurare (0.2 mg/kg, iv) as
necessary for control of ventilation.
The following treatments were applied in random order to each
animal group after establishing baseline values for pressures and flow
and obtaining normal arterial blood gases and pH. (Criteria for rejec¬
tion of hemodynamic data were arterial blood samples with pH < 7.30,
PO2 < 75 mmHg, or PCO2 > 50 mmHg.) 1) Arachidonic acid was infused
directly into the left pulmonary arterial perfusion circuit for 2 min
at varying rates. Doses of arachidonic acid were expressed as amount/
kg body wt.min. 2) Hypoxia was produced by lowering the inspired O2
to 6 % for 3 min. 3) A combination of the two treatments was given,
with hypoxia started for 1 min, and arachidonic acid infused during the
second and third minutes of hypoxia. A recovery period of at least 10
min was allowed between each experimental period in order to return to
control values. Due to deterioration of the animal preparations after
several treatments, each animal did not receive every treatment. The
following variables were sampled at intervals of 1 min, with the first
minute of the treatment sampled at 20-sec intervals: PAP, LAP, Q, mean
SAP and heart rate. A reading was taken 1 min before the start of the
treatment, and this value and the value at the beginning of the treat¬
ment were averaged to give a control value for the variables PVR and
mean SAP. The control values were set to 100 %, and the values at the

22
remaining time intervals were expressed as a percentage of the control
value. Each of the treatments within each animal group was analyzed
using a one-way analysis of variance with repeated measures, and dif¬
ferences between means were tested with the Newman-Keuls test (109).
Differences between groups or between treatments within the same group
were tested for significance using the unpaired Student's t-test. The
level of significance for statistical differences was P < 0.05.
Results
The average responses to 2-min infusions of arachidonic acid are
shown in Fig 2. The average pulmonary vascular response of 8 venti¬
lated fetuses to 9 infusions of arachidonic acid (133.7 ± 11.7
yg/kg.min) was a 26 % increase in PVR, at 2 min, over the control PVR.
The mean SAP at the same point was 96 % of the control value; this was
not significantly different from the control SAP. After the infusion
was turned off, PVR returned to baseline within 60 sec, but at the same
time, mean SAP significantly (P < 0.01) decreased to 91 % of the con¬
trol value and remained close to this level for the next 2 min. A
higher dose range (ave dose = 284.9 ± 12.8 ug/kg.min) was administered
to 10 ventilated fetal lambs, and it resulted in PVR at 40 sec of 156 %
of the control PVR (P < 0.01) (Table 3). Mean SAP at that time was
99 % of control SAP, but it dropped to a low value of 81 % at the fifth
minute. The significant decrease in mean SAP and a longer time for
recovery to baseline at this dose of arachidonic acid were reasons for
not attempting even higher doses.

Figure 2: Average pulmonary and systemic responses to 2-min infusions
of arachidonic acid are shown for 8 ventilated fetuses (9
infusions, ave dose = 133.7 ± 11.7 yg/kg.min) and 6 newborns
(7 infusions, ave dose = 164.0 ± 15.9 yg/kg.min). Data are
expressed as mean ± SEM. Bars indicate the duration of the
treatment. Points marked with asterisks are significantly
different from the value at 0 min; * P < 0.05, ** P < 0.01.

VENTILATED FETAL LAMBS
ARACH ACID-2 min
1 33.7 ^g/kg.hr
TIME (min)
% SAP % PVR
NEWBORN LAMBS
ARACH ACID-2 min
164.0 pg/kg.min
TIME (min)

25
Table 3. Maximal Change in PVR and SAP for all treatments in each group
Ventilated Fetuses
% PVR % SAP
Newborn Lambs
% PVR % SAP
1. Arachidonic Acid
(low dose)-2 min
(inc)
126.33 ±
9.72
191.15 ±
11.87
(dec)
—
91.23 ± 2.92
--
93.15 ± 3.29
(dose)
133.7
± 11.7 pg/kg.min
52.8
± 5.6 pg/kg.min
(n)
9
13
2. Arachidonic Acid
(high dose)-2 min
(inc)
155.71 ±
15.39
301.37 ±
20.24
(dec)
—
80.51 ± 4.08
—
82.90 ± 2.93
(dose)
284.9
± 12.8 pg/kg.min
164.0
± 15.9 pg/kg.min
(n)
15
7
3. Hypoxia
(6 %)-3 min
(inc)
160.25 ±
13.36 120.47 ± 7.22
143.76 ±
4.51 136.48 ± 5.99
(dec)
—
—
--
--
(n)
12
13
4. Combination of 1
+ 3
(inc)
192.33 ±
14.96 136.38 ± 12.39
221.26 ±
9.51 121.84 ± 2.08
(dec)
—
80.78 ± 9.37
—
82.22 ± 6.65
(dose)
155.2
± 14.8 pg/kg.min
39.5
± 4.7 pg/kg.min
(n)
4
5
5. Combination of 2
+ 3
(inc)
234.38 ±
24.71 109.18 ± 15.60
292.04 ±
11.23 126.98 ± 10.65
(dec)
—
72.43 ± 6.21
--
72.74 ± 4.63
(dose)
315.9
± 44.6 pg/kg.min
156.0
± 21.7 pg/kg.min
(n)
4
5
Values shown are maximal increases (inc) or decreases (dec) in PVR or
SAP during the treatment indicated. Data are expressed as mean ± SEM.
The number of treatments in each group is indicated by n. Treatments 4
and 5 are combinations of 3 min hypoxia and 2 min arachidonic acid infu¬
sion at dose ranges as in 1 (for treatment 4) or 2 (for treatment 5).
Locations with a dash indicate the response was in only one direction;
i.e., only vasoconstriction or vasodilatation. Note that the doses of
arachidonic acid which are compared in Fig 2 are from treatment 1 (ven¬
tilated fetuses) and treatment 2 (newborn lambs). Also, Fig 4 shows
doses from treatment 4 for ventilated fetuses and treatment 5 for new¬
born lambs.

26
In 6 newborn lambs, 7 infusions of arachidonic acid (ave dose =
164.0 ± 15.9 pg/kg.min) caused PVR to increase to 301 % of control PVR
(P < 0.01) (Fig 2). Althoug the dose used was comparable to the lower
dose used in the ventilated fetal lambs, the pulmonary response was
markedly greater in the neonate (P < 0.001). The mean SAP at the end
of the 2-min infusion was 84 % of control SAP, and the combination of
the systemic hypotension and severe pulmonary vasoconstriction preven¬
ted further increasing the infusion rate to the same levels used in the
ventilated fetal lambs. Instead, the dose of arachidonic acid infused
was lowered (Table 3), so that 10 lambs received 13 infusions at an
average rate of 52.8 ± 5.6 ug/kg.min. This lower dose caused a signif¬
icant (P < 0.01) increase in PVR to 191 % of baseline PVR, without
causing any significant reductions in mean SAP.
The responses to arachidonic acid observed in ventilated fetal and
neonatal lambs were due to production of PG's, as administration of
indomethacin (2 mg/kg, iv, 1 ventilated fetus) or meclofenamate (2
mg/kg, iv, 1 lamb) prevented any response when arachidonic acid was
infused after 15 min. Also, infusion of a fatty acid, 11,14,17-eicosa-
trienoic acid, which does not undergo conversion to PG's, did not cause
increases in PVR or systemic hypotension in 1 newborn and 1 ventilated
fetus.
The effects of 3 min of alveolar hypoxia (inspired O2 = 6 %) are
illustrated in Fig 3. The pulmonary vascular responses in neonates and
ventilated fetuses were similar with a slightly greater increase in PVR
in the ventilated fetal lambs (160 % of control) than in the newborns
(144 % of control) (Table 3). The increase in mean SAP was also simi¬
lar (ventilated fetuses, 120 %; newborn lambs, 134 %). This period of

Figure 3: Average pulmonary and systemic responses to 3-min periods of
hypoxia (FIq2 = 0.06) are shown for 10 ventilated fetuses
(12 cases) and 12 newborn lambs (13 cases). Symbols are as
in Fig 2.

VENTILATED FETAL LAMBS
HYPOXI A-6%-3 min
TIME (min)
% SAP % PVR
NEWBORN LAMBS
HYPOXIA-6%-3 min
TIME (min)

29
hypoxic exposure produced a consistent response in each group of ani¬
mals and was accompanied by a severe decrease in arterial O2 tension
(Table 4), with recovery to baseline values occurring within 10 min in
most cases.
The results of the combination of 3 min of hypoxia with a 2-min
infusion of arachidonic acid are shown in Fig 4. In 5 newborn lambs,
PVR increased to 292 % of control PVR at the end of the combination,
but this response was not significantly different (P > 0.5) from the
response to a similar dose of arachidonic acid during normoxia (Table
3). In ventilated fetal lambs receiving the lower dose of arachidonic
acid, PVR increased to 192 % of control PVR during alveolar hypoxia and
arachidonic acid infusion. This increase was significantly greater
(P < 0.01) than the response to arachidonic acid alone, but appeared to
be an additive effect to the hypoxic pressor response. Thus, the pul¬
monary response to arachidonic acid was not affected by exposure to
alveolar hypoxia in newborn lambs, but was increased significantly in
ventilated fetal lambs during hypoxia. However, mean SAP was reduced
following the termination of the hypoxia and infusion in both groups of
animals (Fig 4). The fall in SAP to 81 % of control values at 3 min
after the end of the treatment in the ventilated fetuses receiving the
low dose of arachidonic acid was significantly different from the
increase in mean SAP to 136 % of control at the end of the treatment
(P < 0.01). In neonatal lambs, the increases in mean SAP during the
combined hypoxia and arachidonic acid (values at 40 sec, 1, 2 and 3
min) were significantly different from mean SAP following this treat¬
ment (values at 4, 5 and 6 min, P < 0.01). The time course and extent
of the systemic hypotension in the two animal groups during the

Table 4.
Arterial blood gases and pH before, during
and following hypoxia
pH
P02
PC02
(mmHg)
(mmHg)
1. Ventilated Fetal
Lambs
(n = 8)
Before Hypoxia
7.46
+
0.03
123.8 ± 10.0
33.0 ± 1.8
During Hypoxia
7.45
+
0.03
18.6 ± 2.1
34.3 ± 2.1
After Hypoxia
7.42
+
0.02
102.2 ± 13.5
35.2 ± 1.3
2. Newborn Lambs
(n = 12)
Before Hypoxia
7.43
+
0.02
127.7 ± 8.6
33.5 ± 2.1
During Hypoxia
7.43
+
0.02
20.9 ± 0.9
31.8 ± 1.8
After Hypoxia
7.41
±
0.02
112.8 ± 8.5
34.3 ± 1.8
Data are expressed as mean ± SEM.

Figure 4: Average pulmonary and systemic responses to a combination of
3-min periods of hypoxia (FIq2 = 0.06) and 2-min infusions
of arachidonic acid are shown in 2 ventilated fetuses (4
cases, ave dose = 155.2 ± 14.8 ug/kg.min) and 5 newborns (5
cases, ave dose = 156.0 ± 21.7 ug/kg.min). Symbols are as
in Fig 2.

% SAP % PVR
VENTILATED FETAL LAMBS
HYPOXIA-6%-3 min
ARACH ACID-2 min
155.2 pg/kg.min
TIME (min)
* SAP % PVR
NEWBORN LAMBS
HYPOXIA-6%-3 min
ARACH ACID-2 min
156.0 >jg/kg.min
TIME (min)

33
combination of hypoxia and arachidonic acid were markedly different
from the responses to arachidonic acid alone (Fig 2 and 4).
Discussion
Intra-pulmonary infusions of arachidonic acid in 3 dose ranges
caused significant increases in PVR in ventilated fetal and neonatal
lambs. At comparable doses (100-200 ug/kg.min), newborns showed sig¬
nificantly greater increases in PVR than ventilated fetal lambs. This
finding is similar to an earlier report on arachidonic acid in fetal
and newborn goats (91). A possible explanation for the greater respon¬
siveness of the neonate to arachidonic acid is the increase in lung
microsome cyclo-oxygenase activity which occurs after birth (110).
This increase in cyclo-oxygenase activity results in higher concentra¬
tions of PGH2 available for further metabolism in newborns. Addi¬
tionally, PGH2 formed in the lung may exert a local effect on pulmo¬
nary vessels before undergoing metabolism to other PG's. Studies in
perinatal goats indicate that a stable endoperoxide analog caused pul¬
monary vasoconstriction (91). Thus, if similar amounts of arachidonic
acid were infused in newborns and ventilated fetuses, the ability of
newborns to form more PGH2 could result in more severe pulmonary
vasoconstriction (Fig 2).
Pulmonary metabolism of arachidonic acid is dependent on substrate
availability, relative activities of various PG enzymes, and cofactor
levels. Arachidonic acid is converted rapidly to PGH2 by cyclo-oxy¬
genase (15), and the endoperoxide intermediate either is released into
the circulation or is metabolized further. The quantity and nature of

34
PG's formed from PGH2 depend on the PG enzyme activities and the
amount of PGH2 present. In homogenates from adult cat lungs, the
production rates of 6-keto-PGF^a and TXB=2 (metabolites of PGI2 and
TXA2, respectively) were equal at low levels of PGH2 (less than 10 yM)
(111). However, as the concentration of PGH2 was increased, there
was up to 7 times more TXB2 than 6-keto-PGF^a produced. Studies in
fetal and newborn sheep lung homogenates indicate the activity of pros¬
tacyclin synthetase was relatively low and constant throughout gesta¬
tion (110). The enzyme also exhibited saturation at low substrate
levels. However, thromboxane synthetase activity increased during ges¬
tation and continued to rise post-natally, so that at higher concentra¬
tions of PGH2» neonatal lung homogenates formed up to 5 times as much
TXB2 as 6-keto-PGF^a.
A problem with studies using isolated homogenates is a lack of
demonstrated biological activity of the products. Thus, the above stu¬
dies which measure amounts of PG products formed by lungs do not indi¬
cate any vasoactive properties of the compounds produced. The short
half-life of TXA2 makes it difficult to obtain a dose-response curve
in vivo, so that relative potencies for TXA2 and PGI2 cannot be
assessed. Based on the present study, the net effect of arachidonic
acid infusion was pulmonary vasoconstriction, indicating either activi¬
ty due to PGH2, greater formation of TXA2, greater potency of TXA2 on
pulmonary vascular smooth muscle, or a combination of the above. Also,
there could be an interaction with the primary PG's, such as PGF2a
or PGD2» which are pulmonary vasoconstrictors in newborn goats and
lambs (60, 72).

35
The pulmonary vascular effects of exogenous arachidonic acid
infused directly into the pulmonary circulation of neonatal lambs were
not altered by concomitant exposure to alveolar hypoxia. Arachidonic
acid increased PVR over the hypoxic pressor response, but the extent of
pulmonary vasoconstriction was so severe with infusions alone that it
appears the pulmonary vessels may have been unable to increase tone any
further in response to hypoxia. Although ventilated fetuses exhibited
a significantly greater response to arachidonic acid infused during
hypoxia than during normoxia, the increase in PVR was merely additive
to the hypoxic pressor response. Thus hypoxia does not appear to
affect the actions of exogenous arachidonic acid in the perinatal pul¬
monary circulation.
In contrast to these studies, recent reports using adult dogs
demonstrated a reduction in hypoxic pulmonary vasoconstriction during
infusion of arachidonic acid (102, 112). The dose administered in the
study by Gerber et^ al_. (102) was similar to the lower doses infused in
newborns in the present study; however, the only response seen in lambs
was pulmonary vasoconstriction, whether arachidonic acid was infused
during normoxia or hypoxia (Table 3). A recent study in conscious
adult sheep demonstrated that infusions of arachidonic acid at doses of
25, 50 and 100 yg/kg.min caused pulmonary vasoconstriction (113).
Thus, differences in responses may be due to species differences (27).
The hypotensive effects of arachidonic acid on the systemic circu¬
lation were insignificant at the low doses infused into newborns, but
with higher doses mean SAP decreased significantly in both newborn and
ventilated fetal lambs. This is consistent with reports of PGI2
being the predominant PG released by the lung (20, 45, 107). The

36
systemic response to hypoxia was an increase in mean SAP, with a rapid
return to base!in. The addition of arachidonic acid during hypoxia
caused SAP to drpo below control levels at the end of the combined
treatment, and at the end of 5 min mean SAP was still significantly
depressed. These findings are in agreement with studies indicating an
increase in vasodilator PG's during hypoxia (100, 101) and preliminary
data which show an increased efflux of PG's several minutes after in¬
fusing arachidonic acid from lungs of newborn lambs which were perfused
with Krebs solution (Leffler, personal communication).
Although arachidonic acid forms a moderate amount of a systemic
vasodilator when infused during normoxia, the significantly greater
reduction in mean SAP and the prolonged time for recovery from systemic
hypotension during hypoxia and arachidonic acid indicate an increase in
the amount of vasodilator substance coming from the lung. Additional¬
ly, PGI2 may be produced by vascular endothelia in the systemic cir¬
culation. Infusions of arachidonic acid produce only vasoconstriction
in the perinatal pulmonary circulation, during both normoxia and hypox¬
ia. The most likely explanation for these results is that high concen¬
trations of PGH2 are formed in the lung with subsequent release of
TXA2 and/or PGH2. Both of these compounds are presumed to have
vasoconstrictor activity, and both have short half-lives. The predomi¬
nant effect is thus pulmonary vasoconstriction caused by one of these
products of arachidonic acid metabolism, with further metabolism of
PGH2 to PGI2 and degradation of TXA2 to a relatively weak metabolite.
This would result in systemic hypotension due to the predominance of
PGI2 at this time. The effect of hypoxia upon arachidonate metabo¬
lism seems to reflect no change in PG products acting in the lung, but

37
may indicate an enhanced release of PGI2 from the lungs into the
systemic circulation.

CHAPTER THREE
THROMBOXANE SYNTHETASE INHIBITION AND PERINATAL PULMONARY
RESPONSE TO ARACHIDONIC ACID
Introduction
Biological activity of the potent vasoconstrictor and proaggrega-
tory substance TXA2 can be blocked either by inhibition of TX synthe¬
tase or with a TXA2 receptor antagonist (114). Inhibitors of TX syn¬
thetase include imidazole, and its derivatives (114), and pyridine, and
its derivatives (115, 116); TXA2 receptor antagonists include analogs
of PGH2, such as 9,ll-epoxyimino-5,13-dienoic acid (114). Pyridine
and its derivatives have been described as being capable of preventing
release of TXB2 from platelets and from lung microsomes by inhibiting
TX synthetase (115, 116). The pyridine derivative found to be the most
potent inhibitor of TX synthetase inhibition was characterized as £-[4-
(2-carboxy-l-propenyl)benzyl] pyridine hydrochloride (OKY-1555) (115),
and its sodium salt is known as OKY-1581 (sodium (E)-3-[4-(3-pyridyl-
methyl)phenyl]-2-methyl acrylate) (117).
OKY-1581 inhibited platelet aggregation induced by arachidonic
acid in vitro (118), and reduced concentrations of TXB2 in blood of
monkeys (118), rabbits (119), and humans (120) when administered
orally. The incidence of arachidonic acid-induced cerebral infarction
38

39
in rabbits was reduced by pre-treatment with OKY-1581 (118). In dogs
with a partially obstructed coronary artery, intravenous administration
of OKY-1581 inhibited the platelet aggregation caused by the obstruc¬
tion (121). Inhibition of TX synthetase occurred without affecting
synthesis of PGI2 (121).
In fetal and neonatal lambs and goats, infusions of arachidonic
acid into the pulmonary circulation produced dose-related increases in
pulmonary vascular resistance (91, Ch 2). This pressor response may be
due to 1) increased production of the pressor agent TXA2, 2) forma¬
tion of PGH2 or 3) mechanical obstruction due to platelet aggregation.
Thus, inhibition of TX synthesis might prevent pulmonary vasoconstric¬
tion due to arachidonic acid. The purpose of this study was to test
the effect of a specific TX synthetase inhibitor, OKY-1581, on the
response of the perinatal pulmonary circulation to infusions of arachi¬
donic acid and hypoxia.
Materials and Methods
Surgical preparation. The surgical procedure for the in situ
pump-perfused lower left lung preparation in perinatal lambs has been
described in detail in the preceding chapter. Mothers of 12 fetal
lambs (0.91-0.97 gestation, wt = 3.7 ± 0.1 kg) and 13 newborn lambs
(2-9 days of age, wt = 4.6 ± 0.2 kg) were anesthetized with chloralose
and prepared so that left pulmonary arterial pressure (PAP), left
atrial pressure (LAP), systemic arterial pressure (SAP) and heart rate
were monitored continuously under conditions of constant left pulmonary
• •
arterial blood flow (Q). By maintaining Q constant, changes in PAP

40
reflected changes in pulmonary vascular resistance (PVR), which was
calculated as (PAP - LAP) • body wt / Q. In these experiments fetal
lambs were ventilated and the umbilical cord was occluded.
Experimental procedure. The sodium salt of arachidonic acid
(Sigma or NuChek Prep) was stored in dry form at -16°C. Fresh solu¬
tions were prepared daily with saline (final cone = 1 mg/ml) and pro¬
tected from light prior to being infused (Harvard pump) at varying
rates (0.051 - 2.06 ml/min) into the pulmonary arterial circuit.
OKY-1581 (Ono Pharmaceutical Co.) was received and stored in dry form
at 6°C. On the day of the experiment, the compound was diluted with
0.9 % saline to a concentration of 50 mg/ml.
The following treatments were applied in random order to newborn
lambs after establishing baseline values for pressures and flow and
obtaining normal arterial blood gases and pH. (Criteria for rejection
of hemodynamic data were arterial blood samples with pH < 7.30, PO2 <
75 mmHg, or PCO2 > 50 mmHg.) 1) Arachidonic acid was infused direct¬
ly into the left pulmonary arterial circuit for 2 min at varying rates.
2) Alveolar hypoxia was produced by lowering the inspired O2 to 6 %
for 3 min. 3) A combination of the two treatments was given, with
hypoxia started for 1 min, and arachidonic acid infused during the
second and third minutes of hypoxia. A recovery period of at least 10
min was allowed between successive experimental periods in order to
return to control values. Lambs then received 50 mg of OKY-1581,
injected slowly into the pulmonary arterial circuit, and the treatments
were repeated. Thromboxane inhibition lasted at least 2 hours, and
lambs showed responses to arachidonic acid at 2 hours after OKY-1581
which were similar to responses at 15 min after inhibition. Lambs

41
which did not receive OKY-1581 (Ch 2) did not show attenuation of pul¬
monary vascular responses to arachidonic acid after repeated infusions.
Ventilated fetuses were tested for responses to arachidonic acid only,
both before and after TX synthesis inhibition. Due to deterioration of
the animal preparations after several treatments, some animals did not
receive every treatment. The following variables were measured at
intervals of 1 min, with the first minute of the treatment sampled at
20-sec intervals: PAP, LAP, Q, mean SAP and heart rate. A measurement
was made 1 min before the start of the treatment, and this value and
the value at the beginning of the treatment were averaged to give a
control value for the variables PVR and mean SAP. The control values
were set to 100 %, and the values at the remaining time intervals were
expressed as a percentage of the control value. The treatments within
each animal group were analyzed using one-way analysis of variance with
repeated measures, and differences between means were tested with the
Newman-Keuls test (109). Differences between treatments within the
same animal group were tested for significance using the unpaired
Student's t-test. The level of significance for statistical differ¬
ences was P < 0.05.
Results
The effects of treatment with the specific TX synthetase inhibitor
OKY-1581 on infusions of arachidonic acid in neonatal lambs are shown
in Fig 5 and 6. In 8 lambs receiving 2-min infusions of arachidonic
acid (ave dose = 52.4 ± 6.8 pg/kg.min, Fig 5), pre-treatment with
OKY-1581 significantly (P < 0.01) reduced the increase in PVR at 40 sec

Figure 5: Average pulmonary and systemic responses to 2-min infusions
of arachidonic acid are shown for 10 newborn lambs (13
infusions, ave dose = 52.8 ± 5.6 yg/kg.min) and 8 newborns
treated with 50 mg OKY-1581 (13 infusions, ave dose = 52.4 ±
6.8 ug/kg.min). Symbols are as in Fig 2.

NEWBORN LAMBS
ARACH ACID-2 min
52.8 ¿ig/kg.min
a.
<
co
140
120
100
80
80
- 1
TIME (min)
NEWBORN LAMBS
ARACH ACID-2 min
52.4 pg/kg.min
FOLLOWING OKY-1581
i i l i i i i i I i i I i i I i—i—I—i—l—I
00
=- r r—*'
S L-
» 1 1
-1 I I I I I I I I I I l
TIME (min)

Figure 6: Average pulmonary and systemic responses to 2-min infusions
of arachidonic acid are shown for 6 newborn lambs (7
infusions, ave dose = 164.0 ± 15.9 yg/kg.min) and 6 lambs
treated with 50 mg OKY-1581 (6 infusions, ave dose = 141.5 ±
14.5 ug/kg.min). Symbols are as in Fig 2.

NEWBORN LAMBS
ARACH ACID-2 min
184.0 pg/kg.min
TIME (min)
% SAP % PVR
NEWBORN LAMBS
ARACH ACID-2 min
141.5 jig/kg.min
FOLLOWING OKY-1581
TIME (min)

46
as compared with lambs without the inhibitor, and the response remained
lower than the pulmonary response to arachidonic acid (ave dose = 52.8
± 5.6 yg/kg.min) without OKY-1581 until the fifth minute. The peak
increase in PVR due to arachidonic acid following OKY-1581 occurred at
1 min, and was only 17 % of the peak increase produced by arachidonic
acid alone, which occurred at 2 min. At this dose of arachidonic acid
there were no significant differences in the response of the systemic
circulation in the presence or absence of TX synthesis inhibition.
There is a suggestion of a greater fall in systemic pressure. Injec¬
tion of OKY-1581 produced transient (less than 60 sec) decreases in
PAP. Pressure returned to baseline before infusions or hypoxia were
tested.
Figure 6 shows average responses to a higher dose of arachidonic
acid in 6 newborns. Addition of OKY-1581 significantly (P < 0.005)
reduced the pulmonary response to arachidonic acid (ave dose = 141.5 ±
14.5 yg/kg.min) at 40 sec, and PVR remained at significantly lower
values than observed in lambs without OKY-1581 until the sixth minute.
The maximal increase in PVR in lambs receiving OKY-1581 occurred at 1
min, and was 26 % of the peak increase in PVR at 2 min in lambs without
the blocker. Systemic blood pressure decreased significantly from con¬
trol values in both groups of lambs (with and without OKY-1581) at 2
and 3 min (Fig 6), but the fall in SAP in lambs receiving the inhibitor
was more than double the decrease in SAP in lambs without OKY-1581.
This significant (P < 0.025) increase in the systemic hypotensive ef¬
fect of arachidonic acid at 3 min was sustained for the remaining time
intervals, so that at 6 min, mean SAP was only 75 % of control SAP in
treated lambs.

47
The effects of 3 min of hypoxia (inspired O2 = 6 %) in the
presence and absence of TX synthetase inhibition are shown in Fig 7.
There were no significant differences in the responses of the pulmonary
circulation in lambs receiving OKY-1581 prior to hypoxia as compared
with responses in lambs without the inhibitor. The effect of hypoxia
on the systemic blood pressure was also similar in the two groups of
lambs, with a slight, although insignificant, depression of the
increase in SAP during hypoxia occurring in the OKY-treated lambs.
After the hypoxic period ended, SAP in lambs receiving OKY-1581 fell
below baseline values. The decrease observed in SAP at 4, 5 and 6 min
was not significantly different from the control SAP in the TX inhibit¬
ed group; however, the value at 5 min was significantly (P < 0.01) less
than the SAP at 5 min in the lambs without OKY-1581.
Newborn lambs which had received OKY-1581 responded in a similar
manner to combined hypoxia and arachidonic acid infusions as lambs
which were not treated with the blocker (Table 5). In both groups ara¬
chidonic acid caused a further increase in PVR over the hypoxic pressor
response alone, but the combined response was not greater than the ad¬
ditive response of arachidonic acid and hypoxia alone. The pulmonary
response to the lower dose of arachidonic acid during hypoxia in 3 OKY-
treated lambs was significantly (P < 0.05) less than the increase in
PVR in 5 untreated lambs at 2 and 3 min. The peak PVR in lambs with
OKY was 62 % of the peak PVR in lambs without the blocker. At the
higher dose of arachidonic acid infused during hypoxia, 3 lambs receiv¬
ing OKY-1581 showed significantly smaller increases in PVR at the
second and third minutes of the response (P < 0.01) than 5 lambs with¬
out the inhibitor. The difference between the two groups was still

Figure 7: Average pulmonary and systemic responses to 3-min periods of
hypoxia (FIq2 = 0.06) are shown for 12 newborn lambs (13
cases) and b lambs treated with 50 mg OKY-1581 (7 cases).
Symbols are as in Fig 2.

NEWBORN LAMBS
HYPOXIA-6%-3 min
TIME (min)
% SAP % PVR
NEWBORN LAMBS
HYPOXIA-6%-3 min
FOLLOWING OKY-1581
TIME (min)

50
Table 5. Maximal Change in PVR and SAP for all treatments
without OKY-1581
with OKY-1581
% PVR
% SAP
% PVR
% SAP
Venti1ated
Fetuses
1. Arachidonic Acid
(high dose)-2 min
(inc)
155.71 ±
15.39
125.93 ±
5.21
(dec)
96.01 ±
2.33 80.51 ± 4.08
80.68 ±
2.10 69.48 ± 3.62
(dose)
284.9
± 12.8 yg/kg.min
296.9
± 13.1 yg/kg.min
(n)
15
11
Newborn Lambs
1. Arachidonic Acid
(low dose)-2 min
(inc)
191.15 ±
11.87
115.45 ±
4.13
(dec)
--
93.15 ± 3.29
--
86.58 ± 4.01
(dose)
52.8
± 5.6 yg/kg.min
52.4
± 6.8 yg/kg.min
(n)
13
13
2. Arachidonic Acid
(high dose)-2 min
(inc)
301.37 ±
20.24
152.60 ±
9.20
(dec)
—
82.90 ± 2.93
91.92 ±
2.59 62.02 ± 8.00
(dose)
164.0
± 15.9 yg/kg.min
141.5
± 14.5 yg/kg.min
(n)
7
6
3. Hypoxia
(6 %)-3 min
(inc)
143.76 ±
4.51 136.48 ± 5.99
153.37 ±
9.86 124.49 ± 3.66
(dec)
—
—
--
86.77 ± 4.24
(n)
13
7
4. Combination of 1
+ 3
(inc)
221.26 ±
9.51 121.84 ± 2.08
174.58 ±
14.23 130.63 ± 4.44
(dec)
--
82.22 ± 6.65
--
86.88 ± 6.24
(dose)
39.5
± 4.7 yg/kg.min
39.5
± 0.4 yg/kg.min
(n)
5
4
5. Combination of 2
+ 3
(inc)
292.04 ±
11.23 126.98 ± 10.65
189.90 ±
32.69 140.40 ± 26.59
(dec)
—
72.74 ± 4.63
--
75.40 ± 7.28
(dose)
156.0
± 21.7 yg/kg.min
145.8
± 22.9 yg/kg.min
(n)
5
3
Values shown are maximal increases (inc) or decreases (dec) in PVR or
SAP during the treatment indicated. Data are expressed as mean ± SEM.
The number of observations in each treatment is indicated by n. Treat¬
ments 4 and 5 under newborn lambs are combinations of 3 min hypoxia and
2 min arachidonic acid infusion at dose ranges as in 1 (for treatment 4)
or 2 (for treatment 5). Locations with a dash indicate the response was
in only one direction; i.e., only vasoconstriction or vasodilatation.

51
present at 4 and 5 min (P < 0.05). There was a 47 % reduction in peak
PVR in the OKY-treated lambs as compared with the peak PVR in the
untreated group. There were no significant differences between the two
groups in responses of the systemic circulation to combinations of
hypoxia and arachidonic acid at either dose of arachidonate used (Table
5).
Pulmonary and systemic vascular responses to infusions of arachi¬
donic acid in ventilated fetal lambs are shown in Fig 8. The increase
in PVR at 40 sec in 6 fetuses receiving OKY-1581 was 54 % less than the
peak PVR, also at 40 sec, in 10 fetuses without the blocker. Although
the depressed PVR in treated fetal lambs was not significantly differ¬
ent from the peak PVR in the untreated group, the values at 2, 3, 4, 5
and 6 min in fetuses with OKY-1581 were significantly lower than cor¬
responding values in untreated ventilated fetal lambs (P < 0.025, 0.02,
0.001, 0.001, 0.001, respectively). Also, in the OKY-treated fetuses,
decreases in PVR at 3, 4, 5 and 6 min were significantly (P < 0.01)
less than control PVR (Fig 8). Systemic pressure was significantly (P
< 0.05) reduced from control SAP at 1 min in TX-inhibited fetuses, and
values for SAP at 2 and 3 min in the treated group were significantly
(P < 0.01, 0.025, respectively) lower than corresponding values in ven¬
tilated fetuses without OKY-1581.
Discussion
Inhibition of TX synthetase with the pyridine derivative OKY-1581
depressed the increases in PVR which occur with infusions of arachidon¬
ic acid in perinatal lambs. The dose of OKY-1581 used in this study

Figure 8: Average pulmonary and systemic responses to 2-min infusions
of arachidonic acid are shown for 10 ventilated fetal lambs
(13 infusions, ave dose = 284.9 ± 12.8 yg/kg.min) and 6
ventilated fetuses treated with 50 mg OKY-1581 (11 infu¬
sions, ave dose = 296.9 ± 13.1 yg/kg.min). Symbols are as
in Fig 2.

% SAP % PVR
VENTILATED FETAL LAMBS
ARACH ACID-2 min
284.9 pg/kg.min
TIME (min)
VENTILATED FETAL LAMBS
ARACH ACID-2 min
296.9 pg/kg.min
FOLLOWING OKY- 158 1
cn
Go
TIME (min)

54
(50 mg, or 10 - 15 mg/kg) was at least 10 times the effective dose
administered to rabbits to prevent arachidonate-induced sudden death
(115). However, the effectiveness of the inhibition was not estab¬
lished in these studies. When animals were treated with OKY-1581, the
peak increase in PVR in response to infusions of arachidonic acid was
16 %, 26 % and 46 % of the peak pulmonary response without the inhibi¬
tor, depending on the dose of arachidonic acid infused (Table 5). The
hydrochloride compound OKY-1555 has been characterized as a noncompeti¬
tive inhibitor of TX synthetase (115). Presumably, the sodium salt
used in this study, OKY-1581, acts in the same way as the hydrochloride
salt. Therefore, one likely explanation for our findings is that the
increase in PVR seen in the presence of OKY-1581 is caused by PG endo-
peroxides. Alternatively, the enzyme may not have been inhibited com¬
pletely. The pharmacology of OKY-1581 has not been studied with res¬
pect to TX synthetase of developing sheep lung.
Treatment with OKY-1581 also significantly enhanced systemic hypo¬
tension caused by infusions of arachidonic acid in lambs. This could
result from increased production of vasodilator PG's in the lung due to
shunting of PGH2 away from TX synthetase. This is consistent with
studies showing increased formation of 6-keto-PGF^a with TX synthetase
inhibition (120, 121) and may contribute to the "steal" phenomenon pro¬
posed by Gryglewski and others (18). Thus, formation of PG endoperox-
ides could give rise to increased production of PGI2» which is a po¬
tent vasodilator of both systemic and pulmonary circulations in perina¬
tal goats and lambs (78). Additionally, any possible pressor action of
TXA2 itself on the systemic circulation would be missing.

55
Pulmonary vasoconstriction in response to hypoxia was unaffected
by inhibition of TX synthesis (Fig 7). Hypoxic pulmonary vasoconstric¬
tion is not believed to be produced by PG's (100, 101). Inhibition of
cyclo-oxygenase results in potentiation of the hypoxic pressor response
(100, 101), indicating removal of net PG dilator influence during
hypoxia. 0KY-1581 does not interfere with enzymes responsible for
producing dilator PG's (118, 120). PGI2» PGE2 and PGD2 are dilators in
the perinatal pulmonary circulation, and PGI2 and PGE2 are systemic
vasodilators as well (67, 72, 78). Thus, inhibition of TX synthetase
should not affect the production of these dilator PG's in response to
hypoxia. There was a greater reduction in SAP after hypoxia (at 5 min)
in lambs treated with 0KY-1581 as compared to untreated lambs (Fig 7),
perhaps due to increased formation of dilator PG's in the presence of
TX synthesis inhibition.
One of the problems not addressed in this study is the role of
platelets in the pulmonary response to arachidonic acid. It is con¬
ceivable that increases in PVR seen in response to infusions of arachi¬
donic acid are due in part to platelet aggregation leading to plugging
of the pulmonary microvasculature. Thromboxanes are potent platelet
aggregatory compounds, and they also stimulate vascular smooth muscle
contractions in vitro (21). Several authors (93, 122, Leffler, per¬
sonal communication) suggest that platelets are not necessary for pul¬
monary vasoconstriction in response to arachidonic acid. Hyman and
others (93) demonstrated that the pressor response to arachidonic acid
was similar in canine lungs perfused with either blood or platelet-poor
plasma. Leffler (personal communication) has found that neonatal lamb
lungs which were perfused with Krebs solution respond to exogenous

56
arachidonic acid with pulmonary vasoconstriction. Increases in PAP
occurred in isolated rabbit lungs perfused with Krebs-Henseleit albumin
buffer with addition of arachidonic acid to the perfusate (122). In
the above study, arachidonate-induced increases in PAP were substan¬
tially reduced by imidazole, a TX synthetase inhibitor (122). Thus, it
appears likely that platelets are not needed to demonstrate activity of
TX synthetase in producing pulmonary hypertension in response to ara¬
chidonic acid.
The inability of OKY-1581 to completely block the arachidonate-
induced increase in PVR was surprising. In adult sheep pulmonary
hypertension induced by endotoxin infusion was correlated with a sub¬
stantial increase in TXE$2 (123). If TXA2 were solely responsible
for the increase in PAP observed in adult sheep, it would appear that
prevention of TX formation should prevent pulmonary hypertension.
OKY-1581 was unable to completely prevent increases in PVR due to ara¬
chidonic acid, leading to the proposal that the remaining response is
due to formation of PGf^. Prostaglandin H2 is a potent pulmonary
vasoconstrictor in adult dogs (88) and in adult sheep (89). The endo-
peroxide formed in the lung may participate in a "steal" phenomenon,
resulting in increased formation of PGI2• Thromboxanes appear to be
responsible for the majority of the increase in PVR in response to ara¬
chidonic acid in perinatal lambs. Inhibition of TX synthetase with
OKY-1581 resulted in greater than 50 % reduction in pulmonary vasocon¬
striction caused by arachidonic acid, and there was significant en¬
hancement of systemic hypotension in the OKY-treated animals. The
increased systemic hypotension in OKY-treated lambs is probably a
result of increased formation of PGI2 from endoperoxides due to
prevention of formation of TXA2.

CHAPTER FOUR
EFFECTS OF PROSTAGLANDIN H? ON PERINATAL
PULMONARY CIRCULATION
Introduction
Prostaglandin H2 functions as the key intermediate in PG synthe¬
sis from which the stable products, PGD2, PGE2 and PGF2a, as well as
the unstable compounds, PGI2 and TXA2, are derived. The terminal
enzymes which yield these various products are present in varying
amounts and activities in different organs, and this distribution
contributes to the wide range of responses of individual systems to
release of PG's (16, 18, 25-28 ). There is evidence that PGH2 is
capable of eliciting direct responses in biological systems without
undergoing conversion to other PG's (14, 17). Kadowitz et al_. (88)
demonstrated the ability of PGH2 to contract isolated segments of
canine intrapulmonary vein. Additionally, bolus injections of PGH2
caused increases in lobar arterial and small vein pressures in pump-
perfused canine lungs (88). Unanesthetized adult sheep responded to
infusions of PGH2 with dose-related increases of pulmonary arterial
pressure (89). In both of these studies, a stable analog of PGH2
also produced pulmonary vasoconstriction (88, 89). Endoperoxide
analogs have been shown to cause dose-dependent increases in pulmonary
vascular resistance (PVR) and systemic arterial pressure (SAP) in
57

58
perinatal goats (91). Recently it was demonstrated that increases in
PVR produced by infusions of arachidonic acid in perinatal lambs were
not abolished by TX synthetase inhibition (Ch 3). It was proposed that
a portion of the arachidonate-induced increase in PVR was due to PG
endoperoxides. The effects of PGH2 were studied in pump-perfused
lungs of perinatal lambs in the present study.
Materials and Methods
Surgical preparation. The details of the surgical procedure for
the in situ pump-perfused lower left lung preparation in perinatal
lambs have been described in Ch 2. Mothers of 7 fetal lambs (0.93 -
0.97 gestation, wt = 3.6 ± 0.1 kg) were anesthetized with chloralose,
and fetuses were delivered by cesarean section. Fetuses were surgical¬
ly prepared so that left pulmonary arterial pressure (PAP), left atrial
pressure (LAP), SAP and heart rate were monitored continuously under
«
conditions of constant left pulmonary arterial blood flow (Q). Flow
was set at a level at which PAP was equal to or slightly greater than
»
mean SAP. By maintaining Q constant, changes in PAP reflected changes
in PVR, which was calculated as (PAP - LAP) • body wt / Q.
Experimental procedure. Prostaglandin H2 was prepared by Dr.
D.B. McNamara (Tulane Univ., New Orleans) using the method of Egan et
al. (124), as described by She et ajL (111)* The endoperoxide was
shipped on dry ice and stored in a hexane:ether (4:6) solution at
-85°C. Purity was estimated by chromatography of (1-^C) PGH2 on
silica gel plates and by radiochromatographic scan and scraping (111).
Purity of PGH2 was greater than 90 %. Immediately before injection

59
of PGH2, an aliquot of the stock solution was dried under a stream of
nitrogen, diluted with cold saline and drawn into a chilled Hamilton
syringe. An injection of 0.1 ml of various concentrations of PGH2
was performed within 15 sec of dilution with saline. The concentra¬
tions of PGH2 used were 1.0, 10.0, 20.0 and 40.0 yg/ml. OKY-1581
(Ono Pharmaceutical Co), a pyridine derivative, or UK 37,248-01 (Pfiser
Chemical Co), an imidazole derivative, was used to inhibit TX synthe¬
sis. OKY-1581 was diluted with saline to a concentration of 50 mg/ml
and 1.0 ml was injected into the pulmonary arterial circuit. UK
37,248-01 was diluted with saline to a concentration of 6.0 or 10.0
mg/ml, so that 1.0 ml of solution gave a dose of approximately 2 mg/kg.
These doses were greater than the effective doses in rabbits (115).
Following the surgical preparation, baseline values for pressures
and flow in fetuses were established, and arterial blood gases and pH
were analyzed. (Criteria for rejection of hemodynamic data in fetuses
were arterial blood samples with pH < 7.20, PO2 < 15 mmHg or PCO2 >
60 mmHg.) After baseline values were obtained (Table 6), bolus injec¬
tions of PGH2 were made into the pulmonary arterial circuit. Doses
were administered in random order. After several doses were given to
fetuses, ventilation was initiated with a Harvard respirator or a
Healthdyne infant ventilator, and the umbilical cord was occluded.
Following the ventilation-induced fall in PAP, Q was increased to
reflect the normal increase in pulmonary perfusion occurring at birth.
Control values were observed (Table 6), and bolus injections of PGH2
were administered to ventilated fetuses. (Criteria for rejection of
hemodynamic data in ventilated fetuses were arterial blood samples with
pH < 7.30, PO2 < 75 mmHg and PCO2 > 50 mmHg.) After several

Table 6. Control values in unventilated and ventilated fetal lambs
Hemodynamic Data
PAP
SAP
Q
PVR
(mmHg)
(mmHg)
(ml/kg.mi n)
(mmHg.kg.min/ml)
1. Unventilated
70.1
60.6
15.98
4.85
Fetuses
(n = 11)
±2.0
±1.7
±1.85
±0.47
2. Ventilated
27.4
42.3
24.00
1.12
Fetuses
±1.4
±3.0
±1.95
±0.12
(n = 10)
Arterial Blood Samples
pH
P02
(mmHg)
PC0¡
(mmHg'
1. Unventilated
7.31
25.7
52.7
Fetuses
(n = 11)
±0.01
±0.6
±1.3
2. Ventilated
7.44
112.1
33.2
Fetuses
(n = 10)
±0.02
±7.6
±2.0
Data are control values for pulmonary jirterial pressure (PAP), mean systemic arterial
pressure (SAP), pulmonary blood flow (Q), pulmonary vascular resistance (PVR), and
arterial blood gases and pH in 6 unventilated and 7 ventilated fetal lambs. Data are
expressed as mean ± SEM. The number of observations for the control values is indicated
by n.

61
injections, a TX synthetase inhibitor was injected into the pulmonary
arterial circuit, and doses of PGH2 were repeated. One animal
received 2 doses of OKY-1581 (separated by 40 min and 2 injections of
PGH2), 2 received only UK 37,248-01, and 1 received a dose of
OKY-1581, followed by an injection of PGH2, and then a dose of UK
37,248-01, followed by a second injection of PGH2 (separated by at
least 30 min). Due to deterioration of animal preparations after sev¬
eral injections, some animals did not receive all doses of PGH2 and
each of the ventilated fetuses did not receive a TX synthesis blocker.
The following variables were sampled 60 sec before injection, at
•
5-sec intervals for the first minute, and also at 120 sec: PAP, LAP, Q,
mean SAP and heart rate. The readings at 0 and -60 sec were averaged
to give a control value for the variables PVR and mean SAP. The con¬
trol values were set to 100 %, and the values at the remaining time
intervals were expressed as a percentage of the control value. Each of
the treatments within a group (fetus, ventilated fetus, and ventilated
fetus with TX synthetase inhibition) was analyzed using a one-way ana¬
lysis of variance with repeated measures, and differences between means
were tested with the Newman-Keuls test (109). Differences between ven¬
tilated fetuses with and without TX synthetase inhibition were tested
using the unpaired Student's t-test. Differences cited were statisti¬
cally significant at P < 0.05.
Results
The average response of the pulmonary circulation of 6 fetal lambs
to 11 bolus injections of PGH2 is shown in Fig 9. Doses of PGH2

Figure 9: Average pulmonary and systemic responses to bolus injec¬
tions of PGH2 are shown for 6 unventilated fetal lambs
(11 injections, ave dose = 0.50 ± 0.09 yg/kg). Symbols are
as in Fig 2.

SAP % PVR
63
UNVENTILATED FETUSES
PGH2 BOLUSES
0.50 pg/kg
TIME (sec)

64
ranged from 0.24 - 1.18 ug/kg (ave dose = 0.50 ± 0.09 ug/kg); however,
there was not a dose-dependent relationship over this range of doses.
Injections of PGH2 consistently produced decreases in PVR of 10 - 25 %
The fall in PVR was rapid in onset, reached a peak at 10 sec after
injection, and returned to baseline within 35 sec. Values at 5, 10,
15, 20 and 25 sec were significantly (P < 0.01) different from the
value at 0 sec. The peak response was only 79 % of the control PVR.
At the doses of PGH2 injected, there was no effect on SAP.
Figure 10 shows the results of 10 bolus injections of PGH2 in 7
ventilated fetal lambs. The range of doses was 0.24 - 0.61 ug/kg (ave
dose = 0.39 ± 0.05 ug/kg). Within this dose range, injections of PGH2
caused significant pulmonary vasoconstriction, without producing a
systemic effect. Increases in PVR were significantly different from
control PVR at 10, 15, 20, 25, 30, 35, 40 (all P < 0.01), and 45 sec
(P < 0.05). The peak pressor response occurred at 15 sec, and it was
150 % of the baseline value. PVR remained elevated above control
values until 50 sec after injection.
Four ventilated fetal lambs were treated with either OKY-1581 (50
mg) or UK 37,248-01 (2-3 mg/kg) to inhibit TX synthesis. There was no
difference between the two blockers in the pulmonary response to PGH2;
therefore the data for the two drugs were combined (Fig 11). Injec¬
tions of PGH2 (ave dose = 0.31 ± 0.03 ug/kg) following TX synthetase
inhibition were able to cause significant (P < 0.01) increases in PVR
at 10 and 15 sec. The maximal response at 10 sec was 128 % of control
PVR. This peak increase in PVR was 56 % of the maximal increase in PVR
in the lambs without the inhibitors. There were significant differ¬
ences in the increases in PVR in response to PGH2 in ventilated fetal

Figure 10: Average pulmonary and systemic responses to bolus injec¬
tions of PGH2 are shown for 7 ventilated fetal lambs (10
injections, ave dose = 0.39 ± 0.05 yg/kg). Symbols are as
in Fig 2.

% SAP % PVR
66
VENTILATED FETUSES
PGH2 BOLUSES
0.39 ¿jg/kg
TIME (sec)

Figure 11: Average pulmonary and systemic responses to bolus injec¬
tions of PGH2 following thromboxane synthetase inhibition
(TSI) are shown for 4 ventilated fetal lambs (7 injections,
ave dose = 0.31 ± 0.03 wg/kg). Symbols are as in Fig 2.

SAP % PVR
68
VENTILATED FETUSES
PGH2 BOLUSES
0.31 pg/kg
FOLLOWING TSI
TIME (sec)

69
lambs with and without TX synthetase inhibition. In the group treated
with inhibitors, the PGh^-induced increases in PVR were significantly
depressed in comparison with the untreated group at 15, 20, 25, 30, 40,
45 and 55 sec (P < 0.05, 0.01, 0.05, 0.05, 0.05, 0.05 and 0.05, respec¬
tively). There were no differences between the treated and untreated
lambs in response of the systemic circulation to intra-pulmonary injec¬
tions of PGH2*
Control injections of ice-cold saline (0.1 ml) were given to 2
fetal lambs before ventilation and 2 different lambs after ventilation.
These injections did not elicit any changes in PVR or mean SAP. In 3
fetal lambs before and after ventilation injections were given of an
"inactivated" PG^- The endoperoxide was prepared in the same manner
as for an injection, but the syringe containing the PGH£ in saline
was allowed to sit on a warmed surface under light for at least 15 min
before injection. There was no response of the pulmonary circulation
to "inactivated" PGH2 injections in ventilated fetal lambs. There
was a slight, but significant (P < 0.01), decrease in PVR in unventi¬
lated fetal lambs to 94 % of control PVR, perhaps due to degradation of
PGH2 to PGE2* Systemic pressure was not affected by intra-pulmonary
injections of "inactivated" PGH2*
Discussion
The finding that injections of PGH2 into the pulmonary circula¬
tion of ventilated fetal lambs produced increases in PVR confirms ear¬
lier reports on PGH2 and the adult lung (88, 89). Over the dose
range used in this study, PGH2 did not exhibit a dose-dependent

70
relationship with PVR. The range was small primarily due to limited
quantities of PGH2 available. The average dose of PGH2 in the present
study (0.39 yg/kg) was approximately 4 times the dose used in adult
dogs (88). The increase in PVR in ventilated fetal lambs was 50 %
above baseline, while the increase in lobar arterial pressure in adult
dogs was 24 % greater than control. Bowers et al. (89) infused PGH2
into the superior vena cava of unanesthetized adult sheep and measured
pulmonary arterial pressure and cardiac output. They reported a
tripling of PVR at the steady state during an infusion of 0.25
yg/kg.min PGH2 (89). The greater responsiveness of the pulmonary
circulation of adult sheep as compared with the ventilated fetal lambs
could be due to lack of anesthesia in the adults. Also, in the adults
the PGH2 was infused over a period of at least 15 min, and a steady
state increase in PVR was observed. In the present study, PGH2
injections lasted approximately 3 sec, so that comparisons with
responses obtained during infusions are difficult.
Intra-pulmonary injections of PGH2 produced decreases in PVR in
fetal lambs and increases in PVR in ventilated fetuses. The doses of
PGH2 administered to the two groups were similar; thus, there should
be no difference in response due to differing substrate concentrations.
Major differences which existed between the ventilated and unventilated
fetal lambs were decreased PVR, increased Q, increased PO2 and
decreased PCO2 after ventilation (Table 6). These changes reflect
the normal events which occur as a result of ventilation at birth
(68). The decrease in pulmonary vascular tone may be the most impor¬
tant factor in explaining the differing responses to PGH2* Studies
in adult cat lung demonstrated that infusion of the bisenoic PG

71
precursor, arachidonic acid, produced pulmonary vasodilatation when
pulmonary vascular tone was elevated (95). Hyman et a]_. proposed that
the pulmonary vascular response to exogenous arachidonate infusions was
dependent in part on the pre-existing level of PVR. This agreed with
the finding by Gerber et^ al_. (102) that PVR in dogs undergoing a hypox¬
ic pressor response was returned toward pre-hypoxia baseline values by
infusion of arachidonic acid.
In contrast, an earlier study (91) reported only pulmonary vaso¬
constriction when arachidonic acid was infused into the pulmonary cir¬
culation of fetal and neonatal goats. These findings were confirmed in
ventilated fetal and neonatal lambs (Ch 2) which had low pulmonary vas¬
cular tone (comparable to that of the ventilated fetuses in the present
study). However, when PVR was increased above baseline by 6 % hypoxia,
infusion of arachidonic acid at all doses produced only further in¬
creases in PVR (Ch 2). The use of OKY-1581 to inhibit TX synthetase
prior to infusions of arachidonic acid did not prevent pulmonary vaso¬
constriction in ventilated fetal and neonatal lambs (Ch 3). Thus, the
remaining pulmonary pressor response was attributed to formation and
actions of PGH2* While the data in ventilated fetal lambs injected
with PGH2 agree with the above studies, there are differing results
from unventilated fetuses receiving PGH2 and arachidonic acid.
Histamine, a chemically different compound, also produces pulmo¬
nary vasoconstriction and vasodilatation in animals of differing ages
(68, 125). In fetal lambs prior to ventilation, injection of histamine
caused a very large increase in pulmonary blood flow; after ventila¬
tion, the same dose caused little or no increase in pulmonary blood
flow (68). In adult dogs, histamine actively constricted small lobar

72
veins (125). Another prostaglandin, PGD2, has similar actions on the
pulmonary circulation (72). When PGD2 was infused into the pulmonary
circulation of unventilated fetal goats, it caused dose-dependent
decreases in PVR. After ventilation of fetuses and in newborn lambs,
infusions of PGD2 at doses greater than 8.0 yg/kg.min resulted in
increases in PVR (72). It is conceivable that PGD2 could be formed
from the injections of PGH2 in the present study.
Development of PG metabolism by fetal and neonatal lamb lung
microsomes has been described by Friedman et al_. (110). Fetal micro-
somes were capable of producing PGE2, PGI2 and TXA2 enzymatically from
PGH2- Prostacyclin synthetase exhibited enzyme saturation at low
levels of PGH2, and formation of PGI2 was low throughout gestation.
Thromboxane synthetase showed low activity when PGH2 concentrations
were low, but at high PGH2 concentrations (400 ng PGH2/25O yg lung
homogenate protein), TX's were a major product of PG synthesis in late-
term fetal lung. The product formed in greatest quantities by fetal
lamb lung homogenates was PGE2 (110), and formation of PGE2 by
fetal goat lung microsomes was enhanced by addition of reduced gluta¬
thione (GSH) (126). Thus, levels of endogenous GSH in fetal lung may
exert important control over products of PGH2 metabolism, with greater
amounts of TX's formed in the absence of GSH and more PGE2 formed in
the presence of GSH (126). Factors involved in the regulation of GSH
in fetal lung are unknown.
The above studies on PGH2 metabolism in fetal and neonatal lung
microsomes do not indicate whether PGD2 production was assayed. It
has been shown that there is a specific glutathione-S-transferase
present in adult sheep lung which causes large amounts of PGF2a and PGD2

73
to be produced (29). Incubation of the purified enzyme with PGH2
resulted in 3 times more PGD2 than PGE2 produced (29). Thus, if
this enzyme were present and active in fetal sheep lungs, formation of
PGD2 could be a significant factor in the response to PGH2-
Thromboxanes have a potent ability to contract vascular smooth
muscle in vitro (17); PGE2 and PGI2 are vasodilators of fetal and
neonatal pulmonary and systemic circulations (67, 82). In summary,
injections of PGH2 caused increases in PVR in ventilated fetuses.
This is consistent with 1) direct effect of PGH2 on vascular smooth
muscle (17) and 2) formation of products with vasoconstrictor activity,
such as TXA2, PGD2 and PGF2a. Inhibition of TX synthetase with either
OKY-1581 or UK 37,248-01 resulted in a reduction of pulmonary vasocon¬
striction, but did not abolish the response completely (Fig 10). In
contrast, unventilated fetal lambs always responded to injections of
PGH2 with decreases in PVR. These findings suggest metabolism of PGH2
to PGI2, PGD2 and/or PGE2, with little direct action of PGH2 on fetal
pulmonary vessels. It is possible that ventilation of fetal lungs al¬
ters the end products of PGH2 metabolism, perhaps by limiting availa¬
bility of GSH, so that after ventilation more TX's are formed in
response to a bolus of PGH2- Alternatively, the pulmonary vascular
response to PGH2 may depend in part on existing basal pulmonary vas¬
cular tone.

CHAPTER FIVE
ARACHIDONIC ACID AND ADULT PULMONARY CIRCULATION
Introduction
Initial observations by other authors on the effects of arachidon-
ic acid on the pulmonary circulation demonstrated that pulmonary vaso¬
constriction could be produced by infusion or injection of arachidonic
acid into the pulmonary circulation (69, 92, 93). Recent studies have
indicated that arachidonic acid can cause decreases in PVR when admin¬
istered by infusion at low doses (94, 95). Additional reports demon¬
strated the ability of infusions of arachidonic acid to reduce PVR
which had been elevated by hypoxia, PGF2a, or an analog of PGH2
(95, 102, 112). Assay of arterial blood during the infusion of arachi¬
donic acid in the above studies showed formation of 6-keto-PGFia
(95, 102, 112). These reports demonstrated that vasodilator PG's,
PGI2 in particular, were formed from exogenous arachidonic acid when
pulmonary tone was enhanced.
In contrast to these findings in adult animals, infusions of ara¬
chidonic acid into the pulmonary circulation of perinatal goats always
produced pulmonary vasoconstriction (91). The results presented in Ch
2 confirmed that arachidonic acid causes increases in PVR in ventilated
fetal and neonatal lambs. These results were not altered when the
74

75
inspired O2 was reduced to 6 %; instead, a hypoxic pressor response
was observed and the infusion of arachidonic acid caused further pulmo¬
nary vasoconstriction. Consequently, the following studies in adult
sheep were undertaken to determine if the pulmonary pressor actions of
arachidonic acid, when infused during hypoxia, were dependent upon
developmental state of the animal, or if the response was related to
oxygenation or elevated pulmonary vascular tone. Pulmonary vascular
resistance was elevated in two ways: 1) inspired oxygen was reduced to
10 %, or 2) PGF2a was infused while normoxic conditions were main¬
tained. The effects of infusion of arachidonic acid were tested under
both of these conditions.
Materials and Methods
Surgical preparation. The surgical procedure for the isolated
pump-perfused left lower lung preparation, described for perinatal
lambs in Ch 2, was modified slightly for use in adult sheep. Ten non¬
pregnant ewes (52.7 ± 3.5 kg) were anesthetized with chloral ose (50
mg/kg, iv) and tracheotomies were performed. Anesthesia was maintained
with hourly supplements of chloralose (10 mg/kg). Ewes were paralyzed
with d-tubocurare (0.2 mg/kg, iv) and ventilation was initiated with a
Harvard respirator. Colonic temperature was monitored (Yellow Springs
Instrument) and maintained between 38° and 41°C by placement of heating
pads beneath the animal. The femoral artery was cannulated for moni¬
toring SAP (Statham P23DC transducer), heart rate and arterial blood
gases and pH (Instrumentation Laboratory pH/blood gas analyzer 213).
An incision was made along the upper margin of the left fourth rib, and

76
the third and fourth ribs were retracted to permit access to the left
lung. Following isolation and dissection from surrounding tissue of
the left pulmonary artery, the ewe was heparinized (2000 U/kg, iv). A
catheter was placed in the femoral vein and advanced into the inferior
vena cava. Blood was withdrawn from the inferior vena cava and pumped
(Cole-Parmer Masterflex pump no. 7018) into the left pulmonary artery.
Between 35 and 40 % of the total lung tissue, as determined by weight,
was perfused in this manner. Flow to the right lung was not disturbed.
Left pulmonary blood flow (Q) (Statham flowmeter M4401 with Medicon
FloProbe Sensor A2208 electromagnetic flow probe) was maintained con¬
stant at a level that produced approximately 20 mmHg pressure in the
left pulmonary artery. A catheter was placed in the left atrium for
measurement of LAP, and PVR was calculated as (PAP - LAP) • body wt /
Q. Pressures, flow and heart rate were recorded continuously on a
Gould Brush 480 8-channel polygraph. An Apple 11+ computer was con¬
nected to the polygraph with an analog-to-digital interface (AI13,
Interactive Structures, Inc.) for on-line data sampling and calculation
of PVR.
Experimental procedure. The sodium salt of arachidonic acid
(Sigma or NuChek Prep) was stored in dry form at -16°C. Fresh solu¬
tions were prepared daily with saline (final cone = 1 mg/ml) and pro¬
tected from light prior to being infused (Harvard pump) at varying
rates into the pulmonary arterial circulation. Prostaglandin ?2a
(tromethamine salt, Upjohn Co.) was stored in dry form at -16°C, and an
ethanol solution (100 mg/ml) was diluted with saline (final cone = 10.8
mg/ml) and stored at 6°C.

77
Following surgical preparation of the animals, baseline values for
pressures and flow were established and arterial blood gases and pH
were analyzed. (Criteria for rejection of hemodynamic data were arte¬
rial blood samples with pH < 7.30, PO2 < 75 mmHg or PCO2 > 50 mmHg.)
Control values were observed before each experimental period (Table 7),
and the following treatments were administered in random order. 1)
Arachidonic acid was infused into the pulmonary arterial circuit for 5
min at varying rates. Doses of arachidonic acid were expressed as
amount/kg body wt.min. 2) Alveolar hypoxia was produced by lowering
the inspired O2 to 10 % for 15 min. 3) A combination of treatments 1
and 2 was given, with hypoxia started for 5 min, arachidonic acid in¬
fused during the sixth through the tenth minutes, and hypoxia continued
for an additional 5 min after terminating the arachidonate infusion.
4) Prostaglandin F2a was infused into the pulmonary arterial circuit
for 15 min. 5) A combination of treatments 1 and 4 was given, with
PGF2a infused for a total of 15 min and arachidonic acid infused
during the sixth through the tenth minutes. 6) Hypoxia was produced by
lowering the inspired O2 to 10 % for 5 min. 7) Arachidonic acid was
infused directly into the perfusion circuit for 15 min. 8) A combina¬
tion of treatments 6 and 7 was given, with arachidonic acid infused for
15 min and the animal exposed to hypoxia during the sixth through the
tenth minutes. A recovery period of at least 5-10 min was allowed
between experimental periods in order to return to control values. The
minimal duration of an experiment in which an animal received every
treatment was 3 hours. However, due to deterioration of the animal
preparations after several experimental periods, some animals did not
receive every treatment. The following variables were sampled at

Table 7. Control values in adult sheep
A. Hemodynamic Data
PAP
SAP
•
Q
PVR
(mmHg)
(mmHg)
(ml/kg.min)
(mmHg.kg.min/ml)
(n = 55)
19.2
82.4
14.56
1.17
±0.4
±1.9
±0.53
±0.05
B. Arterial Blood Samples
pH
P02
PCOp
(mmHg)
(mmHg)
(n = 55)
7.47
108.4
37.4
±0.01
±3.1
±0.7
Data are control values prior to each treatment for pulmonary.arterial pressure (PAP),
mean systemic arterial pressure (SAP), pulmonary blood flow (Q), pulmonary vascular
resistance (PVR), and arterial blood gases and pH in 10 adult sheep. Data are expressed
as mean ± SEM. The number of observations for each of the control values is given as n.

79
intervals of 1 min, with the first minute of the treatment sampled at
20-sec intervals: PAP, LAP, Q, mean SAP and heart rate. A reading was
taken 1 min before the start of the experimental period, and this value
and the value at the beginning of the treatment were averaged to give a
control value for the variables PVR and mean SAP. The control values
were set to 100 %, and the values at the remaining time intervals were
expressed as a percentage of the control value. Each of the treatments
was analyzed using a oneway analysis of variance with repeated mea¬
sures, and differences between means were tested with the Newman-Keuls
test (109). Differences between treatments were tested for signifi¬
cance using the unpaired Student's t-test. The level of significance
for statistical differences was P < 0.05.
Results
The average pulmonary and systemic responses of 6 adult sheep to
15 min of hypoxia (FIQ2 = 10 %) are shown in Fig 12. Hypoxia pro¬
duced significant (P < 0.01) increases in PVR from 2 min through 16
min. PVR returned to baseline within 3 min of terminating the hypoxic
exposure. Mean SAP was not significantly elevated from the control
value during hypoxia. After returning to normoxia, mean SAP signifi¬
cantly (P < 0.05) declined to 88 % of control SAP at 19 and 20 min.
Thirteen 5-min infusions of arachidonic acid (ave dose = 17.6 ±
2.6 pg/kg.min) were given to 9 ewes. The average vascular responses to
these infusions are shown in Fig 13. Infusions of arachidonic acid in
low doses (as compared to doses used in neonatal lambs, Ch 2) for 5 mi n
caused significant (P < 0.01) increases in PVR at 1 through 6 min.

Figure 12: Average pulmonary and systemic responses to 15-min periods
of hypoxia (FIq2 = 10 %) are shown for 6 adult sheep (6
cases). Symbols are as in Fig 2.

% SAP
ADULT SHEEP
HYPOXIA- 1 0 %- 1 5 min
TIME (min)

Figure 13: Average pulmonary and systemic responses to 5-min infu¬
sions of arachidonic acid are shown for 9 adult sheep (13
infusions, ave dose = 17.6 ± 2.6 yg/kg.min). Symbols are
as in Fig 2.

SAP % PVR
83
ADULT SHEEP
ARACHIDONIC ACID-5 min
17.6 jjg/kg.min
TIME (min)

84
Arachidonate-induced increases in PVR formed a plateau from 2 - 5 min;
PVR was 120 % of control at 4 min. A slight, but significant, reduc¬
tion in mean SAP occurred with the increases in pulmonary pressure.
Mean SAP was significantly decreased from control SAP at 4, 5, 6, 7 and
8 min (P < 0.05, 0.01, 0.05, 0.01 and 0.05, respectively). The maximal
fall in mean SAP was 96 % of control SAP at 5 min.
Arachidonic acid (ave dose = 16.4 ± 2.8 yg/kg.min) was infused
during the sixth through the tenth minutes of hypoxia in 7 adult sheep.
Average pulmonary and systemic vascular responses to 7 combined treat¬
ments are shown in Fig 14. There were significant increases in PVR due
to hypoxia before and after infusion of arachidonic acid. Infusion of
arachidonic acid caused an additional increase in PVR during the sixth
through the tenth minutes. Increases in PVR during the combined treat¬
ments (from 7-10 min) were almost 150 % of control PVR. Values at 7
- 10 min were significantly (P < 0.01) greater than the values at 2, 3,
4 and 5 min, before arachidonic acid was infused. Increases in PVR
during the combined treatments were also significantly greater than the
increases in PVR after the infusion was terminated. Pulmonary vascular
resistance at 10 min was significantly greater than values at 11, 12,
13, 14 and 15 min, while hypoxia was still present (P < 0.05, 0.01,
0.01, 0.01 and 0.01, respectively). The response of the systemic cir¬
culation to the combination of 15 min of hypoxia and 5 min of arachi¬
donic acid progressed in stages (Fig 14). During the first 5 min of
hypoxia, mean SAP was significantly (P < 0.05) increased from control
SAP to 114 % at 5 min. During the sixth through the tenth minutes of
hypoxia, while arachidonic acid was infused, mean SAP returned toward
baseline. Following the termination of the infusion, mean SAP was not

Figure 14: Average pulmonary and systemic responses to a combination of
15-min periods of hypoxia (FIq2 = 10 %) and 5-min infusions
of arachidonic acid are shown for 7 adult sheep (7 cases,
ave dose = 16.4 ± 2.8 yg/kg.min). Symbols are as in Fig 2.

% SAP % PVR
ADULT SHEEP
HYPOXIA- 1 0 %- 1 5 min
ARACHIDONIC ACID-5 min
16.4 jjg/kg.min
TIME (min)

87
significantly different from the value at 0 min. However, the values
at 13, 14, 15, 16, 17, 18, 19 and 20 min were significantly (P < 0.05,
0.05, 0.01, 0.01, 0.01, 0.01, 0.01 and 0.01, respectively) different
from the values at 4 and 5 min.
As an alternative to hypoxia as a means to elevate PVR, PGF2a
was infused for 15 min (ave dose = 4.4 ± 1.3 ug/kg.min) in 5 ewes (Fig
15). The infusion rate used was chosen to produce approximately the
same increase in PVR as hypoxia caused; the two treatments were not
significantly different after the first 4 min. Infusion of PGF2a
produced a rapid increase in PVR; values at 40 sec and 1 min were sig¬
nificantly (P < 0.05) greater than control PVR. Values for PVR at the
remaining time intervals during the infusion were significantly greater
than control PVR at P < 0.01 (from 2 to 15 min). There were increases
in mean SAP during infusion of PGF2a, beginning at 1 min (P < 0.05)
and continuing from 2-18 min (P < 0.01). The value at 19 min was
still significantly (P < 0.05) elevated over control SAP, but mean SAP
had returned to baseline at 20 min.
The effects in 6 ewes of combining 15 min PGF2a (ave dose = 4.1 ±
1.1 ug/kg.min) and 5 min of arachidonic acid (16.1 ± 3.2 yg/kg.min) are
shown in Fig 16. There were significant increases in PVR due to infu¬
sion of PGF2a before and after infusion of arachidonic acid. Infusion
of arachidonic acid caused an increase in PVR during the sixth through
the tenth minutes. Increases in PVR during the combined treatments
(from 7-10 min) were 190 % of control PVR. The values at 7, 8, 9 and
10 min, during the arachidonate infusion, were significantly (P < 0.01)
greater than values during infusion of PGF2a (at 2, 3, 4, 5, 11, 12, 13,
14 and 15 min). The response of the systemic circulation to the

Figure 15: Average pulmonary and systemic responses to 15-min infusions
of PGF¿ are shown for 5 adult sheep (5 infusions, ave
dose = 4.4 ± 1.3 pg/kg.min). Symbols are as in Fig 2.

% SAP % PVR
ADULT SHEEP
PGF2alpha-15 min
4.4 jjg/kg.min
00
LO
TIME (min)

Figure 16: Average pulmonary and systemic responses to a combination of
15-min infusions of PGF2a (ave dose = 4.1 ± 1.1 yg/kg.min)
and 5-min infusions of arachidonic acid are shown for 6
adult sheep (6 infusions, ave dose = 16.1 ± 3.2 pg/kg.min).
Symbols are as in Fig 2.

% PVR
ADULT SHEEP
PGF2alpha-15 min
4.1 pg/kg.min
ARACHIDONIC ACID-5 min
16.1 jjg/kg.min
TIME (min)

92
combined treatment of 15 min of PGF2a and 5 min of arachidonic
acid was not significantly different from the systemic response to
infusion of PGF2a alone. Mean SAP was increased significantly
from the value at 0 min at 2 - 18 min (P < 0.05 at 2, 17 and 18 min; P
< 0.01 at 3 - 16 min). The values for mean SAP during infusion of
arachidonic acid (6 - 10 min) were not significantly different from
values before and after arachidonic acid (2-5 and 11 - 15 min).
To study the effects of arachidonic acid infusion on the hypoxic
pressor response, arachidonic acid was infused for 15 min. After re¬
turning to baseline, responses to 5 min of hypoxia were measured. Then
a combination of the two treatments was given. Pulmonary and systemic
responses to these experimental treatments are summarized in Table 8.
Prolonged infusion of low-dose arachidonic acid (12.3 ± 0.6 pg/kg.min)
caused significant (P < 0.05) increases in PVR at 3 - 15 min. The pul¬
monary hypertension was accompanied by a slight reduction in mean SAP
which was not significantly different from control SAP. The pulmonary
circulation responded to 5 min of reduced oxygen (FIQ2 = 10 %) with
significant (P < 0.01) increases in PVR at 2 - 6 min. There were sig¬
nificant (P < 0.01) increases in mean SAP at 3, 4 and 5 min during
hypoxia. Maximal increases in % PVR and % SAP occurred at 5 min (Table
8); however, the values from 2-5 min were not significantly different
from each other. The two treatments were combined in 5 sheep such that
arachidonic acid was infused for 15 min (ave dose = 12.3 ± 0.6 pg/kg.
min) and inspired oxygen was reduced during the sixth through the tenth
minutes. Arachidonic acid caused significant (P < 0.05) increases in
PVR at 3, 4 and 5 min, and also at 11 - 15 min. Addition of hypoxia
produced further increases in PVR at 6 - 10 min, with values at these

93
Table 8. Maximal change in PVR and SAP for all treatments in adult
sheep
% PVR
% SAP
1. Arachidonic Acid (17.6 ± 2.6 yg/kg.min)-5 min
9 Ewes (inc) 120.38 ± 3.01
13 Treatments (dec)
2. Hypoxia (FIq2 = %)-15 min
6 Ewes (inc) 135.00 ± 7.71
6 Treatments (dec)
95.75 ± 1.75
111.82 ± 3.98
88.35 ± 4.22
3. Hypoxia-15 min and Arachidonic
7 Ewes (inc)
7 Treatments (dec)
4. PGF2a (4.4 ± 1.3 yg/kg.min)-15
5Ewes (inc)
5 Treatments (dec)
5. PGF2a (4.1 ± 1.1 yg/kg.min)-15
(16.1 ± 3.2 yg/kg.min)-
6 Ewes (inc)
6 Treatments (dec)
6. Hypoxia (FIq2 = 10 %)-5 min
7 Ewes (inc)
8 Treatments (dec)
7. Arachidonic Acid (12.3 ± 0.6 yg/kg
5 Ewes (inc)
5 Treatments (dec)
Acid (16.4 ± 2.8 yg/kg.min)-5 min
148.64 ± 10.02 113.97 ± 5.44
89.61 ± 4.17
mi n
142.62 ± 8.96 118.36 ± 1.73
min and Arachidonic Acid
mi n
192.83 ± 15.82 125.82 ± 7.86
120.63 ± 2.53 117.20 ± 7.22
min)-15 min
124.76 ± 12.24
88.16 ± 8.58
8.Arachidonic Acid (12.3 ± 0.6 yg/kg.min)-15 min and Hypoxia-5 min
5 Ewes (inc) 167.58 ± 18.83 119.44 ± 6.33
5 Treatments (dec) -- 91.64 ± 7.39
Values shown are maximal increases (inc) or decreases (dec) in PVR or
SAP during the treatment indicated. Data are expressed as mean ± SEM.
Locations with a dash indicate the response was in only one direction;
i.e., only vasoconstriction or vasodilatation.

94
time intervals significantly greater than control PVR at P < 0.01.
Increases in PVR at 7 - 10 min were significantly greater than values
at 5, 11, 12, 13 (P < 0.05), 3, 4, 14 and 15 min (P < 0.01). Mean SAP
was significantly increased while hypoxia was administered during ara-
chidonate infusion. Values at 7, 8, 9 and 10 min were significantly
increased from control SAP at P < 0.05, 0.01, 0.01 and 0.01, respec¬
tively. The systemic response to arachidonic acid following termina¬
tion of hypoxia was lower than baseline SAP, but decreases in mean SAP
were not significantly different from the value at 0 min. These pulmo¬
nary and systemic vascular responses during the combined treatments are
summarized in Table 8.
Discussion
The results of this study demonstrate that 1) low-dose infusions
of arachidonic acid cause pulmonary vasoconstriction in adult sheep, 2)
the pulmonary response to arachidonate is not altered by the presence
of hypoxia, 3) the pulmonary vasoconstrictor response arachidonate is
not affected by PGF2a-induced elevation of PVR, and 4) the hypoxic
pressor response of the pulmonary circulation is not affected by low-
dose infusion of arachidonic acid. Additionally, arachidonate-induced
pulmonary vasoconstriction is accompanied by a slight systemic hypoten¬
sion. Pulmonary vasoconstriction caused by hypoxia or infusion of
PGF2a is not accompanied by reduction of mean SAP; in both cases,
mean SAP is elevated from baseline during the experimental period. In
animals which were hypoxic, mean SAP decreased to values below baseline
after the hypoxic exposure was terminated. The increase in mean SAP

95
produced during hypoxia is abolished by infusion of arachidonic acid
into the pulmonary circulation, although PVR is increased above hypoxic
values during arachidonate infusion.
These findings suggest the formation of vasoconstrictor substances
from arachidonic acid in the lung, possibly TXA2 and/or PGH2* These
compounds could exert direct vasoconstriction locally, and be either
degraded (from TXA2 to TXB2) or metabolized (from PGH2 to PGI2 and/or
PGE2). The net result in the systemic circulation would be the for¬
mation and action of vasodilator PG's. It appears that these patterns
of arachidonate metabolism and action are not affected by hypoxia.
Infusion of arachidonic acid during hypoxia results in additional pul¬
monary vasoconstriction, while increases in mean SAP caused by hypoxia
are eliminated by arachidonate infusion. Although infusion of arachi¬
donic acid during PGF2a-induced pulmonary vasoconstriction causes
an additional increase in PVR, there is not a reduction in the PGF2a-
induced systemic hypertension by arachidonic acid. Perhaps at the dose
of arachidonate that was infused, the amount of potentially dilatory
PG's formed is insufficient to negate the vasoconstrictor effect of
PGF2a* Alternatively, hypoxia may enhance the activity of enzymes
which form vasodilator PG's. This hypothesis is in agreement with a
study of the effects of hypoxia on formation of TXA2 and PGI2 by
homogenates of dog lung (127). Hamasaki et_ al_. (127) incubated lung
homogenates with arachidonic acid under conditions of high and low
oxygen. No difference was reported in the formation of TXA2, measured
by radioimmunoassay (RIA) as TXB2, between the two levels of oxygena¬
tion; however, there was a significant increase in the amount of PGI2
(measured by RIA as 6-keto-PGF^a) in tissue which had been exposed
to 2.1 % 02 (127).

96
An objective of the present study was to determine the effects of
arachidonic acid on the hypoxic pressor response in adult sheep. Infu¬
sion of arachidonic acid for 15 min causes pulmonary vasoconstriction
which is accompanied by an insignificant reduction in mean SAP. The
arachidonate-induced increase in PVR is observed from the third through
the fifteenth minutes of the response, without exhibiting any tendency
to return to baseline until the end of the infusion. When the inspired
oxygen is reduced to 10 % during the arachidonic acid infusion, there
is a further increase in PVR. There is also a significant systemic
vasoconstriction observed during this period of hypoxia. Thus, the
pulmonary and systemic responses to hypoxia are not altered by concomi¬
tant infusion of arachidonic acid.
These findings are in contrast to a report by Voelkel et a]_. (128)
in which addition of arachidonic acid to the blood which perfused iso¬
lated rat lungs caused a transient pressor response. The increase in
PAP in the above study of 7 - 12 mmHg was immediate, but pressure
returned to baseline within 10 min although arachidonic acid was still
present in the reservoir. The dose of arachidonic acid (15.0
ug/kg.min) used in the isolated lung was similar to doses used in the
present study (12.3 - 17.6 yg/kg.min). In addition, isolated lungs
perfused with blood containing arachidonic acid did not exhibit a pres¬
sor response to hypoxia (128). Voelkel et_ aj_. attributed these actions
of arachidonic acid to formation of PGI2- It is possible that there
was recirculation of blood containing vasodilator PG's without exposure
to peripheral sites for inactivation. Thus, in the blood which per¬
fused isolated rat lungs, there might have been significant amounts of
PGI2 available to cause pulmonary vasodilatation.

97
A study in anesthetized dogs reported that intravenous infusion of
arachidonic acid at a dose of approximately 40 yg/kg.min was capable of
reducing the increase in PVR caused by hypoxia (102). Thin-layer chro¬
matography of arterial blood indicated that PGI2 was produced when
arachidonic acid was infused during hypoxia. The dose used in the
above study was larger than the amount of arachidonic acid infused in
the adult sheep in the present study; however, there are indications
that lower doses of arachidonic acid produce pulmonary vasodilatation
while higher doses produce pulmonary vasoconstriction (95). Pulmonary
vasoconstriction occurred in normoxic cats when the dose of arachidonic
acid infused was approximately 1000 yg/kg.min (95). At lower doses of
arachidonic acid (20 - 400 yg/kg.min), there were insignificant
decreases in PVR under basal conditions (95). However, when pulmonary
tone was enhanced with infusion of PGF2a or an analog of PGH2,
these lower doses of arachidonate caused pulmonary vasodilatation (95).
In the present study, infusion of arachidonic acid during PGF2a
infusion resulted in increases in PVR in addition to the increase
caused by PGF2a« Thus, there are indications of species differ¬
ences in responses to arachidonic acid. Infusion of arachidonic acid
into conscious sheep in doses of 25, 50 and 100 yg/kg.min produced pul¬
monary vasoconstriction (113). Doses of arachidonate infused in the
present study (12.3 - 17.6 yg/kg.min) also produced pulmonary vasocon¬
striction in adult sheep. There were no doses at which pulmonary
vasodilatation was observed in the present study.
The findings of the present chapter extend the results presented
for ventilated fetal and neonatal lambs in Ch 2. The experimental
procedures were different, but the outcomes were qualitatively similar.

98
Thus, infusions of arachidonic acid for 2 min in perinatal lambs and
for 5 and 15 min in adult sheep always resulted in increases in PVR.
These findings were not affected by the presence of hypoxia or by infu¬
sion of PGF2a* In adults, arachidonic acid did not affect the
hypoxic pressor response. The responses to arachidonic acid observed
in adult sheep might be due to formation of PGH2 and/or TXA2, lead¬
ing to pulmonary vasoconstriction. The systemic hypotension which
occurs with arachidonate infusion could result from removal of con¬
strictors by degradation of TXA2 to its relatively weak metabolite,
TXB2> and metabolism of PGH2 to vasodilator PG's such as PGI2 or
PGE2* These patterns of arachidonate metabolism do not appear to be
altered by infusion of PGF2a; however, hypoxia may enhance the
production of vasodilator PG's from arachidonic acid.

CHAPTER SIX
CONCLUSIONS
The findings in these studies confirmed an earlier report (91) on
the effects of exogenous arachidonic acid on the perinatal pulmonary
circulation. In neonatal and ventilated fetal lambs, intrapulmonary
infusions of arachidonic acid produced significant, dose-dependent
increases in PVR. There were also significant reductions in mean SAP
with both dose ranges used in the ventilated fetuses and with the
higher dose used in the newborns. In addition to these findings, the
pulmonary and systemic responses to arachidonate were observed during
hypoxia. In newborn lambs the pulmonary pressor response to arachidon¬
ic acid was not affected by hypoxia. Ventilated fetal lambs showed an
increase in the pulmonary response to arachidonic acid during hypoxia
that was significantly greater than the rise in PVR due to arachidonic
acid during normoxia. However, the total pulmonary response was merely
an additive effect of arachidonic acid on the hypoxic pressor response.
In both groups of animals the presence of hypoxia significantly en¬
hanced the systemic hypotension seen in response to arachidonic acid.
Implications of this study are that in the lungs of perinatal lambs,
arachidonic acid is converted to PGH2 and/or TXA2, which act(s)
locally to produce pulmonary vasoconstriction. The endoperoxide can be
metabolized further to PGI2 and/or PGE2, both of which are vasodilators
99

100
of the systemic circulation, and TXA2 is rapidly degraded to its rela¬
tively weak metabolite, TXB2* Thus, the predominant systemic effect is
vasodilatation. Hypoxia does not appear to alter the perinatal pulmo¬
nary response to arachidonic acid or its metabolism; however, the
increased systemic hypotension may indicate enhanced release of PGI2
or PGE2 from the lungs.
Inhibition of TX synthetase in neonatal and ventilated fetal lambs
blocked over 50 % of the pulmonary response to arachidonic acid. The
remaining response was most likely due to formation of PGH2 from ara¬
chidonic acid. Increases in PVR were dependent on the dose of arachi¬
donic acid infused, perhaps indicating dose-related increases in
amounts of PGH2 formed. Treatment with OKY-1581 also significantly
enhanced systemic hypotension caused by infusion of arachidonic acid
into ventilated fetal and neonatal lambs. A reason for this observa¬
tion could be the shunting of PG endoperoxides away from TX synthetase,
so that more PGH2 is available for prostacyclin synthetase. There was
no change in the response to hypoxia in newborn lambs following TX syn¬
thesis inhibition. Also, the effect of hypoxia on the pulmonary
response to arachidonate in newborns treated with OKY-1581 was similar
to the response observed in untreated lambs, i.e., an additive effect
on the hypoxic pressor response due to arachidonic acid was found. The
elimination of TX formation unmasked the portion of the arachidonate
response that is due to endoperoxide production, and allowed greater
formation and release of dilator PG's from the lungs.
The response of the pulmonary circulation to injections of PGH2
was tested in unventilated and ventilated fetal lambs. In fetal lambs
before ventilation, PGH2 caused decreases in PVR. After ventilation of

101
fetal lambs, the pulmonary response to PGH£ became vasoconstriction.
Inhibition of TX synthetase with either OKY-1581 or UK 37,248-01 in
ventilated fetuses caused a reduction of the pulmonary pressor response
to PGH2* There were no changes in systemic arterial pressure observed
with any of these injections. The differing responses of fetal lambs
before and after ventilation may be related to differences in pulmonary
vascular tone. A similar finding in fetal goats before and after ven¬
tilation was observed with infusion of PGD2 (72). Thus, the pulmonary
response to PGH2 could be a direct effect due to the endoperoxide it¬
self which varies with existing pulmonary tone. Alternatively, the
response to PGH2 could be due in part to active metabolites, which
could be different in unventilated fetuses as opposed to ventilated
fetuses. Differences in metabolism might be related to oxygenation,
acid-base status or availability of cofactors. The pulmonary metabo¬
lism of PGH2 could lead to formation of PGI2, PGE2 and/or PGD2 in un¬
ventilated fetal lambs, and production of TXA2 and/or PGF2a could be
predominant after ventilation. A portion of the pulmonary pressor
response to PGH2 in ventilated fetuses was reduced by inhibition of
TX's, indicating that some metabolism occurs. However, the amounts of
PG's formed from PGH2 by the fetal lung before and after ventilation
have not been determined in an intact animal.
Factors involved in the regulation of PG biosynthesis during the
perinatal period are unresolved. The influence of the level of oxygen¬
ation in the fetus vs. the newborn could be an important regulatory
factor in controlling enzyme activities or cofactor availability.
Also, the role of increased pulmonary perfusion has not been assessed
with regard to location of PG biosynthesis in the lung. The fetus

102
perfuses pulmonary tissue to a lesser extent than the newborn, and this
might limit the site of PG formation and action to a relatively smaller
segment of lung. At birth, there is an increase in the surface area of
lung that is perfused (i.e., capillary surface area), and this could
result in either differing responses to PG's at these additional sites
or differing formation of PG's from newly perfused areas. Additional¬
ly, increased pulmonary blood flow at birth could provide more sub¬
strates and cofactors necessary for the formation of PG's in the lung.
Response of the perinatal pulmonary circulation to exogenous arachidon-
ic acid and PGH2 can be affected by existing pulmonary tone, oxygena¬
tion, and extent of perfusion, and all of these factors should be con¬
sidered in evaluating the role of PG's in regulating pulmonary blood
flow.
Adult sheep responded to infusions of arachidonic acid with pulmo¬
nary vasoconstriction. The doses infused in adults were low in compar¬
ison with doses given to newborns and ventilated fetuses. Infusion of
arachidonic acid in hypoxic sheep also resulted in pulmonary vasocon¬
striction which was in addition to the hypoxic pressor response. Infu¬
sions of PGF2a were used to elevate PVR without changing arterial blood
gases; arachidonic acid was capable of eliciting further increases in
PVR. Thus, elevated PVR does not allow demonstration of pulmonary
vasodilatation in response to arachidonic acid in adult sheep; this is
in contrast to reports in adult dogs (102, 112) and cats (95) in which
infusions of arachidonic acid caused reductions in PVR from elevated
levels. The findings in the adult sheep are in agreement with the
effects of arachidonic acid during normoxia and hypoxia in perinatal
lambs. Thus, differences observed between the present studies and

103
reports by some authors (94, 95, 102, 112) on the pulmonary response to
arachidonic acid might be due to species differences. In support of
this statement, Ogletree and Brigham (113) described pulmonary vasocon¬
striction in response to infusion of arachidonic acid in conscious
adult sheep.
In summary, pulmonary responses to arachidonic acid do not appear
to be age related in sheep. Infusions of arachidonic acid always pro¬
duced increases in PVR; doses ranged from 10 - 20 pg/kg.min in adults
to 300 yg/kg.min in ventilated fetal lambs. The magnitude of the pul¬
monary response to arachidonic acid was not altered by hypoxia; how¬
ever, there was a significant enhancement of systemic hypotension fol¬
lowing arachidonic acid and hypoxia in perinatal lambs. A portion of
the response to arachidonic acid was probably due to formation of TX's;
this was shown by the significant reduction of the pulmonary pressor
response following OKY-1581, a TX synthetase inhibitor. Additionally,
injections of PGH2, a key intermediate in PG biosynthesis, caused
pulmonary vasoconstriction in ventilated fetal lambs. Prior to being
ventilated, fetal lambs responded to PGH2 with decreases in PVR; this
may indicate a ventilation-induced change in the availability of
cofactors necessary for the formation of some PG's. In perinatal and
adult sheep, elevation of PVR by hypoxia or by infusion of PGF2a does
not alter the pressor response to arachidonic acid. Pulmonary vascular
responses to arachidonic acid appear to be species dependent.

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BIOGRAPHICAL SKETCH
I was born in Angleton, Texas, March 5, 1953. I attended
Brazoswood High School, Clute, Texas, and graduated from there in June
1971. I received the Bachelor of Arts degree with a major in biology
from the University of Texas at Austin in December 1974. I was
admitted to graduate study in the Department of Physiology at the
University of Florida in January 1976, but had to postpone my studies
due to the illness of my father. Upon returning to the physiology
program in September 1977, I completed the Master of Science degree in
March 1980. Since that time I have been working towards the completion
of the degree of Doctor of Philosophy. I was married on October 2,
1982 to Calvin Timmerman.
114

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
£2
ssm,
Sidney Cassin, Chairman
Professor of Physiology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Arthur B. Otis
Professor of Physiology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
H.
Wendell N. Stainsby
Professor of Physiology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
â– , / ') :-A \ - â–  \ 1 \
Allen H. NeimS
Professor of Pharmacology
and Therapeutics
This dissertation was submitted
of Medicine and to the Graduate
fulfillment of the requirements
April 1984
to the Graduate Faculty of the College
School, and was accepted as partial
for the degree of Doctor of Philosophy.
Dean,
for Graduate Studies
Dean
and
Research