Table Of ContentProstaglandins
and related substances
Editors
C. PACE-ASCIAK and E. GRANSTROM
Toronto Stockholm
1983
ELSEVI ER
AMSTERDAM. NEW YORK . OXFORD
Elsevier Science Publishers B.V.. 19x3
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Library of Congress Cataloging in Publication Data
Main entry under title:
Pro~taglandinsa nd related substances.
(New comprehensive biochemistry; v. 5)
Includes index.
I. Prostaglandins - Metabolism - Addresses, essays, lectures. 1. Pace-Asciak, C. (Cecil) 11. Granstrom,
E. (Elisabeth) JIJ. Series. [DNLM: 1. Prostaglandins. 2. Thromboxanes. 3. Lipoxygenases. W1 NE372F v.
S/QU 90 P9672a 19831
QD41S.N48 VOI. S [QP801.P68] 574.19'2s [612'.405] 83-1 1491
ISBN 0-444-805 17-6
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PROSTAGLANDINS AND RELATED SUBSTANCES
vii
Preface
Since the chemical structures of the prostaglandins were elucidated and their
biosynthesis from polyunsaturated fatty acids discovered in the early 1960’s, the
following two decades have seen an almost explosive development in prostaglandin
research.
During the last ten years numerous discoveries were made in this field, and
research was initiated in a large number of new areas. Among the mile-stones of this
last decade were the isolation of the potent endoperoxide intermediates; the dis-
covery of non-steroidal anti-inflammatory drugs as inhibitors of the fatty acid
cyclooxygenase; the discovery of the mutually antagonistic endoperoxide products,
5-HPETE+ LTA4 + LTBq,Cq,Dq,Eq +Metabolites
1
8-HPETE
J?
191--HHPPEETTEE
I 12-HPETE --+ EP-ETE 4THETE
ACID
+
15-HPETE -14,15-LTA4 Di HETE
0
= chapter number
the thromboxanes and prostacyclins, whose existence had earlier gone unnoticed
mostly because of their instability and the fact that they were formed only in small
amounts from the precursor fatty acids; the elucidation of prostaglandin metabolism
with the structure determination of a vast number of final break-down products and
the identification of metabolites suitable for monitoring in various biological sys-
tems; the development of sensitive and specific quantitation methods and their
...
Vlll
application in a large number of biological studies; studies on the release of
precursor fatty acids from esterified forms catalysed by various hydrolases as a key
event in prostaglandin biosynthesis; the inhibition of phospholipase-catalysed fatty
acid release by anti-inflammatory steroids and the elucidation of the underlying
mechanism; the discovery of novel pathways in the conversion of polyunsaturated
fatty acids leading to the recently discovered non-prostanoate compounds, the
leukotrienes and their related products; the recognition of numerous biological roles
of the members of the prostaglandin family and their involvement in the pathogene-
sis of a multitude of disorders and diseases; and finally, the beginning of the clinical
use of certain prostaglandins in the treatment of gynecological, gastro-intestinal and
circulatory conditions.
The rapid development of a greatly enhanced volume of published scientific data
has increased the need for comprehensive reviews, written by scientists who are
themselves active in the field, and providing the current state-of-the-science of the
area.
The contributors of this volume in the New Comprehensive Biochemistry series
cover the biosynthesis and metabolism of the prostaglandins, thromboxanes and
leukotrienes; the analytical methods currently in use; the purification and properties
of several enzymes involved in the formation and catabolism of these substances;
activators and inhibitors of these enzymes; as well as the involvement of the
members of the prostaglandin family in numerous physiological and pathological
processes.
Bengt Samuelsson
...
Pace - Asciuk /Granstriim (eds.)P rostaglandins and related substances XI11
01 Elsevier Science Publishers B. K, I983
INTRODUCTION
Physiological implications of products
in the arachidonic acid cascade
MARIE L. FOEGH and PETER W. RAMWELL
a
Departments of Medicine and Physiology and Biophysics ’,
Georgetown University Medical Center, Washington, D. C. 20007, U.S .A.
Physiology is the study of function. The classical procedure used to define physio-
logical roles is by extirpation, ablation or nerve section to reveal inadequate or
inappropriate function in the absence of the postulated mechanism. This approach
cannot be used to study the physiological role of arachidonate metabolites since they
are not organ-localized like the adrenal steroids or concentrated in specific cells like
the adrenergic transmitters. The problem is compounded also by the fact that
arachidonate oxygenation is almost a universal phenomenon. Finally the metabolites
are not stored like histamine or serotonin but are released immediately upon
synthesis. Consequently it is always necessary to initiate synthesis to study release.
Thus release is synonymous with synthesis.
The emphasis on physiology in this section also relates to the nature and quantity
of the arachidonate metabolites released. For example some naive authors state that
“Prostaglandins at physiological concentrations were found to be.. .”. It is ex-
tremely difficult for such concentrations to be defined and such authors are begging
the question as to what is physiological. The other ‘begging’ question is to assume
that the cell or tissue ‘sees’ only one metabolite in vivo, i.e. the one in which the
author is interested. This has been a particular problem in macrophage studies where
the usual product measured is prostaglandin E, and little account has been taken of
the other metabolites. In rodent and human macrophages frequently equimolar
amounts of both TXB, and PGE, are released but little is known of their interaction.
It is possible that the thromboxane released may completely block PGE mediated
elevations in cyclic AMP, or again, because of its transient nature, TXA, may have
little effect. Nevertheless, to avoid consideration of all the products and their
interaction is simplistic.
Two other points are frequently neglected when discussing physiological roles.
The first is that arachidonate metabolite receptors may be subject to regulation.
Thus it is not enough to define the concentration of product formed if there are
marked changes in the receptors. This appears to be the case for PGE compounds in
the myometrium and liver [l]. PGE analogues clearly may down-regulate PGE
receptors in the liver and estrogen down-regulates the myometrial receptors to
PGF,<ra nd PGE,. Other hormones may be involved; recent studies have implicated
prolactin in ovarian receptor regulation and the PGF,, receptors in the corpus
luteum are known to be regulated by luteinizing hormone [2]. There is evidence from
our own laboratory that estrogen and testosterone regulate prostaglandin receptors
in rat aorta [3].
The second point is the role of converting and catabolising enzymes. There is now
evidence for the conversion of PGI, to the stable product 6-keto-PGE, which has
very similar properties [4]. There is also convincing evidence for PGE, to PGF2<,
conversion [5] and vice versa [6]. Finally there is strong evidence that the further
metabolism of PGs by prostaglandin dehydrogenase (PGDH) has a significant role
as demonstrated by PGDH inhibition leading to luteolysis in rodents [7]. These
therefore are points which need to be borne in mind when interpreting data as to the
physiological role of arachidonate metabolites.
An approach to functional ablation has been to evaluate the effects of acute
essential fatty acid (EFA) deficiency. This deficiency has been shown to cause
dermatoses in both humans [8,9] and animals [lo]. Van Dorp (1971) [ 1 I] reported a
marked decrease in PGE, in skin of EFA-deficient rats and Ziboh and Hsia (1972)
[ 121 subsequently found that topical application of PGE, cleared the scaly der-
matoses. However, a potential difficulty is the accumulation of 5,8,1’1-eicosatrienoic
acid (20 : 3, n-9) which may be responsible for some of the symptoms namely loss of
skin elasticity, alopecia and scaliness. Ziboh et al. [13] showed that this trienoic acid
inhibits cyclooxygenase activity. Since this fatty acid accumulates in the skin of EFA
deficient rats it was tested on the skin of nude mice where at only 50 pM it
significantly reduced PGE, and produced the scaly dermatoses. In addition it is
possible that blocking the cyclooxygenase shunts arachidonate through the lipo-
xygenase pathway and products of this pathway are reported elevated in psoriasis
[ 141. Since 5,8,1I -eicosatrienoic acid has proved to be a substrate for 5 lipoxygenase
and thus can yield leukotrienes, it is likely that the dermatoses characteristic of EFA
deficiency may not necessarily result from a PGE, deficiency only. Especially since
lipoxygenase products are associated with psoriasis. Consequently care must be
taken in interpreting data from EFA deficient animals. However it is possible to
avoid the problem of redirection of synthesis by use of receptor antagonists.
Although the attempt to “ablate” or “extirpate” arachidonate metabolites by
using EFA deficiency can be complex, nevertheless it is an approach to the
physiological role of these metabolites which deserves further exploration since it
offers so many experimental models. For example in immunology, evidence is
accruing that the cyclooxygenase products which elevate cyclic AMP are immuno-
suppressive [ 151. Feeding with essential fatty acids also produces immunosuppres-
sion [12] whilst indomethacin abrogates this effect [17]. Moreover there have been
reports that cyclooxygenase inhibitors may increase anti-body response in vivo [ 181.
These EFA feeding experiments raises the interesting question of immune responses
in Eskimos. Does the fish diet which is so rich in eicosapentaenoic acid, lead to
xv
enhanced immune response? One might anticipate this to be the case since this acid
competes with arachidonate for the cyclooxygenase and thus acts as a “nutritional
aspirin”.
An extremely important approach to blocking all arachidonate metabolism is the
use of 5,8,11,16eicosatetraynoica cid [ 191. This acetylenic analogue of arachidonate
was first used to inhibit cyclooxygenase in 1970 and was replaced in 1971 by the non
steroidal anti-inflammatory drugs. Later it returned to favor when it was realized
that tetraynoic acid blocks the lipoxygenase pathways too. Consequently eico-
satetraynoic acid treatment does not involve the complications involved in the use of
either eicosapentaenoic acid or other essential fatty acids.
Eicosatetraynoic acid treatment of rats leads to the same deleterious effect on the
gastro-intestinal mucosa as seen with indomethacin [ZO]. These effects, like the skin
lesions in EFA deficiency, can be prevented by treatment with prostaglandins. The
concept that prostaglandins of the E series are cytoprotective in the stomach has
been suggested in several other body systems. One might also use the term cytopro-
tective to describe the prominent and widespread immunosuppressive effect of these
types of arachidonate metabolites.
A less rigorous approach to evaluating the physiological role of the cycloo-
xygenase products has been to use potent cyclooxygenase inhibitors such as in-
domethacin. These compounds have many side effects and as discussed earlier,
cyclooxygenase inhibition may lead to increased lipoxygenase product formation.
Nevertheless the approach has been effective in revealing the role of the cycloo-
xygenase products. The most significant area has been cardiovascular homeostasis
which will be discussed later in terms of renal and perinatal cardiovascular homeos-
tasis.
The drawback to the use of cyclooxygenase inhibitors respecting arachidonate
diversion to lipoxygenase products can be overcome by specific inhibition of
individual pathways. This more precise approach is proving a useful method of
dissecting out the roles of the individual metabolites. The importance of thrombo-
xane synthase inhibition with the substituted imidazoles and pyrimidine was quickly
appreciated. Inhibition of prostacyclin synthase with 15 hydroperoxy eicosa-
tetraenoic acid or with tranylcypromine has been less successful. By and large the
data indicate in pathophysiological models that inhibition of thromboxane synthase
is protective to some degree but this may be related to an increase in prostacyclin
due to divergence of the endoperoxides. An example of such divergence was seen in
vitro in human peritoneal macrophages [21] as well as in vivo in baboons [22] treated
with the thromboxane synthase inhibitor OKY-1581. Aiken has provided striking
evidence for a physiological role for prostacyclin in the dog coronary circulation
~31.
An even more precise tool is the use of receptor antagonists. In this respect the
evaluation of the role of histamine is particularly instructive. Histamine was clearly
recognised as a mediator of tissue injury by Sir Thomas Lewis in his classical triple
response studies. This is the case now with respect to leukotrienes and thromboxane
in immunological and cardiovascular pathology. But in order for histamine to be
XVI
convincingly shown to have a physiological role in gastric secretion for example, it
was necessary to await the development of the H, receptor antagonist in the early
1970s by Black [24]. In the prostaglandin area an analagous situation is particularly
the case with prostacyclin. The many roles for this important metabolite have been
so strongly asserted that only a specific receptor antagonist will separate the puffery
from reality. The advantage of receptor antagonists is that they do not cause
redirection of synthesis as is seen with thromboxane synthase inhibitors for example.
It is also possible to manipulate prostaglandin receptors like other receptors with
thiol reagents. The prostaglandin receptors are far more sensitive to dithiothreitol for
example than acetylcholine. This approach is useful since it is reversible and
moreover the receptor can be “capped” or protected with prostanoic acid [25].
Valuable clues as to the physiological roles of endogenous substances are fre-
quently derived from glandular failure as for example in Hashimoto’s disease which
involves destruction of the thyroid glandular epithelium. Unfortunately no such
syndrome has yet been identified with respect to arachidonate synthesis. Perhaps the
nearest to such an effect is the essential fatty acid deficiency due to liver failure in
cirrhosis of the liver [26]. Defects in metabolic pathways or in the absence of
receptors also frequently provide valuable clues. A deficiency in platelet cycloo-
xygenase has been reported but this defect apparently involves only a minor
bleeding tendency [27,28]. More such defects are being reported now. For example
attention is being focused particularly on the relation of diabetes to decreased
endothelial prostacyclin synthesis 1291 and possible elevation of platelet thrombo-
xane. Changes in the response of coronary artery preparations have also been
reported in experimentally induced diabetes in the dog [3I ].
A number of these approaches have been applied to evaluating the role of
arachidonate metabolites in perinatal physiology [32-341. The evidence that
arachidonate metabolites have a regulatory role in fetal homeostasis is becoming well
established. This is because attention became directed to the key role of the ductus
arteriosus. The mechanism concerning the role of arachidonic metabolites in
maintaining patency is particularly interesting in that the iipoxygenase pathway does
not appear to be involved and only one cyclooxygenase metabolite may be im-
plicated namely PGE,. However, a role for both PGD, and PGI, has been also
postulated. PGD, increases cardiac output and reduces pulmonary and systemic
resistance but is only a very weak dilator of the ductus arteriosus [35]. PGD, is
particularly interesting as there is evidence that PGD,- receptors appear relatively
late in gestation. Thus it is possible that PGD, and PGE, may be acting synergisti-
cally. The case for PGI, is attractive but more conjectural. It has been suggested that
if PGI, is the primary ductus vasodilator then the high PO, on delivery may destroy
the notoriously susceptible prostacyclin synthase and the loss of PGI, permits the
human ductus to be obliterated [36]. Nevertheless based upon the utility of in-
domethacin to close the patent ductus and the properties of the vasodilator cycloo-
xygenase metabolites on pulmonary vessels, as well as the ductus, there is little doubt
as to the significance of their role in perinatal hemodynamics. One additional aspect
,
is that the cyclooxygenase vasoconstrictor products PGF,, and TXA may exert a
xvii
tonic constrictor effect. This is a biochemical hypothesis with little hemodynamic
support. Nevertheless it would be easy to test in view of the availability of specific
thromboxane synthase inhibitors, as well as receptor agonists to thromboxane and
PG Fz
(I.
A great deal of effort has been invested into determining the role of arachidonate
metabolism in the kidney. As McGiff [37] points out it is useful to consider two roles
for arachidonate metabolites, firstly with respect to the blood compartment and
secondly with respect to tubular mechanisms. The role of the arachidonate metabo-
lites in the regulation of the renal circulation is to protect the kidney against
powerful vasoconstrictor substances such as angiotensin I1 [38]. The renal vascular
vasodilator arachidonate metabolites such as PGE, are released under these circum-
stances and also following renal sympathetic activity [39,40]. Where the renal
circulation is compromised in the diseased kidney or during dehydration then
indomethacin decreases kidney function often in a reversible manner [41]. The
suggestion has been offered that this deterioration may not be entirely due to lack of
arachidonate vasodilator metabolites but may also be due to products with vaso-
constrictor effects. The effect of leukotrienes on renin release is currently being
investigated, but the vasodilator arachidonate metabolites are well known to release
renin [37-391.
The renal kallikrein-kinin [40] system has been suggested as influencing renal
hemodynamic as well as excretory function. This activity may also be linked to
arachidonate metabolites, like PGE,, since increased excretion of cycloovygenase
products are associated with increased kallikrein-kinin excretion. However, the
physiological significance of this relationship is uncertain.
The role of arachidonate metabolites in tubular function [41,42] is more complex
although the PGE compounds were shown early on to block the effect of the
antidiuretic hormone (ADH) on transporting epithelia. The reason for the complex-
ity is the effect of PGE compounds on sodium and water transport on the one hand
and their effect in changing renal blood flow and intrarenal distribution on the
other. It is now generally believed that the role of the vasodilator metabolites is to
preserve renal homeostasis and that their effect becomes apparent when the kidney
function is compromised [43]. The problem with which one is faced is to fit into this
scheme the ADH-like effects of thromboxane [44] and the effects of the leukotrienes,
when they are properly defined. It is possible that thromboxane and leukotrienes
only become prominent in the pathological situation but this remains to be docu-
mented.
In conclusion, the oxygenation of arachidonic acid yields an extensive series of
products which are universally distributed in all animal species and nearly all cells.
These metabolites constitute a modulating system for maintaining homeostasis, e.g.
for preserving hemostasis, hernodynamic and renal function, for signalling pain, for
regulating immunological responsivity etc. One may think of them as a Claude
Bernard homeostatic hormone. The role of such a universal system needs to be
modulatory since so many substances involved in injury and inflammation interact
with it, e.g. vasoactive amines, kallikrein and kinins, clotting factors and thrombin,
