Table Of ContentProstacyclin
and Its Receptors
Prostacyclin
and Its Receptors
Helen Wise
and
Robert L. Jones
The Chinese University ofHong Kong
Hong Kong SAR, China
KLUWER ACADEMIC PUBLISHERS
New York, Boston, Dordrecht, London, Moscow
eBookISBN: 0-306-46822-0
Print ISBN: 0-306-46308-3
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Preface
The 1970's were dramatic times for prostanoid research, with the
discovery of prostacyclin and the elucidation of the mode of action of aspirin
by Sir John Vane and his colleagues. As the most potent endogenous
antiplatelet agent, prostacyclin received much attention, and the race was on
to develop nonhypotensive analogues for thrombosis therapy. Promising
leads were invariably confounded by the possibility of species differences in
prostacyclin (IP-) receptors, and then a molecular biology revolution
occurred in the early 1990's. Shuh Narumiya and his colleagues presented us
with a full hand of cloned prostanoid receptors, and the exact identity of
prostacyclin receptors in blood vessels and platelets finally became clear.
Nevertheless, interest in the biology of prostacyclin remained high, but was
focused more on its excitatory actions. Could prostacyclin exert some of the
pro-inflammatory actions, for example enhancement of pain sensation,
traditionally associated with prostaglandin E ? The demonstration of IP-
2
receptors on central and peripheral neurones, the existence of two neuronal
IP-receptor subtypes with quite different agonist specificities, and the
reduction of pain sensation in the IP-receptor gene-knockout mouse has
triggered a new wave of prostanoid research. In addition, the development
of much-needed IP-receptor antagonists may turn out to be more than
rumours. This monograph has been prepared to put all of these data into
context and to set the scene for progress into the next millenium.
HelenWise
RobertL.Jones
V
Acknowledgments
We wish to thank industrial colleagues who over the years have
supportedour research with generous gifts of prostanoids,and in particular
Professor Helmut Vorbrüggen of Schering AG and Dr. Nick Meanwell of
Bristol-Myers Squibb. RLJ is also appreciative of the camaraderie of Drs.
Norrie WilsonandRomaArmstrong,whowere involvedinthe development
of the EP analogue series at the University of Edinburgh. The excellent
assistance of Mrs. Joresa Ng in the preparation of the manuscript and the
patience of Dr. John Rudd in all-things-Macintosh are gratefully
acknowledged. Finally,we should liketothankthe followingpublishersfor
giving permission to use their copyright material: American Society for
Biochemistry and Molecular Biology, Birkhäuser, Blackwell Science Ltd,
Elsevier Science Ltd, Kluwer Academic / Plenum Publishers, Lippincott
Williams & Wilkins, Macmillan Magazines Ltd, Macmillan Press Ltd, and
ProusScience.
vii
Contents
Chapter 1 Anintroductiontoprostacyclinanditsreceptors 1
Chapter2 Thedevelopmentofprostacyclinanalogues 29
Chapter3 Nonprostanoidprostacyclinmimetics 59
Chapter4 Isolation,cloningandcharacterisationofIP-receptors 79
Chapter5 IP-receptorsonplatelets 109
Chapter6 IP-receptorsinthevasculature 137
Chapter7 IP-receptorsonneutrophils 189
Chapter8 IP-receptorsonmonocytes/macrophages 215
and lymphocytes
Chapter9 IP-receptorsonsensoryneurones 243
Chapter10 IP-receptorsintheentericnervoussystem 271
Chapter11 IP-receptorsinthecentralnervoussystem 285
Index 305
ix
Chapter1
An introduction to prostacyclin and its receptors
1. INTRODUCTION
The platelet inhibitory and hypotensive actions of prostacyclin are by
now so well established that it is surprisingto find that prostacyclin was
actually the last member of the primary (2-series) prostanoid family to be
identified. By coincidence, the prostacyclin (IP-) receptor was also the last
of the prostanoid familyofreceptors to becloned.
Interest in prostacyclin as an antithrombotic agent has resulted in an
intensive search for more clinically useful IP agonists, with high chemical
and metabolic stability, and selectivity of action on platelets. As a result of
these studies, a large amount of structure-activity relationship data is
available,which we have attemptedto summarisein Chapter 2 (prostacyclin
analogues) and in Chapter 3 (nonprostanoid prostacyclin mimetics). The
ready availability of human platelets for screening potential IP agonists, and
the variable choice of agonists used by different groups, has perhapslimited
a thorough assessment of the existence of species-specific IP-receptors.
Therefore, we have attempted to redress the balance by closely examining
the data on the cloned human, mouse and rat IP-receptors (Chapter 4), and
compared this with twenty year’s worth of platelet aggregation studies
(Chapter 5). Despite such a wealth of knowledge on prostacyclin and its
platelet receptors, we are constantly hampered in our interpretation of data
by the lack of IP-receptor antagonists. So we caution the readerto bearthis
in mind, and would encourage a re-examination of our conclusions when IP
antagonistsbecomeavailable.
1
2 Chapter1
The hypotensive property of prostacyclin is generally considered a
drawback to the use of IP agonists as antithrombotic drugs. However, there
are conditions (e.g. pulmonary hypertension) where its vasodilator action
contributes to the therapeutic effect. Prostacyclin can also claim to belong to
the family of endothelium-derived relaxing factors (EDRFs), of which nitric
oxide (NO) is probably the most well known. As an EDRF, prostacyclin
may therefore have an important role in controlling blood flow. These
aspects are discussed in Chapter 6.
Aside from the cardiovascular properties of prostacyclin, its other
functions are less well established. As an inflammatory mediator, the role of
prostacyclin has long been overshadowed by that of prostaglandin E2
(PGE), yet both prostanoids are produced at sites of inflammation, and both
2
have unique profiles of activity. Prostacyclin as an inhibitor of the activity
ofa range of inflammatory cells will be discussed in Chapters 7 and 8, where
it will become clear that the source of cells being studies (e.g. elicited or
nonelicited) has a crucial influence on their responsiveness to IP agonists.
We would also like to draw the reader’s attention to the role ofprostacyclin
in sensory neurones (Chapter 9), and to present evidence showing that IP-
receptors have an important role to play in nociception, as least as important
as PGE receptors.
Of most recent interest is the role of IP-receptors in other neuronal
systems and the possible existence of IP-receptor subtypes. This information
may be found in Chapter 10, where we present evidence for the presence of
IP-receptors on enteric neurones and in Chapter 11, where we look at IP-
receptors in the central nervous system.
Throughout this monograph, we have tried to consider the potential
therapeutic application of drugs acting on IP-receptors, but the topics cannot
be considered exclusive, they simply represent the best studied areas to date.
Because prostacyclin is just one member of an important family of products
derived from arachidonic acid (the eicosanoids), and because many of the
other members are mentioned throughout this monograph, we would like to
set the scene by outlining the metabolic pathways for eicosanoids, the
properties of prostanoid receptors, and how certain anti-inflammatory drugs
interfere with eicosanoid systems.
2. EICOSANOID BIOSYNTHESIS
The 20-carbon polyunsaturated fatty acid arachidonic acid has two quite
different functions in the body. As a component of cell membrane
phospholipids, its cis double bonds disorder the hydrophobic core of the
membrane, thus influencing fluidity, permeability and the behaviour of
An introduction to prostacyclin and its receptors 3
embedded proteins.1 However, following release from internal membranes
by phospholipase A (PLA), arachidonic acid can enter several oxidation
2 2
pathwaystoyieldadiverserangeofproducts, someofwhichhave important
physiological and pathological roles (Fig. 1 and 2). These products are
termed eicosanoidsto indicatethepresence of a 20-carbon backbone (Greek
eicosi = 20); related products derived from 06 fatty acids other than
arachidonate and from some 3 and 9 fatty acids also belong to the
eicosanoidfamily.
Two types of oxygenase enzyme are responsible for the initial steps in
eicosanoid pathways, lipoxygenases (LOXs) and cytochromeP-450 mono-
oxygenases. The basic lipoxygenasereaction involves stereospecific attack
of an hydroperoxy radical on a methylene-interrupted diene unit (-CH=CH-
CH-CH=CH-) in the fatty acid substrate. By attacking one end of one of
2
the three diene units present in arachidonic acid (Fig. 1, inset) individual
LOXs produce unique products. P-450 mono-oxygenasesonthe other hand
appear to show less specificity, producing a range of epoxidesofarachidonic
acid(Fig.2),aswellasits 20-hydroxyderivative.2
2.1 PGHS or COX pathway
The prostaglandin H synthase (PGHS) or cyclo-oxygenase (COX)
pathway, which generates the prostanoids (Fig. 1), was first reported in 1964
by two independent research teams led by Bergström & Samuelsson at the
Karolinska Institute in Sweden3 and by van Dorp & Nugteren at the Unilever
Research Laboratories in Holland.4 They were able to isolate and identify
PGE from homogenates of sheep seminal vesicles incubated with
2
arachidonic acid. The choice of seminal vesicles stemmed from
observations made some 30 years earlier that smooth muscle contractile and
relaxant activities attributable to acidic lipids were present in extracts of
male reproductive tissues.5-8
Prostaglandin H synthase contains both COX and 15-hydroperoxidase
activities. The COX site essentially catalyses two consecutive lipoxygenase
reactions;thefirstoccursatC11 togive 11(R)-hydroperoxy-eicosatetraenoic
acid (11-HPETE).9,10 The second is more complex, involving insertion ofan
hydroperoxy group at C15 and generation of an endoperoxide ring system.
The product PGG is reduced at the 15-hydroperoxidase site to PGH. In
2 2
general, the terms PGHS and COX are used loosely to describe the enzyme
system that forms prostaglandin endoperoxides.
The 9,11-peroxide bond in PGH is chemically labile and spontaneous
2
isomerisation readily occurs to give PGD and PGE. However, specific
2 2
synthase enzymes also generate PGD and PGE and two further isomers
2 2
prostacyclin and thromboxane A (TXA). The defined stereochemistry of
2 2
4 Chapter1
PGH (with oxygens below the plane of the cyclopentane ring, 8 /12
2
side-chains, and S-configuration at C15) is passed on to these primary
prostanoids (see Chapter 2 for further stereochemical information). Both
prostacyclin and TXA are rapidly hydrolysed under physiological
2
conditions with loss of biological activity (t = 3 and 0.5 min respectively).
½
Enzymatic reduction of the 9,11-peroxide bond of PGH yields the fifth
2
primary prostanoid PGF ,, which may also be formed from PGD or
2
PGE.11
2
2.2 Leukotriene pathway
A smooth muscle contractile substance is released from guinea-pig lungs
by cobra venom (a source of PLA)12 and by anaphylactic challenge.13-15
2
The identity of this "slow-reacting substance of anaphylaxis" (SRS-A)
remained elusive for many years until the discovery of the leukotrienes by
Samuelsson's group. It was shown that a 5-lipoxygenase (5-LOX) first
converts arachidonic acid into 5(S)-HPETE and then abstracts water to
generate leukotriene A (LTA) (Fig. 2).16-18 At this point the pathway splits.
4 4
LTA hydrolase generates the 5,12-dihydroxy compound LTB,19 which has
4 4
potent chemotactic activity for polymorphonuclear leukocytes.20
Alternatively, LTC synthase catalyses the opening of the epoxide ring by
4
the SH group of glutathione (y-Glu-Cys-Gly) to yield LTC4;21 metabolism of
the glutathione unit yields LTD and LTE. These three peptidoleukotrienes
4 4
activate specific leukotriene (CysLT) receptors to elicit contraction of
intestinal and bronchial smooth muscle, thus accounting for the bioactivity
of SRS-A.
The leukotriene pathway is well developed in white cell populations,
including neutrophils, eosinophils and pulmonary alveolar macrophages, and
there is good evidence for a role for peptidoleukotrienes in the
bronchoconstriction of human asthma.22-25 Recently, several CysLT
antagonists, (e.g. zafirlukast26 and a 5-LOX inhibitor zileuton27 have been
approved as anti-asthma drugs.28
Whether COX, leukotriene or other arachidonate products are formed
within a cell would be expected to depend on the relative levels of active
oxygenases present. However, more subtle mechanisms such as "substrate
channelling" may have a significant influence. Consecutive enzymes in a
pathway may be located in or directed to internal membranes in such a way
that the product of the first enzyme feeds directly into the catalytic site of the
second. This will increase the substrate concentration of the primary
product, and also protect unstable primary products from chemical reaction
in the surrounding aqueous phase. Substrate channelling appears to be
particularly important in leukotriene biosynthesis.29 During cell activation,
