Pharmac. I'her. Vol. 49, pp. 153-179, 1991 Printed in Great Britain. All rights reserved

0163-7258/91 $0.00+ 0.50 © 1991PergamonPress pie

Specialist Subject Editor: C. W. TAYLOR

PROSTAGLANDIN AND THROMBOXANE BIOSYNTHESIS WILLIAM L. SMITH,* LAWRENCE J. MARNETTt a n d DAVID L. DEWITT* *Department of Biochemistry, Michigan State University, East Lansing, MI 48824, U.S.A. tThe A.B. Hancock, Jr Memorial Laboratoryfor Cancer Research, Department of Biochemistry, Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN 37232-0146, U.S.A. Abstract--We describe the enzymological regulation of the formation of prostaglandin (PG) D 2, PGE2, PGF2,, 9~,1 Ifl-PGF2, PGI2 (prostacyclin), and thromboxane (Tx) A 2 from arachidonic acid. We discuss the three major steps in prostanoid formation: (a) arachidonate mobilization from monophosphatidylinositol involving phospholipase C, diglyceride lipase, and monoglyceride lipase and from phosphatidylcholine involving phospholipase A2; (b) formation of prostaglandin endoperoxides (PGG 2 and PGHz) catalyzed by the cyclooxygenase and peroxidase activities of PGH synthase; and (c) synthesis of PGD 2, PGE2, PGF~, 9a, l 1/~-PGF2, PGI2, and TxA2 from PGH 2. We also include information on the roles of aspirin and other nonsteroidal anti-inflammatory drugs, dexamethasone and other anti-inflammatory steroids, platelet-derived growth factor (PDGF), and interleukin-1 in prostaglandin metabolism.

CONTENTS 1. Introduction 2. An Overview of Prostanoid Biosynthesis 3. Arachidonate Mobilization 3.1. Arachidonate release--an overview 3.2. Agents that will induce prostaglandin formation by intact cells 3.3. Source of arachidonate for prostanoid synthesis 3.4. Platelet arachidonate release 3.5. Platelet phospholipase C and acylglycerol lipase activities 3.6. Platelet phospholipase A2 activities 3.7. General properties of stimulus-induced phospholipid turnover 3.8. Phospholipase A2 activities 3.9. Glucocorticoids and phospholipase inhibition 4. Prostaglandin Endoperoxide Formation 4.1. PGH synthase catalysis--an overview 4.2. Cyclooxygenase catalysis 4.3. Cyclooxygenase inhibition--nonsteroidal anti-inflammatory drugs 4.4. Peroxidase catalysis 4.5. Peroxidase-dependent cooxidation of reducing cosubstrates 4.6. Interdependence of cyclooxygenase and peroxidase reactions 4.7. Suicide reactions 4.8. Physico-chemical properties of PGH synthase 4.9. PGH synthase amino acid sequence 4.10. Heme binding site 4.11. Active site model of PGH synthase 4.12. Regulation of PGH synthase protein concentrations 4.13. Developmental regulation of PGH synthase 4.14. 'Tuning' of PGH synthase 4.15. Intracellular regulation of PGH synthase 4.16. Pools of PGH synthase 4.17. PGH synthase gene structure 4.18. Effects of anti-inflammatory steroids on PGH synthase

154 154 154 154 156 156 156 158 158 159 159 159 160 160 160 161 161 162 162 163 163 163 164 165 165 167 167 167 168 168 168

Abbreviations: PDGF, platelet-derived growth factor; IL-1, interleukin-1; PAF, platelet-activating factor; PG, prostaglandin; Tx, thromboxane; 12-HETE, 12S-hydroxyeicosatetraenoic acid; II-HpEDE, li-hydroperoxy-12-trans.14-ciseicosadienoic acid; 15-HpETE, 15-hydroperoxy-eicosatetraenoic acid; 12-HHTrE (formerly known as HHT), 12-hydroxyheptadecatrienoic acid; MDA, malondialdebyde; LDL, low density lipoprotein; Ptdlns, phosphatidylinositol; PtdCho, phosphatidylcholine; PtdEtn, phosphatidylethanolamine; PLC, phospholipase C; PLC2, phospholipase A2; AVP, arginine vasopressin; PMA, phorbol myristate acetate; PtdlnsP2, phosphatidylinositol-4,5-bisphosphate;PtdSer, phosphatidylserine; PPIX, protoporphyrin IX; cDNA, complementary DNA; LH, luteinizing hormone; FSH, follicle stimulating hormone; PKC, protein kinase C; PKA, protein kinase A; GSH, reduced glutathione; ETYA, eicosatetraynoic acid; for additional details regarding enzyme and leukotriene nomenclature see Smith et aL (1990a). 153

W. L. SMITHet al.

154

5. Metabolism of PGH 2 5.1. PGD synthesis 5.2. PGE synthesis 5.3. PGF synthesis 5.4. PGI synthesis 5.5. Thromhoxane synthesis Acknowledgements References

168 168 169 170 171 171 172 172

1. I N T R O D U C T I O N Summarized in Fig. 1 are the major pathways of the arachidonate cascade which leads to the formation of the oxygenated lipids collectively known as eicosanoids (Smith, 1989). The three major pathways of the cascade are the cyclooxygenase, lipoxygenase and epoxygenase pathways. Prostanoids are formed via the cyclooxygenase pathway, leukotrienes and lipoxins are formed via one or more lipoxygenase reactions (Samuelsson et al., 1987; Smith, 1989), and lipid epoxides and diols are formed through P-450 dependent reactions of the epoxygenase pathway (Fitzpatrick and Murphy, 1989; LaniadoSchwartzman et al., 1988; Capdevila et al., 1990). The cyclooxygenase pathway is so named because the initial step involves a transformation catalyzed by a b/s-oxygenase called cyclooxygenase. The prostanoids formed via the cyclooxygenase pathway include the prostaglandins (PGD, PGE, PGFa, PGI) and thromboxanes (Tx). Their synthesis and its regulation are the major subjects of this review. Prostanoids are local hormones which are synthesized by virtually all mammalian tissues (Smith, 1987) and act at or near their sites of synthesis. These compounds function in both an autocrine and paracrine fashion. For example, PGE synthesized and released by the renal collecting tubule acts both on collecting tubule and neighboring thick limb cells (Smith et al., 1989) to coordinate the synergistic responses of these cells in water reabsorption. The typical responses of a cell to a prostanoid include changes in the intracellular concentration of cAMP (Sonnenburg and Smith, 1988) and Ca 2+ (Negishi et al., 1989) or both. At the molecular level responses to prostanoids are mediated through receptors coupled to guanine nucleotide-dependent regulatory (G) proteins (Smith, 1989). There appear to be a distinct set of receptors for each of the major prostanoids (Coleman et al., 1987); for example, in the case of PGE derivatives, there are at least three pharmacologically distinct PGE receptors coupled to different G proteins to cause activation of adenylyl

cyclase (Sonnenburg and Smith, 1988), inhibition of adenylyl cyclase (Sonnenburg et al., 1990; Watanabe et al., 1986), and stimulation of Ca 2+ mobilization (Negishi et al., 1989). 2. AN OVERVIEW OF PROSTANOID BIOSYNTHESIS The pathway for the formation of prostanoids as it might occur in a generic cell is presented in Fig. 2 (Smith, 1989). Prostanoid formation occurs in three phases: (a) mobilization of arachidonic acid; (b) conversion of arachidonate to the prostaglandin endoperoxides PGG2 then PGH2; and (c) rearrangement or reduction of PGH2 to yield what are considered to be the biologically active prostanoids (PGD2, PGE2, PGF2~, PGI2, and TxA2). In subsequent sections we will deal with each of these processes individually. 3. A R A C H I D O N A T E MOBILIZATION 3.1. ARACHIDONATERELEASE--AN OVERVIEW The paradigm for prostanoid formation is that intact cells mobilize arachidonate from glycerophospholipids in response to extracellular stimuli and that released arachidonate is then converted to PGH2 by an already active PGH synthase; thus, prostaglandin biosynthesis is thought to be regulated, at least acutely, at the level of arachidonate mobilization (Bettazzoli et al., 1990; Dennis, 1987). Experimental support for this model is as follows: (a) in most cells there is either only a low basal level of prostanoid synthesis (e.g. Wightman and Dallob, 1990; Grenier et al., 1982) or no detectable synthesis (Habenicht et al., 1990) until the cells are exposed to an appropriate exogenous stimulus, and various stimuli will then increase the rate of prostanoid formation 2-100-fold; and (b) addition of arachidonate (10-100/~ M) itself to prostaglandin-forming cells will elicit prostanoid formation suggesting that the PGH synthase is already

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EPOXYACIDS(EETs) LEUKOTRIENES(LTs) HYDROXYACIDS(HETEs) DIHYDROXYACIDS LIPOXINS(LXs) FIG. 1. Cyclooxygenase, lipoxygenase, and epoxygenase pathways for the formation of eicosanoids. PROSTAGLANDINS(YGs) rI-IROM]SOX~TES(TXs)

Prostaglandin and thromboxane biosynthesis

155

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FIG. 2. Pathways for the formation of various prostanoids. Adapted from Smith (1989). active, and consistent with this is that PGH synthase is active in isolated membrane preparations. In fact, the intracellular concentration of free arachidonate varies. It is well below the Km for arachidonate of PGH synthase (ca. 5 #M) in some cells (e.g. platelets (Marcus et al., 1969; Bills et al., 1976)) but as high as 20 #M in others (Bazan et al., 1971; Cenedalla et al., !975; Marshall et al., 1987). In cells which have relatively high intracellular levels of arachidonate, the f r ~ arachidonate is apparently unavailable for prostanoid synthesis. Arachidonate destined for conversion to prostanoids is likely to be mobilized in close proximity to PGH synthasc. PGH synthase is associated with several intracellular membranes (Smith, 1986, 1987). The bulk of the enzyme activity and immunereactivity is associated with the endoplasmic reticu-

lure (Rollins and Smith, 1980; DeWitt et al., 1981; Smith, 1986); however, PGH synthase is also present at a roughly equivalent density on the nuclear envelope, presumably on the outer nuclear membrane; and, at least in some cells, PGH synthase is present on the plasma membrane (Fukuoka et al., 1982; Smith et al., 1983; Gerozissis and Smith, unpublished results). With respect to the endoplasmic reticulum, the active site and antigenic determinants of PGH synthase reside on the cytoplasmic surface (DeWitt et al., 1981). It is also thought that the active site of those PGH synthase molecules which are present on the plasma membrane face the cytoplasm because immunochemical staining for the enzyme only occurs following permeabilization of cells. Thus, stimuli of prostanoid formation are likely to effect arachidonate mobilization at the cytoplasmic surface of one or

156

W.L. SMITHet al.

more intracellular membranes. As discussed below, there are soluble phospholipases which become membrane-associated upon Ca 2+ mobilization, and there are phospholipases which are intrinsic membrane proteins. Either or both types of enzymes could be involved in arachidonate mobilization. 3.2. AGENTSTHAT WILL INDUCEPROSTAGLANDIN FORMATIONBY INTACTCELLS

Prostaglandin synthesis can be initiated by a large number of stimuli some of which are 'physiological' and cell specific and others which are physical or pharmacological. Examples of physiological stimuli include hormones such as histamine (Murayama et aL, 1990), bradykinin (Grenier et al., 1981), arginine vasopressin (AVP) (Kirschenbaum et al., 1982), platelet activating factor (PAF) (Glaser et al., 1990), angiotensin II (Gimbrone and Alexander, 1975), intefleukin-I (IL-1) (Albrightson et al., 1985; Burch et al., 1988), leukotrienes (Cramer et al., 1983) and P D G F (Habenicht et al., 1990), and proteases such as thrombin (Weksler et al., 1978). These agents are envisioned as interacting with cell-surface receptors and causing activation of one or more specific phospholipases. In many instances, these effects appear to be mediated through G proteins (e.g. Murayama et al., 1990; Silk et al., 1989). Physiological stimuli differ from other types of stimuli in that they cause a relatively selective release of arachidonate, but not other fatty acids such as linoleate or oleate which are also found at the sn2 position of phospholipids. Physical stimuli which cause prostaglandin formarion include shear forces acting on endothelial cells (Francos et al., 1985; Karwatowska-Prokopczuk et al., 1989) or simply changes of medium for cells grown in culture (Dunn et al., 1976). It is certainly conceivable that shear forces are important in vivo in eliciting vascular PGI 2 formation. Another event which may qualify as a physical stimulus is ischemia (Hsueh et al,, 1977); cardiac ischemia leads to nonspecific release of fatty acids such as oleate from the sn2-position of phospholipids. A phospholipase A 2 has recently been purified from canine cardiac tissue and shown to be specific for plasmalogen phospholipids; this latter enzyme may be responsible for arachidonate released during myocardial ischemia (Hazen et al., 1990). ~3 Pharmacological agents which nonspecifically elicit rostaglandin formation in virtually all cells include the Ca 2+ ionophore A23187, tumor-promoting phorbol esters (e.g. PMA), and arachidonic acid itself. A23187 is thought to nonspecifically activate cellular phospholipases by increasing the [Ca 2÷] in cells; resting intraceilular [Ca 2+ ] are typically 50-100 h i , and virtually all phospholipases operate optimally at higher [Ca:+]. It is important to note that the patterns of phospholipid turnover observed with A23187 are different from those seen with physiological agonists (Rittenhouse-Simmons, 1981; Majerus et al., 1985). PMA apparently activates some phospholipase activities either directly or indirectly via proteins kinase C (PKC)-dependent phosphorylation (Parker et al., 1987; Gronich et al., 1988). In some cases the pattern of glycerophospholipid degra-

dation elicited by PMA may be different from that observed with physiological stimuli (Daniel et al., 1981). Arachidonate itself when used at concentrations of >I 10 gM probably acts as a detergent to permeabilize cells sufficiently so that the exogenous arachidonate becomes accessible to the intracellular P G H synthase. Exogenous arachidonate applied at pharmacological concentrations can elicit prostaglandin synthesis, but there are instances apparently involving normal cell-cell communication in which arachidonate is released by one cell type and is used as a substrate for prostanoid formation by another, neighboring cell type. Specifically, T-lymphocytes can provide arachidonate for macrophage thromboxane synthesis (Goldyne and Stobo, 1983), and adipocytes can supply arachidonate to neighboring endothelial cells which form PGI 2 (Parker et al., 1989). In addition, arachidonate can be delivered to at least some cells via the cholesterol esters or phospholipids of low density lipoprotein (LDL) which are endocytosed, hydrolyzed, and used as prostanoid precursors (Habenicht et al., 1990). 3.3. SOURCEOF ARACHIDONATEFOR PROSTANOID SYNTHESIS

The general question of which pathway(s) is involved in stimulus-induced arachidonate mobilization is unresolved. The information available to date comes from a combination of analyses of stimulusinduced phospholipid turnover (e.g. Bettazzoli et aL, 1990) and characterization of phospholipase activities (e.g. Dennis, 1987). There is not a very complete set of information for any one cell type. However, it is clear that arachidonate can be mobilized from at least two phospholipid classes, phosphatidylinositol (Ptdlns) and phosphatidylcholine (PtdCho), in response to physiological stimuli and that mobilization from these lipids can involve two different pathways (Fig. 3). PI can be cleaved to yield arachidonate by the sequential actions of a Ptdlns-specific phospholipase C, diacylglycerol lipase, and monoacylglycerol lipase (or perhaps alternatively by a phospholipase A2); PtdCho can be cleaved through the action of phospholipase A2. The relative contributions of each pathway probably varies among different cell types. The current trend is to think that the phospholipasc A2 pathway is quantitatively the most important (Dennis, 1987; Bettazzoli et aL, 1990). The cell which has been studied in the most detail with regard to arachidonate release is the human platelet. 3.4. PLATELETARACHIDONATERELEASE

In platelets, arachidonate appears to be mobilized to roughly the same extent from phosphatidylinositol and phosphatidylcholine. However, it is not known whether one of these phospholipids provides arachidonate preferentially for the formation of 12-HETE (formed via 12-1ipoxygenase action) and one phospholipid provides arachidonate for PGH2 formation or whether there is normally a mixing of the pools. It is known that inhibition of P G H synthase by aspirin increases 12-HETE production indicating that arachidonate mobilized for prostanoid production

Prostaglandin and thromboxane biosynthesis

157

A. PHOSPHOLIPASE C PATHWAY: _

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B. PHOSPHOLIPASE A 2 PATHWAY: O R' (ARACI-m~C ACID) O II II (13I20C~ PHOSPI-IOLIPASEA2: a'CO-~H II + O CH2- O-~-o-CH2 CH2 N(CH3)3 OH Ho-~ II + CH2- O-~-O-CH2 CH2 N(CH3)3 PtdCho OH FIG. 3. Two major pathways for the mobilization of arachidonate from phosphatidylinositol and phosphatidylcholine. can be routed to the lipoxygenase pathway under pharmacological conditions (Hamberg et al., 1974). I Platelets contain about 100 nmol of arachidonate per 109 cells virtually all of which is in an esterified fbrm (Marcus et al., 1969; Bills et al., 1977). Following stimulation with an agonist such as thrombin, 5-15 nmol of free arachidonate are released within about 1 rain (Bills et al., 1977; Neufeld and Majerus, 1983). Thrombin-induced arachidonate release is selective; for example, there is no appreciable release of either oleate or linoleate (Bills et al., 1977). Of the total arachidonate found in platelets, approximately 45% is present in phosphatidylethanolamine (PE), 25% in phosphatidylcholine (PC), 14% in phosphatidylserine (PS), and 12% in PtdIns; of the total fatty acid esterified at the sn2 position, arachidonate comprises about 60% of that in PtdEtn, 25% in PtdCho, 45% in PtdSer and 85% in PtdIns. There is considerable circumstantial evidence suggesting that up to half of the arachidonate released when platelets are treated with thrombin comes from phosphatidylinositol in a process initiated by a PtdIns-specific phospholipase C (Rittenhouse-Simmons, 1979; Neufeld and Majerus, 1983). When platelets are stimulated with thrombin,

there is a rapid, transient increase in the levels of diacylglycerol peaking 30 sec after thrombin addition and corresponding to about 0.6 nmol of diacylglycerol/109 cells (Rittenhouse-Simmons, 1979). During this period, there is also a dramatic decrease in phosphatidylinositol levels corresponding to about 10nmol/109 cells (Bell and Majerus, 1980). Thrombin-induced turnover of PtdIns in human platelets fails to show a temporal correlation with phosphatidic acid turnover (Neufeld and Majerus, 1983); this latter result indicates that release of arachidonate comes via formation and hydrolysis of diacylglycerol and does not require the intermediate formation of phosphatidic acid. Degradation of phosphatidylinositol can provide a maximum of about 50% of the arachidonate mobilized when platelets are stimulated with thrombin. When cells are prelabeled with [3H] or [t4C]arachidonate, the released arachidonate has a specific activity intermediate between that found in PtdCho and PtdIns but much lower than that of PtdEtn (Neufeld and Majerus, 1983); these results suggest that arachidonate probably comes from PtdCho as well as PtdIns, but probably not from PtdEtn. Bills et al. (1977) reached a similar conclusion using cells prelabeled with radiolabeled

158

W.L. SmTn et al.

arachidonate. As in most cell types that have been studied, radiolabeled arachidonate incorporated into platelet membranes during short term labeling resides primarily in PtdCho and Ptdlns (Bills et al., 1976).

soluble enzyme, which reportedly represents most of the total soluble platelet PLA2 activity, has several properties expected for an enzyme which could be activated intracellularly upon platelet activation and resulting Ca 2+ mobilization. Perhaps most 3.5. PLATELETPHOSPHOLIPASEC AND ACYLGLYCEROL importantly, this enzyme exhibits half maximal activation with 200-300rim free [Ca~+]; this is in a LIPASE ACTIVITIES range expected to be achieved intracellularly upon Mauco et al. (1979) were the first to describe platelet activation. Moreover, as noted above, the PtdIns-specific phospholipase C (PLC) activity in soluble PLA2 appears to undergo a Ca 2+-dependent human platelets. It is now clear that there are both interaction with platelet membranes. The soluble soluble and membrane-associated forms of the ac- PLA2 from sheep platelets has a subunit molecular tivity, and further, that within each subgroup, there weight of about 30 kDa, but exists as a dimer are multiple isoforms. One of the two PLC activities in solution (Loeb and Gross, 1986). Interestingly, present in platelet membranes has been purified to the soluble PLA 2 consists of a group of closelyhomogeneity (Mr=61,000) (Banno et al., 1988). related isoforms. These forms could conceivably Three soluble forms of the enzyme (Mr = 67,000- result from covalent modification of a parent 140,000) have been partially purified (Banno et al., enzyme, and this is of potential interest for two 1986; Low et al., 1986). All PLC activities require reasons. First, platelet arachidonate release as somewhat higher [Ca 2÷ ] for optimal PtdIns hydroly- well as arachidonate release from other cells is sis than for PtdlnsP2 hydrolysis. It is not known inhibited by cAMP (Minkes et al., 1977; Undem which, if any, of these enzymes are involved in the et al., 1990); thus, inhibition of PLA2 activity hydrolysis of phosphatidylinositol that occurs upon could result from PKA-mediated phosphorylation platelet activation. Thrombin activation of platelets of soluble PLA 2. Second, thrombin activation may involve a G protein coupled to phospholipase C may involve a PKC activation which could also (Banno et al., 1987; Crouch and Lapetina, 1988); but lead to phosphorylation of the enzyme. The soluble again, it is not clear whether this PLC is directly enzyme exhibits a 100-fold greater specificity involved in supplying arachidonate for eicosanoid toward the s n 2 position of 1-(O)-(Z)-dexadecenyl-2synthesis. It is important to note that the amounts of oleoyi-sn-glycero-3-phosphocholine than 1-palmiPtdlnsP and PtdlnsP2 in platelets are relatively in- toyl-2-oleoyl-sn-glycero-3-phosphocholine. That is, significant and could not account for even a fraction it appears to be specific for plasmenylcholines, which are typically enriched at the sn 2 position with of the arachidonate mobilized. Importantly, diacylglycerol lipase and monoacyi- arachidonate (Bettazzoli et al., 1990). The properties of the membrane-bound PLA2 glycerol lipases in human platelet membranes are sufficiently active to account for the generation of purified from human platelets by Kramer et al. 10nmol of arachidonate via breakdown of phos- (1989) are quite different from those of the soluble phatidylinositol following thrombin-induced platelet sheep platelet enzyme. The membrane-bound ctivation (Bell et al., 1979; Prescott and Majerus, enzyme has a molecular weight of about 14 kDa. It 983). A 2-arachidonoyl-glycerol intermediate was is released from platelets upon activation, and maxietected during hydrolysis of diacylglycerol by mal activity requires relatively high [Ca 2+ ] (ca. 10mi). The sequence of the membrane-associated platelet membrane preparations suggesting that the pathway for the mobilization of arachidonate PLA2 has been deduced, and the protein is found to from Ptdlns involves a PLC-mediated cleavage of have a primary structure related to the snake venom Ptdlns to yield diacylglycerol, a diacylglycerol lipase- group II PLA2s (Davidson and Dennis, 1990; Kramer mediated hydrolysis of 1,2-diglyceride to yield a et al., 1989). The mechanism by which extracellular stimuli 2-monoacylglycerol, and cleavage of the 2-acylglycerol by an acyiglycerol lipase to provide free activate platelet PLA 2 to provide arachidonate for prostaglandin endoperoxide formation is unarachidonate. resolved. It is possible that stimuli cause the activation of PLC, PtdInsP2 hydrolysis, and Ca 2+ 3.6. PLATELETPHOSPHOLIPASEA 2 ACTIVITIES mobilization (or simply influx of extracellular Ca 2+ Platelet phospholipase A 2 (PLA2) activities are (Brooks et al., 1989)), and that increases in intrapresent in both the soluble and membrane-associated cellular [Ca 2+] cause soluble PLA 2 to associate fractions of platelet extracts (Kramer et al., 1986). with membranes and hydrolyze PtdCho to liberate When platelets are homogenized in Ca2+-free iso- arachidonate. Alternatively (or additionally) stimuli tonic buffer, two-thirds of the enzyme is found in may activate membrane-associated PLA2 with no the soluble fraction; however, about half of this requisite translocation. In any event, there is good soluble activity becomes membrane-bound when evidence for G protein involvement in PLA2 actiplatelets are homogenized in the presence of 1 mM vation (Murayama et al., 1990; Silk et al., 1989). Ca 2+ (Kramer et al., 1986). Thus, at least a part of Stimulation of PLA2 activity by thrombin is inhibited by pretreatment of permeabilized platelets with the platelet PLA 2 is apparently capable of undergoing a Ca2+-dependent, reversible association with the pertussis toxin (Murayama et al., 1990). Stimulation of PtdCho hydrolysis by platelet membrane membrane. PLA: activities have now been purified from both preparations by GTP analogs does not appear the soluble (Loeb and Gross, 1986) and membrane to involve the intermediate activation of PLC (Kramer et al., 1989) fractions of platelets. The (Silk et al., 1989).

Prostaglandin and thromboxane biosynthesis 3.7. GENERALPROPERTIESOF STIMULUS-INDUCED

PHOSPHOLIPIDTURNOVER Studies on phospholipid turnover associated with prostaglandin formation induced by physiological stimuli in a variety cell types have generally shown the same patterns seen in platelets. Typically, PtdIns and Ptdeho are readily labeled with arachidonate, radioactive arachidonate is rapidly mobilized upon application of a stimulus, and arachidonate release is relatively selective (Hong and Deykin, 1979, 1981; Majerus et aL, 1985). A complicating issue occurs when the cell being examined synthesizes eicosanoids other than prostanoids. For example, many blood ceils and blood cell-derived lines form both leukotrienes and prostanoids. It appears that the arachidonate used to synthesize these two groups of compounds often comes from different precursor pools (Hsueh et al., 1981; Humes et al., 1982; Wightman and DaUob, 1990). Many of the phospholipase activities that have been studied are from macrophage-derived lines. In examining these phospholipases, it is unclear whether certain phospholipases are involved in leukotriene synthesis and others in prostanoid synthesis. 3.8. PHOSPHOLIPASEA2 ACTIVITIES A number of different phospholipase A2 activities have been identified in cell types other than platelets. Of particular note are the soluble phospholipase A2 from the RAW 264.7 macrophage-like cell line (Channon and Leslie, 1990; Leslie et aL, !988) and the membrane-associated PLA 2 from the P388DI macrophage-like line (Dennis, 1987; Lister et al., 1988, 1989; Ross et aL, 1985; Ulevitch et aL, 1988). When RAW 264.7 cells are homogenized in the absence of Ca 2+, about 95% of the activity is in the soluble fraction (Leslie et aL, 1988). This soluble PLA 2 of RAW 264.7 cells has been purified to apparent homogeneity (Channon and Leslie, 1990). It has an alkaline pH optimum and a molecular weight of 60 kDa. The enzyme will act on PtdCho, PtdEtn, and PtdIns, and it exhibits some selectivity for substrates having arachidonatc at the sn 2 position. This soluble PLA 2 is active even at low free [Ca2+] (i.e. 100riM) but more active at higher [Ca2+]. This enzyme has many of the properties of the soluble platelet PLA2 and may also be similar to, or the same as, the P L A 2 activities present in the soluble fractions of P3881 cells (Ross et aL, 1985), mesangiai cells (Gronich et al., 1988), and differentiated premacrophage lines (Suga et al., 1990; Diez and Mong, 1990). In contrast to RAW 264.7 cells, only about one quarter of the total PLA 2 activity of P388D~ cells is found in the soluble fraction (Ross et aL, 1985); again, this activity may be related to the soluble PLA2 of RAW 264.7 cells. In addition to the soluble enzyme, P388D~ cells express two other PLA2 activities which have acidic pH optima and appear to be associated with lysosomes. Finally, a fourth PLA2 activity, which is membraneassociated, Ca2+-dependent, and has an alkaline pH optimum (pH 8.8) has been resolved from

159

other phospholipases and purified about 2500-fold from P388D I cells (Ulevitch et al., 1988); it has a molecular weight of 18 kDa; half-maximal activity is observed with 1.5raM Ca 2+. These properties are similar to those of the membrane-bound PLA 2 of platelets (Kramer et ai., 1989). The purified, membrane-bound PLA2 activity of P388Dt cells shows no apparent specificity for the polar head group and will hydrolyze both 1-O-alkyl,2-acyland diacyl-phosphatidylcholine derivatives at approximately equal rates (Lister et al., 1988, 1989). Interestingly, the enzyme is competitively and selectively inhibited by arachidonate and other unsaturated fatty acids (Lister et al., 1988). Importantly, the enzyme is inhibited by manoalide and its analogs (Deems et al., 1987) and 7,7-dimethyl-5,8-eicosadienoic acid but not p-bromopbenacyl bromide; and there is a good correlation between the ability of these compounds to inhibit (a) the membrane-associated PLA2 and (b) stimulus-induced PGE2 formation by P388Dt cells (Lister et al., 1989). Unfortunately, similar inhibition studies have not been reported for the soluble PLA2. The regulation of phospholipase A2 activity of P388D~ is quite complex. Arachidonate release in these cells is controlled at the level of both transcription and translation (Glaser et al., 1990); moreover, stimulus-induced prostanoid synthesis by these cells is inhibited by a tyrosine-protein kinase inhibitor. Recently, an activated PLA2 activity has been described in rat mcsangial cells (Gronich et al., 1988). Following pretreatment of mesangial cells with arginine vasopressin (AVP) or PMA, cell free extracts of mesangial cells were found to have a three-fold increase in PLA 2 activity assayed by measuring hydrolysis of exogenous PtdCho. This enzyme activity was partially purified and found to have an apparent molecular weight of about 60 kDa, an alkaline pH optimum, and a requirement for Ca 2+. The activated PLA2 appears to be a soluble enzyme which becomes reversibly associated with the membrane in the presence of Ca 2+ . This enzyme from mesangial cells, which apparently undergoes a PKCmediated covalent modification, may be the same as the soluble PLA2 from platelets (Loeb and Gross, 1986), RAW 264.7 cells (Channon and Leslie, 1990), and premacrophage lines (Suga et al., 1990; Diez and Mong, 1990).

3.9. GLUCOCORTICOIDSAND PHOSPHOLIPASE INHIBITION At one time, it was widely accepted that a n t i inflammatory steroids induced the synthesis of lipocortin I which inhibits PLA 2. More recent studies suggest that human recombinant lipocortin I and bovine lung calpactin I (lipocortin II) bind PtdCho vesicles. Apparently they cause inhibition of the PLA 2 by lowering the substrate concentration (substrate depletion) rather than interacting with the enzyme itself (Dennis, 1987; Davidson et al., 1990). As discussed in further detail below, evidence is accumulating that antiinflammatory steroids inhibit the expression of PGH synthase.

160

W.L. SMITHet al. 4. PROSTAGLANDIN ENDOPEROXIDE FORMATION

Arachidonate and related fatty acids are converted to prostaglandin endoperoxides by the action of prostaglandin endoperoxide (PGH) synthase [E.C.I.14.99.1] (Fig. 2) (reviewed by Marnett and Maddipati, 1990; Smith and Marnett, 1991; and DeWitt, 1991). PGH synthase is subject to regulation at both the metabolic and genetic levels. We first describe the catalytic properties of the enzyme and its physical characteristics insofar as possible relating structure to catalysis. We then discuss what is currently known about the regulation of PGH synthase gene expression. 4.1. PGH SYNT~ASECATALYSIS----ANOVERVIEW PGH synthase exhibits both a bis-oxygenase (cyclooxygenase) activity catalyzing PGG2 formation from arachidonate and a peroxidase activity which reduces the 15-hydroperoxyl group of PGG 2 to PGH2 (Fig. 2). The cyclooxygenase and hydroperoxidase activities are associated with the same protein (Miyamoto et al., 1976; van der Ouderaa et al., 1977; Pagels et al., 1983); the two activities occur at distinct albeit interacting sites on the PGH synthase molecule. For example, binding of arachidonate to the cycloxygenase site can be competitively inhibited by ocosahexaenoic acid (Marshall and Kulmacz, 1988) and by aspirin and other nonsteroidal anti-inflammatory agents (van der Ouderaa et al., 1980; Mizuno et al., 1982) without affecting peroxidase activity; moreover, a PGH synthase mutant containing phenylalanine in place of Tyr385 exhibits peroxidase but not cyclooxygenase activity (Shimokawa et al., 1990). Importantly, both cyclooxygenase and peroxidase activities require heme (van der Ouderaa et al., 1977; Kulmacz and Lands, 1984; Roth et al., 1981; Karthein et al., 1987; Lambeir et al., 1985).

~

If sufficient enzyme is available (>/1 #g), cyclooxygenase activity is most accurately assayed by measuring arachidonate-dependent 02 uptake with an oxygen electrode (e.g. DeWitt et al., 1981). With smaller amounts of enzyme, the activity can be measured using radioactive arachidonate (DeWitt et al., 1983). Negative controls employ the use of readily available cyclooxygenase inhibitors (e.g. aspirin or indomethacin). Peroxidase activity is easily measured using H202 and guaiacol as substrates monitoring the change in absorbance at 426 nm due to the oxidation of guaiacol (Marnett et al., 1988). 4.2. CYCLOOXYGENASE CATALYSIS

The substrates for the cyclooxygenase are two molecules of oxygen and one molecule of fatty acid. The K~ for 02 is about 5/~M (Lands et al., 1978). The best fatty acid substrates are twenty carbon co-6 polyunsaturated fatty acids including all cis-5,8,11, 14-eicosatetraenoate (arachidonate) and 8,11,14eicosatrienate (?-homo-linolenate) (Hamberg and Samuelsson, 1967b; Marshall et al., 1987; Odenwaller et al., 1990); with arachidonate, the Km is about 5 #M, and the V~naxis about 1400 molecules arachidonate oxygenated/min/subunit. The cyclooxygenase will also utilize 5,8,11,14,17-eicosapentaenoic acid--the so-called fish oil fatty acid--but the rate of catalysis is relatively low (Culp et al., 1979; Lands and Byrnes, 1981). This may account in part for the putative beneficial effects of including fish oils in the diet (e.g. Yamamoto et al., 1987). The cyclooxygenase will also catalyze the monooxygenation of l l,14-eicosadienoate converting it to 11-hydroperoxy- 12-trans14-cis-eicosadienoate (ll-HpEDE) (Hemler et al.. 1978). Cyclooxygenase catalysis involves an initial activation of substrate fatty acid followed by oxygen insertion, carbon skeleton rearrangement and a second oxygen insertion (Fig. 4) (Hamberg and

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Prostaglandin and thromboxane biosynthesis

Samuelsson, 1967a; Marnett and Maddipati, 1990). A major role of the cyclooxygenase is to position the

161

rapidly. Thus, the net pharmacological effect of ingesting low doses of aspirin is to selectively inactifatty acid substrate with a kink involving rotation vate TxA2 formation without appreciably affecting about the C-9/C-10 bond (Appleton and Brown, PGI2 formation. This is the biochemical basis for the 1979). In the initial step, removal of the 13-pro-S use of aspirin to prevent unstable angina (which hydrogen from arachidonate produces a carbon results from platelet aggregation) (Lewis et aL, 1983; radical which is trapped by 02 at C-11. O2 present Cairns et al., 1985). There is also a growing body of in the aqueous phase likely attacks from the side evidence that aspirin can be used prophylactically to opposite H abstraction. Serial cyclization of the diminish cardiovascular pathologies including myol l-hydroperoxyl radical yields a bicyclic peroxide cardial infarction and stroke (Steering Committee of with trans aliphatic chains. A second O5 molecule the Physicians Health Study Research Group, 1988; then reacts with a carbon radical at C-15. One American-Canadian Co-operative Study Group, electron reduction of the 15-hydroperoxyl radical 1985). yields P G G 2. There is no evidence that other nonsteroidal antiIn order for the cyclooxygenase to abstract the inflammatory drugs exhibit the anti-thrombogenic 13-pro-S hydrogen, it must be activated, a process effects of aspirin. Indomethacin, flurbiprofen, and which requires a peroxide (Smith and Lands, 1972; meclofenamate cause irreversible inactivation of Hemler et al., 1979; Hemler and Lands, 1980). The PGH synthase in vitro (Rome and Lands, 1975) but best peroxide activators are 15-HpETE and PGG2-- apparently not /n vivo (Fitzpatrick and Wynalda, which are also the best substrates for the peroxidase 1976). In the case of indomethacin, binding of the activity o f P G H synthase (Kulmacz and Lands, 1983; drug appears to cause a change in enzyme structure Ohki et al., 1979). Hydroperoxide activation is dis- which leads to tighter binding of indomethacin and cussed in detail below. retention of about 5% of the original cyclooxygenase activity (Kulmacz and Lands, 1985; Kuimacz, 1989). Other nonsteroidal anti-inflammatory drugs includ4.3. CYCLOOXYGENASEINHIBITION--NoNSTEROIDAL ing ibuprofen, naproxen, sulindac, and flufenamate ANTI-INFLAMMATORYDRUGS are simply reversible, competitive cyclooxygenase inThe cyclooxygenase is the target for the action of hibitors (Rome and Lands, 1975; Smith et al., 1990c); aspirin, indomethacin, ibuprofen and most other the rate of decay of their actions corresponds to their nonsteroidal anti-inflammatory drugs (Flower, 1974; rates of metabolism and clearance. Vane and Botting, 1987). These drugs interact with Cyclooxygenase activity is also inhibited by certain the cyclooxygenase active site competing with arachi- fatty acid analogs. Of note are all cis-5,8,11,14,17,20donate for binding. In the case of aspirin, the IDs0 is docosahexaenoic acid (Marshall and Kulmacz, 1988) about 20 mM in the presence of 100/~M arachidonate and 5,8,11,14-eicosatetraynoic acid (ETYA) (Ahem (DeWitt et al., 1990). However, an important sec- and Downing, 1970; Vanderhoek and Lands, 1973). ondary, time-dependent effect of aspirin is to cause Docosahexaenoic acid is a potent competitive inhibiacetylation of the side-chain hydroxyl group of tor of the cyclooxygenase activity with a K~ of 5 #M. Ser530 and concomitant irreversible cyclooxygenase ETYA causes irreversible inactivation of the enzyme (but not peroxidase) inactivation (Van der Ouderaa via a process which requires the presence of both et al., 1980; Mizuno et al., 1982; DeWitt and Smith, oxygen and a p~roxide activator (Vanderhoek and 1988); with 0.1 mM aspirin the half-life of the cyclo- Lands, 1973). Apparently, ETYA interacts with the oxygenase activity is about 30 min at 37°. Recent cyclooxygenase active site, is activated, and reacts studies of PGH synthase mutants with substitutions with the enzyme causing its irreversible inactivation. at Ser530 have established that the Ser530 hydroxyl group is not essential for catalysis and have suggested 4.4. PEROXlDASECATALYSIS that acetylation of this group simply places a bulky group at position 530 which interferes with arachiThe peroxidase activity of PGH synthase is capable donate binding (DeWitt et al., 1990). of catalyzing a net two electron reduction of a variety The irreversible inactivation of PGH synthase by of hydroperoxides utilizing an array of exogenous aspirin is of considerable pharmacological import- electron donors. As noted earlier, peroxidase activity, ance. Once the cyclooxygenase is inactivated, new like cyclooxygenase activity, requires heme (Fe 3÷activity can only occur through new enzyme syn- PPIX). A variety of primary and secondary alkyl thesis. Of particular note is the case of blood platelets. hydroperoxides (e.g. PGG 2 and 15-HpETE) are the Platelet cells cannot synthesize PGH synthase best oxidant substrates whereas H202 is somewhat whereas most other cells can form new enzyme. less active, and tertiary hydroperoxides such as Ingestion of about 70 mg of aspirin--equivalent to a cumene hydroperoxide are relatively inactive (Ohki et 'baby' aspirin tablet is sufficient to inactivate more al., 1979; Kulmacz and Lands, 1983). Presumably, than 95% of the platelet enzyme (FitzGerald et al., the major endogenous substrate is PGG2 generated 1983; Ciabattoni et al., 1987), and the cyclooxygenase by the cyclooxygenase. The peroxidase activity of activity is lost for the life of the platelet--about five PGH synthase resembles other peroxidases in being days. Platelets form TxA2 from PGH2, and TxA2 is a somewhat indiscriminant in its specificity toward prothrombogenic substance. Vascular endothelial electron donors. The endogenous electron donor is cells, on the other hand, form PGI2, an anti-thrombo- not known; however, among naturally occurring genie agent; endothelial cells can synthesize new PGH compounds, epinephrine and uric acid are the most synthase following aspirin treatment, and thus, can probable candidates (Ogino et al., 1979; Markey regenerate their capacity to synthesize PGH2 quite et al., 1987). Uric acid is an inefficient reductant, but

162

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FIG. 5. Model for the mechanism of the peroxidase reaction of PGH synthase. Adapted from Smith and Marnett ( 1991). it is present at relatively high concentrations in plasma (ca. 300/~M) (Ames et al., 1981). The peroxidase of PGH synthase exhibits many ~pectral and catalytic properties of typical hemedependent peroxidases including catalase, cytochrome c peroxidase, and horseradish peroxidase (Dunford and Stillman, 1976; Chance et al., 1984; Yamazaki, 1974). Studies with 10-hydroperoxy-8,12octadecadienoate indicate that the hydroperoxide is reduced by two electrons simultaneously as opposed to in two sequential one electron steps (Markey et al., 1987; Marnett et al., 1988). Consistent with this observation is the finding that incubation of PGH synthase with PGG 2 or 15-HpETE produces a spectral Intermediate I very similar to that of Compound I of horseradish peroxidase (Lambeir et al., 1985). Compound I is a two electron-oxidized heme in which iron is in the + 4 state and the porphyrin ring is oxidized to a radical cation (Fig. 5) (Dolphin and Felton, 1974). When Intermediate I of PGH synthase is allowed to stand for about 100 msec the spectrum changes to one which is virtually identical to that of Compound II of cytochrome c peroxidase (Lambeir et al., 1985). Compound II is a rearranged form of compound I in which iron is in the + 4 state, the porphyrin is neutral, and a protein group has undergone a one electron oxidation (Fig. 5) (Yonetani, 1976). Interestingly, a protein-associated tyrosyl radical is generated within the PGH synthase molecule at the same time that Intermediate I rearranges to form Intermediate II (Karthein et al., 1988). Apparently, the tyrosyl radical is formed by transfer of an electron from a tyrosine to the porphyrin radical cation of Intermediate I. As discussed below, this tyrosine radical may be the active enzyme species involved in the abstraction of the 13-pro-S hydrogen by the cyclooxygenase activity of PGH synthase (Dietz e t a / . , 1988). 4.5. PEROXIDASE-DEPENDENTCOOXIDATIONOF

REDUCINGCOSUBSTRATES Typically, reducing cosubstrates donate a single electron to one of the peroxidase iron-oxo intermediates (e.g. Intermediate I) of PGH synthase (Marnett and Maddipati, 1990). The cosubstrate radical that is produced then undergoes secondary chemical alterations governed by the solution chemistry of the radical (Marnett and Eling, 1983; PaceAsciak and Smith, 1983). For example, epinephrine

undergoes a net dehydrogenation which probably involves a disproportionation reaction involving two epinephrine radicals to produce one epinephrine and one oxidized epinephrine molecule (Ohki et al., 1979). Other cosubstrates such as phenylbutazone are oxidized to radicals which then react with molecular oxygen to form peroxyl radicals (Marnett et al., 1980). In the case of phenylbutazone, the 4-peroxyl radical becomes reduced via an unknown mechanism to 4-hydroxy-phenylbutazone. An unusual group of reducing cosubstrates are the alkylaryl sulfides such as sulindac sulfide which are oxygenated to sulfoxides during the peroxidase reaction (Egan et al., 1980; Pie and Marnett, 1989). ~SO-labeling studies indicate that the oxygen incorporated into the sulfoxide is derived from the hydroperoxide (Egan et al., 1980). This suggests that the sulfide reacts with the oxo-ligand of Intermediate I. Electron transfer either precedes or is concomitant with oxygen transfer. Studies with a series of alkylaryl sulfides of different sizes have suggested that the heme binding pocket of PGH synthase is intermediate in size between those of horseradish peroxidase and cytochrome P-450 (Pie and Marnett, 1989). A final set of interesting oxygenations and cooxidations are those observed when the peroxidase reaction occurs in the presence of certain toxins and carcinogens (Marnett and Maddipati, 1990). Included in this group of compounds are polycyclic hydrocarbons such as benzo[a]pyrene which themselves are not peroxidase cosubstrates. Reactive oxidized species generated during peroxidase catalysis presumably initiate free radical chain reactions which lead to epoxidation or oxidation of these toxins and carcinogens. Several recent reviews are available on peroxidase-dependent cooxidation reactions (Eling et al., 1990; Marnett and Maddipati, 1990). 4.6. INTERDEPENDENCEOF CYCLOOXYGENASEAND

PEROXIDASEREACTIONS Shown in Fig. 6 is a model for the interrelationship between the cyclooxygenase and peroxidase reactions proposed by Ruf and his coworkers (Karthein et al., 1988; Dietz et al., 1988). The peroxidase reaction is envisioned as generating a Compound I-like species which, in turn, rearranges to a Compound II-like species in which there is an iron-oxo intermediate with iron in the + 4 state, a neutral porphyrin, and a tyrosyl radical. The tyrosyl radical is proposed to interact with arachidonate to remove the 13-pro-S hydrogen and to generate a carbon-centered substrate radical at C-11 of arachidonate which can react with the first oxygen molecule. This is followed by rearrangement and a second oxygen addition to form PGG: and to regenerate the tyrosyl radical. In principle, one can imagine that the cyclooxygenase reaction once initiated by the tyrosine oxidation reaction could occur in the presence of an ongoing peroxidase reaction. The Ruf model is attractive because it provides an explanation for (a) the need for a hydroperoxide activator for the cyclooxygenase; (b) the similar substrate specificities of the peroxidase reaction and the peroxide activator; (c) the dependence of both cyclooxygenase and peroxidase activities on heme yet the apparent need for only one heine

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4.7. SUICIDE REACTIONS

In a typical cyclooxygenase assay in which 1-1/~g of enzyme is tested in the presence of 100 #M arachidonate and 200/~i 02, it is observed that the rate of 02 consumption drops to near zero within 5 min and long before significant amounts of arachidonate or 02 are consumed (Smith and Lands, 1972; Hemler et aL, 1979). This process is known as the 'suicide' inactivation of cyclooxygenase, and it apparently occurs because an unstable protein intermediate formed during cyclooxygenase catalysis has a certain probability of rearranging to an inactive enzyme species. On average this occurs about once in every 1300 turnovers (Marshall et al., 1987). The chemical modification which causes the suicide inactivation has not been defined. Importantly, the suicide inactivation appears to occur in vivo as well as in vitro (Lapetina and Cuatrecasas, 1979; Kent et aL, 1983). In addition to the suicide reaction, cyclooxygenase and peroxidase activities can be inactivated by hydroperoxides or hydrogen peroxide (Hemler and Lands,

1980; Kulmacz, 1986; Chen et aL, 1987; Markey et aL, 1987). Loss of peroxidase activity is correlated with loss of cyclooxygenase activity, and this type of inactivation involves changes in the heme spectrum of the enzyme. Presumably, peroxide-dependent inactivation involves a modification of the heme group.

4.8. PHYSICO-CHEMICAL PROPERTIES OF

PGH

SYNTHASE PGH synthase is an integral membrane glycoprotein having an associated heme group. The enzyme can only be solubilized with detergents, and in detergent solution the protein exists as a homodimer. PGH synthase has been purified from ovine (Hemler et al., 1976; van der Ouderaa et al., 1977) and bovine vesicular glands (Miyamoto et al., 1976). The protein has a subunit molecular weight of 72 kDa as determined by its mobility on SDS-PAGE. The molecular weight determined from the deduced amino acid sequences of cDNAs for the sheep (DeWitt and Smith, 1988; Merlie et aL, 1988; Yokoyama et aL, 1988), mouse (DeWitt et aL, 1990), and human (Yokoyama and Tanabe, 1989) enzymes is about 65.5kDa. The difference between the molecular weight determined empirically and that deduced from the protein sequence can be attributed to the presence of two to three high mannose carbohydrate sidechains per subunit (van der Ouderaa et al., 1977; Mutsaers et al., 1985). There are four consensus sites (Asn-X-Ser/Thr) for N-glycosylation including Ash68, Asnl04, Asn144, and Ash410, and there is evidence for glycosylation at three of these sites (Ash68, Asn144, and Ash410) (Marnett, Bienkowski and Chen, unpublished observations). The function of the attached carbohydrate chains has not yet been determined.

4.9. PGH SYNTHASEAMINOACIDSEQUENCE Figure 7 shows the deduced amino acid sequences of the sheep, mouse, and human PGH synthases

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FI~. 7. Deduced amino acid sequences of sheep, mouse, and human PGH synthases. Identical residues are marked with a colon (:); residues involving a one-letter change in the codon (i.e. conservative substitutions) are marked with a period (.); noneonservativechanges involving two- or three-letter changes in the codon are unmarked. Alignments were performed using the FASTP sequencecomparison program. Reprinted from Smith et al. (1990b) with permission of the copyright holder, Raven Press, New York. (Smith et aL, 1990a)*. All have a signal peptide containing 24-26 amino acids. The N-terminus of the mature sheep enzyme (and presumably the mouse and human enzymes) begins with the sequence AlaAspProGlyAlaPro . . . (DeWitt and Smith, 1988) so that with each enzyme sequenced to date, the mature enzyme contains 576 amino acids. 4.10. HEMEBINDINGSITE The current consensus is that there is one heme bound per mole of PGH synthase subunit although *The numbering system used for amino acid residues assigns the number 1 to the methionine at the translational start signal deduced from the eDNA sequence.

values between 0.5 and 2.0 have been reporte (Hemler et al., 1976; Miyamoto et aL, 1976; Rot et al., 1981; van der Ouderaa et al., 1979; R u f e t a~ 1984; Kulmacz and Lands, 1984), The heine stoich ometry has been difficult to determine because c the presence of inactive protein in purified enzyrr preparations and the presence of a nonspecific herr binding site on the enzyme. Heine is only weak] associated with the enzyme (K~= 10-7M), and i assaying the enzyme, exogenous heme in the form c hemin or hemoglobin are added. Both visible absorl tion and epr spectra of PGH synthase suggest th~ heme is bound at both the axial and distal positiot by histidine residues (Lambeir et al., 1985; Kulmac et al., 1987). Although the histidine residues involve in heme binding have not been determined wi!

Prostaglandin and thromboxane biosynthesis

165

XIDE)

FIG. 8. Model for the cyclooxygenase and peroxidase active sites of PGH synthase. Adapted from Smith et al. (1990b). certainty, it is quite likely that His309 is the axial heme ligand (DeWitt et al., 1990; Smith et al., 1990a) and that either His207 or His388 is the distal heme ligand (Shimokawa and Smith, unpublished results). His309 is found in a decameric consensus sequence T I W L R E H N R V (residues 303-312 of the sheep enzyme) present in other mammalian heme peroxidases (Kimura and Ikeda-Saito, 1988); and His309, His207, His388 have all been determined to be essential for both cyclooxygenase and peroxidase activities as determined by site-directed mutagenesis (Shimokawa and Smith, unpublished results). 4.11. ACTIVESITE MODELOF PGH SYNTHASE Depicted in Fig. 8 is a model for the two active sites of PGH synthase (Smith et al., 1990b). A hydroperoxide is envisioned as binding at the peroxidase active site. The heine at the peroxidase site is shown liganded via His309 at the axial position and another histidine (probably His388) at the distal position. Two electron reduction of the peroxide to an alcohol leads to concomitant formation of an Intermediate I which, in turn, rearranges to an Intermediate II with an associated Tyr385 radical. This radical is thought to abstract the 13-pro-S hydrogen atom from arachidonate bound at the cyclooxygenase active site thereby generating a carbon radical at C-11 which reacts with 02. Ser530, which is the site of acetylation by aspirin, is shown neighboring the cyclooxygenase active site. As noted above, acetylation of this residue places a bulky group in the enzyme which inhibits arachidonate binding. 4.12. REGULATIONOF PGH SYNTHASEPROTEIN CONCENTRATIONS Prostaglandin synthesis is regulated acutely at the level of arachidonate suhstrate mobilization, but, of course, prostanoid formation further requires that active PGH synthase be available. There is now abundant evidence that PGH synthase levels vary considerably among different cell types and that the amount o f enzyme present, even in cells which normally form prostaglandins, can fluctuate in response to humoral agents. Table 1 summarizes the

results of some of the studies of PGH synthase in cells and tissues in which various effectors are known to increase overall prostaglandin biosynthetic activity. Figure 9 attempts to localize the effects of various agents which modulate PGH synthase protein levels. Increases in cellular prostaglandin biosynthetic activity are typically accompanied by increases in PGH synthase activity, PGH synthase protein levels, and/or PGH synthase mRNA levels. However, it is important to point out that only with 3T3 cells (Habenicht et al., 1985; Lin et aL, 1989; DeWitt, Meade and Kraemer, unpublished results) have all these parameters been studied, but that even in the case of 3T3 ceils, the rate of gene transcription has not actually been measured directly. While work on the regulation of PGH synthase is in its infancy, regulation of PGH synthase expression can be viewed as falling into three general categories. The first is developmental regulation. Immunohistochemical studies of PGH synthase localization have indicated that only about 15% of all cells have detectable PGH synthase (Smith, 1987); thus, despite the fact that prostanoids are known to be formed by virtually all mammalian tissues and organs, these compounds are not formed by all cell types within an organ. Clearly, then PGH synthase must be developmentally regulated. Furthermore, cells such as ovarian preovulatory follicles express PGH synthase only during certain stages of the estrous cycle (Hedin et al., 1987) and promonocyte lines such as HL60 (Goerig et al., 1987) and U937 cells (Koehler et al., 1990b) express PGH synthase only following differentiation to monocyte/macrophages. As discussed below, these systems have provided models for examining the developmental expression of PGH synthase. Another, apparently different level of regulation of PGH synthase expression, which can be termed 'tuning', occurs in murine 3T3 fibroblasts and vascular endothelial cells which normally contain relatively high levels of enzyme. In these cells, challenge with P D G F or IL-1 causes PGH synthase activity to increase even further. Increases in PGH synthase activity are accompanied by increases in PGH synthase mRNA. And finally, there appears to be an intracellular regulation of PGH synthase expression. As discussed

-

-

-

+ NR NR + NR + ND +

+ + + + + + + + -

+

+ + + + NR + + NR + + NR NR + +

PGHS activity

+

+ + + + + + NE + _ + NR NR NR NE

PG synthesis

- :[:

-

NR + + + NR + ND ND ND

+

NE* + NR + l + + NR + + + NR + + NR

PGHS protein

NR NR NR NR NR NR ND ND ND ND

NR

+ + NR NR + NR NR NR NR NR + + NR

PGHS mRNA

Bailey et al., 1985 Frasier-Scott et al., 1988 Frasier-Scott et al., 1988 Y a m a m o t o et al., 1989 Burch et al., 1986a,b Sumitani et al., 1989 Bienkowski et al., 1989 R a z et al., 1990 R a z et al., 1990 Koehler et al., 1990a R a z et al., 1989

Habenicht et al., 1985; Lin et al., 1989 Simonson et al., 1990 Burch et aL, 1988 Raz et al., 1990, 1989, 1988 Maier et al., 1990; Rossi et al., 1985 R a z et al., 1990 Zakar and Olson, 1988b W u et al., 1988 Goerig et al., 1987; Habenicht et al., 1985 Koehler et al., 1990b Lin et al., 1989 W o n g et al., 1989; Hedin et al., 1987 Eggleston et al., 1990; Huslig et al., 1979 Casey et al., 1987; Z a k k a r and Olson, 1988a; Casey et al., 1988 Yokota et al., 1986

Reference

-- § *NE, has no effect; N R , was not reported; ( + ) , increases synthesis, activity or expression; ( - ), decreases synthesis activity or expression. tlncreased 35S-methionine labeling, but protein levels were not determined. :~Decreased 3SS-methionine labeling, but protein levels were not determined. §Decreased in vitro translatable m R N A levels, total P G H synthase m R N A levels were not determined.

Dexamethasone

TGFfl LPS

Epinephrine

1L-2

LH Progesterone EGF

PMA

MC3T3-EI bovine smooth muscle cells UVECS BAECS MC3T3-E 1 FRTL-5 MC3T3-E 1 U937 h u m a n monocytes h u m a n monocytes U937 dermal fibro.

3T3 cells mesangial SV-T2 3T3 dermal fib. HUVECS dermal fibro. amnion HUVECS HL-60 U937 3T3 cells PO follicles uterus amnion

PDGF/serum

IL-1

Cell system

Effector

TABLE 1. Factors which Affect P G H Synthase Expression In Vitro

t~

Prostaglandin and thromboxane biosynthesis

Postlve Regulatory Agents

r

I£-1 cAMP UPS

SYNTHASE GENE transcription

PGH

Negative I Regulatory Elements

167

LH(cAMP) PGH SYNTHASE mRNA

\._L

SYNTHASE

~'~ PGH translation PROTEIN (degradation) I[

) (?)

JI ~"

Inactivation proteolysis

+

FiG. 9. Regulation of PGH synthase expression. Likely sites of action of factors which modulate PGH synthase protein levels. above, losses of PGH synthase activity occur as a result of both suicide inactivation (Egan et al., 1976; Hemler and Lands, 1980; Kent et al., 1983; Lapetina and Cuatrecasas, 1979; Smith and Lands, 1972) and aspirin therapy (Smith and Marnett, 1991). Accordingly, cells must have some mechanism for monitoring and responding to the presence of inactive PGH synthase molecules. 4.13. DEVELOPMENTALREGULATIONOF PGH

SYNTHASE Developmental regulation has been studied during the maturation of ovarian follicles and the transformation of HL60 and U937 cells to monocyte/ macrophages. Treatment of rats with hCG (Hedin et al., 1987) or preovulatory follicles with LH, FSH, or forskolin (Wong et al., 1989; Hedin et al., 1987) causes an increase in the PGH synthase activity of the follicles and the attendant thecal and granulosa cells. Increases in enzyme activity are correlated temporally with substantial increases in PGH synthase protein. Surprisingly, PGH synthase mRNA levels decrease as PGH synthase protein levels increase; this has been attributed to cotranslational degradation of the PGH synthase mRNA (DeWitt, 1990). LH, FSH, and forskolin all increase PGH synthase expression in preovulatory follicles in vitro, and each of these agents is also known to stimulate cAMP accumulation in follicles (Wong et al., 1989). Thus, one factor which may be involved in regulating PGH synthase expression is cAMP. Another example of cAMPdependent regulation is seen with murine MC3T3-E1 cells, an osteoclast-like line, in which fl-adrenergic stimulated-increases in PGH synthase protein levels are dependent on cAMP formation (Yamamoto et al., 1989). Tumor-promoting phorbol esters, such as PMA, cause differentiation of HL60 (Goerig et al., 1987) and U937 (Koehler et al., 1990b) cells. PGH synthase m R N A levels are below the limits of detection in both HL60 and U937 cells, but it has been observed that PMA-induced differentiation causes increases in both PGH synthase protein and activity. Presumably, increases in PGH synthase induced by PMA involve protein kinase C (PKC). As discussed below, the effects of IL-1 to increase PGH synthase levels in

J~r49/~B

dermal fibroblasts also appear to be mediated in part via PKC (Raz et al., 1990). 4.14. 'TUNING' OF PGH SYNTHASE Murine 3T3 cells and human umbilical vein endothelial cells contain substantial concentrations of PGH synthase. Treatment of 3T3 cells with plateletderived growth factor (PDGF) or serum causes increases in PGH synthase activity and PGH synthase mRNA (Habenicht et al., 1985; Lin et al., 1989; DeWitt, Meade and Kraemer, unpublished results). The peak in mRNA levels occurs 2-3 hr after stimulation. The peak in mRNA levels is closely followed by a peak in PGH synthase activity. The changes (1.5-3-fold) are relatively small but reproducible. Surprisingly, increases in PGH synthase mRNA and activity are not accompanied by detectable changes in total enzyme protein (Lin et al., 1989). Presumably, inactive enzyme is simply replaced by active enzyme but with no net change in the size of the PGH synthase protein pool. These findings suggest that PDGF treatment of 3T3 cells causes a coordinate increase in the rates of PGH synthase protein synthesis and degradation. With cultured endothelial cells, treatment with IL-1 also causes significant increases in PGH synthase mRNA levels, but in this case there are also detectable increases in PGH synthase protein (Maier et al., 1990). Although PGH synthase activity was not measured directly, IL-1 does increase net prostaglandin production by endothelial cells. 4.15. INTRACELLULARREGULATIONOF PGH

SYNTHASE Importantly, cycloheximide potentiates both the effect of IL-1 on the expression of PGH synthase m R N A in cultured endothelial cells (Maier et aL, 1990) and the effect of serum on the expression of PGH synthase in mouse 3T3 cells (DeWitt et al., 1991). This is the phenomenon of superinduction, and it is characteristic of 'immediate-early genes' such as c-fos and c-myc which are expressed shortly after treatment of quiescent cells with mitogenic stimuli (Greenburg et al., 1986; Shaw and Kamen, 1986). Thus, the PGH synthase gene has one important

168

I

W. L. S~TH et al. II II A

B

II I I

,I

II

It

II

I

II

•

i

•

FG

H

I

HI

CDE

J

II

I

I

K

1,4

29.4 KB Al~tlElat~I / K m i l l | I l l II I

cDNA

FIG. 10. Intron--exon structure of PGH synthase. Adapted from Yokoyama and Tanabe (1989) (DeWitt, Meade and Kraemer, unpublished results). characteristic displayed by 'immediate early genes'. In the case of c-fos, superinduction is thought to occur because this protein inhibits transcription of its own gene via a feedback mechanism (Sassone-Corsi et al., 1988). Accordingly, inhibition of c-fos protein synthesis by cycloheximide leads to elevated and continued transcription of the c-fos gene. It is also possible that PGH synthase may act to inhibit the transcription of the PGH synthase gene. This hypothesis is attractive when considered in the context of the suicide inactivation of PGH synthase (Egan et al., 1976; Hemler and Lands, 1980; Kent et al., 1983; Lapetina and Cuatrecasas, 1979; Smith and Lands, 1972). If reaction-inactivated enzyme is structurally altered so that it can no longer inhibit its own transcription, this would provide a means to derepress PGH synthase gene transcription in cells induced to form new prostaglandins. This is not likely to be the only level of modulation, however, because replacement of PGH synthase occurs at a more rapid rate in cells exposed to humoral agents such as PDGF (Habenicht et al., 1985; Lin et al., 1989; Burch et al., 1988; Bailey et al., 1985). 4.16. POOLSOF PGH SYNTHASE Studies on the turnover of PGH synthase have indicated that there may be at least two different pools of the enzyme. In PDGF-treated 3T3 cells incubated with cycloheximide, there is an 80% reduction in enzyme activity within 4 hr, but approximately 20% of the PGE2 biosynthetic capacity of these cells remains even after 10 hr (Habenicht et al., 1985). In cultured endothelial cells it has been found that most of the PGH synthase is degraded with an apparent half-life of less than 10min but that a smaller portion of the enzyme has a half-life of more than 2.5 hr (Tsai et al., 1990; Wu et al., 1988). The biochemical basis for the existence of different pools of PGH synthase is not known. However, it is reasonable to speculate that these pools represent PGH synthase associated with different intracellular membranes (Smith, 1986; Smith et al., 1983). 4.17. PGH SYNTHASEGENE STRUCTURE To understand how PGH synthase is regulated, it will be important to extend present molecular biological studies. The intron--exon structure of the human PGH synthase has recently been described (Fig. 10) (Yokoyama and Tanabe, 1989); the mouse gene has essentially the same structure (DeWitt, Meade and Kraemer, unpublished results). The PGH

synthase gene encompasses about 21 kb and contains 11 exons. Work is currently in progress to determine the transcriptional start site(s) and the sequence of the 5' noncoding region of the gene. In addition, there are ongoing studies in which portions of the Y-end of the gene coupled to reporter genes are being used to determine which 5' sequences are important for regulating transcription. Accordingly, it may soon be possible to reconcile some of the information regarding how PGH gene expression is regulated by PDGF, IL-1, IL-2, PMA, steroids, and cAMP with structural information on hormone-responsive elements in the PGH synthase gene. 4.18. EFFECTSOF ANTI-INFLAMMATORYSTEROIDSON PGH SYNTHASE Anti-inflammatory steroids inhibit the expression of PGH synthase in at least some cells. In dermal fibroblasts dexamethasone prevents the incorporation of 35S-methionine into PGH synthase during treatment of these cells with IL-1 (Raz et al., 1989, 1990). It is not clear whether the effects of dexamethasone seen in these studies are due to inhibition of transcription and/or translation of PGH synthase mRNA. Additionally, Koehler et al. (1990a) have shown that dexamethasone inhibits PGH synthase activity and reduces PGH synthase protein levels by about 50% in PMA-treated U937 cells. Finally, Raz et al. (1990) have shown that dexamethasone inhibits increases in PGH synthase activity in human monocytes treated with bacterial lipopolysaccharide.

5. METABOLISM OF PGH 2 5.1. PGD SYNTHESIS PGD2 is formed by a nonoxidative rearrangement of PGH2 (Fig. 2). Although the physiological roles of PGD2 are incompletely established, it is clear that PGD2 can inhibit platelet aggregation (Whittle et al., 1978) (by interacting with specific receptors (Siegl, 1982; Thierauch et al., 1989)), can cause bronchoconstriction (Wasserman et al., 1977), and may be involved in induction of sleep (Hayaishi, 1989). There are at least three different proteins which exhibit PGD synthase activity [E.C.5.3.99.2]. One enzyme, purified from the soluble fraction of spleen homogenates, catalyzes a reduced glutathione (GSH)-dependent disproportionation of PGH 2 (Christ-Hazelhof and Nugteren, 1982). This enzyme also exhibits GSH-S-transferase activity (Urade et al., 1987b). A second enzyme purified from rat

Prostaglandin and thromboxane biosynthesis

169

H

~"2 o. N

L

OH

\ O~./--~/N/COOH HO' ~,,,N.,,,,~/ b. ~ar~

FIG. 11. Formation of PGE: via a nonoxidative rearrangement of PGH: involving a 1,2 hydride shift facilitated by reduced glutathione (GSH). Adapted from Lands et al. (1971). brain catalyzes a GSH-indepcndent reaction (Urade et al., 1985). Finally, several GSH-S-transferases of

rat liver, which are immunologically unrelated to the spleen enzyme, exhibit GSH-dependent PGD synthase activity (Ujihara et al., 1988). The rat brain GSH-independent PGD synthase present in glial cells is associated with the nuclear membrane and endoplasmic reticulum (Urade et al., 1987a). This distribution is similar to that found for PGH synthase in 3T3 ceUs (Rollins and Smith, 1980), although it is unclear whether glial cells exhibit PGH synthase activity. A cDNA coding for the rat brain PGD synthase has been obtained by Urade et al. (1989). The deduced amino acid sequence is that of a unique protein having a molecular weight of about 20 kDa. The rat brain enzyme appears to contain a signal peptide and probably two Asn-linked oligosaccharide groups. 5.2. PGE SYNTHESIS PGE l was one of the first prostaglandins characterized chemically (Bergstrom et al., 1963). This compound and its homolog PGE 2 can be formed by nonoxidative rearrangements of the endoperoxid¢ groupings of PGHI and PGH2, respectively (Fig. l 1). In fact, PGE derivatives can be formed nonenzymically at relatively rapid rates from PGH precursors (Hamberg and Samuelsson, 1973; Nugteren and Hazelhof, 1973). However, there is also evidence for enzyme-mediated PGE formation catalyzed by PGE synthas¢ [E.C.5.3.99.3]. GSH appears to be an obligatory cofactor for enzyme-mediated PGE synthesis (Ogino et al., 1977; Moonen et al., 1982; Tanaka et al., 1987). There are a number of proteins which can catalyze GSH-dependent disproportionations of PGH 2 to yield PGE2, but it is not known which, if any, of these proteins is actually involved in PGE2 synthesis in vivo.

For example, most of the various GSH-S-transferase isozymes from rat liver exhibit considerable PGE synthase activity (Ujihara et al., 1988), although, in fact, rat liver lacks appreciable PGH synthase activity and synthesizes very little PGE. The PGE synthase activities of bovine (Ogino et al., 1977) and ovin¢ (Moonen et al., 1982) vesicular glands, which form considerable amounts of PGE/n vivo, lack GSH-Stransferase activity. The bovine PGE synthase is a labile protein which is stabilized nonspecifically by thiols; in contrast, the PGH-PGE isomerizing activity of this enzyme is stimulated specifically by GSH and not by other thiols (Ogino et aL, 1977). A solubilized and partially purified enzyme from ovine vesicular glands was reported to have a molecular weight of 60-70kDa (Moonen et al., 1982); however, two different monoclonal antibodies collectively capable of precipitating about 70% of the membrane-associated PGE synthase activity of ovine vesicular glands precipitate two different proteins (Tanaka et al., 1987); one protein had a subunit molecular weight of 17.5 kDa, and another, a subunit molecular weight of about 180 kDa. Again, neither of the immunoprecipitated proteins had detectable GSH-S-transferase activity. The antibody against the smaller protein, which precipitates about 45% of the total PGE synthase activity, coprecipitated PGE and PGH synthase activities from intact vesicular gland microsomes. This finding indicates that the two proteins are associated with the same membrane system. One can speculate, therefore, that this PGE synthase (Mr= 17,500; Km for PGH2=40#M ) is actually involved in PGE: synthesis in vesicular glands. Unfortunately, monoclonal antibodies to this protein fail to stain other tissues such as renal collecting tubules (Tanaka et al., 1987), which also display beat-labile, GSH-dependent PGE synthase activity (Grenier et al., 1981). At this point, it appears that PGE synthesis is catalyzed by different PGE synthase isozymes in

170

W. L. SmTH et al.

\

O,~/%,/~f

POHREDUCTASE

b. ~H 2

i

OH

~ R

PGF2r' EDUCTASE

b. I~E 2

H0,, I

OH POD

2

OH

9o,,11~-PGF2

FIG. 12. Three enzyme-mediated processes through which PGF2 epimers could be formed. NADPH is the electron donor in each case. different tissues and that in some cases GSH-S-transferases could play a role in PGE synthesis. In all cases reported, GSH is required for PGE synthesis from PGH, but, in keeping with the nonoxidative nature of the reaction, GSH is not consumed during POE synthesis (Ogino et al., 1977). Lands et al. (1971) proposed a mechanism for the role of GSH in enzyme-mediated PGE synthesis analogous to the role of GSH in the giyoxylase I reaction; as shown in Fig. 11, GSH is envisioned as facilitating a 1,2 hydride shift involving movement of the C-9 hydrogen of PGH2 to become the incipient C-9 hydroxyl hydrogen upon homologous cleavage of the endoperoxide grouping. The elements of GSH are then lost by elimination. 5.3. PGF SYNTHESIS PGF2 derivatives are the only immediate metabolites of PGH2 that are formed by a net two electron reduction; PGE 2, POD 2, PGI 2, and TxA 2 are all synthesized by nonoxidative rearrangements. There are two epimers of PGF which exhibit biological activity and which are found in biological fluids; one is PGF2~ (Bergstrom et al., 1963; Dunn et al., 1978), and the other is 9~,1 lJ~-PGF2 (Liston and Roberts, 1985; Beasly et al., 1987). There are three potential mechanisms for the formation of PGF 2 derivatives (Fig. 12): (a) reduction of PGH2 to PGF2~ by an endoperoxide reductase, (b) reduction of POD2 to 9~,ll]~-PGF2 by an l l-ketoreductase, and (c) reduction of PGE 2 to PGF2~ by a 9-keto-reductase. There are proteins which will catalyze each of these reactions. However, all of these enzymes exhibit rather broad substrate specificities, and the K ~ d K ~ values for prostaglandin substrates are often unfavorable. It has not yet been established that any of these proteins is involved in PGF synthesis in vivo. A PGF synthase has been purified from bovine lung by Hayaishi and coworkers (Watanabe et al.,

1985). Curiously, this enzyme exhibits both an llketo-PGD2 reductase activity (Kin for P G D 2 = 120pM) and P G H 2 reductase activity (Kin for PGH2 = 10/~M) catalyzing the formation of 9~,ll/~PGF2 and PGF2~, respectively. There is kinetic evidence that these two reactions occur at different sites on the enzyme molecule. NADPH is used in preference to N A D H as a reducing agent; a variety of aldehydes and ketones can substitute for PGD2 as oxidizing substrates. A cDNA for the bovine lung PGF synthase has been isolated; the open reading frame contains 323 amino acids coding for a protein with a molecular weight of about 36 kDa (Watanabe et al., 1988, 1989). There are considerable sequence similarities between the lung PGF synthase, human liver aldehyde reductase, rat lens aldose reductase, and p-crystallin (Hayashi et al., 1989; Watanabe et al., 1989). Thus, the bovine lung PGF synthase appears to be a member of the aldose reductase family of enzymes. Importantly, human liver aldehyde reductase can catalyze the PGH2 reductase activity, but the Km for PGH2 is about ten-fold greater than that of the PGF synthase of bovine lung. Analyses of tissue distributions of 1l-keto-PGD2 reductase activities have revealed high levels of this activity in bovine liver, lung, and spleen (Urade et al., 1990). The lung and spleen enzymes appear to be the same PGF synthases. The liver enzyme is not identical to but is immunologically-related to the lung PGF synthase. It is not clear if the bovine liver enzyme is related to the l l-keto-PGD2 reduetase activity purified earlier from rabbit liver by Wong (1982). A third possible pathway for PGF2~ synthesis involves reduction of PGE 2. 9-Keto-PGE2 reductase activities which will catalyze this reaction have been found in the soluble fractions of renal cortex (Stone and Hart, 1975; Korff and Jarabak, 1982) and placenta (Westbrook et al., 1977; Jarabak et al., 1983). These activities are associated with NADPHdependent (Type II) 15-hydroxyprostaglandin dehydrogenase [E.C.1.1.1.197] (Chang and Tai, 1981;

Prostaglandin and thromboxane biosynthesis

mmmm~

/••"OH I~CO0

H

III

OH 6"ket°'PGF1(z

OH

FIG. 13. Mechanism of formation of PGI2 from PGH2. Jarabak et al., 1983). The Km value for PGE 2 is quite high (ca. 300/~M) suggesting that PGE2 is not the natural substrate and that PGF~ synthesis in vivo is unlikely to involve this enzyme. 5.4. PGI SYNTHESIS

171

suggesting that PGI 2 formation occurs in association with those membranes where PGH2 is generated (Smith et al., 1983). PGI synthase has been purified from bovine aorta by DeWitt and Smith (1983) and from porcine aorta by Graf et al. (1983). The purified enzyme has a subunit molecular weight of about 50 kDa, a specific activity of 1/~mol PGI2/min/mg, and a Km for PGH2 of about 5/~M. PGH], which lacks a double bond at C-5, is not a substrate while both PGH2 (Salmon et al., 1978) and PGH3 (Needleman et al., 1979) can serve as substrates. Associated with the purified PGI synthase is a heme group (0.1-1 heme/subunit), and a peak is present in the visible spectrum of the purified enzyme at 418-420 nm (DeWitt et al., 1983). The peak in the visible spectrum is shifted to 426 nm when the enzyme is incubated with 9,11-azoprosta-5,13-dienoic acid (Azo Analog I), a PGH analog, which is a competitive inhibitor. Graf et al. (1983) have reported that PGI synthase exhibits a reduced CO spectrum similar to that of cytochrome P-450 and have suggested that the axial heme ligand is a thiolate group of cysteine. PGI synthase is rapidly inactivated during catalysis and inactivation is accompanied by bleaching of the heme spectrum (DeWitt and Smith, 1983); a similar change is observed when the enzyme is inactivated by hydroperoxides. Azo Analog I prevents this bleaching which presumably arises from oxidation of the heme group. At this time, only portions of the primary sequence ofPGI synthase are known (Inoue et al., 1987). It has been found that in endothelial cells and murine 3T3 cells PGH synthase and PGI synthase protein levels are coregulated (Weksler, 1987; Goerig et al., 1988). Relatively little work on the properties of this enzyme has been reported in the last five years.

Prostacyclin (PGI2) was discovered in 1976 as an antiplatelet aggregating and smooth muscle relaxing activity extracted from incubates of bovine aorta (Bunting et al., 1976; Johnson et al., 1976). The formation of PGI2 from PGH2 involves an acidcatalyzed beterolytic cleavage of the endoperoxide followed by reaction of a transiently positive oxygen at C-6 (Fig. 13). To account for the endoperoxide cleavage, Ullrich et aL (1981) proposed a mechanism analogous to the oxene transfer mechanism of cytochrome P-450. The hydrogen at C-6 of PGH2 is lost during PGI 2 formation, and an assay for PGI 2 syn5.5. THROMBOXANESYNTHESIS thesis based on this loss of hydrogen has been developed using [5,6-3H]PGH2 (Tai et al., 1980). Thromboxane A 2 (TxAz) was initially discovered as However, PGI 2 synthesis is usually measured by a product of arachidonate metabolism by human following the conversion of uniformly labeled PGH2 platelets (Hamberg et al., 1975). TxA2 is synthesized to radioactive 6-keto-PGFl~ (Salmon and Flower, by lung and macrophages in addition to platelets 1982). This latter product is the stable hydrolysis (Smith, 1987). This product is a potent thrombogenic product of PGI 2 formed during extraction under agent and vasoconstrictor. The half-life of TxA2 is acidic conditions by hydrolysis of the enol-ether approximately 30sec at 37°; its stable hydrolysis linkage of PGI 2 (Pace-Asciak and Wolfe, 1971). product is TxB2 which lacks appreciable biological The enzyme which catalyzes the formation of PGI2 activity. from PGH2 is known as prostaglandin I (PGI) synTxA2 formation is envisioned to be initiated by the thase [E.C.5.3.99.5]. Several monoclonal antibodies transient formation of an electropositive oxygen at have been prepared against the enzyme, and these C-11 and subsequent cleavage of the 9,11-peroxido antibodies cross-react with PGI synthases from most group (Pace-Asciak and Smith, 1983) (Fig. 14). species (Smith et al., 1983). PGI synthase has been Ullrich et al. (1981) proposed that an oxene transfer shown by immunocytochemical techniques to be in mechanism analogous to that catalyzed by a cytohigh concentrations in vascular endothelial cells and chrome P-450 might be involved. Curiously, forin both vascular and nonvascular smooth muscle cells mation of TxA 2 from PGH2 is always accompanied (Smith et al., 1983; DeWitt et al., 1983). In larger by the formation of at least an equivalent amount of vessels the concentrations of PGI synthase are ap- the 17 carbon b ydroxy fatty acid HHTrE (formerly proximately the same in both smooth muscle and called HHT) and malondialdehyde (MDA). With endothelial cells, but smooth muscle contains only PGH analogs other than PGH2, the amount of 17 5% of the PGH synthase of endothelial cells (DeWitt carbon hydroxy acid formed is far in excess of TxA et al., 1983). At the subeellular level, PGI synthase (Diczfalusy et al., 1977). HHTrE has little apparent colocalizes with PGH synthase in the endoplasmic biological activity, and the reason for the coordinate reticulum, nuclear membrane, and plasma membrane formation of TxA 2 and HHTrE is unclear.

172

W. L. SMITHet al.

~

OH

#

,~

~'O

o

i

HOOC PGH2

HHTrE

TxA 2

MDA

III OH

~~__~%/COO H H20 o.

b.

OOH b.

TxB2

FIG. 14. Mechanism of formation of TxA2, HHTrE, and MDA from PGH2. The conversion of PGH 2 to TxA2 is catalyzed by thromboxane A synthase [E.C.5.99.1.3]. TxA synthases have been purified to apparent homogeneity from porcine lung by Shen and Tai (1986b) and from human platelets by Ullrich and coworkers (Haurand and Ullrich, 1985; Nusing et al., 1990). Polyclonal rabbit antiserum raised against the porcine lung enzyme cross-reacts with a protein of Mr = 50,000 from human platelets while an M r = 58,000 has been reported by Nusing et al. (1990); the basis for this apparent size discrepancy has not been resolved. Otherwise the porcine lung and human platelet enzymes have similar properties. Both enzymes exhibit a heme spectrum with a peak at 418 nm and both form HHTrE and TxA 2 from PGH2 in a ratio of about one. The human platelet thromboxane synthase is reported to contain one heine group per mole of subunit (Nusing et al., 1990) and to exhibit a peak in the reduced CO difference spectrum at 450 nm corresponding to a cytochrome P-450. TxA synthase resembles other enzymes of the arachidonate cascade in being inactivated during catalysis (Shen and Tai, 1986b). Unlike the polyclonal antibody, monoclonal antibodies raised against both the porcine (Shen amTai, 1986a) and human enzymes (Nusing et al., 1990) fail to exhibit species cross-reactivity. To date, a cDNA for TxA synthase has not been reported although there is now some protein sequence data available (Nusing et al., 1990). A variety of thromboxane synthase inhibitors have been synthesized for possible use as antithrombogenic agents. Several of these compounds such as UK-37248 are competitive inhibitors of PGH2 binding to TxA synthase (Nusing et al., 1990), but none appear to be more efficacious in vivo than low doses of aspirin. Acknowledgements--Studies from our laboratories reported

here were supported in part by USPHS NIH Grants DK22042 (WLS), DK42509 (WLS), CA47479 (LJM) and GM40713 (DLD) and Grants-ln-Aid from the American Heart Association of Michigan (WLS, DLD). We wish to thank Drs Edward A. Dennis and Robert C. Murphy for helpful discussions and advice in preparing this review.

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