We have recently shown that the synthesis of cyclooxygenase [also called prostaglandin (PG) activity.Previous studies in our laboratory (1) and by others (2) have demonstrated that the cytokine interleukin 1 (IL-1) stimulates formation of prostaglandin E2 (PGE2) in cultured human dermal fibroblasts. We further showed that IL-1-containing monocyte-conditioned medium produced increased Vmas of fibroblast prostaglandin (PG) endoperoxide synthase (also called PG synthase and cyclooxygenase; 8,11,14-icosatrienoate, hydrogen-donor:oxygen oxidoreductase, EC 1.14.99.1), which appeared to be blocked by RNA and protein synthesis inhibitors (3). More recently, we have shown (4) that IL-1 stimulates the synthesis in fibroblasts of PG synthase in a time-and dose-dependent manner. Overall, these results (1-4) suggest an IL-1-dependent transcriptional regulation of the synthesis of PG synthase. Previous studies have shown that the tumor promoter phorbol 12-myristate 13-acetate (PMA) stimulated PGE2 production in a variety of cell types by: (i) increasing phospholipase A2 activity to release arachidonic acid (5, 6), (ii) inhibiting the reacylation enzymes arachidonoyl-CoA synthetase (EC 6.2.1.15) and lysophosphatide acyltransferase (EC 2.3.1.23; refs. 7 and 8), or (iii) increasing the level of cellular PG synthase (9). In the studies reported here, we tested the hypothesis that activation of protein kinase C plays a critical role in the signal transduction mechanism for the IL-1 and PMA induction of PG synthase synthesis.Glucocorticoids are known to inhibit icosanoid production when administered both in vivo and in vitro to cells or tissues (10). Evidence from numerous studies indicated that the mechanism of this glucocorticoid effect involves the synthesis ofa group ofproteins (collectively termed lipocortins) that inhibit the activity of phospholipase A2 and thereby the production of various icosanoids (10). Recent reports, however, have raised some doubts with regard to the validity of this mechanism. Davidson et al. (11) and Aarsman et al. (12) have demonstrated that lipocortins do not directly interact with or inhibit the activity of phospholipase A2 but rather interact with substrate phospholipids. Furthermore, in some studies, glucocorticoids failed to inhibit cellular phospholipase A2 (13) and also failed to induce synthesis of either the mRNA for lipocortin I or the lipocortin mass (14). Other studies (15, 16) have suggested that glucocorticoids may exert their inhibitory action on PG biosynthesis by inhibiting PG synthase activity. In support of this, Pash and Bailey (17) recently reported that dexamethasone inhibited the epidermal growth factor stimulation of PG synthase activity in aortic smooth muscle cells.The present studies were designed to establish incubation conditions that will afford a temporal separation of the transcriptional and translational phases of PG synthase synthesis, so that we could study the regulation of the enzyme synthesis at each of these phases. Lacking a PG synthase cDNA pro...

A B S T R A C T Exogenous arachidonate addition to intact platelets, in the absence or the presence of blood vessel microsomes, results in the production of thromboxane B2 (the stable degradation product of thromboxane A2) only. Prostaglandin (PG) endoperoxides are released from intact platelets only when thromboxane synthetase is inhibited. Thus, addition of exogenous arachidonate to imidazole-pretreated platelets in the presence of bovine aorta microsomes (source of prostacyclin synthetase) results predominantly in the synthesis of 6-keto-PGF,a (the stable degradation product of prostacyclin). Strips of intact aorta were removed from aspirin-treated rabbits, thus the isolated blood vessels were unable to convert endogenous or exogenous arachidonate to prostacyclin. Human platelets, with ['4C]arachidonate-labeled phospholipids, adhered to the blood vessel segments and released some thromboxane B2. The subsequent addition of thrombin facilitated the release of endogenous arachidonate and thromboxane, but no labeled 6-keto-PGF,, was detectable. There is therefore no direct chemical evidence of PG-endoperoxide release from human platelets during either aggregation or adhesion, which therefore precludes the possibility that blood vessels use platelet PG-endoperoxide for prostacyclin synthesis. Imidazole inhibited the thromboxane synthetase in the labeled platelets, and thereafter thrombin stimulation resulted in the release of platelet-derived, labeled PG-endoperoxides that were converted to labeled prostacyclin by the vascular prostacyclin synthetase. The latter result suggests a potential antithrombotic therapeutic benefit might be achieved using an effective thromboxane synthetase inhibitor.

Addition of the 3-series fatty acid precursor (icosapentaenoic acid, IPA), its endoperoxide [prostaglandin (PG) H3J, or thromboxane A3 to human platelet-rich plasma (PRP) does not result in aggregation of the platelets. In fact, preincubation of human PRP with exogenous PGH3 actually inhibited aggregation by increasing platelet cyclic AMP concentrations. PGH3 undergoes rapid spontaneous degradation to PGD3 in human PRP. The PGD3 so formed is adequate to account for the increase of platelet cAMP and inhibition of aggregation. Furthermore, addition of PGD-specific antisera to human PRP blocked the platelet inhibitory activity of exogenous PGH3. PGD3 has considerable potential as a circulating antithrombotic agent. Pretreatment of human PRP with the adenylate cyclase inhibitor 2',5'-dideoxyadenosine blocked the increase of platelet cyclic AMP and the inhibition of aggregation normally produced by PGI2, PGE1, PGD2, PGH3, and PGD3. Furthermore, the dideoxyadenosine unmasked a direct but moderate reversible aggregatory effect in response to the subsequent addition of PGH3. Similarly, the dideoxyadenosine markedly enhanced the aggregation produced by exogenous PGH2. IPA is readily incorporated into tissue lipids but proved to be a poor substrate for kidney, blood vessel, or heart cyclooxygenase. IPA was previously shown to be a poor substrate for platelet cyclooxygenase. IPA is readily deacylated from the renal phospholipid pool in response to bradykinin, a substance that aso stimulates the release of arachidonic acid. A diet that relies primarily on cold-water fish, as in the case of the Greenland Eskimos, lowers endogenous arachidonic acid and markedly increases the IPA content of tissue lipids. Thus, because IPA has the potential to act as an antagonist with arachidonic acid for platelet cyclooxygenase and lipoxygenase, the simultaneous release of IPA could suppress any residual arachidonic acid conversion to its aggregatory metabolites. The normal 2-series prostaglandin (PG) family is derived from arachidonic acid (AA; 5,8,11,14-icosatetraenoic acid). AA and its metabolites PGH2 and thromboxane A2 are potent stimulators of platelet aggregation. The fatty acid precursor of the 3-series PG family, 5,8,11,14,17-icosapentaenoic acid (IPA), can be enzymatically converted by sheep cyclooxygenase into the PG endoperoxide PGH3 (1, 2). Purified PGH3 in turn is converted by the appropriate enzyme source into thromboxane A3 or A'7-prostacyclin (PGI3) (2). The 3-series endoperoxide and thromboxane are less potent contractile agents on rabbit thoracic aorta strips than the corresponding 2-series compounds (i.e., PGH2 and thromboxane A2) (1), and similarly PGI3 relaxes isolated coronary arterial strips but is less potent than PGI2 (3). Surprisingly, the 3-series fatty acid (IPA), endoperoxide (PGH3), and thromboxane (thromboxane A3) do not induce aggregation in human platelet-rich-plasma (PRP) (1) and, furthermore, preincubation of human PRP with exogenous PGH3 or The publication costs of this article were defrayed i...

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A B S T R A C T Both the isolated perfused rabbit heart and kidney are capable of synthesizing prostaglandin (PG) I2. The evidence that supports this finding includes: (a) radiochemical identification of the stable end-product of PGI2, 6-keto-PGF,l, in the venous effluent after arachidonic acid administration; (b) biological identification of the labile product in the venous effluents which causes relaxation of the bovine coronary artery assay tissue and inhibition of platelet aggregation; and (c) confirmation that arachidonic acid and its endoperoxide PGH2, but not dihomo-y-linolenic acid and its endoperoxide PGH, serve as the precursor for the coronary vasodilator and the inhibitor of platelet aggregation. The rabbit heart and kidney are both capable of converting exogenous arachidonate into PGI2 but the normal perfused rabbit kidney apparently primarily converts endogenous arachidonate (e.g., generated by stimulation with bradykinin, angiotensin, ATP, or ischemia) into PGE2; while the heart converts endogenous arachidonate primarily into PGI2. Indomethacin inhibition of the cyclo-oxygenase unmasks the continuous basal synthesis of PGI2 by the heart, and of PGE2 by the kidney. Cardiac PGI2 administration causes a sharp transient reduction in coronary perfusion pressure, whereas the intracardiac injection of the PGH2 causes an increase in coronary resistance without apparent cardiac conversion to PGI1. The perfuLsed heart rapidly degrades most of the exogenous endoperoxide probably into PGE2, while exogenous PGI2 traverses the heart without being metabolized. The coronary vasoconstriction produced by PGH2 in the normal perfused rabbit heart suggests that the endoperoxide did not reach the PGI2 synthetase, whereas