We have evaluated the role of nitric oxide (NO) on the activity of the constitutive and induced forms of cyclooxygenase (COX; COX-1 and COX-2, respectively). Induction of NO synthase (NOS) and COX (COX-2) in the mouse macrophage cell line RAW264.7 by Escherichia coli lipopolysaccharide (1 ,ug/ml, 18 h) caused an increase in the release of nitrite (NOj ) and prostaglandin E2 (PGE2), products of NOS and COX, respectively. Production of both NOj and PGE2 was blocked by the NOS inhibitors NG.monomethyl-L-arinine or aminoguanidine. Production of NO from constitutive NOS is a key regulator of homeostasis, whereas the generation of NO by inducible NOS plays an important role in the host-defense response (1). NOS shares a number of similarities with cyclooxygenase (COX). COX is the rate-limiting enzyme in the biosynthesis of prostaglandins (PGs), thromboxane A2, and prostacycin (PGI2). In addition to the well-characterized constitutive form of COX (COX-1) (4), an inducible isoform of COX (COX-2) is found in endothelial cells (5), fibroblasts (6), and macrophages (7-9) after treatment with proinflammatory agents including LPS and IL-1,3. Antiinflammatory steroids such as dexamethasone inhibit the induction of inducible NOS in vitro and in vivo but have no effect on the expression of constitutive NOS (1). In addition, dexamethasone inhibits IL-13-and LPSstimulated COX-2 protein synthesis in vitro (7,8) and in vivo (8, 9) but has no effect on the constitutive form of COX.Many effectors of NO production lead to the simultaneous release of mediators (such as PGE2 and PGI2) from the COX pathway. This is true for the rapidly acting agonists such as bradykinin (10)(11)(12) and for the longer acting agents such as LPS or 8 (13,14). NO, PGI2, or PGE2 increase the levels of cGMP or cAMP in effector cells (e.g., platelets). This synergistic effect may be one mechanism(s) through which the NOS and COX systems operate to amplify a physiological or pathological response.Another possible interaction is at the level of the enzyme. In this respect, the COX enzymes are potential targets for NO because they contain an iron-heme center at their active site (15)(16)(17), and indeed, the vast majority of effects mediated by NO are a consequence of its interaction with iron or ironcontaining enzymes. For example, the ability of NO to inhibit platelet aggregation and to relax vascular smooth muscle is the result of NO binding to the heme-Fe2+ prosthetic group of the soluble guanylate cyclase leading to its stimulation and subsequent increase in the levels of cGMP (18,19

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Platelets enzymatically convert prostaglandin H 3 (PGH 3 ) into thromboxane A 3 . Both PGH 2 and thromboxane A 2 aggregate human platelet-rich plasma. In contrast, PGH 3 and thromboxane A 3 do not. PGH 3 and thromboxane A 3 increase platelet cyclic AMP in platelet-rich plasma and thereby: ( i ) inhibit aggregation by other agonists, ( ii ) block the ADP-induced release reaction, and ( iii ) suppress platelet phospholipase-A 2 activity or events leading to its activation. PGI 3 (Δ 17 -prostacyclin; synthesized from PGH 3 by blood vessel enzyme) and PGI 2 (prostacyclin) exert similar effects. Both compounds are potent coronary relaxants that also inhibit aggregation in human platelet-rich plasma and increase platelet adenylate cyclase activity. Radioactive eicosapentaenoate and arachidonate are readily and comparably acylated into platelet phospholipids. In addition, stimulation of prelabeled platelets with thrombin releases comparable amounts of eicosapentaenoate and arachidonate, respectively. Although eicosapentaenoic acid is a relatively poor substrate for platelet cyclooxygenase, it appears to have a high binding affinity and thereby inhibits arachidonic acid conversion by platelet cyclooxygenase and lipoxygenase. It is therefore possible that the triene prostaglandins are potential antithrombotic agents because their precursor fatty acids, as well as their transformation products, PGH 3 , thromboxane A 3 , and PGI 3 , are capable of interfering with aggregation of platelets in platelet-rich plasma.

The effect of endogenous glucocorticoids on the expression of the cyclooxygenase enzyme was studied by contrasting cyclooxygenase expression and prostanoid synthesis in adrenalectomized and sham-adrenalectomized mice with or without the concurrent administration of endotoxin. Peritoneal macrophages obtained from adrenalectomized mice showed a 2-to 3-fold induction in cyclooxygenase synthesis and activity when compared to sham controls. Intravenous hj'ection of a sublethal dose of endotoxin (5 jzg/kg) further stimulated cyclooxygenase synthesis, resulting in a 4-fold increase in prostaglandin production. Similar cyclooxygenase induction can be achieved in macrophages obtained from normal mice but only after high doses of endotoxin (2.5 mg/kg) that are 100% lethal to adrenalectomized mice. Restoration of glucocorticoids in adrenalectomized animals with dexamethasone completely inhibited the elevated cyclooxygenase and protected these animals from endotoxin-induced death. In contrast, no signs of cyclooxygenase induction were observed in the kidneys of the adrenalectomized mice, even when treated with endotoxin. Dexamethasone did not affect the constitutive cyclooxygenase activity and prostaglandin production present in normal and adrenalectomized kidneys. These data indicate the existence of a constitutive cyclooxygenase that is normally present in most cells and tissues and is unaffected by steroids and of an inducible cyclooxygenase that is expressed only in the context of inflammation by proinflammatory cells, like macrophages, and that is under glucocorticoid regulation. Under normal physiological conditions glucocorticoids maintain tonic inhibition of inducible cyclooxygenase expression. Depletion of glucocorticoids or the presence of an inflammatory stimulus such as endotoxin causes rapid induction of this enzyme, resulting in an exacerbated inflammatory response that is often lethal.Prostaglandins and glucocorticoids are active endogenous participants in the inflammatory response. Endotoxin (lipopolysaccharide, LPS) administration to human volunteers increases the circulating levels of tumor necrosis factor (TNFa) and corticotropin (ACTH) and elicits hyperthermia. Cyclooxygenase (COX) inhibitors blunt the effect of LPS on body temperature and ACTH but not the rise in TNFa, implicating prostaglandins in some, but not all, of the symptoms associated with endotoxemia (1).Endogenous as well as exogenously administered glucocorticoids act to modulate the natural defense mechanisms that normally follow an inflammatory insult, thus preventing marked changes in homeostasis (2). Adrenalectomized (ADX) animals, which lack glucocorticoids, showed a more severe and often lethal inflammatory reaction to endotoxin (3, 4), which is prevented by the administration of the synthetic glucocorticoid dexamethasone (DEX) (5). Recent evidence suggests that the antiinflammatory effect of glucocorticoids involves inhibition of prostanoid synthesis (6, 7), possibly by the regulation of a cytokine-induced COX. Several investi...

The microsomal fraction of horse and human platelets contains an enzyme which converts prostaglandin cyclic endoperoxides (PGG2 or PGH2) to a substance which is much more potent in contracting strips of rabbit aorta. This substance has the same characteristics as thromboxane A2, and can be distinguished from other products of arachidonic acid metabolism by differential bioassay.