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Cyclooxygenase (COX), first purified in 1976 and cloned in 1988, is the key enzyme in the synthesis of prostaglandins (PGs) from arachidonic acid. In 1991, several laboratories identified a product from a second gene with COX activity and called it COX-2. However, COX-2 was inducible, and the inducing stimuli included pro-inflammatory cytokines and growth factors, implying a role for COX-2 in both inflammation and control of cell growth. The two isoforms of COX are almost identical in structure but have important differences in substrate and inhibitor selectivity and in their intracellular locations. Protective PGs, which preserve the integrity of the stomach lining and maintain normal renal function in a compromised kidney, are synthesized by COX-1. In addition to the induction of COX-2 in inflammatory lesions, it is present constitutively in the brain and spinal cord, where it may be involved in nerve transmission, particularly that for pain and fever. PGs made by COX-2 are also important in ovulation and in the birth process. The discovery of COX-2 has made possible the design of drugs that reduce inflammation without removing the protective PGs in the stomach and kidney made by COX-1. These highly selective COX-2 inhibitors may not only be anti-inflammatory but may also be active in colon cancer and Alzheimer's disease.

The beneficial actions of nonsteroid anti-inflammatory drugs (NSAID) can be associated with inhibition of cyclo-oxygenase (COX)-2 whereas their harmful side effects are associated with inhibition of COX-1. Here we report data from two related assay systems, the human whole blood assay and a modified human whole blood assay (using human A549 cells as a source of COX-2). This assay we refer to as the William Harvey Modified Assay. Our aim was to make meaningful comparisons of both classical NSAIDs and newer COX-2-selective compounds. These comparisons of the actions of >40 NSAIDs and novel COX-2-selective agents, including celecoxib, rofecoxib and diisopropyl fluorophosphate, demonstrate a distribution of compound selectivities toward COX-1 that aligns with the risk of serious gastrointestinal complications. In conclusion, this full in vitro analysis of COX-1/2 selectivities in human tissues clearly supports the theory that inhibition of COX-1 underlies the gastrointestinal toxicity of NSAIDs in man.

Microsomes prepared from rabbit or pig aortas transformed endoperoxides (PGG2 or PGH2) to an unstable substance (PGX) that inhibited human platelet aggregation. PGX was 30 times more potent in this respect than prostaglandin E1. PGX contracted some gastrointestinal smooth muscle and relaxed certain isolated blood vessels. Prostaglandin endoperoxides cause platelet aggregation possibly through the generation by platelets of thromboxane A2. Generation of PGX by vessel walls could be the biochemical mechanism underlying their unique ability to resist platelet adhesion. A balance between formation of anti- and pro-aggregatory substances by enzymes could also contribute to the maintenance of the integrity of vascular endothelium and explain the mechanism of formation of intra-arterial thrombi in certain physiopathological conditions.

Constitutive cyclooxygenase (COX-1; prostaglandin-endoperoxide synthase, EC 1.14.99.1) is present in cells under physiological conditions, whereas COX-2 is induced by some cytokines, mitogens, and endotoxin presumably in pathological conditions, such as inflammation. Therefore, we have assessed the relative inhibitory effects of some nonsteroidal antiinflammatory drugs on the activities of COX-1 (in bovine aortic endothelial cells) and COX-2 (in endotoxinactivated J774.2 macrophages) in intact ceils, broken cells, and purified enzyme preparations (COX-1 in sheep seminal vesicles; COX-2 in sheep placenta). Similar potencies of aspfrin, indomethacin, and ibuprofen against the broken cell and purified enzyme preparations indicated no influence of species. Aspirin, indomethacin, and ibuprofen were more potent inhibitors of COX-1 than COX-2 in all models used. The relative potencies of aspirin and indomethacin varied only slightly between models, although the IC50 values were different.Ibuprofen was more potent as an inhibitor of COX-2 in intact cells than in either broken cells or purified enzymes. Sodium salicylate was a weak inhibitor of both COX isoforms in intact cells and was inactive against COX in either broken cells or purified enzyme preparations. Diclofenac, BW755C, acetaminophen, and naproxen were approximately equipotent inhibitors of COX-1 and COX-2 in intact cells. BF 389, an experimental drug currently being tested in humans, was the most potent and most selective inhibitor of COX-2 in intact cells. Thus, there are clear pharmacological differences between the two enzymes. The use of such models of COX-1 and COX-2 activity will lead to the identification of selective inhibitors of COX-2 with presumably less side effects than present therapies. Some inhibitors had higher activity in intact cells than against purified enzymes, suggesting that pure enzyme preparations may not be predictive of therapeutic action.Cyclo-oxygenase (COX; prostaglandin-endoperoxide synthase, EC 1.14.99.1) converts arachidonic acid to prostaglandin (PG) H2, which is then further metabolized by other enzymes to various PGs, prostacyclin, and thromboxanes (1). COX exists in at least two isoforms with similar molecular weights ("'70 kDa). COX-1 is expressed constitutively and was first characterized, purified, and cloned from sheep vesicular glands (2-7). Activation of COX-1 leads, for instance, to the production of prostacyclin, which when released by the endothelium is antithrombogenic (8) and by the gastric mucosa is cytoprotective (9). COX-2 is induced in cells exposed to proinflammatory agents, including cytokines (10), mitogens (11) and endotoxin (12,13 COX-2 may well explain their therapeutic utility as antiinflammatory drugs, whereas inhibition of COX-1 may explain their unwanted side effects, such as gastric and renal damage.After establishing that bovine aortic endothelial cells in culture contain COX-1 and that endotoxin-activated J774.2 macrophages contain COX-2, we have investigated the inhibitory effects of s...

Endothelin releases prostacyclin and thromboxane A2 from guinea pig or rat isolated lungs and endothelium-derived relaxing factor in the perfused mesentery of the rat. Endothelin is also substantially removed by the pulmonary circulation of the rat in vitro and in vivo and by guinea pig lungs in vitro. In the rat, the effects of endothelin on the blood pressure vary from pressor (in pithed rats) to purely depressor in anesthetized rats where the resting blood pressure is high. It therefore 4ias the characteristics of a local pressor hormone, rather than a circulating one.The endothelial cell (EC) is known to release vasoactive substances such as prostacyclin (PGI2) (1) and endotheliumderived relaxing factor (EDRF) (2), recently identified as nitric oxide (3). Release of endothelium-dependent vasoconstrictor factors has been observed in response to various chemical and physical stimuli such as norepinephrine (4), thrombin (4), hypoxia (5, 6), increased transmural pressure (7), and mechanical stretch (8).Masaki and his colleagues (9) have recently characterized from cultures of porcine aortic ECs a 21-amino acid peptide, which they called endothelin (ET). In the chemically denervated rat, porcine ET is the most potent pressor substance yet described, with a long duration of action. They suggested that ET directly activates dihydropyridine-sensitive calcium channels.We report here that apart from its vasoconstrictor activity, ET can release potent vasodilator substances such as PGI2 and EDRF and is also removed by the pulmonary circulation.MATERIALS AND METHODS Superfusion Bioassay. Spiral strips of de-endothelialized vascular smooth muscle from the rabbit (mesenteric artery, celiac artery, carotid artery, aorta, jugular vein, mesenteric vein) and other smooth muscle preparations (guinea pig trachea, guinea pig ileum, rat stomach strip, rabbit duodenum) were mounted in a cascade (10) and superfused at 5 ml-min-1 with Krebs-Ringer solution containing indomethacin (5.6 ,M). Agonists such as ET (1-50 pmol), bradykinin(1-10 pmol), substance P (1-10 pmol), and angiotensin 11 (1-10 pmol) were injected over the assay tissues.Isolated Lungs. Male Dunkin-Hartley guinea pigs (300-400 g) or male Wistar rats (200-300 g) were anesthetized with sodium pentobarbital (Sagatal, 70 ,umol kg-', i.v.) and a thoracotomy was performed. The pulmonary artery and the trachea were cannulated and the lungs were removed and placed in a warm chamber. The lungs were perfused at S ml-min-' via the pulmonary artery with oxygenated (95% 02/ 5% CO2) and warmed (370C) Krebs-Ringer solution (11). The lungs were left to stabilize for 30 min and ET was infused for 3 min at a flow rate of 0.1 ml-min-' to achieve a final concentration of 1 or 10 nM. The effluent from lungs was collected and analyzed by RIA for 6-oxoprostaglandin F1l (6-oxo-PGF1,) and thromboxane (TX) B2 as measures of prostacyclin and TXA2 release (12). The removal of ET was calculated by comparing the contractions of the assay tissues in response to infusions of ET directly over...

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Cyclooxygeuase (COX) progressively, also peakingatday 14. COX-1 protein remained unaltered throughout. The iNOS activity increased over the first 24 h in the skin, with a further major increase in the granulomatous tissue between days 3 and 7, followed by a decrease at day 14 and a further increase at day 21. The rise in COX and NOS activities in the skin during the acute phase reinforces the proinflammatory role for prostanoids and suggests one also for nitric oxide. However, in the chronic and resolving stages, a dio of COX and NOS activity occurred. Thus, there may be differential regulation of these enzymes, perhaps due to the changing pattern of cytokines during the inflammatory response.