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At low substrate/enzyme ratios, and in the absence of reduced glutathione (GSH), the major prostaglandin (PG) biosynthesised by the ram seminal vesicle cyclo-oxygenase from arachidonic acid was 6-keto-PGF1alpha. The addition of nanomolar amounts of reduced GSH suppressed biosynthesis of this product and stimulated the formation of PGE2; 1-epinephrine enhanced the conversion of the substrate but had no effect on the type of product formed. 15-Hydroperoxy arachidonic acid selectively inhibited formation of 6-keto-PGF1alpha (IC50 100 muM) but blocked synthesis of all cyclo-oxygenase products at concentrations greater than 1 mM. At substrate concentrations of muM or greater, synthesis of 6-keto-PGF1alpha was inhibited and PGE2 and PGF2alpha were the main products formed.

An antiserum was raised in rabbits using 5,6-dihydro prostacyclin, a stable analogue of prostacyclin, as the hapten, conjugated to bovine serum albumin. When added to platelet rich plasma the antiserum neutralised the inhibitory activity of prostacyclin, prostaglandin E 1 and D 2 . The amount of antiserum required to neutralise completely a dose of prostacyclin giving 90–95% inhibition of ADP induced aggregation was 10–30 times less than that required for the other two prostaglandins. Small amounts of antiserum prevented the inhibitory activity of prostacyclin generated from endothelial cells in platelet rich plasma.

Some non-steroid anti-inflammatory drugs which inhibit arachidonate cyclo-oxygenease have been examined for their effects on leukocyte migration, prostaglandin production and oedema formation in carrageenin-induced inflammation in the rat. At doses which inhibited oedeman, all the drugs tested caused a dose-dependent reduction in numbers of leukocytes and prostaglandin concentration in 24-h inflammatory exudates. At lower doses, indomethacin, aspirin, sodium salicylae, flurbiprofen and phenylbutazone isgnificantly potentiated leukocyte migration by 20–70%. Ibuprofen, naproxen and BW755C reversed the indomethacin-induced increase in leukocyte accumulation. BW755C inhibits the generation of chemotactic lipoxygenase products and it is possible that the effects of all these drugs on leukocyte migratoin are mediated though the lipoxygenase pathway of arachidonic acid metabolism.

The pro-inflammatory effects of prostaglandins have been clearly demonstrated with the use of various animal models of inflammation. Furthermore, the anti-inflammatory effects and some of the side effects of aspirin and other non-steroidal anti-inflammatory agents have been shown to depend on their ability to inhibit cyclo-oxygenase. These drugs, therefore, reduce the synthesis of prostaglandins, prostacyclin and thromboxane. They do not affect leukotriene production and there is no firm evidence to suggest that they alleviate inflammation through any other mechanism. In contrast, the corticosteroids facilitate the release of lipocortin which, through inhibition of phospholipase A2 reduces arachidonic acid release. These drugs possess potent anti-inflammatory properties and attempts have been made to develop non-steroidal drugs, such as BW755C, that display similar anti-inflammatory activity through inhibition of the 2 main pathways of the arachidonic acid cascade. Administration of low dose aspirin 40 mg/day selectively inhibits production of thromboxane A2 without affecting prostacyclin. This may be because, firstly, about 60% of an administered dose of aspirin is deacylated to salicylate during first-pass metabolism and, secondly, platelets cannot regenerate cyclo-oxygenase. Thus, absorbed aspirin irreversibly affects platelet thromboxane production in the pre-systemic circulation, but the systemic plasma aspirin concentration is likely to be too low to affect prostacyclin synthesis. Studies in experimental inflammation have shown that after the administration of aspirin, the concentration of salicylate in inflammatory exudate is considerably higher than that of aspirin. In addition, a comparison of prostaglandin synthesis inhibitory potencies shows that the concentration of salicylate, but not of aspirin, at the inflammatory site is high enough to substantially inhibit prostaglandin synthesis.

1. Defibrotide, a single-stranded polydeoxyribonucleotide obtained from bovine lungs, has significant anti-thrombotic, pro-fibrinolytic and prostacyclin-stimulating properties. 2. The present study was designed to evaluate the effects of defibrotide on infarct size and regional myocardial blood flow in a rabbit model of myocardial ischaemia and reperfusion. 3. Defibrotide (32 mg kg-1 bolus + 32 mg kg-1 h-1, i.v.) either with or without co-administration of indomethacin (5 mg kg-1 x 2, i.v.) was administered 5 min after occlusion of the left anterior-lateral coronary artery and continued during the 60 min occlusion and subsequent 3 h reperfusion periods. 4. Defibrotide significantly attenuated the ischaemia-induced ST-segment elevation and abolished the reperfusion-related changes (R-wave reduction and Q-wave development) in the electrocardiogram. In addition, defibrotide significantly improved myocardial blood flow in normal and in ischaemic, but not in infarcted sections of the heart. The improvement in blood flow in normal perfused myocardium, but not in the ischaemic area was prevented by indomethacin. 5. Although the area at risk was similar in all animal groups studied, defibrotide treatment resulted in a 51% reduction of infarct size. Indomethacin treatment abolished the reduction of infarct size seen with defibrotide alone. 6. The data demonstrate a considerable cardioprotective effect of defibrotide in the reperfused ischaemic rabbit myocardium. This effect may be related, at least in part, to a stimulation of endogenous prostaglandin formation. Other possible mechanisms are discussed.

Platelets exposed to thrombogenic surfaces adhere, aggregate and release mitogenic substances from their alpha-granules. Endothelium-derived relaxing factor (EDRF) which has been identified as nitric oxide (NO) has been reported to inhibit platelet aggregation. We now report that NO inhibits mitogen release from stimulated human platelets. Mitogenic activity was assayed on mouse fibroblast 3T3 cells using incorporation of 3H-thymidine. Serum from collagen-aggregated platelets significantly increased 3H-thymidine incorporation by 3T3 cells above that of serum from control platelets. Both this increase and platelet aggregation were blocked by NO and prostacyclin in a dose related manner. The inhibitory activity of NO decayed with time when incubated in platelet-free plasma at 37 degrees C, but was still detectable after 2 minutes.

Imidazole inhibits the enzymatic conversion of the endoperoxides (PGG2 and PGH2) to thromboxane A2 by platelet microsomes (IC50: 22 μg/ml; determined by bioassay). The inhibitor is selective, for prostaglandin cyclo-oxygenase is only affected at high doses. Radiochemical data confirms that imidazole blocks the formation of 14C-thromboxane B2 from 14C-PHG2. Several imidazole analogues and other substances were tested but only 1-methyl-imidadole was more potent that imidazole iteself. The use of imidazole of inhibit thromboxane formation could help to elucidate the role of thromboxanes in physiology or pathophysiology.

The actions of prostacyclin (PGX) and several other derivatives of arachidonic acid were examined on spiral-strips of bovine coronary artery. The strips were contracted by PGE2 and thromboxane A2. Although PGH2 usually cause a transient contraction followed by a relaxation, a few strips were only contracted whilst others were only relaxed. Prostacyclin invariably relaxed coronary artery strips. Sodium arachidonate usually relaxed the strips but occasionally had no effect. Indomethacin increased the resting tone and abolished or substantially reduced the relaxation induced by sodium arachidonate. 15-hydroperoxy arachidonic acid (15-HPAA), a specific inhibitor of prostacyclin synthetase, also increased the resting tone, abolished the effects of sodium arachidonate and the relaxation component of the PGH2 response, but did not greatly modify the relaxation induced by exogenous prostacyclin. These results strongly suggest that prostacyclin mediates the relaxation induced by arachidonic acid in bovine coronary artery strips. As PGH2 is avidly converted into prostacyclin by the vascular tissue of several species including man, prostacyclin is probably involved in the local regulation of the coronary vascular bed.

SummaryProstacyclin (PGI2) infused intravenously into anaesthetized rabbits inhibited electrically-induced thrombus formation in the carotid artery, increased bleeding time and inhibited ex vivo platelet aggregation induced by ADP or arachidonic acid. The increase in bleeding time and the inhibition of ex vivo platelet aggregation lasted for as long as the infusion of PGI2 was maintained but rapidly disappeared after infusion was stopped.Prostacyclin is a more potent inhibitor of platelet function, in vivo than prostaglandin E1 (PGE1) or prostaglandin D2 (PGD2).The effects of prostacyclin on all parameters studied except blood pressure were potentiated by the concomitant administration of theophylline, a phosphodiesterase inhibitor.

1. The aim of the study was to assess and quantify any synergism occurring between the stable analogues of prostacyclin (iloprost) and nitric oxide (sodium nitroprusside) with respect to both relaxation of vascular smooth muscle and inhibition of platelet aggregation in the rabbit. 2. Iloprost (0.3-3 ng ml-1) and sodium nitroprusside (0.3-3 ng ml-1) caused dose-dependent relaxation of the rabbit mesenteric and coeliac arteries. 3. Iloprost (0.3-30 ng ml-1) and sodium nitroprusside (0.03-30 micrograms ml-1) caused dose-dependent inhibition of rabbit platelet aggregation induced by adenosine diphosphate or collagen. 4. In combination, iloprost and sodium nitroprusside caused an inhibition of platelet aggregation that was 2-3 fold greater than would be expected by summation, while no such potentiation was observed on vascular smooth muscle. 5. Thus, our results indicate that under physiological conditions the mediators prostacyclin and endothelium-derived relaxing factor (NO) can exert a synergistic action on platelets, but have only an additive effect on vascular smooth muscle.

1 Gas chromatographic and radio-isotope labelling techniques have been used to establish the origin of the arachindonic acid used by the platelet cyclo-oxygenase for the synthesis of pro-aggregatory prostaglandin endoperoxide derivatives. 2 Measurements of total platelet arachidonate content indicated that more than 95% is esterified in the phosphatide fraction of the cells. 3 During aggregation by collagen or thrombin as much as 80% of the total platelet arachidonate may be liberated and transformed by the platelet enzymes into hydroxyacids and other more polar compounds. 4 The phosphatidylethanolamine, phosphatidylcholine and phosphatidylinositol fractions are major sources of the arachidonate thus used. 5 Indomethacin, which prevents platelet aggregation by inhibiting the cyclo-oxygenase, did not affect this release of arachidonate from the phosphatides but did prevent the transformation of arachidonate to endoperoxide derivatives. 6 Mepacrine, a drug which possesses weak anti-phospholipase activity in platelets, also prevents aggregation by collagen or thrombin, but seems to do so by preventing substrate release from the phosphatide fraction. 7 It is suggested that phospholipase A2 plays a key role in the initial events during platelet aggregation induced by collagen.

Microsomal fractions from arterial walls of pigs and rabbits and fundus of rat stomach generate from prostaglandin endoperoxides (PGG2 or H2) an unstable substance, prostaglandin X (PGX) which is a potent inhibitor of platelet aggregation induced by several different substances. Other microsomal fractions including corpus of stomach, lung and ram seminal vesicles generate smaller amounts of PGX from PGG2 or PGH2. Incubation of microsomes from arterial wall or fundus of stomach with platelet-rich plasma under various conditions shows that the enzyme which generates PGX can utilize endoperoxides liberated from platelets or added to the cuvette, thereby preventing, interrupting or reversing the process of platelet aggregation. The generation of PGX is strongly inhibited (IC50 0.43 mug/ml) by 15-hydroperoxy arachidonic acid. These observations are important in the interpretation of vascular diseases such as atherosclerosis and thrombosis and provide a rational basis for the use of anti-oxidants in the prevention and treatment of these diseases.

The effect of prostacyclin on platelet aggregation and adhesion was investigated in everted pieces of rabbit abdominal aorta, from which the endothelium had previously been removed. Citrated human blood, to which different concentrations of prostacyclin (0.1–100 ng/ml) were added, was perfused through the vessels, after which sections were examined and evaluated by light microscopy. Prostacyclin inhibited thrombus formation at concentrations greater than 0.1 ng/ml, whereas 20 ng/ml were required to reduce the amount of adhesion to the subendothelial surface. Thus prostacyclin prevents thrombus formation at much lower concentrations than are needed to inhibit platelet-vessel wall interaction.

Endothelin-1 (ET-1) has been isolated from cultured endothelial cells and might have a role in cardiovascular regulation. To study the fate of labeled ET-1, we prepared (/sup 125/I)-labeled ET-1 (formerly porcine and human ET) and ET-3 (formerly rat ET). Approximately 0.2 microCi (0.2 pmol) was injected into the left ventricle of anesthetized rats and blood samples analyzed for radioactivity for up to 40 min. The animals were then killed and the distribution of radioactivity determined in various organs. Both ET-1 and ET-3 were rapidly removed from the circulation, with more than 60% of the removal occurring in the first minute. Removal of ET-1 was somewhat faster than that of ET-3. The highest uptake of radioactivity was seen in lung, kidney, and liver. When ET-1 was infused into the isolated perfused lung of the guinea pig, 64 +/- 1.9% of the label was retained. Subcellular fractionation of the lung homogenate following infusion of labeled ET-1 showed that 93% of the label was associated with membranes and intracellular organelles, suggesting internalization of the bound ET-1. Together, the results indicate a high density of ET-1 binding sites in the lung, liver, and kidney and that these organs may be important in removing circulatingmore » ET-1.« less

Prostacyclin (PGI2), the predominant metabolite of arachidonic acid in isolated hearts, relaxes strips of bovine coronary artery and is a potent vasodilator in isolated perfused hearts. We have examined the actions of prostacyclin on coronary blood flow in open chest dogs anaesthetised with chloralose. An electromagnetic flow probe was fitted to the left circumflex artery and phasic coronary flow, mean coronary flow (a measure of coronary volume flow over 4 s intervals), and coronary vascular resistance were recorded together with aortic pressure and heart rate. Intravenous infusion of prostacyclin (0.05 to 1.0 microgram.kg.1.min.1), reduced coronary vascular resistance and aortic pressure according to dose, but had only small effects on phasic coronary flow or mean coronary flow. Both tachycardia and bradycardia occurred during infusion of prostacyclin, but 6-oxo-prostaglandin F1alpha (infused at 10 micrograms.kg-1.min-1), the stable degradation produce of prostacyclin, had no cardiovascular effects. The coronary vasodilator effects of prostacyclin were clear when it was injected into the left circumflex artery via a fine catheter distal to the flow probe. Prostacyclin (0.05 to 0.5 microgram) increased phasic coronary flow and mean coronary flow up to 3 fold and reduced coronary vascular resistance without affecting aortic pressure or heart rate, although higher doses had systemic effects. Prostaglandin E1 (0.1 to 0.5 microgram), which also dilated the coronary vessels, had a longer lasting effect and was 1 to 4 times more potent than prostacyclin. Prostaglandin E2, (0.5 to 4 microgram) was less potent than prostacyclin. In four dogs prostacyclin (20 to 500 micrograms) applied epicardially to the left ventricle caused marked and prolonged coronary vasodilatation. Epicardial application of prostacyclin (10 to 25 micrograms) to the right ventricle increased coronary sinus oxygen content with minimal changes in blood pressure. The endoperoxide prostaglandin H2 was a coronary vasodilator of similar potency to prostacyclin, but its analogue U46619 is a vasoconstrictor. Inhibition of cyclo-oxygenase with indomethacin (5 mg.kg-1 i.v.) or sodium meclofenamate (2 mg.kg-1 i.v.) potentiated the coronary dilator effects of prostacyclin given intravenously or into the coronary artery. Cyclo-oxygenase inhibition did not alter the hypotensive effects and increased the coronary vasodilator potency of prostacyclin relative to prostaglandin E2. Thus the sensitivity of the coronary vascular bed to prostacyclin is enhanced when endogenous biosynthesis of prostaglandin-like substances is inhibited. Although the importance of arachidonic acid metabolites in the coronary circulation still requires validation in vivo, it is clear that prostacyclin, and not prostaglandin E2, is the prostaglandin most likely to be involved.

1 The inactivation of prostacyclin (PGI2) in the circulation of anaesthetized dogs has been studied by the blood-bathed organ bioassay technique. 2 Spiral strips of bovine coronary and rabbit coeliac or mesenteric artery detected concentrations of PGI2 of 2 to 5 ng/ml. These tissues were insensitive to concentrations at least 200 fold higher of 15-oxo-PGI2 and 6-oxo-PGF1alpha. 3 PGI2 assayed on bovine coronary artery, rabbit coeliac artery or rat stomach strip, had a half life in blood of 3.0 +/- 0.3 min, indicating non-enzymatic degradation. 4 No disappearance could be detected by bovine coronary artery when PGI2 was infused across the lungs (0.1 to 0.5 microgram kg-1 min-1). However, PGI2 was partially inactivated in passage through vascular beds of hindquarters and liver. 5 Of PGI2 infused into the aorta 35 to 65% escaped inactivation in one complete circulation. Therefore, endogenous PGI2 released from the lungs may function as a circulating hormone.

The metabolism of arachidonic acid (AA) was studied in perfused isolated hearts from guinea pigs. The coronary effluent was continuously bioassayed for prostaglandin-like substances (PLS) using the cascade technique of Vane. Injections of AA in doses between 1--50 microgram into the perfusion fluid prior to the heart produced vasodilatation of the coronary vascular bed followed by a contraction of the rat stomach strip (RSS), chick rectum (CR) and rat colon (RC) as well as relaxation of the bovine coronary artery (BCA). At the higher doses of AA there was also contraction of the rabbit aorta (RbA). The same pattern of effects on the bioassay tissues was seen when prostaglandin endoperoxide (PGH2) was perfused through the heart. The response of the bank of superfused tissues provided evidence for the formation of prostacyclin (PGX or PGI2), PGE2 and PGF2alpha. Chromatographic studies showed that 6-oxo-PGF1alpha together with other prostaglandins was present in the perfusate after acidification, which suggested that the bovine coronary relaxing substance consists mainly of PGI2. Moreover, the rabbit aorta contracting substance (RCS) released in the perfusate was due to prostaglandin endoperoxides and not to thromboxane (TXA2). The formation of PLS from AA was completely blocked after treatment of the heart with the cyclo-oxygenase inhibitors, indomethacin or meclofenamic acid. Pretreatment of the heart with 15-hydroperoxyarachidonic acid (15-HPAA), a selective inhibitor of prostacyclin synthetase, inhibited the effect of AA on the coronary vasculature and diverted the metabolic transformation of AA towards PGE2 and PGF2alpha.

1. The survival of the endothelium-derived relaxing factor (EDRF) released by acetylcholine (ACh) in the rat isolated perfused mesenteric vascular bed was compared to that of exogenously applied nitric oxide (NO) by direct bioassay of the effluent from the mesenteric bed on rabbit aortic strips (RbAs). 2. NO (0.1-10 nmol) produced dose-related vasodilatations in the mesenteric vascular bed and also survived in the effluent in concentrations sufficient to provoke relaxations of the RbAs. ACh (1.7 pmol-300 nmol) caused dose-related vasodilatations in the mesenteric bed but no EDRF was detected in the effluent. This was true even when ACh provoked much larger vasodilatations in the mesenteric bed than those to doses of NO that survived passage through the bed. 3. Removal of the endothelial cells in the mesenteric vascular bed abolished vasodilatations in response to ACh but did not significantly affect those to NO. Survival of NO through the mesenteric bed was not increased. 4. Superoxide dismutase (SOD, 10 u ml-1) did not significantly affect the vasodilator effects of either ACh or NO. It did increase the survival of NO through the mesenteric vascular bed but native EDRF was still not detected in the effluent. 5. The absence of bioassayable EDRF in the effluent following ACh-induced vasodilatations might be explained by the volume into which EDRF is released abluminally being much smaller than that into which it is released luminally.

Inflammation is caused by release of chemicals from tissues and migrating cells. Most strongly implicated are the prostaglandins (PGs), leukotrienes (LTs), histamine, bradykinin, and, more recently, platelet-activating factor (PAF) and interleukin-1. Evidence for their involvement comes from studies with competitive antagonists for their receptors and inhibitors of their synthesis. H1 histamine antagonists are effective for hay fever and some skin allergies such as urticaria, which indicates the importance of histamine in these conditions. Symptoms of rheumatoid arthritis are alleviated by the aspirinlike anti-inflammatory drugs, which inhibit the cyclo-oxygenase enzyme and reduce synthesis of prostanoids. Corticosteroids prevent the formation of both PGs and LTs by causing the release of lipocortin, which by inhibition of phospholipase A2 reduces arachidonic acid release. They suppress the inflammation of rheumatoid arthritis and asthma. Currently, high doses of nonsedating H1 antihistamines and PAF antagonists are being tested for the treatment of allergic asthma.

Among the nonsteroid antiinflammatory drugs there is generally a close correlation between the potency of their inhibition of arachidonate cyclooxygenase, and thus prostaglandin production, and their antiinflammatory activity. One anomaly in this generalization is that whereas aspirin and salicylate are equipotent as antiinflammatory agents, salicylate is less active than aspirin in inhibiting prostaglandin production in vitro. Using rats, we have now measured the concentrations of aspirin and salicylate in plasma and in inflammatory exudates after their oral administration and determined their effects on thromboxane B2 production in clotting blood and prostaglandin (PG) E2 concentrations in the exudates. We have also investigated the effects of both drugs, at concentrations achieved in the exudates, on PGE2 production by nonproliferative explants of acutely inflamed tissues. Aspirin is rapidly metabolized, resulting in peak concentrations of salicylate in the plasma and exudate that exceeded peak concentrations of aspirin by 30- to 50-fold. Furthermore, concentrations of aspirin rapidly declined, whereas high concentrations of salicylate persisted in the plasma and in the exudate for up to 6 hr after a single administration of aspirin. Both drugs reduced PGE2 concentrations in inflammatory exudates by 50-70%, but aspirin was considerably more potent than salicylate in inhibiting thromboxane B2 production in clotting blood. The concentration of salicylate found in inflammatory exudates 6 hr after the administration of aspirin was sufficient to reduce PGE2 production in explants by more than 50%. We conclude that the antiinflammatory action of both drugs depends on the inhibition of PGE2 synthesis by salicylate.

In the rat paw prostacyclin was 5–10 times less potent than PGE 2 in causing oedema, and 5 times less potent in potentiating carrageenin-induced oedema, which it did in a dose-related manner. Prostacyclin was 5 times more potent than PGE 2 in producing hyperalgesia and as potent as PGE 2 in restoring carrageenin-induced hyperalgesia. The effects on oedema were longer lasting than those on hyperalgesia. 6-oxo-PGF 1α was 500 times less potent than PGE 2 in causing oedema by itself and in potentiating carrageenininduced oedema. It had no hyperalgesic activity in this test.

In cardiopulmonary-bypass experiments in greyhounds the effects of adding prostacyclin, prostacyclin plus heparin, and heparin alone to the extracorporeal circulation were compared. With heparin alone platelet count and function were reduced, the pressure differential across the arterial filters rose, platelet deposits were found on the arterial filters, and plasma fibrinogen levels fell. Plasma from these dogs was toxic to fetal-mouse hearts in culture. With prostacyclin alone, fibrinogen levels fell, but the platelets were preserved. With a combination of prostacyclin and heparin, platelet count and function were maintained, there was no consumption of fibrinogen, and there was little deposition on the arterial filters.

1. By using indomethacin to inhibit their intramural synthesis, we have investigated the contribution of prostaglandins to the maintenance of (a) the intrinsic tone of isolated smooth muscle preparations and (b) contractions produced by drugs or high oxygen concentration. 2. When treated with indomethacin, the rat stomach strip and chick rectum preparation slowly relaxed, whether they were bathed in Krebs solution or blood. Although their sensitivity to added prostaglandin was somewhat enhanced, they became insensitive to changes in oxygen or glucose concentration. However, another smooth muscle preparation, the rat colon, was neither relaxed by indomethacin nor contracted by high oxygen concentration. 3. These results support the hypothesis that intramural generation of prostaglandin maintains the tone of some smooth muscle preparations. 4. Contractions of the guinea-pig isolated colon were induced by histamine. These contractions were normally well maintained but in Krebs solution lacking either oxygen or glucose, only the initial spike contraction remained. In the presence of indomethacin the histamine contraction was also poorly sustained, but maintenance was restored by a low concentration of prostaglandin E2. 5. Thus, the effects on smooth muscle of oxygen or glucose lack may also be mediated by reduction in the synthesis or effects of an intramural prostaglandin. Extension of this hypothesis to intestinal and vascular smooth muscle in vivo is discussed.

1. A search was made for an assay tissue with selective sensitivity to vasopressin. Of those smooth muscle preparations tested, the longitudinal muscle of the isolated rectum of the rabbit was the most satisfactory. 2. The rabbit isolated rectum, bathed in Krebs solution, was contracted by acetylcholine, angiotensin II amide, bradykinin and 5-hydroxytryptamine. It was relaxed by vasopressin, oxytocin and the catecholamines. 3. Vasopressin was active in concentrations of 4-100 μu./ml (0·01-0·25 ng/ml) and was 20-30 times more active than oxytocin. Bretylium had no effect on the relaxant action of vasopressin; nor did concentrations of α- and β-adrenoceptor blocking agents sufficient to abolish the actions of catecholamines. Lignocaine reduced the sensitivity of the rabbit rectum to both vasopressin and oxytocin without altering the actions of adrenaline. High concentrations of either vasopressin or oxytocin desensitized the rabbit rectum to the actions of both hormones, without affecting the actions of adrenaline. It was concluded that vasopressin and oxytocin act on a common population of receptors different from those for catecholamines. 4. Phentolamine, unlike other α-adrenoceptor antagonists, reduced the relaxant action of vasopressin on the rectum. 5. When superfused with blood from an anaesthetized dog, the rabbit rectum maintained a higher tone than in Krebs solution; it retained its sensitivity to vasopressin. Pronethalol, administered intraluminally, reduced spontaneous movement and abolished the actions of low concentrations of catecholamines, thereby increasing the specificity of the assay. No other substance tested relaxed the rectum in concentrations likely to be found in blood. 6. Vasopressin was stable in dog's blood; it survived passage through the pulmonary vascular bed; it had a half-life in the circulation of about 1 min. 7. The half-life of vasopressin in the circulation may depend upon the duration of the infusion.

1. Slices of rabbit spleen generate rabbit aorta contracting substance (RCS) and a prostaglandin of the E series when they are vibrated or stirred. 2. The release of both substances is increased by arachidonic acid and inhibited by aspirin-like drugs. 3. When the spleen effluent containing RCS and prostaglandin E is incubated for a further 3 min, the prostaglandin concentration increases and the RCS concentration declines. 4. RCS may therefore be an unstable intermediate in the biosynthesis of prostaglandins.

Demonstration that the aspirin-like compounds block prostaglandin synthesis has not only provided the long-sought explanation of how these drugs work but is throwing considerable light on the physiologic functions of the prostaglandins. It has also suggested possible new uses for the drugs (perhaps, for example, to prevent unwanted abortions) and new contraindications (e.g., where wound healing might be delayed).

Prostaglandins are released into the splenic venous blood when the spleen contracts. Whatever the significance of this release, the liver and lungs provide an efficient protective mechanism to remove almost all the prostaglandin before it reaches the arterial circulation.

Prostaglandins E1, E2, F2α and A2 are enzymically inactivated to different extents during passage through the pulmonary circulation. The enzyme responsible is probably 15-hydroxyprostaglandin dehydrogenase.

The effects of d-amphetamine (0.25 to 8.0 mg/kg) were studied on key-pecking behavior under variable interval (high response rate) and DRL reinforcement (low response rate) in pigeons. Doses of 2.0 mg/kg and above progressively decreased VI responding, as was the case for DRL with doses 4.0 mg/kg and above. No consistent increases in response output on either reinforcement schedule were observed with any dose of the drug, even though prior studies have suggested that amphetamine ought to increase DRL behavior. When their behavior was reduced by d-amphetamine, DRL birds often earned many more grain reinforcements than usual, but frequently did not consume them. No effects of d-amphetamine were observed on a color discrimination tested in the VI birds.

1. Rabbit aorta contracting substance (RCS) and prostaglandins were released from guinea-pig isolated perfused lungs by gentle massage and also by infusion of Prosparol. 2. RCS and prostaglandins were also released by infusion into the pulmonary artery of bradykinin, arachidonic and dihomo-γ-linolenic acids or shock perfusate (containing RCS-releasing factor). 3. Arachidonic and dihomo-γ-linolenic acids caused a prolonged release of RCS and prostaglandins whereas bradykinin and shock perfusate gave a short-lasting output. 4. RCS and prostaglandins, together with histamine were released when superfused chopped lung tissue was agitated. 5. Challenge of sensitized guinea-pigs in vivo led to the release of an RCS-like substance into the carotid arterial blood. 6. Intravenous injection of bradykinin into guinea-pigs also released an RCS-like substance. 7. The release of RCS and prostaglandins was inhibited by aspirin or indomethacin in all experiments. 8. RCS contracted all vascular tissues investigated and also rat stomach strip. 9. The half-life of RCS was estimated as 1-2 minutes.

Among the active substances released during anaphylaxis in guinea-pig lung is a hitherto unidentified substance which has a contracting effect on rabbit aorta. The release of this compound is antagonized by anti-inflammatory agents.

Present methods of extracting prostaglandins (PGs) give poor recoveries from synovial fluid, probably because the PGs bind to protein and are lost in the precipitation stage of extraction. Addition of the non-polar detergent sodium lauryl sulphate prior to extraction improves recovery of PGs. It is suggested that sodium lauryl sulphate competes with PGs for the binding sites.

Estimation of the relative importance of platelets, lungs, liver and peripheral vascular beds in removing this amine from the circulation of the dog shows that the lungs are most active in this respect, and can remove more than 90 per cent in one circulation. This removal does not depend on monoamineoxidase activity.

Experiments with dogs have shown that both prostaglandin E2 and F2α are released when the spleen is contracted by nerve stimulation or by adrenaline. The prostaglandins are not released from the nerves, but derive from smooth muscle cells or other cells with similar adrenergic innervation. The prostaglandin release is not the cause of splenic contraction.

Nitric oxide (.NO) synthase (NOS) activity in subcellular fractions from cultured endothelial cells (EC) and lipopolysaccharide-activated J774.2 monocyte/macrophages was investigated by monitoring the .NO-mediated increase in intracellular cyclic GMP in LLC-PK1 pig kidney epithelial cells. The constitutive NOS in EC (NOSc) was largely membrane-bound, whereas the inducible NOS in J774.2 cells (NOSi) was equally distributed among cytosol and membrane(s). Both the cytosolic NOSc in EC and the membrane-bound NOSi in J774.2 cells were strictly Ca(2+)-dependent, whereas the membrane-bound NOSc in EC and the cytosolic NOSi in J774.2 cells were not. L-Homoarginine and L-arginine-containing small peptides, such as L-arginyl-L-phenylalanine, replaced L-arginine as a substrate for the NOSc in EC and the Ca(2+)-independent NOSi in J774.2 cells, but not the Ca(2+)-dependent NOSi. Thus, irrespective of their intracellular localisation, at least three isoforms of NOS exist, which can be differentiated by their substrate specificity and Ca(2+)-dependency.

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.

Unlike arachidonic acid (eicosatetraenoic acid, C20:4omega-6, A.A.), eicosapentaenoic acid (C20:5omega-3, E.P.A.) does not induce platelet aggregation in human platelet-rich plasma (P.R.P.), probably because of the formation of thromboxane A3 (T.X.A3) which does not have platelet aggregating properties. Moreover, E.P.A., like A.A., can be utilised by the vessel wall to make an anti-aggregating substance, probably a delta17-prostacyclin (P.G.I3). This finding suggests that, in vivo, high levels of E.P.A. and low levels of A.A. could lead to an antithrombotic state in which an active P.G.I3 and a non-active T.X.A3 are formed. Eskimos have high levels of E.P.A. and low levels of A.A. and they also have a low incidence of myocardial infarction and a tendency to bleed. It is possible that dietary enrichment with E.P.A. will protect against thrombosis.

Genetic engineering now allows biological synthesis and large-scale production of several proteins with therapeutic potential. The principal challenge in this sphere is to identify new, medically and commercially significant targets — the province of cell biologists, physiologists and biochemists. In the future, genetic engineering will surely provide invaluable tools for the study of the molecular basis of cellular control and pathophysiology, which will permit biochemists and medicinal chemists to design novel medicines.

Dans ce rapport on decrit l'activation du systeme fibrinolytique chez le lapin par injection intraveineuse de l'endotheline 3 (ET 3 ). L'ET 3 peut stimuler directement la liberation de l'activateur de plasminogene (PA) a partir de l'endothelium, ou bien en conduisant la liberation du facteur relaxant derive de l'endothelium, peut etre implique dans l'activite fibrinolytique de ce peptide

The effects of three anti-inflammatory drugs, which interfere with arachidonic acid transformation by three different enzymes, have been compared by using a simple model of tissue damage and foreign body rejection. In groups of control rats, subcutaneously implanted polyester sponges were rejected after a mean of 12 days. Indomethacin, which selectively inhibits prostaglandin synthesis, did not significantly change time to rejection but BW755C (3-amino-1-[m-(trifluoromethyl) phenyl]-2-pyrazoline), which is a dual inhibitor of prostaglandin and leukotriene synthesis, prolonged time to rejection to a mean of 22 days. The anti-inflammatory steroid dexamethasone, which reduces arachidonic acid metabolism by stimulating the formation of a phospholipase inhibitor, prolonged time to sponge rejection as BW755C did. Treatment with BW755C or dexamethasone was also accompanied by a reduction in total leukocyte numbers in inflammatory exudates collected at 1-14 days, whereas indomethacin increased leukocyte migration on days 1 and 2 and had no effect at later times. These results suggest that the inhibition of the leukotriene-forming lipoxygenase suppresses leukocyte activation and that this leads to a reduced rate of tissue damage in experimental inflammation.

A major clinical problem encountered in the use of non-steroid anti-inflammatory drugs has been the high incidence of gastrointestinal irritation. The mechanisms underlying damage to the gastric mucosa evoked by these drugs are complex. The normal resistance of the gastric mucosa to the back-diffusion of gastric acid from the lumen into the mucosal tissue can be disrupted by topical administration of aspirin as well as several irritants such as ethanol, bile salts and detergents; this results in gastric mucosal damage1. Such an action, however, cannot be the sole mechanism because many non-steroid anti-inflammatory drugs cause gastrointestinal damage when administered parenterally2,3. It has been proposed that a reduction in prostaglandin biosynthesis by aspirin-like drugs4 in the gastric mucosa leads to a fall in local blood flow which, in turn, gives rise to areas of focal ischaemia ultimately developing into erosions5. However, a combination of the direct topical irritant actions and inhibition of prostaglandin formation can lead to a marked potentiation of gastric damage3. Such a situation is likely to be encountered in the clinical use of these compounds following oral administration. We have investigated the relationship between gastric damage and inhibition of cyclooxygenase in the gastric mucosa following oral administration of anti-inflammatory compounds. For these studies, we have measured the ex vivo formation of prostacyclin (PGI2), the major cyclooxygenase product in the rat gastric mucosa which, like other prostaglandins such as PGE2, has gastro-protective actions6. In the inflammatory exudate induced by carrageenin, PGE2 predominates and has pro-inflammatory actions7. We have, therefore, investigated whether selective inhibition of prostaglandin production, assayed as PGE2-like activity, in the inflammatory exudate can be achieved in vivo. We report here that it can.

EXTRACTS of lung1,2 and of small intestine3 from the pig contain previously unidentified vasoactive peptides which, when given intravenously, increase peripheral blood flow and cause a systemic hypotension, both effects being long lasting. Although the peptide fractions from the lung and small intestine are extractable by similar techniques, the vascular responses they elicit are somewhat different. One peptide from the intestine induced a quicker and more pronounced vasodilatation than either the second peptide from intestine or that from lung3. These substances have not yet been completely purified, and so their activity may be due to a single compound or to several different ones. Because of the short supply of extracted peptides, investigation of their biological activity has been limited largely to their peripheral vasodilator effect.

OUR discovery that aspirin-like drugs inhibit the biosynthesis of prostaglandins1–3 led to the proposal that this was their main mechanism of action1,4. Several observations have added force to the theory. These include the correlation between relative potencies as prostaglandin synthetase inhibitors in vitro and anti-inflammatory activity in vivo5–7, the generality of the inhibition in many species and preparations8, and the demonstration of release of prostaglandins in inflammation9,10 and fever11,12. In addition, prostaglandins of the E series cause an inflammatory response in the skin of rats and man13,14 and a fever in cats15,16 and man17.

THE lung has many important metabolic functions1,2 which include the selective activation or inactivation of endogenous peptides. Bradykinin is inactivated on transit through the pulmonary circulation3,4, probably by at least two endopeptidases5, but other peptides such as angiotensin II (refs. 6–10), polistes kinin11 and eledoisin3,4 pass through without loss.

Aspirin-like drugs inhibit the biosynthesis of prostaglandins in all tissues and species so far studied. The concentrations necessary for this action are achieved after therapeutic dosage. The evidence is reviewed which establishes this biochemical action as the basic mode of therapeutic action of aspirin-like drugs as anti-inflammatory, analgesic, and antipyretic compounds. It may also account for the shared side effects, including the hypersensitivity to aspirin-like drugs shown by some allergic patients.

The effects of arachidonate and its major metabolites were examined in vascular beds perfused via the femoral and mesenteric arteries of chloralose-anaesthetised dogs. Close intra-arterial injection of prostacyclin (PGI2, 0.02--2 microgram), PGE2 (0.05--1 microgram) and their precursors, the endoperoxide PGH2 (0.5--2 microgram) and sodium arachidonate (100--550 microgram), all induced vasodilatation. Sodium linoleate (500 microgram) was inactive. Prostacyclin was equally active in both vascular beds, but PGE2 was more potent in the femoral and less so in the mesenteric bed. PGH2 was of similar potency to prostacyclin in both beds, but 6-oxo-PGF 1 alpha (10--100 microgram) was inactive. Thromboxane A2 (TXA2, 1--2 microgram) was a potent vasoconstrictor of the mesenteric bed, but not the femoral bed, although the endoperoxide analogue U46619 was vasocontrictor in both vasculatures. Fatty acid hydroperoxides did not specifically modify the vasodilator effects of PGH2 or arachidonate, presumably because these inhibitors are rapidly reduced in vivo. Indomethacin and meclofenamate potentiated vasodilatation induced by prostacyclin or endoperoxide, but reduced or abolished that caused by arachidonate. The rise in perfusion pressure induced by TXA2 was potentiated and prolonged by indomethacin. Inhibition of synthesis of endogenous prostacyclin, by exacerbating the vasoconstrictor action of TXA2, may have contributed to this effect.

Despite the use of heparin, activation of platelets on the artificial surface of dialyser membranes results in thrombus formation, microembolisation, and thrombocytopenia. To assess the effects on these events of prostacyclin, the most potent inhibitor of platelet aggregation yet discovered, three groups of healthy greyhounds were dialysed with heparin, heparin plus prostacyclin, or prostacyclin alone. Prostacyclin, either alone or with heparin, abolished microembolisation from the dialyser (as estimated by whole-blood screen filtration pressure) and prevented thrombocytopenia. With prostacyclin, dialysis could be carried out without heparin, and there was no clotting of blood within the extracorporeal circuit nor any change in tests of coagulation.