Prostacyclin Generation Research Articles

Stimulation and inhibition of prostacyclin formation in the gastric mucosa and ileum in vitro by anti-inflammatory agents.

1 Homogenates of rat gastric mucosa, forestomach and ileum and dog gastric mucosa reproducibly generated prostacyclin from endogenous substrate.2 Prostacyclin formation was inhibited by pre-incubation with indomethacin, flurbiprofen, naproxen, ketoprofen or meclofenamate (0.1-10 muM).3 BW755C (3-amino-1[m-(trifluoromethyl)-phenyl]-2-pyrazoline) stimulated prostacyclin production in the rat gastric mucosa and ileum with inhibition occurring only at high concentrations (> 200 muM). The stimulation of prostacyclin production by BW755C in rat forestomach homogenates was less pronounced, with inhibition at concentrations > 20 muM.4 BW755C thus exhibits differential activity on prostacyclin production from different gastric tissues in vitro.5 The antioxidant-lipoxygenase inhibitor, nordihydroguiaretic acid (NDGA, 3-15 muM) likewise augmented rat mucosal prostacyclin formation.6 Paracetamol stimulated and, at higher concentrations, inhibited prostacyclin formation (> 1 mM), and had comparable activity in both rat gastric tissues.7 The ability of NDGA and BW755C to enhance prostacyclin generation may reflect the removal of a modulating influence of lipoxygenase products on prostacyclin formation, the diversion of substrate to the cyclo-oxygenase pathway, or free-radical scavenging.

Effect of dipyridamole of prostaglandin generation by human platelets and vessel walls

These experiments were conducted to determine the effects of dipyridemole on human platelet aggregation, platelet thromboxane A 2 (TXA 2) and human vessel wall prostacyclin (PGI 2) generation. Dipyridamole in varying concentrations (5 to 50 μg/ml) had no direct effect on ADP-induced platelet aggregation in vitro, but it potentiated PGI 2-induced platelet aggregation inhibition at these concentrations. Dipyridamole also inhibited arachidonic acid-induced platelet TXA 2 generation at these concentrations. In continuously perfused umbilical vein segments, dipyridamole treatment resulted in stimulation of PGI 2 release determined by bioassay and by measurement of its stable metabolite 6-keto-PGF 1α. Minimum concentration of dipyridamole causing PGI 2 release was 50 μg/ml. These in vitro studies suggest that anti-thrombotic effects of dipyridamole in man are mediated mainly by potentiation of PGI 2 activity and to some extent by TXA 2 suppression. Stimulation of PGI 2 release by human vessels may not be seen in usual therapeutic concentrations.

Thromboxane and prostacyclin generation by intact human vessels in response to balloon catheter trauma

Recent description of thromboxane (TXA 2) synthesis by endothelial cells in addition to prostacyclin (PGI 2) has stimulated interest in the significance of TXA 2 generation by vessel walls. We studied TXA2 and PGI2 release from human umbilical veins with intact and continuous endothelium. Resting TXB 2 (stable metabolite of TXA 2) concentrations in umbilical vein effluent (mean 0.45 ± 0.07 ng/ml) were lower than those of 6-keto-PGF 1α, (stable metabolite of PGI 2) (mean 92 ± 26 ng/ml). Following mechanical trauma to the umbilical veins with a balloon catheter, documented by adherence of indium 111-labeled platelets, both TXA 2 and PGI 2 increased in the venous effluent. Increase in TXB 2 (65%) was less than that in 6-keto-PGF 1α (199%). These data show that a) human vessel walls generate both TXA 2 and PGI 2, b) both TXA 2 and PGI 2 increase following mechanical trauma, the former less than the latter. Vessel wall TXA 2 generation may become pathologically relevant in conditions of decreased PG1 2 generation.

Deficiency of a plasma factor stimulating vascular prostacyclin generation in patients with lupus nephritis and glomerular thrombi and its correction by ancrod: In-vivo and in-vitro observations

Glomerular thrombi occur frequently in active lupus nephritis. Their presence has been correlated with low platelet counts and with subsequent development of glomerular sclerosis. We have examined the plasma PGI 2 generating capacity of 8 patients with active lupus nephritis with thrombi that were to undergo defibrination therapy with ancrod. PGI 2 generation by these plasma samples was significantly decreased as compared both to normals and to 6 individuals with lupus nephritis and no glomerular thrombi. Significant improvement in the capacity to generate PGI 2 was seen in the post-ancrod treatment plasma samples. The pathogenesis of this defect is discussed.

Dipyridamole and aspirin in relation to platelet aggregation and vessel wall prostaglandin generation.

Modification of platelet function and vessel wall prostaglandin synthesis by pharmacologic intervention has attracted considerable attention. We report our observations on the effects of aspirin and dipyridamole alone and their combination on platelet aggregation and vessel wall prostacyclin (PGI2) generation. Although dipyridamole alone had no effects on platelet aggregation, it potentiated the platelet aggregation inhibitory effects of aspirin in vitro in a dose-related fashion. Dipyridamole also enhanced the platelet aggregation inhibitory effect of synthetic PGI2 in vitro. Potentiation of aspirin- and PGI2-induced platelet aggregation inhibition was observed in therapeutic range (5-10 micrograms/ml). In an isolated umbilical vein model dipyridamole stimulated release of PGI2 at much higher concentration (50-100 microgram/ml). Treatment of umbilical vein with aspirin (180 micrograms/ml) for 10 min blocked the spontaneous release of PGI2. In aspirin-treated umbilical vein segments dipyridamole treatment did not cause PGI2 release as in the untreated segments. These experiments suggest that although dipyridamole enhances both aspirin- and PGI2-induced platelet aggregation inhibition in clinically achieved concentrations, much higher levels are necessary for PGI2 release from intact human vessels. Furthermore, aspirin treatment of human vessels may prevent release of PGI2 in response to dipyridamole by blocking cyclooxygenase enzyme.

A thromboxane synthetase inhibitor reorients endoperoxide metabolism in whole blood towards prostacyclin and prostaglandin E 2

During incubation of citrated blood at 37°C the levels of 6-ketoprostaglandin F 1 α (6-keto PGF 1 α ) and prostaglandin E 2 (PGE 2) remain constant, but rise markedly within one minute after the addition of collagen, particularly when thromboxane synthetase is blocked. The amount of 6-keto PGF 1 α formed is dose-dependent for both collagen and the thromboxane synthetase inhibitor (UK-37,248). Moreover, the number of platelets will determine the extent of the 6-keto PGF 1 α jump, that does not occur when blood is drawn after aspirin ingestion. The production of 6-keto PGF 1 α in function of time is composed of a fast platelet-related (intercept) and a slower probably leukocyte-dependent contribution (slope). In the absence of UK-37,248 the intercept is 115 ± 85 pg/ml, the slope is 12.9 ± 7.7 pg/min/ml whereas in the presence of the thromboxane synthetase inhibitor they are 411 ± 177 pg/ml and 56.2 ± 25 pg/min/ml respectively. The present findings indicate that a thromboxane synthetase inhibitor, by not only reducing thromboxane A 2 production but also enhancing prostacyclin generation when blood is exposed to thrombogenic stimuli such as collagen, should be superior to aspirin as an antithrombotic agent, although possible interference by enhanced PGE 2 production should be taken into account.

Decidual vasculopathy and extensive placental infarction in a patient with repeated thromboembolic accidents, recurrent fetal loss, and a lupus anticoagulant

Evidence exists of an association between the presence of a "lupus" anticoagulant in plasma, recurrent fetal loss, and repeated thromboembolic accidents, also in the absence of systemic lupus erythematosus. Presented is an example of this association, with morphologic and biologic studies to elucidate its pathogenesis. In the case reported, the placenta showed massive infarction. In the spiral arteries of the basal plate of the placenta, lesions of intimal thickening, fibrinoid necrosis, acute atherosis, and intraluminal thrombosis were observed. The plasma of the patient contained a lupus anticoagulant and inhibited the formation of prostacyclin by rat aortic rings. Vascular production of prostacyclin is a major natural defense mechanism against thrombosis. Lack of generation of prostacyclin may account for the decidual vasculopathy and consequent placental infarction and for the generalized thrombotic tendency of some patients with lupus anticoagulant.

Hypoxia stimulates prostacyclin generation by dug lung in vitro

We have investigated the possibility that pulmonary biosynthesis of prostacyclin and thramboxane A 2 (TXA 2) may be affected by variations in PO 2. Fresh lung ha ogenates from 8 dogs were incubated for 1 hour at 37°C in Krebs-Ringer solution, at high (492 mnHg) or low (53 mmHg) PO 2. After incubation, the stable metabolites of prostacyclin (6-keto-PGF 1) and of TXA 2(TXB 2) were measured by radioimunoassay. The basal (“pre-incubtion”) levels of these metabolites, measured in tubes in which biosynthesis was arrested by the addition of indomethacin (10 g/ml), were 0.76 ± 0.15 and 0.76 ± 0.11 ng/mg wet wt. for 6-keto-PGF 1α, in high and low PO 2 respectively, and 0.97 ± 0.09 and 0.82 ± 0.15 × 10 −1 ng/mg wet wt. for TXB 2, in high and low P0 2 respectively. Synthesis during incubation in other tubes was estimated by subtracting basal values from those measured at the end of incubation. More 6-keto-PGF 1α was formed in homogenates exposed to low PO 2 (3.01 ± 0.45) than in those exposed kept at high PO 2 (1.89 ± 0.37, p< 0.001), but equal amounts of TXB 2 were found under both conditions. These results suggest that hypoxia may stimulate pulmonary prostacyclin synthesis; a pulmonary vasodilator, prostacyclin may help modulate hypoxic vasocontriction in the lung.

Dietary administration of eicosapentaenoic and linolenic acid increases arterial blood pressure and suppresses vascular prostacyclin synthesis in the rat

The role of the ‘prostacyclin-thromboxane system’ in the regulation of arterial blood pressure was investigated in rats receiving diets which contained different amounts of eixosapentaenoic (EPA) and linolenic acid (LNA). Forty rats were divided into five groups of 8 animals, each group receiving 25 energy (en) % as fat. All diets contained equal amounts of linoleic acid (5 en%) and oleic acid (5 en%). In the control group I, the remaining 15 en% of fat were given as saturated fat. Two groups of animals received cod liver oil as a source for EPA in amounts of 2.5 (group II)_and 5 en% (group III) while the two remaining groups were given diets supplemented with linseed oil as a source for LNA in amounts of 2.5 (group IV) and 5 en% (group V), respectively. After six weeks of feeding period the animals were sacrificed and portions of their isolated aorta incubated in Tris buffer (pH 9.3) for determination of prostacyclin (PGI 2)-like activity. Arterial blood pressure was uncharged in group I animals, but significantly increased in all rats receiving dietary EPA or LNA supplements. This rise is arterial blood pressure was associated with a marked suppression of the appearance of PGI 2-like activity in the incubation buffer while platelet thromboxane release during blood clotting was unchanged. Our results show that dietary adminis- tration of EPA and LNA increases arterial blood pressure in the rat and that this effect is associated with a suppressed generation of vasodilator prostacyclin by vascular tissue.

Dipyridamole and aspirin in relation to platelet aggregation and vessel-wall prostacyclin generation

Dipyridamole and aspirin in relation to platelet aggregation and vessel-wall prostacyclin generation

Changes of plasma 6-keto-PGF1 alpha and thromboxane B2 levels and platelet aggregation after tourniquet ischemia on the upper limb in normal subjects and patients with ischemic heart disease.

To investigate the pathophysiology of ischemic heart disease (IHD), tourniquet ischemia on the upper limb was one and change in platelet aggregation, plasma 6-keto-PGF1 alpha concentrations and plasma thromboxane B2 (TXB2) concentrations were studied. At rest, platelet aggregability and plasma TXB2 concentrations were significantly increased in IHD patients compared with those in normal subjects (p less than 0.001 and p less than 0.001, respectively). In normal subjects, platelet aggregability, plasma 6-keto-PGF1 alpha concentrations and plasma TXB2 concentrations rose significantly during ischemia (p less than 0.05, p less than 0.02 and p less than 0.05, respectively). In addition, plasma 6-keto-PGF1 alpha concentrations were significantly lower in IHD patients than in normal subjects during ischemia (p less than 0.005), though there was no significant change in the level of either group at rest. These results suggest that increase in prostacyclin synthesis in normal subjects during tourniquet ischemia may be a defense mechanism to maintain the balance between prostacyclin and thromboxane A2 (TXA2) and to prevent platelet aggregation induced by the procedure. Increase in platelet aggregation and TXA2 generation in IHD patients at rest indicates a close correlation between IHD and platelet reactivity. Tourniquet ischemia induced a significant increase in prostacyclin generation in normal subjects but not in IHD patients, which suggests the production of prostacyclin was impaired in IHD patients during ischemia. A marked different was obvious in prostacyclin and TXA2 generation between IHD patients and normal subjects, and this difference may play an important role in the pathogenesis of IHD.

Prostacyclin and thromboxane A 2 release in isolated rat lungs

The influence of platelets and platelet membranes on the generation of prostacyclin (PGI 2) and thromboxane A 2(TXA 2) by isolated rat lung and porcine aortic endothelial cell, as measured by RIA of their stable end-producs, 6-oxo-PGF 1α and TXB 2 respectively, was studied. After introduction of either aspirin-treated platelets or membranes from aspirin-treated platelets to the perfusate, 1 5-fold increase in the amount of 6-oxo-PGF 1α and TXB 2 in the perfusate was observed. Treatment of the lung with aspirin produced a 50% reduction in the platelet-stimulated release of PGI 2 and TXA 2. Treatment of the lung with the phospholipase inhibitor, mepacrine, significantly reduced the platelet-stimulated release of PGI 2 and TXA 2. Incubation of endothelial cells with untreated platelet membranes did not alter the generation of PGI 2. These results suggest that platelet-stimulated release of PGI 2 and TXA 2 occurs via mechanical stimulation of phospholipase A 2, liberating arachidonic acid.

Contractile activity and prostacyclin generation in isolated coronary arteries from diabetic dogs.

The present study was aimed at determining the generation of "prostacyclin (PGI2)-like-material" in coronary arteries from normal and diabetic (pancreatectomized) dogs as well as the contractile responses to prostacyclin of preparations from normal, diabetic and insulin-treated diabetic animals. PGI2 produced a dose-dependent relaxation of coronary arteries from normal dogs. In contrast, those from diabetic animals were not related; indeed, at low concentrations PGI2 failed to evoke any effect but at higher ones it induced a distinct contraction. In arteries from diabetic animals treated with insulin, PGI2 induced a biphasic contractile effect, which lay between that of normal controls and untreated diabetics. In addition the basal generation of "PGI2-like-material" by coronary arteries was significantly higher in the diabetic (141 +/- 0.2 pg/mg, mean +/- SEM) than in normal dogs (59 +/- 0.2 pg/mg). The present experiments demonstrate that the generation of "PGI2-like substance" is significantly increased in coronary arteries from diabetic dogs, but the same vessels are unable to respond to added authentic PGI2 with relaxation; on the contrary they react with a distinct positive contractile response.

Effect of sodium loading and depletion on cyclic nucleotides in plasma and aorta. Interaction between prostacyclin and cyclic nucleotides.

The present study evaluated the effect of sodium loading and sodium depletion on cAMP and cGMP content of rat aorta. Enhanced generation of prostacyclin (PGI2) in aorta was noticed in sodium loading and sodium-depletion. As PGI2 is potent vasoactive agent, the interaction between PGI2 generation in the aorta and cyclic nucleotide accumulation in the aorta was investigated. Sodium depletion of rats induced the elevation of both cAMP and cGMP in the aorta and of cAMP in plasma. No change in cyclic nucleotides was noticed following sodium loading. These changes in cyclic nucleotides were felt to reflect the fluctuation of vasoactive agents under various sodium metabolism conditions. These observations may indicate that PGI2 is not a major factor in the regulation of cyclic nucleotide metabolism under sodium loading and sodium depletion. The increased accumulation of cAMP and cGMP in rat aorta in sodium depletion may be due to the cumulative effects of PGI2, catecholamine and probably angiotension II which are induced by sodium depletion.

Influence of nicotine on basal and stimulated prostaglandin biosynthesis in perfused vascular tissue of the rabbit.

1. The prelabelling technique [incorporation of (1-14C)-arachidonic acid] was used to investigate the influence of nicotine (in comparison with acetylcholine) on release and metabolism of arachidonic acid in the isolated perfused rabbit ear. 2. Injection of increasing amounts of nicotine (up to 300 μg) intra-arterially into the isolated rabbit ear did not influence the small basal release of prostaglandins or arachidonic acid. Injection of acetylcholine or bethanechol dosedependently stimulated the release of prostacyclin. Bethanechol stimulated the release of prostaglandin E2, too. The release of arachidonic acid was not significantly increased by injections of acetylcholine or bethanechol. 3. The release of prostaglandins by bethanechol or acetylcholine was abolished by atropine (5 μg/ml) but remained unaffected by hexamethonium (10 μ/ml). 4. Nicotine (10 μg/ml) strongly reduced a histamine-stimulated release of prostaglandins I2 and E2. Acetylcholine (10 μg/ml) also reduced the histamine-evoked release of prostaglandins but to lesser extent. 5. Labelled arachidonic acid, infused through the ear was incorporated at 75.8%. The rest was immediately converted to a small part into prostaglandins. This conversion into PGI2 and PGE2 was reduced by nicotine (10 μg/ml) but not by acetylcholine (10 μg/ml). 6. Infusion of nicotine (10 μg/ml) did not significantly influence the basal release of prostaglandins. Infusion of acetylcholine (10 μg/ml) led to an immediate increase in the release of prostacyclin and — to a somewhat lower amount —of PGE2. The increase underwent tachyphylaxis and was abolished after 30 min of infusion. The basal release of arachidonic acid was initially increased by infusion of acetylcholine and became lower than the initial basal levels after 30 min of infusion. No other substance significantly in-fluenced the release of arachidonic acid. 7. The results show that acetylcholine first stimulates the release of prostacyclin from peripheral vessels via muscarinic receptors and then reduces the stimulated release of prostaglandins possibly due to tachyphylaxis. Nicotine strongly reduces the stimulated release of prostacyclin and PGE2 possibly due to inhibition of the cyclo-oxygenase. A further influence on the following enzymes, e. g. on prostacyclin synthetase cannot be excluded. A reduced generation of prostacyclin by nicotine may lead to a reduced protection of the endothelium and to an increased tendency of platelet aggregation especially in predamaged vessels.

Serum lipoproteins, lipid peroxides and prostacyclin biosynthesis in patients with coronary hear diseases

Serum low-density lipoproteins (LDL)_and high-density lipoproteins (HDL) were prepared by gradient ultracentrifugation and dialysis from 12 healthy subjects and 15 patients with coronary heart disease and hyperlipoproteinemia. In both lipoprotein fractions cholesterol and lipid peroxides were determined. The effect of these lipoproteins on spontaneous prostacyclin biosynthesis in rat aortic slices was studied. Serum lipoproteins were susceptible to peroxidation during the preparation procedure. LDL were more prone to peroxidation than HDL. Little lipid peroxidase were formed in lipoproteins when calcium ions had been removed by EDTA, and when butylated hydroxytoluene (BHT) was present at all stages of their preparation. LDL when prepared without these precautions either from healthy subjects or from patients with coronary heart diseases markedly suppressed prostacyclin generation by rat aortic slices. This inhibition to LDL-lipid peroxides. Peroxide-low LDL prepared from most of the healthy subjects and patients with coronary heart disease and concomitant hyperlipoproteinemia, did not inhibit prostacyclin biosynthesis. However, in one quarter of the patients. LDL was inhibitory. Consequently, in some patients with coronary heart disease, there operate unknown mechanisms which are responsible for the inhbibitory activity of LDL on prosctacylin generation.

Stimulation of prostacyclin release from the epicardium of anaesthetized dogs.

1 The generation of prostanoids in the hearts of anaesthetized dogs was studied by irrigating in situ the epicardial surface with Krebs solution. Prostanoids were measured by direct bioassay on smooth muscles and by radioimmunoassay of 6-oxo-prostaglandin F1 alpha (6-oxo-PGF1 alpha) and prostaglandin E2 (PGE2) in the epicardial irrigation fluid. 2 The epicardial irrigation fluid contained a prostacyclin-like substance, as indicated by the bioassay tissues, and immunoreactive 6-oxo-PGF1 alpha; PGE2-like materials were also detected. By both methods the output of the prostacyclin-like substance, which decreased with time of epicardial irrigation, was increased by manipulating the heart and by adding arachidonic acid (3 microgram/ml), and decreased by adding indomethacin (1 microgram/ml) to the irrigation fluid. 3 Bioassayed prostacyclin and immunoreactive 6-oxo-PGF1 alpha in the epicardial irrigation fluid increased by about 3-5 ng/ml during and after infusion of isoprenaline (0.1 microgram kg-1 min-1). The substance was not released by isoprenaline when indomethacin was added to the irrigation fluid, or when propranolol (0.5 mg/kg) was given intravenously. 4 Aortic constriction, bilateral carotid artery occlusion and intravenous angiotensin infusion all increased output of the prostacyclin-like substance into the epicardial irrigation fluid. The output was abolished by treating the heart with indomethacin (10 mg/kg intravenously or 1 microgram/ml epicardially). 5 The prostacyclin-like substance was also released by all of the above stimuli after the parietal pericardium had been removed and replaced by a plastic sheet. 6 It is concluded that prostacyclin is continually released from tissues close to the epicardial surface and from the pericardium, and that prostacyclin generation increases when cardiac workload increases. Prostacyclin of epicardial or pericardial origin might therefore contribute to metabolic regulation of coronary blood flow.

Differential effects of itanoxone — A new hypolipidemic and hypouricemic drug — On platelet and vascular prostaglandin generation in rats

Itanoxone ((chloro-2′-diphenyl)-4 oxo-4 methylene 2-butyric acid), a newly developed, hypolipidemic and hypouricemic compound with moderate anti-inflammatory activity, showed a short-lived, dose-dependent (20–200 mg/kg, orally), apparently competitive inhibition of platelet malondialdehyde (MDA), stimulated by either thrombin or arachidonic acid. Repeated doses did not result in any cumulative effect. At doses which completely blocked MDA production, itanoxone also inhibited thrombin-stimulated thromboxane B 2 production in platelets but had no measurable effect on vascular prostacyclin generation. Pretreatment with itanoxone partially prevented the inhibitory effect of aspirin on both platelet and vascular prostaglandin synthesis. This suggests that itanoxone — like aspirin — acts at the level of cyclo-oxygenase but with greater selectivity on the platelet enzyme. This pharmacological activity is of great theoretical interest for the potential use of this compound as an antithrombic drug.

Respiratory movements alter the generation of prostacyclin and thromboxane A 2 in isolated rat lungs: The influence of arachidonic acid-pathway inhibitors on the ratio between pulmonary prostacyclin and thromboxane A 2

The influence of hyperventilation on the spontaneous generation of prostacyclin and thromboxane A 2 by isolated rat lungs was studied. Both prostacyclin and thromboxane A 2, as measured by RIA of their stable end-products, 6-oxo-PGF 1α and TXB 2 respectively, were continuously released into the perfusate. However, the concentration of prostacyclin in the perfusate was higher than thromboxane A 2. Under normal ventilation at a rate 40–50 breaths/min, the ratio between these two compounds was 5:1. Increasing the rate of respiration to 100 breaths/min preferentially stimulated the release of prostacyclin. During hyperventilation, the ratio between 6-oxo-PGF 1α and TXB 2 was 12:1. Aspirin and indomethacin suppressed both basal and hyperventilation-stimulated release of prostacyclin and thromboxane A 2. Hydroperoxy-fatty acids and tranylcypromine inhibited only the release of prostacyclin but did not affect the generation of thromboxane A 2. Our findings confirm that the lung generates prostacyclin predominantly, and provide direct evidence that respiratory movements are involved in generation of pulmonary prostacyclin and thromboxane A 2.

Prostacyclin (PGI 2) generation in lungs of fetal and newborn rabbit

Perfused lungs from fetal (26–28 days of gestation) and newborn rabbits preferentially transform arachidonic acid into a substance which mimics PGI 2 activity on different isolated tissues in cascase. These data support the hypothesis that antiplatelet and vasodilating activity of PGI 2 generated in the lungs may contribute to the characteristics of the fetal circulation.