Effect of prostacyclin (PGI2) on platelet adhesion to rabbit arterial subendothelium

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.     

Effect of indomethacin on airway contraction and the release of LTC4-like material

Ovalbumin (OA) and arachidonic acid (AA) were used to induce contractions of sensitized guinea-pig tracheal and lung preparations in the presence and absence of indomethacin. Leukotriene (LT)C₄-like material released from these tissues was extracted from the bathing fluid and measured by radioimmunoassay. Challenge with either OA or AA induced release of LTC₄-like material from both parenchyma and trachea, AA inducing a greater release than OA although OA induced greater contractions. This suggested that OA-induced the synthesis of other bronchoconstrictor compounds than LTC₄. Although indomethacin enhanced OA- and AA-induced contractions of trachea, there was no enhancement of the release of LTC₄-like material, suggesting enhancement by indomethacin was a result of the inhibition of the synthesis of prostaglandin E₂ and not diversion of AA into the lipoxygenase pathway. Indomethacin had no effect on OA-induced contractions of parenchyma, but attenuated those induced by AA. Indomethacin had no modulatory effect on the release of LTC₄-like material in the parenchyma. The results demonstrate that indomethacin does not result in increased synthesis of LTs in the airways.     

Increased production of PGI2-like activity by frozen-thawed rat aorta

The effects of storage conditions, temperature, and time on the ability of the rat thoracic aorta to produce a platelet aggregation inhibitor were investigated. Aortic fragments were incubated in Tris buffer, aliquots of which were then tested for their ability to inhibit ADP-induced human platelet aggregation. The incubation fluid of samples that had been soaked in Tris buffer at 4°C for 24 hours contained no inhibitor activity, whereas the incubation fluid of similar samples that had been kept at 4°C but not soaked in buffer contained comparable inhibitor activity as that of fresh samples. The incubation fluid of samples that had been kept at ?20°C or ?80°C contained greater inhibitor activity than that of fresh samples, and was maintained in ?20°C samples for 7 days, and ?80° samples for 28 days. The aortic inhibitor had similar properties as PGI₂.     

Hepatic metabolism of prostacyclin (PGI2) in the rabbit: Formation of a potent novel inhibitor of platelet aggregation

Metabolism of [9-³H]-PGI₂ was studied in the isolated Tyrode's perfused rabbit liver. Five products, four radioactive and one non-radioactive, were identified in the perfusate: 19-hydroxy-6-keto-PGF_1α, 6-keto-PGF_1α, dinor-6-keto-PGF_1α, pentanor PGF_1α and a 6-keto-PGE₁-like substance. The first two, 19-hydroxy-6-keto-PGF_1α and 6-keto-PGF_1α, represented 5% and 45% respectively, of the total radioactivity; the last two accounted for 39%. The presence of dinor and pentanor derivatives of 6-keto-PGF_1α indicated that β -oxidation and oxidative-decarboxylation occurs in the liver as the major metabolic pathway of PGI₂. One non-radioactive metabolite which co-migrated with authentic 6-keto-PGE₁ was found to inhibit platelet aggregation, having a potency similar to authentic 6-keto-PGE₁, and its effect can be eliminated by boiling and by alkali treatment. This metabolite, having similar Rf value on TLC and biological behavior as 6-keto-PGE₁, may arise from oxidation of 6-keto-PGF_1α via the 9-hydroxyprostaglandin dehydrogenase pathway, as suggested by recovery of tritiated water in the aqueous phase of the perfusate. This material, a potent inhibitor of platelet aggregation, may arise from PGI₂ or its hydrolysis product, 6-keto-PGF_1α.     

Comparison of the effects of prostacyclin (PGI2), prostaglandin E1 and D2 on platelet aggregation in different species

The activity of prostacyclin (PGI₂), PGE₁ or PGD₂ as inhibitors of platelet aggregation in plasma from human, dog, rabbit, rat, sheep and horse was investigated. Prostacyclin was the most potent inhibitor in all species. PGD₂ was a weak inhibitor in dog, rabbit and rat plasma whereas PGE₁ and prostacyclin were highly active. Theophylline or dipyridamole potentiated the inhibition of human platelet aggregation by prostacyclin, PGE₁ or PGD₂. Compound N-0164 abolished the inhibition by PGD₂ of human platelet aggregation but did not inhibit the effects of PGE₁ or prostacyclin. The results suggest that prostacyclin and PGE₁ act on similar sites on platelets which are distinct from those for PGD₂.     

EG-626: Not a thromboxane A2 antagonist, but a PGI2 potentiator in platelet aggregation

Intracerebroventricular administration of PGI₂ or PGE₂ reduced aconitine-induced cardiac arrhythmia in rats. PGF_2α had no antiarrhythmic effect under the same conditions. The ED₅₀ values of PGI₂ and E₂ were 0.25 μg/kg and 1.1 μg/kg, respectively. Central mechanisms may participate in the antiarrhythmic effect of these PGs.     

Effect of the progesterone antagonist RU486 on human myometrial spontaneous contractility and PGI2 release

We studied the effect of antiprogesterone RU 486 on spontaneous uterine contractility and PGI₂ release with human myometrial strips superfused “in vitro”. A decrease of PGI₂ release into the superfusion medium was observed after 20 min superfusion. The inhibition was dose-dependent and reversible. After 20 min washing with tyrode medium without RU 486, the uterine strips recovered their initial rate of release. R5020, a progesterone agonist, did not affect PGI₂ release nor dexamethasone and testosterone. Parallel to the decrease of PGI₂ observed during RU 486 superfusion, the uterine spontaneous contraction frequency decreased, while the amplitude and duration of contractions increased. The alteration of uterine contractility was also rapid, dose-dependent and reversible. Modifications of uterine strip spontaneous contractility, similar to those induced by RU 486, were also observed with superfusions of R5020 at concentrations as low as 10⁻⁹M, dexamethasone (10⁻⁸M), but not with superfusions of testosterone. These observations are not in favour of a progesterone-receptor mediated effect of RU 486 in our model. The mechanism of action may be related to the antiprogesterone specific structure i.e. the bulky substituent at the C-11 position. The RU 486 effect on uterine strip contractility, mimicked by other steroids, could point to a non-specific lipid/membrane interaction. However, the fact that testosterone did not affect motility, may indicate a possible specificity of steroids having a 3 oxo pregnene structure.     

Prostaglandins and renin release: I. Stimulation of renin release from rabbit renal cortical slices by PGI2

Prostaglandins have been shown to be involved in the mechanism of renin secretion in a variety of situations. Both arachidonic acid and prostaglandin endoperoxide have been shown to release renin from cortical slices and to be converted to PGI₂ by cortical microsomes. In the present studies PGI₂ was found to cause a time dependent increase in renin release from rabbit renal cortical slices, a system isolated from any indirect effects that result from the administration of prostaglandins . The stimulation was linear up to 30 minutes and effective over a range of concentrations from 10⁻⁷ M to 10⁻⁵ M. At similar concentrations 6-keto-prostaglandin F_1α was not active on these slices. Thus, it is proposed that PGI₂ exerts a direct effect on the release of renin from cortical cells and may be the mediator of arachidonate or prostaglandin endoperoxide stimulated renin secretion.     

Effect of synthetic enantiomeric cannabinoids on platelet aggregation.

The effect of a synthetic pair of enantiomeric cannabinoids on platelet function was evaluated. The nonpsychotropic enantiomer, the 1,1-dimethylheptyl homolog of (+)-(3S,4S)-7-hydroxy-delta-6-tetrahydrocannabinol (HU-211), was found to be more active in inhibiting ADP-induced platelet aggregation than the highly psychotropic (-)-enantiomer (HU-210). The related (+)-(3R,4R) cannabinoid, HU-213, which lacks the 7-hydroxy moiety, exerted its inhibitory effect within a wider range of concentrations. The results indicate a differentiation between psychotropic activity and inhibition of platelet aggregation in the cannabinoid group of compounds.     

The use of stable prostaglandins to investigate prostacyclin (PGI2)-binding sites and PGI2-sensitive adenylate cyclase in human platelet membranes

Prostacyclin, (PGI₂) is a potent but unstable inhibitor of platelet aggregation, probably acting through stimulation of adenylate cyclase.A stable analogue of prostacyclin with antiaggregatory properties, 5,6-dihydro-PGI₂ (6β-PGI), and PGE₁ can compete for the binding sites labelled by ³H-PGI₂ in human platelet membranes (the affinity being PGI₂ > PGE₁ > 6β -PGI₁). Both 6β-PGI₁ and PGE₁, as well as PGI₂, bind to two classes of binding sites. 6β -PGI₁ and PGE₁ activate adenylate cyclase to the same extent as PGI₂,with a rank order of potency which parallels that observed in binding experiments. The stimulation of this enzyme is brought about by interaction of each these prostanoids with two different classes of components. The comparison of binding and adenylate cyclase data suggests that the sites to which PGI₂, 6β -PGI₁ and PGE₁ bind might be coupled to the activation of adenylate cyclase. Since 6β-PGI₁ seems to act through the same molecular mechanisms as PGI₂, because of its stability it is an useful tool to investigate the mode of action of prostacyclin in platelets.     

The effects of a 5-lipoxygenase inhibitor, AA-861, on PGI2-like substance production in guinea-pig lungs

To determine the effects of AA-861 on PGI₂ production in guinea-pig lungs, 3 g of guinea-pig lung was chopped in 4 ml of buffer (control group), in buffer with 4 μg/ml indomethacin (indomethacin group) and in buffer with 2.5 × 10⁻⁵M AA-861 (AA-861 group). The chopped lungs were incubated for 30 min. 250 μl of incubation medium from each group was assessed before and after 3, 5, 10, 15, 20, 25 and 30 min of incubation. The incubation medium was centrifuged and the supernatant was tested for a PGI₂-like substance (PGI₂) by platelet aggregation inhibition. PGI₂ was produced mainly during the initial 3–5 min of incubation and was decreased thereafter. PGI₂ production was almost completely inhibited in the indomethacin group at all of the incubation times and was partially inhibited in the AA-861 group during the initial 3–5 minutes. Endogenous 5-lipoxygenase products generated in the early stages of incubation seem to be involved in PGI₂ production in guinea-pig lungs.     

Platelet activating factor (PAF) stimulates release of PGI2 from inflamed rabbit gallbladder cell cultures

This study examines the hypothesis that PAF stimulates release of PGI₂ from inflamed rabbit gallbladder explant cell cultures. New Zealand white rabbits underwent bile duct ligation for 72 h (72 h BDL), or sham operation, Sham and 72 h BDL gallbladder explants were placed in culture, and the cells grown to 75% confluence. The cells were exposed to increasing concentrations of PAF for 60 min. The media analyzed for eicosanoid release by EIA and the cells analyzed for cyclooxygenase and prostacyclin synthase content by immunoblot analysis. PAF increased release of 6-keto-PGF_1α from the 72 h BDL gallbladder cell cultures in a dose-related manner which was inhibited by indomethacin preincubation by 90%. The increased 72 h BDL cell release of 6-keto-PGF_1α was not associated with changes in the content of cyclooxygenase or prostacyclin synthase. PAF did not alter eicosanoid release from sham control cell cultures. These data suggest that PAF can only up-regulate endogenous 6-keto-PGF_1α release from the 72 h BDL cells that had been previously stimulated by inflammation. PAF may thus contribute to gallbladder distention and injury by chronic stimulation of inflamed gallbladder PGI₂ release.     

Prostacyclin (PGI2) 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 PGI₂ activity on different isolated tissues in cascase. These data support the hypothesis that antiplatelet and vasodilating activity of PGI₂ generated in the lungs may contribute to the characteristics of the fetal circulation.     

Actions of prostacyclin (PGI2) on adrenergic neuroeffector transmission in the rabbit kidney

In the Tyrode's perfused rabbit kidney PGI₂ (1.3 × 10⁻⁸-3.3 × 10^−7M) dose-dependently inhibited vasoconstrictor responses to sympathetic nerve stimulation, as did PGE₂. The dose-effect curve of the two compounds differed, making PGI₂ the less potent in the low concentration and the more potent in the high. PGI₂ also inhibited the vasoconstrictor response to exogenous noradrenaline, but it had no effect on transmitter release. The main metabolite of PGI₂, 6-keto-PGF_1α, was ineffective both on noradrenaline release and on vascular responses to nerve stimulation or exogenous noradrenaline. It is suggested that PGI₂,if a significant renal prostaglandin, may modulate renal neuroeffector transmission post-junctionally, thereby forming a complement to the prejunctional action of PGE₂.     

Effect of prostacyclin on platelet-activating factor induced rabbit and platelet aggregation

In this paper, the effect of prostacyclin (PGI2) on the aggregation induced by Platelet-activating factor (PAF), a phospholipid mediator of anaphylaxis, was studied. Synthetic PGI2 and PGI2-like activity generated from rabbit aorta were demonstrated to be effective inhibitors of PAF-induced rabbit platelet aggregation and release of 3H-serotonin (3H-5HT).     

Effect of prostacyclin on platelet-activating factor induced rabbit platelet aggregation

In this paper, the effect of prostacyclin (PGI₂) on the aggregation induced by Platelet-activating factor (PAF), a phospholipid mediator of anaphylaxis, was studied. Synthetic PGI₂ and PGI₂-like activity generated from rabbit aorta were demonstrated to be effective inhibitors of PAF-induced rabbit platelet aggregation and release of ³H-serotonin (³H-5HT).     

Increased production of prostacyclin (PGI2) by vascular intimal smooth muscle cells from atherosclerotic rabbit aorta

PGI₂ synthesis by aortic strips obtained from thoracic aorta of rabbits fed a high cholesterol diet was examined and compared with that of control rabbits fed a normal diet. In this report, the amounts of PGI₂ produced were shown as 6-keto-PGF_1α per μg of aortic tissue DNA instead of per mg wet weight. We also investigated PGI₂ synthesis by cultured smooth muscle cells (SMC) obtained from atherosclerotic intima.Basal PGI₂ production by aortic strips from atherosclerotic rabbit aorta was significantly augmented compared with that of controls. Arachidonic acid (AA)-induced PGI₂ production by atherosclerotic aorta was also significantly higher than that of controls. PGI₂ producing capacities of intimal and medial layers, separated from atherosclerotic aorta, were examined and the intimal layer was found to elicit a significantly greater PGI₂ production than the medial layer.Furthermore, cultured intimal SMC obtained from atherosclerotic rabbit aorta produced a greater amount of PGI₂ than medial SMC from normal rabbit aorta at various cultured conditions. These results suggest that the possibility of enhanced PGI₂ production by atherosclerotic aorta may well be considered as a defence mechanism of the vessel wall against damaging stimuli.     

Effect of taurine on arterial, uterine and cardiac PGI2 and TXA2 synthesis in the rat

The influence of taurine (in drinking water for 6 weeks) on PGI₂ and TXA₂ synthesis by some female rat organs was investigated using radioimmunoassay and platelet antiaggregatory bioassay. Taurine 100 and 200 mg/kg/day increased aortic PGI₂ release from 0.59 ± 0.04 (control) to 0.85 ± 0.05 and 1.01 ± 0.06 ng/mg, respectively and that by the myometrium from 0.24 ± 0.02 (control) to 0.38 ± 0.01 and 0.50 ± 0.04 ng/mg wet tissue, respectively (P < 0.05, n = 6). It did not affect PGI₂ and TXA₂ production in the heart or TXA₂ in the aorta. Taurine 200 mg/kg depressed uterine TXA₂ synthesis from 148.6 ± 9.8 (control) to 85.4 ± 6.8 pg/mg (P < 0.05, n = 6). Furthermore taurine 0.4 and 0.8 mM in vitro stimulated PGI₂ released by the myometrial and aortic tissues from pregnant rats. The stimulant effect of taurine on PGI₂ may be related to its antioxidant effect whereas its inhibitory effect on uterine TXA₂ may result from direction of synthesis towards PGI₂. It is concluded that endogenous taurine may participate in regulation of PGs synthesis and that prostanoids may contribute to its known actions. On broad basis, taurine-induced release of PGI₂ may prove of potential value in those ailments characterised by deficiency in PGI₂ release.     

Angiotensin II: A releaser for PGI2 from fetal and newborn rabbit lungs

Angiotensin II (AII) induces generation of prostacyclin (PGI₂) in rabbit lung in vitro at different ages, i.e., fetus, newborn and adult. Particularly, neonatal rabbit lungs display a pattern of sensitivity to AII in producing PGI₂, measured as 6-oxo-PGF_1α, which seems to be higher than that observed in fetal lungs. The PGI₂ release appears to be specific for AII stimulation since the vasoconstriction induced by noradrenaline and ergotamine was not associated with generation of this lipidic material. The inability of PGI₂ to relax the extralobar pulmonary artery in the newborn suggests that the lung microcirculation is the most likely site where the vasodilating and antiaggregatory functions of PGI₂ may have a physiological role.     

Estrogen increases male rat aortic endothelial cell (RAEC) PGI2 release

Estrogen has been proposed as a negative risk factor for development of peripheral vascular disease yet mechanisms of this protection are not known. This study examines the hypothesis that estrogen stimulates rat aortic endothelial cell (RAEC) release of PGI₂. Male Sprague-Dawley rat abdominal aortic 1-mm rings were placed on 35 mm matrigel plates, and incubated for 1 week. The cells were transferred to a Primaria 60-mm dish and maintained from passage 3 in RAEC complete media and experiments performed between passages 4–10. Cells were incubated with Krebs-Henseleit buffer (pH 7.4) containing carrier or increasing concentrations of β-estradiol or testosterone for 60 min. The effluent was analyzed for eicosanoid release of 6-keto-PGF_1α (6-keto, PGI₂ metabolite), PGE₂ and thromboxane B₂ (TXB₂) by EIA (hormone stimulated — basal). Cells were analyzed for total protein by the Bradford method and for cyclooxygenase-1 (COX-1) and prostacyclin synthase (PS) content by Western blot analysis and densitometry. Testosterone did not alter RAEC 6-keto-PGF_1α release, whereas estrogen increased RAEC 6-keto-PGF_1α release in a dose-related manner. Estrogen preincubation (10 ng/ml) decreased COX-1 and PS content by 40% suggesting that the estrogen-induced increase in male RAEC PGI₂ release was not due to increased synthesis of COX-1 or PS. These data support the hypothesis that estrogen stimulation can increase endogenous male RAEC release of PGI₂.