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.     

Investigation of the vascular actions of arachidonate lipoxygenase and cyclo-oxygenase products on the isolated perfused stomach of rat and rabbit

The vascular actions of several prostanoids and arachidonate lipoxygenase products were investigated on the gastric circulation of rat and rabbit perfused with Kreb's solution. Under resting conditions, prostacyclin and PGE₂ produced small decreases in perfusion pressure with prostacyclin being the more potent. During vasoconstriction induced by infusion of noradrenaline, vasopressin or angiotensin II, prostacyclin was 20–40 times as active as PGE₂ as a gastric vasodilator in rat or rabbit stomach. PGF_2α was a less potent vasoconstrictor than noradrenaline, while the epoxy-methano endoperoxide analogue produced a long-lasting vasoconstriction. The putative metabolite, 6-oxo-PGE₁ was less active than prostacyclin as a vasodilator, having comparable activity to PGE₁, whereas 6-oxo-PGF_1α had very little activity. The endoperoxide, PGH₂ reduced perfusion pressure, this effect being inhibited by concurrent infusion of 15-HPETE. The vasodilation induced by arachidonic acid was likewise reduced by 15-HPETE, and abolished by indomethacin infusion. The arachidonate lipoxygenase hydroperoxides were vasodilator in the gastric circulation, the rank order of potency being 12-HPETE > 11-HPETE > 5-HPETE > 15-HPETE in both rat and rabbit stomach. It is possible that such vasoactive lipoxygenase products, may play modulator roles in the gastric mucosa.     

Inflammatory effects of prostacyclin (PGI2) and 6-oxo-PGF1α in the rat paw

In the rat paw prostacyclin was 5–10 times less potent than PGE₂ 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 PGE₂ in producing hyperalgesia and as potent as PGE₂ 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 PGE₂ in causing oedema by itself and in potentiating carrageenininduced oedema. It had no hyperalgesic activity in this test.     

Platelet and cardiovascular activity of the hydantoin BW245C, a potent prostaglandin analogue

The platelet anti-aggregating, cardiovascular and gastro-intestinal actions of a hydantoin prostaglandin analogue, BW245C have been compared with prostaglandin and PGD₂ several species. In human plasma , BW245C was 0.2 times as active as prostacyclin and 8 times as active as PGD₂ in inhibiting platelet aggregation. In rat and rabbit plasma, BW245C was only weakly active but was more potent in sheep and horse plasma. Since the acivity of PGD₂ varied in a parallel fashion, BW245C may interactive with PGD₂ binding sites on platelets. The potency of BW245C as a vasodepressor also varied between species, being 0.5, 0.1, 0.06 and < 0.02 times as active as prostacyclin in the aneasthetised dog, monkey, rat and rabbit respectively.The relative activity of BW245C as an inhibitor of platelet aggregation was more comparable, being 0.08, 0.04 and 0.05 times as active as prostacyclin following intravenous infusion in the rabbit dog and monkey respectively. In the rabbit, BW245C was a highly selective platelet inhibitor with minimal cardiovascular actions, whereas in the dog and monkey, BW245C lowered BP in anti-aggregating doses. The potent platlet anti-aggregating actions of BW245C following parenteral or oral administration makes this hydantoin a potentially-useful anti-thrombotic prostaglandin analogue.     

Modulation of human platelet adenylate cyclase by prostacyclin (PGX)

Prostacyclin (PGX) ($\text{5Z}\text{)-9-}\text{deoxy}\text{-6,9α-}\text{epoxy}\text{-Δ}{}^5\text{-}\text{PGF}{}_{\mathrm{1\alpha }}$ has been found to be a potent stimulator of cAMP accumulation in human platelet rich plasma (PRP), and a direct stimulator of platelet microsome adenylate cyclase. Prostacyclin is, on a molar basis, at least 10 times more potent a stimulator of cAMP accumulation in platelets than PGE₁. The prostacyclin stimulation of platelet cAMP accumulation can be antagonized by the prostaglandin endoperoxide PGH₂, and a PGH₂-induced platelet aggregation is antagonized by prostacyclin. A model of platelet homeostasis is proposed that suggests platelet aggregation is controlled by a balance between the adenylate cyclase stimulating activity of prostacyclin, and the cAMP lowering activity of PGH₂.     

Actions of prostacyclin (PGI2) and its product, 6-oxo-PGF1α on the rat gastric mucosa in vivo and in vitro

The effects of prostacyclin (PGI₂) and its breakdown product 6-oxo-PGF_1α on various aspects of gastric function were investigated in the rat. PGI₂ increased mucosal blood flow when infused intravenously. PGI₂ was a more potent inhibitor of gastric acid secretion in vivo than PGE₂. Like PGE₂, PGI₂ inhibited acid secretion from the rat stomach in vitro. PGI₂ had comparable activity to PGE₂ in inhibiting indomethacin-induced gastric erosions. Thus prostacyclin shares several of the activities of PGE₂, and may be involved in the regulation of gastric mucosal function.     

Metabolism of arachidonic acid and prostaglandin synthesis in the preadipocyte clonal line OB17

Biosynthesis of prostaglandins in ob₁₇ preadipose cells was studied in culture. Dihomo-γ-linolenic acid is exclusively converted to PGE₁. Arachidonic acid behaves quantitatively as a more potent precursor, leading to the synthesis of PGE₂ and 6-keto-PGF_1α (stable product of prostacyclin). In all cases prostaglandin synthesis was confirmed directly by radioimmunoassay. This synthesis is maximal during the growth phase and decreases dramatically after confluence at a time where adipose conversion occurs, suggesting a possible relationship between both events.     

Respiratory movements alter the generation of prostacyclin and thromboxane A2 in isolated rat lungs: The influence of arachidonic acid-pathway inhibitors on the ratio between pulmonary prostacyclin and thromboxane A2

The influence of hyperventilation on the spontaneous generation of prostacyclin and thromboxane A₂ by isolated rat lungs was studied. Both prostacyclin and thromboxane A₂, as measured by RIA of their stable end-products, 6-oxo-PGF_1α and TXB₂ respectively, were continuously released into the perfusate. However, the concentration of prostacyclin in the perfusate was higher than thromboxane A₂. 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 TXB₂ was 12:1. Aspirin and indomethacin suppressed both basal and hyperventilation-stimulated release of prostacyclin and thromboxane A₂. Hydroperoxy-fatty acids and tranylcypromine inhibited only the release of prostacyclin but did not affect the generation of thromboxane A₂. 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 A₂.     

Prostacyclin analogues inhibit canine parietal cell activity and cyclic AMP formation

To assess further the mechanism by which prostacyclin inhibits acid secretion, the actions of two stable prostacyclin analogues on parietal cell function and cyclic AMP formation were tested using enzymatically dispersed cells from canine fundic mucosa. Accumulation of ¹⁴C-aminopyrine (AP) was used as an index of parietal cell response to stimulation. The 16-phenoxy derivative of PGI₂ inhibited accumulation of AP stimulated by histamine (10 μM), with 50% inhibition (ID₅₀) at 10 nM. 6_β-PGI₁ also inhibited the action of histamine (ID₅₀ 0.5μM) but failed to block stimulation by carbachol or the dibutyryl derivative of cyclic AMP (dbcAMP). In similiar concentrations to those producing inhibition of histamine-stimulated AP accumulation, the 16-phenoxy analogue and 6_β-PGI₁ inhibited histamine-stimulated cyclic AMP generation by parietal cells. At 100 fold higher concentrations, 6_β-PGI₁ stimulated cyclic AMP formation, presumably in non-parietal cells. Even in high concentrations the 16-phenoxy analogue failed to increase cyclic AMP formation by mucosal cells. These data indicate that the stable prostacyclin analogues are potent, direct inhibitors of histamine-stimulated parietal cell function and that it is the inhibition, rather than the stimulation, of cyclic AMP formation that is linked to the antisecretory actions of these prostanoid compounds.     

Effect of the prostacyclin synthetase inhibitor tranylcypromine on uterine blood flow in pregnancy

Prostacyclin is a potent vasodilator in a number of vascular beds including the uterus. However, the role of prostacyclin in maintaining uterine blood flow during pregnancy is not well established. Recent reports have appeared suggesting that tranylcypromine can selectively inhibit prostacyclin synthesis. Thus, the present study was undertaken using an unanesthetized chronically catheterized pregnant sheep preparation to evaluate the effects of direct intra-arterial infusions of tranylcypromine on the uterine vasculature of late-term pregnant ewes. Infusions of 1, 3 and 10 mg/min of tranylcypromine led to dose-related reduction in uterine blood flow (16, 21 and 47 percent, respectively) and increased blood pressure (7, 10 and 23 percent, respectively). However, these alterations were not associated with reductions in the uterine production rates of the prostacyclin metabolite, 6-keto-PGF_1α, as determined by unextracted plasma RIA. In addition, pre-treatment of animals with the α-adrenergic blocking agent, phenoxybenzamine, almost totally abolished uterine and systemic blood pressure responses to tranylcypromine. These data suggest that tranylcypromine either releases or elevates levels of an alpha adrenergic stimulant which constricts the uterine and systemic vasculature and does not alter prostacyclin levels at the dose tested.     

Rat osteogenic sarcoma cells: comparison of the effects of prostaglandins E1, E2, I2 (prostacyclin), 6-keto F1alpha and thromboxane B2 on cyclic AMP production and adenylate cyclase activity.

The effects of prostaglandins (PG's) E₁, E₂, I₂ (prostacyclin), 6-keto F_1α and thromboxane (Tx) B₂ were compared in freshly isolated cells from a rat osteogenic sarcoma and in membranes from cultured cells of the same tumour. Cyclic AMP production was measured in cells and adenylate cyclase activity was measured in cell membranes. In both systems PGI₂ was less potent than either of the PGE's, and both TxB₂ and 6-keto PGF_1α were only weak agonists. These effects on bone-derived cells suggest that PGI₂ is unlikely to be a potent bone resorbing agent. Resitance experiments suggested that all the PG's share the same receptor site which appears distinct from the site of action of parathyroid hormone.     

Dual actions of prostacyclin (PGI2) on the rat pregnant uterus

Using strips of rat pregnant uterus, treated with indomethacin to suppress spontaneous contractility, the oxytocic activity of prostacyclin was compared with other prostaglandins. A prostacyclin concentration of 32 ng/ml elicited uterine contractions in all experiments. In this respect prostacyclin was 80 times more active than 6-oxo-PGF_1α but less active than PGE₂ or PGF_2α. Apart from a direct stimulant effect, prostacyclin also exhibited an indirect potentiating action. In threshould concentrations prostacyclin caused a 3-fold potentiation of threshold doses of oxytocin. A lesser 1.5-fold potentiation of PGF_2α was also observed. The implications of these findings in relation to prostacyclin playing a role in parturition are discussed.     

Mapping of the Human Thromboxane Synthase Gene (TBXAS1) to Chromosome 7q34-q35 by Two-Color Fluorescence in Situ Hybridization

Thromboxane synthase (TS) catalyzes the conversion of the prostaglandin endoperoxide into thromboxane A₂ (TxA₂), a potent vasoconstrictor and inducer of platelet aggregation. In concert with prostacyclin TxA₂ plays a pivotal role in the maintenance of hemostasis. Deficiency of platelet TS activity has been shown to result in bleeding disorders. The potent effect of TxA₂ on platelet function and vascular activity suggests a possible involvement of TS in normal and pathophysiological conditions such as cardiovascular disease. To aid in establishing the correlation of TS to disease states, we localized the human TS gene (TBXAS1) to chromosome 7q34-q35 using dual-color fluorescence in situ hybridization.     

An antiserum to 5,6-dihydro prostacyclin (PGI1) which also binds prostacyclin

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 E₁ and D₂. 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.     

Oxidation of prostacyclin and its analogs by three 15-hydroxyprostaglandin dehydrogenases

A study of the oxidation of prostacyclin and some of its analogs by three 15-hydroxyprostaglandin dehydrogenases was undertaken to determine the structural features of these compounds which might influence their rate of enzymatic inactivation. The effect of some structural changes seemed to be determined by the substrate specificity of individual enzymes. Other changes influenced the rate of oxidation by all three enzymes similarily. Among this latter group it was noted that a 15S hydroxyl group is necessary for oxidation to occur and that steric changes in the carboxy side chain and structural changes in the epoxy ring have a greater effect on the affinity of the substrate for the enzyme than on its maximum rate of oxidation. Certain analogs of prostacyclin are not substrates for one or more of the enzymes tested. Of these, (5S)-9-deoxy-5,9α-epoxy-PGF₁ and its methyl ester are potent inhibitors of only the placental enzyme—an interesting case of apparent selective metabolic regulation.     

Prostacyclin: a potent stimulator of adrenal steroidogenesis.

The relative potencies of various prostaglandins were investigated in trypsin-dispersed cat adrenocortical cells. Prostacyclin proved to be the most potent steroidogenic prostaglandin, being 100-1000 times more potent than PGE2. This stimulant effect of prostacyclin was only partially dependent upon the presence of extracellular calcium and was associated with increased levels of cyclic AMP. These data suggest a possible role for prostacyclin in corticosteroidogenesis.     

Effect of leukotrienes, bradykinin and calcium ionophore (A 23187) on bovine endothelial cells: Release of prostacyclin

Incubation of the bovine endothelial cell line, CPAE, with the calcium ionophore (A23187), bradykinin (BK), leukotriene D₄ (LTD₄) or leukotriene C₄ (LTC₄) resulted in concentration dependent increases in prostacyclin release measured as 6-ketoprostaglandin F_1α. The kinetics of induction of prostacyclin synthesis differed among the agents studied. Statistically significant increases in prostacyclin were observed one minute after treatment with A23187, at slightly longer times with bradykinin and after approximately three minutes with the leukotrienes. Two other leukotrienes were tested. Both leukotriene B₄ and leukotriene E₄ (LTE₄) were inactive at con- centrations up to 10 μM. The induction of prostacyclin synthesis by LTC₄ and LTD₄ was inhibited by cycloheximide and actinomycin-D. The effect of BK was inhibited by cycloheximide but not by actinomycin-D. Induction by A23107 was not inhibited by either actinomycin-D or cycloheximide. The results suggest that these agents induced the increases in prostacyclin synthesis by different mechanisms.     

P2-Purinergic Receptors in Vascular Endothelial Cells: from Concept to Reality

Abstract

ATP exerts at least 2 actions on arterial endothelial cells: it stimulates the release of endothelium-derived relaxing factor, a still unidentified vasodilator, and of prostacyclin, a potent inhibitor of platelet aggregation. A study of agonist specificity indicates that these responses are mediated by P₂-purinergic receptors. We have now demonstrated that in these cells, the P₂-receptors are coupled to a phospholipase C hydrolysing phospha-tidylinositol-bisphosphate and that this coupling involves a pertussis toxin-sensitive GTP-binding regulatory protein.     

Effect of hypoxia on prostacyclin production in cultured pulmonary artery endothelium

Exposure of cultured bovine pulmonary artery endothelial cells to varying levels of hypoxia (10% or 0% O₂) for 4 hours resulted in a significant dose-dependent inhibition in endothelial prostacyclin synthesis (51% and 98%, at the 10% and 0% O₂ levels respectively, p <0.05, compared to 21% O₂ exposure values). Release of ³H-arachidonic acid from cellular pools was not altered by hypoxia. Some of the cells were incubated with arachidonic acid (20 μM for 5 min) or PGH₂ (4 μM for 2 min) immediately after exposure. Endothelium exposed to 0% O₂, but not to 10% O₂, produced significantly less prostacyclin after addition of either arachidonic acid (25 ± 5% of 21% O₂ exposure values, n=6, p <0.01) or PGH₂ (31 ± 3% of 21% O₂ exposure values, n=6, p <0.05). These results suggest that hypoxia inhibits cyclooxygenase at the 10% O₂ level and both cyclooxygenase and prostacyclin synthetase enzymes at the 0% O₂ exposure levels. Exposure of aortic endothelial cells resulted in a 44% inhibition of prostacyclin at the 0% exposure level. No significant alteration in prostacyclin production was found in pulmonary vascular smooth muscle cells exposed to hypoxia. These data suggest that the increased prostacyclin production reported in lungs exposed to hypoxia is not due to a direct effect of hypoxia on the main prostacyclin producing cells of the pulmonary circulation.     

Conversions of prostaglandin endoperoxides by prostacyclin synthase from pig aorta

Partially purified prostacyclin synthase from pig aorta converted the prostaglandin (PG) endoperoxide PGH₂ to prostacyclin (PGI₂), and PGH₁ to 12-hydroxy-8,10-heptadecadienoic acid (HHD). Both reactions were inhibited by 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HP) in a dose-dependent fashion. However, the reactions PGH₂ → PGI₂ and PGH₁ → HHD appeared to differ: substrate availability was rate limiting in the latter reaction, while the enzyme became rapidly saturated with PGH₂ and a steady rate of prostacyclin formation was observed at higher substrate levels.