Enhanced prostacyclin generation by phenolic compounds acting as a tryptophan-like cofactor of PG hydroperoxidase

Isolated rat aortae were incubated at 22 degrees C in tris-buffered saline (pH 7.4). The incubation medium was changed every 10 min, and the amounts of prostacyclin (PGI2) in the medium were immediately bioassayed as an inhibitory activity against rabbit platelet aggregation induced by ADP. The addition of arachidonic acid to the medium increased the generation of PGI2 but this was followed by a gradual decrease even in the presence of the same amount of arachidonic acid. The decrease of PGI2 generation from exogenous arachidonic acid was prevented by tryptophan, which is required by PG hydroperoxidase with heme compound as cofactors. MK-447 and its analogues, which are phenolic compounds and exerted tryptophan-like action on the PG endoperoxide biosynthesis by bovine seminal vesicle microsomes, also prevented the decrease of PGI2 generation in isolated rat aortae. The phenolic compounds enhanced PGI2 generation from endogenous arachidonic acid. These results indicate that the phenolic compounds enhanced PGI2 generation in vascular tissue, acting as a tryptophan-like cofactor of PG hydroperoxidase.     

Low prostacyclin synthetase activity of fetal rat aorta. Progressive increase after birth.

Aortae from fetal or 3 weeks old rats produced very small amounts of PGI₂, prostacyclin. This production increased from 4 weeks on, reaching adult values at about ten weeks. This maturation seemed to be predominantly determined by a change in the PGI₂ synthetase system, rather than in arachidonic acid availability, phospholipase or cyclo-oxygenase activity. The anti-oxidant ascorbic acid stimulated prostacyclin production more strongly in adult than in young rat aortae. This finding suggests that the lower production of PGI₂ by young tissues is not due to an enhanced inhibition of prostacyclin synthetase by lipid peroxides.     

The ability of vascular tissue to produce prostacyclin decreases with age

The ability of aortae from young and mature swine to produce prostacyclin (PGI₂) has been determined. PGI₂ was measured as its hydration product, 6-keto-PGF_1α and assayed by stable isotope dilution GC-MS. There was no significant difference in 6-keto-PGF_1α production between intimal strips from young and mature aortae in the basal state. In the presence of saturating concentrations of arachidonic acid, however, intimal strips from young aortae synthesized twice as much 6-keto-PGF_1α as did older tissues. Fatty acid compositions of young and mature aortae were virtually identical, making dietary differences an unlikely explanation for the age-related decrease in PGI₂ synthesis. Both young and mature vascular tissues produced essentially only PGI₂; insignificant amounts of PGE₂ and PGF_2α were found.     

Quantitative determination of 6-keto-PGF1α released by rat aorta: Comparison of mass fragmentography and high resolution gas chromatography with electron capture detection

Mass fragmentography (MF) and high resolution gas chromatography with electron capture detection (HRGC-ECD) were used for measuring 6-keto-PGF_1α, the stable hydrolysis product of prostacylin (PGI₂) released by fresh rings of rat aorta, incubated in the absence of the precursors arachidonic acid or prostaglandin endoperoxide (PGH₂). The incubation medium was acidified, extracted, chromatographed on silicic acid column and derivatized. Comparable results were obtained analyzing each sample by MF and HRGC-ECD. Both methods proved to be suitable in terms of sensitivity and specificity for the measurement of 6-keto-PGF_1α produced by individual rat aortae.     

Prostaglandins and myogenic control of tension in lower esophageal sphincter in vitro

Active tension is produced by the lower esophageal sphincter (LES) of North American opossum $\text{in vitro}$ by a myogenic mechanism. Strips of LES, but not those from the esophageal body, contracted to prostaglandin (PG)F_2α, stable expoxymethano derivatives of PGH₂ and to thromboxane B₂. Stable endoperoxides were more than 500 times more potent than PGF_2α. PGI₂ and 6-keto PGF_1α were weak relaxants of LES strips. LES strips transformed arachidonic acid into contractile substances. This transformation was prevented by agents which interfere with PG synthesis by inhibiting cyclo-oxygenase [indomethacin (IDM), 5,8,11,14-eicosatetraynoic acid (ETA) or thromboxane synthetase [imidazole]. Tranylcypromine 500 μg/ml also inhibited contractions to arachidonic acid. These agents also reduced muscle tone, so that endogenous PG formation may contribute to active tension in the LES. ETA and IDM increased tone before inhibiting it, and this effect was prevented by prior treatment with ETA or imidazole. There may also be an endogenous PG which inhibits LES tone. The possibility that this may be PGI₂ is discussed.     

Inhibition of prostaglandin synthesis in human endothelial cells treated with metabolic inhibitors

Endothelial cell injury is often associated with increased synthesis of prostaglandin (PG)I₂. We observed, however, that endothelial cells treated with metabolic inhibitors which reduce cellular ATP content develop an injury pattern characterized by reduced PGI₂ synthesis. This study examined the relationship between cell injury, arachidonic acid metabolism and ATP content in human umbilical vein endothelial cells treated with 2-deoxyglucose (2DG), a glycolytic inhibitor, and oligomycin (OG), a respiratory chain inhibitor. Either inhibitor alone significantly reduced cellular ATP concentrations, but only OG reduced basal PG synthesis. The combination of 2DG and OG, however, was more effective than either agent alone in reducing cellular ATP content (≥ 50% of control) and inhibiting basal and agonist-stimulated PGI₂ synthesis. This reduced PGI₂ synthesis preceded ⁵¹ chromium release, lactic dehydrogenase release and was not associated with a net release of arachidonic acid from cell membranes. Histamine, A23187 and bradykinin stimulated PGI₂ synthesis in untreated but not in 2DG and OG treated cells. Exogenous arachidonic acid increased PGI₂ synthesis to a similar extent in both 2DG and OG treated and untreated cells. Therefore, reduced PG synthesis in 2DG and OG treated endothelial cells is not due to inhibition of cyclooxygenase. Furthermore, reduced PG synthesis in these cells occurs prior to cell injury and is not strictly associated with cellular ATP depletion.     

Effect of the protein kinase inhibitors, staurosporine and K-252a, on PGI2 production by rat liver cells (the C-9 cell line)

Staurosporine and K-252a, known inhibitors of several protein kinases, stimulated PGI₂ production (measured as 6-keto-PGF_1α in rat liver cells (the C-9 cell line). Preincubation of the rat liver cells with staurosporine or K-252a enhanced the PGI₂ production stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), platelet activating factor (PAF) and the Ca²⁺-ionophore a-23187, but not the PGI₂ synthesis stimulated by exogeneous arachidonic acid. These results suggest that phosphorylation of some proteins or certain amino acids on a protein can regulate arachidonic acid metabolism probably in the pathway leading to deesterification of phospholipids.     

Regulation of prostaglandin production by osteoblast-rich calvarial cells

The effect of various factors upon prostaglandin (PG) production by the osteoblast was examined using osteoblast-rich populations of cells prepared from newborn rat calvaria. Bradykinin and serum, and to a lesser extent, thrombin, were also shown to stimulate PGE₂ and 6-keto-PGF_1α (the hydration product of PGI₂) secretion by the osteoblastic cells. Several inhibitors of prostanoid synthesis, dexamethasone, indomethacin, dazoxiben and nafazatrom, were tested for their effects on the calvarial cells. All inhibited PGE₂ and PGI₂ (the major arachidonic acid metabolites of these cells) production with half-maximal inhibition by all four substances occuring at approximately 10⁻⁷ M. For dazoxiben and nafazatrom, this was in contrast to published results from experiments which have indicated that the compounds stimulated PGI₂ production. Finally, since the osteoblasts is responsive to bone-resorbing hormones, these were tested. Only epidermal growth factor (EGF) was shown to modify PG production. At early time EGF stimulated PGE₂ release, however, the predominant effect of the growth factor was an inhibition of both PGE₂ and PGI₂ production by the osteoblastic cells. The present results suggest that the bone-resorbing hormones do not act to cause an increase in PG by the esteoblast and that any increase in PG production by these cells may be in response to vascular agents     

Endogenous factors affecting arachidonic acid metabolism: I. Biosynthesis of prostacyclin and prostaglandins by carrageenin granulomas of rats

Arachidonic acid was converted by incubated slices of the rat carrageenin granuloma to prostacyclin (PGI₂), prostaglandins (PGs) E₂ and F_2∞ as detected by bioassay and radiochemical assay. PGI₂ was the major product of arachidonic acid metabolism in the granuloma slices. PGI₂ and PGE₂ formation was dependent on the concentration of the substrate and on the age of the granuloma. Slices obtained from 5-day old granulomas produced significantly more PGI₂ than slices prepared from 3-day old or 8- to 9-day old granulomas while PGE₂ generation was not dependent on the stage of the development of the granuloma. Homogenates of granuloma tissue hardly converted arachidonic acid to PGI₂ at all. This was probably due to the presence of an non- dialysable and heat labile material which, when partially isolated, inhibited PGI₂ production by bovine aortic microsomes.     

Enhanced formation of PGI2, a potent hypotensive substance, by aortic rings and homogenates of the spontaneously hypertensive rat

Intact rings and homogenates of aorta from spontaneously hypertensive rats (SHR) contain enhanced capacity over normal rats (NR) to convert arachidonic acid into PGI₂. The PGI₂ synthetic system in SHR is stimulated to a greater extent than NR by norepinephrine. Indomethacin blocks this stimulation. PGE₂ and PGF_2α were detected in much smaller amounts in homogenates (undetected in rings) but their formation was not enhanced by the hypertensive tissue. The identity of PGI₂ was based on 1) direct pharmacological assay on the rat blood pressure. In this system identical vasodepressor responses to PGI₂ are observed after intracarotid and intrajugular administration 2) indirectly as 6-keto PGF_1α isolated after incubation of aortic homogenates with tritiated arachidonic acid and 3) indirectly by GC-MS assay of PGE₂, PGF_2α and 6-keto PGF_1α formed during incubation of aortic homogenates with excess unlabeled arachidonic acid. These results provide additional support to our recent hypothesis that PGI₂, of aortic origin, might actively participate in the regulation of systemic blood pressure. Its enhanced formation by intact hypertensive vascular tissue reflects an increase in the number of enzyme molecules immediately available to the substrate. This could probably be an adaptive response to the elevated levels of catecholamines in the circulation.     

Effect of various prostaglandins on the release of arachidonic acid from cultured fibroblasts

Effect of various prostaglandins on the release of arachidonic acid from [¹⁴C]arachidonic acid labeled fibroblasts was studied. Prostaglandin(PG) F_2α was found to enhance the release of radioactive arachidonic acid from the cells. The stimulatory effect was dose dependent, and was greater than that of bradykinin. The active compounds can be ranked in potency for the release of arachidonic acid from the pre-labeled cells per cent of control: PGF_2α(200.1%)>PGF_1α (141.8%)>PGD₂ (137.1%)>thromboxane B₂ (113.7%)>PGE₂ (109.4%). On the other hand, PGI₂ showed a strong inhibitory effect on the arachidonic acid release from the pre-labeled cells (the value was only 69% of the control), while 6-ketoPGF_1α, an end metabolite of PGI₂, had no effect.     

The influence of gamma radiation on arachidonic acid release and prostacyclin synthesis

Cultured pulmonary artery endothelial cells produce PGI₂ as their primary prostaglandin. Conditions which inhibit cell division have been shown to accelerate the synthesis of this compound. Exposure of endothelial cells to γ raidation results in an irreversible cessation of growth and enhanced production of PGI₂. The level of PGI₂ measured after radiation exposure exceeds that observed in cultures rendered quiescent by serum reduction. This indicates a role for γ radiation in the elevation of PGI₂ levels which is distinct from its effect on cell division. Result presented indicate that exposure to γ radiation does not, in and of itself, elevate PG levels but capacitates cells for enhanced production when presented with appropriate stimuli. Increased PGI₂ synthesis appears to be a result of an observed increase in arachidonic acid release and an activation of cyclooxygenase.     

Prostanoids and hemostasis in chickens: Anti-aggregating activity of the prostaglandins E1 and E2, but not of prostacyclin and prostaglandin D2

Aggregation of chicken thrombocytes was studied in whole blood using an electronic aggregometer. Serotonin (5-hydroxytryptamine, 5HT), arachidonic acid (AA) and collagen, but not adenosinediphosphate (ADP) induced aggregation. Prostaglandin (PG) endoperoxides were essential for arachidonic acid-induced aggregation, but were not involved in 5HT-induced aggregation, as indicated by inhibitory studies with indomethacin. Similar experiments indicated that biosynthesis of endogenous PG endoperoxides contributed to the aggregation induced by low concentrations of collagen, but was of little importance when high collagen doses were employed. PGE₁ and PGE₂ could abolish all types of aggregation studied, whereas prostacyclin (PGI₂) and PGD₂ were without any anti-aggregatory activity at 1 μg/ml. Between 1 and 100 ng/ml PGE₁ and PGE₂ inhibited arachidonic acid- and 5HT-induced aggregation dose-dependently.The lack of any hemostatic function of PGI₂ in chickens was also indicated by the absence of biosynthesis of endogenous PGI₂ in chicken aorta. PGI₂ was assessed as anti-aggregating activity, released by aortic fragments stirred in rabbit platelet rich plasma. Still, the presence of chicken aortic tissue i chicken whole blood inhibited 5HT-, but not arachidonic acid-induced aggregation. This inhibition was not affected by pretreatment of the aortic fragments with indomethacin or pargyline.     

Prostaglandins do not modulate the positive chronotropic and inotropic effects of sympathetic nerve stimulation and injected norepinephrine in the isolated blood perfused canine atrium

In isolated canine atrium, perfused with blood from a donor dog, the infusions of both prostaglandins (PG)I₂ and E₂ (0.1–1 μg/min) into the sinus node arterial cannula neither altered the sinus rate and developed tension nor the positive chronotropic and inotropic responses elicited by either electrical stimulation or by injected norepinephrine. Infusion of arachidonic acid (10–100 μg/min), a precursor of PGs, or indomethacin (15–20 μg/min), an inhibitor of PG synthesis, into the sinus node arterial cannula also failed to alter the increase in sinus rate or developed tension produced by either adrenergic stimulus in the isolated atria. When arachidonic acid, 100–300 μg/kg or PGI₂, 1 μg/kg, were injected into the jugular vein of the donor dog, they produced a fall in systemic blood pressure; this effect of arachidonic acid but not of PGI₂ was abolished by indomethacin, 1 mg/kg. During administration of either arachidonic acid or indomethacin to the donor dog, the positive chronotripic and inotropic responses to adrenergic stimuli in the isolated atria also remained unaltered. These data indicate that PGs do not modulate adrenergic transmission in the blood perfused canine atrium.     

Contribution of lipoxygenase and cyclooxygenase metabolites in the modulation of cyclic nucleotides in the guinea pig mymotrium. Differential effects of carbachol and ionophore A23187

Accumulation of cyclic GMP in estradiol-treated immature guinea pig myometrium was enhanced by carbachol, ionophore A₂₃₁₈₆, unsaturated fatty acids and their hydroperoxides. Cyclic AMP content was elevated only by arachiodonic acid, A₂₃₁₈₇ and PGI₂. Eicosatetraynoic acid (TYA), but not indomethacin prevented all cyclic GMP responses. The effects of A₂₃₁₈₇ and arachidonate on cyclic AMP were accompanied by a parallel increase (2–3 fold) on the generation of PGI₂ by the myometrium. Both events were similarly reduced by indomethacin, TYA, 15-hydroperoxyarachidonic acid and tranylcypromine, suggesting that PGI₂ was involved. Omission of Ca²⁺ or addition of mepacrine of p-bromophenacylbromide abolished the stimulatory effects of A₂₃₁₈₇ and carbachol on cyclic GMP as well as the A₂₃₁₈₇-induced elevations in both PGI₂ and cyclic AMP generation. Thus, with both exogenous arachidonate as well as with endogenous fatty acid, released through an apparent phospholipase A₂-induced activation process, the lipoxygenase pathway was associated with an activation of the cyclic GMP system and the cyclooxygenase pathway, via PGI₂ generation, with an activation of the cyclic AMP system. Carbachol failed to alter both cyclic AMP content and the release of PGI₂ suggesting a cholinergic receptor-mediated fatty acid release process, selectively coupled to the lipoxygenase route.     

Amplification of arachidonic acid metabolism in rat liver cells (the C-9 cell-line) by tosyl- -phenylalanine chloromethyl ketone and phenylmethyl-sulfonyl fluoride

In the presence of the protease inhibitors phenylmethyl-sulfonyl fluoride and N-tosyl- -phenylalanine chloromethyl ketone, prostacyclin (PGI₂) production by rat liver cells treated with epidermal growth factor, platelet-activating factor, 12-O-tetradecanoylphorbol-13-acetate (TPA), and TPA-type tumor promoters (teleocidin and aplysiatoxin) or 1-oleoyl-2-acetylglycerol is amplified. The PGI2 production stimulated by thapsigargin or exogenous arachidonic acid is not amplified. N-Tosyl- -phenylalanine chloromethyl ketone also amplifies TPA's release of radioactivity from cells isotopically labeled with [³H]arachidonic acid. Indomethacin inhibits the amplification of PGI₂ production but not the release of radioactivity. The presence of the protease inhibitors is not required for the amplification of PGI₂ production. Prior incubation of the cells with these inhibitors, followed by their removal, still results in amplified PGI₂ production by cells subsequently treated with TPA, 1-oleoyl-2-acetylglycerol, or platelet-activating factor but not that stimulated by exogenous arachidonic acid. While phenylmethyl-sulfonyl fluoride's amplification of PGI₂ production by cells treated with TPA was blocked by prior incubation with TPA for 20 h, a similar block of amplification in EGF-treated cells was not observed.     

Prostacyclin production after treatment of rat liver cells with tosyl-L-phenylalanine chloromethyl ketone and 12-O-tetradecanoylphorbol-13-acetate is mediated by a phorbol-ester-stimulated intermediate

The prostacyclin (PGI₂) production in rat liver cells by treatment by N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and 12-O-tetradecanoylphorbol-13-acetate (TPA) is not dependent upon the simultaneous presence of both ligands. Preincubation of the rat liver cells with TPA followed by addition of TPCK, as well as preincubation of the cells with TPCK followed by addition of TPA, results in PGI₂ production. Maximum production is found after a 10 min incubation with TPA or after a 120 min incubation with TPCK. Preincubation with TPA for longer than 10 min or preincubation with TPCK for longer than 2 h results in reduced stimulation of PGI₂ production. Dexamethasone does not eliminate the effects of either preincubation or simultaneous addition of TPCK and TPA. EGTA does not affect either preincubation reaction but does completely inhibit PGI₂ production after simultaneous addition of the agents. Preincubation of the cells for 30 min with aspirin completely inhibits the TPCK-TPA-stimulated PGI₂ synthesis. The PGI₂ production following exogenous addition of arachidonic acid to the cells is unaffected by prior treatment of the cells with TPA, TPCK, or TPA plus TPCK. Taken together the data suggest that TPA stimulates the production of an intermediate which activates a Ca²⁺-dependent phospholipase activity. The intermediate is inactivated by a protease which is inhibited by the SH-reactive agent TPCK. The released arachidonic acid is oxygenated by the constitutively expressed cyclooxygenase (prostaglandin H synthase-1).     

Prostacyclin and thromboxane A2 release in isolated rat lungs

The influence of platelets and platelet membranes on the generation of prostacyclin (PGI₂) and thromboxane A₂(TXA₂) by isolated rat lung and porcine aortic endothelial cell, as measured by RIA of their stable end-producs, 6-oxo-PGF_1α and TXB₂ 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 TXB₂ in the perfusate was observed. Treatment of the lung with aspirin produced a 50% reduction in the platelet-stimulated release of PGI₂ and TXA₂. Treatment of the lung with the phospholipase inhibitor, mepacrine, significantly reduced the platelet-stimulated release of PGI₂ and TXA₂. Incubation of endothelial cells with untreated platelet membranes did not alter the generation of PGI₂. These results suggest that platelet-stimulated release of PGI₂ and TXA₂ occurs via mechanical stimulation of phospholipase A₂, liberating arachidonic acid.     

Prostacyclin (PGI2) mediates hypoxic relaxation of bovine coronary arterial strips

Bovine coronary arterial strips (BCA) exhibiting spontaneous tone, relax in response to a decrease in the PO₂ of the batching medium. Experiments were performed to determine if prostaglandins (PGs) mediate the oxygen-induced changes in tension. BCA were equilibrated in Krebs-bicarbonate solution at 37 °C gassed with 95% O₂, 5% CO₂ and tension was measured isometrically. When the PO₂ of the bathing medium was decreased, BCA exhibited reversible reductions in tension. Switching from 95% O₂, 5% CO₂ to 95% N₂, 5% CO₂ (anoxia) elicited an initial relaxation followed by a contraction. In contrast, a change to 5% O₂, 5% CO₂, 90% N₂ (hypoxia) was followed by a sustained relaxation. Re-introduction of O₂ to anoxic strips produced a biphasic response: relaxation followed by contraction. Indomethacin or eicosatetraynoic acid (EYA) increased tone and inhibited the relaxation produced by anoxia or hypoxia. Indomethacin or EYA did not inhibit the relaxation of anoxic strips during re-introduction of O₂, but did inhibit the contraction partially. Relaxation of arterial strips to arachidonic acid (AA) was similar to relaxation to prostacyclin (PGI₂). Anoxia limited the relaxation to AA but not to PGI₂. We conclude that PG synthesis contributes to the basal tone and the hypoxia-induced relaxation of CBA. In addition, hypoxia, unless severe, does not prevent the conversion of AA to PGI₁.     

Anti-platelet aggregating and disaggregating activities of 6,9-methano PGI2

Anti-platelet aggregating and disaggregating activities of the chemically stable 6,9-methano prostaglandin I₂ (6,9-methano PGI₂) were investigated. 6,9-Methano PGI₂ inhibited ADP-induced platelet aggregation in PRP from humans, rabbits and rats. 6,9-Methano PGI₂ also inhibited rabbit platelet aggregation induced by ADP, collagen, thrombin, arachidonic acid and 11,9-epoxy-methano PGH₂. Antiaggregating activities of 6,9-methano PGI₂ were 0.3 to 2.0 times greater than those of PGE₁. 6,9-Methano PGI₂ facilitated platelet disaggregation in a dose related manner. Antiaggregating and disaggregating activities of 6,9-methano PGI₂ were markedly enhanced by incubation with the phosphodiesterase inhibitor, theophylline.