Cerebral arteriolar damage by arachidonic acid and prostaglandin G2.

Application of arachidonic acid or prostaglandin G(2) to the brain surface of anesthetized cats induced cerebral arteriolar damage. Scavengers of free oxygen radicals inhibited this damage. Prostaglandin H(2), prostaglandin E(2), and 11,14,17-eicosatrienoic acid did not produce arteriolar damage. It appears that increased prostaglandin synthesis produces cerebral vascular damage by generating free oxygen radicals.

Prostaglandin endoperoxides modulate the response to thromboxane synthase inhibition during coronary thrombosis.

Prostaglandin endoperoxides (PGG2/PGH2), precursors of thromboxane (TX) A2 and prostaglandins, may accumulate sufficiently in the presence of a TXA2 synthase inhibitor to exert biological activity. To address whether this modulates the response to TXA2 synthase inhibition in the setting of thrombosis in vivo, we examined the interaction of a TXA2 synthase inhibitor (U63,557a) and a TXA2/prostaglandin endoperoxide receptor antagonist (L636,499) in a canine model of coronary thrombosis after electrically induced endothelial injury. U63,557a exerted little inhibitory effect in this model despite a marked reduction in serum TXB2 and urinary 2,3-dinor-TXB2, an index of TXA2 biosynthesis. Combination of the two drugs was more effective than either drug alone. The enhanced effect achieved upon addition of the TXA2/prostaglandin endoperoxide receptor antagonist to the TXA2 synthase inhibitor suggests that the response to the latter compound was limited by the proaggregatory effects of prostaglandin endoperoxides. The increased effect of the combination over the receptor antagonist alone may reflect metabolism of PGG2/PGH2 to platelet inhibitory prostaglandins. This is supported by the following findings: (a) urinary 2,3-dinor-6-keto-PGF1 alpha, an index of prostacyclin biosynthesis, increased after administration of the synthase inhibitor, an effect that was exaggerated in the presence of thrombosis; (b) inhibition of arachidonate-induced platelet aggregation by U63,557a was dependent on the formation of a platelet-inhibitory prostaglandin; and (c) pretreatment with aspirin abolished the synergism between these compounds. These studies demonstrate that prostaglandin endoperoxides modulate the response to TXA2 synthase inhibition in vivo and identify a drug combination of potential therapeutic efficacy in the prevention of thrombosis.

Synergistic activation by collagen and 15-hydroxy-9 alpha,11 alpha-peroxidoprosta-5,13-dienoic acid (PGH2) of phosphatidylinositol metabolism and arachidonic acid release in human platelets.

Collagen stimulates the activation of phosphatidylinositol (PI)-specific phospholipase C (EC 3.1.4.10) in human platelets, as manifested by the disappearance of PI, the transient formation of diacylglycerol (DG), and release of myoinositol. Platelets exposed to collagen also form lysophosphatidylinositol (LPI). Maximum formation of DG occurs within 60 s of the addition of collagen and is in proportion to the concentration of collagen provided, up to 100 micrograms/2 x 10(9) platelets/ml. Hydrolysis of PI, formation of DG, and release of arachidonic acid are all inhibited approximately 68% by aspirin or indomethacin, both of which inhibit platelet cyclooxygenase. This inhibition is reversed by the product of cyclooxygenase activity, 15-hydroxy - 9 alpha,11 alpha - peroxidoprosta - 5,13 - dienoic acid (PGH2), or by the PGH2 analogue and agonist, U-46619. The counteracting effects of either PGH2 or the PGH2 analogue can be blocked, in turn, by a PGH2 antagonist, U-51605. Neither PGH2 nor its stable analogue is, by itself, an efficient stimulus for PI breakdown to DG and LPI in platelets. However, in conjunction with collagen, these agents synergistically promote the net breakdown of PI and the release of arachidonic acid in aspirin-treated platelets. Our findings thereby imply that PGH2 has an important role in regulating both the release of its precursor, arachidonic acid, and the metabolism of PI induced by collagen. Dibutyryl cyclic AMP or prostaglandin D2 (PGD2), a prostaglandin that elevates concentrations of cAMP in platelets by stimulating adenylate cyclase, inhibits the hydrolysis of PI induced by collagen by 70%. The activation of PI metabolism by collagen appears to be inhibited by cAMP independently of any effects of this inhibitor on the formation of PGH2.

High concentrations of arachidonic acid induce platelet aggregation and serotonin release independent of prostaglandin endoperoxides and thromboxane A2.

We examined platelet aggregation and serotonin release, induced by less than 60 micro M arachidonic acid, using washed platelet suspensions in the absence of albumin. The concentration of arachidonic acid used did not cause platelet lysis. Platelet responses induced by less than 20 micro M arachidonic acid were inhibited by aspirin, whereas those induced by above 30 micro M arachidonic acid were not inhibited, even by both aspirin and 5,8,11,14-eicosatetraynoic acid. Although phosphatidic acid and 1,2-diacylglycerol increased after the addition of arachidonic acid in aspirin-treated platelets, the amounts were not parallel to platelet aggregation. Oleic, linoleic and linolenic acids also induced platelet responses, while palmitic, stearic and arachidic acids did not. EDTA, dibutyryl cyclic AMP, apyrase and creatine phosphate/creatine phosphokinase brought about almost the same effects in platelet responses induced by the unsaturated fatty acids, other than arachidonic acid, as those induced by 40 micro M arachidonic acid. These results suggest that the mechanism of the actions of more than 30 micro M arachidonic acid on platelets is the same as that of the other unsaturated fatty acids and is independent of prostaglandin endoperoxides, thromboxane A2 and, perhaps, phosphatidic acid and 1,2-diacylglycerol.

Potentiation of itch and flare responses in human skin by prostaglandins E2 and H2 and a prostaglandin endoperoxide analog.

The itch and erythematous responses induced by intradermal injection of prostaglandin E2 (PGE2), the unstable prostaglandin endoperoxide PGH2 (t1/2 approximately 5 min at 37 degrees C) and the stable endoperoxide analog (15S)-hydroxy-9alpha, 11alpha-(epoxymethano)prosta-5,13-dienoic acid (EPA) were studied in volunteers. The compounds were given alone or in combination with histamine. All the compounds produced flare reaction in the skin; the order of potency was PGE2 greater than PGH2 greater than EPA. PGE2 and PGH2 evoked a sensation of itch in about half of the subjects whereas the same doses of EPA gave no itch response. In combination with histamine all compounds elicited itch of longer duration and flare of larger area than could be accounted for by simple additive effects of any released histamine. The results indicate that the PGs and PG intermediates formed in skin may potentiate the pruritogenic and flare-inducing effects of inflammagens in man.

Role of pressor prostanoids in rats with angiotensin II-salt-induced hypertension.

This study was designed to assess the contribution of thromboxane A2 to high blood pressure in rats with angiotensin II (Ang II)-salt hypertension. Hypertension was induced in rats drinking 0.15 M NaCl by infusion of Ang II (125 ng/min i.p.) for 12 days. Relative to values in water-drinking rats without Ang II infusion, Ang II-salt hypertensive rats exhibited augmentation (p less than 0.05) of blood pressure (from 129 +/- 3 to 217 +/- 12 mm Hg), urinary thromboxane B2 excretion (from 5.4 +/- 0.9 to 25.4 +/- 2.1 ng/day), and thromboxane B2 release from renal cortex slices (from 71.3 +/- 6.7 to 121.1 +/- 14.4 pg/mg) and aortic rings (from 28.8 +/- 2.9 to 115.8 +/- 12.8 pg/mg). Treatment with an inhibitor of thromboxane A2 synthetase, UK 38485, had no effect on blood pressure in normotensive and Ang II-salt hypertensive rats. Treatment with a thromboxane A2 receptor blocker, SQ 29548, decreased blood pressure in Ang II-salt hypertensive rats from 191 +/- 9 to 152 +/- 9 mm Hg after 3 hours, but it had no effect on blood pressure in normotensive rats. Since SQ 29548 interfered with the pressor effects of the prostaglandin endoperoxide analogue U-46619, prostaglandin F2 alpha, and 9 alpha,11 beta-prostaglandin F2, we suggest that the SQ 29548-induced blood pressure reduction in Ang II-salt hypertensive rats is the manifestation of blockade of the vascular actions of one or more endogenous prostanoids including thromboxane A2 and prostaglandin endoperoxides. If so, pressor prostanoids may be contributory factors in the pathogenesis of severe Ang II-salt hypertension in rats.

9,11-Epoxy-9-homo-14-thiaprost-5-enoic acid derivatives: potent thromboxane A2 antagonists.

A novel bicyclic prostaglandin analogue, (1S)-[1 alpha,2 alpha(Z),3 alpha,4 alpha]-7-[3-[(hexylthio)methyl]-7- oxabicyclo [2.2.1]hept-2-yl]-5-heptenoic acid ((-)-10), and its cogeners were found to be potent antagonists at the TxA2 receptor. Compound (-)-10 was the only stereoisomer out of eight possible structures that was active. Thioether (-)-10 was 30-40-fold more potent than another TxA2 antagonist, BM 13.177, in inhibiting arachidonic acid (AA) induced aggregation of human platelet-rich plasma. Compound (-)-10 was effective (I50 = 0.5 +/- 0.4 microM) in inhibiting 9,11-azo-PGH2-induced (0.1 microgram/mL) contraction of guinea pig tracheal spirals. The bronchoconstriction in anesthetized guinea pigs induced by AA was also effectively antagonized by (-)-10 (1 mg/kg, iv); however, in this assay (-)-10 exhibited some direct agonist activity. Radioligand binding studies in washed (human) platelets revealed that (-)-10 is one of the most potent ligands for the PGH2/TxA2 receptor yet described (Kd = 1.6 +/- 0.4 nM).

Comparison of the actions of U-46619, a prostaglandin H2-analogue, with those of prostaglandin H2 and thromboxane A2 on some isolated smooth muscle preparations.

1 The actions of the prostaglandin H2 (PGH2) analogue, U-46619, have been compared with those of PGH2 on thromboxane A2 (TxA2) on a range of isolated smooth muscle preparations in a superfusion cascade system. 2 U-46619 was a potent agonist on guinea-pig lung strip, dog saphenous vein and rat and rabbit aortae. In contrast, U-46619 was weak or inactive on guinea-pig ileum and fundic strip, cat trachea and dog and cat iris sphincter muscles, preparations on which either PGE2 or PGF2 alpha was the most potent agonist studied. 3 PGH2 was active on all of the preparations and displayed little selectivity. On some of the preparations, the actions of PGH2 may have been mediated indirectly by conversion to other prostanoids. 4 In contrast, TxA2 displayed the same pattern of selectivity as U-46619, being a potent agonist on the lung strip and vascular preparations but weak or inactive on the others. 5 It is suggested that U-46619 is a selective TxA2-mimetic and that it should therefore be a valuable tool in the study of the actions of TxA2.

Renal vascular responsiveness to arachidonic acid in experimental diabetes.

1. Isolated perfused kidneys from diabetic rats (duration 4-6 and 20-24 weeks) were more sensitive to the vasoconstrictor effects of arachidonic acid than kidneys from age-matched control rats. Sensitivity diminished with age in both control and diabetic groups. 2. The enhanced vasoconstrictor effect of arachidonic acid in diabetic rat kidneys was associated with increased conversion to prostaglandins. 3. The renal vasoconstrictor response to arachidonic acid in both groups was reduced by thromboxane A2/prostaglandin H2 receptor antagonism but not by inhibition of thromboxane synthase. 4. Diabetic rat kidneys were also more sensitive to the vasoconstrictor effects of the endoperoxide analogue, U46619, while vasoconstrictor responses to phenylephrine were not markedly different from those of control rat kidneys. 5. In conclusion, prostaglandin endoperoxides appear to mediate arachidonic acid-induced vasoconstriction in diabetic and control rat kidneys. The enhanced renal vasoconstrictor response to arachidonic acid in diabetic rats results from increased sensitivity to endoperoxides and increased formation of endoperoxides from arachidonic acid.

Preparative HPLC purification of prostaglandin endoperoxides and isolation of novel cyclooxygenase-derived arachidonic acid metabolites.

A preparative HPLC purification scheme for the isolation of prostaglandin endoperoxides prepared by short-time incubation of [1-14C]-labelled arachidonic acid (AA) with sheep seminal vesicle microsomes was developed. Milligram quantities of prostaglandin G2 (PGG2) and prostaglandin H2 (PGH2) were obtained in greater than or equal to 95% purity within shortest time. Furthermore, careful application of this HPLC technique led to the isolation of two minor [1-14C]-labelled fractions which according to their spectral and chromatographic characteristics, were identical with 15(S)-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) and 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE). Another HETE substituted at either C11 or C12 was also present. The formation of these products was mediated by cyclooxygenase as evidenced by aspirin (100 microM) and indomethacin (10 microM) inhibition. Sulfhydryl-blocking agents such as p-hydroxymercuribenzoate (1 mM) and/or the 12-lipoxygenase inhibitor esculetin (100 microM) were without effect. In addition to these AA metabolites four other fractions contained arachidonate-derived endoperoxides with antiaggregatory properties, all of which released malondialdehyde upon incubation with thromboxane A2 synthase. No thromboxane formation was observed although turnover numbers were comparable to those of PGG2 and PGH2. The formation of these endoperoxides did not occur via enzymatic or non-enzymatic degradation of PGG2 or PGH2. The exact chemical nature of these endoperoxides remains to be established.

Effects of OKY 1581 on bronchoconstrictor responses to arachidonic acid and PGH2.

The influence of OKY 1581, a thromboxane synthase inhibitor, on airway responses to arachidonic acid and endoperoxide, [prostaglandin (PG) H2], were investigated in anesthetized, paralyzed, mechanically ventilated cats. Intravenous injections of arachidonic acid and PGH2 caused dose-related increases in transpulmonary pressure and lung resistance and decreases in dynamic and static compliance. OKY 1581 significantly decreased airway responses to arachidonic acid but not to PGH2. Sodium meclofenamate, a cyclooxygenase inhibitor, abolished airway responses to arachidonic acid but had no effect on airway responses to PGH2. OKY 1581 or meclofenamate has no effect on airway responses to PGF2 alpha, PGD2, or U 46619, a thromboxane mimic. In microsomal fractions from the lung, OKY 1581 inhibited thromboxane formation without decreasing prostacyclin synthesis or cyclooxygenase activity. These studies show that OKY 1581 is a selective thromboxane synthesis inhibitor in the cat lung and suggest that a substantial part of the bronchoconstrictor response to arachidonic acid is due to thromboxane A2 formation. Moreover, the present data suggest that airway responses to endogenously released and exogenous PGH2 are mediated differently and that a significant part of the response to exogenous PGH2 may be due to activation of an endoperoxide/thromboxane receptor, since responses to PGH2 are blocked by the thromboxane receptor antagonist SQ 29548.

Thromboxane receptor antagonist properties of SQ 27,427 in the anesthetized guinea pig.

The TXA2 receptor antagonist properties of SQ 27,427 (a novel oxabicyclo[2.2.1]heptane derivative) were studied in vivo in the anesthetized guinea pig where changes in pulmonary resistance, dynamic compliance, and mean arterial blood pressure were measured. Both the bronchoconstrictor and pressor responses to arachidonic acid (AA) and to the stable TXA2 mimic 9,11-azoPGH2 (AZO) were taken as indices of in vivo TXA2 receptor activation. The administration of SQ 27,427 (0.1-1.0 mg/kg i.v., and 10.0 mg/kg p.o.) caused dose-related inhibitions of both AA- and AZO-induced bronchoconstriction. Relative specificity of this antagonism was evidenced by the failure of SQ 27,427 (1.0 mg/kg i.v.) to inhibit histamine-induced bronchoconstriction. In the same experiments the pressor response to AA was reversed to a depressor response by SQ 27,427. This reversal was abolished by indomethacin. The pressor response to AZO was antagonized by SQ 27,427, but not by indomethacin. The reversal of the pressor response to AA by SQ 27,427 may be due to the unmasking of the depressor effect of a cyclooxygenase product, i.e., prostacyclin. It is concluded that SQ 27,427 is a relatively specific TXA2 receptor antagonist in vivo in the guinea pig.

Effects of SQ 27,427, a thromboxane A2 receptor antagonist, in the human platelet and isolated smooth muscle.

The TxA2 receptor antagonist properties of SQ 27,427 [a cyclohexylcarbinol-7-oxabicyclo(2.2.1)heptenoic acid analog] were studied in vitro both in the human platelet and various isolated smooth muscle preparations. SQ 27,427 was found to be a potent inhibitor of human platelet aggregation induced by arachidonic acid, ADP, epinephrine, collagen and the stable TxA2 agonists 9,11-azoPGH2 and SQ 26,655. Inhibition of platelet aggregation was achieved at concentrations of SQ 27,427 which did not alter TxB2 levels. SQ 27,427 was found to weakly inhibit the formation of TxB2 from arachidonic acid and had no effect on the synthesis of PGE2 or PGI2 from arachidonic acid. SQ 27,427 was also found to be a weak stimulator of platelet adenylate cyclase, being 1000 times less potent than PGI2. In isolated smooth muscle experiments, SQ 27,427 was shown to be a potent and specific TxA2 receptor antagonist. It caused competitive antagonism of 9,11-azoPGH2-induced contractions of vascular, respiratory and gastrointestinal smooth muscles. This antagonism was specific, as responses to norepinephrine, serotonin, PGE2, PGI2, PGF2 alpha, histamine, carbachol and KCl were not altered by SQ 27,427.

Arachidonic acid induces human platelet-fibrin retraction: the role of platelet cyclic endoperoxides.

Arachidonic acid (0.2-0.8 mM) retracts clots formed in human citrated platelet-rich plasma by batroxobin. Extracellular calcium ions, but not the secretion of ADP by platelets, are required. AA-induced clot-retraction requires cyclo-oxygenase but not thromboxane synthetase activity since the retraction is inhibited by aspirin but not by selective inhibitors of thromboxane synthesis. The data indicate that endogenous cyclic endoperoxides mediate the retraction. Moreover, intact endoperoxide/thromboxane receptors also seem to be necessary because clot retraction is inhibited by thromboxane receptor antagonists.

Prostaglandin H2 directly lowers human platelet cAMP levels.

In the present study we investigated the ability of prostaglandin H2 (PGH2) to lower platelet cAMP levels independent of thromboxane A2 (TXA2) production. Human platelet-rich plasma was incubated (37 degrees C) with 13 nM prostacyclin (PGI2) to increase platelet cAMP levels. Addition of 500 microM arachidonic acid (AA) to PGI2-treated platelets resulted in a return of cAMP to control levels. Inhibition of both PGH2 and TXA2 production with indomethacin (20 microM) blocked the ability of AA to lower PGI2-stimulated increases in cAMP. On the other hand, selective inhibition of TXA2 production with the thromboxane synthetase blocker 7-(1 imidazoyl) heptanoic acid (7-IHA; 10 microM), did not prevent the cAMP lowering effect of added AA. These results provide evidence that PGH2 need not be converted to TXA2 in order to reduce platelet cAMP levels.

Study of the two pathways for arachidonate oxygenation in blood platelets.

During collagen-induced blood platelet aggregation, arachidonic acid is set free from membrane phospholipids and subsequently converted into 12-hydroxyeicosatetraenoic acid by arachidonate lipoxygenase and into thromboxane A2, 12-hydroxyheptadecatrienoic acid (HETE) and malondialdehyde by cyclooxygenase and thromboxane synthase. Lipoxygenase and cyclooxygenase have optimal activity at neutral to basic pH, while the thromboxane synthase is pH-independent between 5 and 9. These enzymes are membrane-bound. The cyclooxygenase is rapidly inactivated upon membrane disruption by nonionic detergents or phospholipid degradation with phospholipase A2. It was found that platelet phospholipase A2 preferentially splits off fatty acid with four double bonds. Eicosatetraynoic acid was used to investigate the physiological function of the arachidonate lipoxygenase during collagen-induced aggregation of rat blood platelets. This fatty acid is a more efficient inhibitor of lipoxygenase than of cyclooxygenase. At an inhibitor concentration of 0.6 microgram/ml, platelet aggreation, 12-hydroxyeicosatetraenoic acid production as well as 15-hydroxytryptamine release are completely inhibited, while there is an apparent stimulation of the cyclooxygenase. These results indicate that arachidonate lipoxygenase is essential for irreversible blood platelet aggregation.