Thromboxane A2 Mimic Research Articles

Hyperoxia and alkalosis produce pulmonary vasodilation independent of endothelium-derived nitric oxide in newborn lambs.

Supplemental oxygen and alkalosis are the most effective treatments used to lower pulmonary arterial pressure in children with pulmonary hypertensive disorders. However, their mechanisms of action are unknown. Endothelium-derived nitric oxide (EDNO) is an important mediator of pulmonary vascular tone and produces potent pulmonary vasodilation during pulmonary hypertension. In vitro evidence suggests that EDNO may mediate the vasodilating effects of oxygen. To investigate whether EDNO synthesis mediates the pulmonary vasodilation produced by hyperoxia [normocarbic ventilation with 100% oxygen, arterial oxygen tension > 450 torr (60 kPa)] or alkalosis (hyperventilation with 21% oxygen, pH > 7.55) in vivo, eight intact newborn lambs were studied during similar degrees of pulmonary hypertension induced either by the infusion of U46619 (a thromboxane A2 mimic) or N omega-nitro-L-arginine (an inhibitor of EDNO synthesis). The lambs were sedated, paralyzed, and mechanically ventilated. Meclofenamic acid was infused to inhibit prostaglandin synthesis. During pulmonary hypertension induced by U46619, pulmonary arterial pressure and pulmonary vascular resistance were significantly decreased by acetylcholine (an EDNO-dependent vasodilator) (23.1 +/- 3.4% and 43.3 +/- 14.5%, respectively), hyperoxia (26.8 +/- 7.8% and 32.9 +/- 10.6%), and alkalosis (32.1 +/- 10.3% and 36.1 +/- 17.0%) (p < 0.05). During pulmonary hypertension induced by N omega-nitro-L-arginine, the decreases in pulmonary arterial pressure and pulmonary vascular resistance produced by acetylcholine (9.6 +/- 6.4% and 23.9 +/- 14.1%, respectively) were significantly attenuated (p < 0.05), but the decreases produced by hyperoxia or alkalosis were unchanged. Therefore, hyperoxia and alkalosis can produce pulmonary vasodilation independent of EDNO synthesis in the intact newborn lamb.

Reconstitution of thromboxane A2 receptor-stimulated phosphoinositide hydrolysis in isolated platelet membranes: involvement of phosphoinositide-specific phospholipase C-beta and GTP-binding protein Gq.

Activation of human platelets by the arachidonic acid metabolite thromboxane A2 and the thromboxane A2 mimic U46619 is mediated through phosphoinositide-specific phospholipase C-catalysed hydrolysis of phosphoinositides. We have established conditions to reconstitute U46619-stimulated phosphoinositide breakdown by addition of guanine nucleotides and soluble platelet phospholipase C activities to isolated 32P-labelled membranes. Receptor-activated phosphoinositide hydrolysis was observed in the presence of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) or GTP plus U46619. Phosphoinositide hydrolysis was dependent on both GTP and U46619, with half-maximal stimulation observed at 5 microM and 500 nM respectively. Phospholipase C isoenzymes beta, gamma 1, gamma 2 and delta were purified from platelet cytosol and their ability to reconstitute GTP[S]-dependent and GTP/U46619-dependent phosphoinositide hydrolysis determined. Phospholipase C-beta and -delta, but not phospholipase C-gamma 1 or -gamma 2, catalysed phosphoinositide breakdown in the presence of GTP[S]. In contrast, only phospholipase C-beta was able to reconstitute GTP-dependent U46619-induced hydrolysis. The participation of GTP-regulatory proteins in the reconstitution of GTP[S]- and GTP/U46619-induced phosphoinositide hydrolysis was examined using antibodies to the C-terminals of the alpha-subunits of three of the heterotrimeric GTP-binding proteins expressed in human platelets Gq, Gi2 and Gi3. Anti-Gq antibody, but not anti-Gi2 or Gi3 antibody, inhibited both GTP[S]- and GTP/U46619-dependent reconstitution of phosphoinositide hydrolysis with phospholipase C-beta. In contrast GTP[S]-stimulated hydrolysis by phospholipase C-delta was not inhibited by any of the G-protein antibodies. These results show the functional specificity of GTP-binding proteins and phospholipase C isoenzymes in mediating agonist-induced phosphoinositide hydrolysis in human platelets.

Effects of prostanoids and indomethacin on isolated smooth muscle from the human lower oesophagus.

Smooth muscle strips representing the longitudinal and the circular muscle layers of the oesophageal body and the oesophagogastric junction of the human oesophagus were mounted in organ baths and isometric tension recorded. Transmural field stimulation was applied to strips from both layers of the oesophageal body and to strips from the longitudinal muscle layer of the oesophagogastric junction. This resulted in contractions after cessation of the stimulus train in circular muscle, and in contractions during stimulation in longitudinal muscle preparations. Indomethacin lowered active tension in strips from the circular muscle layer of the oesophagogastric junction, and decreased responses to transmural field stimulation in all strips from the longitudinal muscle layer. The response to transmural field stimulation was increased in strips from the circular muscle layer of the oesophageal body, suggesting an inhibitory function of endogenous prostanoids in this muscle layer. Prostaglandin (PG) F2 alpha and the thromboxane A2 mimic U-46619 increased the responses to transmural field stimulation in all muscle types and the active tension in circular muscle from the oesophagogastric junction. PGE1 and PGE2 increased the responses to transmural field stimulation strips from the longitudinal muscle layer. In circular muscle strips from the oesophageal body responses to transmural field stimulation were inhibited. PGE1 decreased active tension in circular muscle strips from the oesophagogastric junction, whereas PGE2 produced inconsistent effects. These results suggest a role of prostanoids in the regulation of neuromuscular function in the human lower oesophagus. Moreover, exogenous prostaglandins may modulate oesophageal motor function.

Role of superoxide anion and endothelium in vasoconstrictor action of prostaglandin endoperoxide.

The vasoconstrictor actions of prostaglandin (PG) endoperoxide, PGH2, were examined in isolated rabbit aortic rings suspended for measurement of isometric tension. In aortic rings with an intact endothelium, PGH2 caused concentration-dependent contractions which were blocked by SQ 29548 (a PGH2-thromboxane A2 receptor blocker) or superoxide dismutase (a superoxide anion scavenger) but not by carbethoxyhexyl imidazole (a thromboxane A2 synthase inhibitor) or catalase (a hydrogen peroxide scavenger). In contrast U 46619, a thromboxane A2 mimic, caused contractions, which were blocked by SQ 29548 but not by superoxide dismutase. PGH2 caused significantly greater contractions in aortic rings without endothelium or in those with intact endothelium treated with NG-nitro-L-arginine, a nitric oxide inhibitor; these contractions were inhibited by SQ 29548 but not by superoxide dismutase. In aortic rings with endothelium contracted with phenylephrine, a subthreshold concentration of PGH2, but not U 46619, impaired relaxations to acetylcholine; the inhibition was prevented by treatment with SQ 29548 or superoxide dismutase, indicating that the abnormality of endothelial cell function was specific for PGH2. These observations indicate that PGH2 causes contractions and inhibits endothelium-dependent relaxation by a mechanism involving formation of superoxide anion, which interacts with endothelium-derived nitric oxide.

EDRF inhibition augments pulmonary hypertension in intact newborn lambs.

There is increasing evidence that resting pulmonary vascular tone is mediated by the release of endothelium-derived relaxing factors (EDRF). However, the importance of EDRF release during pulmonary hypertension is unknown. Therefore, in eight newborn lambs we studied the effects of both N omega-nitro-L-arginine (an inhibitor of EDRF synthesis) and L-arginine (a precursor of EDRF synthesis) during pulmonary hypertension induced either by the intravenous infusion of U-46619 (a thromboxane A2 mimic) or by hypoxia. After pretreatment with N omega-nitro-L-arginine, the increases in pulmonary arterial pressure produced by U-46619 (102.0 +/- 34.9% vs. 144.8 +/- 28.6%, P less than 0.05) and by hypoxia (35.6 +/- 17.3% vs. 91.4 +/- 24.8%, P less than 0.05) were significantly augmented. However, after pretreatment with L-arginine, the increases in pulmonary arterial pressure produced by U-46619 (107.0 +/- 21.4% vs. 62.6 +/- 22.6%, P less than 0.05) and hypoxia (44.3 +/- 18.3% vs. 9.2 +/- 11.7%, P less than 0.05) were significantly attenuated. These results suggest that during pulmonary hypertension, EDRF is released to limit the increase in pulmonary arterial pressure and that L-arginine availability becomes rate limiting for further EDRF synthesis and release.

Analysis of responses to sarafotoxin 6a and sarafotoxin 6c in the pulmonary vascular bed of the cat

Pulmonary vascular responses to sarafotoxins 6a and 6c (S6a and S6c) were investigated in the intact-chest cat under constant flow conditions. Injections of S6a and S6c into the perfused lobar artery caused dose-related increases in lobar arterial pressure, increased left atrial pressure, and produced biphasic changes in systemic arterial (aortic) pressure. When left atrial pressure was maintained constant, injections of S6a, S6c, and endothelin 1 (ET-1) caused dose-related increases in lobar arterial pressure. The increases in lobar arterial pressure in response to S6a and S6c were not altered by treatment with a cyclooxygenase inhibitor or a thromboxane receptor blocking agent. Increases in lobar arterial pressure in response to S6a and S6c were not altered when airflow to the left lower lung lobe was interrupted by bronchial occlusion, and pressor responses were not diminished when the left lower lobe was perfused with low-molecular-weight dextran. Under conditions of controlled blood flow and constant left atrial pressure, S6a, S6b, S6c, and ET-1 had similar pressor activity, whereas the thromboxane A2 mimic, U-46619, had far greater activity when compared on a nanomolar basis. The present studies demonstrate that S6a and S6c have significant vasoconstrictor activity in the feline pulmonary vascular bed. These data suggest that pulmonary vasoconstrictor responses to the endothelin peptides are not dependent on release of cyclooxygenase products and the activation of thromboxane A2 receptors, alterations in bronchomotor tone, or interaction with formed elements in blood.

L-Arginine, a precursor of EDRF in vitro, produces pulmonary vasodilation in lambs.

There is increasing evidence that resting pulmonary vascular tone is mediated in part by the release of endothelium-derived relaxing factors (EDRF). Because L-arginine may be a precursor for EDRF synthesis, we studied the pulmonary vasodilating effects of L-arginine at rest and during pulmonary hypertension in 16 intact newborn lambs. At rest, the intravenous infusions of L-arginine (150 mg/kg) had no hemodynamic effects. However, during pulmonary hypertension induced by hypoxia or the infusion of U-46619 (a thromboxane A2 mimic), L-arginine decreased pulmonary arterial pressure by 22 and 27%, respectively (P less than 0.05). The decrease in pulmonary arterial pressure produced by L-arginine was blocked by methylene blue, a guanylate cyclase inhibitor, and augmented by Zapranast, a guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase inhibitor (-17.9 vs. -31.2%, P less than 0.05). In addition, L-arginine partially reversed the pulmonary hypertension induced by N omega-nitro-L-arginine, a competitive EDRF synthesis inhibitor, but D-arginine had no hemodynamic effects. This study suggests that L-arginine produces pulmonary vasodilation by increasing cGMP concentrations, supporting the in vitro hypothesis that L-arginine is a precursor for EDRF synthesis, whose availability may become rate limiting during pulmonary hypertension.

Influence of SQ 30741 on thromboxane receptor-mediated responses in the feline pulmonary vascular bed

The effects of SQ 30741, a thromboxane A2 (TxA2) receptor blocking agent, on responses to the TxA2 mimic, U-46619, were investigated in the pulmonary vascular bed of the intact-chest cat under constant-flow conditions. The administration of SQ 30741 in doses of 1-2 mg/kg iv markedly reduced vasoconstrictor responses to U-46619 without altering responses to prostaglandin (PG) F2 alpha or PGD2 and serotonin. SQ 30741 had no significant effect on mean vascular pressures in the cat, and the dose-response curve for U-46619 was shifted to the right in a parallel manner with a similar apparent maximal response. In addition to not altering responses to PGF2 alpha, PGD2 alpha, or serotonin, SQ 30741 (2 mg/kg iv) was without significant effect on pulmonary vasoconstrictor responses to the PGD2 metabolite 9 alpha, 11 beta-PGF2, norepinephrine, angiotensin II, BAY K 8644, endothelin 1, or endothelin 2. Although responses to vasoconstrictor agents, which act through a variety of mechanisms, were not altered, responses to the PG and TxA2 precursor, arachidonic acid, were reduced significantly. The duration of the TxA2 receptor blockade was approximately 30 and 75 min at the 1- and 2-mg/kg iv doses of the antagonist, respectively. The present data show that SQ 30741 selectively blocks TxA2 receptor-mediated responses in a competitive and reversible manner in the pulmonary vascular bed. These data suggest that responses to arachidonic acid are due in large part to the formation of TxA2 and that discrete TxA2 receptors unrelated to receptors activated by PGD2 or PGF2 alpha are most likely located in resistance vessel elements in the feline pulmonary vascular bed.

In vivo attenuation of endothelium-dependent pulmonary vasodilation by methylene blue.

In vitro evidence suggests that resting pulmonary vascular tone and endothelium-dependent pulmonary vasodilation are mediated by changes in vascular smooth muscle concentrations of guanosine 3',5'-cyclic monophosphate (cGMP). We investigated this hypothesis in vivo in 19 mechanically ventilated intact lambs by determining the hemodynamic effects of methylene blue (a guanylate cyclase inhibitor) and then by comparing the hemodynamic response to five vasodilators during pulmonary hypertension induced by the infusion of U-46619 (a thromboxane A2 mimic) or methylene blue. Methylene blue caused a significant time-dependent increase in pulmonary arterial pressure. During U-46619 infusions, acetylcholine, ATP-MgCl2, sodium nitroprusside, isoproterenol, and 8-bromo-cGMP decreased pulmonary arterial pressure. During methylene blue infusions, the decreases in pulmonary arterial pressure caused by acetylcholine and ATP-MgCl2 (endothelium-dependent vasodilators) and sodium nitroprusside (an endothelium-independent guanylate cyclase-dependent vasodilator) were attenuated by greater than 50%. The decreases in pulmonary arterial pressure caused by isoproterenol and 8-bromo-cGMP (endothelium-independent vasodilators) were unchanged. This study in intact lambs supports the in vitro evidence that changes in vascular smooth muscle cell concentrations of cGMP in part mediate resting pulmonary vascular tone and endothelium-dependent pulmonary vasodilation.

Comparative responses to endothelin-2 and sarafotoxin 6b in the pulmonary vascular bed of the cat

Pulmonary vascular responses to endothelin-2 and sarafotoxin 6b were investigated in the feline pulmonary vascular bed under natural flow and constant flow conditions. Injections of endothelin-2 and sarafotoxin 6b in a dose of 0.3 nmol/kg iv increased pulmonary arterial and left atrial pressures and cardiac output, and caused a biphasic change in calculated pulmonary vascular resistance. Endothelin-2 caused a biphasic change in systemic arterial pressure, while sarafotoxin 6b only decreased arterial pressure. Under constant flow conditions in the intact-chest cat, injections of endothelin-2 and sarafotoxin 6b in doses of 0.1-1 nmol into the perfused lobar artery increased lobar arterial pressure in a dose-related manner but were less potent than the thromboxane A2 mimic, U46619. An ET analog with only the Cys1-Cys15 disulfide bond and an amidated carboxy terminus had no significant activity in the pulmonary vascular bed. The present data show that endothelin-2 and sarafotoxin 6b have significant vasoconstrictor activity in the pulmonary vascular bed of the cat.

Pharmacological regulation of the circulation of bone.

A study was undertaken to investigate the reactivity of the circulation of bone and to pharmacologically characterize the receptor populations that may be present in this poorly described vascular bed. The nutrient artery of the tibia in skeletally mature mongrel dogs was cannulated, under direct vision, through a posterolateral operative approach. An extracorporeal circuit was established so that the nutrient artery of the tibia could be perfused in vivo under conditions of constant blood flow. Diverse vasoactive substances were injected into the perfusion circuit in small volumes as a bolus close to the nutrient artery of the tibia. A range of doses of nitroglycerin, acetylcholine, isoproterenol, methoxamine, U46619 (a thromboxane A2 mimic), dibutyryl cyclic AMP, 8-bromo-cyclic GMP, and endothelin-1 were injected in a randomized sequence for each experiment. The antagonists that were used were atropine (a non-selective muscarinic receptor antagonist), ICI 118551 (a selective beta 2-adrenoceptor antagonist), ONO 3708 (a prostaglandin H2/thromboxane A2 receptor antagonist), and prazosin (an alpha 1-adrenoceptor antagonist). The results of changes in bone-perfusion pressure under conditions of constant blood flow indicated that the vascular bed of bone actively responds to various vasoconstrictor mechanisms, whereas vasodilator mechanisms appear to be considerably less active. Intra-arterial injections of nitroglycerin, acetylcholine, and 8-bromo-cyclic GMP resulted in dose-related decreases in bone-perfusion pressure that were weak relative to concomitant changes in systemic arterial pressure. Intra-arterial administration of methoxamine, U46619, and endothelin-1 resulted in a potent dose-related increase in bone-perfusion pressure. The results of intra-arterial injections of isoproterenol and dibutyryl cyclic AMP were surprising; both substances caused a substantial rise in bone-perfusion pressure. The responses to acetylcholine, methoxamine, and U46619 were blocked in a competitive manner after administration of atropine, prazosin, and ONO 3708, respectively.

WR2721 Ameliorates the Radiation-Induced Depression in Reactivity of Rat Abdominal Aorta to U46619

Previous studies showed that 20 Gy whole-body gamma irradiation results in a decreased response of the abdominal aorta to the stable thromboxane A2 (TXA2) mimic, U46619. The present study evaluated the effect of WR2721 on this radiation-induced decrease in vascular responsiveness. Rats receiving WR2721 (200 mg/kg, i.p.) 20 min before irradiation showed no depression in vascular reactivity to U46619 compared to control. The abolition of the radiation-induced decrease in vascular responsiveness was not caused by a direct vasoconstrictor action of WR2721 or its metabolites. The vascular response of rat abdominal aortic rings to KCl was unchanged after in vivo exposure to ionizing radiation. WR2721 did not alter the vascular response to KCl. These studies confirm that exposure to whole-body ionizing radiation decreased abdominal aortic vascular responsiveness to U46619. This depressed vascular reactivity can be abolished by pretreatment with the radioprotectant, WR2721. These observations may provide a rapid initial screening method for evaluating the in vivo efficacy of radioprotectant drugs.

Vascular responses to vasopressin are tone-dependent in the cerebral circulation of the newborn pig.

The effects of lysine vasopressin (LVP) on pial arteriolar diameter and cortical periarachnoid fluid prostanoid concentrations were investigated in newborn pigs. Chloralose-anesthetized piglets were equipped with closed cranial windows over the parietal cortex for observation of pial arterioles and collection of cerebrospinal fluid (CSF) passing over the cerebral surface. Prostanoids in the CSF were determined by radioimmunoassay. LVP (10-1,000 microU/ml) elicited concentration-dependent increases in pial arteriolar diameter associated with increased levels of 6-keto-prostaglandin (PG)F1 alpha, PGE2, thromboxane B2, and PGF2 alpha. LVP-induced pial arteriolar dilation was unchanged after intravenous indomethacin (5 mg/kg). Conversely, LVP constricts pial arterioles previously dilated by physiological (hemorrhagic hypotension) and pharmacological (topically applied PGE2 or isoproterenol) intervention. This constriction is potentiated by indomethacin. Vascular and biochemical changes elicited by LVP were blocked by intravenous [1-(beta-mercapto-beta beta-cyclopentamethylene propionic acid),2,(O-methyl)-Tyr-AVP] (5 micrograms/kg), a putative V1 receptor antagonist, whereas vascular effects of norepinephrine and U46619, a thromboxane A2 mimic, were unchanged. Therefore, the degree of vascular tone appears to influence responses of the newborn pig cerebral circulation to LVP.

Pharmacological actions of S-145, a novel thromboxane A2 antagonist, in various smooth muscles

Antagonistic actions of S-145 ((+-)-5(Z)-7-[3-endo[(phenylsulfonyl)amino]bicyclo[2.2.1] hept-2-exo-yl]heptenoic acid) against U-46619, a thromboxane A2 mimic, were studied using isolated thoracic aorta of the rat and the trachea, lung parenchyma and ileum of the guinea pig. S-145 as well as SQ-29548 and ONO-3708 inhibited the contraction of aorta induced by U-46619 in a concentration-dependent manner. The IC50 value of each compound was 1.4, 14.5 and 52.6 nM. S-145 also inhibited contractions of the aorta induced by high concentrations of PGE1, PGE2 and PGF2 alpha, but failed to affect the responses to K+, Ca2+, NE, 5-HT, and angiotensin II. Contractions of trachea and lung parenchyma of the guinea pig induced by U-46619 were concentration-dependently inhibited by S-145, but those induced by histamine and leukotriene D4 were not affected. Ileac contractions by PGE2 and PGF2 alpha were not inhibited by S-145. The (+)-isomer of S-145 was more potent and the (-)-isomer was less potent than S-145 for antagonistic action against U-46619. These results suggest that S-145 is a potent and specific antagonist to the thromboxane A2 receptor; and in the aorta, the thromboxane A2 receptor may respond to high concentrations of PGs.

Characterization of Contraction-Mediating Prostanoid Receptors in Human Penile Erectile Tissues

The effects of the thromboxane-receptor antagonist L-636,499 were studied on contractions induced by the thromboxane A2 mimic U44069 and by prostaglandin F2α in isolated preparations of the human corpus cavernosum and corpus spongiosum. The objectives were to characterize prostanoid receptors and, in particular, to elucidate whether more than one receptor was involved in prostanoid-induced contraction of penile erectile tissues. L-636,499 at concentrations 10-6 M to 3 × 10-5 M induced a parallel shift to the right of the concentration-response curve to U44069 in both corpus cavernosum and corpus spongiosum suggesting competitive antagonism. Schild plots using U44069 as the agonist and L-636,499 as the antagonist revealed slope indexes near unity in both tissues, and the pA2 values were almost identical. In contrast, L-636,499 concentration-dependently reduced the maximum response to prostaglandin F2α, indicating a non-competitive action.The results suggest that the main contraction-mediating prostanoid receptor in human penile erectile tissues is a thromboxane A2 sensitive receptor. However, the presence of more than one contraction-mediating prostanoid receptor cannot be excluded.

Further characterization of the contraction‐mediating prostanoid receptors in feline cerebral arteries. Effects of the thromboxane‐receptor antagonist AH 23848

The effects of the thromboxane-receptor antagonist AH 23848 were investigated on isolated feline basilar arteries (BA). AH 23848 (10(-6) mol l-1) had no effect on contractions induced by 5-hydroxytryptamine or potassium, whereas the drug (10(-8)-10(-6) mol l-1) induced a parallel shift to the right in contractions induced by the thromboxane A2 mimic U46619. There was no depression of the maximum contraction, indicating competitive antagonism. The Schild plot revealed a slope index of unity with a pA2 value of 8.46. In contrast, 10(-6) mol l-1 AH 23848 depressed the maximum PGF2 alpha-induced contraction significantly from 100% to 13%. U46619 was able to induce a contraction amounting to 98% if the drug was added on top of the PGF2 alpha-induced contraction in the presence of 10(-6) mol l-1 AH 23848. The results provide strong support for previous suggestions that prostanoid-induced contractions in the feline BA are mediated by two receptor subtypes, one of which can be classified as a thromboxane-sensitive (TP) receptor.

Dihydropyridine agonist Bay K 8644 inhibits platelet activation by competitive antagonism of thromboxane A2-prostaglandin H2 receptor.

The dihydropyridine calcium channel antagonists, such as nifedipine, inhibit platelet aggregation in vitro and ex vivo, but the mechanism by which this occurs is uncertain. Bay K 8644 (BAY) is a substituted dihydropyridine that has effects on voltage-dependent calcium channels in cardiac and smooth muscle that are opposite the effects of nifedipine. To evaluate the mechanism responsible for dihydropyridine-induced inhibition of platelet function, we studied the in vitro effects of BAY on human platelet aggregation and secretion plus several related biochemical parameters, including cytoplasmic ionized calcium ([Ca2+]i). BAY exerted concentration-dependent effects on platelet aggregation and secretion of [14C]serotonin. BAY (1-10 microns) inhibited the second wave of platelet aggregation and secretion stimulated by adenosine diphosphate or epinephrine and blocked shape change, aggregation, and secretion induced by the thromboxane A2 (TXA2) mimic, U46619. BAY also inhibited U46619-induced phosphorylation of the approximately 40,000-dalton cytoplasmic protein substrate of protein kinase C (40K protein), formation of TXA2, and rise in [Ca2+]i, all biochemical consequences of platelet activation. The (+)-(R) enantiomer of BAY [BAY(+)] was predominantly responsible for the inhibitory effects of racemic BAY. Nifedipine had the same inhibitory effects on platelet function and biochemistry, except it was approximately 10 times less potent than BAY. Since these results suggested inhibition of the TXA2-prostaglandin H2 (PGH2) receptor, we measured binding of [3H]U46619 to intact platelets. BAY, BAY(+), and nifedipine all functioned as competitive antagonists of [3H]U46619 binding (BAY Ki = 1.47 microM). They did not inhibit binding of [3H]yohimbine to platelet alpha 2-adrenergic receptors. At 1-10 nM BAY, BAY(+) and the (-)-(S) enantiomer of BAY [BAY(-)] all resulted in slight stimulation of platelet function and biochemical events. No significant increase in [3H]U46619 binding was demonstrable, however. Therefore, dihydropyridines that function as either calcium channel agonists or antagonists in cardiac or smooth muscle exert concentration-dependent effects on platelet function. In nanomolar concentrations, they augment, and in micromolar concentrations, they inhibit platelet activation induced by TXA2 or U46619. These data indicate that dihydropyridines do not inhibit TXA2-induced platelet activation by an effect on voltage-dependent calcium channels; they define the mechanism of inhibition as competitive antagonism of the TXA2-PGH2 receptor. The mechanism responsible for augmentation of platelet activation is uncertain.(ABSTRACT TRUNCATED AT 400 WORDS)

Specificity of FPL 57231 for leukotriene D4 receptors in fetal pulmonary circulation.

The effects of leukotriene D4 and the putative leukotriene receptor antagonist FPL 57231 were studied on the pulmonary circulation of alpha-chloralose-anesthetized fetal lambs close to term. At constant pulmonary inflow leukotriene D4 (LTD4, 0.1-10 micrograms), as bolus injections, caused dose-dependent increases in pulmonary vascular resistance. FPL 57231 (1.0-10 mg/kg) not only blocked the pressor responses to LTD4 but also lowered the normal pulmonary vascular resistance in a dose-dependent fashion. However, FPL 57231 also decreased the pulmonary pressor response to U 46619, a thromboxane A2 mimic, as well as the pressor response to phenylephrine HCl. Furthermore, the pressor responses to LTD4 were markedly reduced by cyclooxygenase inhibition. In preliminary experiments we demonstrated that the phenidone derivative, BW755C, which is a dual inhibitor of both cyclooxygenase and 5-lipoxygenase enzymes, did not block the pressor response to hypoxia. We conclude that FPL 57231 decreases fetal pulmonary vascular resistance by nonspecific mechanisms. Also the action of LTD4, is indirect and by way of the cyclooxygenase system. Although exogenous leukotrienes are able to produce a marked pulmonary pressor response, endogenous leukotrienes are probably not responsible for the hypoxic pulmonary pressor response of the fetal lung or its normally high pulmonary vascular resistance.

Disparate effects of the calcium-channel blockers, nifedipine and verapamil, on alpha 2-adrenergic receptors and thromboxane A2-induced aggregation of human platelets.

Calcium-channel blockers inhibit human platelet aggregation in vitro and ex vivo. To further evaluate the mechanism(s) responsible for the inhibition induced by this structurally heterogeneous group of compounds, we studied the effect of nifedipine and verapamil on human platelet aggregation in vitro. Neither 10 microM nifedipine nor 10 microM verapamil consistently inhibited the aggregation response of platelet-rich plasma to threshold concentrations of ADP, sodium arachidonate, epinephrine, or collagen. However, both 10 microM nifedipine and 10 microM verapamil epinephrine-potentiated, thromboxane A2 (TXA2)-induced aggregation of aspirin-incubated, gel-filtered platelets. Aggregation of similarly prepared platelets induced by TXA2 alone was abolished by 10 microM nifedipine but not by 10 microM verapamil. Even 100 microM verapamil gave only partial and inconsistent inhibition of aggregation. Both drugs had essentially the same effects on platelet aggregation induced by the stable endoperoxide and TXA2 mimic, U46619, with or without epinephrine. Neither 10 microM nifedipine nor 10 microM verapamil elevated platelet cyclic AMP. Verapamil (10 microM) inhibited binding of [3H]-yohimbine (an alpha 2-adrenergic receptor antagonist) to intact human platelets (KD 10.5 nM vs 2.4 nM for control platelets) without altering the number of binding sites. In contrast, 10 microM nifedipine had no effect on KD or number of binding sites. These results indicate that nifedipine and verapamil inhibit epinephrine-potentiated, TXA2-induced human platelet aggregation by different mechanisms. Verapamil inhibits the epinephrine contribution to the aggregation response by blocking alpha 2-adrenergic receptor binding. Nifedipine blocks the platelet response to TXA2 without affecting alpha-adrenergic receptor binding. These observations have potential clinical implications with regard to the mechanisms by which calcium-channel blockers inhibit vascular spasm and myocardial ischemia.

Analysis of responses to leukotriene D4 in the pulmonary vascular bed.

Pulmonary vascular responses to leukotriene D4 were investigated in the intact-chest animal under conditions of controlled pulmonary blood flow. Intralobar injections of leukotriene D4 in the sheep caused dose-dependent increases in lobar arterial and small vein pressures without influencing left atrial or systemic arterial pressure. Leukotriene D4 was very potent in increasing pulmonary vascular resistance in the sheep, with activity similar to that of U-46619, a thromboxane A2 mimic. Pulmonary vascular responses to leukotriene D4 in the sheep were similar when the lung was ventilated and when lobar ventilation was arrested. Responses to leukotriene D4 were similar when the lung was perfused with blood or with dextran. Pulmonary vascular responses to leukotriene D4 but not U-46619 in the sheep were reduced by inhibitors of cyclooxygenase and thromboxane synthesis. In contrast, leukotriene D4 had modest pressor activity in the pulmonary vascular bed of the cat whereas U-46619 had marked activity in this species. Responses to leukotriene D4 in the cat were not altered by cyclooxygenase inhibitors. It is concluded that leukotriene D4 has marked pulmonary vasoconstrictor activity in the sheep, increasing pulmonary vascular resistance by constricting intrapulmonary veins and upstream segments. In this species, responses to leukotriene D4 were independent of changes in ventilation or interaction with formed elements but were dependent on the formation of cyclooxygenase products including thromboxane A2. However, in the cat, leukotriene D4 had very modest pressor activity, and this activity was not dependent on the integrity of the cyclooxygenase pathway. These data suggest considerable species difference in responses to leukotriene D4, a major component of the slow-reacting substance of anaphylaxis, in the pulmonary vascular bed.