Thromboxane A2 Analogue U46619 Research Articles

Atherosclerosis potentiates constrictor responses of cerebral and ocular blood vessels to thromboxane in monkeys.

The goal of our study was to examine the effects of infusion of serotonin and the thromboxane A2 analogue U46619 into one carotid artery to stimulate their release from platelets during aggregation. We measured blood flow to the brain and eye using microspheres and cerebral microvascular pressure in the pial arteries of normal and atherosclerotic cynomolgus monkeys. Unilateral intracarotid infusion of 10-30 micrograms/min serotonin did not affect cerebral blood flow in normal or atherosclerotic monkeys; serotonin did not alter blood flow to the eye in normal monkeys but decreased flow to the retina and choroid in atherosclerotic monkeys by 39 +/- 11% and 44 +/- 10% (mean +/- SEM), respectively. Infusion of 30 ng/min U46619 did not alter cerebral blood flow but increased the pressure gradient from the aorta to the pial artery, which is an index of large-artery resistance, in atherosclerotic monkeys. U46619 had no effect on blood flow to the eye in normal monkeys but decreased blood flow to the retina and choroid by 71 +/- 14% and 53 +/- 13%, respectively, in atherosclerotic monkeys. Thus, atherosclerosis potentiates the constrictor responses of large cerebral arteries to thromboxane and the responses of blood vessels of the eye to thromboxane and serotonin.

Effect of endothelium removal on stimulatory and inhibitory modulation of rat aortic prostacyclin synthesis

1. Removal of the endothelium (DE) enhanced the in vitro release of prostacyclin (PGI2) by rat aortae in response to adrenaline (Ad), noradrenaline (NA), thromboxane A2 analogue U46619, phorbol dibutyrate (PDBU) and sodium fluoride (NaF) when assessed at 3 h post DE. At 6 h post DE, there were no differences between the dose-response curves obtained from aortic rings with or without endothelium. 2. At 3 h post DE the antagonism of Ad- and NA-stimulated PGI2 synthesis by yohimbine and prazosin, and NA-stimulated PGI2 synthesis by nifedipine was markedly reduced in aortae without endothelium when compared with controls. These effects were reversed by protracted incubation of aortic tissue post DE (6 h and 9 h). 3. Acetylcholine, carbachol, substance P and nitroprusside were without effect on de novo or NA-stimulated PGI2 synthesis, whether or not the endothelium was present and irrespective of incubation time, post-DE. 4. These results indicate that: (a) PGI2 synthesis linked to excitatory receptors (alpha-adrenoceptors, thromboxane A2) and associated systems (G proteins, protein kinase C) in the smooth muscle component of the rat aorta is not influenced by endothelium-derived relaxing factor (EDRF); (b) the changes of response to stimulators and inhibitors of PGI2 synthesis may be due to an increased reactivity of the vessels caused by the trauma of DE; and (c) vasodilators (parasympathomimetics, substance P and nitroprusside) that do not act directly on excitatory receptors do not influence PGI2 synthesis.

Diabetes-Induced Enhancement of Prostanoid-Stimulated Contraction in Mesenteric Veins of Mice

We investigated the influence of the diabetic state on the contractile response of longitudinal segments of isolated mesenteric vein to prostanoids and leukotriene (LT), and the contribution of the vascular endothelium to modulation of the contractile response was determined. The normal mesenteric vein and de-endothelialized veins of normal (ddY), diabetic KK-CAy and streptozotocin ddY mice (150 mg/kg, i.v., 6 weeks) were used. In the diabetic state, the contractions produced by noradrendline (60 μM), high K+ solution (143.4 mM), and the thromboxane A2 analogue U-46619 (29 nM–29 mM) were not affected, and LTD4 (0.1 nM-1 μM)-induced contraction was suppressed. Contractions induced by prostaglandin (PG) E2 (0.2 μM-2 mM), PGF2α (0.3 μM-0.3 mM) and the prostacyclin derivatives PGI2-Na (10–100 μM) and TRK-100 (0.2 μM–2 mM) were significantly enhanced in the presence of an intact vascular endothelium, but not in de-endothelialized segments. The increase in PGF2α (0.28 mM) contractions was dependent on age (correlation coefficient r=0.36, significant difference, P< 0.05) and blood glucose (r=0.88, significant difference, P<0.01), but was independent of obesity. The contractile response to PGD2 (0.3–0.9 mM) was enhanced in both intact and de-endothelialized segments. These results indicate that the diabetic state affects prostanoid responses in an endothelium-dependent manner, except for the PGD2 response, which is independent of the endothelium.

Stimulation of thromboxane release by extracellular UTP and ATP from perfused rat liver. Role of icosanoids in mediating the nucleotide responses.

1. In isolated perfused rat liver, infusion of UTP (20 microM) led to a transient, about sevenfold stimulation of thromboxane release (determined as thromboxane B2), which did not parallel the time course of the UTP-induced stimulation of glucose release. An increased thromboxane release was also observed after infusion of ATP (20 microM). Although the maximal increase of portal pressure following ATP was much smaller than with UTP (4.2 vs 11.5 cm H2O), the peak thromboxane release was similar with both nucleotides. 2. Indomethacin (10 microM) inhibited the UTP-induced stimulation of thromboxane release and decreased the UTP-induced maximal increase of glucose output and of portal pressure by about 30%. The thromboxane A2 receptor antagonist BM 13.177 (20 microM) completely blocked the pressure and glucose response to the thromboxane A2 analogue U-46619 (200 nM) and decreased the ATP- and UTP-induced stimulation of glucose output by about 25%, whereas the maximal increase of portal pressure was inhibited by about 50% and 30%, respectively. BM 13.177 and indomethacin inhibited the initial nucleotide-induced overshoot of portal pressure increase, but had no effect on the steady-state pressure increase which is obtained about 5 min after addition of ATP or UTP. 3. The leukotriene D4/E4 receptor antagonist LY 171883 (50 microM) inhibited not only the glucose and pressure response of perfused rat liver to leukotriene D4, but also to leukotriene C4 by about 90%. This suggests that leukotriene D4 (not C4) is the active metabolite in perfused liver and the effects of leukotriene C4 are probably due to its rapid conversion to leukotriene D4. LY 171883 also inhibited the response to the thromboxane A2 analogue U-46619 by 75-80%, whereas the response of perfused liver to leukotriene C4 was not affected by the thromboxane receptor antagonist BM 13.177 (20 microM). The glucose and pressure responses of the liver to extracellular UTP were inhibited by LY 171883 and by BM 13.177 by about 30%. This suggests that the inhibitory action of LY 171883 was due to a thromboxane receptor antagonistic side-effect and that peptide leukotrienes do not play a major role in mediating the UTP response. 4. In isolated rat hepatocytes extracellular UTP (20 microM), ATP (20 microM), cyclic AMP (50 microM) and prostaglandin F2 alpha (3 microM) increased glycogen phosphorylase a activity by more than 100%.(ABSTRACT TRUNCATED AT 400 WORDS)

Hepatocyte heterogeneity in response to icosanoids

1. The metabolic and hemodynamic effects of prostaglandin F2 alpha, leukotriene C4 and the thromboxane A2 analogue U-46619 were studied during physiologically antegrade (portal to hepatic vein) and retrograde (hepatic to portal vein) perfusion and in a system of two rat livers perfused in sequence. 2. The stimulatory effects of prostaglandin F2 alpha (3 microM) on hepatic glucose release, perfusion pressure and net Ca2+ release were diminished by 77%, 95% and 64%, respectively, during retrograde perfusion when compared to the antegrade direction, whereas the stimulation of 14CO2 production from [1-14C]glutamate by prostaglandin F2 alpha (which largely reflects the metabolism of perivenous hepatocytes) was lowered by only 20%. Ca2+ mobilization and glucose release from the liver comparable to that seen during antegrade perfusion could also be observed in retrograde perfusions; however, higher concentrations of the prostaglandin were required. 3. The glucose, Ca2+ and pressure response to leukotriene C4 (20 nM) or the thromboxane A2 analogue U-46619 (200 nM) of livers perfused in the antegrade direction were diminished by about 90% during retrograde perfusion. Sodium nitroprusside (20 microM) decreased the pressure response to leukotriene C4 (20 nM) and U-46619 (200 nM) by about 40% and 20% in antegrade perfusions, respectively, but did not affect the maximal increase of glucose output. 4. When two livers were perfused antegradely in series, such that the perfusate leaving the first liver (liver I) entered a second liver (liver II), infusion of U-46619 at concentrations below 200 nM to the influent perfusate of liver I increased the portal pressure of liver I, but not of liver II. At higher concentrations of U-46619 there was also an increase of the portal pressure of liver II and with concentrations above 800 nM the pressure responses of both livers were near-maximal [19.6 +/- 0.8 (n = 7) cm H2O and 16.5 +/- 1.1 (n = 8) cm H2O for livers I and II, respectively]. There was a similar behaviour of glucose release from livers I and II in response to U-46619 infusion. When liver I was perfused in the retrograde direction, a significant pressure or glucose response of liver II (antegrade perfusion) could not be observed even with U-46619 concentrations up to 1000 nM. 5. Similarly, the perfusion pressure increase and glucose release induced by leukotriene C4 (10 nM) observed with liver II was only about 20% of that seen with liver I.(ABSTRACT TRUNCATED AT 250 WORDS)

Potential role for prostaglandin F2 alpha, D2, E2 and thromboxane A2 in mediating the metabolic and hemodynamic actions of sympathetic nerves in perfused rat liver.

In isolated rat liver perfused at constant pressure perivascular nerve stimulation caused an increase of glucose and lactate output and a reduction of perfusion flow. The metabolic and hemodynamic nerve effects could be inhibited by inhibitors of prostanoid synthesis, which led to the suggestion that the effects of nerve stimulation were, at least partially, mediated by prostanoids [Iwai, M. & Jungermann, K. (1987) FEBS Lett. 221, 155-160]. This suggestion is corroborated by the present study. 1. Prostaglandin D2, E2 and F2 alpha as well as the thromboxane A2 analogue U46619 enhanced glucose and lactate release and lowered perfusion flow similar to nerve stimulation. 2. The extents, the kinetics and the concentration dependencies of the metabolic and hemodynamic actions of the various prostanoids were different. Prostaglandin F2 alpha and D2 caused relatively stronger changes of metabolism, while prostaglandin E2 and U46619 had stronger effects on hemodynamics. Prostaglandin F2 alpha elicited greater maximal alterations than D2 with similar half-maximally effective concentrations. Prostaglandin F2 alpha mimicked the nerve actions on both metabolism and hemodynamics best with respect to the relative extents and the kinetics of the alterations. 3. The hemodynamic effects of prostaglandin F2 alpha could be prevented completely by the calcium antagonist nifedipine without impairing the metabolic actions of the prostanoid. Apparently, prostaglandin F2 alpha influenced metabolism directly rather than indirectly via hemodynamic changes. The present results, together with the previously described effects of prostanoid synthesis inhibitors, suggest that prostanoids, probably prostaglandin F2 alpha and/or D2, could be involved in the actions of sympathetic hepatic nerves on liver carbohydrate metabolism. Since prostanoids are synthesized only in non-parenchymal cells, nervous control of metabolism appears to depend on complex intra-organ cell-cell interactions between the nerve, non-parenchymal and parenchymal cells.

Inhibition of eicosanoid‐mediated coronary constriction during myocardial ischemia 1

Thromboxane A2 and cysteinyl leukotrienes are highly effective microvessel constrictors in normally perfused myocardium. Their release during acute coronary thrombosis might augment myocardial underperfusion. The constrictor action of these substances could be modified substantially, however, by concomitant myocardial ischemia. We compared the effects of the two eicosanoid constrictors in normally perfused and ischemic myocardium of 24 open-chest, pentobarbital-anesthetized pigs. Left anterior descending coronary flow was measured after intracoronary bolus injections of the stable thromboxane A2 analog U46619 (1-10 micrograms) or leukotriene D4 (LTD4, 1-10 micrograms). Each dose was given before and during myocardial ischemia induced by a snare adjusted to produce 63 +/- 2% decrease in coronary flow for 10 min. Marked dose-independent inhibition of eicosanoid-induced coronary flow decrease occurred during ischemia. With 10 micrograms U46619, coronary flow decrease in the unoccluded state (25 +/- 2 from 55 +/- 4 ml/min pretreatment baseline) was virtually eliminated during snare occlusion (1 +/- 1 from 21 +/- 3 ml/min pretreatment baseline, P less than 0.001). Similar results occurred with LTD4. Distal coronary pressure during ischemia indicated a lack of microvessel responsiveness to the eicosanoids rather than a buffering of resistance change by the snare. U46619 and LTD4 did induce transient, small reductions in regional shortening fraction during ischemia. Our data suggest that eicosanoid-induced constriction of myocardial resistance vessels is not a likely complication of acute coronary thrombosis. However, eicosanoids could depress residual contractility in moderately ischemic regions.

Effects of leukotrienes and the thromboxane A2 analogue U-46619 in isolated perfused rat liver. Metabolic, hemodynamic and ion-flux responses.

1) Addition of leukotriene C4 to isolated perfused rat liver led to a stimulation of hepatic glucose output, a slight decrease of 14CO2 production from [1-14C] glutamate, an increase of portal pressure and an inhibition of hepatic oxygen uptake. Withdrawal of leukotriene C4 caused a transient further stimulation of hepatic glucose output. 2) These effects were accompanied by a slow net Ca2+ release from the liver, which was not completed within 8 min. Following leukotriene withdrawal there was a further Ca2+ release for about 1 min superimposing a slow reuptake of Ca2+ of about 10 min duration. 3) Leukotriene C4 induced a characteristic biphasic K+ release from the liver. Withdrawal of the leukotriene resulted in a further net K+ release for about 4 min, being followed by a K+ reuptake over more than 10 min. 4) Effects comparable to those induced with leukotriene C4 (20nM) were obtained with leukotriene D4 (20nM), were as leukotriene B4 and E4 (20nM each) were much less effective. 5) The thromboxane A2 analogue U-46619 produced ionic, metabolic and hemodynamic responses similar to leukotriene C4; however, when given at concentration yielding a comparable glucose release, the thromboxane analogue was much more vasoactive than leukotriene C4. The thromboxane A2 receptor antagonist BM-13.177 (20 microM) blocked the metabolic, hemodynamic and ion flux responses to U-46619 almost completely, but had no effect on the response to leukotriene C4. 6) Each, leukotrienes, U-46619 and UTP led to an inhibition of hepatic oxygen uptake. The extent of inhibition of oxygen uptake induced by these compounds was not exclusively explained by their effects on hepatic circulation: a 30% inhibition of oxygen uptake by leukotriene C4, U-46619 or UTP was accompanied by increases of the portal pressure of 4.9 +/- 0.4 (481 +/- 39 Pa) (n = 7), 16.0 +/- 1.9 (1570 +/- 186 Pa) (n = 7) or 11.4 +/- 0.4 (1118 +/- 39 Pa) (n = 13) cm H2O, respectively. 7) The data show that leukotrienes and possibly also thromboxanes are potent regulators of hepatic metabolism and hemodynamics, probably acting by a Ca2+ mobilizing mechanism and involving different receptor systems. The response of perfused liver to these compounds is qualitatively similar to that obtained with extracellular UTP, but different to that with prostaglandins, extracellular ATP or phenylephrine. The data further support the view that eicosanoids are important modulators of hepatic metabolism and point to a complex regulatory interaction between hepatic parenchymal and non-parenchymal cells.

The kinetics of changes in intracellular calcium concentration in fura-2-loaded human platelets.

We have investigated the sub-second kinetics of changes in cytosolic free calcium, [Ca2+]i, in fura-2-loaded human platelets by stopped-flow fluorimetry. Thrombin, vasopressin, platelet-activating factor, and the thromboxane A2 analogue U46619 all evoked a rise in [Ca2+]i which was delayed in onset by 200-400 ms in the presence of 1 mM external Ca2+. The responses to these agonists in media containing 1 mM EGTA or 1 mM Ni2+, to prevent Ca2+ influx, were delayed by an additional 60-100 ms. These results indicate that agonist-evoked Ca2+ influx precedes the release of Ca2+ from internal stores. The delays in onset of both responses are sufficient for one or more biochemical steps to lie between ligand-receptor binding and Ca2+ flux generation. ADP responses in media containing EGTA or Ni2+ were similar to those evoked by other agonists, but the response in the presence of external Ca2+ was markedly shorter, occurring without measurable delay at optimal ligand concentration. Analysis of this response showed some delay in ADP-evoked influx at lower concentrations, but this delay was markedly less than that observed with thrombin at doses giving the same elevation in [Ca2+]i. These results suggest that ADP evokes influx using a different transduction system, more closely coupled to the Ca2+ entry system than that used by other agonists. Differences between thrombin- and ADP-evoked influx were further demonstrated by the inhibitory actions of cAMP, which reduced and substantially increased the delay in onset of thrombin-evoked influx but did not measurably delay the influx evoked by an optimal concentration of ADP.

Response of human platelets exposed to arachidonate upon stimulation with other agonists: possible role of Ca2+i movement.

Arachidonate (25 microM) induced only 20% maximal platelet aggregation but 80% maximal cytoplasmic ionized calcium movement (as measured by the photoprotein aequorin). Preincubation with 25 microM arachidonate prevented platelet aggregation and cytoplasmic ionized calcium movement induced by threshold concentrations of the thromboxane A2 analogue U-46619, but did not prevent platelet aggregation induced by threshold concentrations of thrombin, despite a marked reduction of cytoplasmic ionized calcium movement. These findings suggest that thrombin may employ pools of cytoplasmic ionized calcium and/or mechanisms to mobilize it different from those utilized by arachidonate and its metabolites.

Stimulation of glycogenolysis and vasoconstriction in the perfused rat liver by the thromboxane A2 analogue U-46619.

Infusion of the thromboxane A2 analogue U-46619 into isolated perfused rat livers resulted in dose-dependent increases in glucose output and portal vein pressure, indicative of constriction of the hepatic vasculature. At low concentrations, e.g. less than or equal to 42 ng/ml, glucose output occurred only during agonist infusion; whereas at concentrations greater than or equal to 63 ng/ml, a peak of glucose output also was observed upon termination of agonist infusion coincident with relief of hepatic vasoconstriction. Effluent perfusate lactate/pyruvate and beta-hydroxybutyrate/acetoacetate ratios increased significantly in response to U-46619 infusion. Hepatic oxygen consumption increased at low U-46619 concentrations (less than or equal to 20 ng/ml) and became biphasic with a transient spike of increased consumption followed by a prolonged decrease in consumption at higher concentrations. Increased glucose output in response to 42 ng/ml U-46619 was associated with a rapid activation of glycogen phosphorylase, slight increases in tissue ADP levels, and no increase in cAMP. At 1000 ng/ml, U-46619 activation of glycogen phosphorylase was accompanied by significant increases in tissue levels of AMP and ADP, decreases in ATP, and slight increases in cAMP. In isolated hepatocytes, U-46619 did not stimulate glucose output or activate glycogen phosphorylase. Reducing the perfusate calcium concentration from 1.25 to 0.05 mM resulted in a marked reduction of the glycogenolytic response to U-46619 (42 ng/ml) with no efflux of calcium from the liver. U-46619-induced glucose output and vasoconstriction displayed a similar dose dependence upon the perfusate calcium concentration. Thus, U-46619 exerts a potent agonist effect on glycogenolysis and vasoconstriction in the perfused rat liver. The present findings support the concept that U-46619 stimulates hepatic glycogenolysis indirectly via vasoconstriction-induced hypoxia within the liver.

Endothelium-derived relaxing factor inhibits in vitro platelet aggregation.

We studied the effects of endothelium-derived relaxing factor (EDRF), bovine retractor penis muscle inhibitory factor and sodium nitroprusside, three stimulants of guanylate cyclase, on the in vitro aggregation of washed human platelets. Platelet aggregation induced either by collagen or by the thromboxane A2 analogue U46619 was inhibited by all three agents. The anti-aggregatory effect of each agent was inhibited by haemoglobin. The anti-aggregatory effect of EDRF was potentiated by superoxide dismutase. These findings are discussed in relation to a potential role for EDRF in haemostasis.

Inositol 1,4,5-triphosphate-induced granule secretion in platelets. Evidence that the activation of phospholipase C mediated by platelet thromboxane receptors involves a guanine nucleotide binding protein-dependent mechanism distinct from that of thrombin.

Phosphoinositide hydrolysis in platelets stimulated by thrombin is thought to be regulated by a pertussis toxin-sensitive guanine nucleotide binding protein (G protein) referred to as Gp. The present studies examine the role of Gp in platelet responses to the thromboxane A2 analogue U46619 and in the pathway by which the phosphoinositide hydrolysis product inositol 1,4,5-triphosphate (IP3) causes secretion. In permeabilized platelets, U46619 caused phosphatidic acid formation and secretion, which were abolished by the G protein inhibitor, guanosine 5'-O-(2-thiophosphate) (GDP beta S). Unlike thrombin, however, U46619-induced phosphoinositide hydrolysis was unaffected by pertussis toxin, and U46619 was unable to inhibit the [32P]ADP ribosylation of the 42-kD pertussis toxin substrate in platelets. IP3-induced secretion, which is known to depend upon intracellular Ca release and subsequent arachidonic acid metabolism, was also inhibited by GDP beta S, as was Ca-induced secretion. These observations suggest that platelet thromboxane A2 (TxA2) receptors are coupled to a toxin-resistant form of Gp distinct from the one that is coupled to thrombin receptors, and that TxA2-stimulated phosphoinositide hydrolysis may serve as a feedback mechanism by which stimuli for arachidonic acid release, such as IP3 and Ca, amplify responses to agonists.