Thromboxane receptor hyper-responsiveness in hypoxic pulmonary hypertension requires serine 324.

BACKGROUND AND PURPOSE: Dysregulation of the thromboxane A2 (TP) receptor, resulting in agonist hypersensitivity and hyper-responsiveness, contributes to exaggerated vasoconstriction in the hypoxic pulmonary artery in neonatal persistent pulmonary hypertension. We previously reported that hypoxia inhibits TP receptor phosphorylation, causing desensitization. Hence, we examined the role of PKA-accessible serine residues in determining TP receptor affinity, using site-directed mutational analysis. EXPERIMENTAL APPROACH: Vasoconstriction to a thromboxane mimetic and phosphorylation of TP receptor serine was examined in pulmonary arteries from neonatal swine with persistent pulmonary hypertension and controls. Effects of hypoxia were determined in porcine and human TP receptors. Human TPα serines at positions 324, 329 and 331 (C-terminal tail) were mutated to alanine and transiently expressed in HEK293T cells. Saturation binding and displacement kinetics of a TP antagonist and agonist were determined in porcine TP, wild-type human TPα and all TP mutants. Agonist-elicited calcium mobilization was determined for each TP mutant, in the presence of a PKA activator or inhibitor, and in hypoxic and normoxic conditions. KEY RESULTS: The Ser324A mutant was insensitive to PKA activation and hypoxia, had a high affinity for agonist and increased agonist-induced calcium mobilization. Ser329A was no different from wild-type TP receptors. Ser331A was insensitive to hypoxia and PKA with a decreased agonist-mediated response. CONCLUSIONS AND IMPLICATIONS: In hypoxic pulmonary hypertension, loss of site-specific phosphorylation of the TP receptor causes agonist hyper-responsiveness. Ser324 is the primary residue phosphorylated by PKA, which regulates TP receptor-agonist interactions. Ser331 mutation confers loss of TP receptor-agonist interaction, regardless of PKA activity.

The function and three-dimensional structure of a thromboxane A2/cysteinyl leukotriene-binding protein from the saliva of a mosquito vector of the malaria parasite.

The highly expressed D7 protein family of mosquito saliva has previously been shown to act as an anti-inflammatory mediator by binding host biogenic amines and cysteinyl leukotrienes (CysLTs). In this study we demonstrate that AnSt-D7L1, a two-domain member of this group from Anopheles stephensi, retains the CysLT binding function seen in the homolog AeD7 from Aedes aegypti but has lost the ability to bind biogenic amines. Unlike any previously characterized members of the D7 family, AnSt-D7L1 has acquired the important function of binding thromboxane A(2) (TXA(2)) and its analogs with high affinity. When administered to tissue preparations, AnSt-D7L1 abrogated Leukotriene C(4) (LTC(4))-induced contraction of guinea pig ileum and contraction of rat aorta by the TXA(2) analog U46619. The protein also inhibited platelet aggregation induced by both collagen and U46619 when administered to stirred platelets. The crystal structure of AnSt-D7L1 contains two OBP-like domains and has a structure similar to AeD7. In AnSt-D7L1, the binding pocket of the C-terminal domain has been rearranged relative to AeD7, making the protein unable to bind biogenic amines. Structures of the ligand complexes show that CysLTs and TXA(2) analogs both bind in the same hydrophobic pocket of the N-terminal domain. The TXA(2) analog U46619 is stabilized by hydrogen bonding interactions of the ω-5 hydroxyl group with the phenolic hydroxyl group of Tyr 52. LTC(4) and occupies a very similar position to LTE(4) in the previously determined structure of its complex with AeD7. As yet, it is not known what, if any, new function has been acquired by the rearranged C-terminal domain. This article presents, to our knowledge, the first structural characterization of a protein from mosquito saliva that inhibits collagen mediated platelet activation.

Cordycepin (3'-deoxyadenosine) inhibits human platelet aggregation induced by U46619, a TXA2 analogue.

Cordycepin (3'-deoxyadenosine), which comes from Cordyceps militaris, the Chinese medicinal fungal genus Cordyceps, is known to have anti-tumour activity. In this study, we investigated the novel effect of cordycepin on human platelet aggregation that was induced by U46619, a thromboxane A(2) (TXA(2)) analogue. TXA(2) is an aggregation-inducing autacoidal molecule that is produced in various agonist-activated platelets. Cordycepin completely inhibited U46619-induced platelet aggregation and simultaneously reduced cytosolic free Ca(2+) ([Ca(2+)](i)), which was increased by U46619 (5 microM) up to 66%. Furthermore, the U46619-stimulated phosphorylation of Ca(2+)-dependent proteins (20 kDa of a myosin light chain and 47 kDa of pleckstrin) was strongly inhibited by cordycepin. These results suggest that cordycepin may have a beneficial effect on autacoidal TXA(2)-mediated thrombotic diseases by inhibiting TXA(2)-induced platelet aggregation via suppression of the Ca(2+) level.

Estrogen potentiates vasopressin-induced contraction of female rat aorta by enhancing cyclooxygenase-2 and thromboxane function.

To determine the roles of estrogen and constrictor prostanoids in vasopressin (VP)-induced contraction of female rat aorta, vascular reactivity to VP was determined in thoracic aortas of intact, ovariectomized, and ovariectomized + estrogen-replaced female rats in the presence of indomethacin (Indo), NS-398, SQ-29,548, or vehicle control. The effects of estrogen on vascular reactivity to the thromboxane A(2) analog U-46619 were also examined. Maximal contractile response to VP in intact female rats (5,567 +/- 276 mg/mg of aortic ring wt) was markedly attenuated by ovariectomy (2,485 +/- 394 mg; P < 0.001) and restored by estrogen replacement with 17beta-estradiol (5,059 +/- 194 mg; P > 0.1). Indo and NS-398 significantly attenuated maximal responses to VP in intact female rats to a similar extent [3,176 +/- 179 (P < 0.0001) and 3,258 +/- 152 mg (P < 0.0001), respectively]. Ovariectomy abolished and estrogen replacement restored the inhibitory effects of Indo, NS-398, and SQ-29,548. Contractile responses of rat aorta to U-46619 were significantly greater (P < 0.0001) in females (5,040 +/- 238 mg) than in males (3,679 +/- 96 mg). Ovariectomy markedly attenuated (3,923 +/- 84 mg; P < 0.01) and estrogen replacement restored (5,024 +/- 155 mg; P > 0.1) responses to U-46619 in female aortas. These data reveal that estrogen is an important regulator of the contractile responses of female rat aorta to VP, which appears to potentiate both cyclooxygenase-2 and constrictor prostanoid function in the vascular wall.

Glibenclamide inhibits thromboxane-mediated vasoconstriction by thromboxane receptor blockade.

Because sulfonylureas, such as glibenclamide, are used to treat Type 2 diabetes and because this disease is associated with various cardiovascular complications that may be mediated by thromboxane (TX), this study was designed to characterize the role of glibenclamide on TX-mediated contractions in isolated ring segments of bovine coronary arteries and rabbit aortas. A series of TXA(2) analogs [9,11 Dideoxy-9alpha, 11alpha-methanoepoxy prostaglandin F(2alpha) (U46619), [1S-(1alpha, 2beta(5Z),3alpha(1E, 3R*),4alpha)]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo [2.2.1]heptan-2-yl]-5-heptenoic acid (I-BOP), carbocyclic TXA(2) (CTA(2)) and 9,11-dideoxy-9alpha,11alpha-epoxymethano prostaglandin F(2alpha) (U44069)], endothelin and phenylephrine contracted both types of blood vessels. Glibenclamide (10 microM) inhibited the contraction to each of the TX agonists but had no effect on endothelin- or phenylephrine-induced contractions. We hypothesized that this effect was due to a direct effect to block the vascular smooth muscle cell TX receptor. Receptor binding studies were performed in rabbit vascular smooth muscle cells and indicated that glibenclamide (10 microM) inhibited (125)I-BOP binding by more than 80%. The inhibition constants or K(i) for glibenclamide was 0.53 microM. These studies provide the first evidence that the ability of glibenclamide to inhibit TX-mediated contractions occurs independent of the vascular K(ATP) channel and is, instead, mediated by the blockade of the vascular TX receptor.

Iloprost inhibits superoxide formation and gp91phox expression induced by the thromboxane A2 analogue U46619, 8-isoprostane F2alpha, prostaglandin F2alpha, cytokines and endotoxin in the pig pulmonary artery.

Since the roles of thromboxane A2 (TXA2), prostacyclin (PGI2) and 8-isoprostane F2alpha in mediating vascular O2*- formation and its relation to adult respiratory distress syndrome (ARDS) is unknown, the effects of these eicosanoids on the expression of gp91phox (catalytic subunit of NADPH oxidase) and O2*- release from cultured pig pulmonary artery (PA) segments, PA vascular smooth muscle cells (PAVSMCs) and PA endothelial cells (PAECs) were investigated. PA segments, PAVSMCs and PAECs were incubated with the TXA2 analogue, U46619, (+/-LPS, tumour necrosing factor-alpha (TNF-alpha) or IL-1alpha), 8-isoprostane F2alpha and+/-iloprost (a stable PGI2 analogue) for 16 h. The formation of superoxide dismutase-inhibitable O2*- was then measured spectrophotometrically and gp91phox expression assessed using Western blotting. In parallel experiments, whole PA segments were treated with LPS, TNF-alpha and IL-alpha after which time TXA2, PGI2, PGF2alpha and 8-isoprostane F2alpha formation was measured using enzyme-linked immunoassays. U46619, PGF2alpha and 8-isoprostane F2alpha promoted the formation of O2*- in PA segments, PAVSMCs and PAECs, an effect inhibited by diphenyleneiodonium and apocynin (both NADPH oxidase inhibitors) and upregulated the expression of gp91phox in PAECs and PAVSMCs. These effects were augmented by LPS, TNF-alpha and IL-1alpha but inhibited by iloprost. Under identical incubation conditions, IL-1alpha, LPS and TNF-alpha all induced an increase in the formation of TXA2, PGF2alpha and 8-isoprostane F2alpha but reduced the concomitant formation of PGI2. These data demonstrate that LPS and cytokines influence the relative balance of TXA2, PGI2, PGF2alpha and 8-isoprostane F2alpha in pig PA, which in turn alter NADPH oxidase expression and O2*- formation. These novel findings have implications in devising effective strategies for treating ARDS.British Journal of Pharmacology (2004) 141, 488-496. doi:10.1038/sj.bjp.0705626

Thrombopoietin increases platelet adhesion under flow and decreases rolling.

Thrombopoietin (TPO) is known to sensitize platelets to other agonists at 20 ng/ml, and above 100 ng/ml it is an independent activator of aggregation and secretion. In studies with a perfusion chamber, TPO, between 0.01 ng/ml and 1 ng/ml, increased platelet adhesion to surface-coated fibrinogen, fibronectin and von Willebrand Factor (VWF) but not to a collagen-coated surface. Increased adhesion was observed at shear rates of 300/s and 800/s in perfusions with whole blood as well as in suspensions of platelets and red blood cells reconstituted in plasma. The by the cyclooxygenase inhibitor, indomethacin, and the thromboxane A2-receptor blocker, SQ30741, abolished the stimulation by TPO. The effect of TPO was mimicked by a very low concentration (10 nmol/l) of the thromboxane TxA2 analogue, U46619. Real-time studies of platelet adhesion to a VWF-coated surface at a shear of 1000/s showed that about 20% of the platelets were in a rolling phase before they became firmly attached. TPO (1 ng/ml) pretreatment reduced this number to < 5%, an effect again abolished by indomethacin. Thus, TPO potentiates the direct and firm attachment of platelets to surface-coated ligands for alphaIIbbeta3, possibly by increasing the ligand affinity of the integrin.

Differential effects of 5,6-EET on segmental pulmonary vasoactivity in the rabbit.

In the rabbit, 5,6-epoxyeicosatrienoic acid (EET) was reported both to dilate and to constrict pulmonary blood vessels. We propose that these seemingly contradictory results could be explained by differences in responses to 5,6-EET in large-conductance pulmonary arteries (PA) compared with smaller PA and resistance vessels. Thus we found that in rings of extralobar PA [>2-mm outside diameter (OD)], in which active tension had been increased with PGF(2alpha), 5,6-EET produced relaxation in a concentration- and cyclooxygenase (COX)-dependent manner. In contrast, 5,6-EET increased tension in intralobar (1- to 2-mm OD) PA. Small extralobar PA (2- to 2.5-mm OD) exhibited intermediate responses. In the intact lung, the net effect of 5,6-EET (1 x 10(-8)-1 x 10(-5) M) was an increase in pulmonary vascular resistance (PVR) from 13.0 +/- 0.5 to 47.8 +/- 4.6 mmHg. 100 ml(-1) x min(-1) (EC(50) 5.9 +/- 1.7 x 10(-7) M). The increase in PVR was accompanied by a 10-fold increase in perfusate thromboxane (TX)B(2) concentration. The 5,6-EET-induced increase in PVR was prevented with indomethacin (100 microM), a cyclooxygenase inhibitor, or ONO-3708 (20 microM), a TX/PGH(2) (TP) receptor antagonist, but not with OKY-046 (700 microM), a TX synthase inhibitor. These results demonstrate that although 5,6-EET dilates large extralobar PA segments in a COX-dependent manner, in the intact rabbit lung 5,6-EET produces constriction that requires synthesis of a COX-dependent agonist of the TP receptor other than TX.

Nitric oxide-induced Cl- secretion in isolated rat colon is mediated by the release of thromboxane A2.

We have shown previously that thromboxane A2 (TXA2), which may be released by the anti-tumour drug irinotecan and by platelet-activating factor (PAF), causes Cl- secretion in the isolated rat colon. In the present study, the involvement of TXA2 in nitric oxide-induced Cl- secretion in isolated rat colon was investigated. In colonic mucosa set between Ussing chambers, the NO donor sodium nitroprusside (SNP; 100 microM) caused Cl- secretion, an effect that was almost completely inhibited by the NO scavenger carboxy-PTIO at 200 microM. The SNP-induced Cl- secretion was inhibited in a concentration-dependent manner by the TXA2 receptor antagonist ONO-3708 (IC50 = 2 microM) and the TX synthase inhibitor Y-20811 (IC50 = 0.4 microM). SNP significantly increased the release of TXA2 (measured as TXB2 release) from the mucosa. The SNP-induced increases in Cl- secretion and TXA2 release were blocked by a NO-sensitive guanylate cyclase inhibitor (ODQ). Dibutyryl cGMP (500 microM) also induced Cl- secretion, which was sensitive to ONO-3708 (10 microM) and Y-20811 (1 microM), and increased the release of TXA2 from the mucosa. PAF-induced (10 microM) Cl- secretion was inhibited by carboxy-PTIO (200 microM) and ODQ (10 microM), whereas irinotecan-induced (500 microM) Cl- secretion was not significantly inhibited by these drugs. A stable TXA2 analogue (STA2) but not SNP (100 microM) changed the membrane potential of epithelial cells in isolated colonic crypts under the whole-cell current-clamp condition. These results indicate that PAF elicits the NO-cGMP pathway and then stimulates the release of TXA2, which is a stimulant of colonic Cl- secretion. In contrast, the NO-cGMP pathway is not involved in the TXA2-mediated Cl- secretion induced by irinotecan.

U-46619-induced potentiation of noradrenergic constriction in the human saphenous vein: antagonism by thromboxane receptor blockade.

OBJECTIVE: We investigated the potentiating effect of U-46619, a synthetic analogue of thromboxane A(2) (TXA(2)), on the adrenergic responses in human saphenous vein. METHODS: Saphenous vein rings were obtained from 35 patients undergoing coronary artery bypass surgery. The rings were suspended in organ bath chambers for isometric recording of tension. RESULTS: U-46619 (10(-10)-3 x 10(-7) mol/l) produced concentration-dependent and endothelium-independent contractile responses. U-46619 (10(-10) mol/l) potentiated the contractions elicited by electrical stimulation and potassium chloride, and produced leftward shifts of the concentration-response curve for noradrenaline. The TXA(2) receptor antagonist SQ-30741 (10(-8) mol/l) prevented the potentiation evoked by U-46619. The dihydropyridine calcium antagonist nifedipine (10(-6) mol/l) did not affect the potentiation of electrical stimulation and noradrenaline induced by U-46619, but abolished the potentiation of U-46619 on KCl-induced contractions. CONCLUSIONS: U-46619 facilitates sympathetic neurotransmission and potentiates constrictor effects of noradrenaline in human saphenous veins through stimulation of TXA(2) receptors. These effects are independent of calcium entry through dihydropyridine calcium channels.

Pathogenetic analysis of three cases with a bleeding disorder characterized by defective platelet aggregation induced by Ca2+ ionophores.

We report three cases of platelet dysfunction characterized by defective Ca2+ ionophore-induced platelet aggregation without impaired production of thromboxane A2 (TXA2). The patients had mild to moderate bleeding tendencies, and their platelet aggregation and secretion induced by ADP, collagen, arachidonic acid, stable TXA2 (STA2) and Ca2+ ionophore A23187 was defective or much reduced. However, ristocetin- or thrombin-induced platelet aggregation was normal. The analysis of second messenger formation showed that inositol 1,4,5-triphosphate formation or Ca2+ mobilization induced by thrombin, STA2 or A23187 was normal. Furthermore, the phosphorylation of 47 kDa protein (pleckstrin) and 20 kDa protein (myosin light chain, MLC) in response to those agonists was normal. These findings suggest that the defective site in the patients' platelets lies in the process distal to or independent of protein kinase C activation, Ca2+ mobilization and MLC phosphorylation.

Adenosine signaling in outer medullary descending vasa recta.

We tested whether dilation of outer medullary descending vasa recta (OMDVR) is mediated by cAMP, nitric oxide (NO), and cyclooxygenase (COX). Adenosine (A; 10(-6) M)-induced vasodilation of ANG II (10(-9) M)-preconstricted OMDVR was mimicked by the cAMP analog 8-bromoadenosine 3',5'-cyclic monophosphate (10(-10) to 10(-4) M) and reversed by the adenylate cyclase inhibitor SQ-22536. Adenosine (10(-4) M) stimulated OMDVR cAMP production greater than threefold. NO synthase blockade with N(G)-nitro-L-arginine methyl ester and N(G)-monomethyl-L-arginine (10(-4) M) did not affect adenosine vasodilation. Adenosine induced endothelial cytoplasmic calcium transients that were small. Indomethacin (10(-6) M) reversed adenonsine-induced dilation of OMDVR preconstricted with ANG II, endothelin, 4-bromo-calcium ionophore A23187, or carbocyclic thromboxane A(2). In contrast, selective A(2)-receptor activation dilated endothelin-preconstricted OMDVR even in the presence of indomethacin. We conclude that OMDVR vasodilation by adenosine involves cAMP and COX but not NO. COX blockade does not fully inhibit selective A(2) receptor-mediated OMDVR dilation.

Combined blockade of endothelin-1 and thromboxane A(2) receptors against postischaemic contractile dysfunction in rat hearts.

1. Endothelin-1 (ET-1) may play a role in myocardial ischaemia/reperfusion injury because both the release and vasoconstrictor effect of ET-1 are increased after ischaemia. Since the increased vasoconstrictor effect of ET-1 can be mediated by ET-1-induced release of thromboxane A(2) (TXA(2)), the aim of this study was to test whether combined blockade of ET and TXA(2) receptors protects the coronary flow, contractile performance, and cardiac energy metabolism during ischaemia and reperfusion. 2. Bosentan (antagonist for ET(A) and ET(B) receptors, 1 microM based on concentration-response curves of ET-1), SQ 30,741 (antagonist of TXA(2) receptors, 0.1 microM), or the combination thereof was administered to isolated perfused rat hearts undergoing 15 min of global ischaemia and 60 min of reperfusion. 3. Neither bosentan or SQ 30,741 alone, nor the combination thereof, improved the incomplete postischaemic recovery of coronary flow, left ventricular developed pressure, phosphocreatine, or ATP. However, they attenuated ischaemia-induced acidosis but this did not translate into a measurable effect on haemodynamic or metabolic variables. 4. Thus, combined blockade of ET and TXA(2) receptors does not protect the coronary flow, contractile performance, and cardiac energy metabolism during ischaemia and reperfusion in isolated perfused rat hearts. This finding suggests that neither ET-1 nor ET-1-induced release of TXA(2) play a major role in the postischaemic recovery of the cardiac contractile function and energy metabolism.

Vasodilatory response of fetoplacental vasculature to adrenomedullin after constriction with the thromboxane sympathomimetic U46619.

OBJECTIVE: This study was undertaken to determine whether adrenomedullin, a hypotensive peptide, decreases vasomotor tone in fetoplacental vasculature that has been constricted with the thromboxane sympathomimetic U46619. STUDY DESIGN: The fetoplacental vascular beds of 20 perfused human placental cotyledons were vasoconstricted with a continuous infusion of U46619 (10(-8) mol/L). The vasculature was then sequentially injected with deionized water, 30 ng adrenomedullin, 300 ng adrenomedullin, and 3000 ng adrenomedullin. Any change in perfusion pressure was noted after each dose. In a separate experiment the fetoplacental vasculature in 2 perfused cotyledons from each of 10 placentas was vasoconstricted with U46619 (10(-8) mol/L). Adrenomedullin was infused continuously at either 200 ng/min (n = 5) or 2000 ng/min (n = 5) for 40 minutes. A corresponding control cotyledon from each placenta had isotonic sodium chloride solution added to its perfusion. Perfusion pressures were recorded every minute during the infusion and for 40 minutes afterward. Analysis of variance was used to compare pressure changes in the cotyledons that received bolus doses of adrenomedullin. Paired t tests of mean percentage pressure changes were used to compare the study and control groups that received the continuous infusions. RESULTS: In the cotyledons that received bolus doses of adrenomedullin, the mean (+/-SEM) percentage perfusion pressure changes from the baseline were -6.7 +/- 0.5 for 30 ng adrenomedullin (P =.0039), -8.5+/- 0.7 for 300 ng adrenomedullin (P <.0001), and -13.1 +/- 1.0 for 3000 ng adrenomedullin (P <.0001). With the continuous adrenomedullin infusion of 200 ng/min, there was no significant difference in the mean percentage pressure change from baseline between the study and control groups (-0.57%). At 2000 ng/min there was a significant difference (-15.34%; P <.0001). CONCLUSION: Adrenomedullin caused vasodilatation of fetoplacental vasculature previously constricted with the thromboxane sympathomimetic U46619 in the isolated perfused placental cotyledon. This vasodilatation occurred in a dose-dependent manner.

Inhibition of thromboxane A(2)-induced Cl(-) secretion by antidiarrhea drug loperamide in isolated rat colon.

The antitumor drug irinotecan clinically causes severe diarrhea as a side effect. Thromboxane A(2) (TXA(2)), released by irinotecan, has been shown to be a novel physiological stimulant of Cl(-) secretion in the rat colon. Herein, we examined the effect of loperamide, an antidiarrhea drug, on Cl(-) secretion induced by irinotecan; 9, 11-epithio-11,12-methano-thromboxane A(2) (STA(2)), a stable TXA(2) analog; and prostaglandin E(2) (PGE(2)) by using isolated mucosae of the rat colon. In the presence of atropine, loperamide in a concentration-dependent manner inhibited the Cl(-) secretion induced by irinotecan, STA(2), and PGE(2). However, the drug inhibited more effectively the irinotecan- and STA(2)-induced secretion (IC(50) = 0. 7 and 1.2 microM, respectively) than the PGE(2)-induced secretion (IC(50) = 23 microM). Naloxone, an opiate antagonist, did not affect the antisecretory action of loperamide. Similar to the case for loperamide, W-7, a specific calmodulin antagonist, inhibited more effectively the STA(2)-induced Cl(-) secretion (IC(50) = 5 microM) than the PGE(2)-induced secretion (IC(50) = 36 microM). W-5, a low-affinity calmodulin antagonist (a dechlorinated control analog of W-7), also inhibited the STA(2)-induced secretion, but this effect was much less than that of W-7. STA(2)-induced increase in the intracellular free Ca(2+) concentration of single colonic crypt cells was not affected by loperamide. We suggest that loperamide efficiently inhibits the TXA(2)-induced secretion by blocking the calmodulin system in the colonic epithelium. The present results may explain why coadministration of loperamide with irinotecan is clinically efficient for avoiding the irinotecan-induced side effect of diarrhea.

Characterization of changes in mechanical responses to histamine in omental resistance arteries in pre-eclampsia.

Changes in the effect of histamine on the smooth muscle of resistance arteries in pre-eclampsia were investigated by measuring isometric contractions in endothelium-denuded strips of omental resistance arteries from pre-eclamptic and normotensive pregnant women (pregnancy-term matched). Histamine (0.03 -1 microM) caused concentration-dependent relaxation of the contraction induced by 9, 11-epithio-11,12-methano-thromboxane A(2) (STA(2)) in strips from both groups. Sensitivity (for pre-eclampsia: pD(2)=6.66+/-0.04, n=5 and for normotensive pregnant women: pD(2)=7.07+/-0.03, n=10, P<0.001) was lower and the maximum response (90.6+/-0.6% vs 95.5+/-1.1%, P<0.05) was smaller in strips from pre-eclamptic women. Although 8-bromoadenosine-3', 5'-cyclic monophosphorothioate (Sp-isomer: Sp-8-Br-cAMPS, 0.1 - 0.3 mM), a phosphodiesterase (PDE)-resistant activator of adenosine-3',5'-cyclic monophosphate (cyclic AMP)-dependent protein kinase, concentration-dependently attenuated the contraction induced by STA(2) in strips from both groups, the sensitivity (for pre-eclampsia: pD(2)=3.68+/-0.04, n=5 and for normotensive pregnant women: 3.94+/-0.09, n=7, P:=0.02) was lower and the maximum response (64.2+/-2.4% vs 74.9+/-4.4%, P:<0.05) was smaller in pre-eclampsia. In beta-escin-skinned strips, the pD(2) value for the contraction-inducing effect of Ca(2+) did not differ significantly between the two groups (for pre-eclampsia, n=6; for normotensive pregnant women, n=6). Thus, omental resistance arteries from human subjects with pre-eclampsia showed (i) a weaker H(2)-receptor-mediated relaxation to histamine and (ii) a weaker cyclic AMP-analogue-induced relaxation, suggesting that the reduced action of histamine may be partly due to a decreased effect of cyclic AMP.

Involvement of phosphatidylcholine-specific phospholipase C in thromboxane A2-induced activation of mitogen-activated protein kinase in astrocytoma cells.

Thromboxane A2 (TXA2) receptor-mediated signal transduction was investigated in 1321N1 human astrocytoma cells. 9,11-Epithio-11,12-methano-TXA2 (STA2), a TXA2 receptor agonist, induced Ca2+ mobilization and phosphoinositide hydrolysis in a concentration-dependent manner. These responses were inhibited by treatment with U73122, an inhibitor of phosphatidylinositol-specific phospholipase C, or by culturing in 0.5% fetal calf serum containing 0.5 mM dibutyryladenosine 3',5'-cyclic monophosphate (dbcAMP) for 2 days. However, the dbcAMP treatment augmented the TXA2 receptor-mediated phosphorylation of mitogen-activated protein kinase (MAPK). These results were confirmed by a functional MAPK assay measuring the incorporation of 32P into the MAPK substrate peptide. The TXA2 receptor-mediated MAPK activation was inhibited by SQ29548, a TXA2 receptor antagonist, and GF109203X, an inhibitor of protein kinase C. Although U73122 did not inhibit or only slightly inhibited the activation of MAPK, D-609, an inhibitor of phosphatidylcholine-specific phospholipase C, potently attenuated the activation in a concentration-dependent manner. Furthermore, STA2 accelerated the release of [3H]choline metabolites from the cells prelabeled with [3H]choline chloride. This release was inhibited by treatment with D-609. These results suggest that phosphatidylcholine-specific phospholipase C and protein kinase C, but not phosphatidylinositol-specific phospholipase C, are involved in TXA2 receptor-mediated MAPK activation in 1321N1 human astrocytoma cells.

The P2Y1 receptor mediates ADP-induced p38 kinase-activating factor generation in human platelets.

U46619, a thromboxane A2 mimetic, but not ADP, caused activation of p38 mitogen activated protein (MAP) kinase in aspirin-treated platelets. In nonaspirinated human platelets ADP activated p38 MAP kinase in both a time-and concentration-dependent manner, suggesting that ADP-induced p38 MAP kinase activation requires generation of thromboxane A2. However, neither a thromboxane A2/prostaglandin H2 receptor antagonist SQ29548 and a thromboxane synthase inhibitor, furegrelate, either alone or together, nor indomethacin blocked ADP-induced p38 kinase activation in nonaspirinated platelets. Other cycloxygenase products, PGE2, PGD2, and PGF2alpha, failed to activate p38 kinase in aspirin-treated platelets. Hence, ADP must be generating an agonist, other than thromboxane A2, via an aspirin-sensitive pathway, which is capable of activating p38 kinase. AR-C66096, a P2TAC (platelet ADP receptor coupled to inhibition of adenylate cyclase) antagonist, did not inhibit ADP-induced p38 MAP kinase activation. The P2X receptor selective agonist, alpha, beta-methylene ATP, failed to activate p38 MAP kinase. On the other hand, the P2Y1 receptor selective antagonist, adenosine-2'-phosphate-5'-phosphate inhibited ADP-induced p38 kinase activation in a concentration-dependent manner, indicating that the P2Y1 receptor alone mediates ADP-induced generation of the p38 kinase-activating factor. These results demonstrate that ADP causes the generation of a factor in human platelets, which can activate p38 kinase, and that this response is mediated by the P2Y1 receptor. Neither the P2TAC receptor nor the P2X1 receptor has any significant role in this response.

Prostanoid stimulation of cytokine production in an amnion-derived cell line: evidence of a feed-forward mechanism with implications for term and preterm labor.

OBJECTIVE: To test the hypothesis that amnion cytokine production might be regulated by prostanoids. METHODS: Amnion-derived WISH cells were treated with a range of prostanoids and their effects on production of interleukin (IL)-6 and IL-8 were determined by enzyme-linked immunosorbent assay and Northern analysis. The effects of thromboxane inhibitors on cytokine production by term primary amnion explants also were examined. RESULTS: Prostaglandin (PG)A2, PGD2, PGF2 alpha, PGE2, PGJ2, and the PGI2 analogue carbaprostacyclin (1-1000 nmol/L) exhibited no significant effects on cytokine production. However, the thromboxane A2 (TXA2) agonist U46619 and carbocyclic (c)TXA2 both stimulated WISH cytokine production with similar potencies under basal or cytokine-stimulated conditions. Significant stimulation of IL-6 production was observed at concentrations > or = 8 nmol/L (P < .05 by analysis of variance), whereas IL-8 production was stimulated significantly but to a lesser extent. The effects of U46619 and cTXA2 were rapid; maximal stimulation of cytokine production occurred within 4 to 8 hours of treatment. U46619 augmented IL-1 beta-stimulated IL-6 and IL-8 mRNA expression within 2 hours of treatment. In amnion explants inhibitors of TX synthesis and action abrogated the stimulatory effects of IL-1 beta on cytokine production. CONCLUSION: These results are consistent with the presence of a feed-forward loop in amnion involving TXA2 and cytokines, which could play a significant role in the progression of the inflammatory response involved in the mechanism of infection-driven preterm labor.