Thromboxane-induced phosphatidate formation in human platelets. Relationship to receptor occupancy and to changes in cytosolic free calcium.

The inter-relationships between receptor occupancy, inositol phospholipid metabolism and elevation of cytosolic free Ca2+ in thromboxane A2-induced human platelet activation were investigated by using the stable thromboxane A2 mimetic, 9,11-epoxymethanoprostaglandin H2, and the thromboxane A2 receptor antagonist, EPO45. 9,11-Epoxymethanoprostaglandin H2 stimulated platelet phosphatidylinositol metabolism as indicated by the rapid accumulation of [32P]phosphatidate and later accumulation of [32P]phosphatidylinositol in platelets pre-labelled with [32P]Pi. These effects of 9,11-epoxymethanoprostaglandin H2 were concentration-dependent and half-maximal [32P]phosphatidate formation occurred at an agonist concentration of 54 +/- 8 nM. With platelets labelled with the fluorescent Ca2+ indicator quin 2, resting cytosolic free Ca2+ was 86 +/- 12 nM. 9,11-Epoxymethanoprostaglandin H2 induced a rapid, concentration-dependent elevation of cytosolic free Ca2+ to a maximum of 300-700 nM. Half-maximal stimulation was observed at an agonist concentration of 80 +/- 23 nM. The thromboxane A2 receptor antagonist EPO45 selectively inhibited 9,11-epoxymethanoprostaglandin H2-induced [32P]phosphatidate formation and elevation of cytosolic free Ca2+, indicating that both events are sequelae of receptor occupancy. Human platelets contain a single class of stereospecific, saturable, high affinity (KD = 70 +/- 13 nM) binding sites for 9,11-epoxymethano[3H]prostaglandin H2. The concentration-response curve for receptor occupancy (9,11-epoxymethano-[3H]prostaglandin H2 binding) is similar to that for 9,11-epoxymethanoprostaglandin H2-induced [32P]phosphatidate formation and for elevation of cytosolic free Ca2+. These observations indicate that human platelet thromboxane A2 receptor occupation is closely linked to inositol phospholipid metabolism and to elevation of cytosolic free Ca2+. Both such events may be necessary for thromboxane A2-induced human platelet activation.     

Thromboxane receptor antagonism combined with thromboxane synthase inhibition. 7. pyridinylalkyl-substituted arylsulfonylamino arylalkanoic acids.

Arylsulfonylamino arylalkanoic acids substituted with a pyridinylalkyl group on the arylalkanoic acid portion of the molecule were synthesized and found to behave as thromboxane receptor antagonists (TxRAs) and thromboxane synthase inhibitors (TxSIs). One of these compounds (11), with a 1,3,5-trisubstituted central aromatic ring was demonstrated to have good functional bioavailability and efficacy as a platelet inhibitor in guinea pigs.     

Interaction of thromboxane A2 and leukotrienes in guinea pig airways in vivo.

Effects of a thromboxane A2 receptor antagonist (S-1452) on bronchoconstriction induced by inhaled leukotriene C4 and a leukotriene receptor antagonist (AS-35) on bronchoconstriction caused by inhalation of a thromboxane A2 mimetic (STA2) were studied in anesthetized, artificially ventilated guinea pigs in order to examine the interaction of thromboxane A2 and leukotrienes in airways. 0.01-1.0 mu g/ml of leukotriene C4 and 0.1-1.0 mu g/ml of STA 2 inhaled from ultrasonic nebulizer developed for small animals caused dose-dependent increase of pressure at the airway opening (Pao) which is considered to be an index representing bronchial response. Pretreatment of the animals with inhaled S-1452 (0.01, 0.033 mg/ml) significantly reduced the airway responses produced by 0.01,0.033,0.1,0.33 and 1.0 mu g/ml of leukotriene C4 in a dose dependent manner. While pretreatment with inhaled AS-35 (1mg) did not affect the STA2 dose-response curve. These findings suggest that leukotriene C4 activates thromboxane A2 generation while thromboxane A2 does not influence 5-lipoxygenase pathway in the airways.     

Physiologic role for enhanced renal thromboxane production in murine lupus nephritis.

To investigate the physiologic significance of enhanced renal thromboxane production in murine lupus nephritis, we measured renal hemodynamics and eicosanoid production in MRL-lpr/lpr mice from 8 to 20 weeks of age. Over this age range, MRL-lpr/lpr mice develop an autoimmune disease with nephritis similar to human systemic lupus erythematosus (SLE). In these studies, glomerular filtration rate (GFR) and PAH clearance (CPAH) decreased progressively with age in MRL-lpr/lpr mice, but not in controls. This impairment of renal hemodynamics was associated with increased renal thromboxane production, as well as increased excretion of both thromboxane B2 (TxB2) and 2,3-dinor TxB2 in urine. There was an inverse correlation between renal thromboxane production in MRL-lpr/lpr mice and both GFR and CPAH. Furthermore, there were positive correlations between thromboxane production by the kidney and both the severity of renal histopathology and serum anti-DNA antibody levels measured in individual animals. Enhanced urinary excretion of TxB2 and the development of renal dysfunction also coincided temporally with the appearance of increased levels of interleukin 1 beta (IL-1 beta) mRNA in renal cortex. Acute administration of the specific thromboxane receptor antagonist GR32191 to MRL-lpr/lpr mice restored GFR to normal in early stages of the autoimmune disease. However, in animals with more advanced nephritis, the effect of acute thromboxane receptor blockade on renal hemodynamics was less marked. We conclude that thromboxane A2 is an important mediator of reversible renal hemodynamic impairment in murine lupus, especially in the early phase of disease.     

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)     

Role of the thromboxane A2 receptor in the vasoactive response to ischemia-reperfusion injury.

Neutrophil-endothelial adhesion in venules and progressive vasoconstriction in arterioles seem to be important microcirculatory events contributing to the low flow state associated with ischemia-reperfusion injury of skeletal muscle. Although the neutrophil CD-18 adherence function has been shown to be a prerequisite to the vasoconstrictive response, the vasoactive substances involved remain unknown. The purpose of this study was to evaluate the role of thromboxane A2 receptor in the arteriole vasoactive response to ischemia-reperfusion injury. An in vivo microscopy preparation of transilluminated gracilis muscle in male Wistar rats (175 +/- 9 g) (n = 12) was used for this experiment. Three experimental groups were evaluated in this study: (1) sham, flap raised, no ischemia (20 venules, 20 arterioles), (2) 4 hours of global ischemia only (19 venules, 22 arterioles), and (3) 4 hours of global ischemia + thromboxane A2 receptor antagonist (ONO-3708) (17 venules, 20 arterioles). ONO-3708 (5 mg/kg), a specific competitive antagonist of thromboxane A2 receptor, was infused at a rate of 0.04 ml/minute into the contralateral femoral vein 30 minutes before reperfusion. Mean arterial blood pressure was not changed at this dose of ONO-3708 (88 +/- 6 mmHg before infusion, 81 +/- 4 mmHg after infusion, n = 3). The number of leukocytes rolling and adherent to endothelium (15-sec observation) were counted in 100-microm venular segments, and arteriole diameters were measured at 5, 15, 30, 60, and 120 minutes of reperfusion. Leukocyte counts and arteriole diameters were analyzed with two-way factorial analysis of variance for repeated measures and Duncan's post hoc mean comparison. Statistical significance was indicated by a p < or = 0.05. The ischemia-reperfusion-induced vasoconstriction was significantly reduced by the thromboxane A2 receptor antagonist (ONO-3708). The mean arteriole diameters at 30, 60, and 120 minutes reperfusion were significantly greater in the treated animals than in the ischemia-reperfusion controls. Despite a significant increase in treated mean arteriole diameters, 30 percent of arterioles still demonstrated vasoconstriction. Neutrophil-endothelial adherence was not reduced by ONO-3708. Thromboxane A2 receptor blockade significantly reduces but does not eliminate ischemia-reperfusion-induced vasoconstriction in this model. This finding suggests that additional and perhaps more important vasoactive mediators contribute to vasoconstriction. Furthermore, thromboxane A2 receptor blockade has no effect on polymorphonuclear endothelial adherence.     

The hepoxilin analog PBT-3 inhibits heparin-activated platelet aggregation evoked by ADP

We have previously shown that PBT-3, a stable synthetic analog of hepoxilins, inhibits the aggregation of human platelets in vitro evoked by collagen through inhibition of thromboxane A(2) formation and action on the TP receptor. We now show that PBT-3 is capable of potently inhibiting the second phase of aggregation evoked by ADP in both washed human platelets and platelet-rich plasma (PRP), a phase associated with thromboxane formation. Aspirin blocks this second phase as well; so does the thromboxane receptor antagonist SQ 29,548. When ADP-evoked aggregation in PRP is activated by heparin through an enhancement of thromboxane formation, PBT-3, aspirin as well as SQ 29,548 block this activation through different mechanisms. These data confirm the inhibitory action of PBT-3 on aggregation of human platelets through inhibition of both thromboxane formation and blockade of thromboxane receptor action and suggest that this family of compounds may be useful in the treatment of thrombotic disorders in combination with heparin.     

Thromboxane induced red blood cell lysis

The two thromboxane A2 mimetics, carbocyclic thromboxane A2 (CTA2) and U-46619 (9,11-methanoepoxy PGH2) at concentrations of 400 ng/ml significantly enhanced the release of hemoglobin from both feline and human erythrocyte suspensions. This effect was significantly attenuated by the thromboxane receptor antagonist BM-13,505 indicating that the membrane leakiness is in some way receptor mediated. The effects also appear to be concentration-dependent over the range of 100-400 ng/ml. The membrane labilizing effect of thromboxane analogs is not due to a non-specific eicosanoid effect since iloprost, the stable prostacyclin analog, actually stabilized erythrocyte membranes. Moreover, synthetic thromboxane A2 exerted similar effects to that of the two TxA2-mimetics. This membrane labilizing action of thromboxanes may be important in propagating the other pathophysiologic effects of thromboxane A2 in cardiovascular disease states.     

Antagonism of the TXA2 receptor by seratrodast: a structural approach.

The crystal structure of seratrodast (AA-2414), a potent thromboxane A2 (TXA2) receptor antagonist, served as starting point to docking studies with the modeled human TXA2 receptor. This structural approach provides rational basis for the design of new antagonists within the aryl sulfonamide family.     

Thromboxane A2/endoperoxide receptors mediate cholinergic constriction of rabbit lung microvessels.

Using an X-ray television system, we directly measured the internal diameter (ID; 100-1,000 microns) of small pulmonary arteries and analyzed the effects of cyclooxygenase inhibition and thromboxane A2/prostaglandin endoperoxide (TP) receptor blockade on the ID reductions in response to vagal nerve stimulation (VNS; 16 Hz) and injection of acetylcholine (ACh; 0.3 micrograms) in anesthetized rabbits. The ID reductions of the small arteries in response to VNS and ACh were completely abolished by pretreatment with cyclooxygenase inhibitors indomethacin and meclofenamate. Those reductions were also eliminated by pretreatment with TP receptor antagonists AA-2414 and Ono 3708. Both TP receptor antagonists abolished the ID reduction to thromboxane A2 mimetic U-46619 but did not affect the reduction to norepinephrine. The ID reductions in response to VNS and ACh were eliminated by atropine. The reduction in response to VNS was abolished by hexamethonium bromide, whereas the reduction in response to ACh was not altered by hexamethonium bromide. The results indicate that vasoconstrictions of the rabbit small pulmonary arteries in response to VNS and exogenous ACh are mediated by TP receptors as well as muscarinic receptors. The data suggest that during VNS endogenous ACh acts on muscarinic receptors to constrict the small arteries mainly by generating thromboxane A2 or prostaglandin endoperoxide.     

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.     

Output and effects of thromboxane produced by the liver perfused with phorbol myristate acetate

The capacity of the perfused rat liver to produce thromboxane after stimulation by phorbol myristate acetate was examined. A total of 109 +/- 20 and 155 +/- 28 pmol/g liver were found in the perfusate and in the bile, respectively, after 40 min. The amount of thromboxane recovered in the perfusate and in the bile accounted for 12.6% of the production calculated from the same number of Kupffer cells in primary cultures, indicating that a major part of thromboxane was taken up and inactivated by hepatocytes. The effect of endogenously synthesized thromboxane on the liver was assessed by using CGS 13080, a thromboxane synthase inhibitor, or BM 13.177, a thromboxane receptor antagonist. 20 nM CGS 13080 in the perfusate inhibited the synthesis of thromboxane and at the same time the elevation of portal pressure and glycogenolysis following administration of phorbol 12-myristate 13-acetate (PMA). The thromboxane receptor antagonist BM 13.177 did not inhibit the synthesis of thromboxane, but reduced the PMA-related elevation of portal pressure and glycogenolysis to the same extent (greater than 60%) as CGS 13080. Sodium nitroprusside, a vasodilator, inhibited the rise in portal pressure caused by PMA to the same extent as CGS 13080 or BM 13.177 but reduced the increase in glycogenolysis only by 25%. These results indicate that thromboxane released by stimulated Kupffer cells of the liver elevates portal pressure and glycogenolysis in the perfused rat liver, although by different mechanisms.     

Thromboxane modulating agents. 1. Design of 1-[(arylsulfonyl)amino] alkylindole derivatives as dual thromboxane synthase inhibitor/thromboxane receptor antagonists.

The design of a series of dual thromboxane synthase inhibitor/thromboxane receptor antagonists based on an indole thromboxane synthase inhibitor template is described. The indole-5-propanoic acid derivatives 17, 22 and 23 were found to be potent dual agents in vitro.     

Molecular mechanism of thromboxane A(2)-induced platelet aggregation. Essential role for p2t(ac) and alpha(2a) receptors.

Thromboxane A(2) is a positive feedback lipid mediator produced following platelet activation. The G(q)-coupled thromboxane A(2) receptor subtype, TPalpha, and G(i)-coupled TPbeta subtype have been shown in human platelets. ADP-induced platelet aggregation requires concomitant signaling from two P2 receptor subtypes, P2Y1 and P2T(AC), coupled to G(q) and G(i), respectively. We investigated whether the stable thromboxane A(2) mimetic, (15S)-hydroxy-9, 11-epoxymethanoprosta-5Z,13E-dienoic acid (U46619), also causes platelet aggregation by concomitant signaling through G(q) and G(i), through co-activation of TPalpha and TPbeta receptor subtypes. Here we report that secretion blockade with Ro 31-8220, a protein kinase C inhibitor, completely inhibited U46619-induced, but not ADP- or thrombin-induced, platelet aggregation. Ro 31-8220 had no effect on U46619-induced intracellular calcium mobilization or platelet shape change. Furthermore, U46619-induced intracellular calcium mobilization and shape change were unaffected by A3P5P, a P2Y1 receptor-selective antagonist, and/or cyproheptadine, a 5-hydroxytryptamine subtype 2A receptor antagonist. Either Ro 31-8220 or AR-C66096, a P2T(AC) receptor selective antagonist, abolished U46619-induced inhibition of adenylyl cyclase. In addition, AR-C66096 drastically inhibited U46619-mediated platelet aggregation, which was further inhibited by yohimbine, an alpha(2A)-adrenergic receptor antagonist. Furthermore, inhibition of U46619-induced platelet aggregation by Ro 31-8220 was relieved by activation of the G(i) pathway by selective activation of either the P2T(AC) receptor or the alpha(2A)-adrenergic receptor. We conclude that whereas thromboxane A(2) causes intracellular calcium mobilization and shape change independently, thromboxane A(2)-induced inhibition of adenylyl cyclase and platelet aggregation depends exclusively upon secretion of other agonists that stimulate G(i)-coupled receptors.     

The role of alternative splicing and C-terminal amino acids in thromboxane receptor stabilization

The thromboxane receptor has two alternatively spliced isoforms, alpha and beta, which differ only in sequences within the cytoplasmic C-terminal domain. Oxidative stress induced by H(2)O(2) in a COS-7 cell model results in stabilization of the thromboxane receptor beta isoform by translocation from the endoplasmic reticulum to the Golgi complex, which in turn results in protection of the receptor from degradation. We now report that both the alpha and beta thromboxane receptor isoforms respond identically to oxidative stress. Further, mutagenesis studies indicate that replacing the normal C-terminus with a nonsense sequence also does not alter stabilization behaviour ruling out a role for the distinct C-termini in this process. Further mutagenesis implicates a cluster of arginine residues within the C-terminal domain as involved in oxidative stress-induced stabilization. These data identify a region of the thromboxane receptor that is responsible for responding to oxidative challenge and open the possibility of identification of the molecular machinery underpinning this response.     

Different calcium pools in human platelets and their role in thromboxane A2 formation.

Activation of human platelets by diverse receptor-transduced signals is followed by an intracellular calcium increase. Calcium liberation from an inositol 1,4,5-trisphosphate-sensitive compartment is recognized to be one of the prime events, followed by further mechanisms to amplify the signal. Among these, the formation of prostaglandin endoperoxides and thromboxane A2 are part of the self-amplificating activation system. Two inhibitors of intracellular Ca(2+)-ATPases, thapsigargin and 2,5-di-(tert-butyl)-1,4-benzohydroquinone have been reported to deplete the intracellular inositol 1,4,5-trisphosphate-responsive stores. In human platelets with EGTA present, we found that these inhibitors of the microsomal Ca2+ sequestration generate quite different Ca2+ transients due to an inherent cyclooxygenase inhibition by the benzohydroquinone derivative compared to thapsigargin, and, therefore, only one-half of the fura-2 signal is generated. For a maximal calcium release, Ca(2+)-ATPase inhibitors depend on the self-amplification system involving thromboxane formation. Following the thapsigargin-induced [Ca2+]i transient, thrombin was unable to raise [Ca2+]i, indicating that thapsigargin mobilizes calcium from the thrombin-responsive store, as long as the self-amplifying system of platelets is intact. With the thromboxane receptor blocked, thapsigargin releases only one-half of the calcium, and, hence, thrombin was able to release additional calcium. Interestingly, in the converse experiment, thrombin did not prevent a raise of [Ca2+]i by thapsigargin at all, although applying thrombin a second time was without any effect. Therefore, we propose two calcium pools in human platelets: receptor activation transiently releases calcium from an inositol-sensitive pool including the thapsigargin-sensitive compartment, followed by reuptake within minutes. Sequestration occurs into the thapsigargin-sensitive compartment from where it can be released even when the endoperoxide/thromboxane receptor is blocked. Calcium release from both compartments allows the formation of thromboxane B2, but not if only the Ca(2+)-ATPase inhibitor-sensitive pool is emptied. In the presence of a protonophor, a calcium accumulation in the Ca(2+)-ATPase-sensitive pool could be observed.     

Blockade of thromboxane responses in the airway of the cat by SQ 29,548

The effects of SQ 29,548, a thromboxane receptor antagonist, on airway responses were investigated in paralyzed, anesthetized, mechanically ventilated cats. Intravenous injections of the thromboxane and prostaglandin precursor, arachidonic acid (AA), and the thromboxane mimic, U 46619, produced dose-related increases in transpulmonary pressure and lung resistance and decreases in dynamic compliance. After administration of SQ 29,548 (0.5 mg/kg iv), bronchoconstrictor responses to AA were reduced by approximately 50%, whereas responses to U 46619 were reduced by approximately 90%. The cyclooxygenase inhibitor, sodium meclofenamate (2.5 mg/kg iv), blocked the component of the airway response to AA remaining after treatment with SQ 29,548. The thromboxane receptor antagonist had no significant effect on bronchoconstrictor responses to prostaglandins F2 alpha, and D2, methacholine, 5-hydroxytryptamine, histamine, or BAY K 8644, an agent that promotes calcium entry. Reductions in systemic arterial pressure in response to AA were enhanced by the thromboxane receptor antagonist and abolished by meclofenamate. SQ 29,548 had no effect on terminal enzyme activity in microsomal fractions from cat lung. These data support the hypothesis that AA-induced bronchoconstriction in the cat is mediated in large part by the actions of thromboxane A2. These data also suggest that U 46619 and U 44069 stimulate the same airway receptor as thromboxane A2 and mimic the bronchomotor effects of this hormone, which has not yet been isolated as a pure substance. These data demonstrate that SQ 29,548 is a selective thromboxane receptor antagonist in the airways of the closed-chest cat and may be a useful probe for studying responses to thromboxane A2 in physiological and pathophysiological processes in the lung.     

Role of thromboxane A2 in airway microvascular leakage induced by inhaled platelet-activating factor.

We studied the effects of OKY-046 (1, 10, and 30 mg/kg iv), a selective thromboxane synthase inhibitor, and of ICI 192605 (0.5 mg/kg), a selective thromboxane A2 receptor antagonist, on airflow obstruction and airway microvascular leakage induced by inhaled platelet-activating factor (PAF). Extravasated Evans blue dye content was measured as a reflection of airway microvascular leakage. In control animals, PAF caused a significantly higher increase in extravasation of dye and significantly less increase in lung resistance (RL) than histamine. OKY-046 significantly inhibited both changes in RL and airway microvascular leakage after PAF in a dose-dependent manner, whereas it inhibited histamine-induced airway microvascular leakage only at main bronchi, without any significant effect on RL. ICI 192605 significantly inhibited both RL and airway microvascular leakage induced by PAF, but not after histamine. After both PAF and histamine, changes in RL correlated significantly with the degree of microvascular leakage. Airway microvascular leakage and airflow obstruction after PAF, but not after histamine, may be dependent on thromboxane A2 generation.     

Thromboxane receptor signalling in human myometrial cells.

We measured the effects of stable thromboxane A2 (TXA2) analogues on signalling in cultured human myometrial cells. U46619 and/or IBOP stimulated total inositol phosphates (IPs) and cAMP production, RhoA-associated protein kinase (ROK) activity and elevated intracellular calcium [Ca2+]i. Pretreatment of the cells with pertussis toxin did not inhibit IPs or [Ca2+]i production but the thromboxane receptor (TP) antagonist SQ-29548 did inhibit IPs and cAMP production, the elevation of [Ca2+]i, and the increase in ROK activity. Pretreatment with thapsigargin inhibited [Ca2+]i elevation. TP receptor-stimulated ROK activity was inhibited by the ROK inhibitor Y27632 while ROK activity was enhanced by the caspase 3 inhibitor, Z-DEVD-FMK. TP receptor-stimulated IPs production is additive to prostaglandin F2alpha (FP) or prostaglandin E (EP) receptor-stimulated IPs production and neither FP nor EP receptor-stimulated IPs production is inhibited by SQ29548. Thus cultured human myometrial cells express at least two functional TP receptor subtypes; TPalpha-like (cAMP-stimulating) and TPbeta-like (IPs, [Ca2+] and ROK-stimulating).