Immune complex induced arthritis in rats: role of lipid mediators on cell infiltration

We investigated the participation of lipid mediators in an experimental immune complex (IC) arthritis model in rats. The animals were subjected to intraarticular injection of anti-bovine sertLm albumin (BSA) IgG antibodies followed by i.v. injection of BSA. Histopathological analysis of the synovial membranes disclosed infiltration of polymorphonuclear (PMN) cells and vascular congestion. Slight increase in vascular permeability, measured by Evans blue dye extravasation into the joints, was detected after 3 h of arthritis. Cellular influx into the articular cavities was most evident at the sixth hour of arthritis with predominance of PMN. Pretreatment with either indomethacin, a cyclooxygenase inhibitor, or L-660,711, a peptido-leukotriene antagonist, did not inhibit cell infiltration, whereas pretreatment with either L-663,536, a 5-lipoxygenase inhibitor, or L-655,240, a thromboxane antagonist, significantly inhibited the phenomenon. Pretreatment with WEB 2170, a platelet activating factor (PAF) antagonist, also significantly inhibited cell influx. These results suggest that thromboxane, LTB(4) and PAF mediate cell infiltration in this IC arthritis model.     

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.     

Biphasic action of phospholipase A in collagen-stimulated rat platelets

The formation of thromboxane A2 (TXA2) in collagen-stimulated rat platelets was successfully divided into two stages, an initial and a second one, by the specific TXA2 receptor antagonist, ONO3708. In the presence of this antagonist, only the initial TXA2 production was observed, without the subsequent platelet shape change and aggregation. Collagen causes the specific cleavage of arachidonic acid from phosphatidylinositol (PI) in the initial stage, whereas in the absence of the antagonist, it caused decrease in the arachidonic acid levels in phosphatidylethanolamine (PE) and PI with concomitant formation of the respective lyso-forms. These results demonstrate that phospholipase A (PLA) preferentially acts on PI to release arachidonic acid which leads to the initial TXA2 production, which might be a trigger for the second release of arachidonic acid from PE and PI.     

Specific binding of the thromboxane A2 antagonist 13-azaprostanoic acid to human platelet membranes

In the present study we characterized the interaction between the thromboxane A2/prostaglandin H2 antagonist, trans-13-azaprostanoic acid (13-APA), and isolated human platelet membranes. In these studies, we developed a binding assay using trans [3H] 13-APA as the ligand. It was found that trans [3H] 13-APA specific binding was rapid, reversible, saturable and temperature dependent. Scatchard analysis of the binding data yielded a curvilinear plot which indicated the existence of two classes of binding sites: a high-affinity binding site with an estimated dissociation constant (Kd) of 100 nM; and a low-affinity binding site with an estimated Kd of 3.5 microM. At saturation, approximately 1 pmol/mg protein of [3H] 13-APA was bound to the high affinity site. In order to further characterize the nature of the [3H] 13-APA binding site, we evaluated competitive binding by cis 13-APA, cis 15-APA, prostaglandin F2 alpha, U46619, 6-ketoprostaglandin F1 alpha and thromboxane B2. It was found that the [3H] 13-APA binding site was stereospecific and structurally specific. Thus, the cis isomer of 13-APA exhibited substantially reduced affinity for binding. Furthermore, the prostaglandin derivatives, thromboxane B2 and 6-ketoprostaglandin F1 alpha, which do not possess biological activity, also did not compete for [3H] 13-APA binding. On the other hand, U46619 which acts as a thromboxane A2/prostaglandin H2 mimetic, and prostaglandin F2 alpha which acts as a thromboxane A2/prostaglandin H2 antagonist, both effectively competed for [3H] 13-APA binding. These findings indicate that trans 13-APA binds to a specific site on the platelet membrane which presumably represents the thromboxane A2/prostaglandin H2 receptor.     

Endothelial thromboxane receptors: biochemical characterization and functional implications

We have identified thromboxane specific receptors in membrane preparations of bovine pulmonary artery endothelial cells using a potent thromboxane specific antagonist, [125I]-PTA-OH in a binding assay. The binding was specific and saturable. Neither thromboxane B2, prostaglandin D2 nor prostaglandin F2 alpha displaced the ligand (0.1 nM) at concentrations up to 10 microM. However, binding was displaced by IPTA-OH greater than SQ29548 greater than U46619. In addition, we observed that thromboxane mimetic U46619 significantly lowered the basal production of prostacyclin and also markedly suppressed bradykinin-stimulated prostacyclin released by endothelial cells. We propose that an important biological effect of thromboxane on vascular endothelial cells may be the suppression of prostacyclin production.     

CD44 Antibodies and Immune Thrombocytopenia in the Amelioration of Murine Inflammatory Arthritis

Antibodies to CD44 have been used to successfully ameliorate murine models of autoimmune disease. The most often studied disease model has been murine inflammatory arthritis, where a clear mechanism for the efficacy of CD44 antibodies has not been established. We have recently shown in a murine passive-model of the autoimmune disease immune thrombocytopenia (ITP) that some CD44 antibodies themselves can induce thrombocytopenia in mice, and the CD44 antibody causing the most severe thrombocytopenia (IM7), also is known to be highly effective in ameliorating murine models of arthritis. Recent work in the K/BxN serum-induced model of arthritis demonstrated that antibody-induced thrombocytopenia reduced arthritis, causing us to question whether CD44 antibodies might primarily ameliorate arthritis through their thrombocytopenic effect. We evaluated IM7, IRAWB14.4, 5035-41.1D, KM201, KM114, and KM81, and found that while all could induce thrombocytopenia, the degree of protection against serum-induced arthritis was not closely related to the length or severity of the thrombocytopenia. CD44 antibody treatment was also able to reverse established inflammation, while thrombocytopenia induced by an anti-platelet antibody targeting the GPIIbIIIa platelet antigen, could not mediate this effect. While CD44 antibody-induced thrombocytopenia may contribute to some of its therapeutic effect against the initiation of arthritis, for established disease there are likely other mechanisms contributing to its efficacy. Humans are not known to express CD44 on platelets, and are therefore unlikely to develop thrombocytopenia after CD44 antibody treatment. An understanding of the relationship between arthritis, thrombocytopenia, and CD44 antibody treatment remains critical for continued development of CD44 antibody therapeutics.     

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.     

Mechanism of the inhibition of platelet aggregation produced by prostaglandin F2 alpha

The inhibition of human platelet aggregation produced by PGF2 alpha is not specific for thromboxane A2 mimetics. Aggregation waves induced by PAF and thrombin are also inhibited by PGF2 alpha (8 microM); ADP is unaffected. These effects are still seen in platelets from aspirin-treated donors and platelets desensitized to thromboxane-like agonists (e.g. 11,9-epoxymethano PGH2). In contrast the thromboxane receptor antagonist EP 045 (up to 20 microM) had no effect on primary aggregation induced by PAF, thrombin and ADP. We have previously shown that EP 045 (IC50 = 0.5 microM), but not PGF2 alpha (28 microM), displaces the specific binding of [3H] 9,11-epoxymethano PGH2 to washed human platelets. PGF2 alpha produces small increases in cAMP levels, and both this effect and the anti-aggregation are diminished by the adenyl cyclase inhibitor SQ 22536. The rise in cAMP induced by PGF2 alpha is inhibited to a greater extent by the presence of ADP than by thrombin, PAF or a thromboxane mimetic. The ability of aggregating agents to inhibit this increase correlates inversely with their sensitivity to inhibition by PGF2 alpha. We suggest that the very weak effect of PGF2 alpha on cyclic AMP production is sufficient to account for its inhibitory activity, and it is unlikely to be a competitive antagonist at the platelet thromboxane receptor as suggested by others.     

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.     

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)     

Involvement of COX-1 in A3 adenosine receptor-mediated contraction through endothelium in mice aorta

We investigated whether A(3) adenosine receptor (A(3)AR) is involved in endothelium-mediated contraction through cyclooxygenases (COXs) with the use of wild-type (WT) and A(3) knockout (A(3)KO) mice aorta. A(3)AR-selective agonist, Cl-IBMECA, produced a concentration-dependent contraction (EC(50): 2.9 +/- 0.2 x 10(-9) M) in WT mouse aorta with intact endothelium (+E) and negligible effects in A(3)KO +E aorta. At 10(-7) M, contractions produced by Cl-IBMECA were 29% in WT +E, while being insignificant in A(3)KO +E aorta. Cl-IBMECA-induced responses were abolished in endothelium-denuded tissues (-E), in both WT and A(3)KO aorta. A(3)AR gene and protein expression were reduced by 74 and 72% (P < 0.05), respectively, in WT -E compared with WT +E aorta, while being undetected in A(3)KO +E/-E aorta. Indomethacin (nonspecific COXs blocker, 10(-5) M), SC-560 (specific COX-1 blocker, 10(-8) M), SQ 29549 (thromboxane prostanoid receptor antagonist, 10(-6) M), and furegrelate (thromboxane synthase inhibitor, 10(-5) M) inhibited Cl-IBMECA-induced contraction significantly. Cl-IBMECA-induced thromboxane B(2) production was also attenuated significantly by indomethacin, SC-560, and furegrelate in WT +E aorta, while having negligible effects in A(3)KO +E aorta. NS-398 (specific COX-2 blocker) produced negligible inhibition of Cl-IBMECA-induced contraction in both WT +E and A(3)KO +E aorta. Cl-IBMECA-induced increase in COX-1 and thromboxane prostanoid receptor expression were significantly inhibited by MRS1523, a specific A(3)AR antagonist in WT +E aorta. Expression of both A(3)AR and COX-1 was located mostly on endothelium of WT and A(3)KO +E aorta. These results demonstrate for the first time the involvement of COX-1 pathway in A(3)AR-mediated contraction via endothelium.     

Role of thromboxane receptors in Forssman shock in guinea pigs

Forssman shock is a bronchospastic reaction mounted in guinea pigs on intravenous administration of an antiserum obtained from rabbits immunized against sheep erythrocytes. The involvement of thromboxane receptors in Forssman shock was determined with SQ 30,741, which was characterized as a selective antagonist of these receptors in guinea pig airways in vitro and in vivo. A volume of antiserum producing consistent, sublethal bronchoconstriction was given either alone (control) or 3 min after SQ 30,741 (0.03, 0.3, or 1.0 mg/kg iv) to urethan-anesthetized guinea pigs. In controls, maximum reductions in dynamic compliance (-59 +/- 6%, P less than 0.01) and increases in airways resistance (383 +/- 97%, P less than 0.01) were detected 1 min after antiserum. Both responses were significantly inhibited by SQ 30,741, either partially at 0.03 mg/kg or completely at 0.3 mg/kg. An accompanying thrombocytopenia was not abated by SQ 30,741. In separate experiments, bronchospasm was reduced by aerosol administration of 0.1% SQ 30,741 and completely prevented by aspirin (10 mg/kg iv). When Forssman antiserum was injected in lethal quantities to other guinea pigs, SQ 30,741 (1 mg/kg iv) attentuated only the resistance component of bronchospasm and did not prevent death. These data demonstrate that thromboxane receptor stimulation is a pivotal step in the pulmonary manifestations of sublethal Forssman shock but is less crucial in more severe forms of the reaction.     

Release of eicosanoids in rat peritoneal cavity stimulated with platelet-activating factor (PAF). Effect of the PAF-antagonist BN-52021

Platelet activating factor (PAF; 10 micrograms) was injected in the peritoneal cavity of rats in the absence or presence of the PAF antagonist BN-52021 (5 mg/kg). Thirty min later, the peritoneal cavity was washed with 3 ml of saline, the fluid was collected and the concentrations of selected eicosanoids were measured using novel enzyme immunoassays. PAF increased by 2.9, 2.8 and 1.7 fold the levels of thromboxane B2, prostaglandin E2 and leukotriene B4 respectively in the peritoneal fluid. The stimulatory effects of PAF was reduced by 42, 51, and 86% for thromboxane B2, prostaglandin E2 and leukotriene B4 respectively by the specific PAF antagonist. These results confirm the presence of specific PAF receptors in tissues and/or cells of rat peritoneal cavity and underline the complex interactions between PAF and eicosanoids.     

Elevation of cytosolic free Ca2+ is directly evoked by thromboxane A2 in human platelets during activation with collagen

The thromboxane A2 antagonist, ONO-3708, completely inhibited the increase in cytosolic free Ca2+ in human platelets during activation with collagen. Half-maximal Ca2+ release and influx required about 3 and 4 nM STA2, a stable thromboxane A2 mimetic, respectively. However, half maximal activation of phospholipase C required about 18 nM STA2. This suggests that thromboxane A2 directly causes Ca2+ mobilization without further activation of phospholipase C during activation of human platelets with collagen.     

Contraction of guinea pig lung parenchyma by pancreatic type phospholipase A2 via its specific binding site.

Porcine pancreatic group I phospholipase A2 (PLA2-I) induced contraction of guinea pig parenchyma in a concentration-dependent manner. Its EC50 value was similar to the Kd value calculated from the specific binding of 125I-labeled porcine PLA2-I in the membrane fraction of guinea pig lung. Type-specific action of PLA2's and homologous desensitization strongly implicated the involvement of PLA2-I-specific sites in the activation process. Thromboxane A2 was found to be the main product from lung tissue by PLA2-I action and the contractile response by PLA2-I was specifically suppressed by thromboxane A2 receptor antagonists and cyclooxygenase inhibitor, but not by leukotriene receptor antagonist and H1 blocker. These findings indicate that PLA2-I-induced contractile response may depend on the secondarily produced thromboxane A2, thus providing a new aspect of PLA2-I from the pathophysiological standpoint.     

Antagonism of thromboxane actions in the isolated perfused rat heart

The effects of SQ-29,548, a novel thromboxane A₂ (TxA₂) receptor antagonist, were studied in the isolated perfused rat heart. SQ-29,548 at concentrations of 2.5 to 50 ng/ml antagonized the increase in coronary perfusion pressure (CPP) in response to the thromboxane agonist, 9,11-methanoepoxy PGH₂. Increases in CPP induced by arginine vasopressin and leukotriene D₄ were not altered by SQ-29,548. We conclude that SQ-29,548 is a very potent and specific TxA₂ receptor antagonist in the coronary vasculature of the rat heart.     

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.     

Pharmacological characterisation of arthritis induced by Bothrops jararaca venom in rabbits: a positive cross talk between bradykinin, nitric oxide and prostaglandin E2

BACKGROUND: Our previous results showed that nitric oxide (NO) and bradykinin (BK) mediate the arthritis induced by Bothrops jararaca venom (BjV) in rabbits. In this study, we investigated the contribution of each receptor of BK as well as the inter-relationship between NO and eicosanoids in BjV-induced arthritis. METHODS: The arthritis was induced in rabbits with 16 microg of BjV injected intra-articularly. Prostaglandin E2 (PGE2), thromboxane B2 (TxB2), leukotriene B4 (LTB4) (radioimmunoassay) and nitrite/nitrate concentrations (NO2/NO3) (Griess reaction) were evaluated in the synovial fluid 4 h later. The animals were prior treated with NO synthase inhibitor (L-NAME; 20 mg/kg/day for 14 days), the B2 antagonist of BK (HOE-140) and the B1 antagonist of BK (des-Arg9[Leu8]-bradykinin), both at a dose of 0.3mg/kg, 30 min prior to the venom injection. RESULTS: Data show that L-NAME and HOE-140 treatment were equally able to reduce PGE2 and NO2/NO3 levels without interfering with TxB2 and LTB4 production. On the contrary, the B1 antagonist of BK inhibited TxB2 and LTB4 production, and did not alter PGE2 and NO metabolites levels in the inflamed joint. DISCUSSIONS: The results presented clarify the contribution of the kinin system, mainly through the B2 receptor, to the local inflammatory response induced by BjV, as well as its positive interaction with PGE2 and NO production.     

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-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.