Different effects of two thromboxane A2/prostaglandin H2 receptor ligands, U46619 and S-145, on rabbit platelets

Stimulation of rabbit platelets with U46619 induced platelet shape change, aggregation and secretion of ATP. However, S-145, which specifically binds to the thromboxane A2/prostaglandin H2 receptor like U46619, induced only shape change. Both compounds rapidly elevated cytoplasmic Ca2+ concentration although only U46619 evoked the formation of inositol phosphates. Chelating external Ca2+ with EGTA did not affect the S-145-induced platelet shape change while intracellular Ca2+ movement was severely reduced. These results suggest an essential role of phospholipase C in the induction of platelet aggregation and secretion and that some factor other than Ca2+ and phospholipase C participates in platelet shape change.     

Na+/H+ exchange modulates Ca2+ mobilization in human platelets stimulated by ADP and the thromboxane mimetic U 46619

According to recent observations ADP stimulates platelets via activation of Na+/H+ exchange which increases cytosolic pH (pHi). This event initiates formation of thromboxane A2 (via phospholipase A2) and, thereafter, inositol 1,4,5-trisphosphate (via phospholipase C) which is known to mobilize Ca2+ from intracellular storage sites. We investigated changes in pHi and cytosolic free Ca2+, [Ca2+]i, activating platelets with ADP and the thromboxane mimetic U 46619. We found that ADP (5 microM) increased pHi from 7.15 +/- 0.08 to 7.35 +/- 0.04 (n = 8) in 2'-7'-bis-(carboxyethyl)-5,6-carboxyfluorescein-loaded platelets, whereas thromboxane A2 formation was inhibited by indomethacin. ADP also induced a dose-dependent Ca2+ mobilization in fura2-loaded platelets which again was not affected by indomethacin. [Ca2+]i increased by 54 +/- 10 nM (n = 8) at 1 microM and by 170 +/- 40 nM (n = 7) at 10 microM ADP above the resting value of 76 +/- 12 nM (n = 47). Inhibition of Na+/H+ exchange by ethylisopropylamiloride (EIPA) reduced ADP-induced Ca2+ mobilization by more than 65% in indomethacin-treated platelets. This inhibition could be completely overcome by artificially raising pHi using either NH4Cl or the Na+/H+ ionophore monensin. We found that U 46619 increased pHi by 0.18 +/- 0.05 at 0.1 microM and by 0.29 +/- 0.07 (n = 7) at 1.0 microM above the resting value via an EIPA-sensitive mechanism. In conflict with the proposed role of the Na+/H+ exchange we found that U 46619 raised [Ca2+]i via a mechanism that for more than 50% depended on intact Na+/H+ exchange. Again, artificially elevating pHi restored U 46619-induced Ca2+ mobilization despite the presence of EIPA. Thus, our data show that Na+/H+ exchange is a common step in platelet activation by prostaglandin endoperoxides/thromboxane A2 and ADP and enhances Ca2+ mobilization independently of phospholipase A2 activity.     

Measurement of thromboxane A2-induced elevation of ionized calcium in collagen-stimulated platelets with the photoprotein, aequorin

Using aequorin-loaded rat platelets stimulated with collagen, we found two phases of Ca2+ mobilization, one coinciding with a shape change and the other with aggregation, which have not yet been detected in quin2-loaded platelets. U46619, a stable analogue of prostaglandin H2, induced only a shape change and a concomitant rapid rise in the cytoplasmic ionized calcium concentration ([Cai2+]). However, upon addition of U46619 to platelets previously stimulated with collagen in the presence of indomethacin, a rapid increase in [Cai2+] and a shape change occurred, and, after about 1 min, second increase in [Cai2+] and aggregation occurred. The actions of U46619 were inhibited by an antagonist for the thromboxane A2 (TXA2) receptor. These results suggest that the collagen-induced shape change is initiated by TXA2-induced Ca2+ mobilization, and aggregation is induced by the secondary Ca2+ mobilization induced by TXA2 and the occupation of the receptor by collagen.     

Effects of thromboxane A2 on lymphocyte proliferation

The main cyclooxygenase-dependent arachidonic acid derivatives produced by monocytes and macrophages have been shown to be thromboxane A2 and prostaglandin E2. The immunomodulatory effects of thromboxane A2 were examined using a specific thromboxane synthase inhibitor (dazoxiben), a thromboxane A2 analog (U46619), and a thromboxane A2 receptor blocker (BM13.177). Dazoxiben inhibited lymphocyte proliferation in response to mitogens (PHA and OKT3), but also reoriented cyclic endoperoxide metabolism towards the production of prostaglandin E2. Prostaglandin E2 has been shown previously to inhibit mitogen-induced lymphocyte proliferation. U46619, a stable thromboxane A2 analog, slightly enhanced lymphocyte responses to mitogens in the presence of dazoxiben and in the presence of a cyclooxygenase inhibitor (indomethacin). This occurred at concentrations of U46619 which are probably supraphysiological in view of the short half-life of natural thromboxane A2. Finally, the thromboxane A2 receptor blocker BM13.177 did not have any effect on mitogen-induced lymphocyte proliferation. It is concluded that thromboxane A2 has no or minimal modulatory effects on lymphocyte proliferative responses to mitogens and that the effect of thromboxane A2 synthase inhibition is rather due to reorientation of cyclic endoperoxide metabolism, resulting in increased prostaglandin E2 production.     

Effects of prostaglandins, cAMP, and changes in cytosolic calcium on platelet aggregation induced by a thromboxane A2 mimic (U46619).

The effects of U46619, a thromboxane mimic, on cytosolic Ca2+ concentration and platelet aggregation were determined in human platelets. Cytosolic Ca2+ concentration was determined by Quin-2 fluorescence and platelet aggregation quantitated with an aggregometer. Addition of U46619 (1 x 10(-7) M) to the platelet suspension produced a rapid increase in cytosolic Ca2+ and platelet aggregation. Pretreatment of platelets with EGTA (3 x 10(-3) M), verapamil (5 x 10(-4) M), a calcium entry blocker, or 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (1 x 10(-3) M), an inhibitor of intracellular Ca2+ release, either blunted or markedly delayed the rate, but not the magnitude, of increase in cytosolic Ca2+ and prevented platelet aggregation by U46619. Pretreatment of platelets with prostaglandin I2 (PGI2) (5 x 10(-8) M), PGD2 (5 x 10(-8) M), PGE1 (5 x 10(-8) M), PGF2 alpha (1 x 10(-5) M), dibutyryl cAMP (5 x 10(-3) M), or forskolin (1 x 10(-6) M) prevented both the increase in cytosolic Ca2+ and the associated platelet aggregation induced by U46619. These data suggest that U46619 may induce platelet aggregation through an increase in cytosolic Ca2+, and that both Ca2+ entry and its release from intracellular storage sites probably contribute to the increase in cytosolic Ca2+. Furthermore, the rate of the increase in cytosolic Ca2+ concentration, as well as the magnitude of the increase, appear to be critical for platelet aggregation induced by U46619. Our data are consistent with the hypothesis that PGs inhibit U46619-induced platelet aggregation by preventing the increase in cytosolic Ca2+, and that these effects may be mediated via an increase in cAMP, since they were induced by PGs and cAMP.     

A photoaffinity label for the thromboxane A2/prostaglandin H2 receptor in human blood platelets

A photoactive iodoarylazide derivative (I-APA-PhN3) of the competitive thromboxane A2/prostaglandin H2 (TXA2/PGH2) antagonist 13-azaprostanoic acid is evaluated. Upon photoactivation, the compound was found to inhibit specifically and irreversibly human platelet aggregation induced by the TXA2/PGH2 mimetic U46619. In receptor-binding studies using [3H]U46619, I-APA-PhN3 exhibited an IC50 of 300 nM for inhibition of U46619 binding. Photoactivation of I-APA-PhN3 resulted in an irreversible 58% reduction in specific binding of U46619. This compound and its corresponding ratio-iodinated form will prove to be useful tools for the isolation and purification of the TXA2/PGH2-binding protein in human platelets.     

Thrombocyte interaction with a collagen substrate: the role of thrombocyte-synthesized prostaglandin endoperoxides and thromboxane A2

The effects of (i) the exogenous arachidonic acid (AA), (ii) stable prostaglandin endoperoxide analogue--U46619, and (iii) cyclooxygenase inhibitor--aspirin on the interaction of platelets with a surface coated with fibrillar calf skin collagen were studied using scanning electron microscopy. AA and U46619 stimulate massive spreading of platelets (on the collagen substrate and formation of surface-bound multilayer (thrombi-like) aggregates. The stimulation of spreading and formation of thrombi-like aggregates by AA correlate with the thromboxane A2 (TXA2) synthesis in platelets. Unlike AA, U46619 induces these processes without transformation into TXA2 and stimulation of its synthesis in platelets. Cyclooxygenase inhibitor--aspirin prevents the AA-induced platelet spreading, formation of the surface-bound thrombi-like aggregates, and TXA2 synthesis. In the absence of soluble platelet inducers, aspirin inhibits the substrate-induced spreading, but doesn't affect the initial attachment of nonactivated platelets to the collagen substrate.     

Leukotrienes increase glucose and lactate output and decrease flow in perfused rat liver

In isolated perfused rat liver leukotriene C4 and D4 but not B4 and E4 enhanced glucose and lactate output and lowered perfusion flow similar to the thromboxane A2 analogue U46619, extracellular ATP and prostaglandin F2 alpha. The kinetics of the metabolic changes caused by leukotriene C4 and D4 resembled those effected by U46619 and ATP but not those elicited by prostaglandin F2 alpha; the kinetics of the hemodynamic changes were similar only to those caused by U46619. The results show that leukotrienes could be important modulators of hepatic metabolism and hemodynamics and point to a complex intra-organ cell-cell communication between non-parenchymal and parenchymal cells.     

Platelet aggregation increases cholinergic neurotransmission in canine airway

To determine whether thromboxane A2 released from aggregating platelets increases the contractile response of airway smooth muscle to cholinergic nerve stimulation and, if so, what the mechanism of action is, we studied in vitro bronchial segments from dogs under isometric conditions. The contractile responses to electrical field stimulation at 30 s and 1 min after the addition of autologous platelets were increased by 11.1 +/- 3.2 (SD) and 20.7 +/- 5.4%, respectively, and were accompanied by the release of thromboxane A2. These effects were inhibited either by pretreatment of platelets with indomethacin or by addition of the thromboxane A2 receptor antagonist SQ 29548. Likewise, the thromboxane A2 mimetic U 46619, in subthreshold doses (i.e., insufficient to increase base-line tension), increased electrical field stimulation-induced contraction by 18.7 +/- 4.8%. The increase was greater in the presence of a concentration of physostigmine that did not cause spontaneous contraction and was blocked by SQ 29548 but not by hexamethonium or by phentolamine. Methacholine-induced contractions were unaffected by U 46619. These results indicate that aggregating platelets, by releasing thromboxane A2, increase the airway contractile response to neural stimulation probably by the accelerated release of acetylcholine.     

Thromboxane A2 stimulates mitogen activated protein kinase and arachidonic acid liberation in rabbit platelets

U46619, a thromboxane A₂ mimetic, caused tyrosine phosphorylation of several proteins in rabbit platelets. Among them, 42 kDa protein was identified as a mitogen-activated protein kinase (MAPK). U46619 activated MAPK in a concentration-dependent manner, measured by incorporation of ³²P to a specific substrate for MAPK. U46619 also liberated [³H)arachidonic acid in a concentration-dependent manner. The U46619-induced MAPK activation and [³H]arachidonic acid liberation were inhibited by SQ29548 and by the removal of external Ca²⁺ ions. This is a first demonstration that TXA₂ activates MAPK accompanied with arachidonic acid liberation in rabbit platelets.     

Enrichment of platelet phospholipids with eicosapentaenoic acid and docosahexaenoic acid inhibits thromboxane A2/prostaglandin H2 receptor binding and function

Human platelet lipids were enriched in vitro with different amounts of either docosahexaenoic acid (22:6n-3), eicosapentaenoic acid (20:5n-3) or linoleic acid (18:2n-6). Of the total fatty acid incorporated, between 82 and 95% was associated with the phospholipid (PL) fraction, with the remainder as either neutral lipid or hydroxy fatty acid. Within the PL fraction, the majority (64% of total) of each fatty acid was incorporated into phosphatidylcholine. It was found that platelet aggregation induced by the thromboxane A2/prostaglandin H2 mimetic (15S)-hydroxy-11,9-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619) was inhibited after PL enrichment with 22:6n-3 or 20:5n-3, but not after 18:2n-6 enrichment. The specificity of 22:6n-3 and 20:5n-3 for U46619 activation was demonstrated by the finding that neither fatty acid significantly inhibited thromboxane A2/prostaglandin H2-independent aggregation induced by A23187 or thrombin. Furthermore, enrichment with 22:6n-3 or 20:5n-3 resulted in inhibition of [3H]U46619 specific binding, while enrichment with 18:2n-6 did not affect binding. Scatchard analysis revealed that thromboxane A2/prostaglandin H2 receptor affinity for [3H]U46619 decreased 4.8-fold following 22:6n-3 incorporation. These results demonstrate that platelet phospholipid enrichment with 22:6n-3 or 20:5n-3 results in a selective inhibition of thromboxane A2/prostaglandin H2 receptor function.     

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.     

"Suicide" inactivation of thromboxane A2 synthase. Characteristics of mechanism-based inactivation with isolated enzyme and intact platelets

"Suicide" inactivation occurs during catalysis by thromboxane synthase. Loss of enzymatic activity, accompanying thromboxane B2 formation, was proportional to the substrate concentration. Inactivation was directly related to product formation: for several different experimental protocols 50% loss of thromboxane synthase activity corresponded with formation of 454 +/- 79 ng of thromboxane B2/mg protein. The time course of inactivation was pseudo-first-order and obeyed saturation kinetics. Inactivation (KI) and first-order rate constants (ki) were 18 microM and 0.18 s-1 for prostaglandin H2. Prostaglandin H1, a poor substrate for turnover, was also a site-directed inactivator with KI = 28 microM and ki = 0.09 s-1. Competitive inhibitors, typified by U63557a and U46619, preserved the enzyme activity by slowing the rate of inactivation from 0.18 to 0.05 s-1. Loss of the hemoprotein Soret absorbance did not correlate quantitatively or temporally with the loss of thromboxane synthase activity. A similar, irreversible inactivation accompanied thromboxane formation by intact platelets. Loss of activity was proportional to substrate concentration and catalytic activity. For a pool of 25 separate donors, thromboxane synthase activity declined exponentially as a function of thromboxane B2 formation: 50% loss of activity corresponded to 23 ng of thromboxane B2/10(7) platelets. The data conform to criteria for a specific, mechanism-based process in which thromboxane synthase participates in two parallel reactions, one leading to thromboxane formation and the other to suicide inactivation. The specific, rather than indiscriminate, nature of the process, and its occurrence in intact platelets may have implications for the cell biology of thrombosis. Depletion of thromboxane synthase activity may be a factor in the choice and effectiveness of antithrombotic agents.     

Inhibition of platelet aggregation by unsaturated fatty acids through interference with a thromboxane-mediated process

cis- and trans-unsaturated fatty acids with 18 carbon atoms (oleic, linoleic, elaidic and linolelaidic acid) inhibited aggregation of washed rabbit platelets stimulated with collagen, arachidonic acid and U46619 when in the same concentration ranges. Thrombin-induced aggregation was not affected by any of them. Saturated fatty acid (stearic acid) had no effect on this response. The inhibition is independent of the induced change in membrane fluidity, since trans-isomers could not induce the change in fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. Unsaturated fatty acids, except linoleic acid, did not interfere with the formation of thromboxane B2 from exogenously added arachidonic acid. All the unsaturated fatty acids only slightly inhibited the arachidonic acid liberation by phospholipase A2 in platelet lysate. This indicates that the unsaturated fatty acids may block a process after formation of thromboxane A2 in response to collagen and arachidonic acid. The increase in phosphatidic acid formation stimulated with U46619 was inhibited dose dependently by each of the unsaturated fatty acids but that stimulated with thrombin was not affected by any of them. Phospholipase C activity measured by diacylglycerol formation in unstimulated platelet lysate was not inhibited by the fatty acids. The elevation of cytosolic free Ca2+ induced by arachidonic acid or U46619 and Ca2+ influx by collagen were inhibited almost completely at the same concentration as that which inhibited their aggregation. These data suggest that the unsaturated fatty acids were intercalated into the membrane and inhibited collagen- and arachidonic acid-induced platelet aggregation by causing a significant suppression of the thromboxane A2-mediated increase in cytosolic free Ca2+, probably due to interference with the receptor-operated Ca2+ channel.     

Phorbol ester-induced phosphoinositide hydrolysis in rat aorta: role of cyclooxygenase products

This study investigates whether phorbol esters increase phosphoinositide hydrolysis in intact vascular smooth muscle, and the mechanism underlying the hydrolysis. Phorbol myristate acetate induced time- and concentration-dependent increases in phosphoinositide hydrolysis, as demonstrated by elevated inositol monophosphate levels, in deendothelialized rat aorta. The phorbol ester-elevated inositol monophosphate levels were abolished by indomethacin, a cyclooxygenase inhibitor, but were only partially decreased by SQ29548, a thromboxane A2/prostaglandin H2 receptor antagonist. SQ29548 also only partially decreased elevated inositol monophosphate levels due to prostaglandin E2, prostaglandin F2alpha, prostaglandin I2 and carbacyclin, a stable prostaglandin I2 analog. SQ29548 abolished elevated inositol monophosphate levels due to U46619, a stable thromboxane A2/prostaglandin H2 receptor agonist. These studies demonstrate that phorbol esters increase phosphoinositide hydrolysis in intact vascular smooth muscle, and that the increase is due, at lease in part, to endogenously released prostaglandins other than prostaglandin H2.     

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 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.     

Binding of an [125I] labelled thromboxane A2/prostaglandin H2 receptor agonist to baboon platelets

To characterize the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor on baboon platelets the binding of [125I]BOP was studied. [125I]BOP bound to washed baboon platelets in a saturable manner. Scatchard analysis of binding isotherms revealed a Kd of 1.12 +/- 0.08 nM and a binding capacity of 54 +/- 5 fmoles/10(8) platelets (326 sites/platelet). Several TXA2/PGH2 agonists and antagonists displaced [125I]BOP from its baboon platelet binding site with a rank order of potency similar to human platelets: I-BOP greater than SQ29548 greater than U46619 = I-PTA-OH greater than PTA-OH. I-BOP aggregated washed baboon platelets with an EC50 of 10 +/- 4 nM. The results indicate that [125I]BOP binds to the TXA2/PGH2 receptor on baboon platelets and that this receptor is similar to its human counterpart.     

Possible involvement of cytoskeleton in collagen-stimulated activation of phospholipases in human platelets

The action of phospholipases A2 and C in the course of collagen-stimulated platelet activation and the effect of cytochalasins on the responses were studied. Stimulation of human platelets with collagen was accompanied by aggregation, Ca2+ mobilization, inositol phosphate formation, and arachidonic acid release. However, in the presence of a cyclooxygenase inhibitor or a thromboxane A2 (TXA2) receptor antagonist, collagen induced only weak arachidonic acid release and weak inositol phosphate formation. The TXA2 mimetic agonist U46619 induced all the responses except for arachidonic acid release, which was induced by synergistic action of collagen and U46619. The result that U46619 did not induce arachidonic acid release despite the activation of phospholipase C suggested that arachidonic acid was not released via phospholipase C but by phospholipase A2. These findings suggested that collagen initially induced weak activation of phospholipases A2 and C and that further activation of phospholipase C as well as Ca2+ mobilization and aggregation were induced by TXA2, whereas further activation of phospholipase A2 required the synergistic action of collagen and TXA2. Platelets pretreated with cytochalasins did not respond to collagen. Further analysis revealed that the initial activation of phospholipases A2 and C was specifically inhibited by cytochalasins, but the responses induced by U46619 or a synergistic action of collagen and U46619 were not inhibited. Therefore, we proposed that interaction of collagen receptor with actin filaments might have some roles in the collagen-induced initial activation of phospholipases.     

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.