prostaglandin-h2 and dazoxiben

Arachidonic acid (AA) is a potent inducer of platelet aggregation in vitro; this activity is due to its conversion to biologically active metabolites, prostaglandin (PG) endoperoxides and thromboxane A2 (TxA2). PG endoperoxides and TxA, are thought to act on the same receptor; however, at least two isoforms of this receptor have been identified. The aim of our work was to clarify whether endoperoxides and TxA2 activate the same or different receptor subtypes to induce aggregation and calcium movements in human platelets. AA-induced aggregation and calcium rises were still detectable in platelets preincubated with thromboxane synthase inhibitors, which suppress TxA2 formation and induce PGH2 accumulation, suggesting that PG endoperoxides can activate platelets. Exogenously added PGH2 was able to induce aggregation and calcium rises. Pretreatment of platelets with GR32191B or platelet activating factor, which desensitize one of the two receptor subtypes identified in platelets, did not prevent calcium rises induced by endogenously generated or by exogenouly added PGH2, indicating that TxA2 and PG endoperoxides share the same receptor subtype(s) to activate platelets. HEK-293 cells overexpressing either of the two thromboxane receptor isoforms cloned to date (TPalpha and TPbeta) and identified in human platelets, stimulated with PGH2, or with the stable endoperoxide analog U46619, formed inositol phosphates. These data show that endoperoxides and TXA2 mediate their effects on platelets acting on both, and the same, receptor isoform(s).

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Aspirin; Biphenyl Compounds; Blood Platelets; Bridged Bicyclo Compounds, Heterocyclic; Calcium Signaling; Cells, Cultured; Enzyme Inhibitors; Fatty Acids, Unsaturated; Heptanoic Acids; Humans; Hydrazines; Imidazoles; Inositol Phosphates; Kidney; Methacrylates; Phenylacetates; Platelet Activating Factor; Platelet Activation; Prostaglandin H2; Prostaglandins H; Protein Isoforms; Receptors, Thromboxane; Recombinant Fusion Proteins; Sulfonamides; Thromboxane A2; Thromboxane B2; Thromboxane-A Synthase

1. Canine jugular and femoral veins were studied to determine the possible importance of thromboxane (TXA2) and prostaglandin endoperoxides (prostaglandin H2, PGH2) in mediating bradykinin(BK)-induced contraction. 2. Isolated vein rings incubated in modified Krebs solution contracted to TXA2/PGH2 analogs SQ26655 and U44069 with potency of contraction exceeding that for BK. The potency ranking for both veins was SQ26655 greater than U44069 greater than BK greater than PGF2 alpha greater than TXB2 much greater than PGD2. 3. The cyclo-oxygenase inhibitors indomethacin (3 x 10(-7) M) and flufenamic acid (10(-5) M) reduced BK contractions without affecting those induced by noradrenaline (NA). 4. TXA2/PGH2 receptor antagonists SQ29548 (10(-8) M) and BM13177 (10(-6) M) strongly inhibited BK-induced tension. The action of antagonists was reversible with negligible influence on NA-elicited contraction. Selective removal of endothelium had no effect on BK-induced contraction or the action of the antagonists. 5. The thromboxane synthase inhibitors dazoxiben (10(-4) M) and CGS 12970 (10(-5) M) had no significant inhibitory effect on BK-induced tension. 6. These results suggest that in canine jugular and femoral vein, the action of BK is largely dependent upon stimulation of the cyclo-oxygenase pathway to produce PGH2 and possibly TXA2, which can activate a smooth muscle TXA2/PGH2 receptor to elicit vasoconstriction.

Topics: Animals; Bradykinin; Bridged Bicyclo Compounds, Heterocyclic; Dogs; Endothelium, Vascular; Fatty Acids, Unsaturated; Female; Hydrazines; Imidazoles; In Vitro Techniques; Muscle Contraction; Muscle, Smooth, Vascular; Prostaglandin Endoperoxides; Prostaglandin Endoperoxides, Synthetic; Prostaglandin H2; Prostaglandin-Endoperoxide Synthases; Prostaglandins H; Pyridines; Receptors, Prostaglandin; Sulfonamides; Thromboxane-A Synthase; Thromboxanes; Veins

Human platelets were prelabelled with [14C]arachidonate and stimulated with thrombin or methyl mercury. [14C]PGH2 and the more stable of the other [14C]eicosanoids formed were rapidly extracted with organic solvent cooled to -30 degrees C and analyzed by radio-TLC. TXB2 and PGH2 were also quantified by radioimmunoassay, the latter as its index metabolite PGE2. PGH2 reached its peak concentration of 12 nmol/l after 20-30 s when it amounted to approximately 2/3 of the TXB2 concentration. In the presence of the thromboxane synthase inhibitor dazoxiben, PGH2 peaked after 60 s and afterwards declined in favour of PGE2, PGD2 and PGF2 alpha. Thirty seconds after stimulation with thrombin 1 IU/ml or methyl mercury 20 mumol/l, PGH2 amounted to 35 or 28% of the cyclooxygenase products in the absence and to 66 or 63% in the presence of dazoxiben, respectively. The platelet-activating potency of PGH2 was evaluated with purified PGH2 in platelets pretreated with acetylsalicylic acid. The EC50 values of PGH2 were 0.69 and 19 nmol/l for shape change and aggregation, respectively. U 46619 produced the same effects at 4.1 and 23 nmol/l. PGH2-induced [3H]serotonin release did not exceed 25%, whereas U 46619 was able to induce approximately 50% [3H]serotonin release. Dazoxiben enhanced the aggregation induced by PGH2. Human serum albumin inhibited the aggregating effect of PGH2, suppressed the enhancing effect of dazoxiben and shifted the metabolism of PGH2 to the inhibitory PGD2. The TXA2/PGH2 receptor antagonist daltroban suppressed the agonistic effects of endogenous or added PGH2, demonstrating that the TXA2/PGH2 receptor was its site of action.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Arachidonic Acid; Arachidonic Acids; Blood Platelets; Humans; Imidazoles; In Vitro Techniques; Methylmercury Compounds; Platelet Activation; Prostaglandin Endoperoxides; Prostaglandin Endoperoxides, Synthetic; Prostaglandin H2; Prostaglandins H; Serotonin; Thrombin; Thromboxane A2; Thromboxane-A Synthase

(1) When platelets form TXA2 from endogeneous AA, PGH2 reaches concentrations very similar to those of TXA2 and high enough to produce strong platelet activation. Therefore, platelet activation by TXA2 appears to go along with an activation by PGH2. (2) PGH2 is a more potent stimulus of platelet shape change and aggregation than U 46619. (3) The agonism of PGH2 is limited by the formation of inhibitory prostaglandins, especially PGD2 at higher concentrations. That is why thromboxane synthase inhibitors in PRP and at a physiological HSA concentration do not augment platelet activation. (4) HSA promotes the formation of inhibitory PGD2 at the expense of agonistic PGH2.

Topics: Blood Platelets; Humans; Imidazoles; In Vitro Techniques; Methylmercury Compounds; Platelet Aggregation; Prostaglandin Endoperoxides; Prostaglandin Endoperoxides, Synthetic; Prostaglandin H2; Prostaglandins H; Thrombin; Thromboxane A2; Thromboxane B2; Thromboxane-A Synthase

The effects of indomethacin, dazoxiben and EPO45 on collagen-induced platelet aggregation in vivo were studied in guinea-pigs and rats to determine the involvement of the prostaglandin endoperoxide/thromboxane A2 pathway in the aggregatory response. Indomethacin and EPO45 (a thromboxane receptor antagonist) partially inhibited platelet aggregation in rats. It was concluded that only one third of the aggregatory response to collagen was mediated by the products of cyclo-oxygenase conversion of arachidonic acid. In rats, dazoxiben was inactive although the conversion of the prostaglandin endoperoxides to thromboxane A2 was inhibited (measured as thromboxane B2). 6-keto PGF1 alpha was detected in plasma after collagen was injected into dazoxiben-treated rats. In this species therefore, the endoperoxides have significant aggregatory activity whilst the apparent increase in the level of prostacyclin was not sufficient to have any anti-aggregatory effect. All three drugs were active in the guinea-pig. About 60% of the aggregatory response to collagen was due to the products of the cyclo-oxygenase pathway, the main mediator being thromboxane A2. In guinea-pigs, dazoxiben also elevated 6-keto PGF1 alpha in the plasma after an injection of collagen. However, this apparent increase in prostacyclin production did not contribute to the anti-aggregatory effect.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Collagen; Female; Guinea Pigs; Imidazoles; In Vitro Techniques; Indomethacin; Male; Platelet Aggregation; Prostaglandin Endoperoxides; Prostaglandin Endoperoxides, Synthetic; Prostaglandin H2; Prostaglandins H; Prostaglandins, Synthetic; Rats; Rats, Inbred Strains; Receptors, Prostaglandin; Receptors, Thromboxane; Species Specificity; Thromboxane A2; Thromboxane B2; Thromboxanes

We and other investigators have recently shown that inhibitors of lipoxygenase reversibly inhibit natural cytotoxic (NC) or natural killer (NK) cell activity, whereas some inhibitors of cyclooxygenase enhance these functions. In addition, exogenous LTB4 augments NC and NK activity, whereas PGE2 depresses it. In the present studies, we sought to investigate the possible role of the TxA2 synthase pathway in NC function. Inhibition of this pathway by OKY-1581 or dazoxiben significantly inhibited NC activity against HSV-infected cells as well as NK function against K562 target cells. The inhibition was dose dependent, reversible, and not due to direct toxicity. NC activity was also significantly inhibited by the addition of PGE2 or PGI2 to the 4-hr assay, whereas addition of 6-keto-PGF1 alpha had no effect. Addition of PGH2, which could be converted to TxA2 or other PG, had no significant effect, but concomitant use of OKY-1581 produced a greater inhibition of NC function than by using OKY-1581 alone. U44069, a TxA2 analog, was inhibitory by itself and could not alter the inhibition caused by OKY-1581 or dazoxiben. In contrast, the TxA2 receptor blocker 13-APA significantly enhanced NC activity and even reversed the inhibitory effect of U44069 at equimolar (10(-7)M) concentrations. Taken together, these data suggest that most of the inhibitory effect of the TxA2 synthase inhibitors on NC and NK cell function derives from their ability to reorient cyclic endoperoxide metabolism toward more inhibitory compounds. In addition, TxA2 itself could exert a negative feedback on NC function through its receptor, as evidenced by the use of a TxA2 analog and a TxA2 blocker.

Topics: Cytotoxicity Tests, Immunologic; Cytotoxicity, Immunologic; Dose-Response Relationship, Immunologic; Humans; Imidazoles; Immunosuppressive Agents; Killer Cells, Natural; Methacrylates; Prostaglandin Endoperoxides, Synthetic; Prostaglandin H2; Prostaglandins H; Thromboxane-A Synthase

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Cattle; Imidazoles; In Vitro Techniques; Lymphatic System; Muscle Contraction; Muscle, Smooth; Prostaglandin Endoperoxides; Prostaglandin Endoperoxides, Synthetic; Prostaglandin H2; Prostaglandins H; Sheep; Thromboxane-A Synthase