thromboxane-a2 and 2--5--dideoxyadenosine

1. Our previous study demonstrated that YC-1, a derivative of benzylindazole, is a novel activator of soluble guanylate cyclase (sGC) in rabbit platelets. This work investigated whether the antiplatelet effect of YC-1 was mediated by a nitric oxide (NO)/sGC/cyclic GMP pathway in human platelets. 2. In human washed platelets, YC-1 inhibited platelet aggregation and ATP released induced by U46619 (2 microM), collagen (10 micro ml(-1)) and thrombin (0.1 u ml(-1)) in a concentration-dependent manner with IC50 values of (microM) 2.1 +/- 0.03, 11.7 +/- 2.1 and 59.3 +/- 7.1, respectively. 3. In a 30,000 g supernatant fraction from human platelet homogenate, YC-1 (5-100 microM) increased sGC activity in a concentration-dependent manner. At the same concentration-range, YC-1 elevated cyclic GMP levels markedly, but only slightly elevated cyclic AMP levels in the intact platelets. 4. MY-5445, a selective inhibitor of cyclic GMP phosphodiesterase, potentiated the increases in cyclic GMP caused by YC-1, and shifted the concentration-anti-aggregation curve of YC-1 to the left. In contrast, HL-725, a selective inhibitor of cyclic AMP phosphodiesterase, did not affect either the increases in cyclic nucleotides or the anti-aggregatory effect caused by YC-1. 5. Methylene blue, an inhibitor of sGC, blocked the increases of cyclic GMP caused by YC-1, and attenuated markedly the anti-aggregatory effect of YC-1. The adenylate cyclase inhibitor, 2',5'-dideoxyadenosine (DDA) did not affect YC-1-induced inhibition of platelet aggregation. 6. Haemoglobin, which binds NO, prevented the activation of sGC and anti-aggregatory effect caused by sodium nitroprusside, but did not affect YC-1 response. 7. These results would suggest that YC-1 activates sGC of human platelets by a NO-dependent mechanism, and exerts its antiplatelet effects through the sGC/cyclic GMP pathway.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Triphosphate; Blood Platelets; Collagen; Cyclic AMP; Cyclic GMP; Dideoxyadenosine; Dose-Response Relationship, Drug; Enzyme Activation; Furans; Guanylate Cyclase; Hemoglobins; Humans; Indazoles; Nitric Oxide; Phosphodiesterase Inhibitors; Phthalazines; Platelet Aggregation Inhibitors; Prostaglandin Endoperoxides, Synthetic; Solubility; Thrombin; Thromboxane A2; Vasoconstrictor Agents

Contraction of glomerular mesangial cells is stimulated in vitro by the vasoconstrictor metabolite of arachidonic acid, thromboxane A2. To establish the role of mesangial prostaglandin (PG) synthesis in the modulation of contractile responses, we studied the effects of the stable thromboxane A2/endoperoxide analogue U-46619 on cultured rat mesangial cells preincubated with 1) four structurally unrelated, nonsteroidal anti-inflammatory drugs, indomethacin, acetylsalicylic acid, meclofenamate, and piroxicam, to inhibit the synthesis of PGE2, the major mesangial metabolite of arachidonic acid; 2) exogenous PGE2 and the stable analogue of PGI2, iloprost; and 3) indomethacin in the presence of exogenous PGE2. Computer-assisted image analysis microscopy demonstrated enhancement of spontaneous and agonist-induced contraction by nonsteroidal anti-inflammatory drugs in individual cells grown on a glass substrate, from 37.2 +/- 7.3% to a maximum of 75.5 +/- 6.4% of the cells with piroxicam, at 1 microM U-46619. PGE2 and iloprost dose-dependently inhibited U-46619-induced contraction, to 5.0 +/- 2.8% and 12.5 +/- 4.7% of the cells, respectively, at 1 microM U-46619. PGE2 also completely reversed the effects of indomethacin. Both PGE2 and iloprost dose-dependently stimulated intracellular cyclic AMP (cAMP) accumulation during 3-minute incubations, an effect that was blocked by the inhibitor of adenylate cyclase, 2',5'-dideoxyadenosine. The latter reversed the inhibitory action of PGE2, enhancing spontaneous and agonist-induced contractility, thus indicating a modulatory role of cAMP. We conclude that endogenous arachidonate metabolism regulates mesangial cell contraction through elevation of intracellular cAMP.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Deoxyadenosines; Dideoxyadenosine; Dinoprostone; Epoprostenol; Glomerular Mesangium; Muscle Contraction; Prostaglandins E; Rats; Thromboxane A2