6533b82cfe1ef96bd128f504

RESEARCH PRODUCT

Synergistic interaction of adenylate cyclase activators and nitric oxide donor SIN-1 on platelet cyclic AMP

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Abstract The molecular mechanism of the synergistic platelet inhibition by activators of adenylate cyclase and guanylate cyclase in human platelets was investigated. The adenylate cyclase activators iloprost and prostaglandin E 1 and the guanylate cyclase activator 3-morpholino-synonimine (SIN-1) dose-dependently inhibited thrombin-induced aggregation of washed human platelets. Furthermore, SIN-1 at a concentration inhibiting platelet aggregation by only 10% shifted the IC 50 values of iloprost and prostaglandin E 1 by one order of magnitude to the left, indicating a synergistic action of adenylate cyclase and guanylate cyclase activators. Iloprost and prostaglandin E 1 dose-dependently elevated platelet cAMP without a significant influence on cGMP. In contrast, the platelet cGMP level was dose-dependently elevated by SIN-1. In addition, SIN-1 markedly increased cAMP level induced by low concentrations of adenylate cyclase activators (0.1–0.3 nM iloprost or 10–150 nM prostaglandin E 1 ). In contrast, the rise in cAMP induced by higher adenylate cyclase activator concentrations (3 nM iloprost or 30 μM prostaglandin E 1 ) was significantly reduced in the presence of SIN-1. The same biphasic mode of action of SIN-1 was observed with forskolin, an adenylate cyclase stimulator acting receptor independently, indicating a prostacyclin-receptor independent mechanism. The cAMP elevating effect of SIN-1 in the presence of low prostanoid concentrations was completely abolished by piroximone, a selective inhibitor of phosphodiesterase type III. Therefore, the inhibition of phosphodiesterase III by cGMP seems to be the mechanism for the elevation of cAMP levels by SIN-1 in the presence of low concentration of adenylate cyclase activators in human platelets. In contrast, the mechanism by which SIN-1 reduces platelet cAMP levels induced by higher concentrations of prostaglandin E 1 , iloprost, and forskolin, remains to be clarified.