Abstract

The kinetics of prostaglandin-regulated cyclic AMP formation by intact human platelets were studied in the presence and absence of phosphodiesterase inhibitors. In the case of iloprost, a chemically stable analogue of prostaglandin I2, the shape of the time course varied with prostaglandin concentration. In the presence of phosphodiesterase inhibitors, low concentrations of iloprost gave a linear rate of cyclic AMP formation. At higher concentrations of iloprost, the initial rate increased as a saturable function of prostaglandin concentration but the curves decayed with time to give new linear rates of cyclic AMP formation with a different prostaglandin concentration dependence from the initial rates. Time courses were simulated using KINSIM [Anal. Biochem. 130: 134-145 (1983)], a kinetic simulation program that employs numerical integration, over a wide range of iloprost concentration (3 nM to 30 microM) by use of a simple model involving rapid activation of adenylate cyclase, followed by slow reversible transition of adenylate cyclase to an inactive form (desensitization) through a distinct inhibitory receptor. The model requires that the affinity for prostaglandins of both the stimulatory and inhibitory receptors declines with prostaglandin concentration, which may be related to the existence of high and low affinity receptor forms depending on the activation state of the appropriate GTP-binding protein. The same two-receptor model can be used to describe cyclic AMP metabolism in the absence of phosphodiesterase inhibitors, giving rise to characteristic peak and plateau effects in the time courses. The putative inhibitory receptor has an apparent affinity for prostaglandin lower than the stimulatory receptor in the case of iloprost and a higher affinity than the stimulatory receptor in the case of prostaglandin E1. The contribution of phosphodiesterase activation to the time course of cyclic AMP formation through phosphorylation by cyclic AMP-dependent protein kinase was assessed. It was shown that phosphodiesterase activation must be rapid. A plausible and perhaps complete description of prostaglandin-regulated cyclic AMP metabolism in platelets is presented.