Effect of cyclic AMP on cytoplasmic free calcium in human platelets stimulated by thrombin: direct measurement with quin2.

With the fluorescent Ca2+ indicator quin2, we measured directly cytoplasmic free Ca2+ [( Ca2+]i) in washed human platelets stimulated by thrombin and examined the effect of cyclic AMP on [Ca2+]i levels and 14C-serotonin release. Thrombin (0.2 U/ml) evoked a rise in [Ca2+]i from the basal level of about 100 nM to integral of 3 microM which was fast enough to trigger serotonin release. This rise was inhibited in a dose-dependent manner by preincubation with prostaglandin E1 (PGE1) (0.1-10 microM) or dibutyryl cyclic AMP (0.01 -1 mM). Parallel to this, serotonin release was also inhibited by these drugs. When added to platelets after stimulation by thrombin, PGE1 caused the rapid decrease of elevated [Ca2+]i. These results provide direct evidence that [Ca2+]i levels in platelets are regulated by cyclic AMP.