Phosphatase inhibitors suppress Ca 2+ influx induced by receptor-mediated intracellular Ca 2+ store depletion in human platelets

Abstract

The effects of three phosphatase inhibitors including okadaic acid, calyculin A and tautomycin were evaluated on platelet Ca 2+ mobilization. Calyculin A and tautomycin at appropriate concentrations appeared to have a selective inhibitory effect on thrombin-induced Ca 2+ influx, but not on [Ca 2+] i release from intracellular Ca 2+ storage sites. In contrast, pretreatment with okadaic acid at concentrations that effectively lowered Ca 2+ influx also suppressed Ca 2+ release from intracellular Ca 2+ stores. In a system that specifically evaluates the effects of agents on Ca 2+ influx induced by the Ca 2+-depleted state of intracellular Ca 2+ storage sites, the three phosphatase inhibitors attenuated Ca 2+ influx in a dose dependent manner and showed complete inhibition at appropriate concentrations. These findings suggest that protein phosphorylation/dephosphorylation plays an important role in mediating signals to open Ca 2+ channels when Ca 2+ depletion in intracellular Ca 2+ stores is caused by thrombin. In contrast, Ca 2+ influx induced by thapsigargin, a Ca 2+-ATPase inhibitor, was only partially suppressed by pretreatment with each of the three phosphatase inhibitors. Based on these findings, we suggest that the Ca 2+-depleted state of intracellular Ca 2+ stores by thapsigargin induces the opening of Ca 2+ channels via phosphatase inhibitor-insensitive pathways. All the phosphatase inhibitors, at the highest concentrations tested in the present study, only partially inhibited Mn 2+ entry induced by thrombin. These findings suggest that there are at least two types of divalent ion channels on platelet plasma membranes and that one of them, that preferentially allows Mn 2+ entry, is resistant to the inhibitory effects of phosphatase inhibitors.