A role for protein phosphorylation in modulating Ca2+ elevation in rabbit platelets treated with thapsigargin Available to Purchase

The effect of modifying protein kinase and phosphatase activity on Ca²⁺ influx induced by inhibition of Ca²⁺-ATPase activity has been investigated in rabbit platelets. Activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) or inhibition of phosphatase type 1/2A (PP1/2A) activity with calyculin A caused a dose-dependent inhibition of cytosolic Ca²⁺ elevation in thapsigargin (Tg)-treated platelets and decreased Ca²⁺ influx into platelets at a time when Ca²⁺ channels had already been opened by pretreatment of cells with Tg. In addition, both activation of PKC and inhibition of PP1/2A activity caused a dose-dependent inhibition of bivalent cation (Mn²⁺) influx (acting as a surrogate for Ca²⁺ influx) in Tg-treated platelets. Inhibition of cyclo-oxygenase activity caused a small decrease in [Ca²⁺]_i elevation in Tg-treated platelets, but had no effect on the ability of PMA or calyculin A to inhibit Tg-induced [Ca²⁺]_i elevation. Unexpectedly, PMA inhibited Tg-induced [Ca²⁺]_i elevation in the absence of extracellular Ca²⁺, and in agreement calyculin A decreased [Ca²⁺]_i elevation almost to basal levels. The results from this study were confirmed with another Ca²⁺-ATPase inhibitor, namely 2,5-di(tert-butyl)hydroquinone (tBHQ). These findings therefore suggest that modification of phosphorylation of target protein(s) on serine/threonine amino acid residues plays a role in the regulation of both Ca²⁺ influx and in the filling state of the intracellular Ca²⁺ pool in platelets treated with Tg.