A role for protein phosphorylation in modulating Ca2+ elevation in rabbit platelets treated with thapsigargin.
The effect of modifying protein kinase and phosphatase activity on Ca2+ influx induced by inhibition of Ca(2+)-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 Ca(2+)-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.
Department of Pharmacology, School of Pharmacy and Pharmacology, University of Bath, U.K.
C T
CT
Murphy
A J
AJ
Bullock
J
J
Westwick
D000818:Animals; D001792:Blood Platelets / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D001798:Blood Proteins / Q000378:metabolism*; D002118:Calcium / Q000097:blood*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000097:blood; D002462:Cell Membrane / Q000378:metabolism; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006493:Heparin / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D007424:Intracellular Fluid / Q000378:metabolism; D008345:Manganese / Q000494:pharmacology; D008387:Marine Toxins; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000097:blood; D011817:Rabbits; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013729:Terpenes / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019284:Thapsigargin
D001798:Blood Proteins; D016861:Cyclooxygenase Inhibitors; D004791:Enzyme Inhibitors; D008387:Marine Toxins; D010080:Oxazoles; D013729:Terpenes; D008345:Manganese; D019284:Thapsigargin; C059041:calyculin A; D015544:Inositol 1,4,5-Trisphosphate; D006493:Heparin; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; D000252:Calcium-Transporting ATPases; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium