Regulation of plasma membrane Ca2+-ATPase by small GTPases and phosphoinositides in human platelets.
We have investigated the restoration of [Ca(2+)](i) in human platelets following the discharge of the intracellular Ca(2+) stores. We found that the plasma membrane Ca(2+)-ATPase is the main mechanism involved in Ca(2+) extrusion in human platelets. Treatment of platelets with the farnesylcysteine analogs, farnesylthioacetic acid and N-acetyl-S-geranylgeranyl-l-cysteine, inhibitors of activation of Ras proteins, accelerated the rate of decay of [Ca(2+)](i) to basal levels after activation with thapsigargin combined with a low concentration of ionomycin, indicating that Ras proteins are involved in the negative regulation of Ca(2+) extrusion. Rho A, which is involved in actin polymerization, was not responsible for this effect. Consistent with this, the actin polymerization inhibitors, cytochalasin D and latrunculin A, did not alter the recovery of [Ca(2+)](i). Activation of human platelets with thapsigargin and ionomycin stimulated the tyrosine phosphorylation of the plasma membrane Ca(2+)-ATPase, a mechanism that was inhibited by farnesylcysteine analogs, suggesting that Ras proteins could regulate Ca(2+) extrusion by mediating tyrosine phosphorylation of the plasma membrane Ca(2+)-ATPase. Treatment of platelets with LY294002, a specific inhibitor of phosphatidylinositol 3- and phosphatidylinositol 4-kinase, resulted in a reduction in the rate of recovery of [Ca(2+)](i) to basal levels, suggesting that the products of these kinases are involved in stimulating Ca(2+) extrusion in human platelets.
Rosado
J A
JA
Department of Physiology, University of Cambridge, Downing Street, Cambridge CB2 3EG, United Kingdom.
Sage
S O
SO
D019870:1-Phosphatidylinositol 4-Kinase / Q000378:metabolism; D000199:Actins / Q000037:antagonists & inhibitors / Q000378:metabolism; D001792:Blood Platelets / Q000201:enzymology*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D027682:Cation Transport Proteins; D002462:Cell Membrane / Q000201:enzymology*; D002867:Chromones / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism*; D010766:Phosphorylation; D053499:Plasma Membrane Calcium-Transporting ATPases; D015539:Platelet Activation / Q000502:physiology; D011233:Precipitin Tests; D015398:Signal Transduction; D012964:Sodium / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism; D018631:ras Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*
D019870:1-Phosphatidylinositol 4-Kinase; D000199:Actins; D002118:Calcium; D000252:Calcium-Transporting ATPases; D027682:Cation Transport Proteins; D002867:Chromones; D004791:Enzyme Inhibitors; D020558:GTP Phosphohydrolases; D015759:Ionomycin; D007476:Ionophores; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D010716:Phosphatidylinositols; D053499:Plasma Membrane Calcium-Transporting ATPases; D012964:Sodium; D014443:Tyrosine; D018631:ras Proteins; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one