Abstract
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.
Highlights
Mediated by two mechanisms, sequestration of Ca2ϩ into intracellular compartments and Ca2ϩ extrusion across the plasma membrane
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 [Ca2؉]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 Ca2؉ extrusion
In the present work we have investigated the possibility that small GTPases of the Ras superfamily are involved in the regulation of plasma membrane Ca2ϩ-ATPase (PMCA) activity in human platelets, so providing a regulatory pathway for a more rapid and sustained increase in intracellular calcium during platelet activation
Summary
Materials—Fura-2 acetoxymethyl ester (Fura-2/AM) was from Texas Fluorescence (Austin, TX). Cells were collected by centrifugation at 350 ϫ g for 20 min and resuspended in HEPES-buffered saline containing (HBS in mM): 145 NaCl, 10 HEPES, 10 D-glucose, 5 KCl, 1 MgSO4, pH 7.45, and supplemented with 0.1% (w/v) bovine serum albumin and 40 g/ml apyrase. After removal of the Protein A-agarose by centrifugation at 16,000 ϫ g for 1 min, 4 g of antiphosphotyrosine monoclonal antibody (PY20) was added followed by 2 h incubation. To detect the primary antibody, blots were incubated with horseradish peroxidase-conjugated ovine anti-mouse IgG antibody diluted 1:10000 in TBST, washed six times in TBST, and exposed to enhanced chemiluminescence reagents for 1 min. Oneway analysis of variance combined with the Dunnett tests was used
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