INTRACELLULAR CALCIUM MOBILIZATION AND ACTIVATION OF THE NA+ H+ EXCHANGER IN PLATELETS

The aim of the present study was to evaluate the regulatory relationship between the cytosolic free calcium concentration ([Ca2+]i) and cytosolic pH (pH(i)). [Ca2+]i and pH(i) were measured using the fluorescent dyes fura-2 and BCECF [2',7'-bis-(carboxy-ethyl)-5,6-carboxyfluorescein] respectively. In a medium with 1 mmol/I extracellular calcium, thrombin (2.5 units/ml) induced an increment in [Ca2+]i of 638 +/- 31 nmol/l (n = 5) and an intracellular alkalinization of 0.14 +/- 0.01 pH units (n = 8). Both responses were dependent on the concentration of thrombin, displaying a sigmoidal dose-response pattern. The intracellular alkalinization was dependent upon extracellular Na+ and was amiloride-sensitive, indicating that it was mediated by activation of the Na+/H+ exchanger. When extracellular calcium was chelated with EGTA prior to the addition of thrombin, the intracellular alkalinization was not affected (0.15 +/- 0.02 at 2.5 units/ml thrombin, n = 8). Under these circumstances, the [Ca2+]i increment represents mobilization from internal stores, reaching 157 +/- 42 nmol/l at 2.5 units/ml thrombin. When platelets were preloaded with the intracellular calcium chelator MAPTAM (1,2-bis-5-methylaminophenoxylethane-NNN'-tetra-acetoxymethyl acetate) to block the increase in [Ca2+]i induced by thrombin, no increment in pH(i) was observed. Moreover, MAPTAM-loaded calcium-depleted platelets had a basal pH(i) that was more acidic than in the presence of 1 mmol/l extracellular calcium (6.93 +/- 0.09 versus 7.14 +/- 0.01, n = 26, P < 0.001). Ionomycin induced an elevation of [Ca2+]i that was accompanied by a concomitant increase in pH(i), which was Na+-dependent and amiloride-sensitive. [Ca2+]i and pH(i) increases induced by ionomycin were both dependent on the concentration of ionomycin. In conclusion, an increase in [Ca2+]i is necessary for the agonist-induced activation of the Na+/H+ exchanger in platelets. Non-agonist-induced increases in [Ca2+]i seems to prompt activation of the exchanger. In addition, Ca2+-depleted platelets have a more acidic basal pH(i), indicating that the basal level of [Ca2+]i is also important for maintaining the basal pH(i).