Objective: In order to elucidate the role of Na(+)-Ca2+ exchange in regulating cytosolic free Ca2+ concentration in human platelets, we investigated the relationship between cytosolic free Na+ and Ca2+ concentrations in human platelets.
Methods: Sodium-binding benzofuran isophthalate and fura-2 were used to monitor cytosolic free Na+ and Ca2+ concentrations, respectively.
Results: Ouabain at a concentration of 100 mumol/l induced an increase in cytosolic free Na+ concentration within 5 min, followed by increases in resting cytosolic free Ca2+ concentration and intracellular Ca2+ store. These three parameters were increased in a time-dependent manner significantly above the timed control over a period of 60 min. Pre-incubation of platelets with 100 mumol/l ouabain for 30 min significantly enhanced the cytosolic free Ca2+ response to thrombin and arginine vasopressin in the absence of extracellular Ca2+. The decrease from peak cytosolic free Ca2+ concentration in response to ionomycin in the absence of extracellular Ca2+ was significantly slower in low-Na+ buffer than in standard buffer. In addition, 5 mumol/ionomycin increased the cytosolic free Na+ concentration markedly in the presence of 0.1 mmol/l extracellular Ca2+, but the rise in cytosolic free Na+ concentration was suppressed by 2 mmol/l Ni2+ (NiCl2) or by removal of extracellular Ca2+.
Conclusions: These results suggest that Na(+)-Ca2+ exchange is important in extruding Ca2+ from the cytosol in human platelets, and inhibition of this exchange leads to the accumulation of intracellular Ca2+ store, which may be responsible for the enhanced responsiveness of cytosolic free Ca2+ to agonists.