ACTIVATION BY CALCIUM OF ERYTHROCYTE NA+ H+ EXCHANGE IN MEN/

Objective To determine whether protein kinase C is necessary for the c alcium activation of the Na+/H+ exchange in human erythrocytes by stud ying activation by calcium of erythrocyte Na+/H+ exchange in control c ells, in protein kinase C-depleted cells after downregulation of prote in kinase C with phorbol-12-myristate-13-acetate and in cells that had been treated beforehand with phorbol-12-myristate-13-acetate with and without the calpain inhibitor E-64d. Methods Erythrocyte Na+/H+ excha nge was measured by determining the initial rates of the influx of Na into Na+-depleted, acid loaded cells. The effects of various concentr ations (0-1 mmol/l) of CaCl2 and the effects of 1 mmol/l CaCl2 on acti vation of the intracellular pH and on the external Na+ activation of N a+/H+ exchange were studied. The effects of 1 mmol/l CaCl2 on Na+/H+ e xchange in control cells and cells that had been incubated beforehand with and without 1 mmol/l phorbol-12-myristate-13-acetate and with E-6 4d and 1 mmol/l phorbol-12-myristate-13-acetate for 1, 2, 3 and 24 h w ere also investigated. Results Addition of Ca2+ to a concentration in the range 0-1 mmol/l in the presence of calcimycin resulted in stimula tion of Na+/H+ exchange: 1 mmol/l CaCl2 increased (P < 0.001) the eryt hrocyte Na+/H+ exchange by 74%. Calcium increased the maximum rate for activations by intracellular pH and by external Na+ of Na+/H+ exchang e, whereas it did not affect the Michaelis-Menten constants for activa tion by intracellular H+ and external Na+. However, calcium did not ac tivate the Na+/H+ exchange in protein kinase C downregulated erythrocy tes and administration of the calpain inhibitor E-64d could not preven t this inactivation. Conclusion Our data indicate that protein kinase C is necessary for the activation by calcium of the erythrocyte Na+/H exchange. (C) 1998 Rapid Science Ltd.