American journal of hypertension : journal of the American Society of Hypertension vol:11 issue:1 Pt 1 pages:81-7
To investigate the interrelationship between erythrocyte Na+/H+ exchange rate and free cytosolic Ca2+ concentration, the effect of the (non)selective protein kinase C inhibitors staurosporine, Ro 31-8220 and CGP 41251 (1 micromol/L) and of the protein kinase C activator phorbol-12-myristate-13-acetate (PMA, 1 micromol/L) was studied in vitro on these variables. PKC depleted erythrocytes were obtained after 24 h PMA down-regulation of the cells and intracellular Ca2+ clamping was obtained using quin-2 AM and fluo-3 AM. PMA increased (P < .05) the erythrocyte Na+/H+ exchange activity and this rise was accompanied by an increase in the free cytosolic Ca2+ concentration. When staurosporine and Ro 31-8220 were added to erythrocytes in suspension, a decrease in free cytosolic Ca2+ concentration was also found, whereas no significant change was observed after CGP 41251 administration. The Na+/H+ exchange rate was decreased in the 24 h PMA down-regulated erythrocytes as well as in Ca2+-clamped cells. Addition of Ca2+ in a concentration range of 0 to 1 mmol/L in the presence of calcimycin resulted (P < .001) in a stimulation of Na+/H+ exchange by 74%. Calcium increased the Vmax for cellular pHi or external Na+ activation of Na+/H+ exchange, whereas it did not affect the Km for H+(i) or external Na+ activation. However, in PKC down-regulated cells, calcium did not activate the Na+/H+ exchange in erythrocytes and the calpain inhibitor E-64d did not prevent this inactivation. Our data show a concomitant increase in free cytosolic Ca2+ concentration and Na+/H+-exchange rate upon protein kinase C activation and a corresponding decrease in both variables upon PKC inhibition, indicating a Ca2+ requirement for protein kinase C activation of Na+/H+ exchange.