Involvement of calcium and protein kinase C in the activation of the Na⁺/H⁺ exchanger in cultured bovine aortic endothelial cells stimulated by extracellular ATP

We have studied the activation of the Na⁺/H⁺ exchanger which leads to the intracellular alkalinization in cultured bovine aortic endothelial cells stimulated by extracellular ATP. The alkalinization induced by ATP was largely dependent on extracellular Ca²⁺ and the rate of alkalinization was decreased by about 60% in the absence of extracellular Ca²⁺. ATP caused a rapid and transient increase and a subsequent sustained increase of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in the Ca²⁺ buffer, while only the rapid and transient increase of [Ca²⁺]_i was observed in the absence of extracellular Ca²⁺. The Ca²⁺-depleted cells prepared by incubation in Ca²⁺-free buffer containing 0.1 mM EGTA showed only a slight increase of [Ca²⁺]_i with no alkalinization on stimulation by ATP. The alkalinization was inhibited by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), an inhibitor of protein kinase C, but not by another isoquinoline analogue (HA1004), which has a less inhibitory effect on the kinase. Phorbol 12-myristate 13-acetate also induced the alkalinization by the activation of the Na⁺/H⁺ exchanger. Neither dibutyryl cyclic AMP nor dibutyryl cyclic GMP affected the alkalinization induced by ATP. Treatment of the cells by pertussis and cholera toxins had no effect on the alkalinization. The results suggest that the increase in [Ca²⁺]_i is essential for the ATP-induced activation of the Na⁺/H⁺ exchanger in cultured bovine aortic endothelial cells and a protein kinase C-dependent pathway is involved in the activation.