Protein kinase C-mediated up-regulation of Na⁺/Ca²⁺-exchanger in rat hepatocytes determined by a new Na⁺/Ca²⁺-exchanger inhibitor, KB-R7943

The regulatory mechanism of the plasma membrane Na⁺/Ca²⁺-exchanger in isolated rat hepatocytes was studied using microspectrofluorometry and ⁴⁵Ca²⁺ uptake methods. Exposure of single hepatocytes to low-Na⁺ solutions induced an increase in the intracellular Ca²⁺ concentration ([Ca²⁺](i)) which depended on the presence of extracellular Ca²⁺. 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate (KB-R7943), a novel selective inhibitor of Na⁺/Ca²⁺-exchangers, inhibited the initial rate of [Ca²⁺](i) increase induced by exposure to the low-Na⁺ solution (IC₅₀=2 μM). KB-R7943 also reduced the initial rate of ⁴⁵Ca²⁺ uptake (IC₅₀=4 μM). The increase in [Ca²⁺](i) induced by exposure to the low-Na⁺ solution was inhibited by pre-incubation with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7, 50 μM), but not with N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8, 60 μM) or a tyrosine kinase inhibitor, genistein (100 μM). Furthermore, taurocholate and phorbol-12,13-dibutyrate, both of which activate protein kinase C, promoted the increase in [Ca²⁺](i). These [Ca²⁺](i) increases were sensitive to KB-R7943. Our results indicate that the Na⁺/Ca²⁺-exchanger is up-regulated via protein kinase C. The activity of Na⁺/Ca²⁺-exchangers is not evident under normal physiological conditions, suggesting that the exchanger may be activated under pathophysiological conditions. Copyright (C) 1998 Elsevier Science B.V.