Activation of Na+, K+, Cl--cotransport mediates intracellular Ca2+ increase and apoptosis induced by pinacidil in HepG2 human hepatoblastoma cells

The role of Na+, K+, Cl--cotransport (NKCC) in apoptosis of HepG2 human hepatoblastoma cells was investigated. Pinacidil (Pin), an activator of ATP-sensitive K+ (K-ATP) channels, induced apoptosis in a dose- and time-dependent manner in HepG2 cells. Pin increased intracellular K+ concentration ([K+](i)). Bumetanide and furosemide, NKCC inhibitors, significantly inhibited the Pin-induced increased [K+](i) and apoptosis, whereas K-ATP inhibitors (glibenclamide and tolbutamide) had no effects. The Pin-induced [K+](i) increase was significantly prevented by reducing extracellular Cl- concentration, and Pin also increased intracellular Na+ concentration ([Na+](i)), further indicating that these effects of Pin may be due to NKCC activation. In addition, Pin induced a rapid and sustained increase in intracellular Ca2+ concentration ([Ca2+](i)), which was completely prevented by the NKCC inhibitors. Treatment with EGTA or BAPTA/AM markedly inhibited the Pin-induced apoptosis, Inhibitors of Na+, Ca2+-exchanger, bepridil, and benzamil significantly prevented both [Ca2+]; increase and apoptosis induced by Pin. Taken together, these results suggest that Pin increases [Na+](i) through NKCC activation, which leads to stimulation of reverse-mode of Na+, Ca2+ exchanger, resulting in [Ca2+](i) increase, and in turn, apoptosis, These results further suggest that NKCC may be a good target for induction of apoptosis in human hepatoma cells. (C) 2001 Academic Press.