Activation of Na(+), K(+), Cl(-)-cotransport mediates intracellular Ca(2+) 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 Ca(2+) concentration (Ca(2+)](i)), which was completely prevented by the NKCC inhibitors. Treatment with EGTA or BAPTA/AM markedly inhibited the Pin-induced apoptosis. Inhibitors of Na(+), Ca(2+)-exchanger, bepridil, and benzamil significantly prevented both Ca(2+)](i) 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(+), Ca(2+) exchanger, resulting in Ca(2+)](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.