Hypertonicity-induced alkalinization of rat hepatocytes is not involved in activation of Na + conductance or Na +,K +-ATPase

Abstract

We investigated whether cell alkalinization via activation of Na +/H + exchange is involved in the stimulation of Na + conductance and Na +,K +-ATPase in rat hepatocytes under hypertonic stress. Osmolarity was increased from 300 to 400 mOsm/l at constant extracellular pH (7.4), whereas osmotically induced cell alkalinization (0.3 pH units in HCO 3 −-free solutions) was mimicked by increasing extracellular pH from 7.4 to 7.8 in normosmotic solutions. In intracellular recordings with conventional and ion-sensitive microelectrodes, hypertonic stress led to a transient shift in the voltage response to low Na + solutions (95% in exchange for choline) by −4.3±0.8 mV and a continuous increase in cell Na + from 13.7±1.8 to 18.6±3.0 mmol/l within 8 min. In the presence of 10 −5 mol/l amiloride, these effects were reduced by 80 and 90%, respectively. In contrast, increasing pH did not change the voltage responses to low Na + or cell Na + concentrations significantly. In addition, application of 2 mmol/l Ba 2+ pulses revealed that a sustained membrane hyperpolarization of 15.6±1.4 mV following intracellular alkalinization exclusively reflects an increase in K + conductance. Increasing osmolarity at pH 7.4 augmented ouabain-sensitive 86Rb + uptake from 5.5±1.1 to 8.5±1.6 nmol mg protein −1 min −1. In normosmotic solution at pH 7.8, 86Rb + uptake equalled 4.9±1.6 nmol mg protein −1 min −1, which is not significantly different from control. We conclude that, in rat hepatocytes, cell alkalinization under hypertonic stress is not responsible for the activation of Na + conductance and probably does not participate in the stimulation of Na +,K +-ATPase.