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

摘要

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 HCO3?-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 Ba2+ 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.