In rat hepatocytes, the hypertonic activation of Na+ conductance and Na+-K+-2Cl− symport - but not Na+-H+ antiport - is mediated by protein kinase C

The initial event in the regulatory volume increase (RVI) of rat hepatocytes is an import of extracellular Na+ via Na+ conductance, Na+-K+-2Cl− symport, and Na+-H+ antiport.Here, the protein kinase inhibitors staurosporine (100 nmol l−1) and bis-indolyl-maleimide I (400 nmol l−1) were used to test for a possible contribution of protein kinase C (PKC) to the hypertonic activation of these transporters in confluent primary cultures.Stimulation of Na+ conductance was monitored: (i) by use of a differential approach based on Na+ fluxes, (ii) by means of cable analysis, and (iii) in experiments with low Na+ pulses. All three experimental protocols in concert demonstrated a block of the activation of Na+ conductance by staurosporine and bis-indolyl-maleimide I.In addition, both compounds significantly reduced the hypertonic activation of Na+-K+-2Cl− symport (quantified on the basis of furosemide-sensitive 86Rb+ uptake) to approximately 30 %.In contrast, neither staurosporine nor bis-indolyl-maleimide I had any detectable effect on the hypertonicity-induced alkalinization of cell pH via Na+-H+ antiport (determined fluorometrically).Staurosporine and bis-indolyl-maleimide I completely blocked the RVI of rat hepatocytes (quantified by means of confocal laser-scanning microscopy). The high efficiency of the block suggests an additional inhibitory effect of both compounds on the activity of Na+/K+-ATPase (determined as ouabain-sensitive 86Rb+ uptake).It is concluded that the hypertonic activation of rat hepatocyte Na+ conductance and Na+-K+-2Cl− symport - but not Na+-H+ antiport - is probably mediated by PKC