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

1. The initial event in the regulatory volume increase (RVI) of rat hepatoc ytes is an import of extracellular Na+ via Na+ conductance, Na+-K+-2Cl(-) s ymport, and Na+-H+ antiport. 2. Here, the protein kinase inhibitors staurosporine (100 nmol l(-1)) and b is-indolyl-maleimide I (400 nmol l(-1)) were used to test for a possible co ntribution of protein kinase C (PKC) to the hypertonic, activation of these transporters in confluent primary cultures. 3. Stimulation of Na+ conductance was monitored: (i) by use of a differenti al approach based on Na+ fluxes, (d) by means of cable analysis, and (iii) in experiments with low Na+ pulses. All three experimental protocols in con cert demonstrated a block of the activation of Na+ conductance by staurospo rine and bis-indolyl-maleimide I. 4. In addition, both compounds significantly reduced the hypertonic activat ion of Na+-K+-2Cl(-) symport (quantified on the basis of furosemide-sensiti ve Rb-86(+) uptake) to approximately 30 %. 5. In contrast, neither staurosporine nor bis-indolyl-maleimide I had any d etectable effect on the hypertonicity-induced alkalinization of cell pH via Na-+H+ antiport (determined fluorometrically). 6. 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 Rb-86(+) uptake). 7. It is concluded that the hypertonic activation of rat hepatocyte Na+ con ductance and Na+-K+-2Cl(-) symport - but not Na+-H+ antiport - is probably mediated by PKC.