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 hepatocytes is an import of extracellular Na(+) via Na(+) conductance, Na(+)-K(+)-2Cl(-) symport, and Na(+)-H(+) antiport. 2. 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. 3. 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. 4. In addition, both compounds significantly reduced the hypertonic activation of Na(+)-K(+)-2Cl(-) symport (quantified on the basis of furosemide-sensitive (86)Rb(+) uptake) to approximately 30 %. 5. 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). 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 (86)Rb(+) uptake). 7. 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.

The hypertonicity-induced Na(+) conductance of rat hepatocytes: physiological significance and molecular correlate.

The initial event in the regulatory volume increase (RVI) of rat hepatocytes is an uptake of extracellular Na(+) that is then exchanged for K(+) via stimulation of Na(+)/K(+)-ATPase. While it was generally assumed that this Na(+) uptake is mediated by the activation of Na(+)/H(+) antiport and Na(+)-K(+)-2Cl(-) symport it could be shown recently that, in addition to these transporters, hypertonic stress also stimulates conductive Na(+) entry. In a quantitative study, it was found that the relative contribution of Na(+) conductance, Na(+)/H(+) antiport, and Na(+)-K(+)-2Cl(-) symport to the initial Na(+) import as well as to the RVI process (at 300 --> 400 mosmol/l) is approximately 4 : 1 : 1. When the osmotic sensitivity of these Na(+) importers was tested (at 300 mosmol/l --> 327, 360, 400, 450 mosmol/l) it became clear that Na(+) conductance is the prominent mechanism of RVI from 360 mosmol/l upwards whereas Na(+)/H(+) antiport is the most sensitive transporter with 65 % of its maximal activation at 327 mosmol/l already. Concerning the intracellular regulation of the Na(+) importers involved in RVI it was found that Na(+) concuctance as well as Na(+)-K(+)-2Cl(-) symport - but not Na(+)/H(+) antiport - are activated via PKC. With respect to the molecular correlate of the volume activated Na(+) conductance it could be shown that it exhibits a rather low affinity to amiloride (IC(50) = 6.0 micromol/l) and an overall sensitivity profile of EIPA > amiloride > benzamil = phenamil that, at first sight, would not speak in favor of a typical epithelial type of Na(+) channel (ENaC). Western-blot analysis and RT-PCR techniques, however, revealed that alpha-, beta-, as well as gamma-ENaC are, in fact, expressed in rat hepatocytes. Moreover, by use of an antisense-DNA based approach it could be shown that at least alpha-ENaC is part of the hypertonicity induced Na(+) conductance.