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.