Effects of L-alanine on membrane potential, potassium (86Rb) permeability and cell volume in hepatocytes from Raja erinacea

Isolated hepatocytes from the elasmobranch Raja erinacea were examined for their regulatory responses to a solute load following electrogenic uptake of L-alanine. The transmembrane potential (Vm) was measured with glass microelectrodes filled with 0.5 M KCl (75 to 208 M omega in elasmobranch Ringer's solution) and averaged -61 +/- 16 mV (S.D.; n = 68). L-Alanine decreased (depolarized) Vm by 7 +/- 3 and 18 +/- 2 mV at concentrations of 1 and 10 mM, respectively. Vm did not repolarize to control values during the 5-10 min impalements, unless the amino acid was washed away from the hepatocytes. The depolarizing effect of L-alanine was dependent on external Na+, and was specific for the L-isomer of alanine, as D- and beta-alanine had no effect. Hepatocyte Vm also depolarized on addition of KCN or ouabain, or when external K+ was increased. Rates of 86Rb+ uptake and efflux were measured to assess the effects of L-alanine on Na+/K+-ATPase activity and K+ permeability, respectively. Greater than 80% of the 86Rb+ uptake was inhibited by 2 mM ouabain, or by substitution of choline+ for Na+ in the incubation media. L-Alanine (10 mM) increased 86Rb+ uptake by 18-49%, consistent with an increase in Na+/K+ pump activity, but had no effect on rubidium efflux. L-Alanine, at concentrations up to 20 mM, also had no measurable effect on cell volume as determined by 3H2O and [14C]inulin distribution. These results indicate that Na+-coupled uptake of L-alanine by skate hepatocytes is rheogenic, as previously observed in other cell systems. However, in contrast to mammalian hepatocytes, Vm does not repolarize for at least 10 min after the administration of L-alanine, and changes in cell volume and potassium permeability are also not observed.