Author:

Keywords:

Animals, Anions, Cell Membrane, In Vitro, Larva, Membrane Potentials, Models, Biological, Mucous Membrane, Nystatin, Patch-Clamp Techniques, Potassium, Rana catesbeiana, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S., Research Support, U.S. Gov't, P.H.S., Skin Physiology, Cardiovascular System & Hematology

Abstract:

Skin from larval bullfrogs was mounted in an Ussing-type chamber in which the apical surface was bathed with a Ringer solution containing 115 mM K+ and the basolateral surface was bathed with a Ringer solution containing 115 mM Na+. Ion transport was measured as the short-circuit current (Isc) with a low-noise voltage clamp, and skin resistance (Rm) was measured by applying a direct current voltage pulse. Membrane impedance was calculated by applying a voltage signal consisting of 53 sine waves to the command stage of the voltage clamp. From the ratio of the Fourier-transformed voltage and current signals, it was possible to calculate the resistance and capacitance of the apical and basolateral membranes of the epithelium (Ra and Rb, Ca and Cb, respectively). With SO4(2-) as the anion, Rm decreased rapidly within 5 min following the addition of 150 U/ml nystatin to the apical solution, whereas Isc increased from 0.66 to 52.03 microA/cm2 over a 60-min period. These results indicate that nystatin becomes rapidly incorporated into the apical membrane and that the increase in basolateral K+ permeability requires a more prolonged time course. Intermediate levels of Isc were obtained by adding 50, 100, and 150 U/ml nystatin to the apical solution. This produced a progressive decrease in Ra and Rb while Ca and Cb remained constant. With Cl- as the anion, Isc values increased from 2.03 to 89.57 microA/cm2 following treatment with 150 U/ml nystatin, whereas with gluconate as the anion Isc was only increased from 0.63 to 11.64 microA/cm2. This suggests that the increase in basolateral K+ permeability produced by nystatin treatment, in the presence of more permeable anions, is due to swelling of the epithelial cells of the tissue rather than the gradient for apical K+ entry. Finally, Cb was not different among skins exposed to Cl-, SO4(2-), or gluconate, despite the large differences in Isc, nor did inhibition of Isc by treatment with hyperosmotic dextrose cause significant changes in Cb. These results support the hypothesis that increases in cell volume activate K+ channels that are already present in the basolateral membrane of epithelial cells.