Regulatory volume decrease in a renal distal tubular cell line (A6). II. Effect of Na+ transport rate.

Abstract

A6 epithelia, a cell line originating from the distal tubular part of the kidney of Xenopus laevis, were cultured on permeable supports and mounted in an Ussing-type chamber. Cell thickness (Tc), short-circuit current (Isc) and transepithelial conductance (Gt) were recorded while tissues were bilaterally incubated in NaCl solutions and the transepithelial potential was clamped to zero. Effects of inhibition and stimulation of transepithelial Na+ transport on cell volume and on its regulation during a hyposmotic challenge were investigated. Under control conditions a slow spontaneous decrease of Tc described by a linear baseline was recorded. The reduction of the apical osmolality from 260 to 140 mosmol/kg did not alter cell volume significantly, demonstrating a negligible water permeability of the apical barrier. The inhibition of Na+ uptake by replacing apical Na+ by N-methyl-d-glucamine (NMDG+) did not affect cell volume under isotonic conditions. An increase of Tc by 12.1% above the control baseline was recorded after blocking active transport with ouabain for 60 min. The activation of Na+ transport with insulin or oxytocin, which is known to activate the apical water permeability in other epithelia, did not alter cell volume significantly. The insensitivity of cell volume to alterations in apical Na+uptake or Na+ pump rate confirms the close coupling between apical and basolateral transport processes. The blockage of basolateral K+ channels by 5 mM Ba2+ elicited a significant increase in Tc of 16.3% above control. Quinine, a potent blocker of volume-activated K+ channels, did not change Tc significantly. Basolateral hypotonicity elicited a rapid rise in Tc followed by a regulatory volume decrease (RVD). An RVD was also recorded after blocking apical Na+ uptake as well as after stimulating apical Na+ uptake with oxytocin or insulin. Inhibition of active transport with ouabain as well as blocking K+ efflux at the basolateral side with Ba2+ or quinine abolished the RVD. The inhibition of the RVD by ouabain seems to be caused by a depletion of cellular K+, whereas the effects of Ba2+ and quinine are most likely due to the blockage of the basolateral K+ pathway.