FEEDBACK-REGULATION OF NA CHANNELS IN RAT CCT .1. EFFECTS OF INHIBITION OF NA PUMP

Na channels in the apical membrane of the rat renal cortical collecting tubule were studied using the patch-clamp technique. Channel activity was monitored in cell-attached patches on tubules that were split open to expose the luminal surface. Channel number (N), open probability (P(o)), and currents (i) were measured at 37-degrees-C during continuous superfusion of the tubule. Addition of ouabain (1 mM) to the superfusate to increase cell Na resulted in a decrease in the mean number of open channels (NP(o)) to less than 20% of control values within 2 min. This effect was not reversible within 5 min after removal of ouabain. There was, in addition, a parallel decrease in i. The mechanism of inhibition appeared to involve increased intracellular Ca (Ca(i)). Ca(i) was measured using the fluorescence of the Ca indicator fura-2 in principal cells of split tubules under conditions identical to those used for electrical measurements. Ca(i) increased from a basal level (153 +/- 36 nM) to a peak level (588 +/- 53 nM) approximately 3 min after the addition of ouabain. When a Ca-free superfusate was used, ouabain did not increase Ca(i) or decrease NP(o), although the decrease in i was similar to that observed in Ca-containing solutions. Similar increases in Ca(i) were elicited by the Ca ionophore ionomycin (5 muM) in the presence of 0.1 mM extracellular Ca. This maneuver also resulted in a decrease in NP(o) which was similar to that observed in the presence of ouabain. Ouabain had no observable effect on cell pH. We conclude that, during inhibition of Na exit from the cell by the ouabain-sensitive Na pump, Ca(i) rises as a result of changes in the driving forces for Na/Ca exchange. The increase in Ca(i) inhibits the apical Na channels, probably by an indirect mechanism. Other factors, such as membrane depolarization, may also contribute to Na channel inhibition under these circumstances.