Functional activity of H-K-ATPase in individual cells of OMCD: Localization and effect of K+ depletion

Recent studies have indicated the presence of hydrogen-potassium-adenosinetriphatase (H-K-ATPase) in the collecting duct. We examined the localization of functional H-K-ATPase activity in individual cells of the outer and inner stripes of outer medullary collecting ducts (OMCD(o) and OMCD(i)). Tubules were isolated from control and K+-depleted rabbits and perfused in vitro. Intracellular pH (pH(i)) of principal cells, intercalated cells, and OMCD(i) cells was monitored by fluorescence ratio imaging using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). An intracellular acid load was induced by NH3/NH4+ prepulse in extracellular Na+-, K+-, and HCO3--free condition, and then 5 mM K+ was added to the lumen or the bath in the presence of Ba2+. Functional activity of H-K-ATPase was estimated by the difference in the rates of pH(i) recovery before and after K+ addition. In the control condition, luminal addition of K+ significantly increased the pH(i) recovery rate by 1.6 +/- 0.4 and 1.9 +/- 0.4 x 10(-3) pH units/s in intercalated cells and OMCD(i) cells, respectively, but not in principal cells. This K+-dependent pH(i) recovery was inhibited by 63% in intercalated cells and 74% in OMCD(i) cells in the presence of luminal Sch-28080 (10 mu M) but was not affected in the presence of luminal bafilomycin-A(1) (10 nM). K+ depletion increased the K+-dependent pH(i) recovery to 2.3-fold in intercalated cells and 2.6-fold in OMCD(i) cells. By contrast, K+-dependent pH(i) recovery was not detected in the basolateral membrane of any cell types in either the control or the K+-depleted condition. These results provide functional evidence that H-K-ATPase is distributed in the luminal membrane of intercalated cells and OMCD(i) cells and that this ATPase is activated by K+ depletion, suggesting the contribution of intercalated cells and OMCD(i) cells to K+ conservation in rabbit OMCD.