Role of H+-K+-ATPase in pH(i) regulation in inner medullary collecting duct cells in culture

Studies in inner medullary collecting duct (IMCD) cells in primary culture have proposed two mechanisms for Na+-independent hydrogen ion transport: an H+-adenosinetriphosphatase (H+-ATPase) and an H+-K+-ATPase. In the present study, we have employed two sources of IMCD cells, cells in primary culture derived from the terminal papilla of the Munich-Wistar rat (IMCD(p)) and an established murine cell line (mIMCD-3), to define the predominant mechanism(s) of Na+-independent intracellular pH (pH(i)) recovery in the IMCD. In confluent monolayers of IMCD(p) and mIMCD-3 cells, pH(i) was measured using the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) following addition and withdrawal of NH4Cl. Removal of K+ completely abolished Na+-independent pH(i) recovery in both IMCD(p) (Delta pH(i)/min = 0.039 +/- 0.006 to 0.005 +/- 0.003; P < 0.001) and in mIMCD-3 (Delta pH(i)/min = 0.055 +/- 0.009 to -0.003 +/- 0.002; P < 0.001) cells, respectively. In mIMCD-3 cells, K+-dependent pH(i) recovery was abolished by either of two specific inhibitors of the H+-K+-ATPase, Sch-28080 (5 or 10 mu M) or A-80915A (10 mu M). In contrast, bafilomycin A(1) (2.5 and 10 nM), an inhibitor of the H+-ATPase, failed to attenuate K+-dependent pH(i) recovery. Moreover, sequence verified mouse gastric and colonic alpha-H+-K+-ATPase probes hybridized to total RNA from mIMCD-3 cells. Based on these findings, we conclude that Na+-independent pH(i) recovery from an acid load in both IMCD(p) and mIMCD-3 cells is critically dependent on extracellular K+. That K+-dependent pH(i) recovery was inhibited by both Sch-28080 and A-80915A but not by bafilomycin A(1) suggests that the predominant mechanism by which Na+-independent pH(i) recovery is accomplished in IMCD is through the H+-K+-ATPase. Expression of both gastric and colonic alpha-H+-K+-ATPase mRNA in mIMCD-3 cells suggests that one or both of these H+-K+-ATPases may be responsible for proton secretion in the IMCD.