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

Abstract

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 (IMCDp) and an established murine cell line (mIMCD-3), to define the predominant mechanism(s) of Na(+)-independent intracellular pH (pHi) recovery in the IMCD. In confluent monolayers of IMCDp and mIMCD-3 cells, pHi 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 pHi recovery in both IMCDp (delta pHi/min = 0.039 +/- 0.006 to 0.005 +/- 0.003; P < 0.001) and in mIMCD-3 (delta pHi/min = 0.055 +/- 0.009 to -0.003 +/- 0.002; P < 0.001) cells, respectively. In mIMCD-3 cells, K(+)-dependent pHi recovery was abolished by either of two specific inhibitors of the H(+)-K(+)-ATPase, Sch-28080 (5 or 10 microM) or A-80915A (10 microM). In contrast, bafilomycin A1 (2.5 and 10 nM), an inhibitor of the H(+)-ATPase, failed to attenuate K(+)-dependent pHi 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 pHi recovery from an acid load in both IMCDp and mIMCD-3 cells in critically dependent on extracellular K(+)-That K(+)-dependent pHi recovery was inhibited by both Sch-28080 and A-80915A but not by bafilomycin A1 suggests that the predominant mechanism by which Na(+)-independent pHi 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.