Inhibition of Na+-independent H+ pump by Na+-induced changes in cell Ca2+

Abstract

Apical membrane H+ extrusion in the renal outer medullary collecting duct, inner stripe, is mediated by a Na+-independent H+ pump. To examine the regulation of this transporter, cell pH and cell Ca2+ were measured microfluorometrically in in vitro perfused tubules using 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein and fura-2, respectively. Apical membrane H+ pump activity, assayed as cell pH recovery from a series of acid loads (NH3/NH4+ prepulse) in the total absence of ambient Na+, initially occurred at a slow rate (0.06 ± 0.02 pH units/min), which was not sufficient to account for physiologic rates of H+ extrusion. Over 15-20 min after the initial acid load, the rate of Na+-independent cell pH recovery increased to 0.63 ± 0.09 pH units/min, associated with a steady-state cell pH greater than the initial pre-acid load cell pH. This pattern suggested an initial suppression followed by a delayed activation of the apical membrane H+ pump. Replacement of peritubular Na+ with choline or N-methyl-D-glucosamine resulted in an initial spike increase in cell Ca2+ followed by a sustained increase in cell Ca2+. The initial rate of Na+-independent cell pH recovery could be increased by elimination of the Na+ removal-induced sustained cell Ca2+ elevation by: (a) performing studies in the presence of 135 mM peritubular Na+ (1 mM peritubular amiloride used to inhibit basolateral membrane Na+/H+ antiport); (b) clamping cell Ca2+ low with dimethyl-BAPTA, an intracellular Ca2+ chelating agent; or (c) removal of extracellular Ca2+. Cell acidification induced a spike increase in cell Ca2+. The late acceleration of Na+-independent cell pH recovery was independent of Na+ removal and of the method used to acidify the cell, but was eliminated by prevention of the cell Ca2+ spike and markedly delayed by the microfilament-disrupting agent, cytochalasin B. This study demonstrates that peritubular Na+ removal results in a sustained elevation in cell Ca2+, which inhibits the apical membrane H+ pump. In addition, rapid cell acidification associated with a spike increase in cell Ca2+ leads to a delayed activation of the H+ pump. Thus, cell Ca2+ per se, or a Ca2+-activated pathway, can modulate H+ pump activity.