Ouabain reduces net acid secretion and increases pHi by inhibiting NH4+ uptake on rat tIMCD Na+-K+-ATPase

In the rat terminal inner medullary collecting duct (tIMCD), Na(+) pump inhibition reduces transepithelial net acid secretion (J(tAMM)) [J(H) = total CO(2) absorption (J(tCO2)) + total ammonia secretion] and increases resting intracellular pH (pH(i)). The increase in pH(i) and reduction in JH that follow ouabain addition do not occur in the absence of NH(4)(+) nor when NH(4)(+) is substituted with another weak base. The purpose of this study was to explore the mechanism of the NH(4)(+)-dependent reduction in J(tCO2) and increase in pH(i) that follow ouabain addition. We hypothesized that NH(4)(+) enters the tIMCD cell through the Na(+)-K(+)-ATPase with proton release in the cytosol. To test this hypothesis, tIMCDs were dissected from deoxycorticosterone-treated rats and perfused in vitro with symmetrical physiological saline solutions containing 6 mM NH(4)Cl. Since K(+) and NH(4)(+) compete for a common binding site on the Nai pump, increasing extracellular K(+) should limit NH(4)(+) (and hence net H(+)) uptake by the Na(+) pump. Upon increasing extracellular K(+) concentration from 3 to 12 mM, the NH(4)(+)- dependent, ouabain-induced increase in pH(i) and reduction in J(tCO2) were attenuated. In the presence but not in the absence of NH(4)(+), reducing Na(+) pump activity by limiting Na(+) entry reduced J(tCO2) and attenuated ouabain-induced alkalinization. Ouabain-induced alkalinization was not dependent on the presence of HCO(3)(-)/CO(2) and was not reproduced with BaCl(2) or bumetanide addition. Therefore, ouabain-induced alkalinization is not mediated by the Na(+)-K(+)-2Cl-cotransporter or a HCO(3)(-) transporter and is not mediated by changes in membrane potential. In conclusion, on the basolateral membrane of the tIMCD cell, NH(4)(+) uptake is mediated by the Na(+)-K(+)-ATPase. These data provide an explanation for the reduction in net acid secretion in the tIMCD observed following administration of amiloride or with dietary K(+) loading.