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Inner medullary collecting duct cells were isolated from rat papillae and grown to confluence on cover slips. H+ secretion was estimated by intracellular pH (pHi) changes measured with the fluorescent probe 2,7-biscarboxyethyl-5(6)-carboxyfluorescein. In buffered NaCl, pHi was 7.14 +/- 0.04 (n = 78). After acidification about 40% of monolayers exhibited Na+-independent alkalinization. In 5 mM glucose, cell alkalinization occurred at a rate of 47 +/- 4 nM H+/min. However, cell alkalinization did not occur in the presence of 2-deoxy-D-glucose (5-15 mM), iodoacetate (5 mM), or KCN (5 mM). All monolayers tested exhibited amiloride-inhibitable Na+-dependent cell alkalinization that appeared to be a first-order kinetic process; Km [Na+] was approximately 52 mM and Vmax was approximately 250 nM [H+]/min. At a constant extracellular [Na+] (110 mM), Na+-dependent H+ efflux was a first-order function of pHi; Km for intracellular [H+] = 321 nM and Vmax = 182 nM H+/min. The data are consistent with the presence of a primary active H+ pump and a secondary active Na+ exchanger. The metabolic energy for the active H+ pump can be provided by glycolysis and oxidative phosphorylation.
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