Effect of increases in cytosolic Ca2+ on inner medullary collecting duct cell pH.

Abstract

Changes in cytosolic calcium concentration (Cai2+) have been implicated in the regulation of intracellular pH (pHi) in several cell types. In the present study we investigated the mechanism by which an increase in Cai2+ stimulates H+ secretion and a rise in pHi in cultured rat inner medullary collecting duct (IMCD) cells. Confluent monolayers were made quiescent by incubation for 24 h in 0.1% serum before study. Changes in pHi and Cai2+ were measured with the fluorescent probes, 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and fura-2. In nominally bicarbonate-free media containing 110 mM Na+ and 1 mM Ca2+, addition of the Ca2+ inophore, ionomycin (10 microM), produced a biphasic response in pHi, a transient acidification from 7.29 +/- 0.07 to 7.12 +/- 0.05 at 2 min followed by a sustained alkalinization to a steady-state value of 7.51 +/- 0.10 at 10 min. The rate of alkalinization was dose dependent. The alkalinization was not affected by 1 mM amiloride, removal of extracellular Na+, or by the proton pump inhibitor, N-ethyl maleimide (NEM). Metabolic energy was not required, but removal of extracellular Ca2+ prevented the alkalinization. By use of the fluorescent probe bisoxonol to assess membrane potential, ionomycin was shown to cause depolarization under the same experimental conditions as those for alkalinization. The Ca2+-induced alkalinization was mimicked by cell depolarization (induced by raising extracellular K+ in the presence of valinomycin 1 microM). We conclude that changes in Cai2+ are important in the regulation of pHi in the IMCD. Ca2+-induced cell alkalinization may be mediated by changes in membrane ionic conductance.