Regulation of intracellular pH (pHi) in snake renal proximal tubules

Previous work on snake proximal-proximal (ppt) and distal-proximal (dpt) tubules with oil-filled lumens in HEPES (25 mM)-buffered medium suggested that basolateral [Na⁺] could affect pH_i. We now evaluated regulation of pH_i, measured with fluorescent dye BCECF, in both absence and presence of HCO₃^-. Resting pH_i was 7.1-7.2 in ppt and dpt in HEPES-buffered medium and in ppt in HEPES (20 mM)/HCO₃^- (5 mM)-buffered medium, but -7.3 in dpt in HEPES/HCO₃^- buffer. pH_i increased to 7.6-7.7 with addition of NH₄Cl (20 mM) and then decreased to 6.9-7.0 with its removal in ppt and dpt in both buffers. Control rate of recovery (dpH_i/dt) from acid level was 3.9 X 10^-3 pH U/s, except for dpt in HEPES/HCO₃^- buffer where it was 5.2 X 10^-3 pH U/s. dpH_i/dt was reduced by Na⁺ removal in dpt in HEPES buffer but was not affected by Na⁺ removal in ppt or dpt in HEPES/HCO₃^- buffer. It was not affected by Cl^-, or both Na⁺ and Cl^- removal or the presence of Ba²⁺ (5 mM) or high K⁺ (75 mM) in either HEPES or HEPES/HCO₃^- buffer. Anion exchange inhibitor DIDS (100 μM) reduced dpH_i/dt only in dpt and only in HEPES/HCO₃^- buffer. Na⁺/H⁺ exchange inhibitor EIPA (100 μM) reduced dpH_i/dt slightly but significantly in dpt, but not ppt in both HEPES and HEPES/HCO₃^- buffers. These data do not clearly support basolateral regulation of pH_i by the most common Na⁺/H⁺, Na⁺-dependent and Na⁺-independent Cl^-/HCO₃^- exchangers, or Na⁺-HCO₃^-CO₃^2- cotransporter.