Apical Na+/H+ antiporter and glycolysis-dependent H+-ATPase regulate intracellular pH in the rabbit S3 proximal tubule.

Abstract

The apical transport processes responsible for proton secretion were studied in the isolated perfused rabbit S3 proximal tubule. Intracellular pH (pH;) was measured with the pH dye, 2'7'-bis(carboxyethyl)-5,6-carboxyfluorescein. Steady state pH; in S3 tubules in nominally HCO3-free solutions was 7.08±0.03. Removal of Na+ (lumen) caused a decrease in pH1 of 0.34±0.06 pH/min. The decrease in pH, was inhibited 62% by 1 mM amiloride (lumen) and was unaffected by 50 ,uM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (lumen) and Cl-removal (lumen, bath). After a brief exposure to 20 mM NH4CIL, pH, fell by -0.7 and recovered at a rate of 0.89±0.15 pH/min in the nominal absence of Na+, HCO3, organic anions, and S042-(lumen, bath). 1 mM N,N'-dicyclohexylcarbodiimide (lumen), 1 mM N-ethylmaleimide (lumen), 0.5 mM colchicine (bath), and 0.5 mM iodoacetic acid (lumen, bath) inhibited the Na+-independent pH; recovery rate by 73%, 55%, 77%, and 86%, respectively, whereas 1 mM KCN (lumen, bath) did not inhibit pH, recovery. Reduction of intracellular, but not extracellular chloride, also decreased the Na+-independent pH, recovery rate. In conclusion, the S3 proximal tubule has an apical Na+/H+ antiporter with a Michaelis constant for Na+ of 29 mM and a maximum velocity of 0.47 pH/min. S3 tubules also possess a plasma membrane H+-ATPase that can regulate pH,, has a requirement for intracellular chloride, and utilizes ATP derived primarily from glycolysis.

Mineralocorticoids and acidosis regulate H+/HCO3- transport of intercalated cells.

“…2). Similar observations were described in S3 proximal tubule (33), OMCDo intercalated cells (6), and OMCDi (8). Recently the presence of H+-K+-ATPase, another H+ pump, has been suggested in OMCDi (29)(30)(31).…”

Topological Location and Structural Importance of the NBCe1-A Residues Mutated in Proximal Renal Tubular Acidosis

“…24 h post-transfection, cells were loaded with fluorescent pH probe BCECF-AM and assayed using a microfluorometer (19 Biotin Maleimide Labeling and Immunoprecipitation-Whole cell labeling with biotin maleimide was proceeded as described previously (20,21). Briefly, transfected HEK 293 cells were incubated with 0.2 mM biotin maleimide in 1 ml of PBSCM (PBS containing 0.1 mM CaCl 2 and 1 mM MgCl 2 , pH 7.0) for 10 min at room temperature.…”

Functional characterization of NBC4: a new electrogenic sodium-bicarbonate cotransporter

“…Functional studies were performed 24 h after transfection. pH i was monitored with the fluorescent probe 2Ј,7Ј-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and a microfluorometer coupled to the microscope (19). Data was obtained from ϳ20 cells per coverslip.…”