title: 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 (pHi) was measured with the pH dye, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. Steady state pHi in S3 tubules in nominally HCO3(-)-free solutions was 7.08 +/- 0.03. Removal of Na+ (lumen) caused a decrease in pHi of 0.34 +/- 0.06 pH/min. The decrease in pHi was inhibited 62% by 1 mM amiloride (lumen) and was unaffected by 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (lumen) and Cl- removal (lumen, bath). After a brief exposure to 20 mM NH4Cl, pHi fell by approximately 0.7 and recovered at a rate of 0.89 +/- 0.15 pH/min in the nominal absence of Na+, HCO3-, organic anions, and SO4(2-) (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 pHi recovery rate by 73%, 55%, 77%, and 86%, respectively, whereas 1 mM KCN (lumen, bath) did not inhibit pHi recovery. Reduction of intracellular, but not extracellular chloride, also decreased the Na+-independent pHi 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 pHi, has a requirement for intracellular chloride, and utilizes ATP derived primarily from glycolysis.
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D002712:Chlorides / Q000378:metabolism; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D006019:Glycolysis; D006863:Hydrogen-Ion Concentration; D007687:Kidney Tubules, Proximal / Q000201:enzymology*; D007700:Kinetics; D008297:Male; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011817:Rabbits; D017923:Sodium-Hydrogen Exchangers
chemical_list: D002352:Carrier Proteins; D002712:Chlorides; D017923:Sodium-Hydrogen Exchangers; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D004024:Dicyclohexylcarbodiimide; D006180:Proton-Translocating ATPases; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid