Kinetics of the Na+-H+ antiporter as assessed by the change in intracellular pH in MDCK cells.
The Na+-H+ antiporter regulates both H+ secretion and cell pH in renal epithelia. The present study was designed to further define the Na+ dependency and kinetics of proton efflux in MDCK cells. Intracellular pH (pHi) was determined with a pH-sensitive fluorescent probe, 2,7-bis-carboxyethyl-5,6-carboxyfluorescein (BCECF). Data were obtained from confluent monolayers grown on plastic cover slips and studied in media free of added CO2 and HCO-3, pH = 7.2 (pHo). Monolayers in NaCl maintain a pHi of 7.48 +/- 0.03 (n = 13). When cells were acidified with NH4Cl, pHi decreased to 6.73 +/- 0.07 (n = 10) and remained stable in Na+-free choline chloride. When monolayers were subsequently exposed to NaCl, pHi increased 0.28 +/- 0.04 pH units/min. Amiloride (2.5 mM) inhibited this Na+-dependent rise in pHi by more than 75%. The rate of pHi recovery after exposure to Na+ exhibited saturation kinetics; the apparent Km(Na) was 30.4 +/- 4.2 mM and maximum velocity was 107.1 +/- 17.1 delta [H+]i nmol X l-1 X min-1. We conclude that pHi regulation in the MDCK cell is in part mediated by a Na+-H+ antiporter, and the kinetics of this process can be reliably assessed by the pH-sensitive fluorometric probe, BCECF.
D000584:Amiloride / Q000494:pharmacology; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D004848:Epithelium / Q000378:metabolism; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007424:Intracellular Fluid; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D012965:Sodium Chloride / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers; D013050:Spectrometry, Fluorescence
D002352:Carrier Proteins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D017923:Sodium-Hydrogen Exchangers; D000643:Ammonium Chloride; D012965:Sodium Chloride; D000584:Amiloride; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein