title: Dual regulatory mechanisms of proton transport in rat papillary collecting duct cells in culture.
abstract: The regulation of proton transport and cytosolic pH was studied in rat papillary collecting duct (PCD) cells in culture using a pH-sensitive fluorescence probe, 2,7-bis-carboxyethyl-5,6-carboxyfluorescein (BCECF). Data were obtained from confluent monolayers grown on glass coverslips and dipped in a HCO3- -free medium, pH 7.40. The resting intracellular pH (pHi) was 7.16 +/- 0.03 (n = 20). When PCD cells had been acidified by pretreatment with NH4Cl, pHi immediately recovered toward the resting value. Two mechanisms participated in this recovery: a Na+-dependent mechanism which could be inhibited by amiloride (indicative of Na+-H+ exchanger) and a Na+-independent process (a proton ATPase). The pHi recovery from acid loading was inhibited by amiloride to about 55% of the control recovery (half-maximal effect at 100 microM). The rate of pHi recovery after the readdition of Na+ to a sodium-free medium exhibited saturation kinetics (half maximal rate at 28 mM). Dicyclohexylcarbodiimide (DCCD), an inhibitor of a plasma membrane proton ATPase, and the depletion of cellular ATP induced by 2 mM potassium cyanide (KCN) also partially inhibited the rate of pHi recovery after cell acidification with a NH4Cl load. When PCD cells were treated with 1 mM DCCD, amiloride almost completely inhibited pHi recovery. Amiloride and the removal of external Na+ had induced a gradual fall in pHi to a new resting value and rapidly recovered when Na+ was added. We conclude that PCD cells grown in culture have at least two proton transport mechanisms: a Na+-H+ exchanger and a plasma membrane proton ATPase. The kinetics of these processes can be reliably assessed by the pH-sensitive fluorescent probe, BCECF. Both the Na+-H+ exchanger and the plasma membrane proton ATPase may contribute to urinary acidification.
authors.affiliation: Department of Medicine, Jichi Medical School, Tochigi, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Takeda
authors.forename: T
authors.initials: T
authors.lastname: Oohara
authors.forename: K
authors.initials: K
authors.lastname: Tabei
authors.forename: Y
authors.initials: Y
authors.lastname: Asano
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007684:Kidney Tubules / Q000378:metabolism*; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D011522:Protons; D051381:Rats; D011919:Rats, Inbred Strains; D017923:Sodium-Hydrogen Exchangers
chemical_list: D002352:Carrier Proteins; D011522:Protons; D017923:Sodium-Hydrogen Exchangers; D004024:Dicyclohexylcarbodiimide; D000584:Amiloride; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases