Characterization of acid-base transporters in cultured outer medullary collecting duct cells.
Cells from the inner stripe of the rabbit outer medullary collecting duct (OMCDi) were grown in primary culture, and their acid-base transport properties were characterized using intracellular pH (pHi) measurements with the fluorescent probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Basal pHi in HCO(3-)-buffered solutions was 7.28 +/- 0.04 (n = 20). The presence of a Cl-/HCO(3-)-antiporter was demonstrated by reversible alkalinization on bath Cl- removal. The mean alkalinization seen on Cl- removal was 0.16 +/- 0.02 pH units (n = 20) and was inhibited 92% by 10(-4) M 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Studies were also performed to determine the presence of an Na+/H+ antiporter and an H(+)-adenosinetriphosphatase (H(+)-ATPase). After an NH4Cl acid load the cells exhibited both Na(+)-dependent and Na(+)-independent pHi recovery mechanisms. The Na(+)-dependent mechanism was inhibited by amiloride. The Na(+)-independent mechanism was completely inhibited by 10(-3) M N-ethylmaleimide or 2.5 x 10(-9) M bafilomycin A1, but was not significantly altered by removal of bathing solution K+. Thus, the Na(+)-dependent recovery mechanism exhibited characteristics of an Na+/H+ antiporter, whereas the Na(+)-independent recovery mechanism was consistent with the presence of an H(+)-ATPase.
Manger
T M
TM
Department of Medicine, University of Connecticut Health Center, Farmington 06030.
Koeppen
B M
BM
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D027963:Chloride-Bicarbonate Antiporters; D005260:Female; D005452:Fluoresceins; D017506:H(+)-K(+)-Exchanging ATPase / Q000378:metabolism*; D007679:Kidney Medulla; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism*; D054328:Proton Pump Inhibitors; D011817:Rabbits; D017923:Sodium-Hydrogen Exchangers
D002352:Carrier Proteins; D027963:Chloride-Bicarbonate Antiporters; D005452:Fluoresceins; D054328:Proton Pump Inhibitors; D017923:Sodium-Hydrogen Exchangers; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D017506:H(+)-K(+)-Exchanging ATPase