H(+)-K(+)-ATPase of rat inner medullary collecting duct in primary culture.
pH recovery in response to addition of and removal from NH4Cl was examined using 2',7'-bis(2-carboxy-ethyl)-5(6)-carboxyfluorescein fluorescence in primary cultures of inner medullary collecting duct (IMCD) cells from rat kidneys. In 0 K+, pH recovery rate was 0.012 +/- 0.010 U/min; in 5 mM K+, the recovery rate was greater at 0.065 +/- 0.013 U/min (P = 0.026). The H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) inhibitors omeprazole and Sch-28080 and the P-type ATPase inhibitor vanadate significantly inhibited pH recovery at 100, 10, and 5 microM, respectively. The vacuolar H(+)-ATPase inhibitor bafilomycin failed to inhibit pH recovery, but N-ethylmaleimide (NEM) did. A range of Sch-28080 concentrations inhibited ouabain-resistant ATPase activity of microsomes from these cells in a reverse sigmoidal manner, with little inhibition < 1 microM, virtually 100% inhibition > 100 microM, and a 50% inhibitory concentration of approximately 20 microM. Bafilomycin only produced significant inhibition of activity at concentrations well in excess of those that are effective against the vacuolar H(+)-ATPase. The ouabain-resistant ATPase activity in cultured IMCD was also sensitive to vanadate (90% inhibition with 5 microM) but relatively resistant to N,N'-dicyclohexylcarbodiimide and NEM. These results indicate that pH regulation in primary cultures of IMCD cells, presumably reflecting H+ transport, is predominantly due to an H(+)-K(+)-ATPase.
Kleinman
J G
JG
Nephrology Division, Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin.
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D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002478:Cells, Cultured; D005033:Ethylmaleimide / Q000494:pharmacology; D017506:H(+)-K(+)-Exchanging ATPase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000494:pharmacology; D007679:Kidney Medulla / Q000201:enzymology*; D007685:Kidney Tubules, Collecting / Q000201:enzymology*; D007700:Kinetics; D018942:Macrolides; D009853:Omeprazole / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D054328:Proton Pump Inhibitors; D051381:Rats; D017207:Rats, Sprague-Dawley; D014638:Vanadates / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D007093:Imidazoles; D018942:Macrolides; D054328:Proton Pump Inhibitors; C035235:Sch 28080; D014638:Vanadates; D010042:Ouabain; D017506:H(+)-K(+)-Exchanging ATPase; D009853:Omeprazole; D005033:Ethylmaleimide; D011188:Potassium