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 rising 2',7'-bis(2-carboxyethyl)-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 muM, 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 muM, virtually 100% inhibition >100 muM, and a 50% inhibitory concentration of approximately 20 muM. 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 muM) 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.