CHARACTERIZATION OF ACID-BASE TRANSPORT MECHANISMS IN THE KIDNEY-CELL LINE RCCT-28A

RCCT-28A cells, a continuous cell line of rabbit cortical collecting tubule origin, have been found to exhibit apical peanut-lectin binding, basal band-3 immunostaining and a transepithelial electrical resistance of 246 +/- 37 OMEGA cm2. For the studies reported, confluent monolayers of RCCT-28A cells were grown on permeable wells and incubated in a control solution or in alkaline solutions by lowering PCO2. Equivalent H+ fluxes (J(H)+) into the apical solution (nmol . min-1 . cm2) were measured in the absence of drugs and in the presence of: amiloride (A,10(-3) M), N-ethylmaleimide (NEM, 5 mM) and omeprazole (OM, 100 muM) in the apical solution. After preincubation in control solutions J(H)+ was 21 +/- 2 while A had no effect. Addition of NEM diminished J(H)+ to 12 +/- 2 (P < 0.005), and OM diminished J(H)+ to 2 +/- 2 (P < 0.001 vs. control). Monolayers incubated at low PCO2 had a basal J(H)+ of 11 +/- 5. No effect on J(H)+ could be demonstrated under these conditions by addition of NEM or OM. Removal of K+ from the apical solution diminished apical acidification by 60%. The inhibitor of H+,K+-ATPase Schering 28080 (SCH) was tested at different concentrations and an inhibitory effect was demonstrated (J(H)+ - 2 +/- 1 vs. 18 +/- 1, SCH vs. control, respectively). Probenecid and bafilomycin-A also decreased apical acidification and an apical base-equivalent extrusion was apparent under the inhibitors effect. J(H)+ was abolished by removal of Cl- from the basolateral solution. These results are compatible with a H+-ATPase coexistent with a H+,K+-ATPase on the apical membrane and a basolateral membrane Cl-/HCO3- exchanger. An apical base extrusion mechanism operating simultaneously with the H+ extrusion is suggested.