HCO3- ABSORPTION IN RABBIT OUTER MEDULLARY COLLECTING DUCT - ROLE OF LUMINAL CARBONIC-ANHYDRASE

Membrane-bound luminal carbonic anhydrase (CA) IV, by catalyzing the d ehydration of carbonic acid into CO2 plus water facilitates H+ secreti on in the renal outer medullary collecting duct from the inner stripe (OMCDi). To examine the role of CA TV on H+ secretion, we measured net HCO3- transport in perfused OMCDi segments and examined the effect on transport of two extracellular CA inhibitors, benzolamide and F-3500, aminobenzolamide coupled to a nontoxic polymer, polyoxyethylene bis(a cetic acid) [synthesized and kindly provided by C. Conroy and T. Maren (C. W. Conroy, G. C. Wynns, and T. H. Maren. Bioorg. Chem. 24: 262-27 2, 1996)]. These agents would inhibit only the luminal CA enzyme. Dose titration curves for net HCO3- flux were performed for each drug. Bas al HCO3- absorptive flux was 12 pmol . min(-1). mm(-1) in control segm ents and significantly increased to 16 pmol . min(-1). mm(-1) in segme nts from 3-day acid-treated animals. The concentrations of benzolamide and F-3500 that inhibited HCO3- absorption by 50% were similar to 0.1 and similar to 5 mu M, similar to the K-i for CA TV inhibition by the se agents (0.2 and 4.0 mu M, respectively; T. Maren, C. W. Conroy, G. C. Wynns, and D. R. Godman. J. Pharmacol. Exp. Ther. 280: 98-104, 1997 ). Adding exogenous CA to the inhibitor in the perfusate nearly restor ed basal HCO3- transport, suggesting that cytosolic CA II was not inhi bited by these impermeant inhibitors. In OMCDi segments from acidotic rabbits, the concentrations of benzolamide and F-3500 that inhibited H CO3- absorption by 50% were 50 and 500 mu M, respectively, >100 times the K-i for CAIV inhibition and for inhibition of HCO3- transport in c ontrol tubules. Thus, in the OMCDi, doses of extracellular CA inhibito rs that inhibited similar to 50% of CA IV activity also comparably inh ibited HCO3- transport, indicating that H+ secretion depends in part o n the availability of luminal CA TV activity. Acidosis substantially d ecreased the sensitivity of HCO3- transport to CA inhibition.