HCO 3 2 absorption in rabbit outer medullary collecting duct : role of luminal carbonic anhydrase

absorption in rabbit outer medullary collecting duct: role of luminal carbonic anhydrase. Am. J. Physiol. 274 (Renal Physiol. 43): F139–F147, 1998.—Membrane-bound luminal carbonic anhydrase (CA) IV, by catalyzing the dehydration of carbonic acid into CO2 plus water, facilitates H1 secretion in the renal outer medullary collecting duct from the inner stripe (OMCDi). To examine the role of CA IV on H1 secretion, we measured net HCO3 2 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(acetic acid) [synthesized and kindly provided by C. Conroy and T. Maren (C. W. Conroy, G. C. Wynns, and T. H. Maren. Bioorg. Chem. 24: 262–272, 1996)]. These agents would inhibit only the luminal CA enzyme. Dose titration curves for net HCO3 2 flux were performed for each drug. Basal HCO3 2 absorptive flux was 12 pmol ·min21 ·mm21 in control segments and significantly increased to 16 pmol·min21 ·mm21 in segments from 3-day acid-treated animals. The concentrations of benzolamide and F-3500 that inhibited HCO3 2 absorption by 50% were ,0.1 and ,5 μM, similar to the Ki for CA IV inhibition by these agents (0.2 and 4.0 μM, 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 restored basal HCO3 2 transport, suggesting that cytosolic CA II was not inhibited by these impermeant inhibitors. In OMCDi segments from acidotic rabbits, the concentrations of benzolamide and F-3500 that inhibited HCO3 2 absorption by 50% were 50 and 500 μM, respectively, .100 times the Ki for CA IV inhibition and for inhibition of HCO3 2 transport in control tubules. Thus, in the OMCDi, doses of extracellular CA inhibitors that inhibited ,50% of CAIV activity also comparably inhibited HCO3 2 transport, indicating that H1 secretion depends in part on the availability of luminal CA IV activity. Acidosis substantially decreased the sensitivity of HCO3 2