AJP - Renal Physiology, Vol 260, Issue 5 738-F748, Copyright © 1991 by American Physiological Society
Distribution of luminal carbonic anhydrase activity along rat inner medullary collecting duct
S. M. Wall, M. F. Flessner and M. A. Knepper
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
The isolated perfused tubule technique was utilized to determine whether
endogenous luminal carbonic anhydrase is present in the initial or terminal
parts of the inner medullary collecting duct (IMCD) of the rat. This was
accomplished by measuring the luminal disequilibrium pH in the presence of
a large luminal proton source created by perfusing the lumen with a
solution containing 10 mM NH4Cl. (NH3 efflux causes H+ to be released from
NH+4 in the lumen). The disequilibrium pH was calculated by subtracting the
equilibrium pH from the measured pH at the end of the tubule lumen. The
end-luminal equilibrium pH was calculated from the total CO2 concentration
in the collected fluid, as measured by microcalorimetry. The end-luminal pH
was determined by measuring the fluorescent signal from the the
pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF),
which was added to the luminal perfusate in its nonesterified form. In the
initial IMCD, there was no measurable disequilibrium pH. With the addition
of the carbonic anhydrase inhibitor acetazolamide to the luminal fluid, a
significant acidic pH disequilibrium was elicited. In the terminal IMCD
under control conditions a statistically significant acidic disequilibrium
pH was measured. The disequilibrium was obliterated when exogenous carbonic
anhydrase was added to the luminal perfusate. These findings were verified
by measuring total ammonia flux by ultramicrofluorometry. The results
demonstrate endogenous luminal carbonic anhydrase activity in the initial
IMCD but a lack of enzyme activity in the terminal IMCD.
