Molecular Pharmacology, Vol 10, 820-836, Copyright © 1974 by the American Society for Pharmacology and Experimental Therapeutics

A New Approach to Measuring the Rate of Rapid Bicarbonate Exchange across Membranes

¹ Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville, Florida 32610

This is a description of a new method for measuring the rate of rapid bicarbonate exchange across membranes, including a description of the inhibition of the bicarbonate exchange across the rat erythrocyte membrane by sodium picrate. The method is based on the measurement, by mass spectrometry, of the rate of depletion of ¹⁸O from labeled bicarbonate dissolved in a solution in which red cells are suspended. The rate of this depletion depends (a) on the rate of diffusion of labeled bicarbonate into the cell, (b) on the rate of chemical reaction of labeled bicarbonate to form labeled carbon dioxide, which then diffuses across the membrane, and (c) on the rate of carbonic anhydrase-catalyzed depletion inside the cell. Expressions are derived for the permeability constant of the cell membrane to bicarbonate and the rate constant for the catalyzed, intracellular dehydration of bicarbonate in terms of the rates of depletion of ¹⁸O from bicarbonate. The contribution of labeled CO₂ diffusion across the membrane, factor (b) above, is estimated by assuming that labeled CO₂ generated intracellularly does not accumulate in the suspending solution, but is depleted of its label inside the cell. This assumption is not rigorously verified and is a possible source of error in separating the bicarbonate flux from the CO₂ flux. This method is most accurate in regions above pH 9 and is restricted to cells containing carbonic anhydrase. At pH 9.6 and 25° the permeability constant of the rat erythrocyte membrane to bicarbonate is (1.81 ± 0.12) x 10^-4 cm/sec, giving a half-time of 0.13 ± 0.02 sec for the exchange of bicarbonate in the cell. The rate constant for the catalyzed, intracellular dehydration of bicarbonate is 3.3 ± 0.4 sec^-1. P, calculated in this way, was found to increase when the pH of the red cell suspension was decreased.

Note:
ACKNOWLEDGMENTS The author thanks Professor Thomas H. Maren for his suggestions and support during this work. He is grateful to Dr. Richard Gerster for his advice on and helpful explanations of ¹⁸O exchange techniques.

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