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Intracellular H(+) mobility was estimated in the rabbit isolated ventricular myocyte by diffusing HCl into the cell from a patch pipette, while imaging pH(i) confocally using intracellular ratiometric SNARF fluorescence. The delay for acid diffusion between two downstream regions approximately 40 microm apart was reduced from approximately 25 s to approximately 6 s by replacing Hepes buffer in the extracellular superfusate with a 5 % CO(2)/HCO(3)(-) buffer system (at constant pH(o) of 7.40). Thus CO(2)/HCO(3)(-) (carbonic) buffer facilitates apparent H(+)(i) mobility. The delay with carbonic buffer was increased again by adding acetazolamide (ATZ), a membrane permeant carbonic anhydrase (CA) inhibitor. Thus facilitation of apparent H(+)(i) mobility by CO(2)/HCO(3)(-) relies on the activity of intracellular CA. By using a mathematical model of diffusion, the apparent intracellular H(+) equivalent diffusion coefficient (D(H)(app)) in CO(2)/HCO(3)(-)-buffered conditions was estimated to be 21.9 x 10(-7) cm(2) s(-1), 5.8 times faster than in the absence of carbonic buffer. Facilitation of H(+)(i) mobility is discussed in terms of an intracellular carbonic buffer shuttle, catalysed by intracellular CA. Turnover of this shuttle is postulated to be faster than that of the intrinsic buffer shuttle. By regulating the carbonic shuttle, CA regulates effective H(+)(i) mobility which, in turn, regulates the spatiotemporal uniformity of pH(i). This is postulated to be a major function of CA in heart.

Type

Journal article

Journal

J Physiol

Publication Date

15/05/2002

Volume

541

Pages

159 - 167

Keywords

Acetazolamide, Algorithms, Animals, Benzopyrans, Bicarbonates, Buffers, Carbon Dioxide, Carbonic Anhydrase Inhibitors, Carbonic Anhydrases, Diffusion, Fluorescent Dyes, Heart Ventricles, Hydrogen, Hydrogen-Ion Concentration, In Vitro Techniques, Microscopy, Confocal, Myocardium, Naphthols, Rabbits, Rhodamines