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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 pHi confocally using intracellular ratiometric SNARF fluorescence. The delay for acid diffusion between two downstream regions ~40 µm apart was reduced from ~25 s to ~6 s by replacing Hepes buffer in the extracellular superfusate with a 5 % CO2/HCO3- buffer system (at constant pHo of 7.40). Thus CO2/HCO3- (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 CO2/HCO3- relies on the activity of intracellular CA. By using a mathematical model of diffusion, the apparent intracellular H+ equivalent diffusion coefficient (DHapp) in CO2/HCO3--buffered conditions was estimated to be 21.910-7 cm2 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 pHi. This is postulated to be a major function of CA in heart.
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