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pH-Dependence of Extrinsic and Intrinsic H⁺-Ion Mobility in the Rat Ventricular Myocyte, Investigated Using Flash Photolysis of a Caged-H⁺ Compound

Pawel Swietach ^*, Kenneth W. Spitzer  and Richard D. Vaughan-Jones ^*

^* Burdon Sanderson Cardiac Science Centre, Department of Physiology, Anatomy and Genetics, Oxford University, Oxford, United Kingdom; and Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, Salt Lake City, Utah

Correspondence: Address reprint requests to Richard D. Vaughan-Jones, E-mail: richard.vaughan-jones{at}physiol.ox.ac.uk.

Passive H⁺-ion mobility within eukaryotic cells is low, due to H⁺-ion binding to cytoplasmic buffers. A localized intracellular acidosis can therefore persist for seconds or even minutes. Because H⁺-ions modulate so many biological processes, spatial intracellular pH (pH_i)-regulation becomes important for coordinating cellular activity. We have investigated spatial pH_i-regulation in single and paired ventricular myocytes from rat heart by inducing a localized intracellular acid-load, while confocally imaging pH_i using the pH-fluorophore, carboxy-SNARF-1. We present a novel method for localizing the acid-load. This involves the intracellular photolytic uncaging of H⁺-ions from a membrane-permeant acid-donor, 2-nitrobenzaldehyde. The subsequent spatial pH_i-changes are consistent with intracellular H⁺-mobility and cell-to-cell H⁺-permeability constants measured using more conventional acid-loading techniques. We use the method to investigate the effect of reducing pH_i on intrinsic (non-CO₂/HCO₃^– buffer-dependent) and extrinsic (CO₂/HCO₃^– buffer-dependent) components of H_i⁺-mobility. We find that although both components mediate spatial regulation of pH within the cell, their ability to do so declines sharply at low pH_i. Thus acidosis severely slows intracellular H⁺-ion movement. This can result in spatial pH_i nonuniformity, particularly during the stimulation of sarcolemmal Na⁺-H⁺ exchange. Intracellular acidosis thus presents a window of vulnerability in the spatial coordination of cellular function.

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