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

Abstract

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-CO 2/HCO 3 − buffer-dependent) and extrinsic (CO 2/HCO 3 − 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.