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

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 (pHi)-regulation becomes important for coordinating cellular activity. We have investigated spatial pHi-regulation in single and paired ventricular myocytes from rat heart by inducing a localized intracellular acid-load, while confocally imaging pHi 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 pHi-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 pHi on intrinsic (non-CO2/HCO3− buffer-dependent) and extrinsic (CO2/HCO3− buffer-dependent) components of Hi+-mobility. We find that although both components mediate spatial regulation of pH within the cell, their ability to do so declines sharply at low pHi. Thus acidosis severely slows intracellular H+-ion movement. This can result in spatial pHi 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.