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 (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.
Swietach
Pawel
P
Burdon Sanderson Cardiac Science Centre, Department of Physiology, Anatomy and Genetics, Oxford University, Oxford, United Kingdom.
Spitzer
Kenneth W
KW
Vaughan-Jones
Richard D
RD
D000818:Animals; D002478:Cells, Cultured; D006352:Heart Ventricles / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D032383:Myocytes, Cardiac / Q000378:metabolism*; D010782:Photolysis; D011522:Protons; D051381:Rats; D017207:Rats, Sprague-Dawley
D011522:Protons