Generation of intracellular pH gradients in single cardiac myocytes with a microperfusion system

This study describes the use of a microperfusion system to create rapid, large regional changes in intracellular pH (pH(i)) within single ventricular myocytes. The spatial distribution of pH(i) in single myocytes was measured with seminaphthorhodafluor-1 fluorescence using confocal imaging. Changes in pH(i) were induced by local external application of NH(4)Cl, CO(2), or sodium propionate. Local application was achieved by simultaneously directing two parallel square microstreams, each 275 microm wide, over a single myocyte oriented perpendicular to the direction of flow. One stream contained the control solution, and the other contained a weak acid or base. End-to-end, stable pH(i) gradients as large as 1 pH unit were readily created with this technique. This result indicates that pH within a single cardiac cell may not always be spatially uniform, particularly when weak acid or base gradients are present, which can occur, for example, in regional myocardial ischemia. The microperfusion method should be useful for studying the effects of localized acidosis on myocyte function, estimating intracellular ion diffusion rates, and, possibly, inducing regional changes in other important intracellular ions.