Relationship between intracellular pH and proton mobility in rat and guinea‐pig ventricular myocytes

Intracellular H + ion mobility in eukaryotic cells is low because of intracellular buffering. We have investigated whether H i + mobility varies with pH i . A dual microperfusion apparatus was used to expose guinea‐pig or rat myocytes to small localized doses (3–5 m m ) of ammonium chloride (applied in Hepes‐buffered solution). Intracellular pH (pH i ) was monitored confocally using the fluorescent dye, carboxy‐SNARF‐1. Local ammonium exposure produced a stable, longitudinal pH i gradient. Its size was fed into a look‐up table (LUT) to give an estimate of the apparent intracellular proton diffusion coefficient ( D app H ). LUTs were generated using a diffusion–reaction model of H i + mobility based on intracellular buffer diffusion. To examine the pH i sensitivity of D app H , whole‐cell pH i was initially displaced using a whole‐cell ammonium or acetate prepulse, before locally applying the low dose of ammonium. In both rat and guinea‐pig, D app H decreased with pH i over the range 7.5–6.5. In separate pipette‐loading experiments, the intracellular diffusion coefficient for carboxy‐SNARF‐1 (a mobile‐buffer analogue) exhibited no significant pH i dependence. The pH i sensitivity of D app H is thus likely to be governed by the mobile fraction of intrinsic buffering capacity. These results reinforce the buffer hypothesis of H i + mobility. The pH i dependence of D app H was used to characterize the mobile and fixed buffer components, and to estimate D mob (the average diffusion coefficient for intracellular mobile buffer). One consequence of a decline in H i + mobility at low pH i is that it will predispose the myocardium to pH i nonuniformity. The physiological relevance of this is discussed.