Modulation of calcium signals by intracellular pH in isolated rat pancreatic acinar cells

Abstract

We have investigated the interactions between intracellular pH (pH_i) and the intracellular free calcium concentration ([Ca²⁺]_i) in isolated rat pancreatic acinar cells. The fluorescent dyes fura-2 and BCECF were used to measure [Ca²⁺]_i and pH_i, respectively. Sodium acetate and ammonium chloride (NH₄Cl) were used to acidify and alkalinize pH_i, respectively. Cytosolic acidification had no effect on [Ca²⁺]_i in resting pancreatic acinar cells, whereas cytosolic alkalinization released Ca²⁺ from intracellular stores. Cytosolic acidification using either acetate or a CO₂-HCO₃⁻-buffered medium enhanced Ca²⁺ signals evoked by acetylcholine (ACh) and cholecystokinin (CCK). In contrast, both NH₄Cl and trimethylamine (TMA) inhibited Ca²⁺ signals during stimulation with either ACh or CCK. This inhibitory effect was also observed in the absence of extracellular Ca²⁺, and was therefore not due to changes in Ca²⁺ entry. Calcium oscillations evoked by physiological concentrations of CCK were enhanced by cytosolic acidification and inhibited by cytosolic alkalinization. In order to determine the effects of pH_i upon Ca²⁺ handling by intracellular Ca²⁺ stores, intraorganellar [Ca²⁺] was monitored using the low affinity Ca²⁺ indicator mag-fura-2 in permeabilized cells. Addition of NH₄Cl, which is expected to alkalinize intraorganellar pH, did not alter intraorganellar [Ca²⁺] in permeabilized cells, suggesting that changing intraorganellar pH does not release Ca²⁺ from intracellular stores. Addition of NH₄Cl or acetate also did not affect the rate of Ca²⁺ release induced by inositol 1,4,5-trisphosphate (InsP₃). Modification of extraorganellar (‘cytosolic’) pH did not affect the rate of ATP-dependent Ca²⁺ uptake into stores, but did modify the rate of Ca²⁺ release evoked by submaximal concentrations of InsP₃. The rate of Ca²⁺ release was increased at more alkaline extraorganellar pHs. These results would suggest that manipulation of intraorganellar pH does not affect Ca²⁺ handling by the intracellular stores. In contrast, extraorganellar (‘cytosolic’) pH does affect InsP₃-induced Ca²⁺ release from the stores. In conclusion, changes in intracellular pH in pancreatic acinar cells can profoundly alter cytosolic [Ca²⁺]. This may shed light on earlier observations whereby cell-permeant weak acids and bases can modulate fluid secretion in epithelia.