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

Abstract

We have investigated the interactions between intracellular pH (pHi) 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 pHi, respectively. Sodium acetate and ammonium chloride (NH4Cl) were used to acidify and alkalinize pHi, 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 pHi 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.

Interdependence of Ca2+ and proton movements in trout hepatocytes

“…] i changes in response to pH e changes Based on previous studies in trout hepatocytes (Walsh, 1986;Krumschnabel et al, 2001b) e , which is consistent with observations in smooth muscle cells (Siskind et al, 1989), rat pheochromocytoma cells (Dickens et al, 1989), bovine lactotrophs (Zorec et al, 1993), rat lacrimal (Yodozawa et al, 1997) and pancreatic acinar cells (Speake and Elliott, 1998) but not with those in rat lymphocytes (Grinstein and Goetz, 1985) ] i (Grinstein and Goetz, 1985 e inhibited this mechanism, resulting in a slower pHi recovery (Fig.·6A). Looking at the peak of pHi increase in response to NH 4 Cl exposure, the difference between the control treatment, on the one hand, and the absence of Ca e was recorded in rat pheochromocytoma cells (Dickens et al, 1989) and vascular smooth muscle (Dickens et al, 1989;Batlle et al, 1993) while no effect on [Ca 2+ ] i has been reported in toxoplasma gondii tachyzoites (Moreno and Zhong, 1996) and rat pancreatic acinar cells (Speake and Elliott, 1998).…”

“…Looking at the peak of pHi increase in response to NH 4 Cl exposure, the difference between the control treatment, on the one hand, and the absence of Ca e was recorded in rat pheochromocytoma cells (Dickens et al, 1989) and vascular smooth muscle (Dickens et al, 1989;Batlle et al, 1993) while no effect on [Ca 2+ ] i has been reported in toxoplasma gondii tachyzoites (Moreno and Zhong, 1996) and rat pancreatic acinar cells (Speake and Elliott, 1998). In the present study, exposure of trout hepatocytes to the weak acid Na-propionate induced a slow increase in [Ca 2+ ] i .…”

Novel Paraoxonase 2-Dependent Mechanism Mediating the Biological Effects of the Pseudomonas aeruginosa Quorum-Sensing Molecule N -(3-Oxo-Dodecanoyl)- l -Homoserine Lactone

“…Therefore, we directly visualized the change in pH i in naive (nontransfected) EA.hy cells after 3OC12 treatment by laser scanning confocal microscopy. Because decreased pH i can induce Ca 2ϩ release and because increased cytosolic Ca 2ϩ [(Ca 2ϩ ) c ] is a common response to 3OC12 in mammalian cells (8,34), we also concomitantly measured [Ca 2ϩ ] c fluxes. The cells were simultaneously loaded with the Ca 2ϩ indicator Fluo-4 AM and the ratiometric pH indicator SNARF-4F and treated with 3OC12, and time-lapse images were acquired.…”

Molecular and Functional Identification of a Ca2+ (Polyvalent Cation)-sensing Receptor in Rat Pancreas

“…Acinar Cell Isolation-Small clusters of rat pancreatic acinar cells were isolated by enzymatic digestion of the pancreas with collagenase as described previously (13,14), except that all media contained a reduced concentration of CaCl 2 (0.5 mM). This value, which is less than half the physiological concentration, was selected to avoid possible activation of CaRs, which is known to occur at [Ca 2ϩ ] concentrations of 1 mM and above (2).…”