Intracellular alkalinization mobilizes calcium from agonist-sensitive pools in rat lacrimal acinar cells.
1. We have investigated interactions between intracellular pH (pHi) and the intracellular free calcium concentration ([Ca2+]i) in collagenase-isolated rat lacrimal acinar cells. The fluorescent dyes fura-2 and 2',7'-bis(carboxyethyl)-5-carboxyfluorescein (BCECF) were used to measure [Ca2+]i and pHi, respectively. 2. Application of the weak base NH4Cl alkalinized the cytosol and caused a dose-dependent increase in [Ca2+]i. Trimethylamine (TMA) also alkalinized the cytosol and increased [Ca2+]i. The increase in [Ca2+]i evoked by NH4Cl or TMA was much smaller than that evoked by the secretory agonist acetylcholine (ACh). 3. Application of NH4Cl also increased [Ca2+]i in cells bathed in Ca(2+)-free medium, indicating that NH4Cl released Ca2+ from an intracellular pool. 4. Ammonium chloride had no effect on [Ca2+]i in cells bathed in Ca(2+)-free medium if agonist-sensitive intracellular Ca2+ pools had been depleted with either ACh or the microsomal Ca(2+)-ATPase inhibitor 2,5-di(tert-butyl)hydroquinone. Treatment of cells with NH4Cl in Ca(2+)-free medium reduced the amount of Ca2+ released by ACh. These results suggest that NH4Cl released Ca2+ from the same intracellular pool released by ACh. 5. Calcium release from the agonist-sensitive pool was also triggered when the cytosol was alkalinized by removing the weak acid acetate. 6. Ammonium chloride caused a modest increase in inositol phosphate production, suggesting that NH4Cl may have released stored Ca2+ via an increase in the intracellular inositol 1,4,5-trisphosphate concentration. 7. The increase in [Ca2+]i evoked by NH4Cl was not sustained even in the presence of extracellular Ca2+. In contrast, when a low dose of ACh was used to evoke intracellular Ca2+ release of similar magnitude, sustained Ca2+ entry was observed. 8. Alkalinizing the cytosol appeared to partially inhibit Ca2+ entry triggered by thapsigargin or by ACh. 9. We suggest that alkalinizing the cytoplasm in unstimulated lacrimal acinar cells can release Ca2+ from the intracellular agonist-sensitive Ca2+ pool. However, releasing stored Ca2+ via alkalinization does not appear to trigger significant Ca2+ entry, perhaps because intracellular alkalinization inhibits either the Ca2+ entry pathway or the mechanism which couples the entry pathway to store depletion.
Yodozawa
S
S
Cell Physiology Group, School of Biological Sciences (G.38), University of Manchester, UK.
Speake
T
T
Elliott
A
A
D019342:Acetic Acid / Q000494:pharmacology; D000109:Acetylcholine / Q000494:pharmacology; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007765:Lacrimal Apparatus / Q000166:cytology / Q000378:metabolism*; D008744:Methylamines / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D019284:Thapsigargin / Q000494:pharmacology
D004791:Enzyme Inhibitors; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008744:Methylamines; D010716:Phosphatidylinositols; D000643:Ammonium Chloride; D019284:Thapsigargin; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; C023336:trimethylamine; D000109:Acetylcholine; D019342:Acetic Acid; D002118:Calcium; D016257:Fura-2