Functional homogeneity of the non-mitochondrial Ca2+ pool in intact mouse lacrimal acinar cells.
In the absence of extracellular Ca2+, treatment of mouse lacrimal acinar cells with maximal concentrations of methacholine released Ca2+ from intracellular stores. No additional Ca2+ was mobilized by subsequent application of the intracellular Ca(2+)-ATPase inhibitor, thapsigargin, the stable inositol 1,4,5-trisphosphate ((1,4,5)IP3) analog, inositol 2,4,5-trisphosphate ((2,4,5)IP3) (by microinjection), or the Ca2+ ionophore, ionomycin. However, following prolonged activation of cells by methacholine in the presence of extracellular Ca2+, Ca2+ accumulated into a pool which was released by ionomycin but not by thapsigargin. This latter accumulation was blocked by prior microinjection of ruthenium red, indicating that it represents mitochondrial uptake. In saponin-permeabilized lacrimal cells, two Ca(2+)-sequestering pools were detected: (i) a ruthenium red-sensitive, thapsigargin-insensitive pool, presumed to be the mitochondria; and (ii) a ruthenium red-insensitive, thapsigargin-sensitive pool. Only the thapsigargin-sensitive pool accumulated Ca2+ at concentrations similar to those in unstimulated cells. The thapsigargin-sensitive Ca2+ pool was sensitive to (1,4,5)IP3; however, in contrast to findings in intact cells, only 44% of this pool was releasable by (1,4,5)IP3 or (2,4,5)IP3. These data indicate that, in intact lacrimal acinar cells, all exchangeable (ionomycin-sensitive) Ca2+ residues in a pool which responds homogeneously to agonists, (1,4,5)IP3, and thapsigargin. Prolonged elevation of [Ca2+]i results in Ca2+ accumulation into a second, ruthenium red-sensitive pool, presumably mitochondria. Finally, permeabilization of the cells fragments the non-mitochondrial pool, resulting in two pools, one sensitive and one insensitive to (1,4,5)IP3.
Bird
G J
GJ
Calcium Regulation Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709.
Obie
J F
JF
Putney
J W
JW
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent; D002478:Cells, Cultured; D005453:Fluorescence; D016257:Fura-2; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D007765:Lacrimal Apparatus / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D016210:Methacholine Chloride / Q000494:pharmacology; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D012430:Ruthenium Red / Q000737:chemistry; D012503:Saponins / Q000737:chemistry; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin
D002413:Cations, Divalent; D007295:Inositol Phosphates; D012503:Saponins; D013729:Terpenes; D016210:Methacholine Chloride; D012430:Ruthenium Red; D015759:Ionomycin; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; C062616:inositol 2,4,5-trisphosphate; D002118:Calcium; D016257:Fura-2