Ca transport by plasma membrane and intracellular stores of gastric cells.
The relative Ca transport activities (i.e., of both pumps and leaks) of carbachol-releasable intracellular stores and the basolateral plasma membrane of gastric parietal cells were studied using digital image processing of fura-2 fluorescence. Cells were treated with either carbachol (a cholinergic agonist) or thapsigargin (an inhibitor of microsomal Ca-adenosinetriphosphatase) or a combination of the two. Ca-free solutions were used to selectively investigate intracellular store release and plasma membrane pump activity, whereas Ca-containing solutions were used to investigate Ca influx and refilling of the intracellular pool. In the resting cell depletion of the intracellular pool in Ca-free solutions was 15-fold faster than control in the presence of thapsigargin, indicating the efficient (> 90%) recycling of leaked Ca by the store Ca pump. Stimulation with carbachol increased the rate of pool depletion by 70-fold, and this Ca flux out of the internal store was ten times larger than the flux across the plasma membrane. Thus the internal store has ten times greater fluxes (both leaks and pumps) than the plasma membrane during resting and stimulated conditions. After carbachol removal (i.e., reloading) the permeability of the internal store decreases, whereas increased influx across the plasma membrane persists until the store is refilled. Cytoplasmic Ca does not increase during refilling because the intracellular store pump operates eightfold faster than the plasma membrane pump, effectively sequestering Ca as quickly as it enters the cell.
Negulescu
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PA
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Machen
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D000818:Animals; D001285:Atropine / Q000494:pharmacology; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism*; D002469:Cell Separation / Q000379:methods; D016257:Fura-2; D005753:Gastric Mucosa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D066298:In Vitro Techniques; D007424:Intracellular Fluid / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D013997:Time Factors
D013729:Terpenes; D019284:Thapsigargin; D001285:Atropine; D002217:Carbachol; D000252:Calcium-Transporting ATPases; D002118:Calcium; D016257:Fura-2