Uptake and intracellular sequestration of divalent cations in resting and methacholine-stimulated mouse lacrimal acinar cells. Dissociation by Sr2+ and Ba2+ of agonist-stimulated divalent cation entry from the refilling of the agonist-sensitive intracellular pool.
The abilities of various divalent cations to enter the cytoplasm of mouse lacrimal acinar cells was examined under resting and agonist-stimulated conditions, by monitoring their effects on the fluorescence of cytosolic fura-2. In vitro, Ni2+, Co2+, and Mn2+ quenched the fura-2 fluorescence, whereas Sr2+, Ba2+, and La3+ produced an excitation spectrum and maximum brightness similar to Ca2+. Stimulation of mouse lacrimal acinar cells with methacholine (MeCh) caused a biphasic elevation of intracellular Ca2+ concentration [( Ca2+]i) resulting from a release of Ca2+ from intracellular pools followed by a sustained entry of extracellular Ca2+. Neither La3+ nor Ni2+ entered the cells under resting or stimulated conditions, but both blocked Ca2+ entry. Although both Co2+ and Mn2+ entered unstimulated cells, this process was not increased by MeCh. Both Sr2+ and Ba2+ were capable of supporting a sustained increase in fura-2 fluorescence in response to MeCh, indicating that these cations can enter the cells through the agonist-regulated channels. However, Sr2+, but not Ba2+, was capable of refilling the agonist-sensitive intracellular stores. These findings demonstrate dissociation of agonist-induced Ca2+ entry from intracellular Ca2+ pool refilling and thereby provide strong support for the recently modified version of the capacitative Ca2+ entry model according to which influx into the cytoplasm occurs directly across the plasma membrane and does not require a specialized cation channel directly linking the extracellular space and the intracellular Ca2+ stores.
Calcium Regulation Section, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
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Kwan
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JW
Putney
D000818:Animals; D001285:Atropine / Q000494:pharmacology; D001572:Benzofurans; D002413:Cations, Divalent / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D005456:Fluorescent Dyes; D016257:Fura-2; D007765:Lacrimal Apparatus / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016210:Methacholine Chloride; D008688:Methacholine Compounds / Q000494:pharmacology*; D051379:Mice; D013050:Spectrometry, Fluorescence
D001572:Benzofurans; D002413:Cations, Divalent; D005456:Fluorescent Dyes; D008688:Methacholine Compounds; D016210:Methacholine Chloride; D001285:Atropine; D016257:Fura-2