Physiological localization of an agonist-sensitive pool of Ca2+ in parotid acinar cells.
Muscarinic stimulation of fluid secretion by mammalian salivary acinar cells is associated with a rise in the level of intracellular free calcium ([Ca2+]i) and activation of a calcium-sensitive potassium (K+) conductance in the basolateral membrane. To test in the intact cell whether the rise of [Ca2+]i precedes activation of the K+ conductance (as expected if Ca2+ is the intracellular messenger mediating this response), [Ca2+]i and membrane voltage were measured simultaneously in carbachol-stimulated rat parotid acinar cells by using fura-2 and an intracellular microelectrode. Unexpectedly, the cells hyperpolarize (indicating activation of the K+ conductance) before fura-2 detectable [Ca2+]i begins to rise. This occurs even in Ca2+-depleted medium where intracellular stores are the only source of mobilized Ca2+. Nevertheless, when the increase in [Ca2+]i was eliminated by loading cells with the Ca2+ chelator bis(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetate (Me2BAPTA) and stimulating in Ca2+-depleted medium, membrane hyperpolarization was also eliminated, indicating that a rise of [Ca2+] is required for the agonist-induced voltage response. Stimulation of Me2BAPTA-loaded cells in Ca2+-containing medium dramatically accentuates the temporal dissociation between the activation of the K+ conductance and the rise of [Ca2+]i. The data are consistent with the hypothesis that muscarinic stimulation results in a rapid localized increase in [Ca2+]i at the acinar basolateral membrane followed by a somewhat delayed increase in total [Ca2+]i. The localized increase cannot be detected by fura-2 but is sufficient to open the Ca2+-sensitive K+ channels located in the basolateral membrane. We concluded that a receptor-mobilized intracellular store of Ca2+ is localized at or near the basolateral membrane.
Foskett
J K
JK
Physiology Department, Armed Forces Radiobiology Research Institute, Bethesda, MD 20814.
Gunter-Smith
P J
PJ
Melvin
J E
JE
Turner
R J
RJ
D000818:Animals; D001572:Benzofurans; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D002449:Cell Aggregation; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005456:Fluorescent Dyes; D016257:Fura-2; D066298:In Vitro Techniques; D008564:Membrane Potentials / Q000187:drug effects; D010306:Parotid Gland / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013050:Spectrometry, Fluorescence
D001572:Benzofurans; D002614:Chelating Agents; D005456:Fluorescent Dyes; D004533:Egtazic Acid; D002217:Carbachol; C058180:5,5'-dimethyl-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium; D016257:Fura-2