Regulation of cytosolic free calcium in fura-2-loaded rat parotid acinar cells.
In order to analyze the factors regulating agonist-stimulated Ca2+ mobilization, cytosolic free [Ca2+] ([Ca2+]i) was measured directly in fura-2-loaded rat parotid acinar cells. Stimulation of muscarinic receptors by carbachol produced a dose-dependent rise in [Ca2+]i. In the presence of external Ca2+, the initial transient rise was followed by a maintained elevation. The maintained elevation is dependent on the presence of external Ca2+. Removal of Ca2+ by addition of EGTA caused a rapid decline in [Ca2+]i back to base line. In the absence of external Ca2+, only an initial transient peak in [Ca2+]i was seen which then declined to base line; the maintained elevation in [Ca2+]i could then be evoked by addition of Ca2+ in the continued presence of carbachol. Muscarinic receptor occupation by carbachol is required to maintain the elevated level of [Ca2+]i; addition of the muscarinic antagonist, atropine, caused [Ca2+]i to decline back to the basal level. The maintained elevation in [Ca2+]i, but not the initial transient peak, can also be blocked by Ni2+ but was unaffected by the organic Ca2+ antagonists. Total substitution of external Na+ with the impermeant cation, N-methyl-D-glucamine, had no effect on either the initial or the maintained response to carbachol; however, total substitution of Na+ with K+ attenuated the maintained response while not affecting the initial peak. Refilling of the intracellular Ca2+ store was also studied and found to take place in the absence of agonist and with no substantial elevation in [Ca2+]i. These experiments also showed that not all of the intracellular vesicular Ca2+ stores can be released by agonists. From these results, we propose a model for the regulation of [Ca2+]i.
Merritt
J E
JE
Department of Cellular Pharmacology, Smith Kline and French Research Ltd., Welwyn, Herts, United Kingdom.
Rink
T J
TJ
D000535:Aluminum / Q000494:pharmacology; D017607:Aluminum Compounds; D000818:Animals; D001285:Atropine / Q000494:pharmacology; D001572:Benzofurans; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism*; D005456:Fluorescent Dyes; D005459:Fluorides / Q000494:pharmacology; D016257:Fura-2 / Q000031:analogs & derivatives*; D008297:Male; D009532:Nickel / Q000494:pharmacology; D010306:Parotid Gland / Q000166:cytology*; D051381:Rats; D011919:Rats, Inbred Strains
D017607:Aluminum Compounds; D001572:Benzofurans; D005456:Fluorescent Dyes; C049925:fura-2-am; D001285:Atropine; D009532:Nickel; D002217:Carbachol; D000535:Aluminum; D005459:Fluorides; D002118:Calcium; D016257:Fura-2; C032311:aluminum fluoride