Characterization of acetylcholine-induced increases in cytosolic free calcium concentration in individual rat pancreatic beta-cells.
The intracellular free Ca2+ ion concentration ([Ca2+]i) was measured using fura-2 microspectrofluorimetry in individual rat pancreatic beta-cells prepared by enzymatic digestion and fluorescence-activated cell sorting. The mean basal concentration of [Ca2+]i in beta-cells in the presence of 4.4 mM glucose and 1.8 mM Ca2+ was 112 +/- 1.6 nM (n = 207). The action of acetylcholine (ACh) was concentration-dependent, and raising the concentration resulted in [Ca2+]i spikes of increasing amplitude and duration in some, but not all of the beta-cells. In addition, the beta-cells demonstrated variable sensitivity to ACh. The increases in [Ca2+]i were rapid, transient and were blocked by atropine at 10(-6) M. A brief exposure to 50 mM K+ resulted in a transient increase in [Ca2+]i similar to that induced by ACh, but resistant to atropine. A high concentration of ACh (100 microL 10(-4) M or 10(-3) M) induced [Ca2+]i oscillations in 11 out of 57 beta-cells in the presence of 4.4 mM glucose. Using calcium channel blockers and Ca2+ free medium, the source of the increase in [Ca2+]i was deduced to be from extra-cellular spaces. Changing the temperature from 22 to 37 degrees C did not affect the action of ACh on [Ca2+]i. These data strongly suggest that ACh exerted a direct action on [Ca2+]i in normal rat pancreatic beta-cells and support a role for Ca2+ as a second messenger in the action of ACh.
Wang
J
J
Dept. of Physiology, University of British Columbia, Vancouver, Canada.
Verchere
C B
CB
McIntosh
C H
CH
Brown
J C
JC
D000109:Acetylcholine / Q000008:administration & dosage / Q000494:pharmacology*; D000818:Animals; D001285:Atropine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D002448:Cell Adhesion; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D008297:Male; D009355:Neomycin / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013696:Temperature
D007328:Insulin; D001285:Atropine; D002217:Carbachol; D009355:Neomycin; D000109:Acetylcholine; D002118:Calcium