title: Video imaging of cytosolic Ca2+ in pancreatic beta-cells stimulated by glucose, carbachol, and ATP.
abstract: In order to define the differences in the distribution of cytosolic free Ca2+ ([Ca2+]i) in pancreatic beta-cells stimulated with the fuel secretagogue glucose or the Ca(2+)-mobilizing agents carbachol and ATP, we applied digital video imaging to beta-cells loaded with fura-2.83% of the cells responded to glucose with an increase in [Ca2+]i after a latency of 117 +/- 24 s (mean +/- S.E., 85 cells). Of these cells, 16% showed slow wave oscillations (frequency 0.35/min). In order to assess the relationship between membrane potential and the distribution of the [Ca2+]i rise, digital image analysis and perforated patch-clamp methods were applied simultaneously. The system used allowed sufficient temporal resolution to visualize a subplasmalemmal Ca2+ transient due to a single glucose-induced action potential. Glucose could also elicit a slow depolarization which did not cause Ca2+ influx until the appearance of the first of a train of action potentials. [Ca2+]i rose progressively during spike firing. Inhibition of Ca2+ influx by EGTA abolished the glucose-induced rise in [Ca2+]i. In contrast, the peak amplitude of the [Ca2+]i response to carbachol was not significantly different in normal or in Ca(2+)-deprived medium. Occasionally, the increase of the [Ca2+]i rise was polarized to one area of the cell different from the subplasmalemmal rise caused by glucose. The amplitude of the response and the number of responding cells were significantly increased when carbachol was applied after the addition of high glucose (11.2 mM). ATP also raised [Ca2+]i and promoted both Ca2+ mobilization and Ca2+ influx. The intracellular distribution of [Ca2+]i was homogeneous during the onset of the response. A polarity in the [Ca2+]i distribution could be detected either in the descending phase of the peak or in subsequent peaks during [Ca2+]i oscillations caused by ATP. In the absence of extracellular Ca2+, the sequential application of ATP and carbachol revealed that carbachol was still able to raise [Ca2+]i after exhaustion of the ATP response. This may be due to desensitization to the former agonist, since the response occurred in the same area of the cell. These results reveal subtle differences in [Ca2+]i distribution following membrane depolarization with glucose or the application of Ca(2+)-mobilizing agonists.
authors.affiliation: Division de Biochimie Clinique, University of Geneva, Switzerland.
authors.forename: J M
authors.initials: JM
authors.lastname: Theler
authors.forename: P
authors.initials: P
authors.lastname: Mollard
authors.forename: N
authors.initials: N
authors.lastname: Guérineau
authors.forename: P
authors.initials: P
authors.lastname: Vacher
authors.forename: W F
authors.initials: WF
authors.lastname: Pralong
authors.forename: W
authors.initials: W
authors.lastname: Schlegel
authors.forename: C B
authors.initials: CB
authors.lastname: Wollheim
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology*; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D007700:Kinetics; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008856:Microscopy, Fluorescence / Q000379:methods; D051381:Rats; D011919:Rats, Inbred Strains; D014741:Video Recording / Q000379:methods
chemical_list: D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D002217:Carbachol; D005947:Glucose; D002118:Calcium