Detection of secretion from single pancreatic beta-cells using extracellular fluorogenic reactions and confocal fluorescence microscopy.
Confocal microscopy with Zinquin, a fluorogenic Zn(2+)-specific indicator, was used for spatially and temporally resolved measurement of Zn2+ efflux from single pancreatic beta-cells. When cells were incubated in buffer containing Zinquin, application of insulin secretagogues evoked an increase in fluorescence around the surface of the cell, indicative of detection of Zn2+ efflux from the cell. The fluorescence increases corresponded spatially and temporally with measurements of exocytosis obtained simultaneously by amperometry. When images were taken at 266-ms intervals, the detection limit for Zn2+ was approximately 0.5 microM. With this image frequency, it was possible to observe bursts of fluorescence which were interpreted as fluctuations of Zn2+ level due to exocytosis. The average intensity of these fluorescence bursts corresponded to a Zn2+ concentration of approximately 7 microM. Since insulin is co-stored with Zn2+ in secretory vesicles, it was concluded that the Zn2+ efflux corresponded to exocytosis of insulin/Zn(2+)-containing granules from the beta-cell. Exocytosis sites identified by this technique were frequently localized to one portion of the cell, indicative of active areas of release.
Qian
W J
WJ
Department of Chemistry, University of Florida, Gainesville 32611-7200, USA.
Aspinwall
C A
CA
Battiste
M A
MA
Kennedy
R T
RT
D000818:Animals; D002478:Cells, Cultured; D005089:Exocytosis; D005110:Extracellular Space / Q000737:chemistry; D005456:Fluorescent Dyes; D007424:Intracellular Fluid / Q000737:chemistry; D007515:Islets of Langerhans / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D015363:Quinolones; D014105:Tosyl Compounds; D015032:Zinc / Q000032:analysis* / Q000378:metabolism
D005456:Fluorescent Dyes; D015363:Quinolones; D014105:Tosyl Compounds; D015032:Zinc; C084731:zinquin