Imaging of Zn2+ release from pancreatic beta-cells at the level of single exocytotic events.
Regulated secretion of Zn2+ from isolated pancreatic beta-cells was imaged using laser-scanning confocal microscopy. In the method, beta-cells were incubated in a solution containing the novel fluorescent Zn2+ indicator FluoZin-3. Zn2+ released from the cells reacted with the dye to form a fluorescent product, which was detected by the confocal microscope. The new dye is much brighter than Zinquin, previously used for this application, allowing detection limits of 10-40 nM and temporal resolution of 16 ms/image. The high temporal resolution allowed imaging of isolated fluorescent transients that occurred at the edge of the cells following stimulation with 20 mM glucose or 40 mM K+. Fluorescent transients took 16-50 ms to reach a peak from the initial rise and returned to baseline after 170 +/- 50 ms (n = 78 transients from 15 cells). It was concluded that the transients correspond to detection of exocytotic release of Zn2+. Analysis of the temporal and spatial dispersion of the transients indicates that the release of Zn2+ is not diffusion limited but is instead kinetically controlled in agreement with previous observations of insulin release detected by amperometry.
Qian
Wei-Jun
WJ
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
Gee
Kyle R
KR
Kennedy
Robert T
RT
D000818:Animals; D005089:Exocytosis; D007202:Indicators and Reagents; D007515:Islets of Langerhans / Q000378:metabolism* / Q000648:ultrastructure; D051379:Mice; D018613:Microscopy, Confocal; D011083:Polycyclic Compounds; D015032:Zinc / Q000737:chemistry / Q000378:metabolism*
C451182:FluoZin-3; D007202:Indicators and Reagents; D011083:Polycyclic Compounds; D015032:Zinc