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.
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
Wei-Jun
WJ
Qian
Kyle R
KR
Gee
Robert T
RT
Kennedy
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