Detection and imaging of zinc secretion from pancreatic beta-cells using a new fluorescent zinc indicator.
A novel Zn2+-selective visible wavelength fluoroionophore (FluoZin-3, 9) was synthesized. The chelating portion of the molecule resembles known EGTA-based Ca2+-selective fluoroionophores, except that one of the N-acetic acid moieties has been deleted in 9. FluoZin-3 is virtually non-fluorescent in the absence of Zn2+, and exhibits a several hundred-fold fluorescence increase upon saturation with Zn2+( approximately 100 nM), with a Kd = 15 +/- 2 nM. A 1:1 binding stoichiometry of 9:Zn2+ was determined, and the fluorescence of the complex is pH-independent at pH > 6. FluoZin-3 was used to monitor Zn2+ that was co-secreted with insulin from pancreatic beta-cells by exocytosis following stimulation with glucose. The total Zn2+ concentration near the cells reached 600 nM, and Zn2+ was detectable at least 15 mum away from secreting cells. Heterogeneity in secretion among cells was indicated in that some cells in a cluster did not release Zn2+. Also, within secreting cells some regions of the cell membrane gave rise to secretion while others did not, suggesting active zones of secretion on the cell surface.
Gee
Kyle R
KR
Molecular Probes, Inc., 4849 Pitchford Avenue, Eugene, Oregon 97402, USA.
Zhou
Zhang-Lin
ZL
Qian
Wei-Jun
WJ
Kennedy
Robert
R
D002413:Cations, Divalent; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D007515:Islets of Langerhans / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D018613:Microscopy, Confocal; D011083:Polycyclic Compounds / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D015032:Zinc / Q000032:analysis / Q000737:chemistry / Q000502:physiology*
D002413:Cations, Divalent; C451182:FluoZin-3; D005456:Fluorescent Dyes; D011083:Polycyclic Compounds; D015032:Zinc