title: Analyzing Ca(2+) dynamics in intact epithelial cells using spatially limited flash photolysis.
abstract: The production of saliva by parotid acinar cells is stimulated by Ca(2+) activation of Cl(-) and K(+) channels located in the apical plasma membrane of these polarized cells. Here we describe a paradigm for the focal photorelease of either Ca(2+) or an inositol 1,4,5 trisphosphate (InsP(3)) analog. The protocol is designed to be useful for investigating subcellular Ca(2+) dynamics in polarized cells with minimal experimental intervention. Parotid acinar cells are loaded with cell-permeable versions of the caged precursors (NP-EGTA-AM or Ci-InsP(3)/PM). Photolysis is accomplished using a spatially limited, focused diode laser, but the experiment can be readily modified to whole-field photolysis using a xenon flash lamp.
authors.affiliation: Department of Pharmacology and Physiology, University of Rochester, Rochester, NY 14642, USA.
authors.forename: Janos
authors.initials: J
authors.lastname: Almassy
authors.forename: David I
authors.initials: DI
authors.lastname: Yule
mesh_terms: D061354:Acinar Cells / Q000502:physiology* / Q000528:radiation effects; D000818:Animals; D002118:Calcium / Q000032:analysis*; D002478:Cells, Cultured; D003584:Cytological Techniques / Q000379:methods*; D015544:Inositol 1,4,5-Trisphosphate / Q000032:analysis*; D007473:Ion Channels / Q000378:metabolism* / Q000528:radiation effects; D008027:Light; D051379:Mice; D010782:Photolysis; D014466:Ultraviolet Rays
chemical_list: D007473:Ion Channels; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium