title: Measurement of calcium fluxes in permeabilized cells using a ⁴⁵Ca²+ uptake and release assay.
abstract: Many cell surface receptors activate phosphoinositidase(s) C, via G proteins that catalyze the hydrolysis of phosphatidylinositol 4,5-biphosphate to produce the second messengers, inositol(1,4,5)trisphosphate [Ins(1,4,5)P(3)] and diacylglycerol. Ins(1,4,5)P(3) interacts with specific receptor populations of ligand-gated channels to mobilize nonmitochondrial intracellular calcium (Ca(2+)) stores. Because Ins(1,4,5)P(3) is very hydrophilic, it cannot readily cross the intact plasma membrane. Consequently, Ins(1,4,5)P(3)-induced Ca(2+) release was initially demonstrated in permeabilized pancreatic acinar cells, and all subsequent studies in cells have involved the introduction of Ins(1,4,5)P(3) by rendering a cell population permeable, using microinjection techniques or by the presentation of chemically modified membrane-permeable Ins(1,4,5)P(3) analogs, such as photolabile "caged Ins(1,4,5)P(3)" (5). An alternative approach involves disruption of the plasma membrane and preparation of microsomes from the intracellular vesicular Ca(2+) stores, however, these preparations exhibit a loss of Ins(1,4,5)P(3) responsiveness compared to cells. The author will describe a (45)Ca(2+)-release assay used to monitor Ins(1,4,5)P(3)-induced Ca(2+) mobilization from nonmitochondrial intracellular Ca(2+) stores using "cytosol-like" buffer (CLB) and permeabilized SH-SY5Y neuroblastoma cell populations.
authors.affiliation: Department of Neurology, Flinders Medical Centre, Bedford Park, Adelaide, Australia.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Wilcox
mesh_terms: D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D002132:Calcium Radioisotopes / Q000378:metabolism*; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism / Q000494:pharmacology*; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism; D009447:Neuroblastoma
chemical_list: D015220:Calcium Channels; D002132:Calcium Radioisotopes; D053496:Inositol 1,4,5-Trisphosphate Receptors; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium