title: Microinjection of myo-inositol(1,4,5)trisphosphate and other calcium-mobilizing agents into intact adherent cells.
abstract: Many receptor tyrosine kinases and seven-transmembrane receptors are directly coupled or coupled via G proteins, respectively, to the activation of phosphoinositidase Cs. These enzymes catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce the second messengers, myo-inositol(1,4,5)trisphosphate [Ins(1,4,5)P(3)] and diacylglycerol. Ins(1,4,5)P(3) interacts with a specific receptor that is a ligand-gated channel that allows mobilization of non-mitochondrial intracellular calcium (Ca(2+)) stores. Because Ins(1,4,5)P(3) is plasma membrane impermeant, this phenomenon was first demonstrated in permeabilized pancreatic acinar cells, and all subsequent studies in cells have involved introduction of Ins(1,4,5)P(3) by rendering a cell population permeable (3), 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)". An alternative approach involves disruption of the plasma membrane and preparation of microsomes from the intracellular vesicular Ca(2+) stores; however, microsomal preparations exhibit a loss of Ins(1,4,5)P(3) responsiveness compared to permeabilized and intact cells.
authors.affiliation: Department of Neurology, Flinders Medical Centre, Bedford Park, Adelaide, Australia.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Wilcox
authors.forename: Ian D
authors.initials: ID
authors.lastname: Forsythe
authors.forename: Terence J
authors.initials: TJ
authors.lastname: McCann
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002448:Cell Adhesion; D002460:Cell Line; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000008:administration & dosage* / Q000031:analogs & derivatives / Q000378:metabolism; D007739:L Cells; D051379:Mice; D008845:Microinjections / Q000379:methods*; D008858:Microscopy, Phase-Contrast / Q000379:methods*; D010006:Osteoblasts / Q000378:metabolism*; D011122:Polyphosphates / Q000008:administration & dosage / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D011122:Polyphosphates; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium