Microinjection of <I>myo</I>-Inositol(1,4,5)Trisphosphate and Other Calcium-Mobilizing Agents Into Intact Adherent Cells<U>

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.