Microinjection of myo-Inositol(1,4,5)trisphosphate and Other Calcium-Mobilizing Agents into Intact Adherent Cells

Many receptor tyrosine kinases and seven-transmembrane receptors are directly coupled or coupled via G proteins, respectively, to the activation of phosphoinositidase Cs (1 ). 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)P₃ ] and diacylglycerol (1 ). Ins(1,4,5)P₃ interacts with a specific receptor that is a ligand-gated channel that allows mobilization of non-mitochondrial intracellular calcium (Ca²⁺ ) stores (1 ). Because Ins(1,4,5)P₃ is plasma membrane impermeant, this phenomenon was first demonstrated in permeabilized pancreatic acinar cells (2 ), and all subsequent studies in cells have involved introduction of Ins(1,4,5)P₃ by rendering a cell population permeable (3 ), using microinjection techniques (4 ), or by the presentation of chemically modified membrane-permeable Ins(1,4,5)P₃ analogs, such as photolabile “caged Ins(1,4,5)P₃ ” (5 ). An alternative approach involves disruption of the plasma membrane and preparation of microsomes from the intracellular vesicular Ca²⁺ stores (6 ,7 ); however, microsomal preparations exhibit a loss of Ins(1,4,5)P₃ responsiveness compared to permeabilized and intact cells.