Measurement of Ca2+ Fluxes in Permeabilized Cells Using 45 Ca2+ and Fluo-3

Many cell-surface receptors via G-proteins activate phosphoinositidase C, which catalyzes the hydrolysis of phosphatidylinositol 4,5-bis-phosphate to produce the second messengers, myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃ or IP₃ ] and diacylglycerol (1 ). Ins(1,4,5)P₃ interacts with a specific receptor populations of ligand-gated channels to mobilize nonmitochondrial intracellular 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(l,4,5)P₃ analogs, such as photolabile “caged-IP₃ ” (5 ). An alternative approach involves disruption of the plasma membrane and preparation of microsomes from the intracellular vesicular Ca²⁺ stores (6 , 7 ). However, these preparations exhibit a loss of Ins(1,4,5)P₃ -responsiveness compared to cells. Here we describe a ⁴⁵ Ca⁴⁵ -release assay and a fluo-3 assay, two methods we use to monitor Ins(l,4,5)P₃ -induced Ca²⁺ mobilization from nonmitochondrial intracellular Ca²⁺ stores using cytosol-like buffer (CLB) and permeabilized SH-SY5Y neuroblastoma cell populations. The ⁴⁵ Ca²⁺ -release assay was similar to those previously described (3 , 8 ), and involves preloading a “spike” of ⁴⁵ Ca²⁺ into the intracellular Ca²⁺ stores of permeabilized cells and then monitoring the resultant release of ⁴⁵ Ca²⁺ induced by concentrations of Ins(1,4,5)P₃ and other agonists. We have utilized this assay to assess the intrinsic activity of Ins(1,4,5)P₃ a wide range of Ins(l,4,5)P₃ analogs, and other Ca²⁺ -mobilizing agents. The assay can be undertaken at a low cell density to minimize cellular metabolism of the inositol polyphosphates, or at high cell density to allow metabolic time-courses to be associated with Ca²⁺ mobilization.