Measurement of Ca2+ Fluxes in Permeabilized Cells Using 45Ca2+ and Fluo-3
Part of the
Methods in Molecular Biology™
book series (MIMB, volume 41)
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 45Ca45-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 45Ca2+-release assay was similar to those previously described (3, 8), and involves preloading a “spike” of 45Ca2+ into the intracellular Ca2+ stores of permeabilized cells and then monitoring the resultant release of 45Ca2+ 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.
KeywordsPermeabilized Cell Release Assay Inositol Polyphosphates Silicon Fluid Bovine Adrenal Chromaffin Cell
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
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