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

  • Robert A. Wilcox
  • James Strupish
  • Stefan R. Nahorski
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.


Permeabilized Cell Release Assay Inositol Polyphosphates Silicon Fluid Bovine Adrenal Chromaffin Cell 
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Copyright information

© Humana Press Inc , Totowa, NJ 1995

Authors and Affiliations

  • Robert A. Wilcox
    • 1
  • James Strupish
    • 2
  • Stefan R. Nahorski
    • 1
  1. 1.Department of Cell Physiology and PharmacologyUniversity of LeicesterUK
  2. 2.Department of AnaesthesiaUniversity of LeicesterUK

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