A miniaturized column chromatography method for measuring receptor-mediated inositol phosphate accumulation.
Inositol phosphates (IPs), such as 1,4,5-inositol-trisphosphate (IP(3)), comprise a ubiquitous intracellular signaling cascade initiated in response to G protein-coupled receptor-mediated activation of phospholipase C. Classical methods for measuring intracellular accumulation of these molecules include time-consuming high-performance liquid chromatography (HPLC) separation or large-volume, gravity-fed anion-exchange column chromatography. More recent approaches, such as radio-receptor and AlphaScreen assays, offer higher throughput. However, these techniques rely on measurement of IP(3) itself, rather than its accumulation with other downstream IPs, and often suffer from poor signal-to-noise ratios due to the transient nature of IP(3). The authors have developed a miniaturized, anion-exchange chromatography method for measuring inositol phosphate accumulation in cells that takes advantage of signal amplification achieved through measuring IP(3) and downstream IPs. This assay uses centrifugation of 96-well-formatted anion-exchange mini-columns for the isolation of radiolabeled inositol phosphates from cell extracts, followed by low-background dry-scintillation counting. This improved assay method measures receptor-mediated IP accumulation with signal-to-noise and pharmacological values comparable to the classical large-volume, column-based methods. Assay validation data for recombinant muscarinic receptor 1, galanin receptor 2, and rat astrocyte metabotropic glutamate receptor 5 are presented. This miniaturized protocol reduces reagent usage and assay time as compared to large-column methods and is compatible with standard 96-well scintillation counters.
Benjamin
Elfrida R
ER
Purdue Pharma Discovery Research, 6 Cedarbrook Drive, Cranbury, NJ 08512, USA. Elfrida.Benjamin@pharma.com
Haftl
Sarah L
SL
Xanthos
Dimitris N
DN
Crumley
Gregg
G
Hachicha
Mohamed
M
Valenzano
Kenneth J
KJ
D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D002852:Chromatography, Ion Exchange / Q000295:instrumentation / Q000379:methods*; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate; D007295:Inositol Phosphates / Q000032:analysis / Q000378:metabolism*; D008904:Miniaturization / Q000295:instrumentation / Q000379:methods; D051381:Rats; D044092:Receptor, Galanin, Type 2 / Q000235:genetics / Q000378:metabolism; D064529:Receptor, Metabotropic Glutamate 5; D043643:Receptor, Muscarinic M1 / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D018094:Receptors, Metabotropic Glutamate / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
D018076:DNA, Complementary; D007295:Inositol Phosphates; D044092:Receptor, Galanin, Type 2; D064529:Receptor, Metabotropic Glutamate 5; D043643:Receptor, Muscarinic M1; D043562:Receptors, G-Protein-Coupled; D018094:Receptors, Metabotropic Glutamate; D011994:Recombinant Proteins; D015544:Inositol 1,4,5-Trisphosphate