Assay of Receptor‐Stimulated Phosphoinositide Turnover

David A. Kendall1, Stephen P.H. Alexander1

1 University of Nottingham Medical School, Nottingham
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 2.7
DOI:  10.1002/0471141755.ph0207s30
Online Posting Date:  October, 2005
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Abstract

The stimulation of phosphoinositide turnover is one of the key means by which receptors evoke responses in target cells and tissues. This is true for both G protein‐coupled receptors and receptors that couple via tyrosine kinase activity. The protocols in this unit allow for pharmacological analysis of receptors coupled to phosphoinositide turnover. In general, the [3H]myo‐inositol prelabeling methodology (described for both tissue slices and cultured cells) is the more widely applicable, since it requires fewer experimental steps and typically gives rise to a better signal‐to‐noise ratio. Individual inositol phosphates can also be determined as described by chromatographic separation on ion‐exchange columns. In some circumstances (for example, when rapid responses to receptor stimulation are to be investigated or when the absolute levels of the active inositol phosphate are to be examined), it is preferable to use the mass assay described here for inositol (1,4,5)‐trisphosphate from either tissue slices and cultured cells. This unit also provides support protocols for the preparation of [3H]myo‐inositol, chromatography columns, tissue slices, and the IP3‐binding protein.

     
 
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Table of Contents

  • Basic Protocol 1: Incorporation of [3H]Inositol into Phospholipids in Tissue Slices and Measurement of [3H]Inositol Phosphates
  • Alternate Protocol 1: Separation of Individual [3H]Inositol Polyphosphates
  • Basic Protocol 2: [3H]MYO‐Inositol Prelabeling Assay in Cultured Cells
  • Support Protocol 1: Purification of [3H]MYO‐Inositol
  • Support Protocol 2: Preparation of Chloride‐Form Dowex AG1X8 Columns
  • Support Protocol 3: Preparation of Formate‐Form Dowex AG1X8 Columns
  • Basic Protocol 3: Estimation of Inositol (1,4,5)‐Trisphosphate in Tissue Slices by Mass Assay
  • Support Protocol 4: Preparation of Tissue Slices
  • Alternate Protocol 2: Inositol (1,4,5)‐Trisphosphate Mass Assay in Cultured Cells
  • Support Protocol 5: Preparation of Inositol Trisphosphate–Binding Protein
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Incorporation of [3H]Inositol into Phospholipids in Tissue Slices and Measurement of [3H]Inositol Phosphates

  Materials
  • Purified [2‐3H]myo‐inositol (see protocol 4)
  • 150 mM LiCl (e.g., Fisher)
  • KHB (see recipe)
  • Chopped tissue slices, pre‐equilibrated with recipeKHB for 60 min (see protocol 8)
  • 95% (v/v) O 2/5% CO 2 (carbogen)
  • Test agonists, antagonists in recipeKHB or distilled water
  • 1.2 M perchloric acid (PCA), ice cold
  • 150 mM KOH
  • 50 mM Tris⋅Cl buffer (pH 7.4 at 20°C; appendix 2A)
  • Dowex AG1X8 columns, chloride form (see protocol 5)
  • 1 M HCl
  • Liquid scintillation fluid
  • 1:2 (v/v) chloroform (CHCl 3)/methanol (stored at room temperature in a stoppered glass bottle)
  • Chloroform
  • 5‐ to 6‐ml flat‐bottom vials (e.g., Hughes & Hughes)
  • Repeating pipettor and pipet tip with ∼3‐mm diameter
  • 20‐ml scintillation vials
  • 5‐ml scintillation mini‐vials

Alternate Protocol 1: Separation of Individual [3H]Inositol Polyphosphates

  • Dowex AG1X8 columns, formate form (see protocol 6)
  • 25 and 200 mM ammonium formate in distilled water
  • 500 mM, 800 mM, and 1 M ammonium formate in 0.1 M formic acid

Basic Protocol 2: [3H]MYO‐Inositol Prelabeling Assay in Cultured Cells

  Materials
  • Cells of interest
  • Cell growth medium (e.g., complete DMEM; unit 3.1)
  • Myo‐inositol‐free cell growth medium
  • Purified [3H]myo‐inositol (see protocol 4)
  • HHB (see recipe), 37°C
  • Test agonists, antagonists in recipeHHB
  • Methanol, −20°C
  • Chloroform
  • 24‐well tissue culture plates
  • 0.45‐µm sterilization filter
  • 1‐ml disposable syringe
  • 5‐ml scintillation mini‐vials
  • 10‐ml plastic test tubes
  • Additional reagents and equipment for column chromatography (see protocol 1 or protocol 2)

Support Protocol 1: Purification of [3H]MYO‐Inositol

  Materials
  • [3H]Myo‐inositol (1 mCi/ml, ∼16 Ci/mmol; Amersham Biosciences, Perkin Elmer Life and Analytical Sciences)
  • 1‐ml plastic pipet tip
  • Glass wool
  • Dowex AG1X8 resin (200 to 400 mesh, chloride form; Bio‐Rad)

Support Protocol 2: Preparation of Chloride‐Form Dowex AG1X8 Columns

  Materials
  • 1 M HCl
  • Dowex AG1X8 resin (200 to 400 mesh, chloride form; Bio‐Rad)
  • Plastic disposable columns (e.g., Econo‐columns, Bio‐Rad)

Support Protocol 3: Preparation of Formate‐Form Dowex AG1X8 Columns

  Materials
  • Dowex AG1X8 resin (200 to 400 mesh, formate form; Bio‐Rad)
  • Solvents:
  •  500 mM, 800 mM, and 1 M ammonium formate in 0.1 M formic acid
  •  25 and 200 mM ammonium formate in H 2O
  • Radiolabeled IP standards (e.g., Amersham Biosciences, Perkin Elmer Life and Analytical Sciences): inositol mono‐, bis‐, tris‐, and tetrakisphosphates
  • 20‐ml glass columns (20‐ml reservoir volume ∼0.5‐cm i.d.)
  • Glass wool

Basic Protocol 3: Estimation of Inositol (1,4,5)‐Trisphosphate in Tissue Slices by Mass Assay

  Materials
  • Chopped tissue slices, pre‐equilibrated with KHB for 60 min (see protocol 8)
  • KHB (see recipe)
  • Test agonist in recipeKHB (when possible)
  • 1.2 M perchloric acid (PCA), ice cold
  • 1.2 M potassium bicarbonate, freshly prepared
  • 1 M NaOH
  • Unlabeled inositol (1,4,5)‐trisphosphate [I(1,4,5)P 3]
  • TE buffer: 100 mM Tris, 4 mM EDTA, pH 8.0, room temperature and ice cold
  • [3H]I(1,4,5)P 3 (Amersham Biosciences, Perkin Elmer Life and Analytical Sciences)
  • 15 to 20 mg/ml IP 3‐binding protein (see protocol 10)
  • Liquid scintillation fluid
  • Flat‐bottom vials
  • 3‐ to 4‐ml polypropylene tubes
  • Whatman GF/B filters
  • Millipore 10‐ or 12‐place vacuum manifold
  • 5‐ml scintillation mini‐vials
  • Graphing and curve‐fitting computer program (e.g., GraphPad Prism or equivalent)
  • Additional reagents and equipment for determining protein concentration (see appendix 3B)

Support Protocol 4: Preparation of Tissue Slices

  Materials
  • Experimental animal or tissue of interest
  • KHB (see recipe)
  • McIlwain tissue chopper (e.g., Mickle Laboratory)

Alternate Protocol 2: Inositol (1,4,5)‐Trisphosphate Mass Assay in Cultured Cells

  • Cells growing in 24‐well tissue culture plates
  • HHB (see recipe), 37°C
  • Test agonists and antagonists in recipeHHB

Support Protocol 5: Preparation of Inositol Trisphosphate–Binding Protein

  Materials
  • Fresh bovine adrenals (local slaughterhouse)
  • 1 mM dithiothreitol (DTT) solution in 20 mM sodium bicarbonate (no pH adjustment), ice cold
  • Polytron tissue homogenizer
  • Additional reagents and equipment for determining protein content ( appendix 3B)
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Figures

Videos

Literature Cited

Literature Cited
   Allison, J.H., Blisner, M.E., Holland, W.H., Hipps, P.P., and Sherman, W.R. 1976. Increased brain myo‐inositol 1‐phosphate in lithium‐treated rats. Biochem. Biophys. Res. Commun. 71:664‐670.
   Batty, I.H., Nahorski, S.R., and Irvine, R.F. 1997. Rapid formation of inositol 1,3,4,5‐tetrakisphosphate following muscarinic receptor stimulation of rat cerebral cortical slices. Biochem. J. 232:211‐215.
   Berridge, M.J. 1993. Inositol trisphosphate and calcium signalling. Nature. 361:315‐325.
   Berridge, M.J. 1997. Elementary and global aspects of calcium signalling. J. Physiol. 499:291‐306.
   Berridge, M.J. 1998. Neuronal calcium signaling. Neuron. 21:13‐26.
   Brown, E., Kendall, D.A., and Nahorski, S.R. 1984. Inositol phospholipid hydrolysis in rat cerebral cortical slices. I. Receptor characterisation. J. Neurochem. 42:1379‐1387.
   Bundey, R.A. and Kendall, D.A. 1999. Inhibition of receptor‐mediated calcium responses by corticotrophin‐releasing hormone in the CATH.a cell line. Neuropharmacology 38:39‐47.
   Challiss, R.A.J. and Nahorski, S.R. 1990. Neurotransmitter and depolarization‐stimulated accumulation of inositol 1,3,4,5‐tetrakisphosphate mass in rat cerebral cortex slices. J. Neurochem. 54:2138‐2141.
   Challiss, R.A.J., Batty, I.H., and Nahorski, S.R. 1989. Mass measurements of inositol (1,4,5)trisphosphate in rat cerebral cortex slices using a radioreceptor assay: Effects of neurotransmitters and depolarization. Biochem. Biophys. Res. Commun. 157:684‐691.
   Foster, D.A. 1993. Intracellular signalling mediated by protein‐tyrosine kinases: Networking through phospholipid metabolism. Cell Signalling 5:389‐399.
   Kennedy, E.D., Challiss, R.A.J., and Nahorski, S.R. 1989a. Lithium reduces the accumulation of inositol polyphosphate second messengers following cholinergic stimulation of cerebral cortex slices. J. Neurochem. 53:1652‐1655.
   Kennedy, E.D., Batty, I.H., Chilvers, E.R., and Nahorski, S.R. 1989b. A simple enzymatic method to separate [3H]inositol 1,4,5‐ and 1,3,4‐trisphosphate isomers in tissue extracts. Biochem. J. 260:283‐286.
   Lohse, M.J. 1993. Molecular mechanisms of membrane receptor desensitization. Biochim. Biophys. Acta 1179:171‐188.
   Nahorski, S.R., Ragan, C.I., and Challiss, R.A.J. 1991. Lithium and the phosphoinositide cycle: An example of uncompetitive inhibition and its pharmacological consequences. Trends Pharmacol. Sci. 12:297‐303.
   Nahorski, S.R., Young, K.W., John Challiss, R.A., and Nash, M.S. 2003. Visualizing phosphoinositide signalling in single neurons gets a green light. Trends Neurosci. 26:444‐452.
   Nash, M.S., Young, K.W., Willars, G.B., Challiss, R.A.J., and Nahorski, S.R. 2001. Single‐cell imaging of graded Ins(1,4,5)P3 production following G‐protein‐coupled‐receptor activation. Biochem. J. 356:137‐142.
   Nash, M.S., Willets, J.M., Billups, B., Challiss, R.A.J., and Nahorski, S.R. 2004. Synaptic activity augments muscarinic acetylcholine receptor‐stimulated inositol 1,4,5‐trisphosphate production to facilitate Ca2+ release in hippocampal neurons. J. Biol. Chem. 279:49036‐49044.
   Paulus, H. and Kennedy, J. 1960. The enzymatic synthesis of inositol monophosphate. J. Biol. Chem. 235:1303‐1311.
   Willars, G.B., McArdle, C.A., and Nahorski, S.R. 1998. Acute desensitization of phospholipase C‐coupled muscarinic M3 receptors but not gonadotropin‐releasing hormone receptors co‐expressed in aT3‐1 cells: Implications for mechanisms of rapid desensitization. Biochem. J. 333:301‐308.
Key References
   Brown et al., 1984. See above.
  Describes the use of the prelabeling methodology in brain slices.
   Challiss et al., 1989. See above.
  Describes the application of the mass assay for I(1,4,5)P3 in brain slices.
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