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Ligand Characterization Using Microphysiometry

Simon Pitchford1

1Molecular Devices Corporation, Sunnyvale, California

Publication Name: 
Current Protocols in Neuroscience
Unit Number: 
UNIT 7.8
DOI: 
10.1002/0471142301.ns0708s02
Print Publication Date: 
February, 1998
Online Posting Date: 
May, 2001
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Abstract

This unit describes the use of a Cytosensor microphysiometer for functional characterization of an agonist and antagonist to a G protein-coupled receptor, the muscarinic M1 receptor. Concentration-response profiles are used to calculate values for the EC50 of the response of cells to the agonist and the pA2 value for the antagonist. Support protocols describe optimization of two aspects of this procedure: the duration of ligand exposure at a given concentration and the length of recovery time between the administration of two different concentrations of ligand to minimize the impact of desensitization. The Cytosensor microphysiometer allows the measurement of receptor activation in both adherent cells, such as the M1WT3 cells used here or in suspension cultures.

     
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Table of Contents

  • Unit Introduction
  • Basic Protocol: Characterizing the Response of Adherent M1-Transfected CHO Cells Using the Cytosensor Microphysiometer
  • Alternate Protocol: Characterizing the Response of Nonadherent Cells Using the Cytosensor Microphysiometer
  • Support Protocol 1: Optimizing Ligand Exposure Time
  • Support Protocol 2: Optimizing Recovery Time
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
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Materials

Basic Protocol: Characterizing the Response of Adherent M1-Transfected CHO Cells Using the Cytosensor Microphysiometer

 Materials
  • Confluent cultures of adherent M1-transfected CHO cells (M1WT3, ATCC CRL-1985) grown in 25-cm2 flasks
  • 0.02% (w/v) EDTA
  • Growth medium (see recipe)
  • Running medium (see recipe), freshly prepared
  • Carbachol (RBI)
  • Pirenzepine dihydrochloride (RBI)
  • Capsule cups (Molecular Devices)
  • Blunt forceps, sterile
  • Capsule spacers (Molecular Devices)
  • Capsule inserts (Molecular Devices)
  • Sensor chambers, sterile
  • Cytosensor microphysiometer workstation, sterile
  • Cytosensor system software
  • 50- and 15-ml polypropylene tubes

NOTE: All solutions and equipment coming into contact with live cells must be sterile, and proper aseptic technique should be used accordingly.

NOTE: All culture incubations are performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified.


Alternate Protocol: Characterizing the Response of Nonadherent Cells Using the Cytosensor Microphysiometer

 Additional Materials (also see Basic Protocol)
  • Agarose entrapment medium (Molecular Devices)
  • Cultures of nonadherent cells expressing the receptor of interest (~1 to 5 × 106 cells total)
  • Heating block or hot plate at >70°C, or microwave oven
  • Incubator or heating block at 37°C
Looking for materials?  Suppliers Guide
     
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Figures

  • Figure 7.8.1
    Assembly of the microphysiometer cell capsule and sensor chamber.

    View Image
  • Figure 7.8.2
    (A) Screen shot from Cytosoft, the Cytosensor system software. Acidification-rate data from eight chambers of M1WT3 cells in response to sequential incubations with gradually increasing concentrations of carbachol ± antagonist, using a similar protocol to that outlined in the text. Duplicate chambers are run in the presence of 0 nM, 30 nM, 300 nM and 3 µM pirenzepine, a selective M1 receptor antagonist. (B) Expanded screen shot of the acidification rate response to 10 µM carbachol (added during the 70-sec period shown by the black vertical bar) in the absence and presence of pirenzepine (PZP). The acidification rate traces have been normalized in this figure to a baseline period selected just prior to carbachol addition.

    View Image
  • Figure 7.8.3
    Calculated concentration-response curves from the data shown in Figure 7.8.2. Each data point represents the mean of the two replicates from a single experiment.

    View Image
  • Figure 7.8.4
    Schild plot calculated from the acidification-rate data. Solid line represents the experimental data points. The broken line is the linear regression through these points. The intercept on the x-axis, the pA2 value, is 8.0.

    View Image

Literature Cited

Literature Cited
    Baxter, G.T., Young, M.-L., Miller, D.L., and Owicki, J.C. 1994. Using microphysiometry to study the pharmacology of exogenously expressed m1 and m3 muscarinic receptors. Life Sci. 55:573-583.
    Buck, M.A. and Fraser, C.M. 1990. Muscarinic acetylcholine receptor subtypes which selectively couple to phospholipase C:Pharmacological and biochemical properties. Biochem. Biophys. Res. Commun. 173:666-672.
    Cytosensor Microphysiometer at Work. 4th ed. Molecular Devices Corporation. Sunnyvale, Calif.
    Denyer, J., Gray, J., Wong, M., Stolz, M. and Tate, S. 1994. Molecular and pharmacological characterization of the human CCKB receptor. Eur. J. Pharmacol. Mol. Pharmacol. Sect. 268:29-41.
    Hafeman, D.G., Parce, W.J. and McConnell, H.M. 1988. Light-addressable potentiometric sensor for biochemical systems. Science 240:1182-1185.
    Johnson, R.M., McNeeley, P.A., DeMoor, K.M., Stewart, G.R., Glaeser, B.S., and Pitchford, S. 1994. Recombinant human ciliary neurotrophic factor stimulates metabolic activity of SH-SY5Y cells as measured by a Cytosensor microphysiometer. Brain Res. 646:327-331.
    Kenakin, T. 1993. Pharmacologic analysis of drug-receptor interaction. 2nd ed. Chapters 9, 10 and 3. Raven Press, New York.
    McConnell, H.M., Owicki, J.C., Parce, W.P., Miller, D.L., Baxter, G.T., Wada, H.G. and Pitchford, S. 1992. The Cytosensor microphysiometer: Biological applications of silicon technology. Science 257:1906-1912.
    Owicki, J.C. and Parce, J.W. 1992. Biosensors based on the energy metabolism of living cells: The chemistry and cell biology of extracellular acidification. Biosens. Bioelectronics 7:255-272.
    Pitchford, S., DeMoor, K. and Glaeser, B.S. 1995. Nerve growth factor stimulates a rapid metabolic response in PC12 cells. Am. J. Physiol. 268:C936-C943.
    Radeka, M.J., Baxter, G.T., Kuo, R., Medina-Selby, A., Coit, D., Valenzuela, P. and Feinstein, S.C. 1994. Signal transduction by the truncated trkB isoform, trkB.T1. Soc. Neurosci. Abstr. 20:37.
    Richards, M.H. 1991. Pharmacology and second messenger interactions of cloned muscarinic receptors. Biochem. Pharmacol. 42:1645-1653.
    Rosser, M.P., Kozlwski, M.R., Neve, R.I. and Neve, K.A. 1992. Effects of D2 and D3 receptor activation measured by microphysiometry. Soc. Neurosci. Abstr. 18:1171.
    Salon, J.A. and Owicki, J.C. 1995. Real-time measurements of receptor activity: Applications of microphysiometric techniques to receptor biology. Methods Neurosci. 25:201-224.
    Samson, M., Labbe, O., Mollereau, C., Vassart, G., and Parmentier, M. 1996. Molecular cloning and functional expression of a new human C-C chemokine receptor gene. Biochemistry 35:3362-3367.
    Wada, H.G., Indelicato, S.R., Meyer, L., Kitamura, T., Miyajima, A., Kirk, G., Muir, V.C. and Parce, J.W. 1993. GM-CSF triggers a rapid, glucose-dependent extracellular acidification by TF-1 cells: Evidence for sodium/proton antiporter and PKC mediated activation of acid production. J. Cell. Physiol. 154:129-138.
     
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