Characterization of Opioid and ORL1 Receptors

Robert N. DeHaven1, Joel A. Cassel1, Rolf T. Windh1, Diane L. DeHaven‐Hudkins1

1 Adolor Corporation, Exton, Pennsylvania
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 1.4
DOI:  10.1002/0471141755.ph0104s29
Online Posting Date:  July, 2005
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Abstract

This unit describes methods for studying activity at opioid receptor subtypes and the ORL1 receptor using conventional radioligand binding assay methods as well as procedures for quantitating functional activity of these receptors by measuring agonist‐induced G protein activation with [35S]GTPγS binding in a manner analogous to traditional receptor‐binding methods. These procedures permit the assessment of the agonist or antagonist character of compounds in a rapid manner that, like most radioligand binding methodologies, is also amenable to high throughput technologies.

     
 
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Table of Contents

  • Receptor‐Binding Assays
  • Basic Protocol 1: Measurement of Opioid Receptor Binding to Cloned Receptors in Membranes
  • Alternate Protocol 1: Measurement of Opioid Receptor Binding in Tissue Membrane Homogenates
  • Basic Protocol 2: Measurement of Binding to Cloned Nociceptin/Orphanin FQ (ORL1) Receptors in Membranes
  • Alternate Protocol 2: FlashPlate Scintillation Proximity Assay to Measure ORL1 Receptor Binding
  • Basic Protocol 3: Testing Inhibitors of Opioid Receptor–Ligand Binding
  • Functional Assays
  • Basic Protocol 4: Measurement of Opioid Receptor‐Mediated [35S]GTPγS Binding using Basic FlashPlates
  • Alternate Protocol 3: Measurement of Opioid Receptor‐Mediated [35S]GTPγS Binding using Wheat Germ Agglutinin (WGA)‐Coated Scintillation Proximity Assay (SPA) Beads
  • Alternate Protocol 4: Measurement of Opioid Receptor‐Mediated [35S]GTPγS Binding using Filtration
  • Support Protocol 1: Saponin Enhancement of Opioid Receptor‐Mediated [35S]GTPγS Binding
  • Basic Protocol 5: Testing Compounds for Functional Activity at Opioid Receptors using [35S]GTPγS Binding
  • Support Protocol 2: Data Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of Opioid Receptor Binding to Cloned Receptors in Membranes

  Materials
  • Cell lines expressing the appropriate cloned human opioid receptor (e.g., CHO‐K1, ATCC no. CCL‐61; HEK‐293, ATCC no. CRL‐1573)
  • PBS (e.g., Life Technologies)
  • Tris assay buffer, pH 7.8 (see recipe), room temperature and 4°C
  • Nonenzymatic cell dissociation solution (e.g., Cellstripper, Mediatech)
  • [3H]Diprenorphine (PerkinElmer Life Sciences)
  • Test compound(s)
  • 100 µM naloxone or other unlabeled ligand to determine nonspecific binding
  • 0.5% (w/v) polyethylenimine/0.1% (w/v) BSA in filter rinse buffer
  • Filter rinse buffer: e.g., 50 mM Tris⋅Cl, pH 7.8 ( appendix 2A), 4°C
  • Water‐compatible scintillation cocktail (e.g., Beckman Coulter ReadySafe for vials or PerkinElmer Microscint‐20 for filter plates)
  • 500‐cm2 culture plates or Cell Factory (Nunc)
  • Polytron homogenizer (Brinkmann)
  • 250‐ml conical centrifuge bottles
  • Deep‐well (1.0 ml) 96‐well microtiter plates (PerkinElmer)
  • Filter‐bottom (glass fiber filter) 96‐well microtiter plates, glass fiber filter mats, or individual glass fiber filters (Whatman)
  • Cell harvester or vacuum filtration device appropriate for the filter style to be used (e.g., Skatron, Brandel, or PerkinElmer)
  • Additional reagents and equipment for Bradford or Lowry protein assay ( appendix 3A)

Alternate Protocol 1: Measurement of Opioid Receptor Binding in Tissue Membrane Homogenates

  • Guinea pig (e.g., male Hartley, 300 to 500 g)
  • HEPES assay buffer (see recipe), ice cold
  • Radiolabeled ligand: [3H]U‐69593 (PerkinElmer Life Sciences)
  • Test compound
  • 10 µM U‐50488H or other unlabeled opioid ligand to define nonspecific binding
  • Filter rinse buffer: 50 mM HEPES, pH 7.4, 4°C
  • Water‐compatible scintillation cocktail (e.g., Beckman Coulter Ready‐Solv)
  • Dissection instruments: operating scissors, bone rongeurs, bone cutting forceps, dissecting knife, microdissecting probes, dissecting plate.

Basic Protocol 2: Measurement of Binding to Cloned Nociceptin/Orphanin FQ (ORL1) Receptors in Membranes

  Materials
  • Cell line expressing the cloned human ORL1 receptor (e.g., HEK‐293, ATCC no. CRL‐1573)
  • PBS (e.g., Life Technologies)
  • Tris assay buffer, pH 7.8 (see recipe), 4°C
  • [3H]Nociceptin (>80 Ci/mmol, PerkinElmer Life Sciences) or [125I]nociceptin (2200 Ci/mmol, PerkinElmer Life Sciences)
  • Test compound(s)
  • 10 µM unlabeled nociceptin (Sigma‐Aldrich) to determine nonspecific binding
  • 0.5% (w/v) polyethylenimine/0.1% (w/v) BSA in filter rinse buffer
  • Filter rinse buffer: e.g., 50 mM Tris⋅Cl, pH 7.8 ( appendix 2A), 4°C
  • Water‐compatible scintillation cocktail (e.g., Beckman ReadySafe or PerkinElmer Life Sciences MicroScint 20)
  • Polytron homogenizer (Brinkmann)
  • Deep‐well (1.0 ml) 96‐well microtiter plates (e.g., PerkinElmer Life Sciences)
  • Filter‐bottom (glass fiber filter) 96‐well microtiter plates (e.g., PerkinElmer Life Sciences), glass fiber filter mats or individual glass fiber filters (Whatman)
  • Cell harvester or vacuum filtration device appropriate for the filter style to be used (e.g., Skatron, Brandel, or PerkinElmer)
  • Additional reagents and equipment for Bradford or Lowry protein assay ( appendix 3A)

Alternate Protocol 2: FlashPlate Scintillation Proximity Assay to Measure ORL1 Receptor Binding

  • Membrane preparation containing opioid receptors (see protocol 1, steps to , or steps to )
  • [125I]nociceptin
  • 96‐ or 384‐well WGA‐coated FlashPlates (PerkinElmer Life Sciences)
  • Sealing tape (e.g., TopSeal, PerkinElmer Life Sciences)
  • Microplate‐compatible scintillation counter (e.g., TopCount, PerkinElmer Life Sciences)

Basic Protocol 3: Testing Inhibitors of Opioid Receptor–Ligand Binding

  Materials
  • Opioid receptor‐containing membrane preparation (see protocol 1, steps to )
  • Tris assay buffer (see recipe)
  • Guanosine‐5′‐(γ‐thio)triphosphate, [35S] ([35S]GTPγS) (see recipe; PerkinElmer Life Sciences)
  • 3 mM guanosine‐5′‐diphosphate, Tris salt (GDP) solution (see recipe)
  • 5 M NaCl stock solution
  • 1 M MgCl 2 stock solution
  • Test compound(s)
  • 100 µM of appropriate standard agonist (loperamide for µ opioid receptor, BW373U86 for δ opioid receptor, and U‐50488H for κ opioid receptor)
  • Polytron homogenizer (Brinkmann)
  • 96‐well Basic FlashPlates (PerkinElmer Life Sciences)
  • 96‐well plate scintillation counter (e.g., TopCount, PerkinElmer Life Sciences)

Basic Protocol 4: Measurement of Opioid Receptor‐Mediated [35S]GTPγS Binding using Basic FlashPlates

  • Opioid receptor‐containing membrane preparation (see protocol 1, steps to )
  • WGA‐coated SPA beads (Amersham Biosciences)
  • 96‐well white, opaque microplates (VWR)

Alternate Protocol 3: Measurement of Opioid Receptor‐Mediated [35S]GTPγS Binding using Wheat Germ Agglutinin (WGA)‐Coated Scintillation Proximity Assay (SPA) Beads

  • Opioid receptor–containing membrane preparation (see protocol 1, steps to )
  • 1% (w/v) bovine serum albumin (BSA) in water
  • Filter rinse buffer: 50 mM Tris⋅Cl, pH 7.8 at 4°C
  • GF/B glass fiber filter mats
  • Deep‐well (1.0 ml) 96‐well microtiter plates (Beckman Coulter)
  • 5‐ml plastic scintillation vials
  • Vial scintillation counter (Beckman Coulter)
  • Water‐compatible scintillation cocktail (e.g., ReadySafe, Beckman Coulter)
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Figures

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Literature Cited

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Key References
   Knapp, R.J., Malatynska, E., Collins, N., Fang, L., Wang, J.Y., Hruby, V.J., Roeske, W.R., and Yamamura, H.I. 1995. Molecular biology and pharmacology of cloned opioid receptors. FASEB J. 9:516‐525.
  Detailed description of binding to cloned opioid receptor subtypes, including an extensive evaluation of reference agents at each cloned receptor subtype.
   Raynor et al., 1994. See above.
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