In Vitro Opioid Receptor Assays

David A. Taylor1

1 The Department of Pharmacology and Toxicology, The Brody School of Medicine at East Carolina University, Greenville, North Carolina
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 4.8
DOI:  10.1002/0471141755.ph0408s55
Online Posting Date:  December, 2011
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Abstract

Although opioid analgesics have been used for centuries, identification of opioid receptors and the ability of an opioid receptor antagonist to block natural pain processes prompted a search for endogenous opioid peptides. In vitro models were needed to characterize opioid activity in biological samples. The longitudinal muscle/myenteric plexus (LM/MP) of the guinea pig ileum was the classical in vitro assay system, but the development of the mouse vas deferens (MVD) assay provided another important model that could be employed. Both assays entail electrical stimulation of intramural nerves to produce muscle contractions of the target organ. The robust contractions of the LM/MP are inhibited by µ and κ opioid receptor agonists, while the more labile contractions of the MVD are inhibited by µ, κ, and δ opioid receptor agonists. These in vitro assay systems are useful for evaluating biological activity of unknown substances and studying the properties of drug tolerance and both are described in this unit. Curr. Protoc. Pharmacol. 55:4.8.1‐4.8.34. © 2011 by John Wiley & Sons, Inc.

Keywords: opioid receptors; mu opioid receptors; kappa opioid receptors; delta opioid receptors; in vitro assays; mouse; guinea pig; longitudinal muscle/myenteric plexus; vas deferens

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Evaluation of Opioid Activity in the Guinea Pig Ileum Longitudinal Muscle/Myenteric Plexus (LM/MP) Preparation
  • Basic Protocol 2: Evaluation of Opioid Activity in the Mouse Vas Deferens (MVD) Preparation
  • Support Protocol 1: Clean the System
  • Support Protocol 2: Analyze the Data
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Evaluation of Opioid Activity in the Guinea Pig Ileum Longitudinal Muscle/Myenteric Plexus (LM/MP) Preparation

  Materials
  • Modified Krebs‐Henseleit solution (see recipe) or other physiological saline solution
  • Carbogen gas cylinders (medical grade, 95% O 2/5% CO 2) connected to a two‐stage regulator
  • Male Hartley guinea pigs (400 to 500 g)
  • Isoflurane
  • Test agents (see recipe; test compounds would be mixtures, chemicals with unknown activity, or NCE being evaluated for activity at MOR or KOR receptors)
  • 1 mM standards in H 2O or appropriate solvent:
    • 1 mM DAMGO ([D‐Ala2, (Me) Phe4, Gly‐ol5] enkephalin) or other MOR agonist (see Table 4.8.1)
      Table 4.8.1   Materials   Relative Receptor Selectivity of Opioid Agents a   Relative Receptor Selectivity of Opioid Agents

      Receptor subtype
      Agonist (+)/Antagonist (−) MOR DOR KOR
      Morphine +++ +
      DAMGO ([D‐Ala2,MePhe4,Gly(ol)5]enkephalin +++
      DADLE, [D‐Ala2, D‐Leu5] enkephalin + ++
      DSLET, [D‐Ser2]‐leucine enkephalin‐Thr + ++
      DPDPE, [D‐Pen2, D‐Pen5] enkephalin ++
      U50, 488H, trans‐(±)‐3,4‐dichloro‐N‐methyl[2‐(1‐pyrrolidinyl) cyclohexyl]‐benzene‐acetamide methan sulfonate +++
      U69, 593, N‐methyl‐2‐phenyl‐N‐[(5R,7S,8S)‐7‐(pyrrolidin‐1‐yl)‐1‐oxaspiro[4.5]dec‐8‐yl]acetamide +++
      Spiradoline +++
      Fentanyl +++
      β‐Endorphin +++ +++
      Met‐enkephalin +++
      Leu‐enkephalin +++
      Methadone +++
      Hydromorphone +++
      Sufentanil +++ + +
      Normorphine +++
      Nalbuphine
      Ethylketocyclazine PA +++
      Dynorphin A ++ +++
      Dynorphin B + + +++
      Nociceptin (Orphanin FQ) NA NA NA
      Buprenorphine PA +++
      Etorphine +++ +++ +++
      Naloxone
      Naltrexone
      CTOP [D‐Phe‐Cys‐Tyr‐D‐Trp‐Orn‐Thr‐Pen‐Thr‐NH 2]
      CTAP [D‐Phe‐Cys‐Tyr‐D‐Trp‐Arg‐Thr‐Pen‐Thr‐NH 2]
      Naltrindole
      Nor‐Binaltorphimine (nor‐BIN)
      β‐funaltrexamine (irreversible) ++
      Diprenorphine
      TIPP[Ψ] (H‐Tyr‐Tic Ψ[CH 2NH]Phe‐Phe‐OH)

       aAgonist activity is denoted as (+) and relative potency defined by the number of (+). Relative selectivity of a particular agent can be identified by comparing the relative potency. Antagonist activity is denoted by (−) with selectivity similarly determined by the relative potency at individual opioid receptor subtypes. Partial agonist activity is denoted by PA. Nociceptin is an opioid peptide whose action is not blocked by naloxone.
    • 1 mM U50,488H (trans‐(±)‐3,4‐dichloro‐N‐methy[2‐(L‐pyrrolidinyl)cyclohexyl]‐benzene‐acetamide methane sulfonate) or other KOR agonist (see Table 4.8.1)
    • 1 mM CTAP (D‐Phe‐Cys‐Tyr‐D‐Trp‐Arg‐Thr‐Pen‐Thr‐NH 2; store at −20°C)
    • 100 µM nor‐BNI (nor‐binaltorphimine)
  • 1 mM morphine (morphine sulfate salt pentahydrate; Sigma, cat. no. M8777)
  • Circulating bath to maintain temperatures of 37°C in tissue baths
  • Tissue bath assembly (see Fig. and protocol introduction, above; product numbers provided are from VWR but any laboratory supply company should be able to provide similar equipment):
    • Support frame to hold tissue baths and force transducers (see Fig. )
    • Base stands (VWR, cat. no. 12985‐074 × 2; or purchase a wooden base of desirable size which will require the use of Lab‐frame feet to support vertical rods (VWR, cat. no. 60079‐257)
    • Stainless steel rod (1/2 in. diameter, 24 in. long × 4; VWR, cat. no. 60079‐535)
    • Aluminum rod (1/2 in. diameter 36 in. long; VWR, cat. no. 60079‐428)
    • Aluminum rod (1/2 in. diameter, 12 in. long; × 4 VWR, cat. no. 60079‐360)
    • Rack and pinion adjustable clamps to hold force transducers (Harvard Apparatus, cat. no. 502609 or 502625 depending on rod size)
    • 10‐ml jacketed glass tissue baths (see Fig. B and/or C) (Radnoti, cat. no. 158311 or 158311‐LL for Luer Locking ports; it is also possible to purchase baths with Quick‐Disconnect connections that appear to be standard on the commercial system)
    • Tissue holders with integral 22‐G platinum‐iridium wire electrodes (see Fig. A, B) (The electrodes we use [shown in Fig. A, B] were fabricated from a drawing provided to Mr. James Rogers at JIM's Instruments, Inc., Iowa City, Iowa). It is possible to purchase tissue holders with stimulating electrodes commercially from suppliers of the tissue bath systems.
    • Clamps, Day pinchcock and screw compressor style (VWR, cat. nos. 21730‐001 and 21710‐026); the commercial tissue bath systems have internal systems for removing and filling the tissue baths.
    • Four 3‐prong extension clamps to hold stimulating electrode (VWR, cat. no. 21570‐125)
    • Wide‐mouth glass bottle with bottle‐top dispenser and adjustable preset volume to deliver at least 10 ml (Barnstead/Labindustries REPIPET II); the commercially available organ bath systems come equipped with a water‐jacketed reservoir and solution distribution system which replaces the need for this dispenser
  • Data acquisition and analysis system (commercially available systems may include the data acquisition system to ensure compatibility of the components):
    • PC (Pentium processor with Windows 2000 or later operating system) or Macintosh computer, with monitor to support an 800 × 600, 256‐color display that is compatible with the PowerLab data acquisition system
    • PowerLab data acquisition and analysis system with a 4‐channel Quad bridge A/D converter interface (AD Instruments, http://www.adinstruments.com)
    • Isometric force transducers (FT‐.03, Grass Technologies; F10 Isometric Force Transducer, Harvard Apparatus; actual product no. depends on the amplifier system employed); if the tissue bath system is purchased commercially, transducers and appropriate connectors should be provided as part of the system
    • 1‐g weight for calibration purposes
    • PowerLab/Chart 5 computer program (AD Instruments, http://www.adinstruments.com)
    • Electrical stimulator (Grass S‐48 stimulator, Grass Technologies)
    • Med‐Lab 4‐Channel stimulator attenuator connected to a Med‐Lab Stimu‐Splitter (Med‐Lab Instruments); this particular item of equipment enables multiple tissues to be electrically stimulated from a single stimulator but provides the ability to independently regulate certain components of the stimulation parameters in individual tissues
  • Disposable absorbent bench protectors (Chux Blue Pads or any absorbent underpad from a biological supply company, such as VWR, cat. no. 56617)
  • Surgical instruments: a basic set of instruments should include large blunt/blunt surgical scissors (1); fine straight or curved iris scissors (1); fine‐tip curved dissecting forceps (2); iris tissue forceps with 1 × 2 teeth (1); and Dumont No. 5 forceps (2); these can be purchased from any biological supply company and the approximate cost of the set of instruments provided above would be $200 from VWR
  • 20‐ml plastic syringe with 18‐G luer‐lok blunt‐end needles or equivalent
  • Cotton balls
  • 3‐0 braided silk surgical thread (Roboz Surgical)
  • Petri dish containing a layer of Sylgard (Dow Corning Sylgard 184 silicone elastomer, Fisher Scientific, cat. no. 9561957); the Sylgard layer is optional for LM/MP but may be useful for pinning tissues when placing surgical thread to connect the tissue to the tissue holder and transducer
  • 150‐ml glass beakers
  • 1‐ml glass pipet with a heat closed end or a solid glass rod of similar size
  • 20‐ or 50‐ml syringes with blunt needles
  • Micropipettors (1‐ to 100‐µl range, with long disposable tips)
  • Polypropylene microcentrifuge tubes with snap caps (Eppendorf or other standard brand)
  • Additional reagents and equipment for anesthesia (Donovan and Brown, ) and euthanasia (Donovan and Brown, ), cleaning the system ( protocol 3), and analyzing the data ( protocol 4)

Basic Protocol 2: Evaluation of Opioid Activity in the Mouse Vas Deferens (MVD) Preparation

  Materials
  • Mg2+‐free Krebs solution (see ), prepared fresh
  • Carbogen gas cylinders (medical grade, 95% O 2/5% CO 2) connected to a two‐stage regulator
  • Male albino mice (35 to 40 g)
  • Isoflurane
  • 1 mM DPDPE ([D‐Pen2, D‐Pen5] enkephalin) or other selective DOR agonist (Table 4.8.1)
  • Test agents (see recipe; test compounds would be mixtures, chemicals with unknown activity, or NCE being evaluated for activity at MOR or KOR receptors)
  • 100 µM naltrindole or other DOR antagonist (Table 4.8.1)
  • Circulating bath to maintain 37°C in tissue baths
  • Tissue bath assembly (see Fig. and protocol introduction, above; product numbers provided are from VWR but any laboratory supply company should be able to provide similar equipment):
    • Support frame to hold tissue baths and force transducers (see Fig. )
    • Base stands (VWR, cat. no. 12985‐074 × 2; or purchase a wooden base of desirable size which will require the use of Lab‐frame feet to support vertical rods; VWR, cat. no. 60079‐257)
    • Stainless steel rod (1/2 in. diameter, 24 in. long × 4; VWR, cat. no. 60079‐535)
    • Aluminum rod (1/2 in. diameter 36 in. long; VWR, cat. no. 60079‐428)
    • Aluminum rod (1/2 in. diameter, 12 in. long; × 4 VWR, cat. no. 60079‐360)
    • Rack and pinion adjustable clamps to hold force transducers (Harvard Apparatus, cat. nos. 502609 or 502625 depending on rod size)
    • 10‐ml jacketed glass tissue baths (see Fig. B and/or Fig. C) (Radnoti, cat. no. 158311 or 158311‐LL for Luer Locking ports; it is also possible to purchase baths with Quick‐Disconnect connections that appear to be standard on the commercial system)
    • Tissue holders with integral 22‐G platinum‐iridium wire electrodes (see Fig. A, B) (The electrodes we use [shown in Fig. A,B] were fabricated from a drawing provided to Mr. James Rogers at JIM's Instruments, Inc.); it is possible to purchase tissue holders with stimulating electrodes commercially from suppliers of the tissue bath systems
    • Clamps, Day pinchcock and screw compressor style (VWR, cat. nos. 21730‐001 and 21710‐026); the commercial tissue bath systems have internal systems for removing and filling the tissue baths
    • Four 3‐prong extension clamps to hold stimulating electrode (VWR, cat. no. 21570‐125)
    • Wide‐mouth glass bottle with bottle‐top dispenser and adjustable preset volume to deliver at least 10 ml (Barnstead/Labindustries REPIPET II); the commercially available organ bath systems come equipped with a water‐jacketed reservoir and solution distribution system which replaces the need for this dispenser
  • Data acquisition and analysis system (commercially available systems may include the data acquisition system to ensure compatibility of the components):
    • PC (Pentium processor with Windows 2000 or later operating system) or Macintosh computer, with monitor to support an 800 × 600, 256‐color display that is compatible with the PowerLab data acquisition system
    • PowerLab data acquisition and analysis system with a 4‐channel Quad bridge A/D converter interface (AD Instruments, http://www.adinstruments.com)
    • Isometric force transducers (FT‐.03, Grass Technologies; F10 Isometric Force Transducer, Harvard Apparatus; actual product no. depends on the amplifier system employed); if the tissue bath system is purchased commercially, transducers and appropriate connectors should be provided as part of the system
    • 100‐mg weight for calibration purposes
    • PowerLab/Chart 5 computer program (AD instruments, http://www.adinstruments.com)
    • Electrical stimulator (Grass S‐48 stimulator, Grass Technologies)
    • Med‐Lab 4‐Channel stimulator attenuator connected to a Med‐Lab Stimu‐Splitter (Med‐Lab Instruments, see Fig. ); this particular item of equipment enables multiple tissues to be electrically stimulated from a single stimulator but provides the ability to independently regulate certain components of the stimulation parameters in individual tissues
  • Disposable absorbent bench protectors (Chux Blue Pads or any absorbent underpad from a biological supply company, such as VWR, cat. no. 56617)
  • Surgical instruments: a basic set of instruments should include large blunt/blunt surgical scissors (1); fine straight or curved iris scissors (1); fine tip curved dissecting forceps (2); iris tissue forceps with 1 × 2 teeth (1); and Dumont No. 5 forceps (2); the Dumont forceps are used to separate connective tissues and blood vessels from the vas deferens and can be purchased from any biological supply company; the approximate cost of the set of instruments provided above would be $200 from VWR
  • Petri dish filled with a layer of Sylgard (Dow Corning Sylgard 184 silicone elastomer, Fisher Scientific, cat. no. NC 9561957)
  • 150‐ml beakers
  • 6‐0 silk thread
  • 30‐G needles (4 to 6)
  • Dissecting microscope
  • Polypropylene microcentrifuge tubes with snap caps (Eppendorf or other brand)
  • Micropipettors (1‐ to 100‐µl range with long disposable tips)
  • Additional reagents and equipment for anesthesia (Donovan and Brown, ) and euthanasia (Donovan and Brown, ), cleaning the system ( protocol 3), and analyzing the data ( protocol 4)

Support Protocol 1: Clean the System

  Materials
  • 0.1 N HCl
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Figures

Videos

Literature Cited

Literature Cited
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