In Vitro Opioid Receptor Assays

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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 ? opioid receptor agonists, while the more labile contractions of the MVD are inhibited by µ, ?, and ? 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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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% O₂ /5% CO₂ ) 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 H₂ O 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‐NH₂ ]	−
CTAP [D‐Phe‐Cys‐Tyr‐D‐Trp‐Arg‐Thr‐Pen‐Thr‐NH₂ ]	−
Naltrindole	−	−	−
Nor‐Binaltorphimine (nor‐BIN)	−	−	−
β‐funaltrexamine (irreversible)	−	−	++
Diprenorphine	−	−	−
TIPP[Ψ] (H‐Tyr‐Tic Ψ[CH₂ NH]Phe‐Phe‐OH)	−

^a Agonist 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‐NH₂ ; 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% O₂ /5% CO₂ ) 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

Figure 4.8.1 Schematic representation of the guinea pig ileum. The various layers of the ileum are identified with the smooth muscle layers and their associated intramural nerve supplies. The myenteric plexus (Auerbach's plexus) provides sensory and motor innervation to the longitudinal muscle while the submucosal plexus (Meissner's plexus) provides similar innervation to the circular muscle. Both muscle‐nerve preparations possess opioid receptors with the longitudinal muscle/myenteric plexus preparation being the most common one employed in evaluating opioid activity in vitro.

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Figure 4.8.2 Representation of the experimental setup. (A ). Schematic of the four‐tissue bath setup employed to hold the tissues. The circulating water bath is depicted to the right and the data acquisition system shown on the left. The temperature of the water baths should be monitored to make certain that they are 37°C for LM/MP and 30°C for the MVD. The output from the force transducer is led to the A/D converter shown on the far left hand side of the figure. (B ) Expanded view of the tissue bath arrangement illustrating the relationship of the tissue to the stimulating ring electrodes, the force transducer, and the carbogen inlet. Note that the force transducer is held by a clamp in an adjustable rack and pinion that is used to establish baseline tension.

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Figure 4.8.3 Removal of the ileum. (A ) Schematic diagram of the opened guinea pig abdomen illustrating the relationship of the ileum to various organs. The ileum can be seen entering the cecum at the ileocecal plica, which provides an important landmark for excising the ileum to obtain the LM/MP preparation. (B ) Photograph of an excised ileum placed in Krebs‐Henseleit buffer. The ileum has been removed from its mesenteric attachment which removes the majority of the serous coat. The site of mesenteric attachment can be seen as a line running the length of the longitudinal muscle upon which most of the blood vessels converge.

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Figure 4.8.4 Preparation of the LM/MP. (A ) Photograph of the ileum placed on the moistened pipet with bathing solution soaked cotton balls being used to remove the longitudinal muscle layer. Note the pipet in the holder and the use of one cotton ball to hold the tissue while the other is used to stroke the longitudinal muscle free from the underlying tissue. (B ) Photograph of the separated longitudinal muscle being removed from the remaining ileum. (C ) The separated longitudinal muscle in bathing solution prior to production of the final preparation for recording. (D ) The longitudinal muscle/myenteric plexus being prepared for the recording session with the two sutures attached to provide the base for holding the tissue and the connection to the force transducer.

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Figure 4.8.5 The tissue holder/stimulating electrode combination. (A ) Photograph of the tissue holder/stimulating electrode illustrating the base for attaching the loop and the ring electrodes through which the tissue must be passed prior to connecting to the force transducer. The ring electrodes serve as the anode and cathode for electrical stimulation. (B ) Photograph of the tissue holder/stimulating electrode with a tissue attached at the base through the loop and extended through the ring electrodes. (C ) Photograph of the tissue holder/electrode combination placed in the tissue bath for connection the force transducer and stimulator.

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Figure 4.8.6 Photograph of the stimulating arrangement that illustrates the Grass S48 stimulator connected directly to the Med‐Lab 4‐Channel Attenuator, which delivers the electrical impulse from the stimulator to the Med‐Lab Stimu‐Splitter II, which delivers the stimulus to the stimulating electrodes. This arrangement allows independent regulation of the output from the attenuator to each of four outputs in the Stimu‐splitter. It is possible to use a single voltage from the stimulator and provide individually regulated output to each one of four stimulating electrodes.

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Figure 4.8.7 Representative tracing of a cumulative concentration‐response curve obtained from the data acquisition system. The effect of increasing concentrations of morphine on the amplitude of the neurogenic contractions of the guinea pig LM/MP is illustrated by the reduction in amplitude of the contractions. Note that the contractions reach a relatively constant level that is reduced in a concentration‐dependent manner. The exposure of the preparation to increasing concentrations causes a relatively rapid reduction in amplitude to a new steady level of contraction. Contractions were elicited at 10 sec intervals, so every 6 contractions equals 1 min.

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Figure 4.8.8 Photographs of the mouse vas deferens (MVD) preparation. (A ) Photograph of the MVD as it is removed from the animal with the surrounding fat tissue still attached. (B ) Photograph of the “cleaned” preparation in which the fat tissue has been removed to expose the vas deferens, the epididymis and the testis. (C ) The separated vas deferens pinned to the Sylgard layer of a Petri dish prior to removal of the tissue sheath and the artery of the deferent duct.

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Figure 4.8.9 Mean concentration‐response curves depicting the ability of morphine (A ) and DAMGO (B ) to inhibit the neurogenic contractions of LM/MP preparations obtained from naïve guinea pigs. The same tissues ( n = 10) were used to construct each concentration response curve which allows comparison of agonist activity. Curves were generated using GraphPad Prism. The calculated mean IC₅₀ values are given in the inset and are consistent with expected IC₅₀ values indicating that DAMGO is a more potent MOR agonist.

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Literature Cited

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In Vitro Opioid Receptor Assays

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited