Nicotinic Acetylcholine Receptor Assays

Brendan J. Canning1

1 The Johns Hopkins Medical Institutions, Baltimore, Maryland
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 4.12
DOI:  10.1002/0471141755.ph0412s04
Online Posting Date:  May, 2001
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Abstract

Nicotinic acetylcholine receptors (nAChRs) in peripheral tissues are localized almost exclusively to autonomic nerves and the motor end plates of striated musculature. Pharmacologic analyses of nicotinic receptor antagonist potencies can be conducted by assessing the ability of these compounds to inhibit responses elicited by preganglionic autonomic nerve stimulation or stimulation of the motor nerves innervating striated muscle in isolated tissue preparations. In addition, in some isolated tissues innervated by autonomic nerves, nicotinic receptor mediated responses can be elicited by exogenously administered agonists, and the effects of antagonists on these responses can be assessed using pharmacologic analyses. This unit describes the guinea pig trachea/esophagus preparation, in which nicotinic receptor pharmacology can be studied at synapses of the parasympathetic and sympathetic nervous system and the striated musculature of the esophagus. In addition, a preparation whereby the nicotinic receptors of the striated musculature of the diaphragm can be studied is described as are techniques for studying exogenous nicotinic agonistmediated effects in two smooth muscle preparations.Nicotinic acetylcholine receptors (nAChRs) in peripheral tissues are localized almost exclusively to autonomic nerves.

     
 
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Table of Contents

  • Basic Protocol 1: The Innervated Guinea Pig Tracheal/Esophageal Isolated Tissue Preparation to Study Receptors on Autonomic Nerves
  • Alternate Protocol 1: Nicotinic Agonist–Mediated Effects: Isolated Guinea Pig Trachealis and Ileum
  • Basic Protocol 2: Nicotinic Receptors in Striated Muscle: The Isolated Diaphragm/phrenic Nerve Preparation
  • Alternate Protocol 2: Nicotinic Receptors in Striated Muscle: The Isolated, Innervated Guinea Pig Esophagus
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: The Innervated Guinea Pig Tracheal/Esophageal Isolated Tissue Preparation to Study Receptors on Autonomic Nerves

  Materials
  • Male or female guinea pigs (200 to 400 g), any strain
  • Krebs buffer (see recipe), 37°C, gassed with Carbogen gas (95% O 2/5% CO 2; also see unit 4.3)
  • Compounds appropriate for pretreatment (see recipe), dissolved in Krebs buffer: indomethacin (required for all studies), atropine, histamine or prostaglandin F , phentolamine, propranolol, papaverine, tetrodotoxin, or barium chloride
  • Test compounds of interest (antagonists, ganglionic blockers) dissolved in Krebs buffer with vehicles (water or ethanol) as controls
  • Deep‐welled dissecting dish with Sylgard‐coated bottom (for crude dissections; see recipe)
  • Surgical instruments:
  •  Large scissors and forceps for cutting bone
  •  Fine scissors and forceps for blunt and fine dissection
  •  4–0, 5–0, or 6–0 surgical silk
  •  18‐ and 30‐G dissecting pins (1/2‐in. or 1.75‐cm hypodermic needles are ideal)
  • Equipment for nerve stimulation (Fig. ) including:
  •  Dissecting microscope with retracting arm
  •  Water‐jacketed dissecting dish with Sylgard‐coated bottom (see recipe)
  •  Small water‐jacketed heating coil
  •  Thermostatted water circulator with tubing (Cole Parmer)
  •  Peristaltic pump with tubing (Cole Parmer)
  •  Bubbler and source of Carbogen gas (95% O 2/5% CO 2)
  •  Fiber‐optic light source
  •  Polygraph with isometric force transducers (also see units 4.3 & 4.4; Grass Instruments)
  •  Stimulator (Grass Instruments Model S44) with suction electrodes (World Precision Instruments Model MPH‐RW 1.2; also see Fig. )
  • Equipment for electrical field stimulation (EFS) including (see units 4.6 & 4.8):
  •  Water‐jacketed organ baths
  •  Tissue holders with platinum ring electrodes
  •  Med‐Lab Stimusplitter (Med‐Lab Instruments)
  • Additional reagents and equipment for maintaining and measuring response in isolated tissue preparations (units 4.3, 4.4 & 4.6)

Alternate Protocol 1: Nicotinic Agonist–Mediated Effects: Isolated Guinea Pig Trachealis and Ileum

  • Isolated tracheal strips (unit 4.6) or ileum (unit 4.8), prepared and placed in an organ bath
  • Nicotine or other nicotinic agonists: e.g., 1,1‐dimethyl‐4‐phenylpiperazine (DMPP)
  • Additional reagents and equipment for maintaining and measuring response in isolated tissue preparations (unit 4.3, & ) and generating dose‐response curves (unit 4.1)

Basic Protocol 2: Nicotinic Receptors in Striated Muscle: The Isolated Diaphragm/phrenic Nerve Preparation

  Materials
  • Male or female guinea pigs (any strain, 400 to 600 g) or rats (any strain, 200 to 400 g)

Alternate Protocol 2: Nicotinic Receptors in Striated Muscle: The Isolated, Innervated Guinea Pig Esophagus

  • 0–0 surgical silk sutures
  • 15‐G cannula
  • Polygraph with pressure transducers
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Figures

Videos

Literature Cited

Literature Cited
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   Bevan, J.A. 1968. Ganglionic blocking drugs. In Selected Pharmacological Testing Methods (A. Berger, ed.) pp. 235‐254. Marcel Dekker, New York.
   Canning, B.J. and Undem, B.J. 1993a. Relaxant innervation of guinea pig trachealis: Evidence for capsaicin‐ sensitive and–insensitive vagal pathways. J. Physiol. (Lond.) 470:719‐739.
   Canning, B.J. and Undem, B.J. 1993b. Evidence that distinct neuronal pathways mediate parasympathetic contractions and relaxations of guinea pig trachealis. J. Physiol. (Lond.) 481:25‐40.
   Cooper, G. and Schiller, A.L. 1975. Anatomy of the Guinea Pig. Harvard University Press, Cambridge, Mass.
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   Watson, N., Barnes, P.J., and Maclagan, J. 1992. Actions of methoctramine a muscarinic M2 receptor antagonist, on muscarinic and nicotinic cholinoceptors in guinea pig airways in vivo and in vitro. Br. J. Pharmacol. 105:107‐112.
   Yamada, S., Kagawa, Y., Takayanagi, N., Nakayama, K., Tsuji, K., Kosuge, T., Hayashi, E., Okada, K., and Inoue, S. 1987. Comparison of antinicotinic activity by neosurugatoxin and the structurally related compounds. J. Pharm. Exp. Ther. 243:1153‐1158.
Key References
   Canning and Undem, 1993a. See above.
  Describes the initial method and use of the isolated, innervated guinea pig tracheal preparation.
   Canning and Undem, 1993b. See above.
  Provides data relating to the potency of selected ganglionic blockers in the guinea pig trachea.
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