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Muscarinic Receptor Assays

Richard M. Eglen1,  Agnes Choppin1,  Nikki Watson1,  Sharath S. Hegde1

1Roche Bioscience, Palo Alto, California

Publication Name: 
Current Protocols in Pharmacology
Unit Number: 
UNIT 4.15
DOI: 
10.1002/0471141755.ph0415s07
Print Publication Date: 
December, 1999
Online Posting Date: 
May, 2001
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Abstract

In the central and peripheral nervous systems, the actions of acetylcholine are mediated by families of ligand gated ion channels and metabotropic receptors. The latter family is composed of muscarinic receptors, since they are selectively activated by muscarine and antagonized by atropine. Five muscarinic receptor subtypes have been identified by molecular biological and cloning studies (M1-M5), however, currently there are only functional preparations for the M1 through M4 subtypes. This unit describes robust preparations for assessing M1, M2 and M3 receptors in isolated tissues. An assay for the M4 is also described although this has not been evaluated extensively. An endogenous assay has not been identified for the M5 receptor and its physiological role is not well understood. In the central and peripheral nervous systems, the actions of acetylcholine are mediated by families of ligand gated ion chann.

     
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Muscarinic M1 Receptors: Canine Saphenous Vein
  • Basic Protocol 2: Muscarinic M2 Receptors: Guinea Pig Paced Left Atrium
  • Basic Protocol 3: Muscarinic M3 Receptors: Rat Urinary Bladder
  • Alternate Protocol 1: Muscarinic M3 Receptors: Guinea Pig Trachea
  • Alternate Protocol 2: Muscarinic M3 Receptors: Guinea Pig Ileum
  • Basic Protocol 4: Muscarinic M4 Receptors: Rabbit Anococcygeus
  • Reagents and Solutions
  • Commentary
  • Bibliography
  • Figures
  • Tables
     
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Materials

Basic Protocol 1: Muscarinic M1 Receptors: Canine Saphenous Vein

 Materials
  • Mongrel dog of either sex, 10 to 30 kg (Charles River)
  • Sodium pentobarbital
  • Modified Krebs-Henseleit solution (see recipe), freshly prepared and continuously oxygenated with 95% O2/5% CO2
  • 3 M KCl
  • Standard muscarinic antagonists (see recipe): e.g., atropine, pirenzepine, methoctramine, tripitramine, himbacine, para-fluorohexahydrosiladifenidol (p-F-HHSiD), darifenacin, and PD 102807
  • Standard muscarinic agonists (see recipe): e.g., acetylcholine (ACh), (+)-muscarine, oxotremorine M, (+)-cis-dioxolane, carbachol
  • Test compound(s)
  • 1 mM physostigmine stock (optional; for use with ACh) in freshly prepared modified Krebs-Henseleit solution, bubbled with 95% O2/5% CO2
  • 95% (v/v) O2/5% CO2 cylinders with regulator capable of delivering 5 to 15 psi
  • Fine forceps and scissors
  • Tissue hooks
  • Standard tissue bath equipment (unit 4.4)
  • Thermostatically regulated water circulator
  • Force-displacement transducer (Grass FT03)
  • Polygraph/chart recorder (Grass 7E)

Basic Protocol 2: Muscarinic M2 Receptors: Guinea Pig Paced Left Atrium

 Materials
  • Male Hartley guinea pig, 250 to 450 g (Charles River)
  • CO2 pellets
  • Modified Krebs-Henseleit solution (see recipe), freshly prepared and continuously oxygenated with 95% O2/5% CO2
  • Standard muscarinic agonists (see recipe): e.g., acetylcholine (ACh), (+)-muscarine, oxotremorine M, (+)-cis-dioxolane, carbachol
  • Standard muscarinic antagonists (see recipe): e.g., acetylcholine (ACh), atropine, pirenzepine, methoctramine, tripitramine, himbacine, para-fluorohexahydrosiladifenidol (p-F-HHSiD), darifenacin, and PD 102807
  • Test compound(s)
  • 1 mM physostigmine stock(optional; for use with ACh) in freshly prepared modified Krebs-Henseleit solution, bubbled with 95% O2/5% CO2
  • 95% (v/v) O2/5% CO2 cylinders with regulator capable of delivering 5 to 15 psi
  • Airtight chamber
  • Guillotine
  • Fine forceps and scissors
  • 1.0 surgical silk
  • Stationary metal rod
  • Force-displacement transducer (Grass FT03)
  • Standard tissue bath equipment (unit 4.4)
  • Thermostatically regulated water circulator
  • Force-displacement transducer (Grass FT03)
  • Polygraph/chart recorder (Grass 7E)
  • Platinum electrodes
  • Electrical stimulator (e.g., Grass S48)
  • Additional reagents and equipment for dissecting the left atrium (unit 4.3)

Basic Protocol 3: Muscarinic M3 Receptors: Rat Urinary Bladder

 Materials
  • Sprague-Dawley rats of either sex, 200 to 300 g
  • CO2 pellets
  • 10 µM indomethacin in modified Krebs-Henseleit solution (see recipe), freshly prepared and continuously oxygenated with 95% O2/5% CO2
  • 3.0 M KCl
  • Standard muscarinic agonists (see recipe): e.g., acetylcholine (ACh), (+)-muscarine, oxotremorine M, (+)-cis-dioxolane, carbachol
  • Standard muscarinic antagonists (see recipe): e.g., atropine, pirenzepine, methoctramine, tripitramine, himbacine, para-fluorohexahydrosiladifenidol (p-F-HHSiD), darifenacin, and PD 102807
  • Test compound(s)
  • 1 mM physostigmine stock (optional; for use with ACh) in freshly prepared 10 µM indomethacin/Krebs-Henseleit solution, bubbled with 95% O2/5% CO2
  • 95% (v/v) O2/5% CO2 cylinders with regulator capable of delivering 5 to 15 psi
  • Airtight chamber
  • Fine forceps and scissors
  • 5.0 surgical silk
  • Standard tissue bath equipment (unit 4.4)
  • Thermostatically regulated water circulator
  • Muscle holder (stationary metal rod; e.g., Fig. 4.5.3)
  • Force-displacement transducer (Grass FT03)
  • Polygraph/chart recorder (Grass 7E)

Alternate Protocol 1: Muscarinic M3 Receptors: Guinea Pig Trachea

 Additional Materials (also see Basic Protocol 3)
  • Hartley guinea pigs of either sex, 250 to 350 g
  • Tissue struts

Alternate Protocol 2: Muscarinic M3 Receptors: Guinea Pig Ileum

 Additional Materials (also see Basic Protocol 3)
  • Hartley guinea pigs of either sex, 250 to 350 g

Basic Protocol 4: Muscarinic M4 Receptors: Rabbit Anococcygeus

 Materials
  • Female New Zealand White rabbits, 2.5 to 3.0 kg (Charles River)
  • CO2 pellets
  • 1 mM phentolamine stock in modified Krebs-Henseleit solution (see recipe), freshly prepared and continuously oxygenated with 95% O2/5% CO2
  • 1 mM histamine in water (optional)
  • Standard muscarinic agonists (see recipe): e.g., acetylcholine (ACh), (+)-muscarine, oxotremorine M, (+)-cis-dioxolane, carbachol
  • Standard muscarinic antagonists (see recipe): e.g., atropine, pirenzepine, methoctramine, tripitramine, himbacine, para-fluorohexahydrosiladifendiol (p-F-HHSiD), darifenacin, and PD102807
  • Test compound(s)
  • 1 µM physostigmine (optional; for use with ACh) in freshly prepared 1 µM phentolamine/Krebs-Henseleit solution, bubbled with 95% O2/5% CO2
  • 95% (v/v) O2/5% CO2 cylinders with regulator capable of delivering 5 to 15 psi
  • Airtight chamber
  • Fine forceps and scissors
  • 4.0 surgical silk
  • Metal support rods
  • Standard tissue bath equipment (unit 4.4)
  • Thermostatically regulated water circulator
  • Force-displacement transducer (Grass FT03)
  • Polygraph/chart recorder (Grass 7E)
  • Platinum electrodes
  • Electrical stimulator (e.g., Grass S48)
Looking for materials?  Suppliers Guide
     
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Figures

  • Figure 4.15.1
    A typical polygraph tracing of responses to the nonselective muscarinic agonist (+)-cis-dioxolane in the canine saphenous vein preparation.

    View Image
  • Figure 4.15.2
    (+)-cis-Dioxolane- and oxotremorine M–induced contractions in the canine saphenous vein measured in grams tension (redrawn from Watson et al., 1995).

    View Image
  • Figure 4.15.3
    A typical polygraph tracing of responses to the nonselective muscarinic agonist, (+)-cis-dioxolane in the guinea pig paced left atrium preparation. Electrical field stimulation was 1 Hz, 1.0 msec, with 1.2× threshold voltage.

    View Image
  • Figure 4.15.4
    Methacholine (MeCh) produces a negative inotropic effect in the guinea pig paced left atrium preparation. Increasing concentrations of the muscarinic M2 receptor antagonist tripitramine result in a rightward shift in the concentration-effect curves (redrawn from Roffel et al., 1997).

    View Image
  • Figure 4.15.5
    A typical polygraph tracing of responses to the nonselective muscarinic agonist (+)-cis-dioxolane in the rat urinary bladder smooth muscle preparation.

    View Image
  • Figure 4.15.6
    Increasing concentrations of the muscarinic agonist 4-DAMP produce parallel rightward shifts of (+)-cis-dioxolane concentration-effect curves in rat urinary bladder smooth muscle preparations (redrawn from Hegde et al., 1997). 4-DAMP, 4-diphenyl-acetoxy-N-methyl-piperidine methiodide.

    View Image
  • Figure 4.15.7
    The anococcygeus muscles of the rabbit. (A) A cross-section of the lower abdominal cavity and (B) the pelvic region, with bladder, uterus, and vaginal/urethral orifice removed.

    View Image
  • Figure 4.15.8
    A typical polygraph tracing of the relaxant response to electrical field stimulation in a histamine-precontracted preparation of the rabbit anococcygeus muscle. Electrical field stimulation parameters: 3 Hz, 40 V, 0.5 msec for 10 sec every 3 min.

    View Image
  • Figure 4.15.9
    Concentration-effect curves for a range of muscarinic agonists in histamine-precontracted preparations of the rabbit anococcygeus muscle (redrawn from Gross et al., 1997b).

    View Image
  • Figure 4.15.10
    Parallel rightward shifts of (+)-muscarine concentration-effect curves produced by increasing concentrations of the muscarinic antagonists pirenzepine and himbacine in rabbit anococcygeus muscle preparations (redrawn from Gross et al., 1997b).

    View Image

Literature Cited

 Literature Cited
    Adem, A. and Karlson, E. 1997. Muscarinic receptor subtype selective toxins. Life Sci. 60:1069-1076.
    Blinks, J.R. 1966. Field stimulation as a means of effecting the graded release of autonomic transmitters in isolated heart muscle. J. Pharmacol. Exp. Ther. 151:221-235.
    Caulfield, M.P. and Birdsall, N.J.M. 1998. International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors. Pharmacol. Rev. 50:279-290.
    Chiarini, A., Budriesi, R., Bolognesi, M.L., Minarini, A., and Melchiorre, C. 1995. In vitro characterization of tripitramine, a polymethylene tetraamine displaying high selectivity and affinity for muscarinic M2 receptors. Br. J. Pharmacol. 114:1507-1517.
    Clague, R., Eglen, R.M., Strachan, A.C., and Whiting, R.L. 1985. Action of agonists and antagonists at muscarinic receptors present on ileum and atria in vitro. Br. J. Pharmacol. 86:163-170.
    Eglen, R.M. and Harris, G.C. 1993. Selective inactivation of muscarinic M2 and M3 receptors in guinea-pig ileum and atria in vitro. Br. J. Pharmacol. 109:946-952.
    Eglen, R.M. and Hegde, S.S. 1997. Muscarinic receptors and genitourinary smooth muscle function. In Muscarinic Receptor Subtypes in Smooth Muscle (R.M. Eglen, ed.) pp. 149-160. CRC Press, New York.
    Eglen, R.M., Montgomery, W.W., Dainty, I.A., Dubuque, L.K., and Whiting, R.L. 1988. The interaction of methoctramine and himbacine at atrial smooth muscle and endothelial muscarinic receptors in vitro. Br. J. Pharmacol. 95:1031-1038.
    Eglen, R.M., Michel, A.D., Montgomery, W.W., Kunysz, E.A., Machado, C.A., and Whiting, R.L. 1990. The interaction of para-fluorohexahydrosiladiphenidol at muscarinic receptors in vitro. Br. J. Pharmacol. 99:637-642.
    Eglen, R.M., Reddy, H., and Watson, N. 1994. Selective inactivation of muscarinic receptor subtypes. Int. J. Biochem. 26:1357-1368.
    Eglen, R.M., Hegde, S.S., and Watson, N. 1996a. Muscarinic receptor subtypes and smooth muscle function. Pharmacol. Rev. 48:531-565.
    Eglen, R.M., Pulido-Rios, M.T., Webber, A.P., Leung, E., and Hegde, S.S. 1996b. Characterization of the interaction of darifenacin at muscarinic receptors subtypes in vitro. Br. J. Pharmacol. 118:35P.
    Ford, A.P.D.W., Levine, W.B., Baxter, G.S., Harris, G.C., Eglen, R.M., and Whiting, R.L. 1991. Pharmacological, biochemical and molecular characterization of muscarinic receptors in the guinea-pig ileum: A multidisciplinary study. Mol. Neuropharmacol. 1:117-127.
    Gillani, S.A.H. and Cobbin, L.B. 1986. The cardioselectivity of himbacine: A muscarinic receptor antagonist. Naunyn-Schmiedebergs Arch. Pharmakol. 332:16-20.
    Gross, J., Augeli-Szafran, C.E., Czeche, S., Friebe, T., Jaen, J.C., Penvose-Yi, J.R., Schwarz, R.D., Mutschler, E., and Lambrecht, G. 1997a. Functional characterization of PD 102807: The first M4-selective muscarinic antagonist. Life Sci. 60:1168.
    Gross, J., Mutschler, E., and Lambrecht, G. 1997b. Evidence for muscarinic M4 receptors mediating nonadrenergic noncholinergic relaxations in rabbit anococcygeus muscle. Naunyn-Schmiedebergs Arch. Pharmakol. 356:505-516.
    Hegde, S.S., Choppin, A., Bonhaus, D., Briaud, S., Loeb, M., Moy, T.M., Loury, D., and Eglen, R.M. 1997. Functional role of M2 and M3 muscarinic receptors in the urinary bladder of rats in vitro and in vivo. Br. J. Pharmacol. 120:1409-1418.
    Jolkkonen, M., van Giersbergen, P.L.M., Hellman, U., Wernstedt, C., and Karlsson, E. 1994. A toxin from the green mamba Dendroaspis angusticeps: Amino acid sequence and selectivity for the muscarinic m4 receptor. FEBS Lett. 352:91-94.
    Roffel, A.F., Hamstra, J.J., Elzinga, C.R., and Zaagsma, J. 1994. Selectivity profile of some recent muscarinic antagonists in bovine and guinea-pig trachea and heart. Arch. Int. Pharmacodyn. Ther. 328:82-98.
    Roffel, A.F., Davids, J.H., Elzinga, C.R.S., Wolf, D., Zaagsma, J., and Kilbinger, H. 1997. Characterization of the muscarinic receptor subtype(s) mediating contraction of the guinea-pig lung strip and inhibition of acetylcholine release in the guinea-pig trachea with the selective muscarinic antagonist tripitramine. Br. J. Pharmacol. 122:133-141.
    Sagrada, A., Duranti, P., Giudici, L., and Schiavone, A. 1994. Himbacine discriminates between putative muscarinic M1 receptor-mediated responses. Life Sci. 54:PL305-310.
    Wallis, R.M. and Napier, C.M. 1999. Muscarinic antagonists in development for disorders of smooth muscle function. Life Sci. 64:395-401.
    Watson, N. and Eglen, R.M. 1994. Effect of muscarinic M2 and M3 receptor stimulation and antagonism on response to isoprenaline of guinea-pig trachea in vitro. Br. J. Pharmacol. 112:179-187.
    Watson, N. and Eglen, R.M. 1996. Selective muscarinic receptor agonists and antagonists. Pharmacol. Toxicol. 78:59-68.
    Watson, N., Reddy, H., and Eglen, R.M. 1995. Pharmacological characterization of the muscarinic receptor mediating contraction of canine saphenous vein. J. Autonom. Pharmacol. 15:437-441.
 Key References
    Blinks, 1966. See above.

Provides an experimental protocol for guinea pig paced left atria.

    Gross et al., 1997a,b. See, above.

Characterization of the rabbit anococcygeus muscle as an M4 preparation.

    Hegde et al., 1997. See above.

Characterization of the rat urinary bladder as an M3 preparation.

    Sagrada et al., 1994. See above.

Comparison of the effects of himbacine (M1-discriminating antagonist) between known (superior cervical ganglia and canine saphenous vein) and proposed (rabbit vas deferens) M1 preparations.

    Watson et al., 1995. See above.

Antagonist affinity estimates for five key antagonists at the canine saphenous vein M1 preparation.

     
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