In Vitro Isolated Tissue Functional Muscarinic Receptor Assays

M. Teresa Pulido‐Rios1, Tod Steinfeld1, Scott Armstrong1, Nikki Watson1, Agnes Choppin1, Richard Eglen1, Sharath S. Hegde1

1 Theravance, South San Francisco
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 4.15
DOI:  10.1002/0471141755.ph0415s48
Online Posting Date:  March, 2010
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Muscarinic receptor (mAChRs) subtypes are viable targets for the design of novel agents for use in a number of central and peripheral disorders. In vitro isolated tissue functional assays for muscarinic receptor subtypes have played an invaluable role in basic research and drug discovery. The availability of biological assays for generation of quantitative estimates of affinity and potency of ligands allows evaluation of the contribution of a given mAChR to the functional end organ response and also enables drug discovery by facilitating the iterative process of screening and optimization of chemical leads. This unit describes isolated tissue functional assays for the quantification of ligand affinity and efficacy at the M1, M2, M3, M4, and M5 muscarinic receptor subtypes in tissues expressing the native receptor using organ bath techniques. Curr. Protoc. Pharmacol. 48:4.15.1‐4.15.29. © 2010 by John Wiley & Sons, Inc.

Keywords: muscarinic; saphenous vein; rat bladder; rat duodenum; rabbit anococcygeus; guinea‐pig ileum; guinea‐pig trachea; basilar artery

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Characterization of Muscarinic M1 Receptors in the Canine Saphenous Vein Preparation
  • Alternate Protocol 1: Characterization of Muscarinic M1 Receptors in Isolated Rat Duodenum
  • Basic Protocol 2: Characterization of Muscarinic M2 Receptors in the Guinea Pig Paced Left Atrium
  • Basic Protocol 3: Characterization of Muscarinic M3 Receptors in the Isolated Rat Urinary Bladder
  • Alternate Protocol 2: Characterization of Muscarinic M3 Receptors in Isolated Guinea Pig Trachea
  • Alternate Protocol 3: Characterization of Muscarinic M3 Receptors in Isolated Guinea Pig Ileum
  • Basic Protocol 4: Muscarinic M4 Receptors: Rabbit Anococcygeus
  • Basic Protocol 5: Muscarinic M5 Receptors: Mouse Basilar Artery
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Characterization of Muscarinic M1 Receptors in the Canine Saphenous Vein Preparation

  Materials
  • Mongrel dog of either sex (10 to 30 kg; Charles River)
  • Sodium pentobarbital
  • Modified Krebs‐Henseleit solution (see recipe), freshly prepared and continuously aerated with carbogen (95% O 2/5% CO 2)
  • 1 mM physostigmine stock (for use with ACh) in freshly prepared modified Krebs‐Henseleit solution, bubbled with carbogen (95% O 2/5% CO 2), optional
  • 3 M KCl aqueous solution
  • Standard muscarinic agonists (see recipe): e.g., acetylcholine (ACh), oxotremorine M, (+)‐cis‐dioxolane, carbachol (CCh), methacholine (MCh), McN‐A‐343
  • Standard muscarinic antagonists (see recipe): e.g., atropine, pirenzepine, methoctramine, tripitramine, himbacine, para‐fluorohexahydrosiladifenidol (p‐F‐HHSiD), 4‐DAMP
  • Test compound(s)
  • Surgical instruments: scalpels, medium and fine forceps, heavy and fine scissors
  • 300‐ml beakers
  • Dissecting dish: Petri dish filled half‐way with silicone (e.g., Sylgard 184 Silicone Elastomer)
  • Tissue hooks
  • Tissue holders (a stationary rigid device, usually metal, used to mount the tissue in the tissue bath chamber, e.g., World Precision Instruments, Danish Myo Technology)
  • Standard tissue bath equipment (unit 4.4)
  • Thermostatically regulated water circulator
  • Force‐displacement transducer (e.g., Grass FT03, Danish Myo Technology Force transducer 750 TOBS, Biopac FT‐100)
  • Electronic data acquisition system (e.g., Biopac Acknowledge, Notocord, DMT) or polygraph/chart recorder (e.g., Grass 7E)

Alternate Protocol 1: Characterization of Muscarinic M1 Receptors in Isolated Rat Duodenum

  • Male Sprague‐Dawley rats (200 to 350 g; Charles River)
  • Source of CO 2 gas (e.g., gas cylinder)
  • Tyrode's solution (see recipe) also containing 10 µM indomethacin and 0.1 µM darifenacin, freshly prepared and continuously oxygenated with carbogen (95% O 2/5% CO 2)
  • Source of carbogen (95% O 2/5% CO 2; e.g., cylinders with regulator capable of delivering 5 to 15 psi)
  • Enclosed euthanasia chamber with inflow line for CO 2 gas delivery and a smaller outflow line for excess gas
  • 10‐ml syringes
  • 4‐0 surgical silk sutures

Basic Protocol 2: Characterization of Muscarinic M2 Receptors in the Guinea Pig Paced Left Atrium

  Materials
  • Male Hartley guinea pig (250 to 450 g; Charles River)
  • Source of CO 2 gas
  • Modified Krebs‐Henseleit solution (see recipe), freshly prepared and continuously aerated with carbogen (95% O 2/5% CO 2)
  • 1 mM physostigmine stock (for use with ACh) in freshly prepared modified Krebs‐Henseleit solution, bubbled with carbogen (95% O 2/5% CO 2), optional
  • Standard muscarinic agonists (see recipe): e.g., acetylcholine (ACh), oxotremorine M, (+)‐cis‐dioxolane, carbachol (CCh), McN‐A‐343
  • Standard muscarinic antagonists (see recipe): e.g., acetylcholine (ACh), atropine, pirenzepine, methoctramine, tripitramine, himbacine, para‐fluorohexahydrosiladifenidol (p‐F‐HHSiD), 4‐DAMP
  • Test compound(s)
  • Enclosed airtight chamber with inflow line for CO 2 gas delivery
  • Guillotine
  • Petri dishes filled half‐way with silicone (e.g., Sylgard 184 Silicone Elastomer)
  • 4‐0 surgical silk sutures
  • Tissue holders (a stationary rigid device, usually metal, used to mount the tissue in the tissue bath chamber, e.g., World Precision Instruments, Danish Myo Technology) with platinum electrodes
  • Force‐displacement transducer (e.g., Grass FT03, Danish Myo Technology Force transducer 750 TOBS, Biopac FT‐100)
  • 10‐ml standard tissue bath equipment (unit 4.4)
  • Electrical stimulator (e.g., Grass S48)
  • Electronic data acquisition system (e.g., Biopac Acknowledge, Notocord, DMT) or polygraph/chart recorder (e.g., Grass 7E)
  • Additional reagents and equipment for dissecting the left atrium (unit 4.3)

Basic Protocol 3: Characterization of Muscarinic M3 Receptors in the Isolated Rat Urinary Bladder

  Materials
  • Sprague‐Dawley or Wistar rats of either sex (200 to 300 g; Charles River)
  • CO 2 source
  • Modified Krebs‐Henseleit solution (see recipe), freshly prepared and continuously aerated with carbogen (95% O 2/5% CO 2)
  • 10 µM indomethacin in modified Krebs‐Henseleit solution (see recipe), freshly prepared and continuously oxygenated with 95% O 2/5% CO 2
  • 1 mM physostigmine stock (for use with ACh) in freshly prepared 10 µM indomethacin/Krebs‐Henseleit solution, bubbled with 95% O 2/5% CO 2, optional
  • 3.0 M KCl
  • Standard muscarinic agonists (see recipe): e.g., acetylcholine (ACh), (+)‐muscarine, oxotremorine M, (+)‐cis‐dioxolane, carbachol (CCh), McN‐A343
  • Standard muscarinic antagonists (see recipe): e.g., atropine, pirenzepine, methoctramine, tripitramine, himbacine, para‐fluorohexahydrosiladifenidol (p‐F‐HHSiD), 4‐DAMP
  • Test compound(s)
  • Enclosed euthanasia chamber with inflow line for CO 2 gas delivery and a smaller outflow line for excess gas
  • Surgical instruments
  • Petri dishes filled half‐way with silicone (e.g., Sylgard 184 Silicone Elastomer)
  • 5‐0 surgical silk sutures
  • 10‐ml standard tissue bath equipment (unit 4.4)
  • Tissue holders (a stationary rigid device, usually metal, used to mount the tissue in the tissue bath chamber, e.g., World Precision Instruments, Danish Myo Technology) with platinum electrodes
  • Force‐displacement transducer (e.g., Grass FT03, Danish Myo Technology Force transducer 750 TOBS, Biopac FT‐100)
  • Electronic data acquisition system (e.g., Biopac Acknowledge, Notocord, DMT) or polygraph/chart recorder (e.g., Grass 7E)

Alternate Protocol 2: Characterization of Muscarinic M3 Receptors in Isolated Guinea Pig Trachea

  • Hartley guinea pigs of either sex (250 to 350 g; Charles River)

Alternate Protocol 3: Characterization of Muscarinic M3 Receptors in Isolated Guinea Pig Ileum

  • Duncan‐Hartley guinea pigs of either sex (250 to 350 g; Charles River)

Basic Protocol 4: Muscarinic M4 Receptors: Rabbit Anococcygeus

  Materials
  • Female New Zealand white rabbits (2.5 to 3.0 kg; Charles River)
  • Source of CO 2 gas
  • 1 mM phentolamine stock in modified Krebs‐Henseleit solution (see recipe), freshly prepared and continuously oxygenated with 95% O 2/5% CO 2
  • 1 µM physostigmine (for use with ACh) in freshly prepared 1 µM phentolamine/Krebs‐Henseleit solution, bubbled with 95% O 2/5% CO 2, optional
  • 1 mM histamine in water, optional
  • Standard muscarinic agonists (see recipe): e.g., acetylcholine (ACh), (+)‐muscarine, oxotremorine M, (+)‐cis‐dioxolane, carbachol (CCh), McN‐A‐343
  • Standard muscarinic antagonists (see recipe): e.g., atropine, pirenzepine, methoctramine, tripitramine, himbacine, para‐fluorohexahydrosiladifendiol (p‐F‐HHSiD), 4‐DAMP
  • Test compound(s)
  • Enclosed airtight chamber with inflow line for CO 2 gas delivery
  • Surgical instruments
  • 4‐0 surgical silk sutures
  • Metal support rods
  • 10‐ml standard tissue bath equipment (unit 4.4)
  • Platinum electrodes
  • Electrical stimulator (e.g., Grass S48)
  • Electronic data acquisition system (e.g., Biopac Acknowledge, Notocord, DMT) or polygraph/chart recorder (e.g., Grass 7E)

Basic Protocol 5: Muscarinic M5 Receptors: Mouse Basilar Artery

  Materials
  • Male C57BL/6J mice (12 to 20 g; Charles River)
  • Source of CO 2 gas
  • Modified Krebs‐Henseleit solution (see recipe) also containing indomethacin (10 µM) freshly prepared and continuously oxygenated with carbogen (95% O 2/5% CO 2) (indomethacin/modified Krebs‐Henseleit buffer), ice cold
  • 1 mM physostigmine stock (for use with ACh) in freshly prepared modified Krebs‐Henseleit solution, bubbled with carbogen (95% O 2/5% CO 2), optional
  • 3 M KCl
  • Thromboxane mimetic U46619 (Sigma or Tocris)
  • Standard muscarinic agonists (see recipe): e.g., acetylcholine (ACh), methacholine (MCh), oxotremorine M, (+)‐cis‐dioxolane, carbachol (CCh), McN‐A‐343
  • Standard muscarinic antagonists (see recipe): e.g., atropine, pirenzepine, methoctramine, tripitramine, himbacine, para‐fluorohexahydrosiladifenidol (p‐F‐HHSiD), 4‐DAMP
  • Test compound(s)
  • Enclosed euthanasia chamber with inflow line for CO 2 gas delivery and a smaller outflow line for excess gas
  • Dissecting microscope
  • Glass micropipets with diameter between 30 and 80 µm
  • 10‐0 nylon monofilament sutures
  • Organ chamber
  • No‐flow tissue bath system with two buffer reservoirs (pressure myograph; Danish Myo Technology)
  • Video camera attached to a microscope
  • Electronic dimension analyzer (e.g., Living Systems Instrumentation)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Blinks, J.R. 1966. Field stimulation as a means of affecting 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.U., 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.
   Dorr, A., Sled, J.G., and Kabani, N. 2007. Three‐dimensional cerebral vasculature of the CBA mouse brain: A magnetic resonance imaging and micro computed tomography study. NeuroImage 35:1409‐1423.
   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 parafluorohexahydrosiladiphenidol 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.
   Fang, X., Faraci, F., Kaduce, T.L., Harmon, S., Modrick, M., Hu, S., Moore, S.A., Falck, J.R., Weintraub, N.L., and Spector, A. 2006. 20‐Hydroxyeicosatetraenoic acid is a potent dilator of mouse basilar artery: Role of cyclooxygenase. Am. J. Physiol.—Heart Circ. 291:H2301‐H2307.
   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.
   Gilani, A.H. and Cobbin, L.B. 1986. Cardio‐selectivity of himbacine: A muscarine receptor antagonist. Naunyn‐Schmiedeberg's Arch. Pharmacol. 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. N‐S Arch. Pharmakol. 356:505‐516.
   Hamrouni, A.M., Gudka, N., and Broadley, K.J. 2006. Investigation of the mechanism for the relaxation of rat duodenum mediated via M1 muscarinic receptors. Autonom. Autacoid. Pharmacol. 26:275‐284.
   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.
   Kenakin, T. 2003. A Pharmacology Primer: Theory, Application and Methods. Academic Press, San Diego.
   Micheletti, R., Schiavone, A., and Giachetti, A. 1988. Muscarinic M1 receptors stimulate a nonadrenergic noncholinergic inhibitory pathway in the isolated rat duodenum. J. Pharmacol. Exp. Ther. 244:680‐684.
   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‐PL310.
   Watson, N. and Eglen, R.M. 1994. Effects of muscarinic M2 and M3 receptor stimulation and antagonism on responses to isoprenaline of guinea‐pig trachea in vitro. Br. J. Pharmacol. 112:179‐187.
   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.
   Watson, N., Daniels, D.V., Ford, A.P., Eglen, R.M., and Hegde, S.S. 1999. Comparative pharmacology of recombinant human M3 and M5 muscarinic receptors expressed in CHO‐K1 cells. Br. J. Pharmacol. 127:590‐596.
   Yamada, M., Lamping, K.G., Duttaroy, A., Zhang, W., Cui, Y., Bymaster, F.P., McKinzie, D.L., Felder, C.C., Deng, C., Faraci, F.M., and Wess, J. 2001. Cholinergic dilation of cerebral blood vessels is abolished in M5 muscarinic acetylcholine receptor knockout mice. Proc. Natl. Acad. Sci. U.S.A. 98:14096‐14101.
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, 2006. See above.
  Provides a summary of the relevant in vitro data for common muscarinic receptor antagonists.
   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.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library