In Vivo Pharmacodynamic Assays for M2 and M3 Muscarinic Receptors

Scott Armstrong1, Tod Steinfeld1, M. Teresa Pulido‐Rios1, Sharath S. Hegde1

1 Theravance, South San Francisco, California
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 12.13
DOI:  10.1002/0471141755.ph1213s48
Online Posting Date:  March, 2010
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M2 and M3 muscarinic receptor subtypes are attractive drug targets for the treatment of pulmonary and urological disorders. Described in this unit is an in vivo pithed rat assay for estimating agonist and antagonist potency at M2 and M3 receptors. In the pithed rat, the muscarinic agonist methacholine induces reduction in heart rate (bradycardia) and blood pressure (depressor response) through interaction with M2 and M3 receptors, respectively. The dissociation of the peripheral and central nervous system in the pithed rat preparation permits the direct assessment of compound effects on the heart and vasculature in the absence of cardiovascular reflexes. Estimates of antagonist potency can be reliably established by quantifying the shift in the agonist dose‐effect curve produced under appropriate equilibrium or non‐equilibrium conditions. Curr. Protoc. Pharmacol. 48:12.13.1‐12.13.10. © 2010 by John Wiley & Sons, Inc.

Keywords: muscarinic; acetylcholine; in vivo; pithed rat; depressor; bradycardia; M2 receptor; M3 receptor

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Table of Contents

  • Introduction
  • Basic Protocol 1: Assessment of Agonist and Antagonist Potency at Muscarinic M2 and M3 Receptors in a Pithed Rat Preparation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Assessment of Agonist and Antagonist Potency at Muscarinic M2 and M3 Receptors in a Pithed Rat Preparation

  • Female Sprague‐Dawley rats (225 to 275 g; Harlan)
  • Sodium pentobarbital (e.g., Nembutal, Ovation Pharma)
  • Povidone‐iodine antiseptic
  • Alcohol pads (70% isopropanol)
  • Physiological saline (0.9% NaCl)
  • Heparin‐saline solution (e.g., 1000 U/ml; see recipe)
  • Angiotensin II (human Ang‐II; see recipe)
  • Standard muscarinic agonists (see recipe): e.g., methacholine (MCh)
  • Standard muscarinic antagonists (see recipe): e.g., atropine, oxybutynin, tolterodine, solifenacin, darifenacin, Ro 320‐6206, and tiotropium
  • Shaver
  • Heated water pad (e.g., Gaymar TP‐500)
  • Surgical instruments: scalpels, heavy and fine scissors, fine forceps (straight and curved), and hemostats
  • Cannulae (PE‐10, PE‐50, and PE‐240; see recipe)
  • 4‐0 silk sutures
  • Surgical staples
  • Gauze pads
  • Electronic data acquisition system (e.g., Biopac Acknowledge, Notocord)
  • Pithing rod, stainless steel, 1.5‐mm diameter (e.g., Susan Bates double‐point knitting needles)
  • Small animal ventilator (e.g., Harvard Apparatus, model 683)
  • Infusion pump (e.g., WPI, model SP220i)
  • Tubing adaptor (e.g., Becton‐Dickenson tubing adaptor with plastic hub removed)
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Literature Cited

   Angeli, P., Cantalamessa, F, Gulini, U., and Melchiorre, C. 1995. Selective blockade of muscarinic M2 receptors in vivo by the new antagonist tripitramine. N‐S Arch. Pharmakol. 352:304‐307.
   Armstrong, S.R., Briones, S., Horger, B., Richardson, C.L., Jaw‐Tsai, S., and Hegde, S.S. 2008. Pharmacological analysis of the interaction of antimuscarinic drugs at M2 and M3 muscarinic receptors in vivo using the pithed rat assay. N‐S Arch. Pharmakol. 376:341‐349.
   Barnes, P.J. 2004. The role of anticholinergics in chronic obstructive pulmonary disease. Am. J. Med. 117:24S‐32S.
   Caulfield, M.P. and Birdsall, N.J.M. 1998. International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors. Pharmacol. Rev. 50:279‐290.
   Eglen, R.M., Hegde, S.S., and Watson, N. 1996. Muscarinic receptor subtypes and smooth muscle function. Pharmacol. Rev. 48:531‐565.
   Gillespie, J.S. and Muir, T.C. 1967. A method of stimulating the complete sympathetic outflow from the spinal cord to blood vessels in the pithed rat. Br. J. Pharmacol. Chemother. 30:78‐87.
   Hegde, S.S. 2006. Muscarinic receptors in the bladder: From basic research to therapeutics. Br. J. Pharmacol. 147:S80‐S87.
   Kenakin, T. 2003. A Pharmacology Primer: Theory, Application and Methods, 2nd ed. pp. 147‐173. Academic Press, San Diego.
   Martin, J.R. 1996. Pressor effect of the putative M1 muscarinic receptor agonist MCN‐A‐343 in the conscious rat. Life Sci. 59:1839‐1852.
   Sagrada, A., Schiavi, G.B., Cereda, E., and Ladinsky, H. 1994. Antagonist properties of McNeil‐A‐343 at 5‐HT4 and 5‐HT3 receptors. Br. J. Pharmacol. 113:711‐716.
   Waynforth, H.B. and Flecknell, P.A. 1999. Experimental and Surgical Technique in the Rat, 2nd ed. pp. 225‐235. Academic Press, San Diego.
Key References
   Armstrong et al., 2008. See above.
  Provides an experimental protocol and pithed rat model characterization using various muscarinic antagonists.
   Gillespie and Muir, 1967. See above.
  Provides additional detail on the pithed rat preparation.
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