Tachykinin Receptor Assays

Stefania Meini1, Carlo Alberto Maggi1

1 Menarini Ricerche, Florence, Italy
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 4.10
DOI:  10.1002/0471141755.ph0410s48
Online Posting Date:  March, 2010
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Described in this unit are methods for obtaining, preparing, and testing smooth muscle preparations bearing tachykinin receptors to study the agonist or antagonist properties of test compounds. Concentration‐response curves to agonists are constructed to measure their ability to produce smooth muscle contractions and thus evaluate the potency and efficacy of the agonists. Antagonists are tested for their ability to shift the agonist concentration‐response curve and to calculate their potency. Two different protocols are described for each of the three tachykinin receptors (NK1, NK2, and NK3). The NK1 receptor assays use guinea pig ileum longitudinal muscle myenteric plexus (GPI) and rat urinary bladder (RUB), the NK2 receptor assays use isolated endothelium‐deprived rabbit pulmonary artery (RPA) and hamster trachea (HT), and the NK3 receptor assays use GPI and rat portal vein (RPV). Curr. Protoc. Pharmacol. 48:4.10.1‐4.10.28. © 2010 by John Wiley & Sons, Inc.

Keywords: tachykinin receptor; smooth muscle; contractility; antagonism; agonism; in vitro bioassay; selectivity

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

Table of Contents

  • Introduction
  • Basic Protocol 1: Evaluation of Tachykinin NK1 Receptors in Isolated Guinea Pig Ileum Longitudinal Muscle Myenteric Plexus (GPI)
  • Alternate Protocol 1: Evaluation of Tachykinin NK1 Receptors in Isolated Rat Urinary Bladder (RUB)
  • Basic Protocol 2: Evaluation of Tachykinin NK2 Receptors in Isolated Endothelium‐Deprived Rabbit Pulmonary Artery (RPA)
  • Alternate Protocol 2: Evaluation of Tachykinin NK2 Receptors in Isolated Hamster Trachea (HT)
  • Basic Protocol 3: Evaluation of Tachykinin NK3 Receptors in Isolated GPI
  • Alternate Protocol 3: Evaluation of Tachykinin NK3 Receptors in Isolated Rat Portal Vein (RPV)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Evaluation of Tachykinin NK1 Receptors in Isolated Guinea Pig Ileum Longitudinal Muscle Myenteric Plexus (GPI)

  Materials
  • Male Hartley guinea pigs, 250 to 350 g body weight (Charles River Laboratories)
  • Krebs solution (see recipe), 37°C, preoxygenated with carbogen gas (95% O 2/5% CO 2)
  • Bath solution 1 (see recipe), 37°C, continuously oxygenated with carbogen gas
  • Agonist stock solutions: 10 mM, 1 mM, 100 µM, 10 µM, 1 µM, and 0.1 µM [Sar9,Met(O 2)11]substance P ([Sar9]SP sulfone; Sigma‐Aldrich, Tocris Bioscience; Table 4.10.1) in water
  • 4 M KCl in water
  • Antagonist stock solution: 0.1 mM FK 888 (Tocris Bioscience) in dimethyl sulfoxide (DMSO)
  • Surgical instruments
  • Petri dishes with silicone elastomer bottoms (e.g., Sylgard 184, Dow Corning)
  • Cotton thread
  • Cotton wool
  • 6‐ to 8‐mm‐diameter glass tubing, bent at a 45° angle (for preparation of longitudinal muscle strips)
  • Organ bath equipment (e.g., World Precision Instruments, Radnoti Glass Technology), consisting of jacketed organ chamber(s) (see note regarding chamber volume) and circulating water heater, with isometric force transducer(s) and potentiometric chart recorder(s) (e.g., Ugo Basile, Grass Instruments, or AD Instruments; see Fig. )
  • 10‐ to 20‐ml plastic syringes
  • 10‐ to 100‐µl glass microsyringes or micropipets in the appropriate volume range

Alternate Protocol 1: Evaluation of Tachykinin NK1 Receptors in Isolated Rat Urinary Bladder (RUB)

  • Male Wistar rats, 300 to 400 g body weight (Charles River Laboratories)
  • Bath solution 2 (see recipe), 37°C, continuously oxygenated with carbogen gas
  • Antagonist stock solution: 1 mM RP 67580 (Tocris Bioscience) in DMSO
  • Organ bath equipment set up as for GPI preparations (see protocol 1) except using isotonic instead of isometric transducers (e.g., Ugo Basile, Grass Instruments, or AD Instruments)

Basic Protocol 2: Evaluation of Tachykinin NK2 Receptors in Isolated Endothelium‐Deprived Rabbit Pulmonary Artery (RPA)

  Materials
  • Male New Zealand White (NZW) rabbits, 2.5 to 3.5 kg body weight (Charles River Laboratories)
  • Krebs solution (see recipe), 37°C, continuously oxygenated with carbogen gas (95% O 2/5% CO 2)
  • 1 mM norepinephrine (Sigma‐Aldrich) in water
  • 100 mM acetylcholine in water
  • Agonist stock solutions: 10 mM, 1 mM, 100 µM, 10 µM, 1 µM, and 0.1 µM neurokinin A (NKA; Sigma‐Aldrich) in water
  • 4 M KCl in water
  • Antagonist stock solution: 0.1 mM MEN 10376 in water (Genscript, ChemPep Inc, Sigma‐Aldrich, Genway, Tocris Bioscience)
  • Surgical instruments
  • Petri dishes with silicone elastone bottom (see protocol 1)
  • Cotton‐tipped applicators
  • Cotton thread
  • Organ bath equipment (see protocol 1), with isometric transducers
  • 10‐ to 100‐µl glass microsyringes

Alternate Protocol 2: Evaluation of Tachykinin NK2 Receptors in Isolated Hamster Trachea (HT)

  • Male Syrian golden hamsters (or LVG hamsters; Charles River Laboratories), 100 to 150 g body weight
  • Antagonist stock solution: 0.1 mM L 659,877 in DMSO (Sigma‐Aldrich)
  • Two stainless steel hooks, each ∼1 cm long, connected to a cotton thread, for each preparation

Basic Protocol 3: Evaluation of Tachykinin NK3 Receptors in Isolated GPI

  Materials
  • Male Hartley guinea pigs, 250 to 350 g body weight (Charles River Laboratories)
  • Krebs solution (see recipe), 37°C, preoxygenated with carbogen gas (95% O 2/5% CO 2)
  • Bath solution 3 (see recipe), 37°C, continuously oxygenated with carbogen gas
  • Agonist stock solutions: 10 mM, 1 mM, 100 µM, 10 µM, 1 µM, 100 nM, and 10 nM senktide (Sigma‐Aldrich, Tocris Bioscience) in water
  • 10 mM carbachol (Sigma‐Aldrich) in water
  • Antagonist stock solution: 0.1 mM SR 142801 (this compound is not commercially available, but can be requested from Sanofi‐Aventis,) in DMSO
  • Surgical instruments
  • Petri dishes with rubber bottom (see protocol 1)
  • Cotton thread
  • Wads of cotton wool
  • 6‐ to 8‐mm‐diameter glass tubing, bent at a 45° angle (for preparation of longitudinal muscle strips)
  • Organ bath equipment (see protocol 1), with isotonic force transducers
  • 10‐ to 20‐ml plastic syringes
  • 10‐ to 100‐µl glass microsyringes

Alternate Protocol 3: Evaluation of Tachykinin NK3 Receptors in Isolated Rat Portal Vein (RPV)

  • Male Wistar rats, 250 to 350 g body weight (Charles River Laboratories)
  • 4 M KCl in water
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Advenier, C., Rouissi, N., Nguyen, Q.T., Emonds‐Alt, X., Breliere, J.C., Neliat, G., Naline, E., and Regoli, D. 1992. Neurokinin A (NK2) receptor revisited with SR 48968, a potent non‐peptide antagonist. Biochem. Biophys. Res. Commun. 184:1418‐2144.
   Almeida, T.A., Rojo, J., Nieto, P.M., Pinto, F.M., Hernandez, M., Martín, J.D., and Candenas, M.L. 2004. Tachykinins and tachykinin receptors: Structure and activity relationships. Curr. Med. Chem. 11:2045‐2081.
   Astolfi, M., Treggiari, S., Giachetti, A., Meini, S., Maggi, C.A., and Manzini, S. 1994. Characterization of the tachykinin NK2 receptor in the human bronchus: Influence of amastatin‐sensitive metabolic pathways. Br. J. Pharmacol. 111:570‐574.
   Bellucci, F., Carini, F., Catalani, C., Cucchi, P., Lecci, A., Meini, S., Patacchini, R., Quartara, L., Ricci, R., Tramontana, M., Giuliani, S., and Maggi, C.A. 2002. Pharmacological profile of the novel mammalian tachykinin, hemokinin 1. Br. J. Pharmacol. 135:266‐274.
   Camarda, V., Rizzi, A., Calò, G., Guerrini, R., Salvadori, S., and Regoli, D. 2002. Pharmacological profile of hemokinin 1: A novel member of the tachykinin family. Life Sci. 71:363‐370.
   Catalioto, R.M., Criscuoli, M., Cucchi, P., Giachetti, A., Gianotti, D., Giuliani, S., Lecci, A., Lippi, A., Patacchini, R., Quartara, L., Renzetti, A.R., Tramontana, M., Arcamone, F., and Maggi, C.A. 1998. MEN 11420 (Nepadutant), a novel glycosylated bicyclic peptide tachykinin NK2 receptor antagonist. Br. J. Pharmacol. 123:81‐91.
   Cialdai, C., Tramontana, M., Patacchini, R., Lecci, A., Catalani, C., Catalioto, R‐M., Meini, S., Valenti, C., Altamura, M., Giuliani, S., and Maggi, C.A. 2006. MEN15596, a novel nonpeptide tachykinin NK2 receptor antagonist. Eur. J. Pharmacol. 549:140‐148.
   D'Orléans‐Juste, P., Dion, S., Drapeau, G., and Regoli, D. 1985. Different receptors are involved in the endothelium‐mediated relaxation and smooth muscle contraction of the rabbit pulmonary artery in response to substance P and related neurokinins. Eur. J. Pharmacol. 125:37‐44.
   Hall, J.M., Flowers, J.M., and Morton, I.K. 1992. A pharmacological study of NK1 and NK2 tachykinin receptor characteristics in the rat isolated urinary bladder. Br. J. Pharmacol. 107:777‐784.
   Lecci, A. and Maggi, C.A. 2008. Tachykinins and their receptors. In Encyclopedia of Molecular Pharmacology, 2nd ed. (S. Offermanns and W. Rosenthal, eds.) pp. 1181‐1191. Springer‐Verlag, New York.
   Lecci, A., Capriati, A., Altamura, M., and Maggi, CA. 2006. Tachykinins and tachykinin receptors in the gut, with special reference to NK2 receptors in human. Auton. Neurosci. 126‐127:232‐249.
   Leffler, A., Ahlstedt, I., Engberg, S., Svensson, A., Billger, M., Oberg, L., Bjursell, M.K., Lindstrom, E., and von Mentzer, B. 2009. Characterization of species‐related differences in the pharmacology of tachykinin NK receptors 1, 2 and 3. Biochem. Pharmacol. 77:1522‐1530.
   Maggi, C.A. 1994. Evidence for receptor subtypes/species variants of receptors. In The Tachykinin Receptors (S.H. Buck, ed.) pp. 395‐470. Humana Press, Totowa, N.J.
   Maggi, C.A. 1995. The mammalian tachykinin receptors. Gen. Pharmacol. 26:911‐944.
   Maggi, C.A. and Schwartz, T.W. 1997. The dual nature of the tachykinin NK1 receptor. Trends Pharmacol. Sci. 18:351‐354.
   Maggi, C.A., Patacchini, R., Rovero, P., and Meli, A. 1989. The hamster isolated trachea: A new preparation for studying NK‐2 receptors. Eur. J. Pharmacol. 166:435‐440.
   Maggi, C.A., Patacchini, R., Rovero, P., and Giachetti, A. 1993. Tachykinin receptors and tachykinin receptor subtypes. J. Auton. Pharmacol. 13:23‐93.
   Meini, S., Patacchini, R., and Maggi, C.A. 1994. Tachykinin NK1 receptor subtypes in the rat urinary bladder. Br. J. Pharmacol. 111:739‐746.
   Patacchini, R., Barthò, L., Holzer, P., and Maggi, C.A. 1995. Activity of SR 142801 at peripheral tachykinin receptors. Eur. J. Pharmacol. 278:17‐25.
   Patacchini, R., Giuliani, S., Turini, A., Navarra, G., and Maggi, C.A. 2000. Effect of nepadutant at tachykinin NK(2) receptors in human intestine and urinary bladder. Eur. J. Pharmacol. 398:389‐397.
   Pennefather, J.N., Lecci, A., Candenas, M.L., Patak, E., Pinto, F.M., and Maggi, C.A. 2004. Tachykinins and tachykinin receptors: A growing family. Life Sci. 74:1445‐1463.
   Petitet, F., Saffroy, M., Torrens, Y., Lavielle, S., Chassaing, G., Loeuillet, D., Glowinski, J., and Beaujouan, J.C. 1992. Possible existence of a new tachykinin receptor subtype in the guinea‐pig ileum. Peptides 13:383‐388.
   Quartara, L. and Altamura, M. 2006. Tachykinin receptors antagonists: From research to clinic. Curr. Drug Targets 7:975‐992.
   Regoli, D., Boudon, A., and Fauchére, J.L. 1994. Receptors and antagonists for substance P and related peptides. Pharmacol. Rev. 46:551‐599.
   Sellers, D.J., Chapple, C.R., Hay, D.P.W., and Chess‐Williams, R. 2006. Depressed contractile responses to neurokinin A in idiopathic but not neurogenic overactive human detrusor muscle. Eur. Urol. 49:510‐518.
   Tousignant, C., Dion, S., Drapeau, G., and Regoli, D. 1987. Characterization of pre‐ and postjunctional receptors for neurokinins and kinins in the rat vas deferens. Neuropeptides 9:333‐343.
   Zhang, Y., Lu, L., Furlonger, C., Wu, G.E., and Paige, C.J. 2000. Hemokinin is a hematopoietic‐specific tachykinin that regulates B lymphopoiesis. Nature Immunol. 1:392‐397.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library