P1 (Adenosine) Purinoceptor Assays

S. Jamal Mustafa1, Habib R. Ansari1, Worku Abebe2

1 Robert C. Byrd Health Science Center, West Virginia University, Morgantown, West Virginia, 2 Medical College of Georgia, School of Dentistry, Augusta, Georgia
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 4.7
DOI:  10.1002/0471141755.ph0407s45
Online Posting Date:  June, 2009
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Abstract

P1 purinoceptors, or adenosine (ADO) receptors, mediate the biological effects of the endogenous nucleoside, ADO and its analogs. ADO works through four receptor subtypes: A1, A2A, A2B, and A3. Isolated tissue assays used for the pharmacological characterization of ADO receptors based on functional responses are described in this unit. The guinea pig atrium, pig coronary artery, guinea pig aorta ,and mouse aorta have been used for the characterization of ADO receptor subtypes. Curr. Protoc. Pharmacol. 45:4.7.1‐4.7.13. © 2009 by John Wiley & Sons, Inc.

Keywords: P1 purinoceptors; adenosine receptors; isolated tissues; guinea pig atrium; pig coronary artery; guinea pig aorta; mouse aorta

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measurement of Tissue Responsiveness to P1 Purinoceptor Agonists
  • Support Protocol 1: Preparation of Guinea Pig Left Atrium
  • Support Protocol 2: Preparation of Pig Left Circumflex Coronary Artery
  • Support Protocol 3: Preparation of Guinea Pig Aorta
  • Support Protocol 4: Preparation of Mouse Aorta
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of Tissue Responsiveness to P1 Purinoceptor Agonists

  Materials
  • Prepared tissue in isolated organ baths: guinea pig atrial strip for A 1 receptors (see protocol 2); pig coronary artery for A 2A receptors (see protocol 3); guinea pig aorta for A 2B receptors (see protocol 4); or mouse aorta (see protocol 5).
  • Bathing medium: Krebs‐Henseleit solution, pH 7.4 (see recipe), 34°C (for atrium) and 37°C (for blood vessels), continuously bubbled with carbogen gas
  • 10 µM isoproterenol hydrochloride (Sigma) in H 2O
  • 1 mM S‐(p‐nitrobenzyl) thioinosine (Sigma) in H 2O
  • 1 mM erythro‐9‐(2‐hydroxy‐3‐nonyl) adenine (EHNA; Sigma) in H 2O
  • 10 mM, 1 mM, 100 µM, 10 µM, 1 µM, 100 nM, and 10 nM adenosine, P1 purinoceptor agonist, or test compound (see recipe and Table 4.7.2)
  • 10 mM, 1 mM, and 100 µM P1 purinoceptor antagonist or test compound (see recipe and Table 4.7.2)
  • 10 mM, 1 mM, 100 µM, 10 µM, and 1 µM carbachol (Sigma) in H 2O
  • 10 mM, 1 mM, 100 µM, 10 µM, and 1 µM sodium nitroprusside (Sigma) in H 2O
  • 10 mM and 10 M KCl
  • 0.1 mM bradykinin (Sigma) in H 2O
  • 10 mM prostaglandin F (Sigma) in H 2O
  • Additional reagents and equipment for maintaining and measuring response in isolated cardiac preparations (unit 4.3) and blood vessel preparations (unit 4.4) and for making dose‐response/antagonism measurements (unit 4.1)

Support Protocol 1: Preparation of Guinea Pig Left Atrium

  Materials
  • Guinea pig (Dunkin Hartley, male, 300 to 500 g, 21 to 40 days; Charles River Labs)
  • Pentobarbital sodium (Sigma)
  • Krebs‐Henseleit solution, pH 7.4 (unit 4.3), ice cold and 34°C
  • Bipolar platinum electrode (Grass)
  • Fixed‐range precision force transducer (BIOPAC System, model TSD125C) connected to differential amplifier (BIOPAC system, model DA100B)
  • 5‐0 silk thread
  • Electrical stimulator (Grass, model SD9)
  • Additional reagents and equipment for making and maintaining isolated cardiac preparations and constructing length‐tension curves (units 4.2& 4.3)

Support Protocol 2: Preparation of Pig Left Circumflex Coronary Artery

  Materials
  • Pig (25 to 45 kg, <1 year old; obtained from local slaughterhouse)
  • Krebs‐Henseleit solution, pH 7.4 (unit 4.3), ice cold and 37°C
  • Triangle‐shaped stainless‐steel wire hooks (made using stainless steel wires purchased from Stainless Steel Products, cat. no. AISI‐36L; see Fig. )
  • Fixed‐range precision force transducer (BIOPAC System, model TSD125C) connected to differential amplifier (BIOPAC System, model DA100B )
  • Digital acquisition system with Acknowledge 3.5.7 software (BIOPAC System)
  • Additional reagents and equipment for making and maintaining isolated cardiac preparations and constructing length‐tension curves (units 4.2& 4.3) and making ring preparations of blood vessels (unit 4.4)

Support Protocol 3: Preparation of Guinea Pig Aorta

  Materials
  • Guinea pig (Dunkin Hartley, male, 300 to 500 g, 21 to 40 days; Charles River Labs)
  • Pentobarbital sodium (Sigma)
  • Krebs‐Henseleit solution, pH 7.4 (unit 4.3), ice cold and 37°C
  • Triangle‐shaped stainless‐steel wire hooks (made using stainless steel wires purchased from Stainless Steel Products, cat. no. AISI‐36L; Fig. )
  • Fixed‐range precision force‐transducer (BIOPAC System, model TSD125C) connected to differential amplifier (BIOPAC System, model DA 100B)
  • Digital acquisition system with Acknowledge 3.5.7 software (BIOPAC System)
  • Additional reagents and equipment for making and maintaining isolated cardiac preparations and constructing length‐tension curves (units 4.2& 4.3) and making ring preparations of blood vessels (unit 4.4)

Support Protocol 4: Preparation of Mouse Aorta

  Materials
  • Mice (10‐ to 12‐weeks‐old, 25 to 30 g; Harlan Sprague Dawley)
  • Pentobarbital sodium (Sigma)
  • Krebs‐Henseleit buffer, pH 7.4 (unit 4.3), ice cold and 37°C
  • Triangular (base = 1.5 cm, side length = 2 cm each) stainless steel wire hooks (Stainless Steel Products)
  • Fixed‐range precision force‐displacement transducer (BIOPAC System, model TSD125C) connected to differential amplifier (BIOPAC System, model DA100B)
  • Digital force transducer acquisition system (BIOPAC)
  • Additional reagents and equipment for making and maintaining isolated aortic preparations and constructing length‐tension curves (units 4.2& 4.3) and making aortic ring preparations (unit 4.4)
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Figures

Videos

Literature Cited

   Abebe, W., Makujina, S.R., and Mustafa, S.J. 1994. Adenosine receptor‐mediated relaxation of porcine coronary artery in presence and absence of endothelium. Am. J. Physiol. 266:H2018‐H2025.
   Ansari, H.R., Nadeem, A., Talukder, M.A., Sakhalkar, S., and Mustafa, S.J. 2007a. Evidence for the involvement of nitric oxide in A2B receptor‐mediated vasorelaxation of mouse aorta. Am. J. Physiol. Heart Circ. Physiol. 292:H719‐H725.
   Ansari, H.R., Nadeem, A., Tilley, S.L., and Mustafa, S.J. 2007b. Involvement of COX‐1 in A3 adenosine receptor‐mediated contraction through endothelium in mice aorta. Am. J. Physiol. Heart Circ. Physiol. 293:H3448‐H3455.
   Collis, M.G. 1991. Adenosine receptor in isolated tissue preparations. Nucleos. Nucleot. 10:1057‐1068.
   Fredholm, B.B., Ijzerman, A.P., Jacobson, K.A., Klotz, K.N., and Linden, J. 2001. International Union of Pharmacology. XXV. Nomenclature and Classification of Adenosine Receptors. Pharmacol. Rev. 53:527‐552.
   Jacobson, K.A., Park, K.S., Jiang, J.L., Kim, Y.C., Olah, M.E., Stiles, G.L., and Ji, X.D. 1997. Pharmacological characterization of novel A3 adenosine receptor‐selective antagonists. Neuropharmacology 36:1157‐1165.
   Jacobson, K.A. and Gao, Z.G. 2006. Adenosine receptors as therapeutic targets. Nat. Rev. Drug Discov. 5:247‐264.
   Makujina, S.R. and Mustafa, S.J. 1993. Adenosine‐5′‐urosamides rapidly desensitize the A2 adenosine receptor in coronary artery. J. Cardiovasc. Pharmacol. 22:506‐509.
   Mustafa, S.J., Nadeem, A., Fan, M., Zhong, H., Belardinelli, L., and Zeng, D. 2007. Effect of a specific and selective A(2B) adenosine receptor antagonist on adenosine agonist AMP and allergen‐induced airway responsiveness and cellular influx in a mouse model of asthma. J. Pharmacol. Exp. Ther. 320:1246‐1251.
   Olsson, R.A. and Pearson, J.D. 1990. Cardiovascular purinoceptors. Physiol. Rev. 70:761‐845.
   Palmer, T.M. and Stiles, G.L. 1995. Adenosine receptors. Neuropharmacology 34:683‐695.
   Ponnoth, D.S., Nadeem, A., and Mustafa, S.J. 2008. Adenosine‐mediated alteration of vascular reactivity and inflammation in a murine model of asthma. Am. J. Physiol. Heart Circ. Physiol. 294:H2158‐H2165.
   Ramagopal, M.V. and Mustafa, S.J. 1988. Effect of adenosine and its analogs on calcium influx in coronary artery. Am. J. Physiol. 24:H1492‐H1498.
   Tabrizchi, R. and Bedi, S. 2001. Pharmacology of adenosine receptors in the vasculature. Pharmacol. Ther. 91:133‐147.
   Tawfik, H.E., Schnermann, J., Oldenburg, PJ., and Mustafa, S.J. 2005. Role of A1 adenosine receptors in regulation of vascular tone. Am. J. Physiol. Heart Circ. Physiol. 288:H1411‐H1416.
   Teng, B., Ledent, C., and Mustafa, S.J. 2008. Up‐regulation of A2B adenosine receptor in A2A adenosine receptor knockout mouse coronary artery. J. Mol. Cell. Cardiol. 44:905‐914.
Key References
   Collis, 1991. See above.
  Describes ADO receptors in isolated tissue preparations and pA2 values for various antagonists.
   Palmer and Stiles, 1995. See above.
  Provides comprehensive information on ADO receptors and their mechanisms of action.
   Tabrizchi and Bedi, 2001. See above.
  Pharmacology of adenosine receptors in the vasculature.
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