α‐Adrenoceptor Assays

Peter W. Abel1, Neha Jain1

1 Department of Pharmacology, Creighton University School of Medicine, Omaha, Nebraska
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 4.5
DOI:  10.1002/0471141755.ph0405s59
Online Posting Date:  December, 2012
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Abstract

α‐Adrenoceptors mediate responses to activation of both peripheral sympathetic nerves and central noradrenergic neurons. They also serve as autoreceptors that modulate the release of norepinephrine (NE) and other neurotransmitters. There are two major classes of α‐adrenoceptors, the α1‐ and α2. Each class is subdivided into three subtypes: α1A, α1B, α1D, and α2A, α2B, α2C. Described in this unit are in vitro isolated tissue methods used to study α‐adrenoceptor functions and to identify novel ligands for these receptors. Detailed protocols describing use of isolated tissues to study the various α1‐ and α2‐adrenoceptor subtypes are provided. Curr. Protoc. Pharmacol. 59:4.5.1‐4.5.32. © 2012 by John Wiley & Sons, Inc.

Keywords: alpha adrenoceptors; bioassay; isolated tissues

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Evaluation of α1A‐Adrenoceptors in Isolated Rat Vas Deferens
  • Support Protocol 1: Setup of Tissue Bath Apparatus for Adrenoceptor Studies
  • Evaluation of α‐Adrenoceptor Subtypes Using Alternative Tissues
  • Alternate Protocol 1: Evaluation of α1A‐Adrenoceptors in Canine Prostate
  • Alternate Protocol 2: Evaluation of α1B‐Adrenoceptors in Rat Spleen
  • Alternate Protocol 3: Evaluation of α1D‐Adrenoceptors in Isolated Rat Aorta
  • Alternate Protocol 4: Evaluation of α2C‐Adrenoceptors in Isolated Human Saphenous Vein
  • Alternate Protocol 5: Evaluation of α2A‐Adrenoceptors in Isolated Rat Vas Deferens Using Electrical Stimulation
  • Basic Protocol 2: Evaluation of α2‐Adrenoceptors by Superfusion of Radiolabeled Isolated Tissues
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Evaluation of α1A‐Adrenoceptors in Isolated Rat Vas Deferens

  Materials
  • Male Sprague‐Dawley rats, ∼250 g
  • Modified Krebs‐Henseleit buffer solution (see recipe)
  • 4.0 M KCl solution
  • Reference compounds (e.g., phenylephrine or prazosin; Sigma or Tocris) dissolved in appropriate solvent at 1000× working concentration
  • 100 µM phenoxybenzamine (Enzo or Sigma) diluted from 10 mM stock in ethanol using 0.3 mM ascorbic acid.
  • 1 N HCl in 95% ethanol
  • Pressurized CO 2 gas source with 2 stage regulator
  • Small‐animal decapitator (Kent Scientific)
  • Water‐jacketed petri dish for preparation of the tissues (Radnoti, http://www.radnoti.com/)
  • Fine forceps
  • Iris scissors
  • 4‐0 surgical silk (Ethicon)
  • Tissue tension apparatus (see protocol 2)

Support Protocol 1: Setup of Tissue Bath Apparatus for Adrenoceptor Studies

  Materials
  • 10‐ml tissue baths (Radnoti)
  • Glass muscle holders supplied with tissue baths (Fig. )
  • Carbogen gas (95% O 2/5% CO 2) and 2 stage regulator capable of delivering 0.1 liter/min
  • Force displacement transducers (Grass FT .03 or equivalent; Grass Technologies)
  • Transducer amplifier (Grass model P11T or equivalent; Grass Technologies)
  • Analog to digital converter for computer recording of responses (Powerlab, ADInstruments)
  • Adjustable tension stands (Kent Scientific or World Precision Instruments)
  • Circulating pump/heater at 37°C (e.g., Isotemp Heated Immersion Circulator, model no. 13‐874‐438, Fisher Scientific)
  • Solenoids (Red Hat II; Automatic Switch Co.) to control buffer flow to tissue baths in conjunction with an electronic or computer controlled timing controller (ChronTrol Timer; ChronTrol)

Alternate Protocol 1: Evaluation of α1A‐Adrenoceptors in Canine Prostate

  • Male dogs, >2 years old
  • Sodium pentobarbital euthanasia solution (Fatal Plus Solution, Webster Veterinary)
  • Antifoam A, a detergent used to reduce foam in the tissue bath (Sigma)

Alternate Protocol 2: Evaluation of α1B‐Adrenoceptors in Rat Spleen

  • Acetylcholine chloride (Sigma)
  • 70% ethanol
  • Prolene monofilament surgical sutures with needles (Ethicon) or stainless steel hooks (Radnoti)
  • PE‐100 polyethylene tubing (Fisher Scientific)
  • Microdissecting scissors with fine tip (4 1/ 2‐in. straight; World Precision Instruments)

Alternate Protocol 3: Evaluation of α1D‐Adrenoceptors in Isolated Rat Aorta

  • Human saphenous veins (can be obtained from patients undergoing coronary bypass surgery or varicose vein surgery; use only health looking vein segments or side branches)
  • Superfusion solution (see recipe)
  • 80 mM KCl
  • 10 µM acetylcholine chloride (Sigma)
  • Norepinephrine bitartrate (Sigma)
  • Various sizes (3‐6 mm outside diameter) polyethylene or other hard plastic tubing (Fisher Scientific)
  • Microdissecting scissors with fine tip (6‐7 1/ 2‐in. straight; World Precision Instruments)
  • Prolene monofilament surgical sutures with needles (Ethicon) or stainless steel hooks (Radnoti)

Alternate Protocol 4: Evaluation of α2C‐Adrenoceptors in Isolated Human Saphenous Vein

  • Reference compounds: (e.g., clonidine and rauwolscine; Sigma or Tocris)
  • Constant‐current device (World Precision Instruments A365 or equivalent)
  • Blunted sewing needle equivalent to 21‐G syringe needle
  • Razor blades or iris scissors
  • Electrical stimulator (Model S88 or equivalent, Grass Technologies)

Alternate Protocol 5: Evaluation of α2A‐Adrenoceptors in Isolated Rat Vas Deferens Using Electrical Stimulation

  Materials
  • Female New Zealand White rabbits, 2 kg
  • Sodium pentobarbital euthanasia solution (Fatal Plus Solution, Webster Veterinary)
  • Modified Krebs‐Henseleit buffer solution (see recipe)
  • [3H]Norepinephrine (PerkinElmer)
  • Superfusion solution (see recipe)
  • Reference compounds (e.g., clonidine and rauwolscine; Sigma or Tocris)
  • 4‐0 surgical silk (Ethicon)
  • Opti‐Fluor scintillation cocktail (PerkinElmer) or equivalent
  • Soluene 350 tissue solubilizer (PerkinElmer) or equivalent
  • Tandem array of water baths (Fig. )
  • Minipuls 3 peristaltic roller pump (Gilson) or equivalent to deliver superfusion solution at 2 ml/min per outlet
  • Heat tapes (Protherm)
  • Syringe pump (Fusion Touch, Chemyx) to deliver 0.02 ml/min
  • Fraction collector and tubes
  • Tissue tension apparatus (see protocol 2) modified as shown in Figure
  • Electrical stimulator (Model S88 or equivalent, Grass Technologies)
  • Platinum wire electrodes
  • Scintillation counter
CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and surroundings. Conduct the experiment and dispose of all waste in appropriately designated areas, following the guidelines provided by your local radiation safety adviser.NOTE: A complete superfusion system to measure [3H]norepinephrine release, but not tension, can be purchased from Brandel
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Figures

Videos

Literature Cited

   Aboud, R., Shafii, M., and Docherty, J.R. 1993. Investigations of the subtypes of α1‐adrenoceptor mediating contractions of rat aorta, vas deferens and spleen. Br. J. Pharmacol. 109:80‐87.
   Bockman, C.S., Gonzalez‐Cabrera, I., and Abel, P.W. 1996. Alpha‐2 adrenoceptor subtype causing nitric oxide‐mediated vascular relaxation in rats. J. Pharmacol. Exp. Ther. 278:1235‐1243.
   Buccioni, M., Kandhavelu, M., Angeli, P., Cristalli, G., Dal Ben, D., Giardinà, D., Lambertucci, C., Lammi, C., Volpini, R., and Marucci, G. 2009. Identification of alpha1‐adrenoceptor subtypes involved in contraction of young CD rat epididymal vas deferens. Eur. J. Pharmacol. 602:388‐394.
   Buckner, S.A., Oheim, K.W., Morse, P.A., Knepper, S.M., and Hancock, A.A. 1996. α1‐Adrenoceptor‐induced contractility in rat aorta is mediated by the α1D‐subtype. Eur. J. Pharmacol. 297:241‐248.
   Bylund, D.B. 2005. Alpha‐2 adrenoceptors subtypes: Are more better? Br. J. Pharmacol. 144:159‐160.
   Bylund, D.B., Eikenberg, D.C., Hieble, J.P., Langer, S.Z., Lefkowitz, R.J., Minneman, K.P., Molinoff, P.B., Ruffolo, R.R. Jr., and Trendelenburg, U. 1994. IV. International Union of Pharmacology nomenclature of adrenoceptors. Pharmacol. Rev. 46:121‐136.
   Connaughton, S. and Docherty, J.R. 1990. Functional evidence for heterogeneity of peripheral prejunctional α2‐adrenoceptors. Br. J. Pharmacol. 101:285‐290.
   Deng, X.F., Chemtob, S., Almazan, G., and Varma, D.R. 1996. Ontogenic differences in the functions of myocardial alpha1‐adrenoceptor subtypes in rats. J. Pharmacol. Exp. Ther. 276:1155‐1161.
   Doxey, JC., Smith, C.F.C., and Walker, J.M. 1977. Selectivity of blocking agents for pre‐ and postsynaptic α‐adrenoceptors. Br. J. Pharmacol. 60:91‐96.
   Eltze, M. 1996. Functional evidence for an α1B‐adrenoceptor mediating contraction of the mouse spleen. Eur. J. Pharmacol. 311:187‐198.
   Eltze, M. and Boer, R. 1992. The adrenoceptor agonist, SDZ NVI 085, discriminates between α1A‐ and α1B‐adrenoceptor subtypes in vas deferens, kidney and aorta of the rat. Eur. J. Pharmacol. 224:125‐136.
   Furchgott, R.F. and Burstyn, P. 1967. Comparison of dissociation constants and of relative efficacies of selected agonists acting on parasympathetic receptors. Ann. N.Y. Acad. Sci. 144:882‐899.
   Furukawa, K., Chess‐Williams, R., Noble, A.J., Rosario, D.J., Chapple, C.R., and Uchiyama, T. 1995. Non‐surmountable antagonist effects of tamsulosin on the α1A‐adrenoceptor‐mediated responses of the rat and human vas deferens. Br. J. Pharmacol. 115:127P.
   Gavin, K.T., Colgan, M.P., Moore, D Shanik, G., and Docherty, J.R., 1997. Alpha2C‐adrenoceptors mediate contractile responses to noradrenaline in the human saphenous vein. Naunyn Schmiedebergs Arch. Pharmacol. 355:406‐411.
   Goetz, A.S., King, H.K., Ward, S.D.C., True, T.A., Rimele, T.J., and Saussy, D.L. Jr. 1995. BMY 7378 is a selective antagonist of the D subtype of α1‐adrenoceptors. Eur. J. Pharmacol. 272:R5‐R6.
   Gornemann, T., von Wenckstern, H., Kleuser, B., Villalon, C.M., 2, Centurion, D., Jahnichen, S., and Pertz, H.H. 2007. Characterization of the postjunctional a2C‐adrenoceptor mediating vasoconstriction to UK14304 in porcine pulmonary veins. Br. J. Pharmacol. 151:186‐194.
   Gray, K., Short, J., and Ventura, S. 2008. The alpha1A‐adrenoceptor gene is required for the alpha1L‐adrenoceptor‐mediated response in isolated preparations of the mouse prostate. Br. J. Pharmacol. 155:103‐109.
   Green, E.C. 1963. Anatomy of the Rat. Hafner Publishing Co., New York.
   Han, C., Abel, P.W., and Minneman, K.P., 1987. Heterogeneity of alpha 1‐adrenergic receptors revealed by chlorethylclonidine. Mol. Pharmacol. 32:505‐510.
   Han, C., Abel, P.W., and Minneman, K.P., 1988. Comparison of alpha 1‐adrenergic receptor subtypes distinguished by chlorethylclonidine and WB 4101. Mol. Pharmacol. 33:509‐514.
   Hancock, A. 1996. α1‐Adrenoceptor subtypes: A synopsis of their pharmacology and molecular biology. Drug Dev. Res. 36:1‐53.
   Hanft, G. and Gross, G. 1989. Subclassification of α1‐adrenoceptor recognition sites by urapidil derivatives and other selective antagonists. Br. J. Pharmacol. 97:691‐700.
   Kenny, B.A., Chalmers, D.H., Philpott, P.C., and Naylor, A.M. 1995. Characterization of an α1D‐adrenoceptor mediating the contractile response of rat aorta to noradrenaline. Br. J. Pharmacol. 115:981‐986.
   Krinke, G.J., 2000. The Laboratory Rat. Academic Press. San Diego, Calif.
   Lepor, H., Zhang, W., Kobayashi, S., Tang, R., Wang, B., and Shapiro, E. 1994. A comparison of the binding and functional properties of alpha‐1 adrenoceptors and area density of smooth muscle in the human, canine and rat prostates. J. Pharmacol. Exp. Ther. 270:722‐727.
   Magalhaes, A.C., Dunn, H., and Ferguson, S.S.G. 2012. Regulation of GPCR activity, trafficking and localization by GPCR‐interacting proteins. Br. J. Pharmacol. 165:1717‐1736.
   Marshall, I., Green, M., Hussain, M.B., and Burt, R.P. 1996. Differences in affinity for the antagonist RS 17053 at α1A‐adrenoceptors between rat tissues. Br. J. Pharmacol. 117:110P.
   Minneman, K.P. and Esbenshade, T.A. 1994. α1‐Adrenoceptor subtypes. Annu. Rev. Pharmacol. Toxicol. 34:117‐133.
   Muramatsu, I., Ohmura, T., Hashimoto, S., and Oshita, M. 1995. Functional subclassification of vascular α1‐adrenoceptors. Pharmacol. Commun. 6:23‐28.
   Muramatsu, I., Morishima, S., Suzuki, F., Yoshiki, H., Anisuzzaman, A.S., Tanaka, T., Rodrigo, M.C., Myagmar, B.E., and Simpson, P.C. 2008. Identification of alpha 1L‐adrenoceptor in mice and its abolition by alpha 1A‐adrenoceptor gene knockout. Br. J. Pharmacol. 155:1224‐1234.
   Nishimune, A., Yoshiki, H., Uwada, J., Anisuzzaman, A., Umada, H., and Muramatsu, I. 2012. Phenotype pharmacology of lower urinary tract α1‐adrenoceptors. Br. J. Pharmacol. 165:1226‐1234.
   Noble, A.J., Rosario, D.J., Korstanje, C., Chapple, C.R., and Chess‐Williams, R. 1995. The affinity of tamsulosin (YM‐617) for functional α1‐adrenoceptor subtypes. Br. J. Pharmacol. 114:26P.
   Oshita, M., Kigoshi, S., and Muramatsu, I. 1993. Pharmacological characterization of two distinct α1‐adrenoceptor subtypes in rabbit thoracic aorta. Br. J. Pharmacol. 108:1071‐1076.
   Rizzo, C.A., Ruck, L.M., Corboz, M.R., Umland, S.P., Wan, Y., Shah, H., Jakway, .J, Cheng, L., McCormick, K., Egan, R.W., and Hey, J.A. 2001. Postjunctional alpha2C‐adrenoceptor contractility in human saphenous vein. Eur. J. Pharmacol. 413:263‐269.
   Smith, K., Connaughton, S., and Docherty, J.R. 1992a. Investigations of prejunctional α2‐adrenoceptors in rat atrium, vas deferens and submandibular gland. Eur. J. Pharmacol. 211:251‐256.
   Smith, K., Connaughton, S., and Docherty, J.R., 1992b. Investigations of the subtype of α2‐adrenoceptor mediating contractions of the human saphenous vein. Br. J. Pharmacol. 106:447‐451.
   Starke, K., Montel, H., Gayk, W., and Merker, R. 1974. Comparison of the effects of clonidine on pre‐ and postsynaptic adrenoceptors in the rabbit pulmonary artery. Naunyn‐Schmiedebergs Arch. Pharmacol. 285:133‐150.
   Starke, K., Endo, T., and Taube, H.D. 1975. Relative pre‐ and postsynaptic potencies of α‐adrenoceptor agonists in the rabbit pulmonary artery. Naunyn‐Schmiedebergs Arch. Pharmacol. 291:55‐78.
   Testa, R., Guarneri, L., Ibba, M., Strada, G., Poggesi, E., Taddei, C., Simonazzi, I., and Leonardi, A. 1993. Characterization of α1‐adrenoceptor subtypes in prostate and prostatic urethra of rat, rabbit, dog and man. Eur. J. Pharmacol. 249:307‐315.
   Testa, R., Seroni, G., Colombo, D., Greto, L., and Leonardi, A. 1994. REC 15/2739, a new α1‐antagonist selective for the lower urinary tract: In vivo studies. Neurourol. Urodyn. 13:471‐473.
   Trendelenburg, A.‐U., Limberger, N., and Rump, L.C. 1994. α2‐Adrenergic receptors of the α2c subtype mediate inhibition of norepinephrine release in human kidney cortex. Mol. Pharmacol. 45:1168‐1176.
   Trendelenburg, A‐U., Philipp, M., Meyer, A., Klebroff, W., Hein, L., and Starke, K., 2003. All three α2‐adrenoceptor types serve as autoreceptors in postganglionic sympathetic neurons Naunyn‐Schmiedebergs Arch. Pharmacol. 368:504‐512
   Van der Graaf, P.H., Shankley, N.P., and Black, J.W. 1996. Analysis of the activity of α1‐adrenoceptor antagonists in rat aorta. Br. J. Pharmacol. 118:299‐310.
   VonRossum, J.M. 1963. Cumulative dose‐response curves. II. Technique for the making of dose‐response curves in isolated organs and the evaluation of drug parameters. Arch. Int. Pharmacodyn. 143:299‐330.
   Yamamoto, Y. and Koike, K. 2001. Characterization of α1‐adrenoceptor‐mediated contraction in the mouse thoracic aorta. Eur. J. Pharmacol. 424:131‐140.
Key References
   Brown, D.A., Docherty, J.R., French, A.M., MacDonald, A., McGrath, J.C., and Scott, N.C. 1983. Separation of noradrenergic and non‐noradrenergic contractions to field stimulation in the rat vas deferens. Br. J. Pharmacol. 79:379‐393.
  Pharmacological characterization of prostatic, epididymal or whole vas deferens to clarify adrenoceptor localization and function.
   Doxey et al., 1977. See above.
  Seminal paper showing pharmacological differences between prejunctional (α2) adrenoceptors and postjunctional (α1) adrenoceptors in isolated rat vas deferens, establishing a framework for differentiation among these receptor types and their subtypes.
   Eltze, 1996. See above.
  Pharmacological characteristics of several compounds used in mouse spleen and other tissues comparing receptor subtypes.
   Endo, T., Starke, K., Bangerter, A., and Taube, H.T. 1997. Presynaptic receptor systems on the noradrenergic neurones of the rabbit pulmonary artery. N. ‐S. Arch. Pharmacol. 296:229‐247.
  Detailed characterization of [3H]norepinephrine release from presynaptic terminals of the rabbit pulmonary artery, documenting the metabolism of radioactive norepinephrine and the substantial reduction of metabolism by superfusion using reuptake blockers (cocaine and corticosterone), allowing the calculation of tritium release to reflect almost exclusively the release of [3H]norepinephrine. The concepts and analytical procedures used have broad applications to all overflow fractional methods of measuring neurotransmitter release.
   Minneman and Esbenshade, 1994. See above.
  Review of pharmacological and molecular biological analyses of α1‐adrenoceptors.
   Trendelenburg et al., 2003. See above.
  Using α2‐adrenoceptor subtype knockout mice, this paper showed that the α2A‐adrenoceptor was the primary subtype of prejunctional/presynaptic autoreceptor modulating neurotransmitter release. This paper also found that both the α2C‐ and α2B‐adrenoceptor subtype could contributed to this response. The contribution of the various α2‐adrenoceptor subtypes varied when comparing brain tissue, atria and vas deferens.
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