β‐Adrenoceptor Assays

Terry Kenakin1, James M. Lenhard1, Mark A. Paulik1

1 Glaxo Wellcome Research and Development, Research Triangle Park, North Carolina
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 4.6
DOI:  10.1002/0471141755.ph0406s00
Online Posting Date:  May, 2001
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Abstract

This unit describes the most commonly used isolated‐tissue and cellular preparations for studying the β‐adrenoceptor subtypes.

     
 
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Table of Contents

  • Basic Protocol 1: β1‐Adrenoceptors: Guinea Pig Left Atria
  • Alternate Protocol 1: β1‐Adrenoceptors: Guniea Pig Right Atria
  • Basic Protocol 2: β2‐Adrenoceptors: Guinea Pig Trachea
  • Basic Protocol 3: β2‐Adrenoceptors: Rat Uterus
  • Basic Protocol 4: Measuring β‐Adrenoceptor‐Stimulated Lipolytic Activity
  • Support Protocol 1: Isolation and Culture of Primary Preadipocytes and Adipocytes
  • Support Protocol 2: Measuring Adipocyte Differentiation by Nile Red Staining or Triglyceride Accumulation
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: β1‐Adrenoceptors: Guinea Pig Left Atria

  Materials
  • Physiological salt solution (Krebs‐Henseleit solution; see reciperecipe in unit 4.3), continuously bubbled with Carbogen gas (unit 4.3)
  • Ascorbic acid and/or EDTA (added as antioxidants if catecholamines are to be tested)
  • Inhibitors of neuronal and extraneuronal catecholamine uptake (optional): desmethylimipramine or cocaine⋅HCl for neuronal uptake and 17β‐estradiol for extraneuronal uptake (all available from Sigma)
  • Phentolamine (Sigma) or other blockers of β‐adrenoceptors
  • Male Hartley guinea pig, 250 to 400 g (Charles River Labs)
  • Standard β‐adrenoceptor agonists: e.g., isoproterenol, epinephrine, or norepinephrine (Table 4.6.1)
  • Standard β 1‐adrenoceptor antagonists: e.g., atenolol (Table 4.6.1)
  • Test compound(s)
  • Distilled H 2O containing 100 µM ascorbic acid
  • Additional reagents and equipment for preparing cardiac muscle and maintaining and measuring response in isolated cardiac preparations (units 4.2 & 4.3)
NOTE: Keep all drug solutions on ice during the course of the experiment.
Table 4.6.1   Materials Sensitivities of Guinea Pig Left Atria to β‐Adrenoceptor Agonists and Antagonists

Drug a pD 2 b Max (α) c pK B d Myocardial depletion (µM) e Time (min) f
Agonists
Isoproterenol 8.5 1.0
Epinephrine 7.5 1.0
Norepinephrine 7.5 1.0
Prenalterol 7.2 0.28
Dobutamine 6.4 1.0
Antagonists
Atenolol 7.2 100 30
Propranolol 8.4 3 60
Timolol 9.4 1 90
Pindolol 9.1 1 90
Nadolol 8.5 1 60

 aCompounds available from Sigma (see suppliers appendix).
 bNegative logarithm of the molar concentration of agonist producing half the maximal response.
 cIntrinsic activity defined as the fractional maximal response to a full agonist (in this case, isoproterenol).
 dNegative logarithm of the equilibrium dissociation constant of the antagonist‐receptor complex (also the negative logarithm of the molar concentration of antagonist that occupies half the receptor population).
 eBeyond this concentration, depression of normal cardiac function may occur.
 fTime required for equilibration of antagonist with β‐adrenoceptors.

Alternate Protocol 1: β1‐Adrenoceptors: Guniea Pig Right Atria

  • Standard agonists and antagonists for receptor classification (Table 4.6.2)
    Table 4.6.2   Additional Materials (also see protocol 1)   Additional Materials    Sensitivities of Guinea Pig Right Atria to β‐Adrenoceptor Agonists g    Sensitivities of Guinea Pig Right Atria to β‐Adrenoceptor Agonists

    Drug h pD 2 j Max (α) j
    Isoproterenol 9.2 1.0
    Epinephrine 8.0 1.0
    Norepinephrine 7.5 1.0
    Prenalterol 6.9 0.4
    Pirbuterol 6.7 0.4
    Dobutamine 6.5 1.0

     gFor antagonists see Table 4.6.1
     hCompounds available from Sigma (see suppliers appendix).
     JIntrinsic activity defined as the fractional maximal response to a full agonist (in this case isoproterenol).

Basic Protocol 2: β2‐Adrenoceptors: Guinea Pig Trachea

  Materials
  • Male Hartley guinea pig, 250 to 400 g (Charles River Labs)
  • Physiological salt solution (modified Krebs‐Henseleit solution; see recipe), continuously bubbled with Carbogen gas (unit 4.3)
  • Ascorbic acid and/or EDTA (if catecholamines are to be tested)
  • Standard agonists and antagonists for receptor classification: e.g., isoproterenol, ICI 118,551, and propranolol (Table 4.6.3; available from Sigma)
  • Test compound(s)
  • Petri dish
  • Surgical instruments (fine scissors and forceps)
  • 5–0 silk thread
  • Additional reagents and equipment for maintaining and measuring response in isolated tissue preparations (units 4.2 & 4.3)
NOTE: Keep all drug solutions on ice during the course of the experiment.
Table 4.6.3   MaterialsSensitivities of Guinea Pig Trachea to β‐Adrenoceptor Agonists and Antagonists

Drug k pD 2 l Max (α) m pK B n
Agonists
Isoproterenol (spontaneous muscle tone) 9.6 1.0
Prenalterol (spontaneous muscle tone) 7.5 0.7
Isoproterenol (contracted with 1 µM carbachol) 9.0 1.0
Prenalterol (contracted with 1 µM carbachol) 7.1 0.4
Isoproterenol (contracted with 10 µM carbachol) 8.15 1.0
Isoproterenol (contracted with 10 µM bethanecol) 7.9 1.0
Norepinephrine (contracted with 10 µM bethanecol) 6.54 1.0
Salbutamol (contracted with 10 µM bethanecol) 6.5 1.0
Antagonists
Atenolol 5.5
Propranolol 8.7
ICI 118,551 9.5
Pindolol 9.6
Timolol 10.1

 kCompounds available from Sigma (see suppliers appendix).
 lNegative logarithm of the molar contration producing half the maximal response to the agonist.
 mIntrinsic activity defined as the fractional maximal response to a full agonist (in this case isoproterenol).
 nNegative logarithm of the equilibrium dissociation constant of the antagonist‐receptor complex (also the negative logarithm of the molar concentration of antagonist that occupies half the receptor population).

Basic Protocol 3: β2‐Adrenoceptors: Rat Uterus

  Materials
  • Female Sprague‐Dawley rats (150 to 200 g; Charles River Labs)
  • 2 mg/ml diethylstilbestrol (Sigma) in 100% ethanol
  • Physiological salt solution (De Jalon's solution, calcium‐free; see recipe), continuously bubbled with Carbogen gas (unit 4.3)
  • Ascorbic acid and/or EDTA (if catecholamines are to be tested)
  • CaCl 2 (most conveniently and accurately added as liquid stock—e.g., Fisher—as dry salt is hygroscopic)
  • Phenoxybenzamine (Sigma)
  • Standard agonists and antagonists for receptor classification: e.g., isoproterenol, ICI 118,551, and propranolol (Table 4.6.4)
  • Test compounds
  • Petri dish
  • Surgical instruments (fine scissors and forceps)
  • 5–0 silk thread
  • Additional reagents and equipment for maintaining and measuring response in isolated tissue preparations (units 4.2 & 4.3)
    Table 4.6.4   Materials   Sensitivities of Rat Uterus to β‐Adrenoceptor Agonists   Sensitivities of Rat Uterus to β‐Adrenoceptor Agonists

    Drug pD 2 Max (α)
    Isoproterenol 9.5 1.0
    Prenalterol 7.2 1.0
    Dobutamine 6.4 1.0
    Terbutaline 7.7 1.0
    Tazolol 6.4 1.0

     For antagonists see Table 4.6.3.
     Compounds available from Sigma (see suppliers appendix).
     Negative logarithm of the molar contration producing half the maximal response to the agonist.
     Intrinsic activity defined as the fractional maximal response to a full agonist (in this case isoproterenol).

Basic Protocol 4: Measuring β‐Adrenoceptor‐Stimulated Lipolytic Activity

  Materials
  • Mature adipocytes growing in tissue culture (see protocol 6)
  • DMEM/F12/1% BSA (see recipe)
  • β‐adrenoceptor agonists/antagonists to be tested (Table 4.6.5)
  • Glycerol standards (Sigma)
  • Triglyceride reagent A (GPO‐Trinder; Sigma)
  • 96‐well tissue culture plates
  • Gelatin‐coated tissue culture vessels (see recipe)
  • Microtiter plate reader/spectrophotometer
  • Additional reagents and equipment for culture of adipocytes (see protocol 6)
NOTE: All reagents and equipment coming into contact with live cells must be sterile, and proper sterile technique must be followed accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 1: Isolation and Culture of Primary Preadipocytes and Adipocytes

  Materials
  • Krebs‐Ringer bicarbonate buffer (KRB; Sigma)
  • Bovine fraction V albumin (Sigma)
  • Source of fat pads: rat or human subject
  • 2 mg/ml collagenase type 1 stock solution (see recipe)
  • Matrigel or Matrigel‐coated tissue culture vessels (Becton Dickinson)
  • Culture media A,B and C (see reciperecipes)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 0.25% trypsin in HBSS (Life Technologies)
  • Freezing medium: DMEM (Life Technologies) containing 20% FBS and 10% DMSO
  • 0.5 M (1000× stock) 1‐methyl‐3‐isobutylxanthine (IBMX; Sigma) in DMSO (store at −20°C)
  • 2.5 mM (10,000× stock) dexamethasone (Sigma) in DMSO (store at −20°C)
  • Dissecting instruments
  • 20‐ml plastic vials (e.g., large scintillation vials; Wheaton)
  • Shaking water bath
  • 250‐µm nylon mesh
  • 50‐ml conical polypropylene centrifuge tubes
  • Tabletop centrifuge
  • 75‐cm2 and 162‐cm2 tissue culture flasks (gelatin‐coated; see recipe)
  • Cryovials
  • Additional reagents and equipment for monitoring differentiation (see protocol 7)
NOTE: All reagents and equipment coming into contact with live cells must be sterile, and proper sterile technique must be followed accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: Except where otherwise indicated, all reagents should be warmed to 37°C prior to use.

Support Protocol 2: Measuring Adipocyte Differentiation by Nile Red Staining or Triglyceride Accumulation

  Materials
  • Differentiated adipocytes (see protocol 6, steps and )
  • 10 mM Nile red (9‐diethylamino‐5H‐benzo[α] phenoxazine‐5‐one; Molecular Probes) in DMSO (store up to 6 months protected from light at −20°C)
  • 0.01% (w/v) digitonin
  • GPO‐Trinder kit (Sigma) consisting of:
    •  Triglyceride reagent A (glycerol kinase, glycerol phosphate oxidase, and peroxidase)
    •  Triglyceride reagent B (lipase)
  • Fluorimeter with 550‐nm excitation filter and 635‐nm emission filter or fluorescence microscope with Zeiss filter set 48‐77‐11 or 48‐77‐14
  • Shaker
  • Spectrophotometer
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Figures

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Literature Cited

Literature Cited
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Key References
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  Discussion of the cardiac preparation.
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  Adipocyte isolation.
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   Fowler, S.D. and Greenspan, J. 1985. Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections: comparison with oil red O. J. Histochem. Cytochem. 33:833‐836.
  Nile red staining.
   Greenspan, P., Mayer, E.P., and Fowler, S.D. 1985. Nile red: A selective fluorescent stain for intracellular lipid droplets. J. Cell Biol. 100:965‐973.
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  Example dose‐response curves to β‐adrenergic agonists and effects of β‐adrenergic antagonists.
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  Experiments involving blockade of catecholamine uptake.
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  Examples of dose‐response curves to β‐adrenergic agonists and effects of β‐adrenergic antagonists.
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  Example of dose‐response curves to β‐adrenergic agonists and effects of β‐adrenergic antagonists.
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  Preadipocyte differentiation.
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