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β‐Adrenoceptor Assays

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

1Glaxo 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
Print Publication Date: 
March, 1998
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 -adrenoceptor subtypes.

     
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: 1-Adrenoceptors: Guinea Pig Left Atria
  • Alternate Protocol: 1-Adrenoceptors: Guniea Pig Right Atria
  • Basic Protocol 2: 2-Adrenoceptors: Guinea Pig Trachea
  • Basic Protocol 3: 2-Adrenoceptors: Rat Uterus
  • Basic Protocol 4: Measuring -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
  • Bibliography
  • Figures
  • Tables
     
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Materials

Basic Protocol 1: 1-Adrenoceptors: Guinea Pig Left Atria

 Materials
  • Physiological salt solution (Krebs-Henseleit solution; see recipe 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 17-estradiol for extraneuronal uptake (all available from Sigma)
  • Phentolamine (Sigma) or other blockers of -adrenoceptors
  • Male Hartley guinea pig, 250 to 400 g (Charles River Labs)
  • Standard -adrenoceptor agonists: e.g., isoproterenol, epinephrine, or norepinephrine (Table 4.6.1)
  • Standard 1-adrenoceptor antagonists: e.g., atenolol (Table 4.6.1)
  • Test compound(s)
  • Distilled H2O 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     Sensitivities of Guinea Pig Left Atria to -Adrenoceptor Agonists and Antagonists
DrugapD2bMax ()cpKBdMyocardial depletion (µM)eTime (min)f
Agonists
Isoproterenol8.51.0———
Epinephrine7.51.0———
Norepinephrine7.51.0———
Prenalterol7.20.28———
Dobutamine6.41.0———
Antagonists
Atenolol——7.210030
Propranolol——8.4360
Timolol——9.4190
Pindolol——9.1190
Nadolol——8.5160
 a Compounds available from Sigma (see suppliers appendix).
 b Negative logarithm of the molar concentration of agonist producing half the maximal response.
 c Intrinsic activity defined as the fractional maximal response to a full agonist (in this case, isoproterenol).
 d Negative 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).
 e Beyond this concentration, depression of normal cardiac function may occur.
 f Time required for equilibration of antagonist with -adrenoceptors.

Alternate Protocol: 1-Adrenoceptors: Guniea Pig Right Atria

 Additional Materials (also see Basic Protocol 1)
  • Standard agonists and antagonists for receptor classification (Table 4.6.2)
     
    Table 4.6.2     Sensitivities of Guinea Pig Right Atria to -Adrenoceptor Agonistsa
    DrugbpD2cMax ()c
    Isoproterenol9.21.0
    Epinephrine8.01.0
    Norepinephrine7.51.0
    Prenalterol6.90.4
    Pirbuterol6.70.4
    Dobutamine6.51.0
     a For antagonists see Table 4.6.1
     b Compounds available from Sigma (see suppliers appendix).
     c Intrinsic activity defined as the fractional maximal response to a full agonist (in this case isoproterenol).

Basic Protocol 2: 2-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 Sensitivities of Guinea Pig Trachea to -Adrenoceptor Agonists and Antagonists
DrugapD2bMax ()cpKBd
Agonists
Isoproterenol (spontaneous muscle tone)9.61.0—
Prenalterol (spontaneous muscle tone)7.50.7—
Isoproterenol (contracted with 1 µM carbachol)9.01.0—
Prenalterol (contracted with 1 µM carbachol)7.10.4—
Isoproterenol (contracted with 10 µM carbachol)8.151.0—
Isoproterenol (contracted with 10 µM bethanecol)7.91.0—
Norepinephrine (contracted with 10 µM bethanecol)6.541.0—
Salbutamol (contracted with 10 µM bethanecol)6.51.0—
Antagonists
Atenolol——5.5
Propranolol——8.7
ICI 118,551——9.5
Pindolol——9.6
Timolol——10.1
 a Compounds available from Sigma (see suppliers appendix).
 b Negative logarithm of the molar contration producing half the maximal response to the agonist.
 c Intrinsic activity defined as the fractional maximal response to a full agonist (in this case isoproterenol).
 d Negative 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: 2-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)
  • CaCl2 (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 Sensitivities of Rat Uterus to -Adrenoceptor Agonistsa
    DrugbpD2cMax ()d
    Isoproterenol9.51.0
    Prenalterol7.21.0
    Dobutamine6.41.0
    Terbutaline7.71.0
    Tazolol6.41.0
     a For antagonists see Table 4.6.3.
     b Compounds available from Sigma (see suppliers appendix).
     c Negative logarithm of the molar contration producing half the maximal response to the agonist.
     d Intrinsic activity defined as the fractional maximal response to a full agonist (in this case isoproterenol).

Basic Protocol 4: Measuring -Adrenoceptor-Stimulated Lipolytic Activity

 Materials
  • Mature adipocytes growing in tissue culture (see Support Protocol 1)
  • DMEM/F12/1% BSA (see recipe)
  • -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 Support Protocol 1)

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% CO2 incubator unless otherwise specified.


 
Table 4.6.5 Pharmacological Characteristics of the Human 3-Adrenoceptor
Ligands aBinding Ki (nM) bActivity (EC50, nM)
-adrenoceptor agonists
(–)Isoproterenol620 ± 2203.9 ± 0.4
(–)Noradrenaline475 ± 756.3 ± 0.7
BRL37344287 ± 9215 ± 3
(–)Adrenaline20,650 ± 2,81049 ± 5
SM110441,300 ± 20084 ± 10
3-adrenoceptor-selective agonists
GR219803B6.180.3 ± 0.1
CL31624314,0001.3 ± 0.3
GR265261X7.274.6 ± 1.7
Bucindolol23 ± 107.0 ± 1.2
Carazolol2.0 ± 0.211.3 ±1.2
GR230127A5.8217.3 ± 7.8
ICI201651257 ± 3420 ± 9
CGP12177A2,300 ± 450139 ± 44
Pindolol11.2 ± 2153 ± 12
Alprenolol110 ± 30219 ± 46
-Adrenoceptor partial agonists/antagonists
(–)Propranolol257 ± 34—
(–)Bupranolol50 ± 14—
-Adrenoceptor antagonists
ICI 118551357 ± 28—
CGP20712A2,300 ± 450—
 a Chemical names: GR219803B, (4-2R-2-(3-Chlorophenyl)-2R-hydroxy-ethylamino]propylamino-phenyl)-acetic acid, dihydrochloride; GR265261X, (4-2R-[2-(3 -Chloro-phenyl)-2R-hydroxyl-ethylamino]-propylamino-2,3-difluoro-phenylacetic acid; GR230127A, (4-2-[2-(3-Chlorophenyl)-2R-hydroxy-ethylamino]-ethylamino-phenyl)-acetic acid, dihydrochloride; BRL37344, (RR,SS)-(±)-4-(2-[2-hydroxy-2(3-chlorophenyl) ethylamino]propyl) pheoxyacetate sodium salt sesquihydrate; CGP12177A, (±)-4-(3-t-butylamino-2hydroxypropoxy)-benz-imidazol-2-one; CGP20712A, (±)-[2-(3-carbomyl-4-hydroxyphenoxy)-ethylamino]-3-[4-(1-methyl-4-trifluormethyl-2-imidazolyl)-phenoxy]2-propanol methane sulfonate; CL316243, disodium (R,R)-5-[2[[2-(chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate; ICI118551, (±)-D-1-(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol hydrochloride; ICI201651, (R)-4-(2-hydroxy-3-phenoxypropylaminoethoxy)-N-(2-methoxyethyl) phenoxy acetic acid; SM11044, L-3-(3,4-dihydroxyphenyl)-N-[3-(4-fluorophenyl) propyl] serinepyrrolidine amidehydrobromide.
 b Data obtained from Strosberg and Pietri-Rouxel (1996).

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 recipes)
  • 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 Support Protocol 2)

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% CO2 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 Support Protocol 1, steps and )
  • 10 mM Nile red (9-diethylamino-5H-benzo[] 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

  • Figure 4.6.1
    Measurement of atrial rate. A constantly declining electrical signal is emitted from the rate meter upon which the inotropic twitch contraction of the atrium is superimposed. When the inotropic signals intersect, the declining signal is monitored and the difference in the declining signal values used to denote the interval between beats. This interval is converted to a rate of beats per minute. It is essential that the sensitivity of the inotropic signal be correctly positioned in the declining rate signal to allow correct measurement of atrial rate. The meter notes the magnitude of the decaying internal signal between pulses from the transducer, and the difference between these values forms one side of a right triangle (side a in inset). The hypotenuse of this triangle is given by the rate of decay of the internal signal (side c); thus the base of the triangle (side b), which is the time between heartbeats, is calculated by the Pythagorean theorem.

    View Image
  • Figure 4.6.2
    Dissection of guinea pig trachea. The trachea is cut into rings (~2 rings of cartilage each), opposing cuts are made partway into the cartilage on either side of the smooth muscle strip, thread is tied into the cuts, the cartilage ring behind is removed, and the strip is hung by the opposing ridges of cartilage.

    View Image
  • Figure 4.6.3
    Dissection of uterine horns from rat (orientation in peritoneum). The horns are split open and divided in half for a total of four strips. These are tied against a platinum punctate electrode on the tissue holder and an isometric transducer.

    View Image
  • Figure 4.6.4
    Lipolytic and lipogenic assays. (A) Triglyceride assay. (B) Glycerol assay. Abbreviations: ADP, adenosine diphosphate; ATP, adenosine triphosphate; ESPA, sodium N-ethyl-N-(3-sulfopropyl)-m-anisidine.

    View Image
  • Figure 4.6.5
    Relative 1-adrenoceptor-mediated responses of guinea pig left atria (filled symbols) and right atria (open symbols). Responses to isoproterenol (circles) and prenalterol (squares).

    View Image
  • Figure 4.6.6
    The interplay of muscle contraction and relaxation in the guinea pig tracheal preparation. Four doses (designated 1, 2, 3, and 4) of contractile agonist (carbachol) are chosen. Their relationship to the maximal active force capability of the tissue is shown in (A), the contractile dose-response curve. A relaxant can reverse this contraction (i.e., produce relaxation) at the steady-state contractile level reached by each of the doses 1 to 4. The tracing for this relaxation is shown in (B). The resulting relaxation dose-response curves, at each level of contraction, are shown in (C). Note that increasing contraction results in a shift to the right of the relaxation dose-response curve (i.e., an increasing resistance to relaxation) until a point is reached whereby the maximal relaxant effect of the agonist is depressed.

    View Image
  • Figure 4.6.7
    Dose-response curves to the high efficacy -adrenoceptor agonist isoproterenol (filled circles) and lower efficacy -adrenoceptor agonist prenalterol (open circles) at various contractile levels of guinea pig trachea. Dose-response curves in trachea (A) under spontaneous muscle tone; (B) contracted with 1 µM carbachol; and (C) contracted with 10 µM carbachol. (D) Correlation of maximal relaxant effect of prenalterol (ordinate) with ED50 of isoproterenol (abscissa) comprising the data from panels A to C.

    View Image
  • Figure 4.6.8
    Regulation of lipolysis in 3-adrenoceptor-expressing adipocytes. The 3-adrenoceptor interacts with the G-protein stimulatory subunit (Gs) of adenylate cylase resulting in cyclic AMP (cAMP) production. cAMP stimulates the cAMP-dependent protein kinase A (PKA) which phosphorylates hormone sensitive lipase (HSL). Phosphorylated HSL translocates to the lipid droplet and stimulates lipolysis (conversion of triglycerides into glycerol and fatty acids). Negative regulators of the 3-adrenoceptor-stimulated lipolysis include (1) three different Gi forms (Gi 1,2,3) coupled to the adenosine receptor, (2) the cAMP-phosphosdiesterase (cGI-PDE), and (3) the insulin receptor.

    View Image
  • Figure 4.6.9
    Effects of isoproterenol on electrically stimulated twitch contractions of guinea pig left atria. (A) -adrenoceptor agonists such as isoproterenol produce an increased peak height and a shortening of the contraction (i.e., faster relaxation, positive lusitropy). (B) Dose-response curves for guinea pig left atrial inotropic response to cumulatively added isoproterenol (filled circles) and prenalterol (open circles). (C) Dose-response curves for guinea pig left atrial lusitropic responses to cumulatively added isoproterenol (filled circles) and prenalterol (open circles).

    View Image
  • Figure 4.6.10
    Chronotropic responses of guinea pig right atria to isoproterenol. Tracing of heart-rate responses with cumulative addition of isoproterenol. After the effects of 3 µM isoproterenol come to steady state, the preparation is washed and fresh medium is added containing the -adrenoceptor blocking drug atenolol (10 µM). After the atrial rate returns to baseline, another cumulative dose-response curve to isoproterenol is obtained in the presence of atenolol. The resulting dose-response curves can be used to calculate the potency of atenolol as a competitive antagonist of -adrenoceptors.

    View Image
  • Figure 4.6.11
    Mechanical problems resulting in apparent arrythmia in guinea pig isolated right atrial preparations. (A) The inotropic signal from the tissue weakens to a point where it does not bisect the constant rate signal from the meter. (B) The baseline of the preparation falls such that the inotropic signal does not bisect the rate signal. (C) Bubbles or other disturbances in the organ bath pull the string to cause erratic isometric signals to bisect the rate signal.

    View Image
  • Figure 4.6.12
    -adrenoceptor mediated responses to agonists. (A) Tracing for effects of isoproterenol on electrically stimulated uterine twitch contractions. (B) Dose-response curves to isoproterenol (filled circles), terbutaline (filled triangles), prenalterol (open squares), and dobutamine (open circles).

    View Image

Literature Cited

 Literature Cited
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    Donovan, J. and Brown, P. 1995b. Parenteral injections. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. 1.6.1-1.6.10. John Wiley & Sons, New York.
    Foster, R.W. 1967. The potentiation of responses to noradrenaline and isoprenaline in guinea pig isolated tracheal chain preparation by desipramine, cocaine, phentolamine, phenoxybenzamine, guanethidine, metanephrine, and cooling. Br. J. Pharmacol. Chemother. 31:466-482.
    Hazen, S.A., Rowe, W.A., and Lynch, C.J. 1995. Monolayer cell culture of freshly isolated adipocytes using extracellular basement membrane components. J. Lipid Res. 36:868-875.
    Iversen, L.L. 1973. Neuronal and extraneuronal uptake mechanisms. In Frontiers in Catecholamine Research. Proc. 3rd Int. Catecholamine Symposium. (E. Usdin and S.H. Snyder, eds.) pp. 403-418. Pergamon Press, New York.
    Lehmann, J.M., Lenhard, J.M., Oliver, B.O., Ringold, G.M., and Kliewer, S.A. 1997. Peroxisome proliferator-activated receptors and are activated by indomethacin and other non-steroidal anti-inflammatory drugs. J. Biol. Chem. 272:3406-3410.
    Lenhard, J.M., Kliewer, S.A., Paulik, M.A., Plunkett, K., and Weiel, J.L. 1997. Effects of troglitazone and metformin on glucose and lipid metabolism: Alterations of two distinct molecular pathways. Biochem. Pharm. 54:801-808.
    Lenhard, J.M., Lancaster, M., Hull-Ryde, E., and Paulik, M.A. 1998. Differentiation of a human osteosarcoma cell line (SAOS-2) into brown adipocytes. Submitted for publication.
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    Paulik, M.A. and Lenhard, J.M. 1997. Thiazolidinediones inhibit alkaline phosphatase activity while increasing expression of uncoupling protein, deiodinase and increasing mitochondrial mass in C3H10T1/2 cells. Cell Tissue Res. 290:79-87.
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    Strosberg, D.A. and Pietri-Rouxel, F. 1996. Function and regulation of the 3-adrenoceptor. TIPS 17:373-381.
 Key References
    Blinks, J.R. 1967. Evaluations of the cardiac effects of beta adrenergic blocking agents. Ann. N.Y. Acad. Sci. 139:673-685.

Discussion of the cardiac preparation.

    Carter, J.R. and Crofford, O.B. 1985. Methods for assessing hormone effects on glucose transport in isolated fat cells. Meth. Enzymol. 109:269-276.

Adipocyte isolation.

    De Jalon, P., Bayo, M.S., and De Jalon, M. 1945. The rat smooth muscle preparation. Farmacoter Act. 3:313.

General methods for setting up rat uterus preparation.

    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.

Nile red staining.

    Ho, R.J., Bomboy, J.D., Wasner, H.K., and Sutherland, E.W. 1985. Preparation and characterization of a hormone antagonist from adipocytes. Meth. Enzymol. 100:431-438.

Adipocyte isolation.

    Howe, R. 1993. 3-Adrenergic agonists. Drugs Future 18:529-549.

3-adrenoceptors.

    Hughes, I.E. 1978. The stability of noradrenaline in physiological salt solutions. J. Pharm. Pharmacol. 30:124-126.

The effects of antioxidants on degradation of catecholamines.

    Kenakin, T.P. 1981. An in vitro quantitative analysis of the alpha adrenoceptor partial agonist activity of dobutamine and its relevance to inotropic selectivity. J. Pharmacol. Exp. Ther. 216:210-219.

Example dose-response curves to -adrenergic agonists and effects of -adrenergic antagonists.

    Kenakin, T.P. 1981. A pharmacological method to estimate the pKi of competitive inhibitors of agonist uptake processes in isolated tissues. Naunyn-Schmiedebergs Arch. Pharmacol. 316:89-95.

Experiments involving blockade of catecholamine uptake.

    Kenakin, T.P. and Beek, D. 1980. Is prenalterol (H133/80) really a selective beta 1 adrenoceptor agonist Tissue selectivity resulting from differences in stimulus-response relationships. J. Pharmacol. Exp. Ther. 213:406-413.

Examples of dose-response curves to -adrenergic agonists and effects of -adrenergic antagonists.

    Kenakin, T.P. 1982. Theoretical and practical problems with the assessment of the intrinsic efficacy of agonists: Efficacy of the reputed beta-1 selective adrenoceptor agonists for beta-2 adrenoceptors. J. Pharmacol. Exp. Ther. 223:416-423.

Example of dose-response curves to -adrenergic agonists and effects of -adrenergic antagonists.

    Kenakin, T.P., Ambrose, J.R., and Irving, P.E. 1991. The relative efficiency of beta adrenoceptor coupling to myocardial inotropy and diastolic relaxation: Organ-selective treatment for diastolic dysfunction. J. Pharmacol. Exp. Ther. 257:1189-1197.

The relationship between myocardial inotropy and relaxation in guinea pig atria.

    MacDougald, O.A. and Lane, M.D. 1995. Transcriptional regulation of gene expression during adipocyte differentiation. Annu. Rev. Biochem. 64:345-73.

Preadipocyte differentiation.

    McGowan, M.W., Artiss, J.D., Strandbergh, D.R., and Zak, B. 1983. A peroxidase-coupled method for the colorimetric determination of serum triglycerides. Clin. Chem. 29:538.

Triglyceride determination.

    Negrel, R., Grimaldi, P., Forest, C., and Ailhaud, G. 1985. Establishment and characterization of fibroblast-like cell lines derived from adipocytes with the capacity to redifferentiate into adipocyte-like cells. Meth. Enzymol. 109:377-385.

Adipocyte isolation.

    Salt, P.J. 1972. Inhibition of noradrenaline uptake in the isolated rat herat by steroids, clonidine, and methoxylated phenylethylamines. Eur. J. Pharmacol. 20:329-340.

Experiments involving blockade of catecholamine uptake.

    Sigma, 1990. Quantitative enzymatic determination of glycerol, true triglycerides, and total triglycerides in serum or plasma at 540 nm. Sigma Diagnostics procedure no. 337, Sigma Chemical Company, St. Louis.

Triglyceride determination.

    Rodbell, M. 1964. Metabolism of isolated fat cells. J. Biol. Chem. 239:375-380.

Adipocyte isolation.

    Smas, C.M. and Sul, H.S. 1995. Control of adipocyte differentiation. Biochem. J. 309:697-710.

Preadipocyte differentiation.

     
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