Adrenoceptor Assays

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UNIT 4.6 â-Adrenoceptor Assays

The neurotransmitter norepinephrine (NE, also known as noradrenaline) and the chemi-

cally related hormone epinephrine (also known as adrenaline) control a myriad of

physiological functions. They do so by activating adrenoceptors, of which there are two

major classesα-adrenoceptors, which are discussed in UNIT 4.5, and β-adrenoceptors,

which are discussed in this unit. These receptor classes were first distinguished pharma-

cologically in 1949 by Ahlquist, who observed varying catecholamine potency ratios in

different tissues. The discovery of pronethalol by Black and colleagues provided the firstdefinitive separation of α- and β-adrenoceptors. Subsequently, the discovery of selective

agonists and antagonists has led to the further subclassification of β-adrenoceptors into

those that mainly control cardiac function (β1-adrenoceptors), those that control smooth-

muscle relaxation and skeletal-muscle tremor (β2-adrenoceptors), and those that control

metabolic function (β3-adrenoceptors).

This unit describes the most commonly used isolated-tissue and cellular preparations for

studying the β-adrenoceptor subtypes. Guinea pig left atria (see Basic Protocol 1) and

right atria (see Alternate Protocol) are used to study β1-adrenoceptors, while guinea pig

trachea (see Basic Protocol 2) and rat uterus (see Basic Protocol 3) are used for

β2-adrenoceptors.

The β3-adrenoceptor is expressed primarily in fat (adipocytes; brown and white adipose

tissue), where it regulates norepinephrine-induced changes in energy metabolism and

thermogenesis. The β3-adrenoceptor is also expressed in smooth muscle, gastrointestinal

tract, gall bladder, and heart; however, its function and relevance in these tissues is not

well understood. This unit includes an assay for measuring β3-adrenoceptor-induced

lipolysis to characterize a functional β3-adrenoceptor response in adipocytes (see Basic

Protocol 4). Also described are methods for isolating and culturing primary adipocytes

(see Support Protocol 1) as well as for differentiating preadipocytes for studying β3-ad-

renoceptors and their respective ligands (see Support Protocol 2).

NOTE: All protocols using live animals must first be reviewed and approved by an

Institutional Animal Care and Use Committee (IACUC) or must conform to governmental

regulations regarding the care and use of laboratory animals.

BASIC

PROTOCOL 1

â1-ADRENOCEPTORS: GUINEA PIG LEFT ATRIA

The guinea pig isolated left atrium is a classical preparation for the assessment of

β1-adrenoceptor function (Blinks, 1966). The responses of this tissue to β1-adrenergic

agonists are rapid in onset and sustained, allowing for the generation of cumulative

dose-response curves. The β1-adrenoceptors in guinea pig left atria are moderately well

coupled (see UNIT 4.1) yielding submaximal responses to partial β1-adrenoceptor agonists

such as prenalterol.

The key to successful studies of β1-adrenoceptor function in left atrium is to be aware of

the tendency of cardiac tissue to desensitize with respect to inotropic function. Thus, whilecumulative dose-response curves can be obtained, the exposure time to β1-adrenoceptor

agonists should be kept to a minimum, with at least a 30 to 60 min wash in drug-free

medium before repeated testing of the preparation with β1-adrenoceptor agonists.

Contributed by Terry Kenakin, James M. Lenhard, and Mark A. PaulikCurrent Protocols in Pharmacology (1998) 4.6.1-4.6.36

Copyright © 1998 by John Wiley & Sons, Inc.

4.6.1

Isolated Tissues



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 betested)

Inhibitors of neuronal and extraneuronal catecholamine uptake (optional):desmethylimipramine or cocaine⋅HCl for neuronal uptake and 17β-estradiol forextraneuronal 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, ornorepinephrine (Table 4.6.1)

Standard β1-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 maintainingand measuring response in isolated cardiac preparations (UNITS 4.2 & 4.3)

NOTE: Keep all drug solutions on ice during the course of the experiment.

Prepare bathing medium

1. Prepare the Krebs-Henseleit solution, begin bubbling with Carbogen gas, and allow

temperature to equilibrate to 31°C (see UNIT 4.3). If catecholamine agonists are to be

used, add ascorbic acid to a final concentration of 100 µM and/or EDTA to a final

concentration of 10 µM at this point to reduce chemical degradation.

Because of the chemical instability of catecholamines, the solution should contain antioxi-

dants or metal chelators to chelate trace amounts of heavy metal ions that catalyze

Table 4.6.1 Sensitivities of Guinea Pig Left Atria to β-Adrenoceptor Agonists andAntagonists

Druga pD2b Max (α)c pKB

d Myocardialdepletion (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 90Pindolol 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).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).eBeyond this concentration, depression of normal cardiac function may occur.f Time required for equilibration of antagonist with β-adrenoceptors.

Current Protocols in Pharmacology

4.6.2

â-AdrenoceptorAssays



oxidation. This is especially necessary in Krebs-Henseleit solution bubbled with Carbogen

gas as required for cardiac preparations.

See UNIT 4.3 for the apparatus discussed here. The tissue holder is made of an unreactive

substance such as Plexiglas and must fix one end of the tissue in the organ bath while

allowing the other end of the t issue to be attached to a recording device. The holder must

have a platinum electrode milled flush with the surface resting against the tissue for

delivering electrical stimulation. A heated circulating water bath is needed with a thermo-

stat capable of controlling the temperature of pumped water to within 0.1°C. A heated

isolated organ bath is needed that is capable of being bubbled continuously with Carbogen

gas and being rapidly filled and emptied using physiological salt solution. Also required are a physiological recorder (standard chart recorder), an isometric force transducer for

recording of muscle tone, and an electrical stimulator capable of producing trains of

square-wave pulses at a precise current strength.

2. If catecholamine agonists (e.g., norepinephrine or epinephrine) are to be used, add

desmethylimipramine to the bathing medium at a final concentration of 0.2 µM or

cocaine⋅HCl at a final concentration of 10 µM (to inhibit neuronal uptake) as well as

17β-estradiol to a final concentration of 5 µM (to inhibit extraneuronal uptake).

Neuronal and extraneuronal uptake processes cause large differences between the concen-

trations of drug added to the medium and concentrations reaching the receptor, resulting

in a rightward shift in the agonist dose-response curves that can range from 2-fold to

>100-fold. Accordingly, it is important that uptake be inhibited in the isolated tissue if

receptor agonists are used that are transported into cells (Foster, 1967; Iversen, 1973).

While there are numerous antagonists of neuronal catecholamine uptake, many have

secondary effects that interfere withβ1-adrenoceptor function. The uptake inhibitors may

be added directly to the organ bath before conducting dose-response experiments or to the

stock solution of bathing medium.

3. If the β1-adrenoceptor agonists to be studied are known to activate α-adrenoceptors

(e.g., epinephrine and norepinephrine), add phentolamine at a final concentration of

3 µM (or other blockers of α-adrenoceptors).

It is crucial to includeα-adrenoceptor antagonists in the assay if nonselective adrenergic

agonists are usede.g., norepinephrine, epinephrine, or certain synthetic agents. Epineph-

rine, phenylephrine, and norepinephrine activate α-adrenoceptors, which can result in

weak inotropic effects in this t issue.The α-adrenoceptor blocker can be added when the medium is prepared initially, or can

be added to the organ bath at the time of experimentation.

Set up tissue preparation

4. Sacrifice a male Hartley guinea pig by CO2 asphyxiation.

Donovan and Brown (1995a) describe this procedure in detail.

5. Prepare left atrium from guinea pig, place in the 31°C organ bath, and set up the

apparatus to maintain 0.5 g resting tension with readjustment to this limit throughout

the experiment (UNITS 4.2 & 4.3).

Once the initial stretching of the preparation has waned (because of wetting of the thread

and stretching of the elastic components in the t issue), the baseline tension should remain

constant throughout the remainder of the experiment. However, if this is not the case, the

resting tension should be adjusted, since the responsiveness of the tissue will vary as a

function of the resting tension (seeUNIT 4.2).

6. Set the electrical stimulation parameters at 0.5 Hz frequency of stimulation and 5

msec duration of square-wave stimulation, at threshold voltage + 30%.

The tissue should begin twitching at a frequency of 0.5 Hz immediately.


4.6.3

Isolated Tissues



7. Wash the preparation with the bathing medium prepared in steps 1 to 3, above,

according to the general procedures outlined in UNIT 4.3, Basic Protocol, steps 10 and

11. Allow at least 30 min for the tissue to equilibrate with the additives in the bathing

medium.

Test compounds

8. Make up dilutions of standard agonists, antagonists, and test compound(s) in deion-

ized water containing 100 µM ascorbic acid. Keep them wrapped in aluminum foil

to reduce degradation by ultraviolet light.

Begin dilutions at 1 nM. Since dose-response curves are semilogarithmic, a convenient

range is 3-fold dilutions between points, as this results in evenly spaced data points on the

log concentration scale.

Catecholamines such as isoproterenol, epinephrine, and norepinephrine are chemically

unstable and are degraded rapidly by molecular oxygen in solution. This process is

accelerated by traces of heavy metals, alkaline pH, and ultraviolet light. When they degrade

they form a vivid pink chromophore. For this reason prepare all stocks and all subsequent

dilutions in deionized water containing 100ìM ascorbate and wrap in aluminum foil.

9. Optional: Test the responsiveness of the isolated left atrial preparation to standard

β1-adrenoceptor agonistse.g., isoproterenol, norepinephrine, or epinephrine.

A concentration producing∼50% of the maximal response is best for this test since it willnot cause significant desensitization of theβ1-adrenoceptors.

Shown in Table 4.6.1 are sensitivities of guinea pig left atria toβ1-adrenoceptor agonists

and antagonists. The data are given as the negative logarithm of molar concentrations

producing 50% of the maximal response (pD2). Therefore, addition of this concentration

of agonist to the preparation can be used as an indicator of the maximal scale of

β1-adrenoceptor response and of the viability of the preparation.

Verification that the observed agonist response is indeed mediated by activation of

β1-adrenoceptors can be obtained by using low concentrations of β-adrenoceptor antago-

nists to block the responses. For example, 3 mM atenolol added to the tissue 30 min before

addition of agonist should produce complete inhibition of a concentration of agonist

producing submaximal responses (i.e., the pD2 concentration of β1-adrenoceptor agonists

shown in Table 4.6.1), provided that the agonist is producing response through activationof β1-adrenoceptors. Similar results should be obtained with 0.1 mM propranolol added

60 min before agonist challenge.

Natural β-adrenoceptor agonists such as norepinephrine and epinephrine are rapidly

removed from the the receptor compartment by neuronal uptake mechanisms. Both of these

agonists, as well as isoproterenol, are also removed from the receptor compartment by

extraneuronal uptake mechanisms and are degraded by catechol-O-methyl transferase. It

is best to use agonists that are quickly removed from the receptor compartment when testing

tissue responsiveness, since this reduces the risk of desensitization.

10. Wash the preparation with drug-free physiological salt solution until a stable baseline

is achieved (UNIT 4.3).

“Drug-free” refers to the medium composition used just before addition of either agonist

or antagonisti.e., if uptake inhibitors are being used then they should be added again at

this step, but agonists and/or antagonists must be omitted.

11. Measure tissue response to a test compound using the same procedure as for the

standard agonists.

While the concentrations needed to produceβ1-adrenoceptor–mediated responses in this

preparation are known from previous study for a number of compounds, test compounds

are of unknown activity. A reasonable starting point for suspected agonists is to prepare

10-fold dilutions in deionized water ranging from 0.1 nM to 100 mM. Beginning with the


4.6.4




lowest concentration, a 1⁄ 100 volume of the drug stock solution (i.e., 0.1 ml added to a 10

ml organ bath) will equilibrate the tissue with a 1 pM solution of test drug. The most potent

known β1-adrenoceptor agonists produce responses in this concentration range. Cumula-

tive addition of 10-fold higher concentrations thereafter should expose the preparation to

a concentration range of 1 pM to 10 mM. β1-adrenoceptor–mediated responses are rapid,

so a 5-min exposure of the tissue to each concentration is sufficient.

12. Calculate results.

Dose-response curves usually are sigmoid in nature and can be fit to the general logistic

function of the form: Response = ([A]

n

× Max)/([A]

n

+ K

n

), where Max refers to themaximal response to a full agonist such as isoproterenol, [A] refers to the concentration

of test agonist, and n and K are fitting parameters. The location parameter of the

dose-response curve (K) is the concentration of agonist producing 50% maximal response

to that particular agonist. This scales the responses observed as fractions of the maximal

response to the full agonist isoproterenol. These ideas are discussed further inUNIT 4.1.

ALTERNATE

PROTOCOL

â1-ADRENOCEPTORS: GUINEA PIG RIGHT ATRIA

The guinea pig right atrium is similar to the left except that it is the influence upon the

rate of contraction of the right atrium that constitutes the β1-adrenoceptor-mediated

response. In effect, the right atrium is a life-support system for the A-V (atrioventricular)

node, a specialized group of cells that automatically send an electrical impulse to the rest

of the cardiac muscle, initiating contraction. The right atrium offers a certain advantageover the left in that there is a built-in control of tissue viabilityi.e., there is a range of

automatic cardiac rates that indicate a healthy as opposed to an unhealthy tissue. Thus,

the spontaneous rate of beating of a guinea pig right atrium ranges from 120 to 180 beats

per minute (bpm), with a good preparation generally having a spontaneous rate of 140 to

160 bpm. Spontaneous rates outside of this range may indicate abnormal in vitro

conditions.

As with the guinea pig left atrium (see Basic Protocol 1), cumulative dose-response curves

can be obtained with the right atrium. However, the contact time between the tissue and

β1-adrenoceptor agonists should be kept to a minimum, with at least a 30 to 60 min wash

in drug-free medium allowed before repeated testing of the preparation with β1-adreno-

ceptor agonists, to minimize possible desensitization.

Additional Materials (also see Basic Protocol 1)

Standard agonists and antagonists for receptor classification (Table 4.6.2)

1. Prepare Krebs-Henseleit solution, begin bubbling with Carbogen gas, and allow

temperature to equilibrate to 31°C (see UNIT 4.3). If catecholamines are to be used, add

antioxidants and metal chelators to the medium (see Basic Protocol 1, step 1). If

required, add inhibitors of neuronal/extraneuronal uptake (see Basic Protocol 1, step

2).

2. Sacrifice a male Hartley guinea pig (250 to 400 g) by CO2 asphyxiation.

Donovan and Brown (1995a) describe this procedure in detail.

3. Prepare right atrium from guinea pig, place in 31°C organ bath, and set up the

apparatus to maintain 0.5 g resting tension with readjustment to this limit throughout

the experiment (UNITS 4.2 & 4.3).

The setting of the resting tension is critical for this preparation, since the contractile signal

produced by tissue contraction is used to bisect an internal signal produced by the rate

meter. This internal signal has an extremely accurate time course of decay. The isometric

twitch of the right atrium produces a pulse of electric current that is fed into the rate meter.


4.6.5

Isolated Tissues



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 Fig. 4.6.1 inset). The hypotenuse of this triangle is given by the rate of decay of the

internal signal (side c in Fig. 4.6.1 inset); thus the base of the triangle (side b in Fig. 4.6.1

inset), which is the time between heartbeats, is calculated by the Pythagorean theorem.

This time between beats is processed as a reciprocal quantity to give the rate of beating

(usually in beats per minute). Thus, when the contraction of the tissue interferes with the

decaying internal signal, the strength of the signal at that explicit time point can be used

to relay a rate according to a previous calibration of rate versus strength of internal signal.

Thus, the meter essentially measures the interval between beats, which is then converted

to the reciprocal frequency value. If the starting point for the atrial signal changes (i.e., if the resting tension changes), then it is possible that the contraction will not be in a suitable

position to bisect the internal signal of the rate meter (see Fig 4.6.1). The strength of

contraction is also relevant to this mechanism, since, if the contractile signal is not of

sufficient strength to interfere with the internal rate signal, then no measure of atrial rate

will ensue. For this reason, the actual contractile activity of the right atrium should be

monitored in the initial stages of the experiment to insure that the inotropic activity is stable

and of uniform strength and that the resting tension does not change with time. Also see

UNIT 4.2.

4. Once the basal inotropic activity of the right atrium is stable, introduce the rate meter

into the signal.

At this time, the rate of the spontaneously beating atrium (in bpm) should be registered and

stable.

5. Measure tissue responsiveness to various dilutions of β1-adrenoceptor agonists,

antagonists, and test compounds (see Basic Protocol 1).

Shown on Table 4.6.2 are sensitivities of guinea pig right atria toβ1-adrenoceptor agonists.

The data are given as the negative logarithm of the molar concentrations producing 50%

∆ current

∆ current measures

interval betweencontractions

constantly

declining

rate meter

signal

c

b

atwitch contractions

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.


4.6.6




of the maximal response (pD2). Therefore, addition of this concentration of agonist to the

preparation can be used an an indicator of both the maximal scale of β1-adrenoceptor

response and of the viability of the tissue preparation.

BASIC

PROTOCOL 2

â2-ADRENOCEPTORS: GUINEA PIG TRACHEA

Tracheal smooth muscle is a very versatile preparation. Its spontaneous tone can be used

to measure receptor-mediated relaxation or it can be eliminated with indomethacin,

increasing the utility of this preparation as a contractile smooth muscle. Tracheal muscle

function can be measured either isometrically as tension or isotonically as shortening.

The steps below are for measurement of isometric tension.

Because this is a slowly contracting and relaxing muscle, long equilibration times are

often required to obtain dose-response data. The intrinsic muscle tone develops slowly

and can obscure drug-induced events unless it attains steady-state (i.e., the muscle may

be slowly contracting in accordance with endogenous tone while a drug-induced relaxa-

tion is being studied). For this reason, the preparation must be equilibrated for 1 to 2 hr

for the spontaneous tone to come to equilibrium. Alternatively, indomethacin (1 µM) is

used to suppress the spontaneous tone.

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 inisolated tissue preparations (UNITS 4.2 & 4.3)

NOTE: Keep all drug solutions on ice during the course of the experiment.

1. Prepare the Krebs-Henseleit solution, begin bubbling with Carbogen gas, and allow

temperature to equilibrate to 37°C (see UNIT 4.3). If catecholamines are to be used, add

Table 4.6.2 Sensitivities of Guinea Pig Right Atriato β-Adrenoceptor Agonistsa

Drugb pD2c Max (α)c


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

aFor antagonists see Table 4.6.1.bCompounds available from Sigma (see SUPPLIERS APPENDIX ).cIntrinsic activity defined as the fractional maximal response to a

full agonist (in this case isoproterenol).


4.6.7

Isolated Tissues



antioxidant (ascorbic acid) and metal chelator (EDTA) to the medium (see Basic

Protocol 1, step 1).



allowing the other end of the tissue to be attached to a recording device. The holder must




isolated organ bath is needed which is cablable of being bubbled continuously with

Carbogen gas and which has the capability of being rapidly filled and emptied withphysiological salt solution. Also required are a physiological recorder (standard chart

recorder), an isometric force transducer for recording of muscle tone (or isotonic displace-

ment transducer for recording muscle length), and an electrical stimulator capable of

producing trains of square-wave pulses at a precise current strength.

2. Sacrifice the guinea pig by CO2 asphyxiation. Make a midline incision to the chest,

being careful not to sever the trachea. Using forceps, gently spread the muscle layers

covering the trachea and insert the curved blade of the forceps under the preparation.

Run the forceps under the length of the trachea to free the tissue of connections, then

sever the trachea at the top and bottom and remove it from the chest cavity. Place the

isolated trachea into a petri dish containing modified Krebs-Henseleit solution.

Donovan and Brown (1995a) describe the CO2 asphyxiation procedure in detail.

3. Carefully trim away the thin adventitial layer surrounding the strip of smooth muscle

joining the rings of cartilage.

The trachea may now be prepared in different ways (step 4) to measure mechanical

function.

4. Cut the trachea into ring segments consisting of two natural ridges of cartilage, ∼2

mm wide (for a convenient preparation). For a more responsive preparation, attach

threads to the cartilage on either side of the smooth muscle and remove the intervening

ring of cartilage (split-ring approach; Fig. 4.6.2).

The tracheal preparation is a stiff semicircular tube of cartilage joined by contractile

smooth muscle. It is this thin strip of muscle from which pharmacological responses aremeasured. If the ring is left intact, the stiff cartilage semicircle may hinder the smooth

muscle relaxation. For this reason, it is much better to use the split-ring approach (Fig.

4.6.2), although this may not be possible with very small tracheal preparations. With young

animals, the stiffness of the cartilage may be minimal and therefore it may be possible to

use a complete ring.

To measure isotonic shortening, use several tracheal ring preparations, since there is very

little shortening in the thin strip of muscle. For this type of measurement, several split rings

may be tied together end-to-end in series.

5. Secure one end of the preparation to the tissue holder with 5–0 silk thread and insert

the tissue into the 37°C organ bath (UNIT 4.3). Tie the other end of the preparation to

a transducer (either isometric or isotonic) and adjust the resting tension to 1 g (or as

appropriate for the particular protocol).

A 1-g resting tension is the minimum requirement for the trachea and will be exceeded by

the spontaneous tone of the tissue as the experiment progresses. The magnitude of the

resting tension varies with the type of preparation and is provided in the individual

protocols.

Tracheal muscle spontaneously releases leukotrienes and prostaglandins, which cause

contraction of a magnitude that is almost always submaximal, making it possible to

enhance contraction further with receptor agonists. However, the level of spontaneous tone


4.6.8




may vary since it involves endogenous enzymatic reactions. To minimize this problem, the

preparation may be exposed to indomethacin (1 ìM; Sigma) for 60 min to inhibit the

production of leukotrienes and prostaglandins. The assay of relaxant effects on such

preparations requires either spontaneous muscle tone or the addition of a spasmogen to

stimulate muscle tone. If endogenous basal tension is not required for the experiments, as

when indomethacin is present, this and the following wash steps should still be performed,

to remove unwanted substances from the medium.

6. Wash the preparation with at least six changes of fresh modified Krebs-Henseleit

solution within the first 20 min (UNIT 4.3).

At this point, the resting basal tension of the preparation should increase. If this does not occur within 30 min, wash the tissue again with fresh medium. After a period of 40 min,

the tension should begin to increase.

7. Once the tension has begun to increase, wash the tissue every 15 to 20 min until the

resting tension attains a steady state (equilibration period).

8. Measure tissue response to various dilutions of standard agonists, antagonists, and

test compounds (also see Basic Protocol 1).

Standard agonists and antagonists useful for receptor classification include isoproterenol

(a useful standard agonist for β2-adrenoceptors with high efficacy and potency, which can

easily be removed by washing with drug-free medium); ICI 118,551 (a potent and selective

β2-adrenoceptor antagonist); and propranolol (a good β2-adrenoceptor antagonist). Also

see Table 4.6.3.

smooth muscle

cartilage

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.


4.6.9

Isolated Tissues



BASIC

PROTOCOL 3

â2-ADRENOCEPTORS: RAT UTERUS

The rat uterus is a classical β2-adrenoceptor preparation. This tissue has the specific

advantage of being exceedingly sensitive to β2-adrenoceptor agonists. It is not clear

whether this is due to a high concentration of β2-adrenoceptors or to a highly efficient

receptor-effector coupling system. In either case, low-efficacy β2-adrenoceptor agonists

produce robust responses in this preparation.

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 stocke.g., Fisherasdry salt is hygroscopic)

Phenoxybenzamine (Sigma)

Standard agonists and antagonists for receptor classification: e.g., isoproterenol,ICI 118,551, and propranolol (Table 4.6.4)

Table 4.6.3 Sensitivities of Guinea Pig Trachea to β-AdrenoceptorAgonists and Antagonists

Druga pD2b Max (α)c pKB

d

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


7.1 0.4

Isoproterenol


8.15 1.0

Isoproterenol

(contracted with 10 µM bethanecol)

7.9 1.0

Norepinephrine


6.54 1.0

Salbutamol


6.5 1.0

Antagonists

Atenolol 5.5

Propranolol 8.7

ICI 118,551 9.5

Pindolol 9.6

Timolol 10.1

aCompounds available from Sigma (see SUPPLIERS APPENDIX ).bNegative logarithm of the molar contration producing half the maximal response to

the agonist.cIntrinsic 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 thatoccupies half the receptor population).


4.6.10




Test compounds

Petri dish

Surgical instruments (fine scissors and forceps)

5–0 silk thread

Additional reagents and equipment for maintaining and measuring response inisolated tissue preparations (UNITS 4.2 & 4.3)

1. Administer 1 mg/kg diethylstilbestrol to rats by subcutaneous injection (e.g., 0.1 ml

of 2 mg/ml diethylstilbestrol in ethanol for a 200-g rat) on each of the two consecutivedays before sacrifice.

This step will ensure stable preparations resulting from having the rat in a state of estrus.

The concentration must be such that only a small volume needs to be administered to the

rat to deliver a dose of 1 mg/kg.

Donovan and Brown (1995b) describes the procedure for subcutaneous injection in rats.

2. Prepare calcium-free De Jalon’s solution. If catecholamines are to be used, add

antioxidant (ascorbic acid) and metal chelator (EDTA) to the medium (see Basic

Protocol 1, step 1).

3. After 2 days of treatment with diethylstilbestrol, sacrifice rat and open abdomen.

Remove the two uterine horns (Fig 4.6.3A) and place in calcium-free De Jalon’ssolution that is being continuously gassed with Carbogen gas.

4. Trim away the fatty tissue from each horn, bisect it transversely, and split the horns

open longitudinally (Fig. 4.6.3B). Place 5–0 silk ties on each end of the split uterine horn.

Each horn should yield two isolated tissue preparations.

5. Prepare the organ bath (UNIT 4.3) using the calcium-free De Jalon’s solution with

antioxidants and metal chelators (step 2) and allow the temperature to equilibrate to

31°C. Tie one end of the tissue to the tissue holder in such a way that the tissue rests

flush with the platinum electrode (see Fig 4.6.3C and UNIT 4.3). Place the tissue in the

organ bath, tie the other thread to an isometric transducer and adjust the resting

tension to 1 g.



allowing the other end of the t issue to be attached to a recording device. The holder must




isolated organ bath is needed that is capable of being bubbled continuously with Carbogen

gas and being rapidly filled and emptied using physiological salt solution. Also required

are a physiological recorder (standard chart recorder), an isometric force transducer for

recording of muscle tone, and an electrical stimulator capable of producing trains of

square-wave pulses at a precise current strength.

6. Wash the tissue with fresh calcium-free De Jalon’s solution. Administer electricalstimulation via the punctate electrode and an external platinum electrode, using a

train of square waves 2 sec in duration with pulses every 100 sec at 10 Hz, 10 msec

in duration, at threshold voltage + 30%.

See UNIT 4.3 for details of washing and electrical stimulation procedure.

7. Allow the preparation to equilibrate for 10 min (with two to three washes of fresh

calcium-free De Jalon’s solution during this period), then add CaCl2 to the medium

to a final concentration of 1.25 mM.


4.6.11

Isolated Tissues



Within a few minutes a uniform set of contractions in response to each train of stimuli

should be observed.

8. Add phenoxybenzamine to the bathing medium at a final concentration of 10µM and

pretreat tissue for 20 min to block extraneuronal uptake of catecholamines and

stimulation of α-adrenoceptors. After this time, wash tissue three times with drug-free

De Jalon’s solution to remove residual phenoxybenzamine. At this time, and for the

duration of the experiment, increase the calcium concentration in the bath to 2.5 mM

and add this after every wash or change the bathing medium to De Jalon’s solutioncontaining 2.5 mM CaCl2.

An alkylating agent, phenoxybenzamine reacts with various chemical groups, including

hydroxyl groups of water molecules. Therefore, the solution must be prepared fresh and

used immediately or else the reactive species will form an alcohol, becoming inactive.

Solutions may be kept on ice for a few hours if prepared in acid medium, where the reaction

with water occurs exceedingly slowly. Phenoxybenzamine is a chemically reactive alkylat-

ing agent that forms an aziridinium ion in aqueous solution, which goes on to form alkyl

bonds with many other chemical groups. Since it attaches irreversibly, the tissue should be

A

uterine

horns

four preparations

B

C

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.


4.6.12




exposed to it for a given period of time and then the drug removed from the medium. If not,

phenoxybenzamine can cause damage to the tissue by alkylating cellular proteins. A

complete removal of the active aziridinium ion from the medium is achieved by washing

the tissue with De Jalon’s solution containing sodium thiosulfate (3 mM); this ion readily

forms an inactive Bunte salt with the aziridinium ion, rendering the drug inactive.

9. Measure tissue response to various dilutions of standard agonists, antagonists, and

test compounds (also see Basic Protocol 1).

Standard agonists and antagonists useful for receptor classification include isoproterenol

(a useful standard agonist for β2-adrenoceptors with high efficacy and potency, which can

easily be removed by washing with drug-free medium); ICI 118,551 (a potent and selective

β2-adrenoceptor antagonist); and propranolol (another good β2-adrenoceptor antagonist

and β1-adrenoceptor blocker). Also see Table 4.6.4.

BASIC

PROTOCOL 4

MEASURING â-ADRENOCEPTOR-STIMULATED LIPOLYTIC ACTIVITY

The accumulation of cellular triglycerides is dependent on a balance between lipogenesis

and lipolysis. Thus, the success of measuring β-adrenoceptor-mediated lipolysis andtriglyceride accumulation requires culturing the cells under conditions that maintain high

lipogenic activity and low basal lipolytic activity. Once the cells have differentiated into

adipocytes and accumulated substantial substrate (i.e., triglycerides), any antilipolytic

agentse.g., insulin, thiazolidinediones, or adenosineshould be removed from the

cells. Subsequently, selective agonists for the β1, β2 and β3 receptors can be used to

stimulate lipolysis and determine the β-receptor species present on the adipocytes. In

order to test a compound selectively for β3 adrenergic activity, one needs to assay in the

presence of β1 and β2 antagonists (see Table 4.6.5) which will selectively block both

receptors, enabling measurement of only β3 activity.

The assays for measuring β-adrenoceptor-mediated lipolysis and triglyceride accumula-

tion within the cells involve detecting the glycerol that is liberated from the cells as aresult of triglyceride hydrolysis. By activating endogenous lipases with β-adrenoceptor

agonists or adding exogenous lipases, the triglycerides within the adipocyte can be

hydrolyzed into glycerol and free fatty acids. The glycerol that is released into the medium

is converted into a colorimetrically quantifiable dye via coupled enzyme reactions

involving glycerol kinase, glycerol phosphate oxidase, and peroxidase (see Figure 4.6.4).

The pink dye is easily visible and can be measured spectrophotometrically.

Table 4.6.4 Sensitivities of Rat Uterus toβ-Adrenoceptor Agonistsa

Drugb pD2c Max (α)d


Prenalterol 7.2 1.0

Dobutamine 6.4 1.0

Terbutaline 7.7 1.0

Tazolol 6.4 1.0

aFor antagonists see Table 4.6.3.bCompounds available from Sigma (see SUPPLIERS APPENDIX ).cNegative 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).


4.6.13

Isolated Tissues



Materials

Mature adipocytes growing in tissue culture (see Support Protocol 1)

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 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.

1. Wash the mature adipocytes once with DMEM/F12/1%BSA. Add 100 µl of

DMEM/F12/1% BSA per cm2 of gelatin-coated culture vessel and incubate 5 hr in

the presence and absence of β-selective agonist/antagonist to be tested.

Standard adrenoceptor agonists and antagonists are used to test for functional responses

in the adipocytes. Several compounds can be used as β3-selective agonists, such as

GR219803B or CL316243 (see Table 4.6.5). Isoproterenol and catecholamines can be used

as nonselective agonists. SR 59230A and ICI 118,551 can be used as β3-adrenoceptor

antagonists. Depending upon the agonist/antagonist used, dose curves should be set in the

range of 2 to 3 orders of magnitude on either side of the EC 50 value for the particular

compound being tested (see Table 4.6.5).

Since isoproterenol and other catecholamines (e.g., epinephrine and norepinephrine)

degrade when exposed to alkaline pH, trace heavy metals, and/or ultraviolet light, prepare

triglycerides

glycerol + ATP

glycerol-1-phosphate + O2

H2O2 + 4-aminoantipyrine + ESPA

glycerol + fatty acids

glycerol-1-phosphate + ADP

dihydroxyacetone phosphate + H2O2

quinoneimine dye + H2O

lipoprotein lipase

glycerol kinase

glycerol phosphate oxidase

peroxidase

glycerol + ATP

glycerol-1-phosphate + O2

H2O2 + 4-aminoantipyrine + ESPA

glycerol-1-phosphate + ADP

dihydroxyacetone phosphate + H2O2

quinoneimine dye + H2O

glycerol kinase

glycerol phosphate oxidase

peroxidase

A

B

Figure 4.6.4 Lipolytic and lipogenic assays. (A) Triglyceride assay. (B) Glycerol assay. Abbrevia-tions: ADP, adenosine diphosphate; ATP, adenosine triphosphate; ESPA, sodium N -ethyl-N -(3-sul-

fopropyl)-m -anisidine.


4.6.14




all stocks and subsequent dilutions in deionized water containing 100ìM ascorbate and

10 ìM EDTA and wrap vessels in aluminum foil to reduce ultraviolet exposure.

Gelatin coating promotes cell adhesion to the culture vessel surface. For reproducible and

robust β-adrenoceptor assays, use gelatin-coated plates.

2. After treatment of the cells with agonists and/or antagonists, transfer 50 µl of the

culture medium from each culture vessel to a well in a new 96-well culture plate. Also,

construct a standard curve of glycerol concentrations in neighboring wells or in a

separate plate using glycerol standards according to the manufacturer’s instructions.

Since there will be variations in the amount of glycerol released from the mature adipocytes,it is recommended that the cells be pretested to determine exactly how much glycerol is

being released. This enables one to gauge the appropriate glycerol standard curve. If one

is only doing a comparative study (i.e., comparing the efficiency of one lipolytic agent

Table 4.6.5 Pharmacological Characteristics of the Human β3-Adrenoceptor

Ligandsa Binding Ki (nM)b Activity (EC50, nM)

β-adrenoceptor agonists

(−)Isoproterenol 620 ± 220 3.9 ± 0.4

(−)Noradrenaline 475 ± 75 6.3 ± 0.7BRL37344 287 ± 92 15 ± 3

(−)Adrenaline 20,650 ± 2,810 49 ± 5

SM11044 1,300 ± 200 84 ± 10

β3-adrenoceptor-selective agonists

GR219803B 6.18 0.3 ± 0.1

CL316243 14,000 1.3 ± 0.3

GR265261X 7.27 4.6 ± 1.7

Bucindolol 23 ± 10 7.0 ± 1.2

Carazolol 2.0 ± 0.2 11.3 ±1.2

GR230127A 5.82 17.3 ± 7.8

ICI201651 257 ± 34 20 ± 9

CGP12177A 2,300 ± 450 139 ± 44Pindolol 11.2 ± 2 153 ± 12

Alprenolol 110 ± 30 219 ± 46

β-Adrenoceptor partial agonists/antagonists

(−)Propranolol 257 ± 34

(−)Bupranolol 50 ± 14

β-Adrenoceptor antagonists

ICI 118551 357 ± 28

CGP20712A 2,300 ± 450

aChemical names: GR219803B, (4-{2R-{2-(3-Chlorophenyl)-2R-hydroxy-ethylamino]propyl-

amino}-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-Chlo-

rophenyl)-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.bData obtained from Strosberg and Pietri-Rouxel (1996).


4.6.15

Isolated Tissues



versus another), then a glycerol standard curve may not be necessary and the data can be

expressed in relative terms (i.e., absorbance at 540 nm).

3. Add 100 µl triglyceride reagent A (GPO-Trinder) to each well, avoiding the creation

of air bubbles. Incubate 5 to 60 min at 37°C.

If color development is rapid, then shorter incubation times should be used (∼5 min).

Conversely, a faint signal requires longer incubation times (≥60 min).

If air bubbles are formed when adding the GPO-Trinder working reagent, at the end of the

incubation add 95% ethanol to a final concentration of 5% to 10% to break the surface

tension of the air bubbles. The addition of ethanol also serves to stop the reaction.

GPO-Trinder reagent A contains glycerol kinase, glycerol phosphate oxidase, and peroxidase.

4. Read the absorbance at 540 nm using a microtiter plate reader/spectrophotometer.

The optical density at 540 nm is directly proportional to the triglyceride concentration in

the samples. The standard curve is used to calculate the concentration of glycerol (and

extent of lipolysis) in each sample.

Lipolysis is linear for the first 5 hr. Although cumulative dose-response curves can be

obtained, changes in β3-adrenoceptor activity are undetectable after 24 hr of agonist

treatment.

SUPPORT

PROTOCOL 1

ISOLATION AND CULTURE OF PRIMARY PREADIPOCYTES AND

ADIPOCYTES

This protocol describes a versatile method for preparing primary cultures of preadipocytes

and adipocytes from various tissue sources for measuring adrenoceptor-mediated lipoly-

sis (see Basic Protocol 4). Careful preparation of the tissue is important for obtaining a

functional adrenergic response. The protocol employs various sources of tissue ranging

from rodents to biopsies obtained from human subjects. The fat depots containing the

largest amount of β3-adrenoceptors are the hibernating glands (i.e., intrascapular brown

fat) in rodents and the visceral fat depots in human subjects. These depots, as well as bone

marrow stromal cells, also serve as good sources of preadipocytes, which can be made to

express the various adrenoceptors depending on the culture conditions.

CAUTION: Human tissue is a biohazard and should be handled according to the Occu-

pational Safety and Health Administration (OSHA) regulations for bloodborne pathogens

(29CFR1910-1030). This document is available athttp://www.osha-slc.gov/OshStd_data/

1910_1030.html. Institutional guidelines must be strictly followed.

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 (storeat −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)


4.6.16




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 SupportProtocol 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.

Prepare tissue

1. Prepare 100 ml KRB containing 1% (w/v) bovine fraction V albumin. Warm to 37°Cand adjust pH to 7.4 with 0.1 M HCl. Maintain at 37°C.

2. Excise fat pads or hibernating gland from exsanguinated rat or use fat sample from

human subject(s). Rinse sample in sterile KRB repeatedly to remove any blood.

Fat pads (e.g., epididymal, perirenal, perithymus, or intrascapular fat) may be isolated

from Sprague-Dawley rats (Charles River Labs). A 180-g rat will yield 0.5 g intrascapular

fat and 1 g epididymal fat. A sample of human fat may be obtained from a fat depot (e.g.,

visceral or perirenal). These samples may be obtained from patients undergoing surgery

at a local hospital. Young subjects, subjects with pheochromocytoma symptoms, and

subjects treated with troglitazone have increased brown adipocyte mass and β3-receptor

expression.

3. Add 3 ml KRB to 3 g adipose tissue in a sterile 20-ml plastic vial.

It is important to keep the ratio between adipose tissue mass and KRB volume at 1:1 (w/v).

4. Mince fat with a very sharp pair of dissecting scissors into pieces ∼2 mm in diameter,

using a swift cutting motion with the scissors so as to avoid rupturing the adipocytes.

Dissect out any fibrous material and/or blood vessels before proceeding.

5. Add 2 ml of 2 mg/ml collagenase type 1 stock per 3 mg tissue. Swirl and digest for

∼1 hr in a 37°C shaking water bath set at 100 strokes/min, swirling every 15 min

during the digestion and every 5 min near the end of the digestion.

The endpoint is reached when the buffer becomes creamy and runs down the sides of the

vials in sheets when gently swirled. Do not stop incubation while buffer still has a red color,

because cells will probably rupture when filtered.

From this step on, handle the cells carefully. Excessive cell lysis, which will interfere with

the sensitivity of β3 receptor assays, is detected by the formation of an oil interface on the

surface of the medium.

6. Add an equal volume of KRB to the vial containing the digested cells. With a rubber

band, secure a 250-µm piece of nylon mesh over the top of the scintillation vial and

gently squeeze contents into a sterile 50-ml conical centrifuge tube, gently tipping

the vial back and forth if necessary.


4.6.17

Isolated Tissues



Separate adipocytes and preadipocytes

7. Centrifuge the 50-ml collection tube 15 min at 800 × g, room temperature. Turn off

the centrifuge and use brake to stop.

Upon centrifugation, the cells separate into a pellet containing preadipocytes, a KRB

interface (to be discarded), and a top floating layer containing the mature adipocytes.

8. Using a wide-mouthed plastic pipet or a plastic transfer pipet, carefully remove the

floating adipocytes from the top and place them into a new 50-ml conical tube with

25 ml fresh KRB. Remove the KRB interface layer and discard, taking care not to

disrupt the pellet.

9. Carefully resuspend the preadipocyte pellet in 1 ml KRB and transfer to a new 50-ml

tube. Gently break up clumps of cells in the pellet by mixing up and down with an

additional 25 ml of KRB.

10. Separately wash the resuspended mature adipocytes and preadipocytes three times,

each time by centrifuging 15 min at 800 × g, room temperature, removing the

supernatant, and adding 25 ml KRB. Finally, centrifuge one more time at 1500 × g,

and remove the supernatant.

At this point, the floating adipocytes can be used to measure adrenoceptor-mediated

lipolysis (see Basic Protocol 4). The preadipocytes in the pellet must be cultured on sterile

plastic plates and differentiated into adipocytes before measuring adrenergic responses

(steps 24 and 25).

11. Plate the mature adipocytes on Matrigel according to the manufacturer’s instructions.

The floating adipocytes are difficult to maintain in culture and should be used as soon as

possible for measuring β-adrenoceptor mediated lipolysis. If long-term incubations (e.g.,

>24 hr) of adipocytes are required, the cells should be cultured in the presence of Matrigel

(Hazen et al., 1995). Matrigel does not interfere withβ-adrenoceptor-mediated lipolysis.

A variety of plates (6, 24, and 96-well) precoated with Matrigel are also available (Becton

Dickinson).

Culture and differentiate preadipocytes

12. Remove buffer from the preadipocyte pellet and carefully resuspend cells in culture

medium A.

If the sample is contaminated with red blood cells, this will markedly decrease cell

adherence and proliferation. Red blood cells can be removed by incubating the preadipo-

cyte sample for 10 min with an erythrocyte-lysing buffer consisting of 0.5 M NH 4Cl, 10 mM

KHCO3, and 0.1 M EDTA at room temperature. These conditions will lyse >95% of the red

blood cells without damaging the preadipocytes. The amount of cell damage may be

assessed by trypan blue exclusion (Phelan, 1996).

13. Remove an aliquot of the cells and determine the total cell number using a hemacy-

tometer.

Phelan (1996) provides details of counting cells.

14. Plate the preadipocytes on gelatin-coated plastic culture vessels at a density of 4–6

×10

3

cells/cm

2

. Add 0.2 ml culture medium A per cm

2

of surface and begin incubation.Feed cultures once or twice a week with culture medium A. When cells reach a density

of 1⁄ 3 to 2⁄ 3 confluency, split at a ratio of 1:10 to 1:20 into 162-cm2 gelatin-coated tissue

culture flasks.

Gelatin coating promotes cell adhesion to the culture vessel surface. For reproducible and

robust β-adrenoceptor assays, use gelatin-coated plates.

At this point the growing preadipocytes may be differentiated into adipocytes (steps 24 and

25) or stored frozen (steps 15 to 18), and later thawed (steps 19 to 23) and differentiated

into adipocytes (steps 24 and 25).


4.6.18




Freeze preadipocytes for storage

15. Remove culture medium from one 90% to 100% confluent 162-cm2 flask and rinse

once with sterile PBS.

16. Add 3 ml of 0.25% trypsin (enough to cover the bottom of the flask). Incubate 5 min

at 37°C, then terminate trypsinization by adding 20 ml culture medium A.

Treatment with trypsin will cause the cells to round up and detach from the culture surface.

17. Using a sterile pipet, transfer cell suspension to a centrifuge tube and centrifuge 5

min at 800 × g, room temperature, then remove medium. Resuspend cell pelletcarefully in freezing medium at a final cell density of 8 × 105 to 1 × 106 cells/ml.

18. Rapidly transfer the cell suspension to cryovial at 1 ml/vial. Place cells at −20°C for

2 to 4 hr, then transfer frozen cells to a liquid nitrogen storage container.

Recover preadipocytes from storage

19. Warm vial at 37°C immediately after removal from frozen storage. As soon as cell

suspension has defrosted, wipe vial with ethanol and transfer cells to a sterile plastic

tube. Add 15 ml culture medium A and centrifuge 5 min at 800 × g, 4°C, and remove

supernatant to eliminate residual DMSO.

20. Using a sterile pipet, carefully resuspend cell pellet in culture medium A and transfer

to a gelatin-coated 75-cm2 flask containing sufficient medium A for a total volumeof 25 ml.

21. Incubate 24 hr and replace medium with fresh medium A. Incubate until confluence

(passage 1).

These conditions yield an 80% to 90% recovery of viable cells, which are ready to

propagate up to six passages.

22. Plate cells on gelatin-coated plates at a density of 3000 cells/cm2. Allow cells to

adhere onto the plates for 16 to 20 hr.

At this density cells are usually in a preconfluent stage after the 20-hr attachment period.

23. Wash cells carefully with PBS to remove nonadhering material (e.g., cell debris andwhite blood cells), then add culture medium A to the cells and continue incubating.

Differentiate preadipocytes

24. Remove the medium from semiconfluent preadipocytes (1 day after plating) and add

0.2 ml differentiation medium (culture medium B for human cultures or culture

medium C for rodent cultures) per cm2 of culture vessel. Feed cells with fresh

differentiation medium once per week. Monitor differentiation by measuring

triglyceride accumulation or with Nile red staining (see Support Protocol 2).

Refeeding cells too often will result in cells coming off the plates. One approach to refeeding

is to carefully replace half of the medium at each feeding, so as not to disturb the adherent

cells.

After the first week of differentiation, there should be signs of lipid-droplet formation with

rodent cultures, whereas human cultures will display noticeable lipid accumulation to-

wards the end of the second week. After 3 to 4 weeks in culture, the cells should have

accumulated enough lipid to measure β-adrenoceptor-mediated lipolysis. Furthermore,

the β3-adrenoceptor message will have increased significantly after 3 to 4 weeks of

differentiation.

25. Optional:If there are problems in differentiating the preadipocytes into adipocytes,

supplement the differentiation medium by adding 0.5 M (1000× stock) dexametha-


4.6.19

Isolated Tissues



sone to a final concentration of 250 nM and 2.5 mM (10,000× stock) IBMX to a final

concentration of 500 µM to facilitate differentiation.

Under these conditions, the cells will express moreβ2-adrenoceptors and fewer β3-adreno-

ceptors.

SUPPORT

PROTOCOL 2

MEASURING ADIPOCYTE DIFFERENTIATION BY NILE RED STAININGOR TRIGLYCERIDE ACCUMULATION

Several methods can be employed to monitor the course of lipid accumulation and the

differentiation of adipocytes upon treatment with defined media (see Support Protocol 1).

These include using the fluorescent histochemical stain Nile red, as well as measuring

total triglyceride accumulation. Furthermore, morphological criteria can be judged by

viewing the cells under an inverted microscope. Differentiated adipocytes acquire a

rounded shape and their cytoplasm is completely filled with multiple lipid droplets. These

lipid-containing droplets are further identified by staining with the lipid-specific stain,

Oil-red O (Novikoff et al., 1980).

Materials

Differentiated adipocytes (see Support Protocol 1, steps 24 and 25)

10 mM Nile red (9-diethylamino-5H-benzo[α] phenoxazine-5-one; MolecularProbes) 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, andperoxidase)

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

To stain accumulated lipid with Nile red

1a. Add 10 mM Nile red stock solution to the cell medium at a final concentration of 5µM and incubate cells 5 to 10 min at 37°C.

Since Nile red fluorescence is quenched in aqueous solutions, the dye in the medium does

not affect background signals.

2a. Determine cellular fluorescence using a fluorimeter equipped with a 550-nm excita-

tion filter and a 635-nm emission filter. Alternatively, observe the cells under a

fluorescence microscope using either of the following spectral settings:

1. Yellow-gold fluorescence, 450- to 500-nm band-pass exciter filter; 580-nm cen-

ter-wavelength chromatic beam splitter; and 528-nm long-pass barrier filter (Zeiss

filter set 48-77-11)

2. Red fluorescence, 515- to 560-nm band-pass exciter filter; 580-nm center-wave-length chromatic beam splitter; and 590-nm long-pass barrier filter (Zeiss filter

set 48-77-14).

The yellow-gold filter set will allow for a more robust fluorescent signal than the red

filter.


4.6.20




To measure triglyceride accumulation in the cell

1b. Carefully aspirate off the medium from the differentiated cells. Add 40 µl of 0.01%

digitonin per cm2 of tissue culture vessel and incubate 30 min at room temperature

with shaking.

2b. Mix 4 parts GPO-Trinder reagent A and 1 part GPO-Trinder reagent B. Carefully add

50 µl of this mixture to the cell lysate, mix, and read the absorbance at 540 nm.

REAGENTS AND SOLUTIONS

Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, seeAPPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX .

Collagenase type 1 stock solution, 2 mg/ml

Prepare a 2 mg/ml solution of collagenase type 1 (Worthington) in DMEM (Life

Technologies) containing 4% (w/v) BSA.

Culture additive stock solutions

Troglitazone (10,000× stock solution): 100 mM dissolved in DMSO

Human insulin (1000× stock solution): 10 mg/ml dissolved in 0.01 N HCl, steril-

ized by filtration through 0.22-µm Millipore filter

Biotin (1000× stock solution): 100 µg/ml dissolved in distilled water, sterilized

by filtration through 0.22-µm Millipore filterStore stock solutions at −20°C

Biotin and human insulin are available from Sigma; troglitazone is available from Parke-

Davis.

Culture medium A

Dulbecco’s Minimum Essential Medium (DMEM), high-glucose formulation

(Life Technologies)

10% fetal bovine serum

10 mM HEPES

100 U/ml penicillin

0.1 mg/ml streptomycin

25 µg/ml Fungizone

Store up to 1 week at 4°C

Culture medium B


(Life Technologies)


10 mM HEPES

33 µM biotin (see recipe for culture additive stock solutions)

17 µM pantothenate (Sigma)

500 nM human insulin (see recipe for culture additive stock solutions)

1 nM triiodothyronine (Sigma)

10 µM troglitazone (see recipe for culture additive stock solutions)

1 µM 9-cis-retinoic acid (Sigma)100 U/ml penicillin


25 µg/ml Fungizone



4.6.21

Isolated Tissues



Culture medium C


(Life Technologies)


10 µg/ml human insulin (see recipe for culture additive stock solutions)

10 µM troglitazone (see recipe for culture additive stock solutions)

1 µM 9-cis retinoic acid

100 U/ml penicillin


25 µg/ml Fungizone


De Jalon’s solution

20× stock:

180 g NaCl

8.4 g KCl

Dilute to 1 liter with ultrapure deionized water

Store at 4°C

The most convenient method for preparing liter quantities of physiological salt solutions is

to keep a refrigerated concentrated stock solution of all of the ingredients except calcium,

glucose and bicarbonate.

Only the purest water can be used for in vitro isolated tissue experiments since trace amounts

of heavy-metal ions can lead to cell death.

1× working solution (calcium-free):

50 ml 20× stock solution (see above)

0.5 g NaHCO3

0.5 g D-(+)-glucose (C6H12O6⋅H2O)

Dilute to 1 liter with ultrapure deionized water

Add 0.06 g anhydrous CaCl2 to prepare De Jalon’s solution with 2.5 mM calcium.

To achieve the proper pH, this solution must be bubbled vigorously with Carbogen gas (95%

O2/5% CO2) for at least 30 min (see UNIT 4.3). The composition of De Jalon’s solution is:

Na+, 165.6 meq/liter; K + 5.6 meq/liter; Ca2+ (optional), 2.5 meq/liter; Cl−, 163 meq/liter;

HCO3−, 5.95 meq/liter, and glucose, 2.78 meq/liter.

DMEM/F12/1% BSA

DMEM/F-12 (Life Technologies) supplemented with:

1% (w/v) bovine serum albumin

100 U/ml penicillin


Gelatin-coated vessels

Prepare a 2% (w/v) stock solution of type B gelatin (from bovine skin; Sigma) and

store up to 1 year at 4°C. Dilute with PBS (see recipe) to a final concentration of

0.2%. Add 50 µl of this solution per cm2 of culture plate or flask surface area and

incubate 1 hr at room temperature. Aspirate the gelatin solution. Store coated vessels

up to 2 weeks under sterile conditions at 4°C.

Krebs-Henseleit solution (modified), 1×

Prepare Krebs-Henseleit solution (see recipe in UNIT 4.3), except add half as much

calcium (0.14 g CaCl2 per liter of 1× working solution).


4.6.22




Phosphate-buffered saline (PBS)

2.7 mM KCl

1.5 mM KH2PO4

8.1 mM Na2HPO4

137 mM NaCl

Store up to 1 year under sterile conditions at room temperature

This buffer is also known as Ca2+- and Mg2+-free Dulbecco’s phosphate-buffered saline.

COMMENTARY

Background InformationThe study of adrenoceptors by Dale and

others showed a heterogeneity in catecho-

lamine responses that suggested different re-

ceptor subtypes. These ideas were formalized

by Ahlquist, who classified adrenoceptors into

two general categories: α and β. However, the

tools available at that time were inadequate to

characterize these receptors further until Black

and colleagues described the first β-adrenocep-

tor blocker, pronethalol. The widespread avail-

ability of a pronethalol analog, propranolol,produced by the same group, led to the formal

classification of β-adrenoceptors. In sub-

sequent years, the development of potent and

selective agonists and antagonists has led to the

classification of β-adrenoceptors into three

subclasses: β1, β2, and β3 (see UNIT 1.5).

â -adrenoceptors: guinea pig atria

The sensitivity of guinea pig right atria to

β1-adrenoceptor stimulation (see Alternate

Protocol) is greater than that of left atria (see

Basic Protocol 1). This is reflected by a lower

comparative ED50 in right atrial preparations

(∼5-fold; compare Tables 4.6.1 and 4.6.2) and

a higher maximal response for the partial

agonists. This is illustrated by the relative re-

sponses to the β1-adrenoceptor partial agonist

prenalterol in the two preparations (Fig 4.6.5).

This increased sensitivity to weak agonists can

be advantageous when screening for β1-ad-

renoceptor agonists.

â 2-adrenoceptors: guinea pig trachea

The guinea pig trachea preparation (see Ba-

sic Protocol 2) was crucial in defining subtypes

of β-adrenoceptors and also in the discovery of

β2-adrenoceptor agonists for treatment of

asthma. It is a highly responsive preparation

that is able to detect responses to low-efficacy

β2-adrenoceptor agonists. It is versatile because

the potency and intrinsic activity of β2-adreno-

ceptor agonists can be controlled by changing

–10 – 9 – 8 –7 – 6 – 5

0

50

100

log[agonist] (M)

Maximum response (%)

Figure 4.6.5 Relative β1-adrenoceptor-mediated responses of guinea pig left atria (filled symbols)

and right atria (open symbols). Responses to isoproterenol (circles) and prenalterol (squares).


4.6.23

Isolated Tissues



the magnitude of the contraction placed on the

tissue. This manipulation of responsiveness can

be extremely useful when investigating the ef-

ficacy and affinity of agonists.

Guinea pig trachea assumes a spontaneous

tone, and frequent washing with bathing me-

dium hastens the onset of spontaneous contrac-

tion. Usually, spontaneous tone assumes be-

tween 20% and 50% of the total maximal tra-

cheal tension and takes 30 to 60 min to achievesteady state. β2-adrenoceptor agonists produce

relaxation of spontaneous tracheal tone. Since

this is the lowest level of contraction that can

be used to visualize relaxation,β2-adrenoceptor

agonists are most potent in relaxing spontane-

ous tone. Tracheas can be contracted to produce

a greater degree of tone; muscarinic receptor

agonists, such as carbachol, are used to increase

tone. Figure 4.6.6 shows the interplay between

contractile tone, potency, and observed maxi-

mal responses to β2-adrenergic agonists. Thus,low levels of tone, such as spontaneous tone,

1 2 3 4Maxim

um contractions (%)

log[carbachol]

Maximum relaxation (%)

log[carbachol]

1 2 3 4

1 2

3

4

relaxation

contraction

A

B

C

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.


4.6.24




make the tissue sensitive to β2-adrenergic

agonists, whereas high degrees of contractile

tone produce a dextral (rightward) shift in the

β2-adrenoceptor agonist dose-response curves,

with further contraction producing depression

of the relaxant response.

The opposing forces of muscarinic receptor

agonist–induced contraction and β2-adreno-

ceptor agonist–induced relaxation are useful in

the study of β2-adrenoceptor agonists in tra-cheal preparations. Thus, weak β2-adrenocep-

tor agonists can be used as complete antago-

nists for the measurement of their affinity (see

UNIT 4.1) by the production of strong muscarinic

contractions. Figure 4.6.7 shows the effects of

the low-efficacy β2-adrenoceptor agonist,

prenalterol, on guinea pig trachea that is weakly

contracted by spontaneous tone, and under con-

ditions of increasing contraction with carba-

chol, the muscarinic agonist. In the presence of

10 µM carbachol, prenalterol produces no

agonism, but rather appears to be an antagonist

of isoproterenol. Under these circumstances,the blockade of isoproterenol responses can be

used to estimate the affinity of prenalterol on

β2-adrenoceptors. This ability to control the

sensitivity of the tissue to β2-adrenoceptor

agonism is a unique feature of guinea pig trachea.

â 2-adrenoceptors: rat uterus

The most common problems encountered

with the rat uterus preparation are either lack

of responsiveness or an inordinate amount of

random spontaneous contraction. The former

problem usually stems from the rat not being

in a state of estrus. If the uterine horns are not

plump white tubes but are shriveled, this prob-

ably is the case. The second problem stems

from the fact that the uterus is naturally aspontaneously contracting organ. The removal

of calcium ion from the medium while the

preparation is being set up is designed to make

the preparation quiescent, but in some cases

there are sufficient internal calcium stores to

produce lasting spontaneous activity. Other

than frequent washing with calcium-free me-

dium, there is no solution to this problem other

than to set up another preparation.

Measuring â -adrenergic-stimulated lipolytic

activity

The stimulation of β3-adrenoceptors acti-vates adenylate cyclase through coupling with

G-stimulatory subunits (UNIT 2.1). The activation

of adenylate cyclase promotes the accumula-

tion of intracellular cAMP, which in turn acti-

vates a cAMP-dependent protein kinase A

–10 – 9 – 8

A

C

B

D

1.0

0

1.0

0 0

1.0

0

1.0

Fractional maximum

relaxation

Figure 4.6.7 Dose-response curves to the high efficacy β-adrenoceptor agonist isoproterenol

(filled circles) and lower efficacy β-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.


4.6.25

Isolated Tissues



(PKA). Subsequently, PKA phosphorylates

hormone sensitive lipase (HSL) and induces its

translocation to lipid droplets where HSL cata-

lyzes hydrolysis of triglyceride to glycerol and

free fatty acids in a process termed lipolysis

(Fig. 4.6.8). Thus, lipolysis can be utilized as a

functional readout of β3-adrenoceptor activa-

tion.

It should be pointed out thatβ3-adrenoceptor

expression may not necessarily translate intoβ3-adrenoceptor function (e.g., adenylate cy-

clase activity, cAMP production, and lipolysis

activation). Therefore, determination of both

expression and functional assays for the β3

receptor are critical for fully characterizing the

β3 receptor in tissue samples. Expression of the

β3 receptor is determined at the level of either

RNA or protein using the appropriate probes

(i.e., oligonucleotides and/or antibodies). Also,

binding assays utilizing radiolabeled ligands

allow determination of receptor expression and

number (see UNIT 1.5). Functionality of β3-ad-

renoceptors in both brown and white adipo-

cytes may be demonstrated by the use of a

number of selective and partial agonists for the

β3 receptor. Furthermore, antagonists for both

β1- and β2-adrenoceptors may be added to the

β3 assay, eliminating some of the potential

noise in the signal created by either receptor;the remaining activity is then attributed solely

to the β3-adrenoceptor either in the presence or

absence of β3 agonist.

The β3-adrenoceptor displays distinct func-

tional characteristics. Unlike the β1- and β2-ad-

renoceptors, theβ3-adrenoceptor interacts with

both Gs and Gi proteins. This interaction results

in activation of thermogenesis in brown adipo-

cytes. In white adipocytes, this interaction re-

β3

receptoradipocyte plasma membrane

adenosine

receptor

insulin

receptor

GS

cGI

PDE

adenylate

cyclase

Gi

ATP cAMP 5′ AMP

PKA

hormone

sensitive

lipase

hormone

sensitive

lipase

PO4

triglycerides glycerol + fatty acids

Figure 4.6.8 Regulation of lipolysis in β3-adrenoceptor-expressing adipocytes. The β3-adreno-

ceptor 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 β3-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.


4.6.26




sults in the oxidation of free fatty acids as fuel

and activation of lipolysis, releasing free fatty

acids into the circulation to supply the fuel for

the increased thermogenesis in brown adipo-

cytes. Further, brown adipose tissue (BAT) and

white adipose tissue (WAT) are different with

regard to their lipolytic responses to β-adreno-

ceptor agonists. This is a result of differences

in the relative numbers of the β1, β2, and β3-ad-

renoceptors in these tissues. There is strongevidence that the β1- and β2-adrenoceptors are

abundant in WAT, while β3-adrenoceptors are

abundant in BAT (Muzzin et al., 1991). Diet

and in vitro culture conditions can alter the

response of adipocytes to the various β-adreno-

ceptor agonists.

Critical Parameters

â -adrenoceptors: guinea pig left atria

The isolated left atrium preparation must be

stable to accurately measure drug effects (also

see UNIT 4.3, Critical Parameters, for isolatedcardiac muscle). An assessment of the sensitiv-

ity of the preparation to β1-adrenoceptor stimu-

lation can be gained from the location of the

dose-response curve with respect to standard

β-adrenoceptor agonists. The response of this

preparation toβ1-adrenoceptor agonists is gen-

erally sustained, making it possible to obtain

cumulative concentration-response curves. A

measure of sensitivity is obtained from a com-

plete concentration-response curve within 30

min (Fig. 4.6.9A). In this example, the individ-

ual lines represent a slow-chart-speed scan of

isometric twitch contractions. If the twitch isstudied on a faster time scale, β1-adrenoceptor

stimulation produces an increased inotropy (in-

creased maximal peak height) and increased

speed of contraction with higher rate of relaxa-

tion (inset to Fig 4.6.9A). Inotropy and diastolic

relaxation produced by β1-adrenoceptor stimu-

lation are not equally responsive, with the re-

laxation effects being observed at lower con-

centrations than inotropic effects. This is prob-

ably due to a higher sensitivity of the

sarcoplasmic reticulum calcium-removal sys-

tem to cytosolic cyclic AMP generated by β1-

adrenoceptor stimulation. Therefore, certainresponses to β1-adrenoceptor stimulation can

be used to study low-efficacy β1-adrenoceptor

agonists. For example, while the β-adrenocep-

–10 – 9 – 8 –7 –6 – 5

B

–10 – 9 – 8 –7 –6 – 5

C

Apositive

inotropy

positive

lusitropy

Tension (g)

Time

log[agonist]

1.0

0

1.0

0Fractional

maximum

response

Figure 4.6.9 Effects of isoproterenol on electrically stimulated twitch contractions of guinea pig

left atria. (A) β-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).


4.6.27

Isolated Tissues



tor partial agonist prenalterol produces a low

level of inotropic response in this tissue (Fig

4.6.9B), a larger, maximal scale of responsive-

ness can be obtained by measuring myocardial

relaxation (Fig 4.6.9C). A similar effect is ob-

tained with blockade of phosphodiesterases.

Typical sensitivities of guinea pig left atria

to β1-adrenoceptor agonists are provided on

Table 4.6.1 as the negative logarithm of molar

concentrations producing half the maximal re-sponse (pD2 values). The maximal responses

are shown as a fraction of that obtained with

isoproterenol in the same preparation.

A further measure of receptor classification

is gained from the study of receptor antagonists.

This is accomplished using Schild analysis

(UNIT 4.1), whereby cumulative agonist dose-re-

sponse curves are shifted to the right by a simple

competitive antagonist. For example, a cumu-

lative dose-response curve to isoproterenol is

obtained and the baseline response regained by

washing with drug-free medium. Following

this, the tissue is equilibrated with a set concen-tration of antagonist, after which a second dose-

response curve to the agonist is obtained. The

magnitude of the rightward shift of the dose-

response curve is used to calculate the potency

of the antagonist, and from this estimation it is

possible to determine whether the agonist pro-

duced its response by activation of β1-adreno-

ceptors. Antagonists can also produce depres-

sion of inotropic responses in guinea pig left

atria at concentrations greater than those re-

quired for receptor blockade. Accordingly,

these concentrations should be avoided since

such an effect would block non-β1-adrenocep-tor–mediated inotropy. Table 4.6.1 shows a list

of simple competitive receptor antagonists with

pKB values (negative logarithm of the molar

concentration of antagonist that produces a

two-fold rightward shift of the agonist dose

response curve) for potency estimation, deter-

mination of times required to reach equilib-

rium, and concentrations where myocardial de-

pression is observed. Atenolol is a rapid, con-

venient antagonist to use for identification of

β1-adrenoceptors.

â -adrenoceptors: guinea pig right atria

The stability of the isolated right atrium

preparation can be assessed by the lack of

arrhythmia in the spontaneous signal (also see

UNIT 4.3, Critical Parameters, for isolated cardiac

muscle). The response of this preparation to

β1-adrenoceptor agonists is generally sus-

tained, making it possible to obtain cumulative

concentration-response curves. A measure of

sensitivity may be obtained from a complete

concentration-response curve within 30 min

(Fig 4.6.10). In this example a dose-response

curve to the β1-adrenoceptor agonist isoproter-

enol is shown. The β1-adrenoceptor–mediated

chronotropy is readily blocked by β1-adrener-

gic blockers such as atenolol. Also shown in

Figure 4.6.10 is the time course and extent of

blockade of prenalterol β1-adrenergic re-

sponses by 10 µM atenolol.As with left atria, Schild analysis is used to

further classify receptors in this preparation

(see UNIT 4.1). Thus, cumulative dose-response

curves to an agonist are shifted to the right by

a simple competitive receptor antagonist. A

cumulative dose-response curve to isoproter-

enol is obtained, and the baseline response

regained by washing with drug-free medium

for 30 min. Following this, the tissue is equili-

brated with a set concentration of antagonist for

a predetermined time, then a second agonist

dose-response curve is generated. The magni-

tude of the rightward shift of the dose-responsecurve is used to calculate the potency of the

antagonist and to determine whether the agonist

produced the response by activation of β1-ad-

renoceptors. Antagonists also may depress

inotropic responses in guinea pig left atria at

concentrations greater than those required to

block receptors. Such concentrations should be

avoided because this may drive the inotropic

signal below the threshold needed to trigger the

rate-meter response. Table 4.6.2 shows a list of

simple competitive antagonists, with pKB val-

ues for potency estimation, times needed to

reach equlibrium, and concentrations wheremyocardial depression is observed. Atenolol is

a rapid, convenient antagonist to use for iden-

tificaton of β1-adrenoceptors.


Typical absolute potencies of β-adrenocep-

tor agonists are not shown, since the sensitivity

of this preparation to β-adrenoceptor agonists

is inversely proportional to the degree of con-

tractile tone on the tissue. Table 4.6.3 shows an

example of the pD2 values (the negative log of

the molar concentration producing 50% maxi-

mal effect) for isoproterenol and prenalterol on

trachea under spontaneous tone and contracted

by various concentrations of carbachol. Of

great value are the relative potencies of β-ad-

renoceptor agonists on this preparation. Thus,

while absolute potencies vary with contractile

tone, relative potencies do not, and are therefore

used for receptor classification and as a test of

tissue viability.


4.6.28




â 3-adrenoceptor-stimulated lipolytic activityAdipocyte isolation and culturing. Cell in-

tegrity is a major concern when isolating pri-

mary adipocytes and maintaining functional

β3-adrenergic responses. Since adipocytes, and

to a lesser extent preadipocytes, are fragile,

great care must be exercised in handling these

cells. A large-orifice pipet should be used for

resuspension and the pelleted cells should be

resuspended slowly. A simple, qualitative

measure for assessing the integrity of the cells

is to observe the amount of oil floating on the

top of the medium. There will always be some

oil floating during the collagenase incubation

and even during the initial washes; however,

the final washes should have very little oil

floating on the top. Cell integrity can also be

monitored by trypan-blue exclusion.

Another concern is the variability between

different batches of collagenase. This variabil-

ity can influence the amount and duration of

incubation for proper digestion of the fat tissue

as well as the response of the cells toβ3-adreno-ceptor agonists. Therefore, each batch of col-

lagenase must be tested individually for both

digestion efficiency and effects on β3-adreno-

ceptor sensitivity, which is key towards obtain-

ing functional adipocytes.

Measuring adipocyte differentiation. It is

important to culture the adipocytes under con-

ditions allowing for maximal fat accumulation

(lipogenesis) in the cell before testing for ad-

renergic activity, so that when lipolysis is meas-

ured as a function of adrenoceptor activity,

endogenous substrate (i.e., triglycerides) does

not limit the sensitivity of the assay. Nile red

(9-diethylamino-5H-benzo[α] phenoxazine-5-

one) is a versatile vital stain for detecting the

accumulation of intracellular lipids by fluores-

cence techniques (i.e., microscopy, fluorimetry,

and flow cytometry). This dye can be applied

to cells in an aqueous medium and does not

dissolve the lipids that it reveals. It is highly

fluorescent, but only in a hydrophobic environ-

10 nM 30 nM 0.1 µM 0.3 µM wash + 0.3 µM 1 µM 3 µM 10 µM 30 µM100

200

300

400

isoproterenol atenolol (10µM) isoproterenol

100

50

– 9 –8 –7 – 6 –5

% Maximum

response to isoproterenol

log[isoproterenol]

add

atenolol

A

B

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 isoproter-

enol come to steady state, the preparation is washed and fresh medium is added containing the

β-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 β-adrenoceptors.


4.6.29

Isolated Tissues



ment. This staining method is more versatile

than Oil-red O staining, since it can be readily

quantitated and is reversible. Staining can be

carried out on either fixed (1.5% formaldehyde,

5 min) or unfixed live cells. The main advantage

of Nile red is that it is not cytotoxic. Thus, after

Nile red staining the cells can be used to meas-

ure β3-adrenoceptor responses.

The advantage of using the GPO-Trinder

reagents over Nile red staining for measuringtotal triglyceride accumulation is that GPO-

Trinder can be quantitated using a conventional

spectrophotometer. Further, it allows for a di-

rect measure of cellular substrate (triglyceride)

and product (glycerol) involved in lipolysis.

However, the GPO-Trinder assay is not revers-

ible. Thus, the cells cannot be reused for meas-

uring β-adrenergic responses.

Measuring β-adrenoceptor-induced lipoly-

sis in isolated preadipocytes and mature adipo-

cytes. There are some advantages to using ma-

ture adipocytes (floating fat) versus differenti-

ating preadipocytes (pellets) when measuringlipolytic activity. Mainly, the floating cells have

accumulated the substrate (triglycerides)

needed for measuring lipolytic activity and will

be ready to assay on the day that they are

harvested. In contrast, the preadipocytes will

need time to differentiate and accumulate lipid.

However, the advantage of culturing preadipo-

cytes is that these cells are viable for longer

periods of time than isolated mature fat cells.

Further, these cells can be manipulated to ex-

press the β-adrenoceptor of choice depending

upon the pharmacological agents that are pre-

sent during differentiation. For example, treat-ment of preadipocytes with various compounds

(dexamethasone, dibutyryl cyclic AMP, PMA,

butyrate, or insulin) will up-regulate β1 or β2

while down-regulating β3-adrenoceptor ex-

pression through a transcriptional effect. How-

ever, treatment with triiodothyronine, and in

some instances thiazolidinedione (e.g., trogli-

tazone), will result in increased β3-adrenocep-

tor expression/responses.

Troubleshooting

â 1-adrenoceptors: guinea pig left atria

General problems with the isolated left atrial

preparation can be found in the Troubleshoot-

ing section of UNIT 4.3.

No observable response to β1-adrenoceptor

stimulation. The preparation may be releasing

endogenous catecholamines, causing maximal

β1-adrenoceptor stimulation in the absence of

added agonist. See UNIT 4.3, Troubleshooting, for

a suggested course of action.

Low sensitivity to β1-adrenoceptor agonism

but sufficient maximal asymptotic response.

Errors in drug concentration are usually the

cause of apparently low potency. If the drug is

not sufficiently dissolved in the stock solution,

subsequent dilutions will only compound the

error. As a first check, discard the drug solutions

and prepare fresh stocks.


General problems with the isolated right

atrial preparation can be found in the Trou-

bleshooting section of UNIT 4.3.

Loss of rate-response reading.The inotropic

twitch must bisect the internal temporal elec-

tronic signal of the rate meter in this assay. If

the preparation becomes weaker with washing,

or a negative inotropic drug such as a calcium-

channel blocking agent is used, then the maxi-

mal peak height may diminish below the thresh-

old for reading the rate response (Fig 4.6.11A).Because the strength of the inotropic response

can be augmented electronically, an amplifica-

tion of the inotropic signal entering the rate

meter will correct for this. Another way this

may occur is if the resting tension on the tissue

diminishes. Under these circumstances, either

the peak height will decline or the complete set

of contractions (from basal to peak) will fall

below the necessary rate signal of the meter (Fig

4.6.11B). If the transducer signal is not balanced,

changing the amplification of the inotropic sig-

nal midway through an experiment may alter

the baseline position. This should be correctedby electronic balancing of the transducer.

Cardiac arrhythmia. Heavy-metal ions can

cause serious arrhythmias in spontaneous car-

diac preparations. If this occurs, the solution

should be discarded. A fresh solution should be

prepared with deionized water and the prepa-

ration washed extensively. An apparent ar-

rhythmia may be due to erratic waveforms

entering the rate meter. If the inotropic signal

is exceedingly weak, or the aeration of the

solution is so high that large bubbles cause

irregular straining on the transducer, the inter-

ference with the temporal electronic signal of

the rate meter may be irregular (Fig 4.6.11C)

and will register as an apparent arrhythmia. The

ideal situation is one where the right atrium

produces a robust and strong signal. If artifac-

tual arrythmia is suspected, the rate meter

should be removed from the system and the

actual inotropic twitch responses visualized to

detect erratic baseline and peak responses.


4.6.30




No observable response to β1-adrenoceptor

stimulation. The preparation may be releasing

endogenous catecholamines causing maximal

β1-adrenoceptor stimulation in the absence of

added agonist. See UNIT 4.3, Troubleshooting, for

a suggested course of action.

Low sensitivity to β1-adrenoceptor agonism

but sufficient maximal asymptotic response.The easiest parameter to check is drug concen-

tration. Errors in drug concentration are most

commonly responsible for apparently low po-

tency. If the drug is not sufficiently dissolved

in the stock solution, subsequent dilutions will

only compound the error. To assess this possi-

bility, discard the drug solution and prepare

fresh stocks.

â -adenoceptors: guinea pig trachea

Guinea pig trachea does not attain a spon-

taneous tone. The tissue may be damaged or

otherwise compromised, resulting in spontane-

ous release of endogenous catecholamines.

Frequent washing may produce spontaneous

contraction, which would indicate removal of

endogenous agonists and/or recovery from in-

jury. If spontaneous tone is not observed, a new

preparation should be made.

The resting muscle tone is not constant. If a

fading baseline is obtained, the preparation

should be washed repeatedly to remove endo-

genous catcholamines. Alternatively, the rest-

ing tension on the tissue may be declining due

to slippage of the securing thread. The resting

tension should be readjusted and the tissue

washed repeatedly until a stable tone is ob-

tained.


Basal peak height begins to fade.A uniform

electrical stimulation is required for this prepa-

ration to be stable. If the strength of the signal

diminishes, it might reach the point where com-

plete recruitment of the muscle is not achieved

and the peak height will wane. This can occur

if the resistance of the system increases during

the experiment. The original peak height can

be recovered by increasing the voltage from the

amplifier. However, care must be taken not to

increase the voltage too much to avoid burning

the tissue at its interface with the tissue holder.

It also is possible that salts may encrust the

punctate electrode on the holder, thus increas-

ing resistance. This can be corrected by clean-

ing the surface of the electrode.

Spontaneous arrhythmic contractions.This

is probably the most common problem with this

preparation. If the rat is in a state of estrus,

spontaneous arrhythmic contractions can be

A loss of signal due

to declining inotropyB loss of signal due

to falling baseline

C loss of signal due to

mechanical abnormalities

rate

rate

rate

arrhythmia

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 notbisect 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.


4.6.31

Isolated Tissues



minimized. When it does occur, the electrical

stimulation should be switched off and the fluid

replaced with calcium-free De Jalon’s (several

wash cycles may be required to remove residual

calcium from the preparation). After 20 to 30

min in calcium-free medium, calcium should

be reintroduced gradually beginning with 1.25

mM for 15 min followed by the full 2.5 mM,

after which the electrical stimulation is reinsti-

tuted at threshold voltage (after 20 min). If arrhythmia persists, the experiment should be

terminated.

â -adrenoceptor-stimulated lipolytic activity

Lack of functional β3 response. Variability

in β-adrenoceptor expression is observed from

species to species, patient to patient, depot to

depot, and with conditions used to isolate and

culture the cells/tissue. For example, the β3

response is usually greater in rodent than in

human adipocytes. Similarly, the β3 response

is usually greater in neonatal than adult fat.

Often, the best responses can be obtained em-pirically by examining the effects of adrenergic

agonists on multiple fat depots. Responses to

β3 agonists may be undetectable if the tissue

contains mostly white adipocytes (i.e., human

subcutaneous fat contains predominantly β2

adrenoceptors). The β3-adrenergic responses

will be greatest in brown adipocytes (e.g., the

rodent hibernating gland). The amount of

brown adipocytes present in any depot can be

determined by the expression of markers that

are expressed predominantly in this cell type

(i.e., uncoupling protein or Type II-5-deiodi-

nase). Some fat depots (e.g., perirenal fat) mayexpress all of the β-receptor subtypes.

No observable lipolytic response to adren-

ergic stimulation.The cells may not have accu-

mulated enough substrate (triglycerides) to de-

tect generation of the product (glycerol) upon

stimulation with a β3-adrenoceptor agonist.

This problem can be remedied by allowing the

cells to differentiate for a longer period (>3

weeks) before measuring an adrenergic re-

sponse. Optimal adipogenesis requires the ad-

dition of insulin and thiazolidinedione (trogli-

tazone) or indomethacin (Lehmann et al., 1997;

Lenhard et al., 1997). Although glucocorticoids

will also stimulate lipid accumulation, they will

increase the amount of β1 and β2 receptors and

decrease the β3 receptor in fat. If accumulation

of endogenous triglyceride is not attainable,

then one can measure cAMP levels after stimu-

lation with β-adrenoceptor agonist/antagonist.

There also may be antilipolytic activity re-

sulting from endogenous insulin or adenosine.

Further washings of the cells with PBS and

treating the cells with adenosine deaminase

should remove these problems.

Endogenous phosphodiesterase may inhibit

the lipolytic response. The addition of 10 to 100

µM IBMX can help improve the adrenergic

response. Caution should be used, as the addi-

tion of too much phosphodiesterase inhibitor

(e.g., 1 mM IBMX) will maximally stimulate

lipolysis and mask the lipolytic response toβ3-agonists.

Low cell viability may result from inade-

quate culture conditions. Matrigel can be used

to help keep the adipocytes intact in culture for

longer periods of time. Follow the manufac-

turer’s instructions for optimal usage of Ma-

trigel (Hazen et al., 1995).

If the desired β3-adrenergic response is not

attainable using isolated primary adipocytes,

then immortalized cell lines may provide an-

other option. One cell line, the C3H10T1/2

pluripotent mesenchymal stem cell (available

from ATCC; see SUPPLIERS APPENDIX ), can differ-entiate into β3-adrenoceptor-expressing cells

given the proper differentiation conditions

(Paulik and Lenhard, 1997; Lenhard et al.,

1998). Furthermore, SAOS-2 cells, a human

osteosarcoma cell line, can also differentiate

into adipocytes that express the β3-adrenocep-

tor (Lenhard et al., 1998).

Anticipated Results


This preparation should yield a cumulative

dose-response curve to isoproterenol in 30 min,with a pD2 of 8.5. This response should be

readily blocked by 0.1µM atenolol. The relaxa-

tion effects of β1-adrenoceptor agonists are

observed at concentrations of agonist approxi-

mately one-fifth those required for positive

inotropy (except for partial agonists).


This preparation should yield a cumulative

dose-response curve to isoprerenol in 30 min,

with a pD2 of 9 to 9.2. This response should be

readily blocked by 0.1 µM atenolol.


A stably contracting preparation should be

exquisitely sensitive to β2-adrenoceptor stimu-

lation. These preparations should produce a

stable train of contractions within 1 hr. Once

the peak height of the contractions is stable, the

tissue is ready for experimentation. The tissue

should be stable for at least 4 hr with no desen-


4.6.32




sitization to β2-adrenoceptor stimulation if

dose-response curves are separated by 30 to 45

min washings in drug-free medium.

One feature of this tissue is the extremely

rapid onset of response and attainment of

steady-state (Fig. 4.6.12A). Figure 4.6.12B

shows a dose-response curve to isoproterenol.

In addition to being rapid, the response is sus-

tained, thus allowing for the generation of cu-

mulative dose-response curves (Fig. 4.6.12A).Another feature of this tissue is that recovery

after β2-adrenoceptor stimulation is evident

once the twitch response returns to basal levels

after removal of isoproterenol and washing

with drug-free medium.

â 3-adrenoceptors

Brown adipocytes, which express predomi-

nately β3-adrenoceptors, should yield dose-re-

sponse curves to isoproterenol and β3-selective

agonists, such as GR219803B or CL316243,

but not to β1 or β2-selective agonists (e.g.,

RO363 and albuterol, respectively). The EC50

for lipolysis induced by GR219803B or

CL316243 and isoproterenol should be be-

tween 1 to 4 nM using both human and rodent

adipocytes expressing β3 receptors. The lipo-

lytic response to β3-selective agonists should

not be blocked by 10 nM of the β1 antagonist

CGP20712A or the β2 antagonist ICI 118,551.Approximately 1 nM of glycerol should be

liberated from the cells after treating 1 cm2 of

confluent adipocytes for 1 hr at 37°C.

Time Considerations


The tissue preparation can be operative

within 90 min (30 min for setup and 60 min for

equilibration). Cumulative dose-response

A

B

1g

5 min

03 10 30 100

nM isoproterenol

–10 – 9 – 8 –7 – 6 – 5

Figure 4.6.12 β-adrenoceptor mediated responses to agonists. (A) Tracing for effects of isopro-

terenol on electrically stimulated uterine twitch contractions. (B) Dose-response curves to isopro-

terenol (filled circles), terbutaline (filled triangles), prenalterol (open squares), and dobutamine

(open circles).


4.6.33

Isolated Tissues



curves to standard agonists require ∼30 min to

obtain, followed by a 30-min washout period.

Repeated dose-response curves can be obtain-

ed, since the preparation is stable for 6 to 8 hr.

â 1-adrenoceptors: guinea pig right atria

The tissue preparation can be operative

within 90 min (30 min for setup and 60 min for

equilibration). Cumulative dose-response

curves to standard agonists require ∼30 min toobtain, followed by a 30-min washout period.

Repeated dose-response curves may be ob-

tained and the preparation is stable for 6 to 8

hr.


The rat must be pretreated with diethylstil-

bestrol (1 mg/kg) for two consecutive days

(using a single subcutaneous injection each

day). The dissection is rapid and the preparation

can be completed in 20 min. Dose-response

curves can be obtained 60 min later. A series of

dose-response curves to β2-adrenoceptoragonists can be generated in 4 to 5 hr.

â 3-adrenoceptor-stimulated lipolytic activity

Separation and preparation of primary

adipocytes and preadipocytes can be performed

in 2 hr. The primary adipocytes can be used

immediately for measuring β3-adrenoceptor-

mediated lipolysis and the assay completed

within 5 hr. The length of time for differentiat-

ing primary preadipocytes into adipocytes may

take from 2 to 3 weeks for the rodent cultures

and 3 to 4 weeks for the human cultures. Once

the primary cells have differentiated into adipo-cytes in culture, they can be used any time over

the next 2 to 3 months for testing β-adrenocep-

tor-mediated lipolysis. Feeding the cells fresh

medium requires only a few minutes. Time

courses can be run to determine the best times

for measuring β-adrenergic responses. Typi-

cally, dose-response curves of β3-agonists in

the lipolysis assay can be performed for 3 to 24

hr without significant differences in the calcu-

lated EC50 values. However, significant de-

creases in the rate of glycerol accumulation in

the medium are observed after 8 hr because of

desensitization of the lipolytic response. The

GPO assay for measuring glycerol release in

the medium can be completed in 1 hr. Extended

incubation times (>16 hr) for the GPO assay

will result in decreased optical density. Since

the quinoneimine dye is unstable, extended

incubation times are not recommended.

Literature CitedBlinks, J.R. 1966. Field stimulation as a means of

effecting the release of autonomic transmitters inisolated heart muscle. J. Pharmacol. Exp. Ther.151:221-235.

Donovan, J. and Brown, P. 1995a. Euthanasia. InCurrent Protocols in Immunology (J.E. Coligan,A.M. Kruisbeek, D.H. Margulies, E.M. Shevach,and W. Strober, eds.) pp. 1.8.1-1.8.4. John Wiley& Sons, New York.

Donovan, J. and Brown, P. 1995b. Parenteral injec-tions. 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 responsesto noradrenaline and isoprenaline in guinea pigisolated tracheal chain preparation by desi-pramine, cocaine, phentolamine, phenoxyben-zamine, guanethidine, metanephrine, and cool-ing. Br. J. Pharmacol. Chemother. 31:466-482.

Hazen, S.A., Rowe, W.A., and Lynch, C.J. 1995.Monolayer cell culture of freshly isolated adipo-cytes using extracellular basement membrane

components. J. Lipid Res. 36:868-875.Iversen, L.L. 1973. Neuronal and extraneuronal up-

take mechanisms. In Frontiers in CatecholamineResearch. Proc. 3rd Int. Catecholamine Sympo-sium. (E. Usdin and S.H. Snyder, eds.) pp. 403-418. Pergamon Press, New York.

Lehmann, J.M., Lenhard, J.M., Oliver, B.O., Rin-gold, G.M., and Kliewer, S.A. 1997. Peroxisomeproliferator-activated receptors γ and α are acti-vated by indomethacin and other non-steroidalanti-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 lipidmetabolism: Alterations of two distinct molecu-lar pathways. Biochem. Pharm. 54:801-808.

Lenhard, J.M., Lancaster, M., Hull-Ryde, E., andPaulik, M.A. 1998. Differentiation of a humanosteosarcoma cell line (SAOS-2) into brownadipocytes. Submitted for publication.

Muzzin, P., Revelli, J.P., Kuhne, F., Gocayne, J.D.,McCombie, W.R., Venter, J.C., Giacobino, J.P.,and Fraser, C.M. 1991. An adipose tissue–spe-cific beta-adrenergic receptor: Molecular clon-ing and down-regulation in obesity. J. Biol.Chem. 266:24053-24058.

Novikoff, A.B., Novikoff, P.M., Rosen, O.M., and

Rubin, C.S. 1980. Organelle relationships in cul-tured 3T3-L1 preadipocytes. J. Cell Biol.87:180-196.

Paulik, M.A. and Lenhard, J.M. 1997. Thiazolidi-nediones inhibit alkaline phosphatase activitywhile increasing expression of uncoupling pro-tein, deiodinase and increasing mitochondrialmass in C3H10T1/2 cells. Cell Tissue Res.290:79-87.


4.6.34




Phelan, M.C. 1996. Techniques for mammalian tis-sue culture. In Current Protocols in MolecularBiology (F.M. Ausubel, R. Brent, R.E. Kingston,D.D. Moore, J.G. Seidman, J.A. Smith, and K.Struhl, eds.) pp. A.3F.1-A.3F.14. John Wiley &Sons, New York.

Strosberg, D.A. and Pietri-Rouxel, F. 1996. Func-tion and regulation of the β3-adrenoceptor. TIPS17:373-381.

Key ReferencesBlinks, J.R. 1967. Evaluations of the cardiac effectsof 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 forassessing hormone effects on glucose transportin 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, forthe detection of neutral lipid deposits in tissuesections: comparison with oil red O. J. Histo-chem. Cytochem. 33:833-836.

Nile red staining.

Greenspan, P., Mayer, E.P., and Fowler, S.D. 1985.Nile red: A selective fluorescent stain for intra-cellular lipid droplets. J. Cell Biol. 100:965-973.

Nile red staining.

Ho, R.J., Bomboy, J.D., Wasner, H.K., and Suther-land, E.W. 1985. Preparation and character-ization of a hormone antagonist from adipocytes.Meth. Enzymol. 109:431-438.


Howe, R. 1993. β3-Adrenergic agonists. Drugs Fu-ture 18:529-549.

β3-adrenoceptors.

Hughes, I.E. 1978. The stability of noradrenaline inphysiological salt solutions. J. Pharm. Pharma-col. 30:124-126.

The effects of antioxidants on degradation of catecholamines.

Kenakin, T.P. 1981. An in vitro quantitative analysisof the alpha adrenoceptor partial agonist activityof dobutamine and its relevance to inotropicselectivity. J. Pharmacol. Exp. Ther. 216:210-219.

Example dose-response curves to β-adrenergic

agonists and effects of β-adrenergic antagonists.

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

Experiments involving blockade of catecholamineuptake.

Kenakin T.P. and Beek, D. 1980. Is prenalterol(H133/80) really a selective beta 1 adrenoceptoragonist? Tissue selectivity resulting from differ-

ences in stimulus-response relationships. J.Pharmacol. Exp. Ther. 213: 406-413.

Examples of dose-response curves to β-adrener-

gic agonists and effects of β-adrenergic antago-nists.

Kenakin, T.P. 1982. Theoretical and practical prob-lems with the assessment of the intrinsic efficacyof agonists: Efficacy of the reputed beta-1 selec-tive adrenoceptor agonists for beta-2 adrenocep-tors. J. Pharmacol. Exp. Ther. 223:416-423.

Example of dose-response curves to β-adrenergic

agonists and effects of β-adrenergic antagonists.

Kenakin, T.P., Ambrose, J.R., and Irving, P.E. 1991.The relative efficiency of beta adrenoceptor cou-pling to myocardial inotropy and diastolic re-laxation: Organ-selective treatment for diastolicdysfunction. 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. Tran-scriptional regulation of gene expression duringadipocyte 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 methodfor the colorimetric determination of serum tri-glycerides. Clin. Chem. 29:538.

Triglyceride determination.

Negrel, R., Grimaldi, P., Forest, C., and Ailhaud, G.1985. Establishment and characterization of fi-broblast-like cell lines derived from adipocyteswith the capacity to redifferentiate into adipo-cyte-like cells. Meth. Enzymol. 109:377-385.


Salt, P.J. 1972. Inhibition of noradrenaline uptake inthe isolated rat herat by steroids, clonidine, andmethoxylated phenylethylamines. Eur. J. Phar-macol. 20:329-340.

Experiments involving blockade of catecholamineuptake.

Sigma, 1990. Quantitative enzymatic determinationof glycerol, true triglycerides, and total tri-glycerides in serum or plasma at 540 nm. SigmaDiagnostics procedure no. 337, Sigma ChemicalCompany, St. Louis.


4.6.35

Isolated Tissues



Triglyceride determination.

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


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

Preadipocyte differentiation.

Contributed by Terry Kenakin,James M. Lenhard, and Mark A. Paulik

Glaxo Wellcome Research and DevelopmentResearch Triangle Park, North Carolina

â-Adrenoceptor

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Adrenoceptor Assays