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â-Adrenoceptor Assays UNIT 4.

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 classes—α-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 first
definitive 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.

â1-ADRENOCEPTORS: GUINEA PIG LEFT ATRIA BASIC


PROTOCOL 1
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, while
cumulative 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.

Isolated Tissues

Contributed by Terry Kenakin, James M. Lenhard, and Mark A. Paulik 4.6.1


Current Protocols in Pharmacology (1998) 4.6.1-4.6.36
Copyright © 1998 by John Wiley & Sons, Inc.
Materials
Physiological salt solution (Krebs-Henseleit solution; see recipe in UNIT 4.3),
continuously bubbled with Carbogen gas (UNIT 4.3)
Ascorbic acid and/or EDTA (added as antioxidants if catecholamines are to be
tested)
Inhibitors of neuronal and extraneuronal catecholamine uptake (optional):
desmethylimipramine or cocaine⋅HCl for neuronal uptake and 17β-estradiol for
extraneuronal uptake (all available from Sigma)
Phentolamine (Sigma) or other blockers of β-adrenoceptors
Male Hartley guinea pig, 250 to 400 g (Charles River Labs)
Standard β-adrenoceptor agonists: e.g., isoproterenol, epinephrine, or
norepinephrine (Table 4.6.1)
Standard 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 maintaining
and measuring response in isolated cardiac preparations (UNITS 4.2 & 4.3)
NOTE: Keep all drug solutions on ice during the course of the experiment.

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 and
Antagonists

Myocardial
Druga pD2b Max (α)c pKBd Time (min)f
depletion (µM)e
Agonists
Isoproterenol 8.5 1.0 — — —
Epinephrine 7.5 1.0 — — —
Norepinephrine 7.5 1.0 — — —
Prenalterol 7.2 0.28 — — —
Dobutamine 6.4 1.0 — — —
Antagonists
Atenolol — — 7.2 100 30
Propranolol — — 8.4 3 60
Timolol — — 9.4 1 90
Pindolol — — 9.1 1 90
Nadolol — — 8.5 1 60
aCompounds available from Sigma (see SUPPLIERS APPENDIX).
bNegative logarithm of the molar concentration of agonist producing half the maximal response.
cIntrinsic activity defined as the fractional maximal response to a full agonist (in this case, isoproterenol).
dNegative logarithm of the equilibrium dissociation constant of the antagonist-receptor complex (also the
negative logarithm of the molar concentration of antagonist that occupies half the receptor population).
â-Adrenoceptor eBeyond this concentration, depression of normal cardiac function may occur.
Assays fTime required for equilibration of antagonist with β-adrenoceptors.

4.6.2
Current Protocols in Pharmacology
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 tissue 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 with β1-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 include α-adrenoceptor antagonists in the assay if nonselective adrenergic
agonists are used—e.g., norepinephrine, epinephrine, or certain synthetic agents. Epineph-
rine, phenylephrine, and norepinephrine activate α-adrenoceptors, which can result in
weak inotropic effects in this tissue.
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 tissue), 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 (see UNIT 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.
Isolated Tissues

4.6.3
Current Protocols in Pharmacology
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 100 ìM ascorbate and wrap in aluminum foil.
9. Optional: Test the responsiveness of the isolated left atrial preparation to standard
β1-adrenoceptor agonists—e.g., isoproterenol, norepinephrine, or epinephrine.
A concentration producing ∼50% of the maximal response is best for this test since it will
not cause significant desensitization of the β1-adrenoceptors.
Shown in Table 4.6.1 are sensitivities of guinea pig left atria to β1-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 activation
of 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 antagonist—i.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 produce β1-adrenoceptor–mediated responses in this
preparation are known from previous study for a number of compounds, test compounds
â-Adrenoceptor are of unknown activity. A reasonable starting point for suspected agonists is to prepare
Assays 10-fold dilutions in deionized water ranging from 0.1 nM to 100 mM. Beginning with the
4.6.4
Current Protocols in Pharmacology
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 + Kn), where Max refers to the
maximal 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 in UNIT 4.1.

â1-ADRENOCEPTORS: GUINEA PIG RIGHT ATRIA ALTERNATE


PROTOCOL
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 advantage
over the left in that there is a built-in control of tissue viability—i.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. Isolated Tissues

4.6.5
Current Protocols in Pharmacology
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 to β1-adrenoceptor agonists.
The data are given as the negative logarithm of the molar concentrations producing 50%

c
a
twitch contractions

∆ current

∆ current measures
interval between constantly
contractions declining
rate meter
signal

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
â-Adrenoceptor the rate of decay of the internal signal (side c); thus the base of the triangle (side b), which is the
Assays
time between heartbeats, is calculated by the Pythagorean theorem.
4.6.6
Current Protocols in Pharmacology
Table 4.6.2 Sensitivities of Guinea Pig Right Atria
to β-Adrenoceptor Agonistsa

Drugb pD2c Max (α)c


Isoproterenol 9.2 1.0
Epinephrine 8.0 1.0
Norepinephrine 7.5 1.0
Prenalterol 6.9 0.4
Pirbuterol 6.7 0.4
Dobutamine 6.5 1.0
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).

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.

â2-ADRENOCEPTORS: GUINEA PIG TRACHEA BASIC


Tracheal smooth muscle is a very versatile preparation. Its spontaneous tone can be used PROTOCOL 2
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 in
isolated 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
Isolated Tissues

4.6.7
Current Protocols in Pharmacology
antioxidant (ascorbic acid) and metal chelator (EDTA) to the medium (see Basic
Protocol 1, step 1).
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 tissue 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 which is cablable of being bubbled continuously with
Carbogen gas and which has the capability of being rapidly filled and emptied with
physiological 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 are
measured. 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
â-Adrenoceptor
Assays enhance contraction further with receptor agonists. However, the level of spontaneous tone

4.6.8
Current Protocols in Pharmacology
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.

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.

Isolated Tissues

4.6.9
Current Protocols in Pharmacology
Table 4.6.3 Sensitivities of Guinea Pig Trachea to β-Adrenoceptor
Agonists and Antagonists

Druga pD2b Max (α)c pKBd

Agonists
Isoproterenol 9.6 1.0 —
(spontaneous muscle tone)
Prenalterol 7.5 0.7 —
(spontaneous muscle tone)
Isoproterenol 9.0 1.0 —
(contracted with 1 µM carbachol)
Prenalterol 7.1 0.4 —
(contracted with 1 µM carbachol)
Isoproterenol 8.15 1.0 —
(contracted with 10 µM carbachol)
Isoproterenol 7.9 1.0 —
(contracted with 10 µM bethanecol)
Norepinephrine 6.54 1.0 —
(contracted with 10 µM bethanecol)
Salbutamol 6.5 1.0 —
(contracted with 10 µM bethanecol)
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).
dNegative logarithm of the equilibrium dissociation constant of the antagonist-receptor
complex (also the negative logarithm of the molar concentration of antagonist that
occupies half the receptor population).

BASIC â2-ADRENOCEPTORS: RAT UTERUS


PROTOCOL 3 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 stock—e.g., Fisher—as
dry salt is hygroscopic)
Phenoxybenzamine (Sigma)
â-Adrenoceptor Standard agonists and antagonists for receptor classification: e.g., isoproterenol,
Assays ICI 118,551, and propranolol (Table 4.6.4)
4.6.10
Current Protocols in Pharmacology
Test compounds
Petri dish
Surgical instruments (fine scissors and forceps)
5–0 silk thread
Additional reagents and equipment for maintaining and measuring response in
isolated tissue preparations (UNITS 4.2 & 4.3)
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 consecutive
days 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’s
solution 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.
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 tissue 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.
6. Wash the tissue with fresh calcium-free De Jalon’s solution. Administer electrical
stimulation 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. Isolated Tissues

4.6.11
Current Protocols in Pharmacology
A B

four preparations

uterine
horns

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.

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 10 µM 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 solution
containing 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-
â-Adrenoceptor ing agent that forms an aziridinium ion in aqueous solution, which goes on to form alkyl
Assays bonds with many other chemical groups. Since it attaches irreversibly, the tissue should be
4.6.12
Current Protocols in Pharmacology
Table 4.6.4 Sensitivities of Rat Uterus to
β-Adrenoceptor Agonistsa

Drugb pD2c Max (α)d


Isoproterenol 9.5 1.0
Prenalterol 7.2 1.0
Dobutamine 6.4 1.0
Terbutaline 7.7 1.0
Tazolol 6.4 1.0
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.
dIntrinsic activity defined as the fractional maximal response to
a full agonist (in this case isoproterenol).

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.

MEASURING â-ADRENOCEPTOR-STIMULATED LIPOLYTIC ACTIVITY BASIC


PROTOCOL 4
The accumulation of cellular triglycerides is dependent on a balance between lipogenesis
and lipolysis. Thus, the success of measuring β-adrenoceptor-mediated lipolysis and
triglyceride 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
agents—e.g., insulin, thiazolidinediones, or adenosine—should 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 a
result 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.

Isolated Tissues

4.6.13
Current Protocols in Pharmacology
A
lipoprotein lipase
triglycerides glycerol + fatty acids

glycerol kinase
glycerol + ATP glycerol-1-phosphate + ADP

glycerol phosphate oxidase


glycerol-1-phosphate + O2 dihydroxyacetone phosphate + H2O2

peroxidase
H2O2 + 4-aminoantipyrine + ESPA quinoneimine dye + H2O

B
glycerol kinase
glycerol + ATP glycerol-1-phosphate + ADP

glycerol phosphate oxidase


glycerol-1-phosphate + O2 dihydroxyacetone phosphate + H2O2

peroxidase
H2O2 + 4-aminoantipyrine + ESPA quinoneimine dye + H2O

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.

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 EC50 value for the particular
compound being tested (see Table 4.6.5).
â-Adrenoceptor Since isoproterenol and other catecholamines (e.g., epinephrine and norepinephrine)
Assays degrade when exposed to alkaline pH, trace heavy metals, and/or ultraviolet light, prepare
4.6.14
Current Protocols in Pharmacology
all stocks and subsequent dilutions in deionized water containing 100 ìM 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 ± 0.7
BRL37344 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 ± 44
Pindolol 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).
Isolated Tissues

4.6.15
Current Protocols in Pharmacology
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 ISOLATION AND CULTURE OF PRIMARY PREADIPOCYTES AND


PROTOCOL 1 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 at http://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 (store
at −20°C)
2.5 mM (10,000× stock) dexamethasone (Sigma) in DMSO (store at −20°C)
â-Adrenoceptor Dissecting instruments
Assays 20-ml plastic vials (e.g., large scintillation vials; Wheaton)
4.6.16
Current Protocols in Pharmacology
Shaking water bath
250-µm nylon mesh
50-ml conical polypropylene centrifuge tubes
Tabletop centrifuge
75-cm2 and 162-cm2 tissue culture flasks (gelatin-coated; see recipe)
Cryovials
Additional reagents and equipment for monitoring differentiation (see Support
Protocol 2)
NOTE: All reagents and equipment coming into contact with live cells must be sterile,
and proper sterile technique must be followed accordingly.
NOTE: All culture incubations are performed in a humidified 37°C, 5% CO2 incubator
unless otherwise specified.
NOTE: Except where otherwise indicated, all reagents should be warmed to 37°C prior
to use.

Prepare tissue
1. Prepare 100 ml KRB containing 1% (w/v) bovine fraction V albumin. Warm to 37°C
and 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.

Isolated Tissues

4.6.17
Current Protocols in Pharmacology
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 with β-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 NH4Cl, 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
× 103 cells/cm2. Add 0.2 ml culture medium A per cm2 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
â-Adrenoceptor 25) or stored frozen (steps 15 to 18), and later thawed (steps 19 to 23) and differentiated
Assays into adipocytes (steps 24 and 25).

4.6.18
Current Protocols in Pharmacology
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 pellet
carefully 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 volume
of 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 and
white 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- Isolated Tissues

4.6.19
Current Protocols in Pharmacology
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 more β2-adrenoceptors and fewer β3-adreno-
ceptors.

SUPPORT MEASURING ADIPOCYTE DIFFERENTIATION BY NILE RED STAINING


PROTOCOL 2 OR 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; Molecular
Probes) in DMSO (store up to 6 months protected from light at −20°C)
0.01% (w/v) digitonin
GPO-Trinder kit (Sigma) consisting of:
Triglyceride reagent A (glycerol kinase, glycerol phosphate oxidase, and
peroxidase)
Triglyceride reagent B (lipase)
Fluorimeter with 550-nm excitation filter and 635-nm emission filter or
fluorescence microscope with Zeiss filter set 48-77-11 or 48-77-14
Shaker
Spectrophotometer

To stain accumulated lipid with Nile red


1a. Add 10 mM Nile red stock solution to the cell medium at a final concentration of 5
µM 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.

â-Adrenoceptor
Assays

4.6.20
Current Protocols in Pharmacology
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, see
APPENDIX 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 filter
Store 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
Dulbecco’s Minimum Essential Medium (DMEM), high-glucose formulation
(Life Technologies)
10% fetal bovine serum
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
0.1 mg/ml streptomycin
25 µg/ml Fungizone
Store up to 1 week at 4°C

Isolated Tissues

4.6.21
Current Protocols in Pharmacology
Culture medium C
Dulbecco’s Minimum Essential Medium (DMEM), high-glucose formulation
(Life Technologies)
10% fetal bovine serum
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
0.1 mg/ml streptomycin
25 µg/ml Fungizone
Store up to 1 week at 4°C
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
0.1 mg/ml streptomycin
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).

â-Adrenoceptor
Assays

4.6.22
Current Protocols in Pharmacology
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 Information Protocol) is greater than that of left atria (see
The study of adrenoceptors by Dale and Basic Protocol 1). This is reflected by a lower
others showed a heterogeneity in catecho- comparative ED50 in right atrial preparations
lamine responses that suggested different re- (∼5-fold; compare Tables 4.6.1 and 4.6.2) and
ceptor subtypes. These ideas were formalized a higher maximal response for the partial
by Ahlquist, who classified adrenoceptors into agonists. This is illustrated by the relative re-
two general categories: α and β. However, the sponses to the β1-adrenoceptor partial agonist
tools available at that time were inadequate to prenalterol in the two preparations (Fig 4.6.5).
characterize these receptors further until Black This increased sensitivity to weak agonists can
and colleagues described the first β-adrenocep- be advantageous when screening for β1-ad-
tor blocker, pronethalol. The widespread avail- renoceptor agonists.
ability of a pronethalol analog, propranolol,
produced by the same group, led to the formal â2-adrenoceptors: guinea pig trachea
classification of β-adrenoceptors. In sub- The guinea pig trachea preparation (see Ba-
sequent years, the development of potent and sic Protocol 2) was crucial in defining subtypes
selective agonists and antagonists has led to the of β-adrenoceptors and also in the discovery of
classification of β-adrenoceptors into three β2-adrenoceptor agonists for treatment of
subclasses: β1, β2, and β3 (see UNIT 1.5). asthma. It is a highly responsive preparation
that is able to detect responses to low-efficacy
â-adrenoceptors: guinea pig atria β2-adrenoceptor agonists. It is versatile because
The sensitivity of guinea pig right atria to the potency and intrinsic activity of β2-adreno-
β1-adrenoceptor stimulation (see Alternate ceptor agonists can be controlled by changing

100
Maximum response (%)

50

0
–10 –9 –8 –7 –6 –5
log[agonist] (M)

Figure 4.6.5 Relative β1-adrenoceptor-mediated responses of guinea pig left atria (filled symbols)
Isolated Tissues
and right atria (open symbols). Responses to isoproterenol (circles) and prenalterol (squares).
4.6.23
Current Protocols in Pharmacology
the magnitude of the contraction placed on the relaxation of spontaneous tracheal tone. Since
tissue. This manipulation of responsiveness can this is the lowest level of contraction that can
be extremely useful when investigating the ef- be used to visualize relaxation, β2-adrenoceptor
ficacy and affinity of agonists. agonists are most potent in relaxing spontane-
Guinea pig trachea assumes a spontaneous ous tone. Tracheas can be contracted to produce
tone, and frequent washing with bathing me- a greater degree of tone; muscarinic receptor
dium hastens the onset of spontaneous contrac- agonists, such as carbachol, are used to increase
tion. Usually, spontaneous tone assumes be- tone. Figure 4.6.6 shows the interplay between
tween 20% and 50% of the total maximal tra- contractile tone, potency, and observed maxi-
cheal tension and takes 30 to 60 min to achieve mal responses to β2-adrenergic agonists. Thus,
steady state. β2-adrenoceptor agonists produce low levels of tone, such as spontaneous tone,

A
Maximum contractions (%)

1 2 3 4
log[carbachol]

B 4
contraction
relaxation

3
2
1

C
Maximum relaxation (%)

1 2 3 4

log[carbachol]

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
â-Adrenoceptor
Assays 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
Current Protocols in Pharmacology
make the tissue sensitive to β2-adrenergic â2-adrenoceptors: rat uterus
agonists, whereas high degrees of contractile The most common problems encountered
tone produce a dextral (rightward) shift in the with the rat uterus preparation are either lack
β2-adrenoceptor agonist dose-response curves, of responsiveness or an inordinate amount of
with further contraction producing depression random spontaneous contraction. The former
of the relaxant response. problem usually stems from the rat not being
The opposing forces of muscarinic receptor in a state of estrus. If the uterine horns are not
agonist–induced contraction and β2-adreno- plump white tubes but are shriveled, this prob-
ceptor agonist–induced relaxation are useful in ably is the case. The second problem stems
the study of β2-adrenoceptor agonists in tra- from the fact that the uterus is naturally a
cheal preparations. Thus, weak β2-adrenocep- spontaneously contracting organ. The removal
tor agonists can be used as complete antago- of calcium ion from the medium while the
nists for the measurement of their affinity (see preparation is being set up is designed to make
UNIT 4.1) by the production of strong muscarinic the preparation quiescent, but in some cases
contractions. Figure 4.6.7 shows the effects of there are sufficient internal calcium stores to
the low-efficacy β2-adrenoceptor agonist, produce lasting spontaneous activity. Other
prenalterol, on guinea pig trachea that is weakly than frequent washing with calcium-free me-
contracted by spontaneous tone, and under con- dium, there is no solution to this problem other
ditions of increasing contraction with carba- than to set up another preparation.
chol, the muscarinic agonist. In the presence of
10 µM carbachol, prenalterol produces no Measuring â-adrenergic-stimulated lipolytic
agonism, but rather appears to be an antagonist activity
of isoproterenol. Under these circumstances, The stimulation of β3-adrenoceptors acti-
the blockade of isoproterenol responses can be vates adenylate cyclase through coupling with
used to estimate the affinity of prenalterol on G-stimulatory subunits (UNIT 2.1). The activation
β2-adrenoceptors. This ability to control the of adenylate cyclase promotes the accumula-
sensitivity of the tissue to β2-adrenoceptor tion of intracellular cAMP, which in turn acti-
agonism is a unique feature of guinea pig trachea. vates a cAMP-dependent protein kinase A

A B
1.0 1.0
Fractional maximum

0 0
relaxation

C D
1.0 1.0

0 0
–10 –9 –8

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)
Isolated Tissues
comprising the data from panels A to C.
4.6.25
Current Protocols in Pharmacology
(PKA). Subsequently, PKA phosphorylates binding assays utilizing radiolabeled ligands
hormone sensitive lipase (HSL) and induces its allow determination of receptor expression and
translocation to lipid droplets where HSL cata- number (see UNIT 1.5). Functionality of β3-ad-
lyzes hydrolysis of triglyceride to glycerol and renoceptors in both brown and white adipo-
free fatty acids in a process termed lipolysis cytes may be demonstrated by the use of a
(Fig. 4.6.8). Thus, lipolysis can be utilized as a number of selective and partial agonists for the
functional readout of β3-adrenoceptor activa- β3 receptor. Furthermore, antagonists for both
tion. β1- and β2-adrenoceptors may be added to the
It should be pointed out that β3-adrenoceptor β3 assay, eliminating some of the potential
expression may not necessarily translate into noise in the signal created by either receptor;
β3-adrenoceptor function (e.g., adenylate cy- the remaining activity is then attributed solely
clase activity, cAMP production, and lipolysis to the β3-adrenoceptor either in the presence or
activation). Therefore, determination of both absence of β3 agonist.
expression and functional assays for the β3 The β3-adrenoceptor displays distinct func-
receptor are critical for fully characterizing the tional characteristics. Unlike the β1- and β2-ad-
β3 receptor in tissue samples. Expression of the renoceptors, the β3-adrenoceptor interacts with
β3 receptor is determined at the level of either both Gs and Gi proteins. This interaction results
RNA or protein using the appropriate probes in activation of thermogenesis in brown adipo-
(i.e., oligonucleotides and/or antibodies). Also, cytes. In white adipocytes, this interaction re-

β3 adenosine insulin
adipocyte plasma membrane receptor
receptor receptor
cGI
Gi PDE
GS

adenylate
cyclase

ATP cAMP 5′ AMP

PKA

PO4

hormone hormone
sensitive sensitive
lipase lipase

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)
â-Adrenoceptor coupled to the adenosine receptor, (2) the cAMP-phosphosdiesterase (cGI-PDE), and (3) the insulin
Assays receptor.
4.6.26
Current Protocols in Pharmacology
sults in the oxidation of free fatty acids as fuel dose-response curve with respect to standard
and activation of lipolysis, releasing free fatty β-adrenoceptor agonists. The response of this
acids into the circulation to supply the fuel for preparation to β1-adrenoceptor agonists is gen-
the increased thermogenesis in brown adipo- erally sustained, making it possible to obtain
cytes. Further, brown adipose tissue (BAT) and cumulative concentration-response curves. A
white adipose tissue (WAT) are different with measure of sensitivity is obtained from a com-
regard to their lipolytic responses to β-adreno- plete concentration-response curve within 30
ceptor agonists. This is a result of differences min (Fig. 4.6.9A). In this example, the individ-
in the relative numbers of the β1, β2, and β3-ad- ual lines represent a slow-chart-speed scan of
renoceptors in these tissues. There is strong isometric twitch contractions. If the twitch is
evidence that the β1- and β2-adrenoceptors are studied on a faster time scale, β1-adrenoceptor
abundant in WAT, while β3-adrenoceptors are stimulation produces an increased inotropy (in-
abundant in BAT (Muzzin et al., 1991). Diet creased maximal peak height) and increased
and in vitro culture conditions can alter the speed of contraction with higher rate of relaxa-
response of adipocytes to the various β-adreno- tion (inset to Fig 4.6.9A). Inotropy and diastolic
ceptor agonists. relaxation produced by β1-adrenoceptor stimu-
lation are not equally responsive, with the re-
Critical Parameters laxation effects being observed at lower con-
centrations than inotropic effects. This is prob-
â-adrenoceptors: guinea pig left atria ably due to a higher sensitivity of the
The isolated left atrium preparation must be sarcoplasmic reticulum calcium-removal sys-
stable to accurately measure drug effects (also tem to cytosolic cyclic AMP generated by β1-
see UNIT 4.3, Critical Parameters, for isolated adrenoceptor stimulation. Therefore, certain
cardiac muscle). An assessment of the sensitiv- responses to β1-adrenoceptor stimulation can
ity of the preparation to β1-adrenoceptor stimu- be used to study low-efficacy β1-adrenoceptor
lation can be gained from the location of the agonists. For example, while the β-adrenocep-

A
positive
inotropy

positive
lusitropy Tension (g)

Time

B C
1.0 1.0
Fractional
maximum
response

0 0
–10 –9 –8 –7 –6 –5 –10 –9 –8 –7 –6 –5

log[agonist]

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
Isolated Tissues
to cumulatively added isoproterenol (filled circles) and prenalterol (open circles).
4.6.27
Current Protocols in Pharmacology
tor partial agonist prenalterol produces a low sensitivity may be obtained from a complete
level of inotropic response in this tissue (Fig concentration-response curve within 30 min
4.6.9B), a larger, maximal scale of responsive- (Fig 4.6.10). In this example a dose-response
ness can be obtained by measuring myocardial curve to the β1-adrenoceptor agonist isoproter-
relaxation (Fig 4.6.9C). A similar effect is ob- enol is shown. The β1-adrenoceptor–mediated
tained with blockade of phosphodiesterases. chronotropy is readily blocked by β1-adrener-
Typical sensitivities of guinea pig left atria gic blockers such as atenolol. Also shown in
to β1-adrenoceptor agonists are provided on Figure 4.6.10 is the time course and extent of
Table 4.6.1 as the negative logarithm of molar blockade of prenalterol β1-adrenergic re-
concentrations producing half the maximal re- sponses by 10 µM atenolol.
sponse (pD2 values). The maximal responses As with left atria, Schild analysis is used to
are shown as a fraction of that obtained with further classify receptors in this preparation
isoproterenol in the same preparation. (see UNIT 4.1). Thus, cumulative dose-response
A further measure of receptor classification curves to an agonist are shifted to the right by
is gained from the study of receptor antagonists. a simple competitive receptor antagonist. A
This is accomplished using Schild analysis cumulative dose-response curve to isoproter-
(UNIT 4.1), whereby cumulative agonist dose-re- enol is obtained, and the baseline response
sponse curves are shifted to the right by a simple regained by washing with drug-free medium
competitive antagonist. For example, a cumu- for 30 min. Following this, the tissue is equili-
lative dose-response curve to isoproterenol is brated with a set concentration of antagonist for
obtained and the baseline response regained by a predetermined time, then a second agonist
washing with drug-free medium. Following dose-response curve is generated. The magni-
this, the tissue is equilibrated with a set concen- tude of the rightward shift of the dose-response
tration of antagonist, after which a second dose- curve is used to calculate the potency of the
response curve to the agonist is obtained. The antagonist and to determine whether the agonist
magnitude of the rightward shift of the dose- produced the response by activation of β1-ad-
response curve is used to calculate the potency renoceptors. Antagonists also may depress
of the antagonist, and from this estimation it is inotropic responses in guinea pig left atria at
possible to determine whether the agonist pro- concentrations greater than those required to
duced its response by activation of β1-adreno- block receptors. Such concentrations should be
ceptors. Antagonists can also produce depres- avoided because this may drive the inotropic
sion of inotropic responses in guinea pig left signal below the threshold needed to trigger the
atria at concentrations greater than those re- rate-meter response. Table 4.6.2 shows a list of
quired for receptor blockade. Accordingly, simple competitive antagonists, with pKB val-
these concentrations should be avoided since ues for potency estimation, times needed to
such an effect would block non-β1-adrenocep- reach equlibrium, and concentrations where
tor–mediated inotropy. Table 4.6.1 shows a list myocardial depression is observed. Atenolol is
of simple competitive receptor antagonists with a rapid, convenient antagonist to use for iden-
pKB values (negative logarithm of the molar tificaton of β1-adrenoceptors.
concentration of antagonist that produces a
two-fold rightward shift of the agonist dose â2-adrenoceptors: guinea pig trachea
response curve) for potency estimation, deter- Typical absolute potencies of β-adrenocep-
mination of times required to reach equilib- tor agonists are not shown, since the sensitivity
rium, and concentrations where myocardial de- of this preparation to β-adrenoceptor agonists
pression is observed. Atenolol is a rapid, con- is inversely proportional to the degree of con-
venient antagonist to use for identification of tractile tone on the tissue. Table 4.6.3 shows an
β1-adrenoceptors. example of the pD2 values (the negative log of
the molar concentration producing 50% maxi-
â-adrenoceptors: guinea pig right atria mal effect) for isoproterenol and prenalterol on
The stability of the isolated right atrium trachea under spontaneous tone and contracted
preparation can be assessed by the lack of by various concentrations of carbachol. Of
arrhythmia in the spontaneous signal (also see great value are the relative potencies of β-ad-
UNIT 4.3, Critical Parameters, for isolated cardiac renoceptor agonists on this preparation. Thus,
muscle). The response of this preparation to while absolute potencies vary with contractile
β1-adrenoceptor agonists is generally sus- tone, relative potencies do not, and are therefore
â-Adrenoceptor tained, making it possible to obtain cumulative used for receptor classification and as a test of
Assays concentration-response curves. A measure of tissue viability.

4.6.28
Current Protocols in Pharmacology
A
400
300
200
100
10 nM 30 nM 0.1 µM 0.3 µM wash + 0.3 µM 1 µM 3 µM 10 µM 30 µM
isoproterenol atenolol (10 µM) isoproterenol

B
100
response to isoproterenol

add
% Maximum

atenolol
50

–9 –8 –7 –6 –5
log[isoproterenol]

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.

â3-adrenoceptor-stimulated lipolytic activity as well as the response of the cells to β3-adreno-


Adipocyte isolation and culturing. Cell in- ceptor agonists. Therefore, each batch of col-
tegrity is a major concern when isolating pri- lagenase must be tested individually for both
mary adipocytes and maintaining functional digestion efficiency and effects on β3-adreno-
β3-adrenergic responses. Since adipocytes, and ceptor sensitivity, which is key towards obtain-
to a lesser extent preadipocytes, are fragile, ing functional adipocytes.
great care must be exercised in handling these Measuring adipocyte differentiation. It is
cells. A large-orifice pipet should be used for important to culture the adipocytes under con-
resuspension and the pelleted cells should be ditions allowing for maximal fat accumulation
resuspended slowly. A simple, qualitative (lipogenesis) in the cell before testing for ad-
measure for assessing the integrity of the cells renergic activity, so that when lipolysis is meas-
is to observe the amount of oil floating on the ured as a function of adrenoceptor activity,
top of the medium. There will always be some endogenous substrate (i.e., triglycerides) does
oil floating during the collagenase incubation not limit the sensitivity of the assay. Nile red
and even during the initial washes; however, (9-diethylamino-5H-benzo[α] phenoxazine-5-
the final washes should have very little oil one) is a versatile vital stain for detecting the
floating on the top. Cell integrity can also be accumulation of intracellular lipids by fluores-
monitored by trypan-blue exclusion. cence techniques (i.e., microscopy, fluorimetry,
Another concern is the variability between and flow cytometry). This dye can be applied
different batches of collagenase. This variabil- to cells in an aqueous medium and does not
ity can influence the amount and duration of dissolve the lipids that it reveals. It is highly
incubation for proper digestion of the fat tissue fluorescent, but only in a hydrophobic environ- Isolated Tissues

4.6.29
Current Protocols in Pharmacology
ment. This staining method is more versatile added agonist. See UNIT 4.3, Troubleshooting, for
than Oil-red O staining, since it can be readily a suggested course of action.
quantitated and is reversible. Staining can be Low sensitivity to β1-adrenoceptor agonism
carried out on either fixed (1.5% formaldehyde, but sufficient maximal asymptotic response.
5 min) or unfixed live cells. The main advantage Errors in drug concentration are usually the
of Nile red is that it is not cytotoxic. Thus, after cause of apparently low potency. If the drug is
Nile red staining the cells can be used to meas- not sufficiently dissolved in the stock solution,
ure β3-adrenoceptor responses. subsequent dilutions will only compound the
The advantage of using the GPO-Trinder error. As a first check, discard the drug solutions
reagents over Nile red staining for measuring and prepare fresh stocks.
total triglyceride accumulation is that GPO-
Trinder can be quantitated using a conventional â-adrenoceptors: guinea pig right atria
spectrophotometer. Further, it allows for a di- General problems with the isolated right
rect measure of cellular substrate (triglyceride) atrial preparation can be found in the Trou-
and product (glycerol) involved in lipolysis. bleshooting section of UNIT 4.3.
However, the GPO-Trinder assay is not revers- Loss of rate-response reading. The inotropic
ible. Thus, the cells cannot be reused for meas- twitch must bisect the internal temporal elec-
uring β-adrenergic responses. tronic signal of the rate meter in this assay. If
Measuring β-adrenoceptor-induced lipoly- the preparation becomes weaker with washing,
sis in isolated preadipocytes and mature adipo- or a negative inotropic drug such as a calcium-
cytes. There are some advantages to using ma- channel blocking agent is used, then the maxi-
ture adipocytes (floating fat) versus differenti- mal peak height may diminish below the thresh-
ating preadipocytes (pellets) when measuring old for reading the rate response (Fig 4.6.11A).
lipolytic activity. Mainly, the floating cells have Because the strength of the inotropic response
accumulated the substrate (triglycerides) can be augmented electronically, an amplifica-
needed for measuring lipolytic activity and will tion of the inotropic signal entering the rate
be ready to assay on the day that they are meter will correct for this. Another way this
harvested. In contrast, the preadipocytes will may occur is if the resting tension on the tissue
need time to differentiate and accumulate lipid. diminishes. Under these circumstances, either
However, the advantage of culturing preadipo- the peak height will decline or the complete set
cytes is that these cells are viable for longer of contractions (from basal to peak) will fall
periods of time than isolated mature fat cells. below the necessary rate signal of the meter (Fig
Further, these cells can be manipulated to ex- 4.6.11B). If the transducer signal is not balanced,
press the β-adrenoceptor of choice depending changing the amplification of the inotropic sig-
upon the pharmacological agents that are pre- nal midway through an experiment may alter
sent during differentiation. For example, treat- the baseline position. This should be corrected
ment of preadipocytes with various compounds by electronic balancing of the transducer.
(dexamethasone, dibutyryl cyclic AMP, PMA, Cardiac arrhythmia. Heavy-metal ions can
butyrate, or insulin) will up-regulate β1 or β2 cause serious arrhythmias in spontaneous car-
while down-regulating β3-adrenoceptor ex- diac preparations. If this occurs, the solution
pression through a transcriptional effect. How- should be discarded. A fresh solution should be
ever, treatment with triiodothyronine, and in prepared with deionized water and the prepa-
some instances thiazolidinedione (e.g., trogli- ration washed extensively. An apparent ar-
tazone), will result in increased β3-adrenocep- rhythmia may be due to erratic waveforms
tor expression/responses. entering the rate meter. If the inotropic signal
is exceedingly weak, or the aeration of the
Troubleshooting solution is so high that large bubbles cause
irregular straining on the transducer, the inter-
â1-adrenoceptors: guinea pig left atria ference with the temporal electronic signal of
General problems with the isolated left atrial the rate meter may be irregular (Fig 4.6.11C)
preparation can be found in the Troubleshoot- and will register as an apparent arrhythmia. The
ing section of UNIT 4.3. ideal situation is one where the right atrium
No observable response to β1-adrenoceptor produces a robust and strong signal. If artifac-
stimulation. The preparation may be releasing tual arrythmia is suspected, the rate meter
endogenous catecholamines, causing maximal should be removed from the system and the
â-Adrenoceptor β1-adrenoceptor stimulation in the absence of actual inotropic twitch responses visualized to
Assays detect erratic baseline and peak responses.

4.6.30
Current Protocols in Pharmacology
C loss of signal due to
mechanical abnormalities

A loss of signal due B loss of signal due


to declining inotropy to falling baseline

arrhythmia
rate
rate

rate

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

No observable response to β1-adrenoceptor should be washed repeatedly to remove endo-


stimulation. The preparation may be releasing genous catcholamines. Alternatively, the rest-
endogenous catecholamines causing maximal ing tension on the tissue may be declining due
β1-adrenoceptor stimulation in the absence of to slippage of the securing thread. The resting
added agonist. See UNIT 4.3, Troubleshooting, for tension should be readjusted and the tissue
a suggested course of action. washed repeatedly until a stable tone is ob-
Low sensitivity to β1-adrenoceptor agonism tained.
but sufficient maximal asymptotic response.
The easiest parameter to check is drug concen- â2-adrenoceptors: rat uterus
tration. Errors in drug concentration are most Basal peak height begins to fade. A uniform
commonly responsible for apparently low po- electrical stimulation is required for this prepa-
tency. If the drug is not sufficiently dissolved ration to be stable. If the strength of the signal
in the stock solution, subsequent dilutions will diminishes, it might reach the point where com-
only compound the error. To assess this possi- plete recruitment of the muscle is not achieved
bility, discard the drug solution and prepare and the peak height will wane. This can occur
fresh stocks. if the resistance of the system increases during
the experiment. The original peak height can
â-adenoceptors: guinea pig trachea be recovered by increasing the voltage from the
Guinea pig trachea does not attain a spon- amplifier. However, care must be taken not to
taneous tone. The tissue may be damaged or increase the voltage too much to avoid burning
otherwise compromised, resulting in spontane- the tissue at its interface with the tissue holder.
ous release of endogenous catecholamines. It also is possible that salts may encrust the
Frequent washing may produce spontaneous punctate electrode on the holder, thus increas-
contraction, which would indicate removal of ing resistance. This can be corrected by clean-
endogenous agonists and/or recovery from in- ing the surface of the electrode.
jury. If spontaneous tone is not observed, a new Spontaneous arrhythmic contractions. This
preparation should be made. is probably the most common problem with this
The resting muscle tone is not constant. If a preparation. If the rat is in a state of estrus,
fading baseline is obtained, the preparation spontaneous arrhythmic contractions can be Isolated Tissues

4.6.31
Current Protocols in Pharmacology
minimized. When it does occur, the electrical Further washings of the cells with PBS and
stimulation should be switched off and the fluid treating the cells with adenosine deaminase
replaced with calcium-free De Jalon’s (several should remove these problems.
wash cycles may be required to remove residual Endogenous phosphodiesterase may inhibit
calcium from the preparation). After 20 to 30 the lipolytic response. The addition of 10 to 100
min in calcium-free medium, calcium should µM IBMX can help improve the adrenergic
be reintroduced gradually beginning with 1.25 response. Caution should be used, as the addi-
mM for 15 min followed by the full 2.5 mM, tion of too much phosphodiesterase inhibitor
after which the electrical stimulation is reinsti- (e.g., 1 mM IBMX) will maximally stimulate
tuted at threshold voltage (after 20 min). If lipolysis and mask the lipolytic response to
arrhythmia persists, the experiment should be β3-agonists.
terminated. Low cell viability may result from inade-
quate culture conditions. Matrigel can be used
â-adrenoceptor-stimulated lipolytic activity to help keep the adipocytes intact in culture for
Lack of functional β3 response. Variability longer periods of time. Follow the manufac-
in β-adrenoceptor expression is observed from turer’s instructions for optimal usage of Ma-
species to species, patient to patient, depot to trigel (Hazen et al., 1995).
depot, and with conditions used to isolate and If the desired β3-adrenergic response is not
culture the cells/tissue. For example, the β3 attainable using isolated primary adipocytes,
response is usually greater in rodent than in then immortalized cell lines may provide an-
human adipocytes. Similarly, the β3 response other option. One cell line, the C3H10T1/2
is usually greater in neonatal than adult fat. pluripotent mesenchymal stem cell (available
Often, the best responses can be obtained em- from ATCC; see SUPPLIERS APPENDIX), can differ-
pirically by examining the effects of adrenergic entiate into β3-adrenoceptor-expressing cells
agonists on multiple fat depots. Responses to given the proper differentiation conditions
β3 agonists may be undetectable if the tissue (Paulik and Lenhard, 1997; Lenhard et al.,
contains mostly white adipocytes (i.e., human 1998). Furthermore, SAOS-2 cells, a human
subcutaneous fat contains predominantly β2 osteosarcoma cell line, can also differentiate
adrenoceptors). The β3-adrenergic responses into adipocytes that express the β3-adrenocep-
will be greatest in brown adipocytes (e.g., the tor (Lenhard et al., 1998).
rodent hibernating gland). The amount of
brown adipocytes present in any depot can be Anticipated Results
determined by the expression of markers that
are expressed predominantly in this cell type â-adrenoceptors: guinea pig left atria
(i.e., uncoupling protein or Type II-5-deiodi- This preparation should yield a cumulative
nase). Some fat depots (e.g., perirenal fat) may dose-response curve to isoproterenol in 30 min,
express all of the β-receptor subtypes. with a pD2 of 8.5. This response should be
No observable lipolytic response to adren- readily blocked by 0.1 µM atenolol. The relaxa-
ergic stimulation. The cells may not have accu- tion effects of β1-adrenoceptor agonists are
mulated enough substrate (triglycerides) to de- observed at concentrations of agonist approxi-
tect generation of the product (glycerol) upon mately one-fifth those required for positive
stimulation with a β3-adrenoceptor agonist. inotropy (except for partial agonists).
This problem can be remedied by allowing the
cells to differentiate for a longer period (>3 â-adrenoceptors: guinea pig right atria
weeks) before measuring an adrenergic re- This preparation should yield a cumulative
sponse. Optimal adipogenesis requires the ad- dose-response curve to isoprerenol in 30 min,
dition of insulin and thiazolidinedione (trogli- with a pD2 of 9 to 9.2. This response should be
tazone) or indomethacin (Lehmann et al., 1997; readily blocked by 0.1 µM atenolol.
Lenhard et al., 1997). Although glucocorticoids
will also stimulate lipid accumulation, they will â2-adrenoceptors: rat uterus
increase the amount of β1 and β2 receptors and A stably contracting preparation should be
decrease the β3 receptor in fat. If accumulation exquisitely sensitive to β2-adrenoceptor stimu-
of endogenous triglyceride is not attainable, lation. These preparations should produce a
then one can measure cAMP levels after stimu- stable train of contractions within 1 hr. Once
lation with β-adrenoceptor agonist/antagonist. the peak height of the contractions is stable, the
â-Adrenoceptor There also may be antilipolytic activity re- tissue is ready for experimentation. The tissue
Assays sulting from endogenous insulin or adenosine. should be stable for at least 4 hr with no desen-

4.6.32
Current Protocols in Pharmacology
sitization to β2-adrenoceptor stimulation if agonists, such as GR219803B or CL316243,
dose-response curves are separated by 30 to 45 but not to β1 or β2-selective agonists (e.g.,
min washings in drug-free medium. RO363 and albuterol, respectively). The EC50
One feature of this tissue is the extremely for lipolysis induced by GR219803B or
rapid onset of response and attainment of CL316243 and isoproterenol should be be-
steady-state (Fig. 4.6.12A). Figure 4.6.12B tween 1 to 4 nM using both human and rodent
shows a dose-response curve to isoproterenol. adipocytes expressing β3 receptors. The lipo-
In addition to being rapid, the response is sus- lytic response to β3-selective agonists should
tained, thus allowing for the generation of cu- not be blocked by 10 nM of the β1 antagonist
mulative dose-response curves (Fig. 4.6.12A). CGP20712A or the β2 antagonist ICI 118,551.
Another feature of this tissue is that recovery Approximately 1 nM of glycerol should be
after β2-adrenoceptor stimulation is evident liberated from the cells after treating 1 cm2 of
once the twitch response returns to basal levels confluent adipocytes for 1 hr at 37°C.
after removal of isoproterenol and washing
with drug-free medium. Time Considerations
â3-adrenoceptors â-adrenoceptors: guinea pig left atria
Brown adipocytes, which express predomi- The tissue preparation can be operative
nately β3-adrenoceptors, should yield dose-re- within 90 min (30 min for setup and 60 min for
sponse curves to isoproterenol and β3-selective equilibration). Cumulative dose-response

nM isoproterenol
03 10 30 100

A
5 min

1g

–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
Isolated Tissues
(open circles).
4.6.33
Current Protocols in Pharmacology
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Repeated dose-response curves can be obtain- effecting the release of autonomic transmitters in
isolated heart muscle. J. Pharmacol. Exp. Ther.
ed, since the preparation is stable for 6 to 8 hr.
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Foster, R.W. 1967. The potentiation of responses
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isolated tracheal chain preparation by desi-
The rat must be pretreated with diethylstil- pramine, cocaine, phentolamine, phenoxyben-
bestrol (1 mg/kg) for two consecutive days zamine, guanethidine, metanephrine, and cool-
(using a single subcutaneous injection each ing. Br. J. Pharmacol. Chemother. 31:466-482.
day). The dissection is rapid and the preparation Hazen, S.A., Rowe, W.A., and Lynch, C.J. 1995.
can be completed in 20 min. Dose-response Monolayer cell culture of freshly isolated adipo-
curves can be obtained 60 min later. A series of cytes using extracellular basement membrane
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adipocytes and preadipocytes can be performed
Lehmann, J.M., Lenhard, J.M., Oliver, B.O., Rin-
in 2 hr. The primary adipocytes can be used gold, G.M., and Kliewer, S.A. 1997. Peroxisome
immediately for measuring β3-adrenoceptor- proliferator-activated receptors γ and α are acti-
mediated lipolysis and the assay completed vated by indomethacin and other non-steroidal
within 5 hr. The length of time for differentiat- anti-inflammatory drugs. J. Biol. Che m.
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troglitazone and metformin on glucose and lipid
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metabolism: Alterations of two distinct molecu-
cytes in culture, they can be used any time over lar pathways. Biochem. Pharm. 54:801-808.
the next 2 to 3 months for testing β-adrenocep-
Lenhard, J.M., Lancaster, M., Hull-Ryde, E., and
tor-mediated lipolysis. Feeding the cells fresh Paulik, M.A. 1998. Differentiation of a human
medium requires only a few minutes. Time osteosarcoma cell line (SAOS-2) into brown
courses can be run to determine the best times adipocytes. Submitted for publication.
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the lipolysis assay can be performed for 3 to 24 and Fraser, C.M. 1991. An adipose tissue–spe-
hr without significant differences in the calcu- cific beta-adrenergic receptor: Molecular clon-
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lated EC50 values. However, significant de- Chem. 266:24053-24058.
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Rubin, C.S. 1980. Organelle relationships in cul-
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Paulik, M.A. and Lenhard, J.M. 1997. Thiazolidi-
incubation times (>16 hr) for the GPO assay nediones inhibit alkaline phosphatase activity
will result in decreased optical density. Since while increasing expression of uncoupling pro-
the quinoneimine dye is unstable, extended tein, deiodinase and increasing mitochondrial
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290:79-87.
â-Adrenoceptor
Assays

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sue culture. In Current Protocols in Molecular estimate the pKi of competitive inhibitors of
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Carter, J.R. and Crofford, O.B. 1985. Methods for
assessing hormone effects on glucose transport Kenakin, T.P. 1982. Theoretical and practical prob-
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Example dose-response curves to β-adrenergic Sigma, 1990. Quantitative enzymatic determination


agonists and effects of β-adrenergic antagonists. of glycerol, true triglycerides, and total tri-
glycerides in serum or plasma at 540 nm. Sigma
Diagnostics procedure no. 337, Sigma Chemical
Company, St. Louis. Isolated Tissues

4.6.35
Current Protocols in Pharmacology
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Preadipocyte differentiation.

â-Adrenoceptor
Assays

4.6.36
Current Protocols in Pharmacology