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INTRODUCTION |
-Adrenergic receptors
(
-ARs)1 are members of the
superfamily of G-protein-coupled receptors that are stimulated by the
naturally occurring catecholamines, epinephrine and norepinephrine. As
part of the sympathetic nervous system,
-ARs have been shown to have important roles in cardiovascular, respiratory, metabolic, central nervous system, and reproductive functions. Using techniques of molecular cloning, three distinct
-AR subtypes have been identified (
1-AR,
2-AR, and
3-AR) (1-3). All three of these
-AR
subtypes are believed to signal by coupling to the stimulatory
G-protein Gs
leading to activation of adenylyl cyclase
and accumulation of the second messenger cAMP (1-3).
Because of the diverse physiological functions mediated by
-ARs,
much effort has been spent in understanding the roles of individual
-AR subtypes. In the past, researchers have relied on
pharmacological tools such as subtype-selective agonists and antagonists to probe the function of the different
-AR subtypes. The
presence of multiple
-AR subtypes was first suggested by Lands and
co-workers (4, 5) who divided
-ARs into
1-ARs and
2-ARs.
According to Lands' classification,
1-ARs mediate cardiac
stimulation, and
2-ARs mediate smooth muscle relaxation in the
peripheral vasculature and respiratory system. The presence of a third
-AR subtype was suggested when some of the effects of
-AR
agonists could not be efficiently blocked by typical
-AR antagonists. This third
-AR subtype is now known as the
3-AR and
has been shown to have important roles in adipose tissue and the
gastrointestinal tract (6).
Although both
1-ARs and
2-ARs are expressed in the heart of most
mammalian species,
1-ARs are expressed at higher levels and are
recognized as playing the major role in regulating cardiac function.
Functional studies have confirmed that activation of
1-ARs leads to
increased heart rate and force of contraction (7). Although they
represent a smaller population in the heart than
1-ARs,
2-ARs
have also been shown to play a role in regulating cardiac function in a
variety of species (7-9). In studies using subtype-selective agonists
and antagonists in the human heart,
2-AR stimulation leads to
activation of adenylyl cyclase and contributes to both inotropic and
chronotropic responses (7). In the murine heart, however,
2-ARs do
not appear to couple to inotropic or chronotropic responses. When
isolated cardiac muscle from
1-AR knockout mice is stimulated with
the non-subtype-selective
-AR agonist isoproterenol, neither
inotropic nor chronotropic responses are observed (10).
In addition to their roles in the heart,
-ARs also regulate
peripheral vascular tone. Stimulation of peripheral
-ARs leads to
relaxation of vascular smooth muscle, thereby controlling the distribution of blood flow to different tissues. During exercise, for
example, stimulation of
-ARs contributes to the increased blood flow
to skeletal muscle. Based on the studies of Lands and co-workers (4,
5), the
-AR in the peripheral vasculature have been classified as
the
2-AR. Some reports, however, have shown roles for the other
-AR subtypes,
1-ARs and
3-ARs, in the peripheral vasculature
(11-13).
Although much has been learned about the role of individual
-AR
subtypes using classical pharmacological techniques, these studies are
complicated by the fact that subtype-selective ligands are never
perfectly selective. Moreover, at the doses required to block
-ARs
in vivo, most
-AR ligands lose much of their subtype selectivity and may bind to other G-protein-coupled receptors such as
serotonin receptors and dopamine receptors. Studies with
-AR ligands
are especially difficult to interpret in vivo where it is
hard to estimate the concentration of ligands and their metabolites in
target tissues. In order to further investigate the roles of the
different
-AR subtypes in physiology, we have selectively
inactivated the
2-AR gene in mice using gene-targeting techniques.
The knockout (
2-AR
/
) mice appear grossly normal and are
fertile. Resting cardiovascular physiology is remarkably unperturbed in
2-AR
/
mice. The major effects of
2-AR gene disruption were
observed only during the stress of exercise.
2-AR
/
mice were
able to exercise farther and with a lower respiratory exchange ratio at
any given workload than wild type controls. However, they are
hypertensive during exercise, suggesting an imbalance between the
vasoconstrictive and vasorelaxant effects of endogenous catecholamines.
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MATERIALS AND METHODS |
Targeting Vector Construction--
The targeting vector was
constructed using sequence that had been cloned from a C57BL/6 mouse
genomic library (14). In total, the targeting vector contained 11.4 kb
of homology to the endogenous
2-AR genomic locus. The gene for the
2-AR was disrupted in the targeting vector by placing a neomycin
(neo) resistance gene cassette into the coding sequence at a unique
ClaI site (15). This insertion disrupts the
2-AR at the
end of the fourth transmembrane segment and should produce a
nonfunctional receptor. The short arm of the targeting vector was a
2.6-kb fragment from a 5' EcoRI site to the ClaI
in the receptor. The long arm of the targeting vector (8.8 kb) extended
from the ClaI site in the receptor to a downstream SalI site. Also included in the vector was the herpes
simplex virus thymidine kinase cassette to allow for negative selection when isolating ES cell clones (15). In order to screen for homologous recombinants a 5' external probe was used. This probe is a 300-base pair BamHI/EcoRI fragment that detects a 4.9-kb
fragment after mouse genomic DNA is digested with BamHI and
then subjected to Southern blot analysis. In cases where the targeting
vector has homologously recombined with the endogenous locus, the same
probe would detect an additional band at 6.6 kb.
Transfection of ES Cells--
R1 embryonic stem (ES) (16) cells
were transfected using standard techniques (17). ES cells were grown on
a monolayer of mouse embryonic fibroblasts in Dulbecco's modified
Eagle's medium (UCSF tissue culture facility, San Francisco)
supplemented with 20% fetal bovine serum (HyClone, Logan, UT), 1 mM sodium pyruvate (Life Technologies, Inc.), non-essential
amino acids, and penicillin/streptomycin (UCSF Cell Culture Facility,
San Francisco, CA), 10
4 M
-mercaptoethanol
(Specialty Media, Lavallette, NJ), and 2,000 units/ml of leukemia
inhibitory factor (ESGRO; Life Technologies, Inc.). Cells were grown in
an incubator at 37 °C in 95% air, 5% CO2. For the
transfection, a 10-cm2 dish of ES cells was transfected via
electroporation with 20 µg of targeting vector previously linearized
with NotI. After selecting ES cells for 9 days in media
containing G418 (Life Technologies, Inc.) and gancyclovir (Syntex, Palo
Alto, CA), individual clones were picked and subcloned in 96-well
plates. BamHI-digested DNA from clones was analyzed by
Southern blot analysis with the 5' external probe. Nine homologous
recombinants were isolated from 300 ES cell clones. Homologous
recombinants were also screened with a neo probe to confirm that a
single integration of the targeting vector had occurred.
Morula Aggregation--
Chimeric mice were generated using the
morula aggregation technique described previously (18). Briefly,
embryos at morula stage (2.5 days pc) were isolated from oviducts of
superovulated CD-1 mice by flushing the oviducts with M2 medium
(Specialty Media, Lavallette, NJ). After removing the zona pellucida
with an acidic Tyrode's solution (Specialty Media, Lavallette, NJ),
the embryos were placed in depressions in a 6-cm tissue culture dish
and covered with a droplet of M16 medium (Specialty Media, Lavallette,
NJ). A protective layer of mineral oil (Sigma) was placed over the droplets. Clumps of ES cells with the targeted disruption (10-20 cells) were then seeded into the depression and placed in contact with
the embryos. After an overnight incubation at 37 °C in 95% air, 5%
CO2, the chimeric embryos were transferred to the uteri of
pseudopregnant CD-1 hosts (20-25 embryos per host). Chimeric mice were
identified in the resulting offspring by the presence of dark coat
color patches. Chimeric males were then mated to FVB/N female mice to
screen for germ line transmission of the ES cell DNA. After achieving
germ line transmission,
2-AR +/
mice were intercrossed to generate
2-AR +/+ and
/
mice for use in binding studies. For in
vivo studies, the knockout allele was placed on a FVB/N background
by backcrossing
2-AR +/
mice to wild type FVB/N mice for four
additional generations (5 backcrosses to FVB/N in total).
Binding Assays--
Whole lungs were dissected from wild type
and knockout littermates, placed in a lysis buffer (10 mM
Tris-HCl, 1 mM EDTA, pH 7.4), and homogenized with a
Polytron (4 × 20-s bursts). The membrane fraction was isolated by
centrifugation at 10,000 × g and resuspended in
binding buffer (75 mM Tris-HCl, 12.5 mM
MgCl2, 1 mM EDTA, pH 7.4). Binding reactions
were carried out by incubating membranes with the radioligand
[125I]iodocyanopindolol (125I-CYP) (NEN Life
Science Products) in 500-µl volumes. After a 2-h incubation at room
temperature, vacuum filtration was performed, and the filters were
counted in a gamma counter. For saturation experiments, 3 µg of
membrane protein was incubated with increasing amounts of
125I-CYP (1-300 pM). Nonspecific binding was
determined in the presence of 1 µM
DL-propanolol (Sigma). For competition experiments, binding reactions were set up with 50 pM 125I-CYP, 3-6
µg of membrane protein, and varying concentrations (50 pM-13 µM) of the
2-AR-selective antagonist
ICI 118,551 (Tocris Cookson, Ballwin, MO). Saturation and competition
data were analyzed with GraphPAD software (GraphPAD Software Inc., San
Diego, CA).
In Vivo Cardiovascular Physiology--
In vivo
studies were carried out as described previously (19). Adult male mice
(12-16 weeks of age) were anesthetized with isofluorane using a
vaporizer (Airco Inc., Madison, WI), and a stretched Intramedic PE10
polyethylene catheter (Clay Adams, Parsippany, NJ) was inserted into
the left carotid artery. The catheter was tunneled through the neck and
then placed in a subcutaneous pouch in the back. After a minimum of
16 h recovery, the saline-filled catheter was removed from the
pouch and connected to a Spectramed DTX Plus pressure transducer
(Spectramed, Oxnard, CA). Output from the pressure transducer was
amplified using a Gould 8-channel recorder and digitized using a Data
Translation Series DT2801 analog-digital converter (Marlboro, MA). The
digital signal was analyzed using Crystal Biotech Dataflow data
acquisition software (Crystal Biotech, Hopkinton, MA) on a Gateway 2000 486DX2 microcomputer (Sioux City, SD). Baseline heart rate and mean
arterial blood pressure were recorded after a 1-h equilibration period
when the animals were awake but not active. In order to examine drug
responses, drugs were administered through the carotid artery catheter.
(
)-Isoproterenol hydrochloride (3 µg/kg) and epinephrine bitartrate
(3 µg/kg) were purchased from Sigma and dissolved in saline for
injection. In order to measure heart rate and blood pressure during
exercise, cannulated mice were challenged with a graded treadmill
exercise protocol (19) on a Simplex II rodent treadmill (Columbus
Instruments, Columbus, OH). Treadmill activity was initiated at 3.5 m/min, 0° inclination, and increased to 5 m/min, 2° inclination 3 min later. Treadmill speed and inclination were then increased by 2.5 m/min and 2° inclination every 3 min thereafter. Exercise was
terminated after the mice had completed 3 min at 20 m/min, 14°
inclination. Mice that failed to complete the exercise protocol were
excluded from the study.
In Vivo Metabolic Responses to Exercise and Total Exercise
Capacity--
In order to measure metabolic responses to exercise and
exercise capacity, non-instrumented mice were challenged with the graded treadmill exercise protocol described above. Treadmill activity
was initiated after the mice had equilibrated in the exercise chamber
for 30-60 min. During the exercise protocol, oxygen consumption and
carbon dioxide production were continuously monitored with an Oxymax
gas analyzer (Columbus Instruments, Columbus, OH). Stepwise increases
in treadmill speed and inclination were made every 3 min until the mice
stopped running from exhaustion. Exercise capacity was calculated as
the total distance run by the animals during the exercise protocol.
Body Weight, Epididymal Fat Pad Weight, Density, FFA Levels, and
Glycerol Levels--
Male mice, 12-13 weeks old, were used for these
studies. Mice were maintained in 12-h light/dark cycles. On the day of
study, food was removed from the cage at the beginning of the light
cycle, and mice were studied 3-5 h later. Each mouse was weighed and then anesthetized with 5% isofluorane for 45 s in an anesthesia induction box. The mouse was quickly removed from the box, and blood
was collected by cardiac puncture with a 22-guage needle. The mouse was
then sacrificed via cervical dislocation. The volume of the mouse was
determined by attaching a weight to the mouse and measuring the water
displacement. Density was calculated as the body weight divided by the
volume. After the volume measurement, both epididymal fat pads were
dissected from the animal and weighed. The proportional weight of the
fat pads was calculated by dividing the fat pad weight by the total
body weight. After the blood samples had clotted in serum separator
tubes (Becton Dickinson, Franklin Lakes, NJ), the samples were spun at
17,000 × g for 5 min to isolate the serum. Free fatty
acid levels were determined with an enzymatic colorimetric kit (Wako
Chemicals, Germany). Glycerol levels were determined with an enzymatic
colorimetric kit (Roche Molecular Biochemicals).
Locomotor Activity--
Locomotor activity of male mice, 12-13
weeks old, was measured by a photobeam cage system (San Diego
Instruments, San Diego, CA). Mice were studied in pairs with a
2-AR
+/+ mouse and a
2-AR
/
mouse placed in individual cages (30 × 50 cm). A frame containing 4 × 6 infrared photobeams was
placed around each cage. Mice were placed in the cages at 5 p.m.,
and their activity was monitored as the number of beam breaks in a 48-h
period. The mice used for these studies had not been used for any
previous experiments.
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RESULTS |
2-AR Gene Targeting--
Using standard ES cell techniques, the
R1 ES cell line was transfected with the
2-AR targeting vector shown
in Fig. 1A. Homologous recombinants were identified by performing Southern blot analysis using
the 5' external probe. Targeted clones were rescreened with a probe to
the neomycin resistance gene to ensure that a single integration of the
targeting vector had occurred (data not shown). Chimeric mice were
generated with the targeted ES clones using the morula aggregation
technique. Following germ line transmission of the knockout allele,
heterozygous knockout pairs were intercrossed to generate
2-AR +/+,
2-AR +/
, and
2-AR
/
mice. Shown in Fig. 1B is a
Southern blot using DNA from the offspring of a
2-AR +/
intercross.

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Fig. 1.
Gene targeting strategy for the
2-AR gene. A, shown from the
top is the targeting vector, the endogenous locus of the
2-AR gene, and the result of homologous recombination. B,
BamHI site; C, ClaI site;
E, EcoRI site; N, NotI
site; S, SalI site; Neo, neomycin
resistance cassette; HSVTK, thymidine kinase cassette. The
black box represents coding sequence of 2-AR gene, and
the gray line represents untranslated genomic sequence. The
5' external probe and the expected fragments from Southern blot
analysis are also shown. B, Southern blot of tail DNA from
offspring of a heterozygous knockout intercross. Wild type (+/+),
heterozygous knockout (+/ ), and homozygous knockout ( / ) mice are
recovered from this mating.
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Results of Intercrosses--
After backcrossing
2-AR +/
mice
to wild type FVB/N mice for 5 generations,
2-AR +/
mice were
intercrossed. From 171 intercross progeny screened at weaning, 36
2-AR +/+ mice, 91
2-AR +/
mice, and 44
2-AR
/
mice were
identified. These results are consistent with the ratio predicted by
Mendelian genetics (
-squared = 1.45, p > 0.4).
Thus, there is no embryonic or postnatal lethality associated with
disruption of the
2-AR gene in mice. After maturing into adults,
2-AR
/
mice appear grossly normal and do not exhibit overtly
abnormal behavior. Both
2-AR
/
males and females are fertile.
2-AR Expression and Pharmacology in
2-AR
/
Mice--
In
order to verify that the genetic modification prevents expression of
the
2-AR gene, ligand binding experiments were performed using lung
tissue isolated from
2-AR +/+ and
/
littermates. Saturation
binding experiments with the radioligand
[125I]iodocyanopindolol (125I-CYP)
demonstrate a reduction in total binding in the
2-AR
/
mice
(Fig. 2A). The
Bmax is reduced from 990 fmol/mg in
2-AR +/+
mice to 360 fmol/mg in the
2-AR
/
mice (36% of the wild type
value). Competition binding experiments were performed using the
2-AR-selective antagonist ICI 118,551 to characterize the residual
125I-CYP binding in the
2-AR
/
mice (Fig.
2B). In lung membranes from wild type mice, the data were
best fit by a biphasic curve with 62% high affinity ICI 118,551 binding (
2-AR) sites and 38% low affinity (
1-AR) sites. The
competition binding data from
2-AR
/
mice were best fit with a
one-site curve that has low affinity for ICI 118,551. Thus, the
residual 125I-CYP-binding sites in
2-AR
/
lung are
due to
1-ARs, confirming the loss of
2-AR-binding sites in
2-AR
/
mice. These data also demonstrate that there has not been
a compensatory change in
1-AR expression in the lung as a result of
the
2-AR gene disruption;
1-AR expression in
2-AR
/
mice
is 330 fmol/mg protein, whereas in
2-AR +/+ mice,
1-AR expression
is 380 fmol/mg protein (0.38 × 990 fmol/mg protein).

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Fig. 2.
Pharmacological characterization of
2-AR +/+ and / mice. A,
saturation binding on membranes prepared from lungs of 2-AR +/+ and
/ mice. Studies were performed by incubating membranes with varying
concentrations of the -AR antagonist 125I-CYP.
Nonspecific binding was determined in the presence of 1 µM DL-propanolol and subtracted from total
binding. Values shown represent the mean ± S.E. for three
individual experiments. B, competition binding on membranes
prepared from lungs of 2-AR +/+ and / mice. Experiments were
performed by incubating 3-6 µg of membranes with 50 pM
125I-CYP and varying concentrations of the 2-AR
selective antagonist ICI 118,551. Nonspecific binding was determined in
the presence of 1 µM DL-propanolol. Values
shown represent the mean ± S.E. for three individual
experiments.
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Cardiovascular Physiology--
In order to examine the effects of
the gene disruption on whole animal physiology,
2-AR +/+ and
2-AR
/
mice were instrumented with carotid catheters to allow
measurements of mean arterial blood pressure and heart rate in awake,
non-anesthetized, and non-restrained mice. Under baseline resting
conditions, mean blood pressure and heart rate were not significantly
different between
2-AR +/+ and
2-AR
/
mice (Table
I). In order to examine the effects of
-AR stimulation,
2-AR +/+ and
2-AR
/
mice were given an
intra-arterial bolus of 3 µg/kg of the non-selective
-AR agonist
isoproterenol, a dose previously shown to produce maximal increases in
heart rate and maximal reductions in blood pressure in wild type mice.
Fig. 3 shows the typical response of a
2-AR +/+ and a
2-AR
/
mouse to isoproterenol. In
2-AR +/+
mice, isoproterenol produced a rapid onset tachycardia and hypotension.
In
2-AR
/
mice, the tachycardic response to isoproterenol was
preserved, but the hypotensive response was significantly blunted (Fig.
3 and Table I).
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Table I
Cardiovascular indices at rest and changes in cardiovascular indices
after isoproterenol (Iso) and epinephrine (Epi) administration in
2-AR +/+ and / mice
Values shown represent the mean ± S.E. The number of mice studied
is shown in parentheses. The isoproterenol-stimulated values represent
the maximum changes in blood pressure (BP) and heart rate (HR) after
drug administration. The epinephrine-stimulated values represent the
maximum blood pressure change and the corresponding heart rate change
after drug administration. The unpaired t test was used for
statistical comparison between groups.
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Fig. 3.
In vivo cardiovascular responses
to isoproterenol in 2-AR +/+ and /
mice. After an overnight recovery from surgery, isoproterenol (3 µg/kg) was administered intra-arterially. Representative tracings
from a 2-AR +/+ and / mouse are shown. The upper
trace represents heart rate, and the lower trace
represents blood pressure. Isoproterenol was administered at the 1-min
time mark.
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Responses to the endogenous catecholamine, epinephrine (a combined
-AR and
-AR agonist), were also significantly different between
2-AR
/
and wild type mice (Fig.
4). In both
2-AR
/
and wild type
mice, administration of epinephrine produced a transient hypertensive
response (blood pressure typically returned to baseline within 1 min).
However, the hypertensive response was significantly greater in
2-AR
/
mice than in wild types (Table I and Fig. 4). Heart rate
responses in both
2-AR
/
and wild type mice to epinephrine were
variable (Table I). Although there was a trend for wild type mice to
show heart rate increases while
2-AR
/
showed heart rate
decreases, these heart rate responses were not significantly different
between genotypes.

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Fig. 4.
In vivo cardiovascular responses
to epinephrine in 2-AR +/+ and /
mice. After an overnight recovery from surgery, epinephrine (3 µg/kg) was administered through the arterial catheter. Representative
tracings from a 2-AR +/+ and / mouse are shown. The upper
trace represents heart rate, and the lower trace
represents blood pressure. Epinephrine was administered at the 1-min
time mark.
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The effects of exercise on heart rate and blood pressure are shown in
Fig. 5. For these experiments,
catheterized mice were tested using a graded exercise treadmill
protocol.
2-AR
/
and wild type mice showed similar heart rate
increases during the exercise protocol. A significant difference,
however, was observed in the blood pressure response to exercise.
During the exercise protocol,
2-AR
/
mice became hypertensive
compared with wild type mice. At the peak exercise level of 20 m/min,
2-AR
/
mice had a mean blood pressure of 139.3 ± 4.4 mm Hg
(mean ± S.E.), whereas wild type mice had a mean blood pressure
of 126.3 ± 3.3 mm Hg (mean ± S.E.).

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Fig. 5.
Cardiovascular response to exercise in
2-AR +/+ and / mice. During a graded
treadmill exercise program, heart rate (A) and blood
pressure (B) were monitored in mice through the arterial
catheter. Values shown represent the mean ± S.E. for 2-AR +/+
(n = 8) and 2-AR / (n = 7) mice.
¥, significance at p < 0.001 for comparing blood
pressure from 2-AR +/+ mice to 2-AR / mice by 2-way analysis
of variance with repeated measures (treadmill speed × genotype
interaction); R, recovery phase defined as 10 min after
stopping exercise.
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Metabolic Response to Exercise--
In a separate set of
experiments, metabolic responses to exercise and exercise capacity were
measured in uncatheterized mice. Oxygen consumption and carbon dioxide
production were continuously monitored while the mice exercised
according to a graded treadmill exercise protocol (Fig.
6). Oxygen consumption and carbon dioxide production were not significantly different between the two genotypes. However, there was a trend for
2-AR
/
mice to have greater levels of oxygen consumption at any given workload.
2-AR
/
mice
had a significantly lower respiratory exchange ratio during exercise
than did wild type mice (Fig. 6C). There was also a
significant difference between
2-AR +/+ mice and
2-AR
/
mice
in exercise capacity. Interestingly,
2-AR
/
mice exercised
significantly longer than wild type control mice (Fig. 6D).
Wild type mice covered 471 ± 22 meters (mean ± S.E.),
whereas
2-AR
/
mice covered 582 ± 15 meters (mean ± S.E.) during the graded exercise protocol.

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Fig. 6.
Metabolic response to exercise and exercise
capacity in 2-AR +/+ and / mice.
During a graded treadmill exercise protocol, O2 consumption
(A), CO2 production (B), and the
respiratory exchange ratio (C) were determined for each step
in the exercise protocol. Values shown represent the mean ± S.E.
for 2-AR +/+ (n = 5) and 2-AR /
(n = 5) mice. O2 consumption and
CO2 production are reported in units of ml/min/kg. The RER
represents the ratio of CO2 production to O2
consumption. Exercise capacity (D) was measured as the total
distance covered during the exercise protocol. Values shown represent
the mean ± S.E. for 2-AR +/+ (n = 5) and
2-AR / (n = 6) mice. ¥, significance at
p = 0.0062 for comparing exercise RER curves from
2-AR +/+ mice to 2-AR / mice by 2-way analysis of variance
with repeated measures (treadmill speed × RER interaction); *,
significance at p = 0.0031 for comparing 2-AR +/+ to
/ mice using an unpaired t test.
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Body Weight, Body Fat, and Serum Free Fatty Acids--
To
investigate possible mechanisms for the greater exercise capacity in
2-AR
/
mice, we examined body weight, epididymal fat pad weight,
body density, and serum levels of free fatty acid (FFA) and glycerol in
wild type and
2-AR
/
mice. As shown in Table
II,
2-AR
/
mice weigh
significantly less than wild type mice. Epididymal fat pads from
2-AR
/
mice also represent a smaller proportion of total body
weight than fat pads from wild type mice. Previous studies have shown
that the epididymal fat pad weight as a proportion of total body weight
is highly correlated with total body fat in mice (20, 21). Body
density, serum FFA levels, and serum glycerol levels were not
significantly different between the two genotypes under base-line
conditions.
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Table II
Body weight, proportional weight of the epididymal fat pads, density,
free fatty acid (FFA) levels, and glycerol levels in 2-AR +/+ and
2-AR / mice.
Values shown represent the mean ± S.E. for 2-AR +/+ and
2-AR / male mice. The unpaired t test was used for
statistical comparison between groups. The numbers in parentheses
represents the number of mice used for the
study.
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Locomotor Activity--
Activity studies were performed to
determine if the observed differences in exercise capacity, body fat,
and body weight in
2-AR +/+ and
2-AR
/
mice can be explained
by differences in the level of daily activity. As shown in Fig.
7, there was no significant difference in
locomotor activity between the two genotypes over a 48-h period. No
significant differences were observed when the 48-h period was broken
down into day and night segments (data not shown).

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Fig. 7.
Locomotor activity of
2-AR +/+ and / mice. The total number of
beam breaks for 2-AR +/+ (n = 5) and 2-AR /
(n = 5) mice is shown. Values shown represent the
mean ± S.E. The total number of beam breaks is not significantly
different between the two genotypes (p > 0.05 using an
unpaired t test).
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 |
DISCUSSION |
Using gene targeting techniques we have generated mice that have a
disruption in the
2-AR gene. Based on ligand binding data this
mutation blocks expression of the
2-AR.
2-ARs do not appear to
play a critical role in prenatal development since there is no
embryonic lethality associated with the mutation. Furthermore,
2-AR
/
mice appear grossly normal and are fertile, demonstrating that
2-ARs are not required for postnatal development or for normal
reproductive function. There is no apparent compensatory up-regulation
of
1-ARs in the lungs of
2-AR
/
mice.
Cardiovascular Effects of the
2-AR Gene Disruption--
One of
the goals in generating
2-AR
/
mice was to define further the
roles of the three different
-AR subtypes in the cardiovascular
system. A genetic approach has been used previously to show that
1-AR and not
2-AR stimulation regulates cardiac inotropy and
chronotropy in the murine heart (10). Even though
2-ARs are present
in the myocardium of
1-AR
/
mice, stimulation of
2-ARs does
not lead to improvements in cardiac function either in vivo
or in vitro. In contrast, in
2-AR
/
mice, normal
heart rate responses are observed in response both to isoproterenol and
to exercise, further demonstrating that the
1-AR plays the major
role in regulating cardiac function in the mouse.
Classical pharmacological studies have suggested that the
2-AR is
the
-AR subtype that mediates vascular smooth muscle relaxation (4,
5). Vascular relaxation leads to a decrease in total peripheral
resistance and is manifested by a hypotensive blood pressure response.
In
2-AR
/
mice, the hypotensive response to isoproterenol is
significantly blunted compared with wild type mice, confirming that
2-ARs play a significant role in mediating peripheral vascular
relaxation. The fact that hypotensive responses are still present in
2-AR
/
mice, however, suggests that other
-AR subtypes also
play a role, albeit a smaller one, in regulating peripheral vascular
tone in the mouse. This response could also be due to an up-regulation
of other
-AR subtypes in response to deletion of the
2-AR gene;
however, there was no evidence for compensatory up-regulation of the
1-AR in the lung, the tissue with the highest density of
2-ARs in
wild type mice.
Although
1-ARs are considered to be the cardiac
-AR, some
previous studies support our conclusion that
1-ARs can mediate vascular relaxation.
1-ARs have been shown to be involved in vascular relaxation in the isolated rat aorta and rat pulmonary artery
(22).
1-ARs have also been implicated in vascular relaxation in the
coronary arteries from a variety of species (23-26). Although these
larger vessels are not expected to make a significant contribution to
the total peripheral resistance, studies on whole animals have also
suggested that
1-ARs may regulate resistance vessels in the
peripheral vasculature (13).
3-ARs, which are abundant in adipose
tissue, have recently been shown to play a role in regulating
peripheral vasodilation (11), although this was highly species-dependent (12). By generating double
1-AR
/
2-AR
/
mice, further insight into
3-AR subtype function in
the peripheral vasculature has been obtained (35).
The Role of
2-AR Receptors in Essential Hypertension--
In
generating
2-AR
/
mice, we were also interested in the possible
role of
2-ARs in hypertension (27). Hypertension is a complex
disease with many environmental and genetic influences. Impaired
-AR-mediated vascular relaxation may contribute to this disease by
preventing dynamic reductions in total peripheral vascular resistance.
In animal models of hypertension,
-AR-mediated vascular relaxation
has been shown to be attenuated (28). It is not clear from these
studies, however, whether decreased
-AR signaling is a primary cause
of the hypertension or is a secondary effect, perhaps due to
desensitization of
-ARs by chronically elevated catecholamines (29).
-AR-mediated vascular relaxation is impaired not only in animal
models but also in humans with hypertension (30). Interestingly, humans
with borderline hypertension have impaired
-AR-mediated vasodilation
suggesting that defective
-AR signaling occurs early in the disease
process and may be involved in its pathogenesis (30). A recent human
population study has demonstrated that genetic variation of the
2-AR
is associated with a predisposition to develop hypertension (31). Defects in
2-AR-mediated vasodilation may thus be a contributing factor to the development of hypertension.
In generating the
2-AR
/
mouse we had the opportunity to test
whether or not defective
2-AR signaling can lead to a hypertensive state. Under baseline conditions we found that
2-AR
/
mice are
normotensive compared with wild type control mice. Thus,
2-AR stimulation may not be involved in regulating resting blood pressure in
mice. Given that
2-AR
/
mice have had the disruption of the
2-AR gene since conception, it is also possible that there have been
other compensatory changes that allow the maintenance of resting blood
pressure homeostasis. During the stress of treadmill exercise, however,
2-AR
/
mice become hypertensive compared with wild type mice. As
an endogenous stimulus for catecholamine release, exercise may lead to
unopposed
-adrenergic mediated vasoconstriction in the
2-AR
/
mice. In support of this hypothesis, administration of epinephrine to
the
2-AR
/
mice reproduced the hypertensive phenotype. As a
result of the disruption of the
2-AR gene,
-adrenergic receptor
stimulation may predominate and predispose the animals to develop
hypertension in states where endogenous catecholamines are elevated. In
humans, hypertension worsens with the process of aging. In the present
study, we studied young adult animals at 12-16 weeks of age. Future
studies will be required to examine whether the
2-AR
/
mice
develop hypertension as they age.
Metabolic Effects of the
2-AR Receptor Gene
Disruption--
2-ARs are known to play a role in the metabolic
response to stress (32). To investigate the effects of the knockout
mouse on physiologic stress, metabolic responses to exercise and
exercise capacity were measured in
2-AR
/
and wild type mice.
Surprisingly,
2-AR
/
mice exercised for a longer duration than
wild type mice did. One explanation for this difference in exercise
capacity is that there are alterations in energy metabolism secondary
to ablation of the
2-AR gene. In support of this hypothesis is the finding that
2-AR
/
mice have a lower body fat content, and the
respiratory exchange ratio (RER) was significantly lower in
2-AR
/
mice at any given workload than in wild type mice during exercise. Although carbon dioxide production was similar between wild
type and
2-AR
/
mice, there was a trend toward higher oxygen
consumption in the
2-AR
/
mice. This trend for increased oxygen
consumption during exercise may be explained by the lower body fat in
2-AR
/
mice. Fat can be considered to be metabolically inert;
therefore, changes in body fat content can also have an impact on
oxygen consumption and carbon dioxide production when these parameters
are normalized to total body weight. A change in body composition,
however, does not explain the decrease in exercise RER in the
2-AR
/
mice. RER is a ratio of oxygen consumption to carbon dioxide
production; therefore, body composition factors are cancelled out by
taking a ratio.
RER is an indicator of metabolic state and substrate utilization. Since
more oxygen is required to burn fat than carbohydrate, a lower RER in
2-AR
/
mice suggests that they may use a greater ratio of fat to
carbohydrate than do wild type mice during exercise. This could also
explain the lower body fat content in
2-AR
/
mice. Previous
studies (33, 34) have demonstrated that
-AR stimulation leads to
glycogenolysis during exercise. If the knockout mice were to result in
a defect in
2-AR-mediated mobilization of glycogen,
2-AR
/
mice may preferentially metabolize fat during exercise. Reduced
utilization of the glycogenolysis pathway during exercise would
conserve muscle glycogen and may be responsible for increasing the
duration of exercise before glycogen depletion. We did not observe
significant differences in serum-free fatty acids and glycerol in
2-AR
/
mice under basal conditions; however, it is possible that
differences in fat metabolism occur during exercise. Lactate production
may also be influenced by the
2-AR gene disruption. It has been
shown that catecholamines stimulate lactate production, possibly
through
2-AR receptors (33, 34). Thus,
2-AR
/
mice may have
lower lactate levels for a given workload.
Other mechanisms may contribute to the increase in exercise capacity of
2-AR
/
mice. There may be differences in the redistribution of
cardiac output between visceral and peripheral muscular beds during
exercise because of alterations in
2-AR-mediated vasorelaxation. In
the
2-AR
/
mouse, the defect in
-AR-mediated vasorelaxation may attenuate the increase in flow to non-exercising tissues thereby allowing a larger percentage of the cardiac output to be diverted to
skeletal muscle. The increased exercise capacity and reduced body fat
content of
2-AR
/
mice could reflect an elevated basal level of
activity in these mice. However,
2-AR +/+ and
2-AR
/
mice
displayed similar 48-h activity levels (Fig. 7).
In summary, we have generated mice that have a targeted disruption in
the
2-AR gene. These mice have normal resting heart rate and blood
pressure but manifest hypertension in response to epinephrine infusion
or to the cardiovascular stress induced by exercise.
2-AR knockout
mice should prove to be a useful model for further defining the roles
of
-AR subtypes in cardiovascular, respiratory, metabolic, central
nervous system, and reproductive functions.