Cardiovascular and Metabolic Alterations in Mice Lacking Both
1- and
2-Adrenergic Receptors*
Daniel K.
Rohrer
,
Andrzej
Chruscinski§¶,
Eric H.
Schauble
,
Daniel
Bernstein
, and
Brian K.
Kobilka§**
From the
Department of Molecular Pharmacology, Roche
Bioscience, Palo Alto, California 94304, the § Howard
Hughes Medical Institute, Stanford University, and the
Division
of Pediatric Cardiology, Department of Pediatrics, Stanford
University, Stanford, California 94305
 |
ABSTRACT |
The activation state of
-adrenergic receptors
(
-ARs) in vivo is an important determinant of
hemodynamic status, cardiac performance, and metabolic rate. In order
to achieve homeostasis in vivo, the cellular signals
generated by
-AR activation are integrated with signals from a
number of other distinct receptors and signaling pathways. We have
utilized genetic knockout models to test directly the role of
1-
and/or
2-AR expression on these homeostatic control mechanisms.
Despite total absence of
1- and
2-ARs, the predominant
cardiovascular
-adrenergic subtypes, basal heart rate, blood
pressure, and metabolic rate do not differ from wild type controls.
However, stimulation of
-AR function by
-AR agonists or exercise
reveals significant impairments in chronotropic range, vascular
reactivity, and metabolic rate. Surprisingly, the blunted chronotropic
and metabolic response to exercise seen in
1/
2-AR double
knockouts fails to impact maximal exercise capacity. Integrating the
results from single
1- and
2-AR knockouts as well as the
1-/
2-AR double knock-out suggest that in the mouse,
-AR
stimulation of cardiac inotropy and chronotropy is mediated almost
exclusively by the
1-AR, whereas vascular relaxation and metabolic
rate are controlled by all three
-ARs (
1-,
2-, and
3-AR).
Compensatory alterations in cardiac muscarinic receptor density and
vascular
3-AR responsiveness are also observed in
1-/
2-AR
double knockouts. In addition to its ability to define
-AR
subtype-specific functions, this genetic approach is also useful in
identifying adaptive alterations that serve to maintain critical
physiological setpoints such as heart rate, blood pressure, and
metabolic rate when cellular signaling mechanisms are perturbed.
 |
INTRODUCTION |
The
-adrenergic receptors (
1-,
2-, and
3-AR)1 belong to the
superfamily of G-protein-coupled receptors (1). Both sequence comparisons and functional studies suggest that these three receptors share many structural and mechanistic features (2). Agonist stimulation
of cloned and exogenously expressed
-ARs has demonstrated that all
three subtypes can couple through G
s to stimulate
adenylate cyclase activity (3-5). Despite these common structural and
functional properties, however, individual
-AR subtypes in
vivo remain as distinct therapeutic targets due to a number of
factors that actually serve to distinguish them. These distinctions
include tissue-specific expression patterns, the ability to couple to
different G-proteins, pharmacological heterogeneity, and differences in
agonist-dependent desensitization (6, 7).
-AR subtypes can be distinguished pharmacologically by synthetic as
well as natural ligands. The
1-AR subtype shows little preference
for epinephrine or norepinephrine, whereas the
2-AR preferentially
interacts with epinephrine (8, 9). More recent experiments demonstrate
that the
3-AR (previously termed "atypical") preferentially
interacts with norepinephrine over epinephrine. Synthetic
subtype-selective agents have been developed which display much greater
selectivity than these endogenous catecholamines. Some typical examples
of these would include the antagonists CGP20712A (
1-AR-selective)
and ICI118551 (
2-AR-selective) and the agonist CL316243
(
3-AR-selective). Such synthetic compounds have proven invaluable
for studying
-AR pharmacology and function (2, 10).
In vivo,
-ARs are known to modulate a wide range of
physiological processes, from cardiac chronotropy and inotropy to
vascular and smooth muscle tone, metabolism, and behavior. Functional
assignment of
-AR subtype functions using pharmacological tools
suggests that the
1-AR is the predominant subtype regulating heart
rate and contractility, although at least in the human,
2-ARs are also thought to participate.
2-ARs have been thought to be the predominant subtype mediating the vascular smooth muscle relaxant properties of
-AR agonists. The
3-AR was initially identified and
proposed to be the major
-AR subtype controlling lipolysis in
adipose tissue. Although these functional divisions are not absolute,
they appear to be well conserved across species and serve as a
convenient framework for
-AR classification. However, defining
-AR subtype-specific functions in vivo can present
significant challenges. Some subtype-selective agents display non-ideal
behavior in vivo, either due to poor biodistribution or
cross-reactivity with unrelated receptors. Gene disruption, or
"knockout" experiments, has proven to be a useful approach in
defining adrenergic receptor function in vivo. To date, this
technique has been used to disrupt expression of all three
2-AR
subtypes, the
1b-AR, the
1-, and the
3-ARs (11-16), and most
recently, the
2-AR (17). When the pharmacologic tools outlined above
are used in conjunction with genetic techniques, the power to reveal
novel functions and mechanisms of action can be greatly enhanced.
Given the prominent role of
-AR signaling in the maintenance of
normal physiology in vivo, we sought to test the functional consequences of
-AR gene disruption via a combinatorial approach. In
the companion article (17), the functional consequences of
2-AR
disruption are described. We have previously described the functional
consequences of
1-AR gene disruption (13, 18). We have now produced
mice that lack both
1- and
2-ARs. The role of these two
-AR
subtypes and the inferred role of the remaining
3-AR subtype in
cardiovascular physiology and metabolism are reported here.
 |
MATERIALS AND METHODS |
Generation of
-AR Knockout Mice--
The generation of
1-AR knock-out mice has been previously described (13). Briefly,
disruption of the
1-AR gene was achieved using a positive-negative
selection strategy to effect homologous recombination in the R1
embryonic stem cell line, using a targeting construct in which over
90% of the coding sequence was deleted. The strain background of
1-AR knockout mice was a mixture of 129SvJ, C57Bl6/J, and DBA/2
which is less prone to the prenatal mortality previously described
(13). The targeting strategy used to create
2-AR knockout mice is
described in the companion article (17) and is based on a similar
positive-negative selection scheme and homologous recombination in the
R1 embryonic stem cell line. Combination
1/
2-AR double knockouts
were generated by mating
2-AR homozygous knockouts (on a combined
129SvJ and FVB/N mouse strain background) to homozygous
1-AR
knockouts. The resulting F1 generation of compound heterozygotes was
subsequently intercrossed to generate F2 mice with all possible
combinations of
1- and
2-AR gene disruptions. According to
Mendelian inheritance, 1/16 of progeny were predicted to be
homozygous-deficient for
1- and
2-AR, and 1/16 of progeny were
predicted to be wild type for both
1- and
2-AR (see Table I). The
F2
1/
2-AR double knockouts were bred to produced to double
knockouts used in our experiments. The wild type F2 mice were bred to
produce wild type controls. Thus, the overall strain contributions
between wild type and
1/
2-AR double knockouts were equivalent.
Mice were genotyped for both
1- and
2-AR disruptions by Southern
blotting of mouse tail biopsies (13, 17).
Mouse Instrumentation--
Catheters were surgically implanted
in either the left carotid artery or the left carotid artery plus the
left jugular vein under isoflurane anesthesia. Briefly, anesthesia was
induced with 3% (v/v) isoflurane in oxygen using an isoflurane
vaporizer (Airco Inc., Madison, WI), and then induction was maintained
at 1.25-1.75% while monitoring the responsiveness of the animal. The
vessels were cannulated with a stretched Intramedic PE10 polyethylene catheter (Clay Adams, Parsippany, NJ), which was filled with
heparinized normal saline, sutured in place, and tunneled to the back.
Blood pressure was measured using a DTX Plus pressure transducer
(Spectramed, Oxnard, CA) amplified with a Gould 8-channel recorder, and
the analog pressure was digitized using a Data Translation Series DT2801 analog-digital converter (Marlboro, MA). Digital signals were
analyzed and stored using Crystal Biotech Dataflow data acquisition software (Crystal Biotech, Hopkington, MA). Heart rate measurements were determined on-line and were derived from the pressure recordings. Drugs were infused through the arterial catheter as a bolus in a volume
of 1-3 µl/g. (
)-Isoproterenol hydrochloride (3 µg/kg), atropine
sulfate (1 mg/kg), epinephrine bitartrate (3 µg/kg), and sodium
nitroprusside (30 µg/kg) were purchased from Sigma. CL316243 (100 µg/kg) was a kind gift of Wyeth Ayerst Laboratories (Philadelphia, PA).
Assessment of Ventricular Function--
A 1.4 French
micromanometer-tipped Millar pressure transducer (Millar Instruments,
Houston, TX) was advanced into the left ventricle via the right carotid
artery under isoflurane anesthesia (see above). Correct placement of
the catheter in the ventricle was judged by loss of the arterial
waveform and transition to a waveform with similar peak systolic
pressure, but diastolic pressures with minima in the 0-5 mm Hg range.
Following correct placement, a jugular venous catheter was placed via
the left jugular vein and advanced ~1 cm. The surgical incision was
then sutured closed with 4-0 silk, and the mouse was allowed to
stabilize for 10-15 min at 2.5% isoflurane. Pressure recordings were
measured using a MacLab 8S digitizer/amplifier (MacLab, Milford, MA),
recorded, and analyzed using MacLab/s version 3.5 software on a
Macintosh 3400c. "Anesthetized" recordings of ventricular function
were taken during a 1-min interval at the end of this 10-15-min
equilibration period, at 2.5% isoflurane. Isoflurane anesthesia was
then reduced in a stepwise fashion, from 2.5 to 1.25%, and mice were
allowed to stabilize for 10 min. Following this period, isoflurane
anesthetic was turned off, and the mouse was removed from the
anesthetic nose cone and placed on its back. Upon self-righting (or the
"awakening" state), mice were quickly euthanized with an
intravenous dose of avertin. Awakening recordings of ventricular
function were taken in the 30-60 s prior to the righting response.
Exercise Protocols, Metabolic Measurements--
Mice were
subjected to either constant or graded treadmill exercise, using a
Columbus Instruments Simplex II metabolic rodent treadmill, fitted with
Oxymax oxygen and carbon dioxide gas analyzers (Columbus Instruments,
Columbus, OH). For graded exercise, mice were placed in the exercise
chamber and allowed to equilibrate (usually 30-60 min). 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. Pre-operatively, mice were initially subjected to this
protocol, with regular stepwise increases until mice stopped running
from exhaustion. Post-operatively, mice were run to a final end point
of 20 m/min and 14° inclination. We have previously shown linear
relationships between heart rate, VO2 and VCO2
during graded treadmill exercise in mice (19).
In Vitro Cardiac Physiology--
The right ventricular free wall
was dissected away from the left ventricle and interventricular septum,
and silk sutures were tied at both ends of the long axis. Ventricles
were placed in an oxygenated 32 °C tissue bath containing modified
Krebs solution (118 mM NaCl, 5.4 mM KCl, 2.5 mM CaCl2, 0.57 mM
MgSO4, 1.0 mM Na2HPO4,
2.5 mM NaHCO3, 11.1 mM
D-glucose). Ventricles were paced at 3.3 Hz by use of a
Grass stimulator (30-ms pulse duration, 8-15 V). Signals from
isometric force transducers were amplified and digitized with a MacLab
8S series amplifier and fed to MacLab/s version 3.5 software running on
a Macintosh 3400c to determine twitch amplitude.
For spontaneously beating atria, right and left atria were dissected
free of ventricular tissue, and both atrial appendages were tied with
4-0 silk sutures. These were placed in an oxygenated 32 °C tissue
bath, where isometric force transduction and rate were monitored as above.
Radioligand Binding Assays--
Ventricular homogenates were
prepared by Polytron homogenization of whole organs in 5 mM
Tris-Cl, 5 mM EDTA, pH 7.4, followed by centrifugation at
10,000 × g. The resultant pellet was resuspended in
1× binding buffer (for
-adrenergic receptor binding, 150 mM NaCl, 50 mM Tris-Cl, 5 mM EDTA,
pH 7.4; for muscarinic receptor binding, 75 mM Tris-Cl,
12.5 mM MgCl2, 1 mM EDTA, pH 7.4),
and protein concentration was determined. For saturation binding, 50-100 µg of homogenate protein was used in a 500-µl reaction containing 300 pM
125I-2-[
-(4-hydroxyphenyl)-ethyl-aminomethyl]tetralone
or 300 pM [3H]N-methyl scopolamine
(both from NEN Life Science Products). Nonspecific binding was
performed in duplicate with 20 µM prazosin (Research
Biochemicals, Natick, MA) or 5 µM atropine sulfate, respectively (Sigma). All binding reactions were carried out at room
temperature for ~2 h prior to vacuum filtration onto Whatman GF-C
filters and determination of membrane-bound radioligand.
 |
RESULTS |
Generation and Recovery of
-AR Knockout Mice--
The
generation and viability of
1-AR knockout mice (
1-AR
/
) have
been described previously (13). Briefly, homozygous
1-AR knockouts
derived from heterozygote:heterozygote matings (
1-AR +/
×
1-AR
+/
) are recovered at an unexpectedly low frequency as predicted from
Mendelian inheritance, although this effect can be ameliorated if the
1-AR gene disruption is bred onto a multiple strain background. As
described by Chruscinski et al. (17), the recovery of
homozygous
2-AR knockouts (
2-AR
/
) is in accord with expected
Mendelian frequencies.
Crosses were carried out between homozygous
1-AR knock-outs and
homozygous
2-AR knockouts to generate compound heterozygotes (
1-AR +/
:
2-AR +/
, see "Materials and Methods"), and these in turn were intercrossed to generate homozygous
1- and
2-AR double knockout mice (
1-AR
/
:
2-AR
/
). The expected
frequency of recovering double knockout mice from compound heterozygote matings is 1 out of 16 or 6.25%. The observed frequency among weanlings was 7.23%, well within the expected range. Table
I lists the expected and observed
frequencies among the nine possible genotypes arising from the compound
heterozygote intercrosses. The
2 distribution suggests
that there are no significant deviations from Mendelian expectations
either among individual genotypes or the group as a whole
(
2 = 9.38 with 8 degrees of freedom, p = 0.29), although
1-AR knockouts (
1-AR
/
:
2-AR +/+) appear to
be less well represented, in accord with our previous findings (13).
Double knockout:double knockout matings were subsequently performed to
generate mice for the studies reported here. Litter size, maternal
behavior, and pup viability all appeared to be normal in this
group.
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Table I
Frequency of viable pups at weaning from 1 +/ : 2 +/ × 1
+/ : 2 +/ intercrosses
The nine possible genotypes are listed together with the number of
recovered viable pups. Expected values are derived from Mendelian
inheritance patterns; 2 = d2/E, where d is expected
number observed number, and E is expected number,
with 8 degrees of freedom.
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Basal Cardiovascular Function--
Basal cardiovascular parameters
were measured in awake, unrestrained mice by use of indwelling carotid
arterial catheters. As seen in Fig. 1,
neither baseline heart rate (range 400-470 beats/min) nor mean
arterial blood pressure (range 115-125 mm Hg) are significantly
different when comparing wild type mice (
1-AR +/+:
2-AR +/+) to
double knockouts (
1-AR
/
:
2-AR
/
).

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Fig. 1.
Basal cardiovascular indices in wild type
and 1/ 2 knockout
mice. Conscious, unrestrained mice instrumented with carotid
arterial catheters were monitored for both heart rate (in beats per min
(bpm)), and mean arterial blood pressure (mm Hg). 12 mice of
each genotype were studied. 1/ 2KO refers to 1/ 2-AR double
knockout mice.
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|
Response to Catecholamines--
Both isoproterenol and epinephrine
were administered to wild type and
1/
2-AR double knockout mice.
The grouped response to these agents is shown in Fig.
2A. Whereas the non-selective
-AR agonist isoproterenol (3 µg/kg) elicits robust chronotropic and hypotensive responses in wild types, both of these responses are
severely attenuated in
1/
2-AR double knockout mice. Of note, both
responses are also significantly time-delayed in
1/
2-AR double
knockouts in comparison to wild type responses. Furthermore, the small
but significant increase in heart rate seen in double knockout mice in
response to isoproterenol was attenuated by 93% in mice pretreated
with the muscarinic antagonist atropine (1 mg/kg, data not shown),
suggesting that the majority of this effect is due to the baroreflex,
mediated by the vagus in response to the drop in blood pressure.

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Fig. 2.
Cardiovascular response to
catecholamines. A, isoproterenol (3 µg/kg) or
epinephrine (3 µg/kg) were injected intra-arterially as a bolus to
conscious unrestrained wild type mice (squares) or 1/ 2
knockouts (filled circles) at the 2.5-min time point. The
effects on heart rate and blood pressure are shown for these two
agents, expressed as the change ( ) in beats/min (bpm) or
mm Hg, respectively (wild type, n = 10; 1/ 2KO,
n = 9). B, the percentage contribution of
individual -AR subtypes is inferred from a comparison of 1-,
2-, and 1/ 2-AR knockouts. As above, the response to
isoproterenol (1-3 mg/kg intra-arterially) is shown as a percentage of
the wild type response for either the increase in heart rate or the
decrease in mean blood pressure. The dotted line indicates
100% of the wild type response ( , 1 knockouts, n = 24; , 2 knockouts, n = 16; , 1/ 2
knockouts, n = 9). Data for 1 knockouts were adapted
from Rohrer et al. (18); data for 2 knock-outs were
adapted from Chruscinski et al. (17). Responses in all
groups except heart rate in 2 knockouts are significantly decreased
(p 0.01) in comparison to the wild type response.
For heart rate responses, all genotypes display significantly different
heart rate responses from each other (p 0.02). For
blood pressure response, both 1 and 2 knock-outs are
significantly different from 1/ 2 knockouts (p 0.01).
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The effect of epinephrine (3 µg/kg) on
1/
2-AR double knockouts
is seen in the right-hand panel of Fig. 2A. This
endogenous catecholamine is a mixed, non-selective
-AR and
-AR
agonist. In wild types, this dose of epinephrine elicits tachycardia
and a biphasic blood pressure response consisting of an initial brief hypertension followed by a more prolonged hypotensive response. In
contrast, ablation of
1- and
2-AR signaling in the double knockout appears to convert this mixed
- and
-AR agonist into a
selective
-AR agonist; these mice display concomitant bradycardia and a monophasic hypertensive blood pressure response. Again, the heart
rate response to epinephrine seen in double knockouts appears to be
predominantly due to baroreflex stimulation, as atropine pretreatment
blocks 60% of this response (data not shown).
Fig. 2B is a compilation of the chronotropic and hemodynamic
effects of isoproterenol on conscious and unrestrained
1-AR knockouts,
2-AR knockouts, and
1/
2-AR double knockouts. These are all displayed relative to the response seen in wild type mice (dotted line at 100%) and represent the peak chronotropic
and vasodilatory responses obtained in each genotype, respectively. Based on these data, ~50% of the chronotropic response to
isoproterenol is lost when the
1-AR is knocked out, whereas there is
no detrimental effect on heart rate in
2-AR knockouts. The combined
1- and
2-AR deficiency reduces the chronotropic response by over
85%. In terms of the vasodilatory response to isoproterenol, there appears to be a graded and additive attenuation of the hypotensive response with loss of the
1-AR (20% reduction),
2-AR (35%
reduction), and combined
1-/
2-AR (71% loss).
Hemodynamic Responses to the
3-AR Agonist CL316243--
The
hemodynamic response to the
3-AR agonist CL316243 was tested in
-AR knockout mice to clarify the role of the
3-AR in the
regulation of peripheral vasodilatory responses in vivo.
Infusion of the
3-AR-selective agonist was followed by infusion of
the non-selective
-AR agonist isoproterenol, to ascertain residual
1- and/or
2-AR responsiveness. Both drugs were used at doses that
elicit maximal responses in vivo (19, 20). As can be seen in
the top panel of Fig. 3,
administration of CL316243 at 100 µg/kg to a wild type mouse leads to
a gradual but sustained hypotensive response. Near-maximal responses to
this agonist were observed after 10 min, at which time isoproterenol
was infused. These results clearly show that a residual
1- and/or
2-AR vasodilatory response can be elicited by isoproterenol even
while
3-ARs are maximally stimulated. The time course of
3-AR-mediated vasodilatation suggests that the duration of action as
well as the time interval to peak response is much longer for the
3-AR response to CL316243 than for the
1/
2-AR response to
isoproterenol. This appears to be unique to the response mediated by
3-ARs and not specific to CL316243, as isoproterenol given to
1/
2-AR double knockouts also exhibits a similar time lag
(Fig. 2A).

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Fig. 3.
Hemodynamic responsiveness to the
3-AR agonist CL316243 and the non-selective agonist
isoproterenol. The upper panel is a representative
blood pressure tracing of a wild type mouse given a single bolus
injection of CL316243 (100 µg/kg). Ten minutes following injection of
the 3-AR agonist, isoproterenol was given (3 µg/kg
intra-arterially). The bar graph in the lower
panel summarizes identically performed experiments for wild type,
1-, 2-, and 1/ 2-AR knockouts. The hemodynamic effect of
CL316243 at 10 min (just prior to Iso administration) is shown by the
cross-hatched bars, and the additional effect of Iso
(compared with the period just preceding Iso administration) is shown
by the black bars. Wild type, n = 16; 1
knockouts (KO), n = 6; 2 knockouts,
n = 9; 1/ 2 knockouts, n = 7. For
2 knockouts, * indicates p 0.01 versus
wild type. For 1/ 2 knockouts, indicates p 0.01 versus wild type.
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The bottom panel of Fig. 3 summarizes identically performed
experiments on wild type,
1-,
2-, and combination
1-/
2-AR knockout mice. Interestingly, the response to the
3-AR agonist CL316243 alone was significantly augmented in the
1/
2-AR double knockouts. Furthermore, the effect of isoproterenol following
3-AR
stimulation (residual response) revealed that mice lacking
1-ARs
showed no deficit in the residual vasodilatory response, whereas loss
of
2-ARs had a large impact on further vasodilatory responses.
Surprisingly, when both
1- and
2-ARs were lacking, isoproterenol
infusion actually had a small hypertensive effect. Such a response can
be due to either injection artifact or cross-reactivity to
-ARs. In
either case, these results suggest that all three
-ARs can mediate
vasodilatory responses in vivo and that an enhancement of
3-AR responsiveness is seen in mice lacking both
1- and
2-ARs.
Cardiovascular and Metabolic Responses to Exercise in the
1-/
2-AR Double Knockout--
We also tested the role of
1-
and
2-AR signaling on the response to the physical stress of
exercise. Knowing that
-ARs are recruited during exercise to
modulate heart rate, hemodynamics, airway conductance, and metabolic
rate, we hypothesized that mice lacking both
1- and
2-ARs would
be compromised in both exercise capacity as well as the cardiovascular
and metabolic response to exercise. Using graded treadmill exercise
(GTE) as a stimulus, where both speed and angle of inclination are
progressively increased, both wild type mice and
1/
2-AR double
knockouts were tested for total exercise capacity as well as the
physiological response to fixed end point GTE. Total exercise capacity
was measured as cumulative distance run in non-instrumented mice, with
treadmill speed and angle of inclination increasing by 2.5 m/min and
2° every 3 min until mice stopped running from exhaustion.
Physiological responses to fixed end point GTE were obtained by running
instrumented mice to a final end point of 20 m/min and 14°
inclination (see "Materials and Methods").
Experiments designed to test total exercise capacity showed no
significant differences between wild types and
1/
2-AR double knockouts with respect to cumulative distance run. Wild type mice ran a
total distance of 578.8 ± 33.3 m (n = 7),
whereas
1/
2-AR double knockouts ran a total distance of
545.2 ± 30.0 m (n = 5). The metabolic
response to GTE in the maximal exercise capacity experiment is shown in
Fig. 4B, demonstrating that whereas both wild types and
1/
2-AR double knockouts have virtually identical levels of O2 consumption and CO2 production at
rest, consistent deficits in both of these indices are revealed at all
exercise levels in the double knockout. This metabolic deficit appears to result from the combined deficiency of
1- and
2-ARs, since neither the
1-AR knockout nor the
2-AR knockout display such deficits (17, 18). Interestingly, however, there are differences in
total exercise capacity between wild type mice and
2-AR knockouts, with the
2-AR knockout demonstrating a slight enhancement of total
exercise capacity and reduced respiratory exchange ratios (17). In
contrast,
1-AR ablation has no effect on total exercise capacity
(18).

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Fig. 4.
Cardiovascular and metabolic response to
exercise. Both wild type and 1/ 2-AR knockouts were subjected
to a GTE regimen (see "Materials and Methods"). A, the
cardiovascular response to GTE was determined in instrumented mice,
which were run to a final end point of 20 m/min and 14° inclination.
Rec, recovery (10 min post-exercise). Wild type,
n = 7. 1/ 2 knockouts,, n = 6. For
heart rate, wild type versus 1/ 2 knockouts is
significantly different (p 0.01 by two-way analysis
of variance with repeated measures, excluding recovery). B,
the metabolic response to GTE was monitored in non-instrumented mice,
which were run to their voluntary limit. O2 consumption and
CO2 production are expressed as ml/min/kg. Wild type,
n = 7. 1/ 2 knockouts, n = 5. Wild
type mice are significantly different than 1/ 2 knockouts in both
O2 consumption and CO2 production
(p 0.01 by two-way analysis of variance with
repeated measures, up to 27.5 m/min treadmill speed).
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The physiological response to GTE is seen in Fig. 4A. Both
blood pressure and heart rate were monitored in resting and exercising mice by use of indwelling carotid arterial catheters. The normal response to increasing exercise workloads is a corresponding increase in heart rate (up to the maximally achievable rate of ~800 beats/min in the mouse). Both wild type and
1/
2-AR knockouts show
workload-dependent increases in heart rate; however, the
heart rate of
1/
2-AR mice was lower than the heart rate of wild
type mice at all exercise levels (at rest, heart rate differences are
not statistically significant between the two genotypes). The effect of
exercise on mean peripheral arterial blood pressure is not different
between these two groups. The loss of exercise-induced tachycardia is most likely the result of
1-AR ablation, as a virtually identical behavior is seen in
1-AR knockout mice (18), whereas
2-AR knockouts show no deficit in exercise-induced tachycardia (17).
Muscarinic and
1-Adrenergic Receptor Density in Cardiac
Membranes--
There is a well known functional interdependence
between
-ARs and muscarinic receptors in the heart, which represent
the two major targets of cardiac sympathetic and parasympathetic
stimulation, respectively. Additionally, the role of
1-ARs either
alone or in combination with
-ARs is thought to be critical for both
acute responsiveness to catecholamines, as well as in longer term
adaptive or remodeling responses in the heart. We thus sought to test
whether any gross alterations in either of these receptor families was apparent in
1/
2-AR double knockouts in comparison to wild types. Whereas muscarinic receptor density displayed a mild but significant reduction in double knockouts in comparison to wild types (28.9 ± 1.6 fmol/mg protein versus 33.6 ± 1.0 fmol/mg protein;
n = 4 for both, p
0.05),
1-AR
density was not significantly affected by loss of both
1- and
2-ARs in comparison to wild types (50.3 ± 3.4 fmol/mg protein
versus 54.1 ± 4.1 fmol/mg protein, respectively; n = 4 for both, p = not significant).
In Vitro Cardiac Responsiveness to G-protein-coupled Receptor
Agonists--
The effect of various G-protein-coupled receptor
agonists was tested in either spontaneously beating atrial preparations
(chronotropic assay) or paced right ventricular strips (inotropic
assay). These experiments were performed to investigate the efficacy of
these compounds relative to
-AR agonists, as well as to reveal any potential compensation for loss of
-AR signaling that could be manifested as an altered response relative to wild type preparations. An additional utility of these experiments was the potential to reveal
any compensation with an indirect mechanism of action, since several
potential compensatory signaling pathways could exert their effects
through modulating the release of catecholamines and subsequent
activation of
-ARs (21-23). The agonists tested included serotonin,
angiotensin II, the
3-AR agonist CL316243, dopamine, histamine, the
-AR agonist isoproterenol (Iso), and the
-AR agonist
phenylephrine. As can be seen in Fig. 5,
there were no significant differences between wild type and
1/
2-AR double knockout preparations in response to any of these
drugs, with the exception of isoproterenol, which has robust effects on
both atrial rate and ventricular contractility in wild type preparations but no effect in
1/
2-AR double knockout
preparations. The lack of effect of isoproterenol in the double
knockout is virtually identical to that seen in
1-AR knockout
preparations (13), supporting the predominant role of
1-ARs in the
regulation of murine heart rate and contractility. The concentration of
agonist used in each of these experiments (see Fig. 5 legend) was
designed to elicit a maximal effect, based on prior experiments. It is notable that neither CL316243 nor isoproterenol has any appreciable effect on either atria or ventricles from
1-/
2-AR double
knockouts, given the purported negative coupling behavior of the
3-AR in human cardiac preparations (24).

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Fig. 5.
In vitro chronotropic and
inotropic responsiveness of wild type and
1/ 2-AR knockout
preparations. A variety of pharmacological agents was tested for
their ability to alter chronotropy or inotropy in spontaneously beating
atrial preparations or paced right ventricular preparations,
respectively. The upper panel shows the chronotropic
response to serotonin (5-HT, 1 µM),
angiotensin II (A II, 0.1 µM), the 3-AR
agonist CL316243 (CL, 1 µM), dopamine
(DA, 1 µM), histamine (Hist, 1 µM), isoproterenol (Iso, 1 µM),
and phenylephrine (Phen, 1 µM), and the
lower panel displays the inotropic response to the same
agents. Wild type, n = 3. 1/ 2 knockouts,
n = 5. All comparisons between wild type and 1/ 2
knockouts are not significant with the exception of Iso
(p 0.05 for both atrial rate and ventricular twitch
response).
|
|
In Vivo Left Ventricular Contractility in Anesthetized and
Awakening Mice--
Based on the observation that exercising
1/
2-AR double knockout mice can achieve similar workloads at
reduced heart rates, we sought to test whether corresponding deficits
were also present in the inotropic component of heart function in
vivo, given the well known effects of
-AR agonists to regulate
cardiac contractility. These studies were performed in both
anesthetized and awakening mice in an attempt to reduce the
cardiodepressant effects of anesthesia, using micromanometer-tipped
left ventricular catheters (see "Materials and Methods"). Fig.
6 shows representative tracings from two
mice in both anesthetized and awakening states. Clearly both wild types and
1/
2-AR double knockouts show depressed cardiac contractility, both in terms of developed pressure and dP/dt
(the first derivative of left ventricular pressure) in the anesthetized
versus the awakening state. Furthermore,
1/
2-AR double
knockouts show diminished positive and negative peak
dP/dt values relative to wild type mice whether
measured during anesthesia or during awakening. Table II summarizes the data from these
experiments, with the finding that while both
+dP/dt and
dP/dt were
reduced in
1/
2-AR double knockouts relative to wild type mice (in
both anesthetized and awakening states), there were no differences
between wild types and double knockouts with respect to peak developed
pressures in either state. There were also significant differences in
heart rate between wild type and
1/
2-AR double knockouts both in
the anesthetized and awakening states, which in turn may influence dP/dt values. Monitoring of left ventricular
pressure was terminated after mice righted themselves. At this time,
the intraventricular pressure measurements became very unreliable and
prone to position effects making these measurements in fully awake mice
untenable.

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Fig. 6.
Left ventricular contractility in
anesthetized and awakening mice. Micromanometer-tipped
pressure-sensing catheters were advanced from the carotid to the left
ventricle under anesthesia, measurements were taken, and mice were
allowed to recover until self-righting became evident. Representative
tracings from two mice are shown, in both the anesthetized and
awakening state. Both the left ventricular pressure tracings
(upper panels) and the first derivative of pressure,
dP/dt (lower panels), are shown for a
wild type and 1/ 2-AR knockout mouse. Time axis is in
seconds.
|
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Table II
Summary of left ventricular contractility measurements
Heart rate (HR), maximum left ventricular pressure (Max LVP), minimum
left ventricular pressure (Min LVP), maximum
dP/dt (Max dP/dt), and
minimum dP/dt (Min dP/dt)
are shown for both wild types and 1/ 2-AR knockouts under
anesthetized and awakening conditions. Wild type, n = 5; 1/ 2 knockouts, n = 6.
|
|
 |
DISCUSSION |
The
-ARs are recognized as important components of the
sympathetic nervous system, playing critical roles in the maintenance of cardiac, vascular, and metabolic homeostatic mechanisms. The purpose
of these studies was to delineate the subtype-specific contributions of
the
1-AR and
2-AR on these physiological processes by genetic
knockout techniques, where
1- and
2-ARs were knocked out
individually as well as in combination. By inference, we have also
investigated cardiovascular functions specific to the remaining
3-AR. Surprisingly, total elimination of both
1- and
2-ARs has
little impact on resting cardiovascular tone or basal metabolism, although functional deficits are clearly revealed when mice are stimulated by
-AR agonists or maximal exercise. Such results underscore the notion that the
-ARs are modulators of these
physiologic functions but not intrinsic to or required for the
functions themselves.
Basal Cardiovascular Function--
Loss of both
1- and
2-ARs
has minimal impact on basal heart rate and blood pressure. Based on the
phenotype of both the
1-AR knockout (13, 18) and the
2-AR
knockout (17), these results are not surprising. Such results would be
unexpected, however, when considered in the context of numerous
pharmacological studies using either non-selective or selective
-AR
antagonists that are commonly used to lower heart rate and blood
pressure. Why do mice lacking both adrenergic receptors fail to exhibit abnormalities at rest? First, there may be fundamental differences between animals that lack a given receptor from conception onwards and
animals treated with antagonists at a discrete point in time. Furthermore, the bulk of evidence from knock-outs of the
1b-,
2a-,
2b,
2c-, and
3-ARs also reveals that basal
physiological functions are not significantly perturbed, again failing
to reproduce the phenotypes observed by acute subtype-specific blockade
in normal animals (25). Our data suggest that there are alternative control points for such critical physiological functions such as
cardiac rate and contractility, vascular tone, and metabolic state,
which can be altered to compensate for the lack of
-AR signaling.
The parasympathetic nervous system, which acts in functional opposition
to signals generated by the sympathetic nervous system (26, 27), has
the potential to compensate for absent
-AR signaling, as do a
variety of other hormone or neurotransmitter systems. Our demonstration
that cardiac muscarinic receptor density is reduced in the
1/
2-AR
double knockout may reflect a counterbalancing reduction in a receptor
that is known to functionally antagonize stimulatory
-ARs.
3-AR Function--
Another example of a compensatory change in
G-protein-coupled receptor signaling resulting from
1- and
2-AR
deficiency is the supranormal response of
1/
2-AR knockouts to the
3-AR agonist CL316243. Stimulation of
3-ARs by this agonist (and
37344 from Life Technologies, Inc.) in rats and dogs elicits sustained
decreases in both blood pressure and total peripheral resistance (20). In wild type mice, we have demonstrated that
3-AR stimulation also
results in a robust and sustained hypotensive response. Mice deficient
in both
1- and
2-ARs show an exaggerated response to CL316243.
There are several possible explanations for such altered responses.
First, vascular
3-ARs may be up-regulated in the
1-/
2-AR
double knockout. The demonstration that
1-ARs are up-regulated in
adipose tissue of
3-AR knockout mice (12) supports the contention
that deficiencies in
-AR signaling can be counteracted by increases
in the density and/or signaling efficiency of other
-AR subtypes.
Another possibility is that there is an up-regulation of the signaling
machinery distal to
-ARs in the vascular beds of
1-/
2-AR
double knockouts, secondary to disuse. The phenomenon of
-AR
supersensitization following prolonged
-AR antagonist therapy is
well known (28-30) and may be analogous to the situation in mice when
both
1- and
2-ARs are absent.
Experiments with the
3-AR agonist CL316243 demonstrate that residual
responses (defined as the additional isoproterenol-induced vasodilatory
response during full
3-AR stimulation) of
1-ARs differ from that
of
2-ARs in mice which lack one or the other subtype. In these
experiments,
1-AR knock-outs possess identical residual
isoproterenol responses in comparison to wild types, whereas
2-AR
knockouts showed attenuated residual responses. This could be due to
either increased efficacy of the
2-AR in mediating vasodilatation,
different mechanism(s) of receptor activation, or to differences in the
distribution among the three
-ARs within vascular beds. As an
example, preferential distribution of
2-ARs in small resistance
arterioles and
1-ARs in large conductance vessels would tend to
favor
2-ARs in the primary control of peripheral vascular
resistance. Additionally, the attenuated response of
2-AR knockouts
to isoproterenol in these experiments could indicate an overlap of
1- and
3-AR expression in the same vascular beds, such that
3-AR stimulation with CL316243 precludes a maximal response through
1-ARs co-expressed in the same resistance vessels.
Metabolic and Physiologic Response to Exercise--
The ability of
-AR antagonists to alter the metabolic response to exercise is well
known (31-33).
-AR activation normally stimulates glycogenolysis as
well as lipolysis, reflected in the rise of plasma glucose and free
fatty acids during exercise. Whereas free fatty acid mobilization is
impaired in
-AR-blocked exercising subjects, glycogen utilization is
unimpaired, and there appears to be no change in the glucagon response
(31-33). In addition to the increased mobilization of metabolic fuels,
there are a variety of downstream effectors of
-AR stimulation that
can contribute to increased metabolic demands as follows: adenylate
cyclase, the Na+/K+-ATPase, and the
L-type Ca2+ channel are all activated by
-AR
stimulation (34-36). Thus, it is not surprising that metabolic demands
are reduced in
1-/
2-AR knockout mice during exercise, although it
is surprising that these mice show no impairment of total exercise
capacity when run to exhaustion. It is interesting to note that the
metabolic alteration seen in
1-/
2-AR double knockouts appears to
result from combined
1- and
2-AR deficiency, as neither single
1- nor
2-AR knockouts display the same degree of metabolic
hypo-responsiveness during exercise. In humans, such metabolic
responses were traditionally thought to be regulated primarily by the
2-AR (31), although there are instances where both
1- and
2-ARs appear to be involved in the metabolic response to exercise
(37, 38). Our results in mice suggest that both the
1- and
2-AR
normally subserve redundant metabolic functions in vivo and
that both receptor subtypes must be eliminated before significant
defects are manifested.
The study of receptor pharmacology in conscious animals is frequently
complicated by responses that are either indirect or reflexive in
nature. In the present study, the chronotropic effect of isoproterenol
in the
1-/
2-AR double knockout is almost completely eliminated by
pretreatment with atropine and thus must be primarily a reflex response
to
3-AR-mediated vasodilatation. In a previous study using atropine,
we demonstrated that ~50% of the typical heart rate response to
isoproterenol in conscious mice is due to parasympathetic
withdrawal, and ~50% is due to direct
1-AR stimulation (18).
A reflex mechanism for
3-AR stimulation of heart rate is supported
by studies in dogs (20) and by our own studies on isolated atria, in
which
1-/
2-AR knockout preparations fail to demonstrate any
direct inotropic or chronotropic effects from either isoproterenol or
the
3-AR agonist CL316243. In some instances, we were not able to
block completely the baroreflex with atropine, as evidenced by the
bradycardic effect of epinephrine in the
1-/
2-AR double
knockouts. These results suggest that either atropine dosage was not
sufficiently high to block the increase in vagal tone following a
hypertensive stimulus or that an
-AR component of the action of
epinephrine has negative chronotropic effects. The failure to
demonstrate significant chronotropic or inotropic effects of agents
such as histamine or serotonin, despite their ability to stimulate
cardiac adenylate cyclase in vitro (39, 40), suggests that
-ARs are the primary G-protein-coupled receptors regulating cardiac
function in vivo.
Given that both
1-AR knockouts (18) and the
1-/
2-AR double
knockout show normal exercise capacities at submaximal heart rates, we
wanted to investigate whether there were any significant differences in
cardiac inotropic state that could represent an adaptation to the loss
of
-ARs. Based on our previous findings with
1-AR knockouts (13,
41), the inotropic state is largely determined by the presence or
absence of the
1-AR and any underlying sympathetic tone. Our results
here suggest that there may be some residual sympathetic tone in
anesthetized mice, which in wild type mice manifests itself as an
increased +dP/dt and decreased
dP/dt in comparison to the
1/
2-AR double
knockout; this is even further accentuated in the awakening state.
Alternatively, the difference in heart rates between wild types and
double knockout mice during anesthesia and upon awakening could be
responsible for this difference in dP/dt.
Interestingly, inotropic state is greatly enhanced even in
1/
2-AR
double knockouts upon transition from anesthetized to awakening states.
Depression of myocardial contractility while under isoflurane
anesthesia can be due to indirect effects to reduce sympathetic
outflow, as well as direct inhibitory effects on cardiac muscle
(42-44). Despite the reduced positive and negative
dP/dt values in
1/
2-AR double knockouts, these mice develop equivalent peak left ventricular pressures in
comparison to their wild type counterparts. Taken together with the
exercise studies, our results would suggest that the
-AR-mediated
increases in heart rate and contractility in the mouse are not
necessary for maximal performance during a stress such as exercise. In
fact, at the extremely high heart rates typical for a mouse at maximal
exercise, reduced diastolic filling time may serve to limit any further
increases in cardiac output. The demonstration that humans under
-AR
blockade can increase stroke volume via the Frank-Starling mechanism
(while heart rate remains depressed) supports the idea that
chronotropic and inotropic stimulation through
-ARs are not required
for maximal exercise performance (45). Other studies have shown that
during exercise, intrinsic mechanisms such as increased venous return
enhances diastolic filling and hence cardiac output (46, 47) and can be
preferentially utilized over heart rate changes in some pathological
states to maintain cardiac output (48). Together with our data in
genetically altered mice, such results underscore the importance of
intrinsic preload and afterload mechanisms and tend to mitigate the
requirement for
-AR signaling in the adaptive responses to exercise.
In summary, the present study has further characterized the
physiological role of
1- and
2-ARs in mice by means of genetic knockout techniques. The impact of
1-,
2-, or
1/
2-AR loss on basal physiological functions such as heart rate, blood pressure, or
metabolic rate is remarkably minor; however, striking differences between these knockouts and their wild type counterparts can be seen
following
-agonist administration or during the stresses of
exercise. Given what is known about the important role that
1- and
2-ARs play in both physiological and pathophysiological processes,
one can speculate as to whether the gene knockout technique has
revealed the "true" role of these receptor subtypes. More thorough
studies involving different types of stresses (such as induced heart
failure) and longitudinal studies will help to clarify this issue.
Despite the limitations of the model systems studied to date, certain
-AR-modulated functions such as heart rate and contractility are
well defined and can be attributed to single
-AR members (in this
case the
1-AR). Other functions, such as
-AR-mediated
vasodilatation, are additive and integrated, with all three
-AR
subtypes contributing at some level. For other functions, such as
metabolic rate,
-AR actions appear to be redundant, and deficiencies
are not apparent until both
-AR subtypes are knocked out. Mice
lacking
1- and/or
2-ARs represent useful model systems for the
study of
-AR-modulated function in vivo, as well as the
role that
-ARs play in pathophysiology.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Supported in part by Medical Scientist Training Program
Training Grant GM07365 from the NIGMS, National Institutes of Health.
**
To whom correspondence should be addressed: Howard Hughes Medical
Institute, Stanford University, Stanford, CA 94305. Tel.: 650-723-7069;
Fax: 650-498-5092; E-mail: kobilka{at}cmgm.stanford.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
-Ar(s),
-adrenergic receptor(s);
GTE, graded treadmill exercise;
Iso, isoproterenol.
 |
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