From the Department of Molecular Cardiology, The
Lerner Research Institute, the ** Department of Cardiology, The
Cleveland Clinic Foundation, Cleveland, Ohio 44195, the
¶ Autonomic Physiology Unit, Division of Neuroscience and
Biomedical Systems, Institute of Biomedical and Life Sciences,
University of Glasgow, Glasgow, Scotland G12 8QQ, United
Kingdom, and the § Department of Pharmacology and the
Vascular Biology Research Group, The University of Kentucky College of
Medicine, Lexington, Kentucky 40536
Received for publication, September 22, 2000, and in revised form, January 30, 2001
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ABSTRACT |
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The adrenergic receptor family, which includes 3 The progress toward elucidating the distinct regulatory role of each
Mice--
The generation and genotyping of transgenic mice
possessing systemic Echocardiography--
Echocardiographic measurements were
performed on mice according to a previously published transthoracic
echocardiographic method (19). The mice were anesthetized via
intraperitoneal injection of 0.05 mg/g ketamine HCl and 0.1 mg/g
thiobutabarbital. The chest area was shaved and ultrasonic gel was
applied. Measurements were made as previously described using the
Acuson Sequoia 512 system (Mountain View, CA) that employed a
dynamically focused symmetrical annular array transducer (13 MHz) for
two-dimensional and M-mode imaging. The parasternal long and short axes
and apical four chamber views were visualized. Five consecutive cycles
of each parameter were measured. Cardiac output was calculated from echocardiographic data using the following equation,
[ Mean Carotid Artery Pressure in the Conscious Mouse--
The
measurement of the mean carotid artery blood pressure in conscious mice
was performed as described previously (20). The mice were anesthetized
via intraperitoneal injection of 0.1 mg/g ketamine and 2 µg/g
acepromazine maleate. A carotid catheter was inserted and connected to
a low compliance COBE CDXIII pressure transducer (Cobe Cardiovascular,
Arvada, CO). Blood pressure readings were collected on a Model 7D
Polygraph (Grass Instrument Division, West Warwick, RI). The recording
began immediately after surgery and continued for a 7-h period.
Mean Femoral Artery Pressure in the Anesthetized
Mouse--
The mean femoral artery pressure was determined in mice
using a modified version of a previously described method (21). The
mice were anesthetized with an initial intraperitoneal injection of
ketamine (0.05 mg/g) followed 5 min later by an injection of thiobutabarbital (0.1 mg/g). Supplemental doses of thiobutabarbital were delivered only when necessary to maintain stable anesthesia. A
femoral catheter was inserted and connected to a low compliance COBE
CDXIII pressure transducer (Cobe Cardiovascular) interfaced with an AH
60-9315 universal oscillograph (Harvard Apparatus, Holliston, MA). The
right femoral vein was cannulated similarly for intravenous
administration of increasing amounts of phenylephrine at a delivery
rate of 0.1 µl/g/min using a microinfusion pump.
Ex Vivo Arterial Contractile Studies--
Four strains of mice
were used for this experiment. The Catecholamine and Cortisol Determination--
Mice were
anesthetized via intraperitoneal injection of thiobutabarbital (0.125 mg/g). An abdominal incision was made, and blood samples were obtained
via venipuncture of the vena cava either 5 min after application of the
anesthetic or after 1 h of stable anesthesia. Total plasma
epinephrine and norepinephrine levels were determined in 100 µl of
plasma samples using the commercially available plasma catecholamines
by high pressure liquid chromatography kit (Bio-Rad). Plasma
cortisol levels were determined in parallel in 100 µl of plasma
samples using the commercially available fluorescence polarization
immunoassay kit (Abbott).
Statistical Analyses--
All reported errors and error bars
represent S.E., and significance was determined using either an
unpaired two-tailed Student's t test (p < 0.05) or a one-way analysis of variance (see Figure and Table Legends).
General Characterization of Mice Possessing
It should be noted that when bred to homozygosity, mice overexpressing
constitutively active mutant forms of the Cardiac Hypertrophy in Mice Possessing
Molecular confirmation of cardiac hypertrophy was attempted by
measuring ANF message levels via Northern blot
analysis of poly(A) mRNA purified from 8-month-old NT and T2 mouse
hearts. ANF, a gene often associated with cardiac
hypertrophy (27), was not up-regulated in T2 mice relative to the NT
controls (data not shown), suggesting that the morphologic and
echocardiographic findings are indicative of an early stage
hypertrophy. Besides our model, the hypertrophic cardiomyopathy mouse
(28) also shows hypertrophy in the absence of ANF
up-regulation, suggesting that the progression of cardiac hypertrophy
is not always strictly associated with the up-regulation of
ANF (29) and/or other fetal genes. Another more likely
reason for the lack of ANF up-regulation is the low level of
Despite an increased ventricular diameter in both diastole and systole,
the cardiac output in the transgenic lines was lower than that seen in
NT mice (Table I). This probably is attributed to the decreased heart
rate and increased isovolumetric relaxation time displayed by
transgenic animals (Table I). The decreased heart rate, which was
confirmed via a tail cuff measurement in conscious mice (Table I), may
be the result of a direct effect on Purkinje fiber automaticity, which
is thought to be controlled by the
Interestingly, a robust myocardial overexpression (>40-fold) of the
wild type Hypotension in Mice Possessing
4-6-month-old S1 and T2 mice were hypotensive relative to age-matched
NT control mice. Whereas T2 mice showed modest hypotension while still
under the influence of the anesthetic agents (Fig. 3, A and B), fully
conscious and unrestrained S1 and T2 mice showed a more significant
decrease in basal pressure compared with the NT control (Fig. 3,
A and C). The mean arterial pressure in conscious W2 mice was lower than that in NT animals (Fig. 3C);
however, this was not statistically significant. Confirming these
measurements made in conscious animals, the basal mean femoral artery
pressure was also significantly lower in 4-month-old anesthetized S1
mice than in age-matched NT control mice (Fig.
4A). Overall, our observation of basal hypotension in constitutively active
To extend these findings, we compared the potency of the
To confirm that the
Because of the apparent lack of direct
Our transgenic
Overall, our analysis of 1-Adrenergic
receptors (
1A,
1B, and
1D)
are regulators of systemic arterial blood pressure and blood flow.
Whereas vasoconstrictory action of the
1A and
1D subtypes is thought to be mainly responsible for this
activity, the role of the
1B-adrenergic receptor
(
1BAR) in this process is controversial. We have
generated transgenic mice that overexpress either wild type or
constitutively active
1BARs. Transgenic expression was
under the control of the isogenic promoter, thus assuring appropriate
developmental and tissue-specific expression. Cardiovascular phenotypes
displayed by transgenic mice included myocardial hypertrophy and
hypotension. Indicative of cardiac hypertrophy, transgenic mice
displayed an increased heart to body weight ratio, which was confirmed
by the echocardiographic finding of an increased thickness of the
interventricular septum and posterior wall. Functional deficits
included an increased isovolumetric relaxation time, a decreased heart
rate, and cardiac output. Transgenic mice were hypotensive and
exhibited a decreased pressor response. Vasoconstrictory regulation by
1BAR was absent as shown by the lack of
phenylephrine-induced contractile differences between ex
vivo mesenteric artery preparations. Plasma epinephrine, norepinephrine, and cortisol levels were also reduced in transgenic mice, suggesting a loss of sympathetic nerve activity. Reduced catecholamine levels together with basal hypotension, bradycardia, reproductive problems, and weight loss suggest autonomic failure, a
phenotype that is consistent with the multiple system atrophy-like neurodegeneration that has been reported previously in these mice. These results also suggest that this receptor subtype is not involved in the classic vasoconstrictory action of
1ARs that is
important in systemic regulation of blood pressure.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1,
3
2, and 3
-receptor subtypes, is a group of
heptahelical G protein-coupled receptors that mediate the effects of
the sympathetic nervous system. Extensive effort has been spent in
classifying the three known
1-adrenergic receptor
(
1AR)1
subtypes (
1A,
1B, and
1D)
via molecular cloning techniques (1-4) and pharmacological analyses
(5). The most well characterized cardiovascular regulatory actions
associated with
1AR activation include the contraction,
growth and proliferation of vascular smooth muscle cells (6-9),
increased cardiac contractility (10), and regulation of the
hypertrophic program in the myocardium (11, 12). In other
1AR-expressing tissues such as liver and kidney, the
function of these receptors is to regulate metabolic processes (13) and
sodium and water reabsorption (14), respectively. These responses are
transduced primarily via receptor coupling to the
Gq/phospholipase C pathway (5), which leads to the
subsequent activation of downstream signaling molecules including
protein kinase C and inositol 1,4,5-trisphosphate.
1 subtype in the various physiologic responses mentioned above has been constrained by a limited number of subtype-selective agonists and antagonists. This is especially true in the
1B system where there are no selective agonists or
antagonists available. We have alleviated this constraint by examining
the unique attributes of the
1BAR in a transgenic mouse
model that exhibits constitutive
1BAR activity targeted
only to tissues that normally express the receptor. The appropriate
distribution of receptor overactivity was achieved by using the mouse
isogenic
1BAR promoter (15) to drive the overexpression
of a transgene containing cDNAs of either the wild type (W) hamster
1BAR (3) or the constitutively active mutant forms of
the receptor. Two such mutants were employed, a C128F single mutant (S)
and a C128F/A204V/A293E triple mutant (T), both of which spontaneously
couple to Gq (16, 17). The systemic expression of
constitutively active
1BARs in these transgenic mice has
already led to the identification of a pathology similar to multiple
system atrophy suggesting that overstimulation of these
receptors leads to neurodegeneration (18). In the present study, we
extend this examination of phenotype to the cardiovascular system.
Discrete overexpression of constitutive
1BAR
activity in the cardiovascular system makes these mice well suited to
address questions regarding
1BAR regulation of
cardiovascular homeostasis. Our findings not only confirm the
involvement of the
1BAR in cardiac hypertrophy but
suggest that this subtype is not involved with blood pressure-related
vasoconstriction. Rather, the hypotension seems to be a manifestation
of autonomic failure and not the result of a direct action of the
1B subtype in the peripheral vasculature. Understanding
the
1BAR control over these processes and the
manifestation of disease will further define the therapeutic potential
that would come from the development of
1BAR-selective
antagonists and will have an impact on the future development of novel
gene therapies.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1BAR overactivity has been described
elsewhere (18). Tissue-specific distribution of systemic
1BAR overactivity was achieved by using the mouse
1BAR gene promoter (15) to drive the overexpression of a
transgene containing a cDNA coding for the wild type (W)
1BAR (3) or the constitutively active single mutant (S)
C128F
1BAR (16) or triple mutant (T) C128F/A204V/A293E
1BAR (17). The Cleveland Clinic Foundation Transgenic
Core Facility injected ~200 copies of each transgene into the
pronuclei of one cell B6/CBA mouse embryos, which
were surgically implanted into pseudo-pregnant female mice. 3 W, 5 single mutant, and 3 triple mutant founder mice were identified, and
subsequent generations were genotyped by Southern analysis
of genomic DNA extracted from tail biopsies. All phenotypic studies
detailed below are carried out using equal proportions of male and
female mice.
× (PA)2 × VTI × HR]/4,
where PA was the diameter of the pulmonary artery, VTI was the doppler
velocity time integral in the pulmonary artery, and HR was the
echocardiographically determined heart rate.
1B knockout mouse
(KO, C57-black) and its non-knockout control (NK mice) were bred in
Glasgow from breeding pairs supplied by Professor S. Cotecchia
(University of Lausanne, Lausanne, Switzerland) (for review
see Ref. 8). Tissues were also taken from the W2
1BAR overexpressor in this study and its appropriate non-transgenic control
(NT mice). Mice weighing 25-35 g (KO and their age-matched controls)
or 35-55 g (overexpressed and their age-matched controls) were killed
by an overdose of CO2. The mesentery was removed, and the
branches of first order mesenteric artery were dissected and cleared of
connective tissue before mounting on a wire myograph for isometric
force recording. The arteries were bathed in Krebs solution, and
the temperature was maintained at 37 °C at a pH of 7.4 with a gas
mixture of 95% O2, 5% CO2 throughout
the experiment. Preliminary studies employed the normalization
technique of Mulvany and Halpern (22) in 1977 to obtain vessel internal
diameter and normalized resting tension. Thereafter, the vessel
segments were set at 0.17 mg of resting tension before construction of concentration response curves. After a "priming" protocol involving challenges of norepinephrine (10 µM) and/or KCl (50 mM), tissues were washed and allowed 30 min before
beginning construction of a cumulative concentration response curve to
phenylephrine (1-10 µM). The potency of the agonist was
determined by comparing EC50 values (concentration required
to produce 50% of maximum response) obtained in each tissue.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1BAR
Overactivity--
We have previously described the genotypic and
initial phenotypic analysis of systemic
1BAR mice (18),
confirming transgene integration. The tissue-specific overexpression of
wild type and mutant
1BARs was confirmed via saturation
binding analysis of various tissues from F1 and F2 generation of
heterozygous mice. Of the seven transmitting founder lines, five
exhibited significant transgene overexpression including two W lines
(W1 and W2), one single mutant line (S1), and two triple mutant lines
(T1 and T2). The distribution and magnitude of receptor overexpression
were not significantly different among the various lines as expected for the housekeeping nature of the promoter. The level of
1BAR overexpression was ~2-fold in the heart with
greater overexpression seen in the liver, lung, brain, and spleen (18).
Confirming constitutive signaling of these overexpressed receptors in
the transgenic lines, inositol 1,4,5-trisphosphate levels were
significantly higher in kidneys from W2+/
, S1+/
, and T2+/
mice
than in age-matched NT mice (Fig. 1).
Similar constitutive stimulation of inositol 1,4,5-trisphosphate
metabolism has been previously shown in the liver (18). The rank order
increase in inositol 1,4,5-trisphosphate pool size seen among the
various lines (T2 > S1 > W2) corresponds with the strength
of constitutive signaling that was found for these receptors in
vitro (16, 17).
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Fig. 1.
IP3 levels were determined in
kidneys from 6-month-old NT, W2+/ , S1+/
, and T2+/
mice as
described under "Experimental Procedures" (n = 3). The asterisk indicates the significance from the NT
group. The dagger indicates significant increases compared
with the W2+/
group. The double dagger indicates
significant increases compared with the S1+/
group. Significance was
determined using a two-tailed Student's t test
(p < 0.05).
1BAR (S1 and
T2) displayed reproductive problems. This was not seen in the W2 line,
suggesting that reproductive failure was unlikely the result of
breeding artifacts. Therefore, all phenotypic analyses were performed
on heterozygotes. All transgenic lines also displayed a 20-30%
reduction in body weight, but this was only apparent in older mice that
were more than 12 months of age (18).
1BAR
Overactivity--
1ARs have been shown to evoke a
hypertrophic response in cultured cardiac myocytes (23, 24) with the
regulation of this process predominated by the
1A
subtype (25, 26). Because myocardial-targeted overexpression of
constitutively active
1BARs has also been shown to cause
cardiac hypertrophy in mice (11), we performed morphologic and
echocardiographic analyses in the context of our systemic transgenic
model. Indicative of a hypertrophic phenotype, W2, S1, T1, and T2 mice
showed an increased heart to body weight (heart/Bw) ratio
compared with age-matched (4-6 months) NT control mice (Fig.
2). Body weight was not significantly
different among the lines at 4-6 months of age. It should be noted
that other organs including the liver, kidneys, lungs, and brain did not exhibit a change in mass relative to body weight (data not shown).
Increases in heart mass ranged between 12 and 41% with S1 mice showing
the largest increase. These findings were confirmed echocardiographically in W2, S1, and T2 mice, which showed
an increased thickness of the
posterior wall and interventricular septum compared with age-matched NT
control mice (Table I).
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Fig. 2.
Heart to body weight
(Heart/Bw) ratios were calculated in NT, W2+/ ,
S1+/
, T1+/
, and T2+/
mice at 4-6 months of age (n > 12 for each line). The
asterisk indicate the significance from the NT group based
on a two-tailed Student's t test (p < 0.05).
Echocardiographic analyses
LVIDs)/LVIDd]. Cardiac output (CO) was calculated as
described under "Experimental Procedures." The asterisks
denote significance from NT (n
5) based on a
two-tailed Student's t test (p < 0.05).
1BAR overexpression present in our model. For example,
the G
q overexpression mouse model of cardiac hypertrophy (30)
displayed no change in ANF expression with a 2-fold increase in the G
q protein, a circumstance similar to the 2-fold
overexpression of cardiac
1BARs in our mice. However, a
4-fold increase in G
q was sufficient to invoke ANF
transcription. These findings collectively indicate that a threshold of
expression may be necessary to evoke changes in fetal gene transcription.
1BAR (31) and is
consistent with the overexpression of the receptor. A similar decrease
in heart rate was also found in the heart-targeted G
q-overexpressing
mouse (30). Overall, because
1BARs are coupled to G
q,
the decrease in heart rate may be directly related to signaling events
downstream of
1BAR activation, or it may be part of the
autonomic dysfunction, which we describe later.
1BAR has been shown to cause increased
diacylglycerol content and ANF mRNA without inducing the
morphological hallmarks of hypertrophy (12). One conclusion that can be
drawn from this earlier study is that only constitutively active
1BARs can induce hypertrophy. This raises the
possibility that constitutively active receptors may signal through
different pathways than wild type receptors. However, in arguing
against this possibility, modest developmental and tissue-specific
overexpression (2-fold in the heart) of wild type
1BARs
in our mice caused a cardiac hypertrophy that was less robust but
similar to that seen in the heart-targeted constitutively active
1BAR mouse. Unlike the heart-targeted model, our model
may be exhibiting a phenotype that more genuinely represents the end
point impact of
1BAR action in the heart because our use
of the isogenic
1BAR promoter facilitates transgene
overexpression in all
1BAR-expressing cardiac cell
types, not just cardiac myocytes. Overall, because several
experimentally distinct approaches to genetically induce
1BAR overactivity have independently led to the
manifestation of a somewhat similar cardiac phenotype, the emergence of
that phenotype must be
1BAR-dependent and
not simply the spurious outcome of transgenic manipulation. Based on
this assumption, we assert that in addition to the
1AAR,
the
1BAR plays an important regulatory role in the
progression of the hypertrophic program in cardiac tissue.
1BAR
Overactivity--
The
1ARs are widely expressed in the
peripheral arteries (4, 5) and possess the capacity to regulate
vasoconstriction (32-36), thus implicating them in the control of
blood pressure. Regarding the
1BAR, however, the bulk of
the literature suggests that this subtype does not play a significant
role in the direct regulation of the peripheral vascular tone (5, 9).
Rather, the predominant role of
1BARs expressed in the
vasculature has been proposed to include the regulation of growth and
metabolic activity (6, 37-39). Contrary to these studies, however, the
1BAR knockout mouse showed a decreased pressor response
to phenylephrine infusion (8), which indicates the participation of the
receptor in the regulation of peripheral vasoconstriction. Based on
these findings from the knockout model, if
1BARs
participate in vasoconstriction, our constitutively active
1BAR mice should display hypertension because of the
constitutive activation of this process. However, our mice displayed
the opposite phenotype, a significantly reduced systemic arterial blood pressure.
1BAR mice
contradicts the idea that activation of the
1BAR can
induce vasoconstriction and is the first report to indicate that an
1AR can affect resting arterial blood pressure. It
should be noted that although all transgenic lines demonstrated a
hypertrophic phenotype in the heart, only the two constitutively active
lines (S1 and T2) demonstrated hypotension. This was probably
attributed to the intermediate level of constitutive signaling (see
Fig. 1) and variability in the data collected from W2 mice. For
example, some parameters of hypertrophy were not significant for the W2
line, and blood pressure was reduced but was highly variable and not
significant.
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Fig. 3.
Mean carotid pressure (Basal Carotid
MAP) was determined under basal conditions in conscious NT,
W2, S1, and T2 mice via an in-dwelling catheter as described under
"Experimental Procedures." A, time course of
carotid MAP in NT (open circles) and S1 (closed
circles) recovering from anesthesia (n > 8 for
each point). B, a summary of carotid MAP in NT, W2, S1, and
T2 mice immediately after surgery while still under anesthesia
(n > 8 for each line). C, a
summary of carotid MAP in fully conscious NT, W2, S1, and T2 mice
7 h after surgery (n > 8 for each
line). The asterisks in each part of the figure
indicate the significance from the NT group based on a two-tailed
Student's t test (p < 0.05).
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Fig. 4.
Mean femoral artery pressure (Basal
Femoral MAP) was determined in NT and S1 mice via an
in-dwelling catheter under basal conditions and following intravenous
presentation of phenylephrine as described under "Experimental
Procedures." A, basal femoral MAP in NT and S1 mice
under anesthesia (n > 6 for each line). The
asterisk indicates the significance from the NT group based
on a two-tailed Student's t test (p < 0.05). B, phenylephrine dose effect on femoral MAP in NT
(open circles) and S1 (closed circles) mice under
anesthesia (n > 6 for each line). Dose
response data was analyzed using the non-linear regression functions of
the non-iterative curve fitting program GraphPad Prism.
1AR-selective agonist phenylephrine to evoke a pressor
response in NT and transgenic mice. Phenylephrine produced a
dose-dependent increase in systemic arterial blood pressure
in the NT and all transgenic groups. The pressor response in the
transgenic group was no greater than that seen in NT animals. Indeed
the pressor dose response curve in transgenic animals was shifted to
the right of that seen in NT mice (Fig. 4B), arguing that
the
1BAR does not transduce the phenylephrine pressor
response. This rightward shift seen in the transgenic lines was
probably because of the decrease in basal blood pressure. It should be
noted that the dose-response curves could not be completed to
saturation because of the lethal effect of high doses of phenylephrine.
Because the expression of
1BARs has been identified in
peripheral arteries via the use of an
1B-specific
antibody (36), these results suggest that vascular
1BARs
are not directly involved with the regulation of vasoconstriction.
1BAR is not directly involved in
blood pressure regulation either via vasoconstriction or somehow via a
negative influence on the pressor response (i.e.
vasodilation, Fig. 4B), contractile-response curves were
generated using ex vivo segments of the mesenteric artery
prepared from several lines of mice. The vasoconstrictory action of
phenylephrine was tested in artery segments from
1BAR
knockout mice (8) from our W2 line of mice and from the appropriate
non-knockout and non-transgenic control groups. The phenylephrine dose
response curves generated for each of these groups were not
significantly different from each other (Fig.
5), demonstrating that the
1BAR does not participate in blood pressure-related
vasoconstriction and confirming that the hypotension seen in our
transgenic animals is not the result of an arterial event.
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Fig. 5.
Concentration-response curves for the
1AR agonist phenylephrine in isolated
segments of mouse mesenteric artery (first order branches, external
diameter 200-220 µm) taken from non-knockout
(NK, closed circles),
1BAR knockout (KO,
open circles), non-transgenic (NT,
closed triangles), and W2 transgenic mice
(W2, open triangles). Data
points represent the mean for each group (n = 11 for NK
and n = 5 for KO, NT, and W2 each).
log(EC50) (log[M]) values were
5.53 for NK
mice,
5.54 for KO mice,
5.84 for NT mice, and
6.0 for W2 mice.
Dose response data were analyzed using the non-linear regression
functions of the non-iterative curve fitting program GraphPad Prism.
Groups were determined to not be significantly different from each
other based on a one-way analysis of variance.
1BAR control over
vasoconstriction, the question remains how does systemic
1BAR overactivity lead to a reduction in blood pressure?
It is well established that peripheral vascular tone is partially
regulated by sympathetic nervous system activity (40). Lower
sympathetic activity, as measured by a reduction of plasma
catecholamines, could lead to a lower blood pressure because of a
reduced activation of all vascular
1-adrenergic targets.
This hypothesis was tested by assessing sympathetic function via the
measurement of total blood levels of norepinephrine, epinephrine, and
cortisol in the transgenic lines. Indicating reduced sympathetic output
in transgenic animals, 6-month-old S1 and T2 mice showed a roughly 50%
reduction of total blood catecholamine compared with age-matched NT
control mice (Fig. 6A). A
similar reduction in total catecholamines was seen after a 1-h period
of stable anesthesia (Fig. 6B), suggesting that the
reduction was not a result of indirect effects of anesthesia or of
altered reactivity/stress induced by handling. It should be noted that
catecholamine levels seen in our mice (ng/ml) are in the same range as
those reported in other transgenic mouse models (41). As expected,
because cortisol is released from the adrenal medulla in response to
sympathetic stimulation, we also found a corresponding reduction in
plasma cortisol (50%) in S1 and T2 mice relative to the NT control
(Fig. 6C). Because the reduced plasma catecholamine and
cortisol levels were correlated to blood pressure effects, it is
possible that these two events may be linked. These data suggest that
the hypotension seen in our transgenic mice may be, at least in part,
because of a reduction in the sympathetic nerve activity. It is also
possible that the decrease in heart rate and cardiac output may also
contribute to the hypotension seen in our transgenic mice. This
possibility does not seem probable given the fact that W2 mice, which
displayed a reduced heart rate and cardiac output, were not
hypotensive.
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Fig. 6.
Total plasma epinephrine and norepinephrine levels
in NT, W2, S1, and T2 mice were determined as described under
"Experimental Procedures" either 5 min after application of
anesthesia (A) or after 1 h of stable anesthesia
(B) (n = 5 for each line).
(C) total plasma cortisol levels in NT, W2, S1, and T2 mice
were also determined 5 min after application of anesthesia as described
under "Experimental Procedures" (n = 3 for each
line). The asterisks in each part of the figure
indicate the significance from the NT group based on a two-tailed
Student's t test (p < 0.05).
1BAR mice display a Parkinsonian-like
syndrome termed multiple system atrophy with associated
neurodegeneration in the substantia nigra, olive pontine, thalamus, and
locus coerulus (18). Symptomatically, the presence of multiple system
atrophy often involves autonomic failure because of these extensive
neurodegenerative lesions in the brain. Therefore, a probable reason
for the hypotension seen in our mice is a lowered sympathetic output
caused by autonomic dysfunction. Although some patients with autonomic
failure have hypertension (42), autonomic neuropathy is a common cause
of orthostatic hypotension (43) and is also responsible for the hypotension commonly seen in Parkinson's disease and multiple system
atrophy patients (44). Accordingly, these patients also exhibit low
plasma levels of norepinephrine (44). Besides hypotension, our
transgenic mice displayed reproductive problems, weight loss (at 12 months of age), bradycardia, depressed heart function, and low cortisol
and catecholamine levels that are all associated with autonomic
dysfunction. Autonomic failure produces distinct abnormalities
depending upon the location of the lesions (44). Therefore, our model
is probably the outcome of autonomic dysfunction that is caused by
1BAR-induced neurodegeneration.
1BAR control of blood pressure
from a systemic perspective has led us to conclude that
1BAR overactivity does not cause an elevation in
pressure but rather induces a net hypotension. The mechanism driving
this hypotension is probably rooted in an autonomic failure because
many of the symptoms displayed by our mice are consistent with this
diagnosis. The data presented in this study are also counterindicative
of a vasoconstrictive role for
1BARs. Our findings
support an emerging hypothesis, which predicts that
1BARs do not play a major role in contractile regulation
in vascular smooth muscle (9) but rather are predominately coupled to
various metabolic and cellular processes at vascular sites where the
receptor is expressed (6, 37-39). This realization has important
implications in the pharmacotherapeutic approach to the manipulation of
blood pressure.
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ACKNOWLEDGEMENTS |
---|
We thank the Cleveland Clinic Foundation Transgenic Core Service for performing the transgene injections, which produced founder mice. Blood catecholamine and cortisol levels were determined by the Laboratory Medicine Section of the Cleveland Clinic Foundation.
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FOOTNOTES |
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* This work was funded by National Institutes of Health Grants RO1HL61438 (to D.M.P.), RO1HL31820 (to M.T.P.), and F32HL10004 (to M.J.Z.), EC FP5 Program Grant QLG1-1999-00084 and Medical Research Council Innovation Award G0000042 (to J.C.M.), and an American Heart Established Investigator Award and an unrestricted research grant from Glaxo Wellcome (to D.M.P.).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.
Recipient of British Heart Foundation Postgraduate Scholarship
FS/97053.
** To whom correspondence should be addressed: Dept. of Molecular Cardiology NB50, The Lerner Research Inst., The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-444-2058; Fax: 216-444-9263; E-mail: perezd@ccf.org.
Published, JBC Papers in Press, February 1, 2001, DOI 10.1074/jbc.M008693200
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ABBREVIATIONS |
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The abbreviations used are:
1AR,
1-adrenergic receptor;
1BAR,
1B-adrenergic receptor;
W, wild
type
1BAR;
S, C128F single mutant
1BAR;
T, C128F/A204V/A293E triple mutant
1BAR;
NT, non-transgenic;
NK, non-knockout;
KO, knockout;
ANF, atrial natriuretic
factor;
MAP, mean arterial pressure;
gBw, gram body weight.
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