(Received for publication, April 8, 1997, and in revised form, June 3, 1997)
From the Departments of Surgery and
Medicine
and the ** Howard Hughes Medical Institute, Duke University Medical
Center, Durham, North Carolina 27710 and the § Department of
Medicine, University of California at San Diego,
La Jolla, California 92093
Transgenic mice were generated with
cardiac-specific overexpression of the wild-type (WT)
1B-adrenergic receptor (AR) using the murine
-myosin heavy chain gene promoter. Previously, we described
transgenic mice with
-myosin heavy chain-directed expression of a
constitutively active mutant
1B-AR that had a phenotype of myocardial hypertrophy (Milano, C. A., Dolber, P. C.,
Rockman, H. A., Bond, R. A., Venable M. E., Allen,
L. F., and Lefkowitz, R. J. (1994) Proc. Natl. Acad.
Sci. U. S. A. 91, 10109-10113). In animals with >40-fold WT
1-AR overexpression, basal myocardial diacylglycerol
content was significantly increased, indicating enhanced
1-adrenergic signaling and phospholipase C activity. In
contrast to the mice overexpressing constitutively active mutant
1B-ARs, the hearts of these mice did not develop cardiac
hypertrophy despite an 8-fold increase in ventricular mRNA for
atrial natriuretic factor. In vivo physiology was studied
in anesthetized intact animals and showed left ventricular
contractility in response to the
-agonist isoproterenol to be
significantly depressed in animals overexpressing WT
1B-ARs. Membranes purified from the hearts of WT
1BAR-overexpressing mice demonstrated significantly attenuated adenylyl cyclase activity basally and after stimulation with
isoproterenol, norepinephrine, or phenylephrine. Interestingly, these
in vitro changes in signaling were reversed after treating the mice with pertussis toxin, suggesting that the extraordinarily high
levels of WT
1B-ARs can lead to coupling to pertussis
toxin-sensitive G proteins. Another potential contributor to the
observed decreased myocardial signaling and function could be enhanced
-AR desensitization as
-adrenergic receptor kinase (
ARK1)
activity was found to be significantly elevated (>3-fold) in
myocardial extracts isolated from WT
1B-AR-overexpressing mice. This type of altered signal transduction may become critical in disease conditions such as heart
failure where
ARK1 levels are elevated and
-ARs are
down-regulated, leading to a higher percentage of cardiac
1-ARs. Thus, these mice serve as a unique experimental
model to study the in vivo interactions between
- and
-ARs in the heart.
There have been numerous in vitro studies
characterizing the role of 1-adrenergic receptor
(AR)1 signaling in cardiac
myocytes. Agents that stimulate
1-ARs, leading to the
activation of the guanine nucleotide-binding protein Gq,
have been shown to induce nuclear transcription factors such as
c-myc, c-fos, and c-jun and to mediate
morphological changes including increases in myocyte size and volume
(1). Signaling through the
1-AR/Gq pathway
leads to the activation of the effector enzyme phospholipase C and
protein kinase C, both of which may act as biochemical initiators of
myocardial hypertrophy (2). In addition to Gq-mediated
hypertrophy, recent studies have implicated a p21ras
(Ras)-dependent hypertrophic pathway initiated by
1-AR activation (3). Other in vitro
1-AR/Gq-mediated signaling events reported
to exist in myocytes include positive inotropy and chronotropy and
induction of the egr-1 gene (4). In addition to coupling to
Gq,
1-ARs have also been reported to
activate pertussis toxin (PTx)-sensitive G proteins, leading, in
myocytes, to negative chronotropy, positive inotropy,
Na+-K+-ATPase activation, and modulation of
intracellular calcium transients and cell shortening (5).
In contrast to these studies, very few reports have investigated
in vivo cardiac 1-AR signaling particularly
with respect to physiological sequelae and the potential in
vivo significance of dual G protein coupling.
1A-
and
1B-ARs have been shown to exist in neonatal myocytes
(6), whereas in adult human myocardium, the
1A-AR
appears to predominate (7). Both the
1A and
1B subtypes have been implicated in myocyte growth and
hypertrophy (6). One possible functional role of
1-ARs
is as a source of inotropic reserve in pathophysiological conditions
where the
-AR system is down-regulated and uncoupled (8). Thus, an
interrelationship may exist in the heart between
1- and
-ARs. Previous work from our laboratory revealed that
cardiac-specific expression of a constitutively active mutant (CAM) of
the
1B-AR in transgenic mice leads to myocardial
hypertrophy, demonstrating that cardiac
1-adrenergic
signaling in vivo can trigger responses similar to
myocytes in culture (9).
In this study, we continue our characterization of in vivo
myocardial adrenergic signaling in transgenic mice (9-13) by
describing animals with cardiac overexpression of the wild-type (WT)
1B-AR. As in our previous studies, cardiac expression
was targeted by using the murine
-MyHC promoter (9-13). To
determine the consequences of WT
1B-AR overexpression,
we studied both biochemical signaling and in vivo
physiology. We have previously observed for the WT
2-AR
that when overexpression of these WT receptors reaches extraordinarily high levels, agonist-independent signaling can occur due to a small
percentage of spontaneously activated receptors that is significant at
high levels of receptor density (10, 14). Both
1- and
-AR signal transduction was assessed, including measurements of
myocardial diacylglycerol (DAG) content, ventricular atrial natriuretic
factor (ANF) mRNA levels, adenylyl cyclase activity, and G
protein-coupled receptor kinase (GRK) activity. In addition, the
presence of myocardial hypertrophy was assessed. Finally, in
vivo basal and
-AR-mediated cardiac function was assessed by
catheterization of anesthetized mice. The results from these studies
reveal findings that point to potentially important interactions between
- and
-adrenergic signaling in the heart.
A 5.5-kilobase
SalI-SacI fragment containing the murine -MyHC
promoter (15) was ligated into a previously described plasmid containing the SV40 intron poly(A) signal (9-13) to generate a new
plasmid, pGEM-
-MyHC-SV40. A 2.0-kilobase
SalI-SalI fragment containing the coding sequence
for the wild-type hamster
1B-AR was then ligated into
pGEM-
-MyHC-SV40 to generate pGEM-
-MyHC-
1B-AR-SV40. The transgene was then linearized and purified before pronuclear injections done by the Duke Comprehensive Cancer Center Transgenic Facility (9-13). Two lines of mice were established, TG
43 and TG
47. Litter sizes and postnatal development were indistinguishable from nontransgenic littermate controls. Offspring were screened by
Southern blot analysis with a probe to the SV40 sequences. Second
generation adult animals (2-5 months of age) were used for all
studies. Institutional Review Board approval for all mouse experiments
was obtained from the University of California at San Diego and from
Duke University Medical Center.
Membrane fractions were prepared from
hearts and resuspended in binding buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, and 5 mM EDTA or 75 mM Tris-HCl, pH 7.4, 12.5 mM MgCl2,
and 2 mM EDTA). Binding assays were performed on 25 µg of
membrane protein using saturating amounts of 125I-HEAT (300 pM), an 1-AR-specific ligand, or
125I-CYP (300 pM), a
-AR-specific ligand.
Nonspecific binding was determined in the presence of 50 µM prazosin for
-binding and 20 µM
alprenolol for
-binding. Reactions were conducted in either 250 or
500 µl of binding buffer at 37 °C for 1 h and then terminated by vacuum filtration through glass-fiber filters. All assays were performed in triplicate, and receptor density (fmol) was normalized to
mg of membrane protein following the method of Bradford (29).
Lipid fractions were extracted from 50 mg
of homogenized myocardial tissue as described (6, 9). Aliquots of lipid
and DAG standards were dried under nitrogen, resuspended in detergent micelles, and then completely phosphorylated using Escherichia coli DAG kinase and [-32P]ATP.
32P-Labeled phosphatidic acid (phosphorylated DAG) was
isolated by silica gel thin-layer chromatography and quantitated with a PhosphorImager (Molecular Dynamics, Inc.). DAG content was normalized to tissue phospholipid, and the final DAG concentration was expressed as pmol of DAG/nmol of lipid phosphate as described previously (9).
Ventricular tissue was separated from the atria under a dissecting microscope. Total RNA was extracted using RNAzol (Biotecx Laboratories, Houston TX) in a single-step guanidinium-based isolation procedure (16). Total RNA was then fractionated on a 1% formaldehyde-agarose gel and transferred to nitrocellulose as described (9). Blots were prehybridized in a 50% formamide solution for 4 h at 42 °C and then hybridized overnight with a random primer, radiolabeled ANF cDNA probe (9). Blots were washed three times in 0.2 × SSC at 65 °C for 30 min before exposure to x-ray film. All blots were then stripped in water at 95-100 °C for 15 min and reprobed with a rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe. The ANF and GAPDH bands were quantitated with the PhosphorImager, and the ANF/GAPDH signal intensity ratio was determined (9).
Adenylyl Cyclase ActivityCrude myocardial membranes were
prepared as described above from both transgenic and nontransgenic
control hearts. Membranes (20-30 µg of protein) were incubated for
15 min at 37 °C with [-32P]ATP under basal
conditions or in the presence of one of the following: 100 µM isoproterenol, 100 µM norepinephrine,
100 µM phenylephrine, or 10 mM NaF. Cyclic
AMP was quantitated by standard methods described previously (17).
Cytosolic
extracts were prepared as described previously (11). These were
concentrated using a Centricon microconcentrator (Amicon, Inc.).
Concentrated cytosolic extracts (300 µg of protein) were incubated
with rhodopsin-enriched rod outer segments in 75 µl of lysis buffer
(25 mM Tris-HCl, pH 7.4, 5 mM EDTA, 5 mM EGTA, 10 µg/ml leupeptin, 20 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride) with 10 mM
MgCl2 and 0.1 mM ATP containing
[-32P]ATP. The reactions were incubated in white light
for 15 min and quenched with 300 µl of ice-cold lysis buffer and then
centrifuged for 15 min at 13,000 × g. Sedimented
proteins were resuspended in 25 µl of protein gel loading dye and
electrophoresed through SDS-12% polyacrylamide gels. Phosphorylated
rhodopsin was visualized by autoradiography of dried polyacrylamide
gels and quantified using the PhosphorImager.
Mice were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (2.5 mg/kg) given intraperitoneally. After endotracheal intubation, mice were connected to a rodent ventilator. Following bilateral vagotomy, the chest was opened, and a 1.8-French high-fidelity micromanometer catheter (Millar Instruments, Inc., Houston, TX) was inserted into the left atrium, advanced through the mitral valve, and secured in the left ventricle (LV). Hemodynamic measurements were recorded at base line and 45-60 s after injection of incremental doses of isoproterenol. Doses of isoproterenol were specifically chosen to maximize the contractile response but to limit the increase in heart rate. Continuous high-fidelity LV pressure and fluid-filled aortic pressure were recorded simultaneously on an eight-channel chart recorder and in digitized form at 2000 Hz for later analysis. Experiments were then terminated with an overdose of pentobarbital. Hearts were rapidly excised, and individual chambers were separated, weighed, and then frozen in liquid N2 for later analysis. Parameters measured were heart rate, LV systolic and end diastolic pressure, and the maximal and minimal first derivative of LV pressure (LV dP/dtmax and LV dP/dtmin). Ten sequential beats were averaged for each measurement.
Statistical AnalysisData are expressed as mean values ± S.E. Student's t test was used to analyze all biochemical data. Two-way repeated measure analysis of variance was used to evaluate the in vivo hemodynamic measurements under basal conditions and with isoproterenol stimulation. When appropriate, post hoc analysis was performed with a Newman-Keuls test. For all analyses, p < 0.05 was considered significant.
Two transgenic lines were established expressing the WT
1B-AR and were named TG
43 and TG
47.
Cardiac-specific transgene expression was documented by Northern
analysis of RNAs from different tissues, including heart, lung,
diaphragm, quadriceps muscle, kidney, and liver (data not shown). This
cardiac-specific expression is consistent with the previously
documented pattern of transgene expression achieved with the murine
-MyHC promoter (9-13). At 10 weeks of age, transgene expression was
quantitated by radioligand binding assays performed on purified
myocardial membranes using 125I-HEAT, and the results are
shown in Fig. 1. Total cardiac
1-AR density in TG
43 animals was 43-fold greater
than in nontransgenic littermate controls (NLC) and 26-fold greater in
the TG
47 line compared with NLC. Unless otherwise stated, all
subsequent studies were performed on TG
43 animals.
Classical 1-AR/Gq coupling leads to
stimulation of phospholipase C, generating the second messengers
inositol trisphosphate and DAG, which subsequently leads to the
activation of protein kinase C (18, 19). To assess the functional
coupling of overexpressed WT
1B-ARs, myocardial DAG
content was quantitated, and as shown in Fig.
2, base-line DAG content in TG
43
hearts was significantly higher than in control hearts. This indicates
that
1B-AR/Gq signaling is enhanced under
basal conditions.
Since signaling through 1-ARs has been shown to evoke a
hypertrophic response in cultured myocytes including activation of fetal gene expression (2, 20), we investigated the levels of ANF
mRNA present in the ventricles of these transgenic mice. ANF is a
gene normally inactive in the ventricles after maturation and has been
shown to be associated with cardiac hypertrophy (21). To examine ANF
gene activation present in TG
43 mice, Northern blots of ventricular
RNA were generated and probed with a mouse ANF cDNA (Fig.
3A). Control ventricles showed
minimal or undetectable ANF signals, which is consistent with the
inactivation of this gene in normal adult ventricular myocytes (21). In
contrast, there was a strong ANF signal in RNA isolated from TG
43
ventricles, which, when normalized to the control GAPDH mRNA, was
~9-fold higher compared with controls (Fig. 3B). The
increase in ventricular ANF mRNA in TG
43 mice was twice that
seen in our previously described CAM
1B-AR-overexpressing mice (9). Surprisingly, despite the
extraordinarily high ANF mRNA levels, TG
43 animals did not have
significantly different LV/body weight ratios or increased LV myocyte
cross-sectional areas compared with controls (data not shown).
Nonsignificant changes in heart mass were also found in TG
47
animals (data not shown). This is unlike the phenotype in the CAM
1B-AR transgenic animals, which had increased heart mass
and increased cross-sectional areas of ventricular myocytes that
accompanied the increased ANF signal (9). The lack of a hypertrophic
phenotype in these animals is not clearly understood since signaling
through
1-ARs is clearly elevated, and ventricular ANF
expression is high (Figs. 2 and 3). This suggests that signaling through CAM
1-ARs is somehow different from that through
WT receptors.
In addition to investigating a possible hypertrophic phenotype in TG
43 mice, the primary aim of this study was to determine the in
vivo physiological effects of enhanced signaling through myocardial
1-ARs. In vivo measurements of
1-AR-mediated cardiac hemodynamics using a
pharmacological approach is difficult since
-agonists are potent
vasoconstrictors that can change the loading conditions and secondarily
affect cardiac function. In TG
43 mice, we chose to initially study
basal cardiac physiological parameters to determine whether the
enhanced
1-AR/Gq signaling indicated by
increased DAG content can affect basal function. In addition, we
examined cardiac responses to the
-agonist isoproterenol to
determine if enhanced
1-AR signaling affects this
response, which is the primary mechanism for increasing performance of
normal hearts. Following catheterization of anesthetized mice (10-13), we measured several hemodynamic parameters, including heart rate and LV
dP/dtmax and LV dP/dtmin,
measures of cardiac contractility and relaxation, respectively. The
results found in TG
43 animals and NLC mice under basal conditions
and in response to isoproterenol are shown in Fig.
4. There was no difference in LV systolic
pressure between the two groups (Fig. 4A). Base-line and
-agonist-stimulated heart rates were significantly depressed in TG
43 mice compared with NLC mice (Fig. 4B). There was no
statistically significant difference in basal
dP/dtmax or dP/dtmin in
TG
43 mice versus NLC mice (Fig. 4, C and
D), although the trend was for lower values in TG
43
animals. There was, however, a significant decrease in these parameters
in TG
43 animals compared with NLC mice in response to progressive
isoproterenol infusion. Thus,
-AR-mediated LV function is depressed
in these animals, suggesting that there is significant cross-talk
between the signaling of
1-ARs and
-ARs in the hearts
of these transgenic animals. The increased constitutive
1-AR signaling present in TG
43 mice has a
significant effect on cardiac physiological responses elicited by
-AR stimulation. Although previous studies have demonstrated that
1-AR stimulation can lead to negative chronotropy (5),
1-AR-mediated negative inotropy is a novel finding. To
further study
1-AR-mediated effects on cardiac
contractility, it would be relevant to study these in vivo
parameters in the presence of an
-agonist such as phenylephrine. However, the use of phenylephrine presents major fundamental problems due to its predominant peripheral effects on systolic pressure, which
would influence myocardial function independent of any myocardial
1-AR signaling. The data presented above could have
important clinical significance since, in pathophysiological conditions such as heart failure, there is significant loss of both
-AR density
and functional coupling, which could potentially increase the role of
1-AR signaling in response to endogenous
catecholamines.
To investigate possible molecular mechanisms involved in the altered
cardiac physiology seen in TG 43 animals, we carried out in
vitro biochemical assays on heart extracts to examine the
-AR
system. Signaling through
-ARs involves the activation of the G
protein Gs, which activates adenylyl cyclase, leading to increases in intracellular cAMP and activation of
cAMP-dependent protein kinase A (22). Activated protein
kinase A phosphorylates several myocardial proteins, leading to
positive inotropy as well as chronotropy. The
-AR system as well as
several other G protein-coupled receptors undergo rapid
desensitization, which is the loss of response in the continued
presence of agonist. This is initiated by targeted receptor
phosphorylation via a family of serine/threonine kinases known as the
GRKs, of which the
-AR kinase (
ARK1) is a prototypic member (23).
We first examined
-AR density in myocardial membranes purified from
TG
43 hearts and control nontransgenic myocardial membranes using
125I-CYP and found no significant difference in total
-AR density (46.5 ± 0.8 fmol/mg of membrane protein in TG
43 mice versus 44.3 ± 1.6 in controls). Therefore,
changes in myocardial
-AR density cannot account for the differences
in cardiac contractility seen in TG
43 mice.
We then studied myocardial membrane adenylyl cyclase activity. There
was significantly lower basal adenylyl cyclase activity in membranes
purified from TG 43 hearts compared with nontransgenic myocardial
membranes (Table I). This could account
for the decreased basal heart rate seen in these animals. As shown in
Table I, agonist-stimulated adenylyl cyclase activity in TG
43
membranes compared with control membranes was also significantly
depressed following addition of isoproterenol or norepinephrine.
Interestingly, the depressed response to norepinephrine, a mixed
/
-agonist, was greater than with the strict
-agonist
isoproterenol. These results indicate that the depressed in
vivo cardiac function shown above (Fig. 4) is likely due, at least
in part, to the lower adenylyl cyclase activity and an attenuated
-AR-mediated cAMP response. To examine whether decreased adenylyl
cyclase activity in TG
43 membranes was secondary to enhanced
1-AR signaling, we studied adenylyl cyclase activity
following addition of an
-agonist. Surprisingly, the addition of 100 µM phenylephrine resulted in significant lowering of
basal activity in TG
43 membranes, whereas control membranes had no
1AR-mediated cAMP response (Table I). This strongly
suggests that further
1-AR stimulation leads to inhibition of membrane adenylyl cyclase activity. This also suggests that the significant decrease in basal adenylyl cyclase activity is due
to enhanced basal
1-AR signaling present in TG
43
hearts. Strengthening these conclusions of a receptor-mediated
phenomenon, NaF-stimulated adenylyl cyclase activities were similar in
membranes from TG
43 animals and nontransgenic controls (Table
I).
|
One hypothesis for the decrease in adenylyl cyclase activity in TG
43 myocardial membranes is coupling of WT
1B-ARs to
the adenylyl cyclase inhibitory G protein, Gi. As mentioned
above, dual coupling of
1-ARs to Gq and
Gi has been demonstrated in vitro, but not yet
investigated in vivo. To examine the potential involvement
of Gi in the
1-AR-mediated cyclase
responses, we intraperitoneally injected TG
43 and NLC mice with
either 100 µg/kg PTx or 150 µl of saline and sacrificed the animals
24 h later. Myocardial membranes were purified from these animals, and adenylyl cyclase activities were measured. As shown in Table II, PTx treatment reversed the depressed
basal cyclase activity in TG
43 membranes, and in fact, the percent
increase in TG
43 basal activity was significantly higher compared
with PTx-treated controls (Table II), indicating the enhanced
Gi coupling of overexpressed WT
1B-ARs.
Isoproterenol- and norepinephrine-stimulated adenylyl cyclase
activities were also significantly increased in TG
43 membranes
following PTx treatment. Additionally and in contrast to findings in
saline-treated transgenic animals, phenylephrine did not decrease
membrane adenylyl cyclase activity (Table II). PTx treatment also
enhanced NaF-stimulated activities compared with saline treatment, as
expected, but TG
43 membranes did not differ from controls,
indicating that there is no change in the levels or function of
myocardial G proteins. To confirm this, we carried out protein
immunoblotting of membranes for G
s and G
i
and found no difference in protein levels in TG
43 versus
control hearts (data not shown). Overall, PTx treatment converted TG
43 membranes to having the biochemical characteristics of control
membranes. Thus, these results indicate that the involvement of
PTx-sensitive G proteins is significantly higher in hearts overexpressing the WT
1B-AR, which could lead to the
dampened myocardial performance seen in TG
43 animals (Fig. 4).
|
Potentially, there could be additional contributors to the decreased
-AR-mediated myocardial signaling and function seen in TG
43
animals. Since
-AR density is unaltered in TG
43 hearts, desensitization and functional uncoupling may be enhanced. In fact, the
attenuated adenylyl cyclase activities and in vivo
-AR cardiac responses are similar to the phenotype we have previously described for transgenic mice with cardiac overexpression of either the
ARK1 (11) or GRK5 (12). These two members of the GRK family are
expressed in the heart and can produce desensitization and functional
uncoupling of myocardial
-ARs (11, 12). Interestingly, it has
recently been reported that
ARK1 can be regulated by protein kinase
C (24, 25). This regulation involves the enhancement of
ARK activity
following the phosphorylation of
ARK1 by protein kinase C (24, 25).
This is of significance in the present study since protein kinase C
activity is apparently increased in the hearts of TG
43 animals as
indicated by the measured increase in myocardial DAG content (Fig. 2).
To investigate any in vivo regulation of
ARK in the
hearts of these transgenic mice, we carried out in vitro
phosphorylation assays using myocardial extracts and the G
protein-coupled receptor substrate rhodopsin (11, 12). Fig.
5 contains our findings using soluble
myocardial fractions that represent GRK activity primarily attributable
to
ARK1. As shown, GRK activity was increased 3-fold in soluble
extracts from the hearts of TG
43 animals compared with NLC heart
extracts. Thus, this enhanced GRK activity seen in TG
43 hearts
could contribute to the attenuated
-AR signaling observed in these
animals, and interestingly, the increase in
ARK activity is in the
same range as that in transgenic mice overexpressing
ARK1 (11) and
following the development of pressure overload cardiac hypertrophy
(26). The increased soluble GRK activity appears not to involve
ARK1 up-regulation as protein immunoblots revealed no changes in the levels
of
ARK1 in the hearts of TG
43 animals compared with NLC (data
not shown). Thus, these data are the first to suggest possible
enhancement of
ARK activity as an in vivo consequence of
increases in the
1-AR/Gq/protein kinase C
cascade. Thus, previous in vitro findings of protein kinase
C regulation of
ARK1 (24, 25) may have important in vivo
implications. In additional studies, we examined the protein content of
GRK5, which is found exclusively in the membrane fraction of extracts
(27), and found that the levels of this GRK are unaltered in TG
43
hearts (data not shown).
In summary, we have demonstrated that -MyHC-directed cardiac
overexpression of the WT
1B-AR can have profound effects
on adrenergic signaling and in vivo cardiac physiology. This
is evident even under basal conditions, suggesting that WT
1-ARs can signal spontaneously at this level of
overexpression (>40-fold), much like the findings in mice
overexpressing the WT
2-AR (10, 14). In TG
43
animals, Gq signaling was enhanced as assessed by
myocardial DAG content and ventricular ANF mRNA expression, but in
contrast to our previously described transgenic mice overexpressing a
CAM
1B-AR, myocardial hypertrophy was not associated
with this enhanced Gq signaling. In vivo
hemodynamic evaluation of TG
43 mice revealed significantly dampened
left ventricular function in response to
-agonist stimulation
compared with nontransgenic controls. Probing possible mechanisms for
altered myocardial function revealed that dual coupling of the
1B-ARs exists as PTx-sensitive G protein-mediated attenuation of adenylyl cyclase was seen in myocardial membranes purified from TG
43 animals. The surprising lack of hypertrophy in
TG
43 and TG
47 animals suggests that the agonist-independent signaling of the CAM
1B-AR differs from the enhanced
signaling through WT
1B-ARs. One possible explanation
for this phenomenon is the significant dual G protein coupling seen in
animals overexpressing the WT
1B-AR, which greatly
alters
-AR signaling in these mice. This
1-AR
regulation of
-AR signal transduction is not evident in the CAM
1B-AR transgenic
mice,2 which suggests a true
difference in the biochemical phenotype of these mice. Interestingly,
we have recently observed significant coupling of overexpressed WT
2-ARs to Gi in the hearts of transgenic animals.3 We also observed
enhanced GRK activity, presumably
ARK1, in the hearts of TG
43
mice, which also could contribute to the observed phenotype. Enhanced
ARK activity is probably due to elevated protein kinase C activity
as a result of enhanced
1-AR/Gq signaling,
in as much as protein kinase C activation of
ARK1 has been observed
in vitro (24, 25). In support of the notion that this is a
Gq-mediated phenomenon, CAM
1B-AR animals
have a similar increase in soluble GRK activity.2
These results demonstrate that myocardial 1-AR signaling
can significantly alter the signaling through myocardial
-ARs via two distinct receptor-mediated mechanisms. First,
1-AR
coupling to PTx-sensitive G proteins can occur in vivo, and
second, enhanced
1-AR signaling can lead to the enhanced
activity of
ARK1, which can cause functional uncoupling of
-ARs.
Such cross-talk between different adrenergic signaling pathways could
have important implications in organs like the heart that contain
-
and
-ARs and might become critical in pathological conditions where
additional signaling alterations take place. For example,
ARK1
expression and activity have been shown to be increased in end-stage
human congestive heart failure (28). Thus, the TG
43 animals
represent a unique model to study specific in vivo
interactions between
1- and
-AR signaling as well as
other pathways that can be regulated by GRK activity.