From the
Stimulation of
Cultured neonatal ventricular cardiomyocytes are a useful model
for studying changes in gene expression associated with myocardial cell
hypertrophy. The hypertrophic response, characterized by an increase in
cell size, is accompanied by up-regulation of the myosin light chain-2
(MLC-2)
In order to determine how
G-protein-coupled receptors regulate cardiac gene expression, we have
heterologously expressed wild-type and chimeric receptors that have
altered G-protein/effector coupling properties. The M
The studies reported here demonstrate that heterologously
expressed mAChRs of the M
We first determined whether endogenous M
The specificity
of M
Heterologous expression of wild-type and chimeric
G-protein-coupled receptors provides a new approach for studying
signaling pathways that mediate hypertrophic changes in gene expression
in neonatal ventricular myocytes. This approach can be used because the
functional response of interest, induction of the cardiac-specific ANF
gene, can be monitored in transiently transfected cells using a
sensitive ANF promoter/luciferase reporter gene and by
immunofluorescent detection of ANF protein. The M
In order to
demonstrate that transcriptional activation of the ANF gene by
carbachol was occurring through the heterologously expressed
M
Muscarinic
cholinergic receptors have been extensively characterized in terms of
their specificity for coupling to PLC and adenylate cyclase. Receptors
of the M
The loss of function
seen with the chimeric M
Remarkably, a 21-amino acid fragment was sufficient to allow the
M
The role of Ras in
Neonatal ventricular myocytes were co-transfected with an
RSV/luciferase expression plasmid along with either the pCMV backbone
or M
We thank Elliott M. Ross, Stephen K.-F. Wong, and Kirk
U. Knowlton for supplying plasmids and advice concerning experimental
protocols and David Goldstein for technical assistance.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-adrenergic receptors in
neonatal ventricular cardiomyocytes induces hypertrophic changes
including activation of the atrial natriuretic factor (ANF) gene. This
receptor couples to G
to activate phospholipase C (PLC) and
protein kinase C, which have been implicated as mediators of the
hypertrophic response. To directly determine whether receptor coupling
to G
/PLC is sufficient to induce ANF expression, we
expressed wild-type and chimeric muscarinic cholinergic receptors
(mAChRs) with altered G-protein coupling properties in cardiac myocytes
and examined their ability to activate an ANF promoter/luciferase
reporter gene. The cholinergic agonist carbachol failed to induce
transcriptional activation of the ANF reporter gene through endogenous
G
-linked M
mAChRs or in cells transfected with
M
mAChRs. In contrast, in cells transfected with
M
mAChRs, which effectively couple to G
/PLC,
carbachol increased ANF reporter gene expression 10-fold and also
increased ANF protein, as determined by immunofluorescence.
Carbachol-mediated ANF gene expression was inhibited by the mAChR
antagonist pirenzepine with a K
value
characteristic of an M
mAChR. Studies using chimeric
M
- and M
mAChRs demonstrated that the N-terminal
21 amino acids of the third intracellular loop of the
M
mAChR were required for receptor coupling to ANF gene
expression. This region, previously shown to specify receptor coupling
to G
/PLC, also conferred partial activity to a chimeric
M
receptor. We further demonstrated that M
mAChR
coupling to ANF gene expression was Ras-dependent since co-expression
of dominant-interfering Ala-15 Ras inhibited M
mAChR-induced
ANF expression by 60%. In contrast, ANF expression induced by the
chimeric M
receptor was not blocked by dominant-interfering
Ras. We suggest that receptor coupling to G
/PLC is
sufficient to induce ANF expression and that a Ras-dependent pathway
contributes additional signals required for maximal
M
mAChR-mediated ANF gene expression.
(
)
gene and reactivation of the atrial
natriuretic factor (ANF) and other genes
(1, 2, 3, 4, 5) . Signals that
lead to cardiac cell growth and gene expression can be generated
through activation of G-protein-coupled
-adrenergic
receptors (
AdrRs)
(6, 7) . In cardiac
cells, the
AdrR stimulates phospholipase C (PLC)
through the guanine nucleotide (GTP)-binding protein G
(8, 9) and activates protein kinase C
(10, 11) . Both G
and protein kinase C have
been implicated as mediators of the genetic response induced by
AdrR activation
(9, 12, 13, 14, 15) . In
addition Ras, a small GTP-binding protein involved in growth factor
signaling pathways, has been shown to be required for
AdrR-induced increases in cardiac cell size and gene
expression
(16) . It is not known whether an agonist needs to
activate G
as well as Ras to regulate myocardial cell
growth and gene expression.
muscarinic cholinergic receptor (M
mAChR) is a
G-protein-coupled receptor that, like the
AdrR,
activates PLC through its interaction with G
(17) .
M
mAChRs are not present in cardiomyocytes
(18, 19) , which predominantly express
M
mAChRs that regulate adenylyl cyclase through interaction
with G
(20, 21) . The M
mAChR and
M
mAChR are highly conserved in their transmembrane spanning
regions, but vary significantly in their third intracellular (i3) loop,
a region shown to confer G-protein/effector coupling specificity
(22, 23, 24, 25) . The determinants for
G-protein recognition and activation appear to be shared among other
receptors that utilize similar signaling pathways. For example,
peptides representing the i3 loop of the M
mAChR have
recently been shown to compete with the i3 loop of the
AdrR for activation of PLC
(26) , indicating
that these receptors interact with G-proteins in functionally similar
ways.
subtype, like endogenous
AdrRs, transduce signals leading to cardiac-specific
gene expression. In contrast, expression of M
mAChRs does
not transduce muscarinic receptor agonist binding into ANF gene
expression. Using chimeric receptors, we demonstrate that a 21-amino
acid region of the i3 loop critical for coupling M
mAChR to
G
/PLC is necessary for receptor-mediated ANF expression and
can induce a partial response when exchanged for the homologous region
in the M
mAChR. Expression of dominant-interfering Ala-15
Ras inhibits M
mAChR-induced ANF gene expression but does
not block the response mediated by the chimeric M
receptor,
suggesting that the 21-amino acid region conferring G-protein
selectivity is not sufficient to couple the mAChR to a Ras-dependent
pathway.
Cell Culture Procedure
Neonatal rat ventricular
cardiomyocytes from 1-3-day-old Sprague-Dawley rats were isolated
and cultured as described previously
(15, 27) . Cells
were plated at a density of 3.5-4.0
10
/cm
on 60-mm gelatin-coated tissue culture
dishes or on 25-mm etched coverslips (Bellco) and maintained overnight
in 4:1 Dulbecco's modified Eagle's medium/medium 199 (Life
Technologies, Inc.) containing 10% horse serum, 5% fetal calf serum,
and antibiotics (100 units/ml penicillin and 100 µg/ml
streptomycin).
Plasmid Constructs
The following promoters fused
to firefly luciferase cDNA were used as reporter gene constructs: a
638-base pair fragment of the rat ANF promoter
(2) , a 2700-base
pair fragment of the MLC-2 promoter
(28) , and the Rous sarcoma
virus (RSV) long term repeat
(15) . A series of muscarinic
receptors cloned into the cytomegalovirus promoter-driven (pCMV)
expression vector
(29, 30) were provided by Drs.
Elliott Ross and Stephen Wong. These are: MmAChR (cDNA
encoding the human M
mAChR); M
mAChR (cDNA
encoding the human M
mAChR); M
:NM
(cDNA of human M
mAChR containing amino acids
208-230 from the human M
mAChR); and
M
:NM
(cDNA of human M
mAChR
containing amino acids 210-230 from the human
M
mAChR). The dominant-interfering Ala-15 Ras expressed in
the pZip backbone was provided by Dr. Kirk Knowlton
(9) .
Transient Transfection Assay
Myocytes were
transfected in serum-containing medium using a modified calcium
phosphate transfection technique as described previously
(15) .
A total of 8-10 µg of DNA was used, which consisted of the
appropriate luciferase reporter gene (3 µg) and either the pCMV
backbone or pCMV vector containing various receptor cDNAs (0.1-6
µg). Following transfection, cells were washed extensively,
cultured in the presence of carbachol or phenylephrine for 48 h, and
harvested in a 0.5% Triton X-100 buffer. Luciferase activity and
protein concentration were determined for each sample as described
(15) .
Measurement of Phosphoinositide
Hydrolysis
Myocytes were labeled overnight with 3 µCi/ml
[H]inositol in serum-free medium and
[
H]inositol phosphate formation assayed in the
presence of 10 m
M LiCl as described
(31) .
Immunofluorescence Analysis
Indirect
immunofluorescence analysis was performed using a modification of a
previously described procedure
(7) . Briefly, cells plated on
etched coverslips were transfected with the pCMV backbone or
MmAChR expression plasmid along with an RSV/luciferase
reporter gene. After a 48-h treatment with carbachol or phenylephrine,
cells were fixed with 3% paraformaldehyde and permeabilized with 0.3%
Triton X-100. Transfected cells were identified by immunostaining with
a polyclonal anti-luciferase antibody (Cortex Biochem) followed by a
fluorescein-conjugated, goat anti-rabbit antibody. ANF expression was
detected by using a mouse monoclonal antibody against ANF (provided by
Dr. C. Glembotski, San Diego State University) and a
rhodamine-conjugated, goat anti-mouse antibody. Luciferase and ANF
protein expression were analyzed by fluorescence microscopy using a
Plan-neofluar 40
objective (Zeiss).
Radioligand Binding Experiments
Neonatal rat
cardiomyocytes were transfected with the pCMV backbone or
MmAChR cDNA as described above, and membrane fractions were
prepared. To determine mAChR density, membranes were incubated in a
Hepes-buffered salt solution
(32) with 1 n
M (-)-[ N- methyl-
H]QNB
(a concentration 10 times its K
) at 30
°C for 60 min. Nonspecific binding, determined in the presence of 1
µ
M atropine, was <10% of total binding. For competition
experiments, the assay contained 0.6 n
M (-)-[ N- methyl-
H]QNB
and varying amounts of atropine (0.01 n
M to 100
µ
M) or pirenzepine (0.1 n
M to 30
µ
M). The results of competition experiments were
com-puter-analyzed by using EBDA/LIGAND to obtain
K
values for the antagonists
(32) .
mAChRs in
neonatal cardiomyocytes regulate ANF gene expression by transiently
transfecting cells with an ANF promoter/luciferase reporter gene and
stimulating cells with the stable acetylcholine analog carbachol for 48
h. Carbachol failed to increase luciferase expression while the
-adrenergic receptor agonist phenylephrine caused 10-fold
activation of the ANF promoter (Fig. 1 A). Subsequent
experiments examining PLC activation demonstrated that carbachol caused
only modest stimulation of [
H]inositol phosphate
formation when compared to the robust response to phenylephrine (Fig.
1 B). This is consistent with evidence that mAChRs of the
M
subtype couple poorly to PLC
(23, 33, 34, 35) . Thus, endogenous
cardiac mAChRs show low efficacy for activation of PLC and do not
couple to effectors regulating ANF expression.
Figure 1:
Differential
effects of carbachol and phenylephrine treatment on ANF gene expression
and phosphoinositide formation. A, neonatal rat ventricular
myocytes were transfected with an ANF promoter/luciferase reporter gene
and then incubated for 48 h with either no drug, 300 µ
M carbachol, or 100 µ
M phenylephrine (with 2 µ
M propranolol to block -adrenergic receptors). Luciferase
activity was normalized to micrograms of protein for each sample and
agonist-induced increases expressed as -fold stimulation relative to no
drug treatment. B, inositol monophosphate accumulation was
assayed in [
H]inositol-labeled myocytes which
were treated with agonists plus 10 m
M LiCl for 20 min at the
concentrations tested above. Data are the mean ± S.E. of
quadruplicate samples from a representative
experiment.
To determine whether
ANF gene expression could be induced if G/PLC-linked mAChRs
or a greater number of M
mAChRs were present, we transfected
myocytes with the ANF reporter gene along with either the pCMV backbone
vector or with human M
or M
mAChR cDNA and
subsequently treated cells with carbachol for 48 h (Fig. 2). In
cells transfected with the vector alone or M
mAChR cDNA,
carbachol failed to induce ANF gene expression. However, in cells
expressing the M
mAChR, carbachol treatment resulted in a
10-fold increase in luciferase activity. To examine the concentration
dependence of this response, cardiomyocytes were transfected with
various concentrations of M
mAChR cDNA. As shown in Fig. 3,
concentrations of the M
mAChR cDNA as low as 0.01 µg
conferred maximal responsiveness to carbachol. Furthermore, the
carbachol dose-response curve was shifted to the right as the amount of
receptor cDNA was decreased, suggesting a relationship between the
response and the number of expressed receptors (Fig. 3).
Figure 2:
Transcriptional activation of ANF gene
expression in myocytes transfected with wild-type muscarinic
cholinergic receptors. Neonatal rat ventricular myocytes were
co-transfected with the backbone pCMV expression vector or the pCMV
vector containing Mor M
receptor cDNA, along
with an ANF promoter/luciferase reporter gene. Cells were then
incubated for 48 h in the absence ( hatched bars) or presence
( solid bars) of 300 µ
M carbachol. Luciferase
activity was normalized to micrograms of protein. The data are
expressed as -fold stimulation by carbachol relative to untreated (no
drug) samples and represents the mean ± S.E. of triplicate or
quadruplicate samples from 2-8
experiments.
Figure 3:
Dose dependence of ANF gene expression on
receptor and carbachol concentrations. Cardiomyocytes were
co-transfected with either 0.01, 0.1, or 3.0 µg of
MmAChR cDNA along with the ANF promoter/luciferase reporter
gene. Cells were then incubated for 48 h with varying concentrations of
carbachol. Luciferase activity was measured and normalized to
micrograms of protein. The data are expressed as percent of maximal ANF
luciferase measured at the highest carbachol concentration and
represent the mean ± S.E. of duplicate or triplicate samples
from 1-3 experiments.
Because a relatively small fraction of the cardiomyocytes express
exogenous DNA (<5% based on -galactosidase staining), we could
not detect an increase in mAChR expression using radioligand binding
techniques. Thus, in binding experiments using a saturating
concentration of [
H]QNB, pCMV-transfected cells
had specific [
H]QNB binding of 189 ± 9
fmol/mg of protein, and M
mAChR-transfected cells had
essentially the same amount of specific binding (201 ± 8 fmol/mg
of protein; data not shown). We were, however, able to demonstrate that
heterologously expressed M
mAChRs regulate ANF expression by
using pharmacological characterization. Cells transfected with the
M
mAChR and ANF reporter gene were incubated with various
concentrations of the mAChR antagonists atropine and pirenzepine prior
to the addition of 10 µ
M carbachol. The IC
values for inhibition of carbachol-stimulated ANF expression were
determined from the curves shown in Fig. 4and corrected to give
estimated K
values for the antagonists.
K
values were calculated from radioligand
binding competition experiments (Fig. 4). The binding constants
determined for atropine in experiments examining ANF expression and
radioligand binding were similar ( K
= 0.8 n
M; K
= 1.2 n
M) while those for pirenzepine were
markedly different ( K
= 11
n
M; K
= 600 n
M).
The mAChR mediating ANF gene expression therefore has about a 50-fold
higher affinity for pirenzepine than does the endogenous receptor
assessed by radioligand binding. The estimated
K
value for pirenzepine antagonism of ANF
expression (11 n
M) is characteristic of an M
mAChR
(36, 37, 38) .
Figure 4:
Inhibition of carbachol-stimulated ANF
gene expression and of [H]QNB binding by atropine
and pirenzepine. Ventricular myocytes transfected with
M
mAChR cDNA and the ANF promoter/luciferase reporter gene
were treated with increasing concentrations of either atropine
( open squares) or pirenzepine ( open circles) 30 min
prior to the addition of 10 µ
M carbachol. After a 48-h
drug treatment, luciferase activity was assayed. Data are expressed
relative to maximal carbachol-stimulated ANF luciferase measured in the
absence of antagonist. Each point represents the mean ± S.E. of
triplicate samples from two experiments. The IC
values for
atropine and pirenzepine (13 n
M and 200 n
M,
respectively) were corrected by the Cheng-Prusoff equation (using an
EC
for carbachol of 0.63 µ
M) to give the
estimated K. For radioligand binding experiments, myocyte
membranes were incubated with 0.6 n
M [
H]QNB and varying amounts of atropine
( solid squares) or pirenzepine ( solid circles). The
results shown are for the specific binding and are the mean ±
S.E. of triplicate samples from two experiments. The results were
analyzed by EBDA/LIGAND to give the calculated K values.
In ventricular cardiomyocytes,
AdrR activation increases ANF protein
(9, 16) . To determine whether carbachol induces ANF
protein expression in M
mAChR-transfected myocytes, cells
were transfected with M
mAChR cDNA or the pCMV backbone
along with an RSV/luciferase expression vector and analyzed by
immunofluorescence. Luciferase expression was used to identify
transfected cells and ANF protein expression was examined for each
experimental condition. About 60% of the cells transfected with the
pCMV backbone vector showed increased ANF immunoreactivity when treated
with phenylephrine, whether or not the cells expressed luciferase
(). In contrast, stimulation with carbachol increased ANF
expression only in myocytes transfected with M
mAChR cDNA
and exclusively in cells expressing luciferase. These data confirm
results obtained from transient transfection experiments by
demonstrating that ANF protein expression is induced by carbachol only
in cells transfected with M
mAChR cDNA.
mAChR-mediated gene expression was further investigated
by examining the transactivation of the cardiac-specific MLC-2 promoter
and the nonspecific viral (RSV) promoter/luciferase reporter genes. In
cells transfected with M
mAChR cDNA, carbachol increased
MLC-2 gene expression 12-fold (Fig. 5). In contrast, carbachol
did not activate the RSV promoter in these cells. These results
indicate that activation of expressed M
mAChRs does not have
a nonspecific effect on gene expression but is selectively coupled to
activation of at least two cardiac genes known to be induced during
myocardial cell hypertrophy.
Figure 5:
Effect of M muscarinic
cholinergic receptor activation on cardiac-specific and nonspecific
gene expression. Neonatal rat ventricular myocytes were transfected
with expression constructs containing luciferase reporter genes driven
by either the ANF, MLC-2, or RSV promoter along with the wild-type
M
mAChR. Cells were incubated without ( hatched
bars) or with ( solid bars) 300 µ
M carbachol
for 48 h, and luciferase activity was determined and normalized to
micrograms of protein. The data are expressed as -fold stimulation by
carbachol relative to untreated (no drug) samples and represents the
mean ± S.E. of triplicate or quadruplicate samples from
1-3 experiments.
To examine regions of the receptor
responsible for transducing signals leading to cardiac gene expression,
we tested chimeric receptors in which regions of the i3 loop were
altered. Initially, we tested a chimeric
M:
I
I
receptor in which the
entire i3 loop and part of the i2 loop of the M
receptor
were replaced with those of the
-adrenergic receptor
(29, 30) . This chimeric receptor failed to transduce
carbachol binding into increased ANF gene expression at any cDNA
concentration tested
(39) . Similarly, a chimeric
M
:NM
receptor which differs from the native
M
mAChR by replacement of the N-terminal 21 amino acids of
the i3 loop with the homologous region of the M
mAChR was
unable to induce ANF expression even at a concentration 60 times that
of the wild-type M
mAChR (compare 0.1 µg of M
versus 6 µg of M
:NM
in
Fig. 6
). Both of these chimeric M
receptors are
expressed at levels similar to those of the wild-type receptor in COS
or A293 cells
(29) .
(
)
We also tested a
chimeric M
:NM
receptor in which the N-terminal
23 amino acids of the M
mAChR i3 loop are replaced with
those of the M
mAChR i3 loop (Fig. 6). Activation of
this chimeric receptor with carbachol significantly increased ANF gene
expression, although the maximal response was 50% of that seen with the
M
mAChR even at the highest concentrations of receptor cDNA
(6 µg) or carbachol (300 µ
M) tested. These data
demonstrate that as little as 21 amino acids from the
M
mAChR confers receptor specificity for coupling to ANF
gene expression.
Figure 6:
Effects of wild-type and chimeric
muscarinic cholinergic receptors on ANF gene expression. Ventricular
myocytes were transfected with cDNA for MmAChR (0.1 or 3
µg), M
mAChR (3 or 6 µg), M
:NM
chimera (3 or 6 µg), or M
:NM
chimera
(0.1, 3, or 6 µg) together with the ANF luciferase reporter gene.
Cells were stimulated with 10 µ
M carbachol for 48 h, and
luciferase activity was measured and normalized to micrograms of
protein. The data are expressed as -fold stimulation relative to no
drug treatment and represent the mean ± S.E. of triplicate
samples from 2-3 experiments. The same results were obtained when
cells were stimulated with 300 µ
M carbachol.
Ras has been shown to be involved in
AdrR-mediated ANF gene expression
(9, 16) . To determine whether Ras was similarly
involved in mAChR-mediated ANF gene expression, we transiently
co-expressed either the M
or chimeric
M
:NM
receptor cDNA along with a
dominant-interfering Ala-15 Ras cDNA to block Ras function.
M
mAChR-mediated ANF gene expression was significantly
inhibited (60%) by the dominant-interfering Ras (Fig. 7). Thus, Ras
appears to be a mediator of M
mAChR-induced ANF expression,
as previously shown for the
AdrR. In contrast, ANF
expression induced by the chimeric M
:NM
receptor was not inhibited by the dominant-interfering Ras.
Therefore, the 21 amino acids of the M
mAChR i3 loop appears
to specify coupling to G
/PLC but not to a Ras-dependent
pathway.
mAChR is a
structural and functional homolog of the endogenous
AdrR which, we reasoned, would mimic
AdrR-mediated hypertrophic responses and which could
be experimentally modified to evaluate the functional determinants
required for coupling receptors to gene expression.
mAChRs, we first showed that carbachol-stimulated ANF gene
expression was blocked by the muscarinic receptor antagonist
pirenzepine with a K
similar to that
determined for M
mAChRs in other systems
(36, 37, 38) . We also demonstrated that the
dose-response curve for carbachol shifts to higher concentrations as
the amount of transfected M
mAChR cDNA is decreased
(Fig. 3), suggesting that the response depends on the number of
heterologous receptors expressed. Finally, only individual myocytes
that expressed exogenous cDNA showed increased ANF immunoreactivity in
response to carbachol (). These data indicate that
exogenous M
mAChRs are expressed and generate signals that
lead to transcriptional activation of the ANF gene.
and M
subtypes preferentially couple
to the regulation of PLC via the pertussis toxin-insensitive G protein
G
, whereas M
- and M
mAChRs inhibit
adenylate cyclase through interaction with G
(21, 40, 41) . Specificity for effectors
is largely conferred by the third intracellular loop of the
G-protein-coupled receptor
(22, 23, 24, 25) . We demonstrate that
there is a clear pattern of selectivity in the requirements for
coupling mAChR to ANF gene expression since the M
mAChR,
which couples to G
/PLC, is effective while the endogenous
and heterologously overexpressed G
-linked
M
mAChRs are not (Figs. 2 and 6).
:
I
I
and M
:NM
receptors in cardiomyocytes is
further evidence that receptor coupling to G
/PLC is
required for ANF expression, because these and homologous
M
:NM
chimeras are ineffective at stimulating
PLC through G
-dependent pathways
(23, 24, 29) .
Thus, there appears
to be a high degree of selectivity and little promiscuity in mAChR
receptor subtype coupling to the regulation of ANF gene expression.
receptor to function like an M
mAChR in
regulating ANF gene expression. This finding is consistent with the
observation that this region is critical for conferring specificity for
receptor coupling to PLC
(21, 40) . The
M
:NM
chimera, however, showed only about
one-half the efficacy of the wild-type receptor for increasing ANF
expression, even at the highest concentration tested. This could
reflect a lower efficacy of the N-terminal i3 loop chimera for PLC
activation, as shown for M
:NM
receptors stably
expressed in A9L cells
(24) ; however, the
M
:NM
and homologous M
:NM
chimeras are as effective as the wild-type mAChRs for inducing
inositol phosphate formation in transiently transfected COS and A293
cells
(23, 30) .
An alternative possibility
is that the M
:NM
chimeric receptor has a
submaximal effect on ANF expression because, while it couples
efficiently to G
/PLC, it lacks regions needed for coupling
to additional downstream effectors. The observation that the
dominant-interfering Ras significantly inhibited M
but
failed to block M
:NM
receptor-mediated ANF gene
expression suggests that the chimeric receptor lacks the ability to
couple to Ras-signaling pathways.
AdrR-mediated myocardial hypertrophy has been
previously demonstrated by the ability of a dominant-interfering Ras
expression vector to block phenylephrine-mediated increases in cell
size and ANF gene expression
(16) . It has also been shown that
Ras is activated in response to
AdrR stimulation in
cardiac myocytes
(42, 43) . We report here that mAChRs
must interact with a G
-dependent pathway to induce
transcriptional activation of ANF and that Ras function is also
required for maximal ANF gene expression in response to
M
mAChR stimulation. While it is not possible to directly
demonstrate that the heterologously expressed M
mAChRs
activate Ras in cardiomyocytes, the same receptor stably expressed in
fibroblasts has been shown to activate Ras
(44) . We are
currently exploring the use of adenoviral vectors to increase the
transfection efficiency of cardiac myocytes so that biochemical
responses such as Ras and G
/PLC activation can be directly
determined in cells expressing wild-type and chimeric mAChRs. Further
studies using expression of heterologous receptors should provide
insight into the question of how pathways initiated by
G-protein-coupled receptors collaborate with those involving Ras to
regulate cardiac cell growth and gene expression.
Table:
MmAChR-transfected cells express ANF
protein
mAChR cDNA. Cells were subsequently stimulated with 300
µ
M carbachol or 100 µ
M phenylephrine (with 2
µ
M propranolol) for 48 h. Transfected cells were
identified by their expression of luciferase using an anti-luciferase
antibody and a fluorescein-conjugated secondary antibody. ANF protein
expression was detected with an antibody against ANF and a
rhodamine-conjugated secondary antibody. The percent of ANF positive
cells was calculated by dividing the number of ANF-expressing cells
either by the number of cells that also express RSV/luciferase cDNA
(luciferase-positive) or by the number of cells that do not express
luciferase (luciferase-negative). Approximately 150 luciferase-negative
and 30 luciferase-positive cells were scored for ANF immunoreactivity
for each experimental condition.
AdrR,
-adrenergic receptor; QNB,
(±)-3-quinuclidinyl benzoate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.