From the
The three cloned -adrenergic receptor (AR)
subtypes,
,
, and
, can all couple to the same effector, phospholipase
C, and the reason(s) for conservation of multiple subtypes remain
uncertain. All three
-ARs are expressed natively in
cultured neonatal rat cardiac myocytes, where chronic exposure to the
agonist catecholamine norepinephrine (NE) induces hypertrophic growth
and gene transcription. We show here, using RNase protection, that the
-AR subtype mRNAs respond in distinctly different ways
during prolonged NE exposure (12-72 h).
and
mRNA levels were repressed by NE, whereas
mRNA was induced. Changes in mRNA levels were
mediated by an
-AR, were not explained by altered mRNA
stability, and were reflected in receptor proteins by
[
H]prazosin binding.
-AR-stimulated phosphoinositide hydrolysis and myocyte
growth were not desensitized. Three other hypertrophic agonists in
culture, endothelin-1, PGF2
, and phorbol 12-myristate 13-acetate,
also induced
mRNA and repressed
mRNA. In myocytes from hearts with pressure overload hypertrophy,
mRNA changes were identical to those produced by NE
in culture. These results provide the first example of a difference in
regulation among
-AR subtypes expressed natively in
the same cell. Transcriptional induction of the
-AR
could be a mechanism for sustained growth signaling through this
receptor and is a common feature of a hypertrophic phenotype in cardiac
myocytes.
The natural catecholamines norepinephrine (NE) ()and
epinephrine activate adrenergic receptors (ARs) in three families,
,
, and
. Multiple subtypes have
been cloned within each family: three
-ARs (B, C, and
D), (
)three
-ARs (A, B, and C; also called
C10, C2, and C4, respectively), and three
-ARs (1, 2, and
3)(1) . The reason(s) for conservation of multiple subtypes
remain uncertain, since all subtypes in each family couple
preferentially to the same effector when overexpressed,
-ARs to activation of phospholipase C (PLC),
-ARs to inhibition of adenylyl cyclase, and
-ARs
to activation of adenylyl cyclase(1) .
An intriguing
difference among -AR and
-AR subtypes has been
suggested recently, in the regulation of receptor levels during
prolonged agonist exposure. Levels of the
-AR (2, 3, 4) and the
-AR (5, 6) are not down-regulated during long term agonist
exposure, at least in some cells, in contrast with down-regulation of
- and
-ARs, and
-
and
-ARs. Down-regulation of receptor expression is
thought to be a major determinant of desensitization when catecholamine
exposure is prolonged(7) . Conversely, a receptor that is
induced by agonist might be adapted to mediate catecholamine responses
when sympathetic activity is increased chronically, a condition that
occurs frequently in the intact organism.
We have been studying a
physiological response that develops over long periods of catecholamine
exposure, -adrenergic induction of hypertrophic growth
and gene transcription in primary cultures of neonatal rat cardiac
myocytes(8, 9, 10, 11, 12, 13) .
We have found recently that the cardiac myocytes express the mRNAs for
all three cloned
-AR subtypes, the
,
the
, and the
, whereas cardiac
fibroblasts do not express any
-AR(14) . This
system thus provides the opportunity to study regulation of all three
-AR subtypes expressed natively in the same cell. Most
prior studies of chronic agonist regulation of the
-
and
-ARs have been in transfected cells
overexpressing these
subtypes(3, 4, 5, 6) , where
transcriptional regulation of receptor expression would not occur.
In the present study, we report that the -AR
subtype mRNAs respond in distinctly different ways during chronic
exposure to NE. The mRNAs encoding the
and the
were repressed, whereas
mRNA was
induced. These mRNA changes were mediated through an
-AR, were likely transcriptional in origin, and were
followed by changes in receptor protein by radioligand binding.
-AR-stimulated phosphoinositide hydrolysis and myocyte
growth were not desensitized. Three other hypertrophic agonists,
endothelin-1 (ET-1)(15) , PGF2
(16) , and phorbol
12-myristate 13-acetate (PMA)(17) , also induced
mRNA and repressed
mRNA. Changes identical to
those produced by NE in culture were seen in myocytes isolated from
hearts with hypertrophy produced by abdominal aortic banding. These
results provide the first example of a difference in regulation among
-AR subtypes expressed natively in the same cell.
Transcriptional induction of the
-AR could be a
mechanism for sustained growth signaling through this receptor and is a
common feature of a hypertrophic phenotype in cardiac myocytes.
In this neonatal rat cardiac myocyte culture model,
-AR-induced growth and gene expression are
half-maximum at
12-18 h and maximum at
24-48 h of
catecholamine exposure(9, 10) ; and these responses
require continuous receptor occupancy by
agonist(25, 26, 27) . Thus, continuous
-AR signaling appears to occur over long times,
seemingly counter to the well established concept of AR desensitization
by agonist(7) .
To determine if prolonged catecholamine
exposure produced atypical regulation of one or more of the three
-AR subtypes in the myocytes, the
-AR
mRNAs were quantified by RNase protection after treatment with NE. A
dose of NE was used (2 µM) that is maximum for myocyte
growth and gene transcription(9, 10) . After 24 h of
NE exposure,
and
mRNA levels were
reduced to about 26 and 42%, respectively, of those in control myocytes
treated concurrently with vehicle (Fig. 1). Repression of
and
mRNAs was also evident when
normalized to the levels at time 0, when NE was added (19 ± 1%
and 14 ± 1% for
and
,
respectively, n = 5, p < 0.05). (
)In direct contrast to the decrease of
and
mRNAs, NE increased the abundance of
mRNA, by over 3-fold (Fig. 1). Induction of
mRNA was also observed when normalized to the level
at time zero (4.4 ± 1.2-fold, n = 5, p < 0.05). As a control, NE had no significant effect on the mRNA
for the ``housekeeping'' gene GAPDH (at 24 h, 1.16 ±
0.10-fold versus vehicle, 1.10 ± 0.08-fold versus time 0, n = 3, p = not
significant). The nonselective
-AR antagonist prazosin
inhibited regulation of the
-AR mRNAs by NE, whereas
the
-AR antagonist timolol did not (Fig. 1). Thus
activation of an
-AR induced
mRNA
and repressed
and
mRNAs.
Figure 1:
NE induces mRNA and
represses
and
mRNAs in cardiac
myocytes through an
-AR. Cultured cardiac myocytes
were treated with 2 µM NE or vehicle control in the
absence or presence of the
-AR antagonist prazosin (Pzn) (2 µM) or the
-AR antagonist timolol (Tim) (2 µM), added 30 min before NE. After 24 h,
the
-AR subtype mRNAs were quantified by RNase
protection. Values are the mean ± S.E. treated/control ratios of
mRNA band density/µg of total RNA for the number of experiments
indicated below the bars, with seven 100-mm dishes used for
each group in each experiment. Prazosin or timolol alone had no
significant effect on any mRNA (data not shown). *, p <
0.05 versus vehicle control.
As
shown in Fig. 2, the effects of NE on each mRNA were sustained
for up to 72 h of continuous NE exposure. The effects were detectable
at the earliest time studied (2 h; 1.4-fold
control,
76% of control, and
37%
of control; means from two experiments), were submaximum at 12 h (two
experiments as in Fig. 3), and were maximum at
24 h (four
experiments as in Fig. 2).
Figure 2:
NE regulation of -AR
mRNAs is sustained over 72 h. Cultured myocyte RNA was harvested for
RNase protection assay at the time of 2 µM NE addition (0 h), and at 24, 48, and 72 h after the addition of NE
(+) or vehicle(-). Protected fragments of the following
sizes (bases) are shown:
, 432;
,
315;
, 217; and GAPDH, 316(21) . Yeast
transfer RNA (tRNA) was a control for nonspecific
hybridization, and GAPDH was a control for input RNA. The same results
were observed in three additional experiments, and the numbers were
similar to those quantified in Fig. 1. At 72 h, the level of
mRNA in cells treated with NE alone was 25% of the
vehicle control, whereas it was higher, 70% of control, with NE in the
presence of timolol (2 µM), suggesting a
-AR
contribution to persistent
mRNA
down-regulation (mean from 3-4
experiments).
Figure 3:
NE has no effect on -AR
mRNA degradation. Cultured cardiac myocytes were treated with
actinomycin D (+Act D) (0.05 µg/ml) or with its
vehicle (-) in minimal essential medium with 1% calf serum for 3
h, and then 2 µM NE (+NE) or its
vehicle(-) were added at time 0 (0 h). RNA was harvested
at the times indicated (-3 to 12 h), and the
-AR mRNAs and GAPDH mRNA were assayed by RNase
protection. Protected fragments are as in Fig. 2, except a
longer
riboprobe, 474 bases, was used to protect 446
bases of
mRNA (nucleotides
1752-2197(52) ). The same results were obtained in an
additional experiment, and receptor mRNA degradation half-lives (see
``Results'') were estimated from semilogarithmic plots of
mRNA levels versus time in the presence of actinomycin
D.
To test if the NE-induced changes
in mRNA levels were transcriptional in origin, the myocytes were
incubated with actinomycin D, at a concentration (0.05 µg/ml) that
inhibited transcription by >95% within 3 h, as assayed by
[H]uridine incorporation into total RNA, but had
no effect on cell viability over 12 h (data not shown). As shown in Fig. 3, all three
-AR mRNAs disappeared rapidly
in the presence of actinomycin D, with apparent degradation half-lives
of
2.5, 3, and 1 h for
,
, and
mRNAs, respectively (Fig. 3, lanes
1-3 and 5). Treatment with NE when transcription
was inhibited by actinomycin D had no effect on the abundance of any
-AR mRNA (Fig. 3, lanes 3-6). In
the absence of actinomycin D, in contrast, the characteristic effects
of NE on all three mRNAs were observed over the same time interval (12
h; Fig. 3, lanes 7 and 8). These results
indicated that NE did not alter
-AR mRNA stability and
suggested that
-AR stimulation regulated transcription
of the
-AR genes.
Radioligand binding was used to
test whether the changes in receptor subtype mRNAs were accompanied by
parallel changes in receptor proteins. In competition binding assays
with [H]prazosin, the cloned rat
-AR has much higher affinity for the antagonist 5MU
(4 nM) (28) than does the rat
-AR
(122 ± 4 nM) (28, 29) or the rat
-AR (140 ± 120 nM) (28, 29, 30, 31, 32) . In
the cultured cardiac myocytes, competition binding with
[
H]prazosin distinguished an
-AR
population with high affinity for 5MU (4.4-7.6 nM) and a
population with low affinity (261-277 nM) (Table 1). The high affinity sites were assumed to reflect the
-AR, and the low affinity sites were assumed to
reflect the
-AR and/or the
-AR. (
)Exposure to NE for 72 h decreased the number of low
affinity sites to 60% of control, consistent with down-regulation of
the
and/or the
(Table 1).
In contrast, NE exposure for 72 h doubled the number of high affinity
sites, consistent with up-regulation of the
-AR (Table 1) and in agreement with the 3-fold increase in
mRNA (Fig. 1). Thus in the cardiac myocytes
exposed chronically to NE, the
-AR became the
predominant receptor, increasing from 26 to 55% of total
-ARs (Table 1). It was noteworthy that total
receptor number did not change, despite the significant shift in
subtype proportions (Table 1).
PLC activation was used to test
for desensitization of -AR signaling.
-AR coupling to PLC in cultured neonatal rat cardiac
myocytes is through an
-AR with high affinity for 5MU
(2 nM)(33) , probably the
(see
above for 5MU affinities of cloned
-ARs). In myocytes
exposed to 2 µM NE for 48 h, the EC
for
NE-stimulated total [
H]IP production was
unchanged (0.7 µM for NE-treated cells versus 0.9
µM for control cells, mean of four experiments). Fractions
corresponding to IP
, IP
, and IP
were increased equally in NE-treated and control cells (data not
shown), and maximum total [
H]IP responses were
not different (3.6-fold for NE-treated cells versus 3.3-fold
for controls). Thus there was no desensitization of
-AR-mediated PLC activation with 72 h of NE exposure,
consistent with prior studies in cultured myocytes (34, 35) and with unimpaired
-AR
signaling. It was not possible to test for desensitization of
and/or
signaling, since
biochemical responses coupled to these
-ARs in
myocytes have not been identified conclusively.
There was also no
desensitization of a physiological response to chronic
-AR activation, NE-induced myocyte growth. In myocytes
pretreated with 2 µM NE for 72 h, the EC
for
NE-stimulated protein accumulation over the subsequent 72 h was the
same as in control myocytes (0.8 ± 0.3 µM for
NE-treated cells versus 0.7 ± 0.3 µM for
controls, n = 5, p = not significant).
To test if induction and repression of the -AR
subtype mRNAs required
-AR occupancy, mRNA levels were
measured after treatment of the myocytes with three other hypertrophic
growth factors, ET-1(15) , PGF2
(16) , and
PMA(17) . As shown in Fig. 4, ET-1, PGF2
, and PMA
were all similar to NE, inducing
mRNA and repressing
mRNA. Interestingly, unlike NE, PGF2
tended to
induce
mRNA, and PMA had no effect on
(Fig. 4).
Figure 4:
Hypertrophic stimuli induce
mRNA and repress
mRNA in culture
and in vivo. Cultured neonatal rat cardiac myocytes were
treated for 24 h with 2 µM NE, 100 nM ET-1, 10
µM PGF2
, 100 nM PMA, or their vehicle
controls. Adult cardiac myocytes were isolated from the intact rat
heart after 10-12 weeks of aortic banding. Total RNA was prepared
and the
-AR subtype mRNAs were quantified by RNase
protection. For the growth factors in culture, values are the mean
± S.E. treated/control ratios for three separate experiments
(five for PMA). For aortic banding, values are the mean banded/sham
ratios for four banded rats and five shams; statistical analyses were
done on the absolute phosphor imaging values. *, p < 0.05 versus vehicle control.
The pattern of -AR mRNA
regulation produced by NE in culture was also observed with a pressure
overload stimulus for hypertrophy in the intact animal. Myocytes were
isolated from the adult rat heart after 10-12 weeks of abdominal
aortic banding. Banding stimulated myocyte hypertrophy, increasing the
mean volume of isolated myocytes by
20%, from 36,000 ± 300
µm
/myocyte with sham operation to 43,000 ± 900
µm
with banding (n = 4-5 hearts, p < 0.001). As shown in Fig. 4, aortic banding
increased
mRNA level by almost 3-fold and decreased
the levels of
and
significantly,
to 71 and 56% of sham, respectively. As a control, banding did not
change the level of myocyte
-actin mRNA (1.14-fold versus sham).
These results provide the first example of a difference in
regulation among -AR subtypes expressed natively in
the same cell. The
- and
-AR mRNAs
were repressed by chronic
-AR stimulation of cultured
cardiac myocytes, whereas
mRNA was induced, and
there were coordinate changes in
-AR binding activity.
A similar pattern of mRNA induction and repression was seen with other
hypertrophic agonists in culture, ET-1, PGF2
, and PMA, and with a
pressure overload stimulus for hypertrophy in the intact animal, aortic
banding. Our results differ from overexpression studies in rat 1
fibroblasts, where all three
-AR subtypes are
down-regulated equally by agonist(36) , and thus emphasize the
importance of transcriptional control of native
-AR
expression.
The observed differences in subtype regulation might be
physiologically important. Induction of the -AR is a
potential mechanism for sustained signaling during hypertrophic growth,
a chronic physiological response to catecholamines in cardiac myocytes.
-AR-stimulated growth and transcription in cultured
myocytes requires sustained
-AR signaling over long
times, since the trophic effects decay whenever agonist is removed or
antagonist is added(25, 26, 27) . Consistent
with these earlier results, we found here that exposure of cultured
myocytes to NE for 3 days did not desensitize
-AR-stimulated PLC activation or growth. Both
responses have been attributed to an
-AR with
-AR-like affinity for 5MU(33) , and it thus
can be proposed that the responses are sustained because of
-AR induction. Enhanced sensitivity of PLC
activation or growth was not observed with
-AR
up-regulation. A possible explanation can be found in recent
overexpression studies, where increases in
-AR number
in the 2-3-fold range, the increase in
-ARs we
found in the present study, do not change PLC activation
significantly(37) , possibly because
G
/G
are concurrently
down-regulated(36) . Thus
-AR induction might
sustain PLC activation and growth despite down-regulation of G proteins
or other changes that could desensitize the responses.
Induction of
mRNA and repression of
were
observed with several hypertrophic agonists in culture and with
pressure overload in vivo, suggesting that these mRNA changes
could be one feature of a specific hypertrophic transcriptional
program, analogous to the induction in hypertrophy of genes expressed
preferentially during fetal cardiac development, such as the
contractile protein isogenes,
-myosin heavy chain, and skeletal
-actin (for a review see (38) ). A transcription factor
has been identified in cardiac myocytes, transcriptional enhancer
factor-(TEF-1), that is involved somehow in
-adrenergic activation of the
-myosin heavy chain
and skeletal
-actin
promoters(11, 12, 13) . The rat
promoter, the only
-AR promoter studied in
detail so far, contains a potential TEF-1 binding site(39) .
Thus it will be interesting to see if the
-AR is
expressed preferentially during fetal cardiac development and if TEF-1
plays a role in
-AR gene transcription. It will also
be interesting to test whether
-AR induction is a
required mechanism for sustaining cardiac growth induced by various
hypertrophic stimuli.
The mechanisms for transcriptional control of
-AR genes are likely to vary in different cell types.
Expression of
-AR subtype mRNAs is tissue- and
cell-specific(14, 21) , and regulation of
-AR mRNAs by agonist is also different in different
cells. For example, in cultured smooth muscle cells,
mRNA is reduced minimally (40) or not at all (41) with prolonged catecholamine exposure, in contrast to the
marked repression of
mRNA in cardiac myocytes
observed in this study.
Even in myocytes, transcriptional controls
might not be identical for all hypertrophic agonists, since PMA and
PGF2 differed from NE in their failure to repress
mRNA. In this regard, it is notable that the pattern of
mRNA induction and
and
mRNA repression was identical with NE treatment in
culture and pressure overload in vivo. We did not test whether
NE was the proximate stimulus for the mRNA changes with aortic banding,
but sympathetic activity is increased in animals and humans with
hypertrophy (for examples see (42, 43, 44, 45) ).
In the
transgenic mouse heart, overexpression of an activated
-AR induces cardiac myocyte hypertrophy(46) ,
possibly in conflict with the idea that the
-AR
transduces growth in myocytes under native conditions(33) . On
the other hand, this activated
-AR couples
efficiently to PLC activation when overexpressed in cell
lines(47, 48) , and the same appears to be true when
it is overexpressed in myocytes, as judged by increased diacylglycerol
accumulation(46) . Thus the activated
-AR
might produce hypertrophy simply because it is able to stimulate PLC in
myocytes. In addition, our results here suggest that stimulation of PLC
by the activated
could induce the native
-AR in the transgenic hearts, and thus the native
might mediate the hypertrophic response. It will be
important to test whether the
has some structural
property that is required for growth, in addition or alternate to PLC
activation.