(Received for publication, May 24, 1995; and in revised form, August 17, 1995)
From the
The dihydropyridine (DHP) and ryanodine (RY) receptors play a
critical role in depolarization-induced calcium release in skeletal
muscle, yet the factors which govern their expression remain unknown.
We investigated the roles of electrical activity and trophic factors in
the regulation of the genes encoding the ,
, and
subunits of the DHP receptor as well as
the RY receptor in rat skeletal muscle in vivo. Muscle
paralysis, induced by denervation, had no effect on the DHP receptor
mRNA levels while the RY receptor mRNA was decreased. In contrast,
chronic superfusion of tetrodotoxin onto the sciatic nerve resulted in
a marked increase in mRNA levels and transcriptional activity of both
DHP and RY receptor genes. Since nerve can induce changes in second
messenger pathways which modulate muscle gene expression, we attempted
to identify factors which regulate DHP and RY receptor expression using
cultured myotubes. Elevated cAMP levels specifically inhibited the
expression of RY receptor mRNA while
12-O-tetradecanoylphorbol-13-acetate, an activator of protein
kinase C, increased the transcripts encoding the RY receptor and the
subunit of the DHP receptor. Changes in the level of
mRNAs were paralleled by altered receptor numbers. Neither cAMP nor
protein kinase C altered transcriptional activity of the DHP and RY
receptor genes. These results demonstrate that neural factor(s)
regulate DHP and RY receptor mRNA levels in vivo via
transcriptional mechanisms while protein kinase C and cAMP can modulate
DHP and RY receptor transcript levels by a transcription-independent
process.
Nerve-induced muscle contraction is preceded by depolarization
of the sarcolemmal membrane and elevation of intracellular calcium
levels in muscle cells. Several sarcolemmal proteins, namely the
nicotinic acetylcholine receptor (nAChR) ()and the
voltage-gated sodium channel, play important roles in nerve-induced
depolarization of the sarcolemma. The depolarization of the sarcolemmal
membrane is transmitted via the transverse tubules across the triad
junction to the sarcoplasmic reticulum. The dihydropyridine (DHP)
receptors of transverse tubules respond to changes in membrane polarity
by acting as voltage sensors/voltage-sensitive calcium channels and
trigger calcium release by interacting with the ryanodine (RY)
receptors/calcium release channels of the sarcoplasmic reticulum.
A large body of information is available regarding the structure, function, and subunit composition of DHP and RY receptors in adult skeletal muscle (reviewed in Fleischer and Inui(1989), Catterall (1991), McPherson and Campbell(1993), and Meissner(1994)). The cDNAs of all five subunits of the DHP (Tanabe et al., 1987; Ellis et al., 1988; Ruth et al., 1989; Jay et al., 1990) and the RY receptor (Takeshima et al., 1989; Zorzato et al., 1990) in skeletal muscle have been cloned. However, very little is known about the factor(s) that induce and regulate the expression of the genes encoding these receptors.
Expression of many
muscle-specific genes, like the nAChR subunits (Buonanno and Merlie,
1986), voltage-sensitive sodium channel (Offord and Catterall, 1989),
and more recently the DHP receptor subunits and RY receptor (Kyselovic et al., 1994), are switched on when mononucleated myoblasts
fuse to form multinucleated myotubes. As myotubes mature and acquire
nerve supply, nerve-induced electrical activity and nerve-derived
trophic factors play important roles in the regulation of
muscle-specific gene expression. Electrical stimulation of developing
muscle and myotubes in culture represses the expression of mRNA for the
nAChR and the -subunit of the voltage-sensitive sodium channel
(Witzemann et al., 1991; Chahine et al. 1993). On the
other hand, expression of the
- and
-subunit genes of the
nAChR is regulated by neurotrophic factors (Witzemann et al.,
1991; Jo et al., 1995). Thus, while nerve-induced electrical
activity and nerve-derived factors play an important role in the
regulation of genes of at least two of the main proteins involved in
neuromuscular transmission, the contribution of these processes in the
regulation of DHP and RY receptor genes remains unknown. Furthermore,
muscle-specific gene expression can be modulated by second messengers
such as cAMP and calcium (Winter et al., 1993; Chahine et
al., 1993). The role of second messenger pathways in modulating
the expression of the DHP and RY receptors remains elusive. In a recent
study it was shown that there was a temporal difference in the
induction of the DHP receptor subunit mRNAs and the RY receptor mRNA in
developing muscle, suggesting that these genes may be under the control
of distinct endogenous factors (Kyselovic et al., 1994). The
objective of the present investigation was to elucidate the mechanisms
that regulate the expression of DHP and RY receptor genes, the two
molecules involved in electrical signal transmission at the triad
junction.
Female Sprague-Dawley rats (16-17 days gestation) were obtained from Charles River Laboratories (St-Constant, Quebec). Animals were housed in temperature-controlled rooms and maintained on a 12-h light/12-h dark cycle. Care and treatment of animals were in accordance with the guidelines presented by the Canadian Council on Animal Care.
The polyclonal antibodies directed
against synthetic peptides of the and
subunit
of the dihydropyridine receptor were a generous gift from Dr. W. A.
Catterall (University of Washington) and those directed against the RY
receptor were kindly obtained from Dr. D. H. MacLennan (University of
Toronto). DHP receptor-enriched preparations from rabbit skeletal
muscle were isolated as described by Tuana et al.(1988) and
Sunahara et al.(1990). Protein concentrations were determined
by the method of Markwell et al.(1978).
Frozen muscles, 3 g each from
control and TTX-paralyzed rats, were homogenized in 10 volumes of lysis
buffer without Nonidet P-40. Following centrifugation (500 g; 5 min), pellets were resuspended in 1 ml of lysis buffer
containing 0.05% Nonidet P-40. Nuclei were isolated as described above,
suspended in storage buffer (5
10
/50 µl) and
used immediately.
Nuclei (50 µl) were suspended, in a final
volume of 200 µl, with 5 reaction mixture (1.5 M(NH
)
SO
, 500 mM Tris-HCl, pH 7.9, 20 mM MgCl
, 20 mM MnCl
, 1 M NaCl, 2 mM EDTA, 0.5
mM phenylmethylsulfonyl fluoride), 0.12 mM dithiothreitol, 1 mM of ATP, GTP, and CTP each, 20%
glycerol, and 250 µCi of [
-
P]UTP
(DuPont). RNA was transcribed at 28 °C for 30 min. Following RQ1
DNase (Promega) treatment, labeled RNA were purified by
phenol-chloroform extraction, gel filtration, and ethanol
precipitation. Labeled RNA were hybridized at 42 °C for 48 h to
nylon membranes (MSI MAGNA) containing 10 µg each of the linearized
,
, and
subunits, RY receptor,
and glyceraldehyde-3-phosphate dehydrogenase plasmids, and
nonlinearized tubulin plasmid. Plasmids containing the
and
subunit DNA fragments were linearized using EcoRI. The
subunit and RY receptor DNA sequence
containing plasmids were linearized using BamHI. The plasmid
containing the glyceraldehyde-3-phosphate dehydrogenase DNA fragment
was linearized using PstI. Following hybridization, membranes
were washed extensively (1
SSC; 0.1% SDS) at room temperature.
Films were exposed for autoradiography at -70 °C for
3-7 days using two intensifying screens.
RNA isolated from skeletal
muscle were size fractionated, transferred to nylon membranes, and
hybridized with cDNA probes for the ,
, and
subunits of the DHP receptor and the RY
receptor to study mRNA expression. In 10-day denervated muscle, levels
of mRNA encoding DHP receptor subunits (transcript of 6.4, 8, and 1.9
kb for
,
and
subunits,
respectively) were essentially unchanged while expression of RY
receptor mRNA (15 kb transcript) was markedly inhibited (Fig. 1A, lane D). In agreement with previous
reports of Michel et al.(1994), the AChE transcript was
severely decreased in denervated muscle (Fig. 1A, lane
D). By contrast, expression of
,
, and
subunit mRNAs of the DHP receptor as well
as the RY receptor mRNA was increased in TTX-paralyzed muscle (Fig. 1A, lane T) as compared with denervated (Fig. 1A, lane D) and untreated matched control muscle (Fig. 1A, lane C). Quantitation of the mRNA data
confirmed that denervation did not affect DHP receptor levels (Fig. 1B) while a 30% reduction in the RY receptor mRNA
level was observed. When expressed as percent of control, in denervated
muscle RY receptor message was 70 ± 15% (n = 5)
of the control level (Fig. 1B). In TTX-paralyzed
muscle, there was 4.5 ± 1.3- (n = 5) fold
induction of the
subunit mRNA compared with a 2
± 0.2- (n = 5) and 1.8 ± 0.44-fold (n = 5) induction of the
and
-subunits
of the DHP receptor, respectively, and a 2.3 ± 0.17-fold (n = 5) induction of the RY receptor (Fig. 1B).
Figure 1:
Gene expression of the DHP receptor
,
, and
subunits and the RY
receptor following denervation and TTX paralysis of adult rat muscle. A, 20 µg of total RNA from muscle of control (lane
C), denervated (lane D), and TTX-paralyzed (lane
T) rats were separated electrophoretically, transferred to a nylon
membrane, and hybridized with
P-labeled cDNA probes
complementary to skeletal muscle calcium channel
,
, and
subunit mRNAs, ryanodine receptor (RyR), and AChE mRNAs. Signals at 6.4 (
), 8
(
), 1.9 (
), 15 (RY receptor), 2.4 (AChE), and 1.8-kb glyceraldehyde-3-phosphate dehydrogenase (GAPD) were visualized by autoradiography. B,
autoradiograms were quantitated by densitometry. Densitometry data were
normalized with respect to the glyceraldehyde-3-phosphate dehydrogenase
mRNA and expressed as percent of control message. Each bar represents the mean ± S.E. of five independent experiments.
*, represents significantly different from the control (p <
0.05).
Primary culture of skeletal myotubes have
been used extensively to investigate the induction and regulation of a
variety of different muscle-specific genes (Offord and Catterall, 1989;
Klarsfeld et al., 1989; Chahine et al., 1993).
Mononuclear myoblasts fuse to form multinucleated myotubes by days 3
and 4 after plating, and spontaneous contractility appears between days
5 and 7 in culture. It can be seen in Fig. 2, A and B, that the ,
, and
subunit mRNAs appeared as early as day 3 in culture, were rapidly
induced during myoblast fusion and reached their respective peaks after
day 7. The
subunit transcript was induced much more rapidly when
compared to the
and
transcripts.
The RY receptor transcript was not induced until day 10, although a
basal level of expression was detectable as early as day 3. The
induction of the mRNA preceded the appearance of the polypeptides in
developing myotubes as described previously (Romey et al.,
1989; Kyselovic et al., 1994). The time course of the
induction of the DHP and RY receptors in tissue culture paralleled
their expression in developing muscle, suggesting that myotubes in
culture were a convenient model for investigating DHP and RY receptor
gene expression. Consequently, we used myotubes on day 7 to study the
effect of muscle inactivity on the expression of the DHP and RY
receptor mRNA. Spontaneous contractions and electrical activity of
skeletal myotubes were blocked with the sodium channel blocker TTX. As
shown in Fig. 3A, pretreatment of cultured myotubes
with 1 µM TTX (lane T) had no effect on the
expression of
,
, and
subunit
transcripts of the DHP receptor or the RY receptor mRNA. Similar
results were obtained with another membrane stabilizing agent,
bupivacaine (data not shown).
Figure 2:
Gene
expression of DHP and RY receptors in development. RNA from developing
myotubes at different days of development was examined in Northern
blots for expression of the ,
, and
subunits of the DHP receptor, RY receptor, and tubulin mRNAs with
specific cDNA probes (A). The mRNA levels were quantified by
densitometry and normalized for RNA loading (B). The data
shown is a typical representation of two different
experiments.
Figure 3:
Gene expression of DHP receptor
,
, and
subunits and the RY
receptor in myotubes treated with TTX and forskolin. A,
myotubes on day 4 were exposed to TTX (1 µM) and forskolin
(1 µM) for 72 h. On day 7, RNA was isolated from untreated
control (lane C), TTX (lane T), and forskolin (lane F) pretreated myotubes. RNA was size separated
electrophoretically, transferred to a nylon membrane, and hybridized
with
P-labeled cDNA probes complementary to the skeletal
muscle 6.4-kb
, 8-kb
, and 1.9-kb
subunit mRNAs of the DHP, the 15-kb RY receptor (RyR)
mRNA, and the 1.8-kb glyceraldehyde-3-phosphate dehydrogenase (GAPD) mRNA. Signals were visualized by autoradiography. B, effects of 8-bromo-cAMP and isobutylmethylxanthine (IBMX) on the expression of
subunit of the
voltage-sensitive calcium channel in skeletal myotubes in culture.
Myotubes on day 4 were exposed to 500 µM 8-bromo-cAMP and
50 µM isobutylmethylxanthine for 72 h. On day 7 RNA was
isolated, size separated, transferred to nylon membrane, and hybridized
with
P-labeled cDNA probe complementary to the
subunit and glyceraldehyde-3-phosphate dehydrogenase mRNAs.
Signals were visualized by autoradiography.
We have reported that during
development, the subunit and RY receptor mRNAs and
DHP and RY binding sites reached their respective peaks at a time when
myotubes were spontaneously contracting (Kyselovic et al.,
1994). Spontaneous contractile activity of skeletal myotubes is
associated with membrane depolarization and activation of protein
kinase C (Vergara et al., 1985). We investigated the effect of
TPA, an activator of protein kinase C, and staurosporine, a protein
kinase C antagonist, on the expression of DHP and RY receptor mRNAs.
Staurosporine inhibited the basal level of expression of both the
subunit of the DHP and RY receptor, while TPA (200
ng) stimulated the expression of
subunit and the RY
receptor mRNA (Fig. 4A). The stimulatory effect of TPA
was antagonized by staurosporine (Fig. 4A).
Staurosporine inhibited the expression of
subunit
(27%) and RY receptor (31%), while TPA stimulated mRNA levels of both
the
subunit and RY receptor by 175 and 145%,
respectively (Fig. 4B). TPA and staurosporine did not
affect the expression of
or
subunit mRNAs of
the DHP receptor in cultured myotubes (not shown).
Figure 4:
TPA and staurosporine modulate the mRNA
expression of subunit of DHP receptor and the RY
receptor. A, myotubes on day 4 were exposed to TPA (200 ng)
and staurosporine (200 ng). RNA was isolated on day 7, size
fractionated, transferred to a nylon membrane, and hybridized with
P-labeled cDNA probes complementary to the 6.4-kb
subunit of the DHP, 15-kb RY receptor (RYR),
and 1.8-kb glyceraldehyde-3-phosphate (GAPD) mRNAs. Signals
were visualized by autoradiography. B, autoradiograms were
quantitated by densitometry. Densitometry data were normalized with
respect to the glyceraldehyde-3-phosphate dehydrogenase message, and
expressed, in arbitrary units, as percent of control response. Each bar represents the mean of three independent
experiments.
Figure 5:
Neural regulation of transcriptional
activity of DHP and RY receptor genes. A, nuclei were isolated
from TTX-paralyzed (TTX) and control (CON) rat
skeletal muscle. Nuclei were isolated and transcribed at 28 °C
using P-labeled UTP as described under ``Materials
and Methods.'' Labeled RNA (1.5
10
cpm) were
hybridized for 48 h at 42 °C to nylon membranes containing 10
µg each of linearized
,
,
subunit, RY receptor (RYR), and glyceraldehyde-3-phosphate
dehydrogenase (GAPD) plasmids and the nonlinearized tubulin (TB) plasmid. Signals were visualized by autoradiography. Data
is the representative of three similar experiments. B,
densitometry data in control and TTX-paralyzed muscle were normalized
with respect to glyceraldehyde-3-phosphate dehydrogenase message.
Changes in gene transcription due to TTX paralysis of muscle were
represented as percent of control response. Each bar represents mean ± S.E. of three separate experiments. *,
represents significantly different from control response (p < 0.05). TUB, tubulin.
Since forskolin and TPA altered the DHP and RY receptor mRNA level following 72 h exposure of myotubes, we attempted to investigate if changes in transcript levels could be attributed to enhanced transcription of the respective genes. We isolated nuclei from myotubes pretreated with forskolin (1 µM) and TPA (200 nM) for 72 h. Nuclei were subjected to in vitro transcription. As shown in Fig. 6A, TPA (TPA panel) and forskolin (Forsk panel) pretreatment of myotubes did not alter the rate of transcription of the DHP and RY receptor genes. No statistically significant difference in the expression of either calcium channel subunit gene was observed when data were normalized with respect to glyceraldehyde-3-phosphate dehydrogenase message and expressed as percent of control (Fig. 6B).
Figure 6:
Effects of forskolin and TPA on
transcriptional activity of DHP and RY receptor genes. A,
myotubes on day 4 were exposed to buffer (CON), TPA (200 ng),
and 1 µM forskolin (FORSK). Nuclei were isolated
on day 7 and transcribed at 28 °C using P-labeled UTP
as described under ``Materials and Methods.'' Labeled RNA
(1.5
10
cpm) were hybridized for 48 h at 42 °C
to nylon membranes containing 10 µg each of linearized
,
,
subunit, RY receptor (RYR), and glyceraldehyde-3-phosphate dehydrogenase (GAPD) plasmids and the nonlinearized tubulin (TB)
plasmid. Signals were visualized by autoradiography. Data is the
representative of three similar experiments. B, densitometry
data in control, TPA, and forskolin-treated cultured myotubes were
normalized with respect to glyceraldehyde-3-phosphate dehydrogenase
message. Changes in transcriptional activity due to TPA or forskolin
treatment of myotubes were represented as percent of control response.
Each bar represents mean ± S.E. of three separate
experiments.
Figure 7:
Dihydropyridine and RY receptor
polypeptide levels in denervated and TTX-paralyzed adult skeletal
muscle. A, muscle proteins (200 µg) from control (C), denervated (D), and TTX-paralyzed (T)
muscle were separated in SDS-PAGE and subjected to Western blot
analysis with specific antibodies raised against the and
subunits of the voltage-sensitive calcium channel (De
Jongh et al., 1989) and the RY receptor. The apparent
molecular mass of the immunoreactive polypeptides were 170-, 56-, and
450-kDa, respectively. The results are a typical representation of
three independent experiments. B, densitometry data were
expressed as percent of control response. Each data point represents
mean ± S.E. of three independent experiments. *, represents
significantly different from control (p <
0.05).
Figure 8:
Forskolin, TPA, and staurosporine affect
the number of DHP and RY receptor binding sites in cultured myotubes.
Cultured myotubes were exposed on day 4 to forskolin (1
µM), TPA (200 ng), and staurosporine (200 ng) for 72 h. On
day 7 myotubes were homogenized and DHP and RY binding were determined
using [H]PN 200-110 and
[
H]ryanodine (Murphy and Tuana, 1990). Each bar represents the mean ± S.E. of three independent
experiments.
The results of the present study demonstrate that nerve plays a critical role in regulating the expression of DHP and RY receptors in vivo. Our data also indicates that the cAMP and protein kinase C pathways can modulate the protein and mRNA levels but not the transcriptional activity of these important genes.
Formation of the
neuromuscular junction is an important step in the course of
development and maturation of skeletal muscle. Muscle-specific
voltage-sensitive sodium channel and nAChR subunits undergo
redistribution from the body of myotubes to the neuromuscular junction
as well as subunit and/or isoform switching under the influence of
nerve supply (Schuetze and Role, 1987; Hall and Sanes, 1993). A common
approach to determine the contribution of nerve-induced electrical
activity on muscle-specific gene expression is to surgically denervate
the muscle. Denervation of adult muscle and blocking spontaneous
contractility of cultured myotubes with TTX reverts myotubes back in
time to the embryonic stage which is manifested by pronounced
up-regulation of the gene expression of the embryonic isoforms of nAChR
and TTX-insensitive sodium channel (Goldman et al., 1989;
Kallen et al., 1990; Offord and Catterall, 1989; Witzemann et al., 1991; Chahine et al., 1993; reviewed in
Schuetze and Role(1987) and Hall and Sanes(1993)). By contrast,
denervation of adult skeletal muscle had no effect on the mRNA levels
of ,
, and
subunits of the DHP
receptor while the RY receptor and AChE messages were down-regulated.
Similarly, blocking spontaneous contractility of cultured myotubes with
TTX and bupivacaine did not have any effect on the DHP and RY receptor
mRNA levels. This lack of effect of muscle inactivity on DHP and RY
receptor transcript levels may be related to the fact that unlike
muscle-specific nAChR and sodium channel proteins, the DHP and RY
receptors do not appear to undergo isoform and/or subunit switching and
redistribution from the body of myotubes to neuromuscular junction in
response to nerve influence.
TTX paralysis of the sciatic nerve
markedly induced the ,
,
subunit transcripts of the DHP receptor as well as the RY receptor
mRNA. Increases in the DHP receptor mRNAs were accompanied by
corresponding increases in transcriptional activity of the respective
genes in TTX-paralyzed skeletal muscle. This suggested that increases
in the DHP and RY receptor transcripts were due, at least in part, to
induction of the respective genes. The increases in the mRNA levels of
subunit and the RY receptor were accompanied by
changes in the two polypeptide levels when assayed with specific
antibodies. In TTX-paralyzed skeletal muscle, there is no neuronal
evoked action potentials and transmitter release but axonal transport
and spontaneous quantal release of neurotrophic factors is maintained
at the neuromuscular junctions (see Witzemann et al.(1991)).
Thus, induction of the DHP and RY receptor genes may be attributed to
the release of some nerve-derived factor(s). In this regard several
nerve-derived factors, including calcitonin gene related peptide (New
and Mudge, 1986), acetylcholine receptor inducing agent (Jo et
al., 1995), and Schwann cell-derived maturation factor (Chapron
and Koenig, 1989) are capable of inducing the expression of muscle
nAChR genes. In addition, it has been shown that growth factors
including transforming growth factors
(Shih et al.,
1990; Giannini et al., 1992) and fibroblast growth factor
(Marks et al., 1991) can regulate the DHP and RY receptor
message levels. We do not know if any of these aforementioned factors
contribute toward the induction of DHP and RY receptor genes observed
in TTX-paralyzed muscle. We did not observe any inducing effect of
brain-derived neurotrophic factor, ciliary neurotrophic factor, or
nerve growth factor on the DHP receptor mRNA level in primary culture
of skeletal myotubes. (
)
Previous studies have shown that
muscle inactivity and denervation lead to increased cAMP levels and
activation of the cAMP-dependent protein kinase pathway in muscle
(Chahine et al., 1993). Furthermore, the induction of nAChR
and TTX-insensitive sodium channel genes are believed to be mediated
via the cAMP pathway in inactive and denervated muscle (Chahine et
al., 1993; Offord and Catterall, 1989). In the present study, we
did not see any effect of forskolin on the ,
, and
subunit mRNA levels of the DHP receptor in
myotube cultures. A very similar effect was seen when 8-bromo-cAMP and
isobutylmethylxanthine were used to elevate cAMP levels in this system,
which is in agreement with the results obtained in denervated skeletal
muscle and TTX-treated myotubes. Forskolin did not alter
transcriptional activity of the
,
,
and
subunit genes. Taken together, these results suggest that the
cAMP pathway may not be a major regulator of DHP receptor expression in
skeletal muscle.
Forskolin had a pronounced inhibitory effect on the level of RY receptor mRNA and expression of the sarcoplasmic reticular ryanodine binding sites, suggesting that the inhibitory effect of forskolin on the RY receptor binding sites was mediated through down-regulation of the transcript. An inhibition of the RY receptor mRNA level was also observed in denervated muscle. Thus elevated cAMP levels may contribute toward the regulation of RY receptor message level. Forskolin, however, did not have any effect on the induction of the RY receptor gene. This suggests that forskolin, and consequent elevated cAMP levels, might be acting by destabilizing the RY receptor transcript. A similar mechanism has been suggested in the down-regulation of the AChE mRNA in skeletal myotubes in culture (Luo et al., 1994). In cultured myotubes, where the electrical activity was depressed with TTX or bupivacaine, there was no change in RY receptor mRNA levels. While we do not know the precise reason for these results, it is possible that the elevation in cAMP levels in TTX-treated myotubes was not sufficient to inhibit RY receptor mRNA levels. It should be noted that, unlike the nAChR and voltage-sensitive sodium channel, the expression of RY receptor mRNA was not induced by blocking sarcolemmal electrical activity and elevated cAMP levels, but was repressed instead.
It has been shown that spontaneous and
electrical stimulation-induced muscle contractions are preceded by
membrane depolarization and activation of protein kinase C (Vergara et al., 1985; Huang et al., 1992), which can modulate
the expression of several muscle-specific genes (Klarsfeld et
al., 1989; Huang et al., 1992; Walke et al.,
1994). Our results demonstrated that TPA, the protein kinase C
activator, increased the level of both DHP receptor subunit and RY receptor transcripts and the respective protein
levels. Since staurosporine, a protein kinase C antagonist, antagonized
TPA-induced responses, we can rule out the possibility that TPA was
acting by down-regulating protein kinase C. Staurosporine also
inhibited the mRNA and protein levels of
subunit and
RY receptor in developing cultured myotubes, at a time when the
endogenous protein kinase C is maximally activated, suggesting that
protein kinase C may play a role in regulation of these transcripts.
Since TPA did not increase transcriptional activity of the DHP receptor
subunit and RY receptor genes, and both TPA and staurosporine responses
were limited only to the
subunit message of the DHP
receptor, it is possible that TPA was acting by modulating the
stability of the
subunit and RY receptor transcripts.
Consistent with this notion, TPA response elements were not detected in
the promoter region of the RY receptor gene. (
)In this
regard, mRNA stabilization has been suggested to play an important role
in the regulation of the AChE mRNA level both during myogenesis and
development (Fuentes and Taylor, 1993).
Despite having a similar onset of induction, the DHP and RY receptor transcripts follow temporally distinct courses to reach their respective peaks. While the DHP receptor subunits reached their peak before the appearance of spontaneous contractility, the RY receptor message began to accumulate when myotubes were spontaneously contracting. This may imply that mRNAs of these receptors are regulated by distinct endogenous factors. Endogenous cAMP can be one such factor. We have shown that the RY receptor transcript level was lower in denervated muscle where basal cAMP levels have been reported to be elevated (Chahine et al., 1993). We have also found that elevation of cAMP levels in myotubes markedly inhibited the RY receptor message without altering transcriptional activity of the gene. Thus it is possible that at the early stages of muscle development the RY receptor transcript is destabilized by high cAMP levels. Subsequently, as myotubes mature and begin to contract spontaneously, activation of protein kinase C (Vergara et al., 1985; Huang et al., 1992) and/or down-regulation of cAMP may stabilize the RY receptor message. Consistent with this we have found that TPA increased RY receptor message level.
In conclusion, in this study we show that nerve plays
an important role in the regulation of DHP and RY receptor genes.
Nerve-derived factor(s) can enhance transcriptional activity of DHP and
RY receptor subunit genes and their respective mRNA levels. DHP
receptor subunit messages are insensitive to muscle denervation and
muscle inactivity while RY receptor mRNA is down-regulated by
denervation of skeletal muscle. TPA and cAMP can also modulate
transcript levels of the subunit of DHP receptor and
RY receptor by a transcription independent mechanism.