The Rowett Research Institute, Bucksburn, Aberdeen, Scotland AB21 9SB
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ABSTRACT |
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Rat soleus muscle
was denervated for 3 or 7 days, and total membrane protein kinase C
(PKC) activity and translocation and immunocytochemical localization of
PKC isoforms were examined. Dietary administration of clenbuterol
concomitant with denervation ameliorated the atrophic response and was
associated with increased membrane PKC activity at both 3 (140%) and 7 (190%) days. Of the five PKC isoforms (,
,
,
, and µ)
detected in soleus muscle by Western immunoblotting, clenbuterol
treatment affected only the PKC-
and PKC-
forms. PKC-
was
translocated to the membrane fraction upon denervation, and the
presence of clenbuterol increased membrane-bound PKC-
and active
PKC-
as assayed by Ser657 phosphorylation. PKC-
protein was downregulated upon denervation, and treatment with
clenbuterol further decreased both cytosolic and membrane levels.
Immunolocalization of PKC-
showed differences for regulatory and
catalytic domains, with the latter showing fast-fiber type specificity.
The results suggest potential roles of PKC-
and PKC-
in the
mechanism of action of clenbuterol in alleviating denervation-induced atrophy.
skeletal muscle; -agonist; kinase activity
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INTRODUCTION |
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DENERVATION-INDUCED
ATROPHY has been used as a model to study the process of muscle
wasting. Clenbuterol is one of a range of -adrenergic agonists that
exhibit a muscle-specific anabolism and effectively increase protein
accretion in both wasting and normal muscle (3,
24, 25). Drug-induced increased protein accretion has been shown to result from both an initial increase in the
fractional rate of protein synthesis and a subsequent decrease in the
rate of protein degradation (21, 23).
Although the precise mechanism of clenbuterol action remains unknown,
several lines of evidence suggest that the drug may act by mimicking or interacting with a neural function(s). First, clenbuterol can reverse
the effects of denervation-induced atrophy (24),
apparently by both maintaining total RNA content and increasing mRNA
translational efficiency (22). Second, clenbuterol can
promote the precocious fusion of myoblasts both in vitro and in vivo
(20, 26), an event associated in vivo with
the onset of innervation. Third, clenbuterol administration, akin to
reinnervation, can repress the expression of the myogenic regulatory
factors myoD and myogenin and decrease acetylcholine receptor number in
denervated muscle (19). The coupling of membrane
excitation to acetylcholine receptor gene inactivation has been shown
to involve myogenin and to be mediated by changes in protein kinase C
(PKC) activity (13). Furthermore, previous studies have
also implicated PKC as an essential component in the regulation of
myoblast fusion (5).
The PKCs are a family of Ser/Thr kinases with a central role in the
transduction of signals elicited by external stimuli (17). They comprise at least eleven isoforms, which are closely structurally related but differ in their cofactor dependencies (18).
PKCs have been classified into three groups. The classical PKC isoforms (cPKC-, -
I, -
II, and -
) are Ca2+ dependent and
diacylglycerol (DAG) activated; the novel PKC isoforms (nPKC-
, -
,
-
, -
, and -µ) are DAG activated but Ca2+
independent; and the atypical PKCs (aPKC-
and -
/
) are both Ca2+-independent and DAG-non-responsive enzymes. The
activity of PKC can be monitored by directly measuring kinase activity
against a specific PKC substrate, in addition to examining the
translocation of specific isoforms from the soluble cytosolic fraction
where they appear inactive to the particulate membrane fraction where activation takes place (16). Moreover, PKC must be
phosphorylated before it is competent to respond to second messengers
(15). For cPKC-
, phosphorylation at Ser657
is required for full catalytic activation and stabilization of the
enzyme when bound to the membrane (10). Additionally, for some PKC isoforms, limited proteolysis can occur, resulting in a free
catalytic fragment that by itself can display distinct signaling roles,
including remodeling and phosphorylation of structural proteins
(27) and regulation of gene expression, as well as induction of apoptosis (4). Differences between tissue,
cellular and subcellular distributions, substrate specificities, and
cofactor dependencies of the PKC isoforms suggest distinct roles for
each in cellular regulation. At the neuromuscular junction, PKC has been shown to play an integral role in synapse formation and signal transduction (13, 14). cPKC-
and nPKC-
have been identified as the two most abundant isoforms in skeletal
muscle (28), and both appear localized at the nerve-muscle
interface (11), strongly suggesting a role(s) at this site.
Because a number of muscle-wasting conditions can be effectively reversed by treatment with clenbuterol, an investigation of the signaling events involved in the reversal of denervation-induced atrophy was carried out. We focused on PKC activity, because PKC has been shown to be intimately involved in nerve-muscle signal transduction, and the evidence suggested that clenbuterol may act by mimicking a function of the nerve.
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MATERIALS AND METHODS |
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Animals and Experimental Design
All animal experimental procedures were carried out under the auspices of the UK Home Office (Scientific Procedures) Act of 1986. Male rats of the Rowett hooded Lister strain were weaned at 19 days postpartum, were divided into eight groups of equal mean body weight, and were designated as follows: group 1, control diet for 3 days; group 2, clenbuterol-containing diet (3 mg/kg) for 3 days; group 3, control diet with unilateral denervation for 3 days; group 4, clenbuterol-containing diet (3 mg/kg) with unilateral denervation for 3 days; group 5, control diet for 7 days; group 6, clenbuterol-containing diet (3 mg/kg) for 7 days; group 7, control diet with unilateral denervation for 7 days; and group 8, clenbuterol-containing diet (3 mg/kg) with unilateral denervation for 7 days.The animals were accustomed to the nutritionally adequate semisynthetic
control diet (PW3: 30) until they were 22 days of age, when they were
housed individually and offered their experimental diet where
appropriate. At the same time, all the animals in groups 3,
4, 7, and 8 were subjected to
unilateral denervation by midsciatic section under halothane
anesthesia. The clenbuterol in the drug-containing diet was thoroughly
mixed into the diet to ensure even distribution, and animals had free
access to food and water at all times. Postoperatively, the animals
were maintained on their respective dietary regimens for 3 or 7 days
and then were weighed and killed by stunning and cervical dislocation.
The heart and the soleus muscles from both hindlimbs were quickly
removed, weighed, and frozen in liquid nitrogen. The samples were
stored at 70°C until analysis was carried out. Contralateral
innervated muscles from denervated animals were not used for any
analysis in this study.
PKC Activity Assay
Isolation of muscle membrane fractions.
Muscle samples (n = 4) were taken from storage at
70°C, rapidly crushed between supercooled aluminum blocks, and then
homogenized on ice for 30 s in buffer (2) containing
250 mM sucrose, 1 µg/ml leupeptin, 2 µg/ml aprotinin, and 1 µg/ml
pepstatin A. The homogenate was incubated for 15 min on ice and then
centrifuged at 500 g for 5 min at 4°C to remove debris and
nuclei. The resultant supernatant was spun at 100,000 g for
45 min at 4°C. The pellet was resuspended in 0.1-0.2 ml of the
above buffer with a hand-held homogenizer and was designated the
membrane fraction. The protein concentration of the membrane fraction
was assayed (BCA assay kit, Pierce, Rockford, IL) before assay of the
PKC activity.
Measurement of PKC activity in isolated membranes. PKC activity was measured in the membrane fraction (see above), as described elsewhere (2), with the following exceptions. Twenty-five micrograms of protein were used for the assay, which was carried out at 30°C for 15 min (conditions previously determined to give linear reaction rates; not shown). The reaction was terminated by spotting 90 µl onto P-81 Whatman paper and washing the paper 5 times for 10 min each in 0.5% phosphoric acid. After a final wash in acetone, the filters were air dried, and the bound radioactivity was measured by liquid scintillation (1900TR Packard counter) with Ultima Gold XR scintillation fluid (Packard, Groningen, Holland). For each assay, the background counts incorporated onto the P-81 paper in the absence of the peptide substrate were subtracted from the total counts measured in the presence of the substrate. Duplicate samples were analyzed for each assay, and the PKC activity measured was inhibited ~95% by the addition of the PKC-specific inhibitor Ro-31-8220 (15 nM, Calbiochem, San Diego, CA) or the PKC inhibitor peptide, RFARKGALRQKNVHEVKN (20 µM, Bachem, Torrance, CA).
Western immunoblotting analysis.
Whole soleus muscles (n = 4) were taken from 70°C,
crushed between two supercooled aluminum blocks, and then homogenized (Polytron) on ice for 30 s in 0.5 ml of 20 mM Tris, pH7.5, 0.25 M
sucrose, 10 mM EGTA, 2 mM EDTA, 1 mM sodium orthovanadate, 25 mM sodium
-glycerophosphate, 50 mM sodium fluoride, 1 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml pepstatin A. After incubation on ice for 1 h with intermittent mixing, the homogenate was spun at 100,000 g for 1 h at
4°C, and the supernatant, designated the cytosolic fraction, was
removed. The pellet was resuspended in the above buffer with the
addition of 1% Triton X-100 and incubated on ice with intermittent
mixing for 1 h. The homogenate was spun at 100,000 g
for 30 min at 4°C, and the supernatant, designated the membrane
fraction, was removed. Equal amounts (20 µg) of cytosolic and
membrane fractions were electrophoresed on SDS-polyacrylamide gels
(4.5% stacking gel and 9% running gel). After 1 h at 100 mA, the
gels were electrotransferred onto Immobilon-P membrane (Millipore,
Bedford, MA). The membrane was blocked for 1 h at room temperature
in TBST [Tris-buffered saline (TBS), pH 7.6, with 0.1% Tween-20]
containing 5% (wt/vol) dried milk (Marvel, Stafford, UK). Primary
antibody [PKC-
, -
, -
, -
, -
, -µ, and -
,
Transduction Laboratories, Lexington KY; PKC-
and phospho-PKC-
,
(Ser657), Upstate Biotechnology, Lake Placid, NY] was then
added at the manufacturer's recommended dilution in TBST + 5%
Marvel for 1 h at room temperature. Membranes were washed six
times for 5 min each in TBST and then incubated for 30 min in secondary
antibody conjugated to horseradish peroxidase (DAKO, Cambridge, UK).
After a further six washes as above in TBST, the membranes were left in
TBS, and chemiluminescent detection was carried out (Pierce, Rockford,
IL). Specificity of the bands was assessed by molecular mass markers
(New England Biolabs, Beverly, MA) and the inclusion of an appropriate
positive control from the manufacturer.
Immunocytochemistry.
Dissected soleus muscle midbelly portions (n = 4) were
oriented on cork for transverse sectioning and stored in liquid
N2 until analysis was carried out. Serial transverse
sections were cut at 6 µm and air dried for 30-45 min at room
temperature and then fixed in acetone for 10 min. The primary antibody
(PKC-, Transduction Laboratories; PKC-
, as stated below) was
applied at a dilution of 1:20 in PBS, pH 7.5, overnight at 4°C. This
was followed by three 10-min washes with PBS. The secondary antibody, anti-mouse or anti-rabbit fluorescein isothiocyanate (FITC), as appropriate (Sigma, Poole, UK), was applied at a 1:100 dilution in PBS,
pH 7.5, and incubated for 30-60 min at room temperature. The
slides were then washed as above and mounted in a Vectashield (Vector
Laboratories, Burlingame, CA), viewed on a Leica DMRBE fluorescence
microscope (Leica, Milton Keynes, UK), and photographed. For PKC-
,
two primary antibodies were used, a rabbit polyclonal raised against
amino acids 656-671 in the catalytic domain of mouse PKC-
(Santa Cruz Biotechnology, Santa Cruz, CA) and a monoclonal antibody
raised against amino acids 21-217 in the regulatory region of
human PKC-
(Transduction Laboratories). Two types of control were
employed in this study. Serial sections were incubated without primary
antibody in PBS alone and were therefore exposed only to the
FITC-labeled secondary antibody. In addition, for the rabbit PKC-
and the PKC-
antibodies, serial sections were incubated with primary
antibody that had been preincubated for 3 h at room temperature
with an excess of antigenic peptide (according to manufacturer's
instructions). This latter treatment completely blocked all fluorescent
signal, and little or no background fluorescence was observed with
secondary antibody alone. Serial cryostat sections were used to show
the localization of the fast isoform of the myosin heavy chain (MHCf)
(Nova Castra Laboratories, Newcastle-upon-Tyne, UK).
Ca2+-activated ATPase staining at pH 9.4 was carried out,
as previously described (24). Photographic exposure times
were the same for all images.
Statistical Analysis
The analysis of changes in PKC activity, immunocytochemical analysis, and Western analysis were carried out in soleus muscle with a minimum of four animals per group. For statistical comparison between groups, the data were analyzed by use of three-factor analysis of variance (innervation condition vs. drug treatment vs. day) with statistical significance set at the 5% probability level. ![]() |
RESULTS |
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Clenbuterol treatment did not affect total body weight but led to
increases in the weight of heart and skeletal muscle (Table 1). Increases in heart muscle weight
served as a control for the presence of the drug in the diets, because
clenbuterol, in common with other 2-agonists, causes
cardiac as well as skeletal muscle hypertrophy. Denervation for 3 or 7 days led to a reduction (P < 0.001) in the weight of
soleus muscle. Clenbuterol increased (P < 0.001)
muscle weight in denervated muscles after 3 and 7 days of treatment
(Table 1). In innervated muscle, although there was a numerical
increase in soleus weight from clenbuterol-treated animals at 3 days,
significance was reached only at 7 days (Table 1).
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Membrane PKC Kinase Activity in Atrophic and Clenbuterol-Treated Soleus Muscle
PKC activity was measured directly in isolated membranes where the active enzyme was present (16). Neither clenbuterol treatment alone nor denervation alone for 3 or for 7 days resulted in any change in total membrane PKC activity (Fig. 1). However, there was a significant increase (P < 0.05, 3 days; P < 0.01, 7 days) in membrane PKC activity in denervated muscle that had been treated with clenbuterol (Fig. 1).
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Detection of PKC Isoforms and Their Translocation in Soleus Muscle
Antibodies for eight PKC isoforms (PKC- was detected mainly in the cytosolic fraction in control
innervated muscle, and the presence of clenbuterol caused no change in
this distribution (Fig. 2). Denervation
of the muscle alone resulted in the translocation of PKC-
protein to
the membrane, whereas clenbuterol treatment of denervated muscle
resulted in the translocation of a consistently greater amount of
PKC-
to the membrane fraction (Fig. 2). PKC-
was found only in
the membrane and was not detected in the cytosol after denervation or
drug treatment or both (Fig. 2). PKC-µ appeared similar to PKC-
in that it was present in the membrane and not in the cytosolic fraction and in that denervation or drug did not alter its localization (Fig.
2). PKC-
was detected in both the cytosolic and membrane fractions
in the innervated muscle, and it was noted that clenbuterol treatment
consistently resulted in increased PKC-
protein in the cytosol (Fig.
2). Denervation resulted in a reduction of both the cytosolic and
membrane PKC-
protein, with a consistently greater reduction
occurring in the cytosolic fraction. The level of PKC-
protein in
both fractions was further decreased in the clenbuterol-treated
denervated muscle (Fig. 2). PKC-
was present in both cytosolic and
membrane fractions, and there was no detectable alteration in its
localization in any of the treatment groups (Fig. 2). Essentially
similar results were obtained at the 7-day time point (not shown).
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Detection of Phosphorylated PKC- Protein in Soleus Membrane
Fractions
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Immunofluorescent Localization of PKC Isoforms in Soleus Muscle
Because both PKC-
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DISCUSSION |
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In the present study, significant muscular atrophy was achieved at
both 3 and 7 days by denervation of the soleus muscle, and clenbuterol
treatment ameliorated the atrophy. Assay of total membrane-PKC activity
at those time points demonstrated a specific increase in PKC activity
exclusively in those muscles undergoing reversal of atrophy. Western
immunoblot analysis identified five PKC isoforms (,
,
,
,
and µ) in soleus muscle, in good agreement with the work of others
(29). Three PKC isoforms (
,
, and µ) showed no
alteration in their cytosol-to-membrane translocation that would be
consistent with the observed increase in membrane PKC activity. Only
PKC-
and PKC-
exhibited clenbuterol-mediated changes, and these
were restricted solely to denervated muscle. PKC-
was translocated
to the membrane fraction upon denervation, as assessed by both Western
and immunofluorescent analysis, and the presence of clenbuterol
increased the amount of membrane-bound PKC-
and phosphorylation on
Ser657. Both membrane translocation and phosphorylation of
PKC correlate with activation (15, 16). In
contrast, PKC-
protein was downregulated upon denervation, as
observed previously (12), and clenbuterol stimulated a
further downregulation in PKC-
protein levels. Therefore, activation
of PKC-
alone appeared to account for the increased membrane PKC
activity associated with the amelioration of denervation-induced atrophy by clenbuterol.
The atrophic response of muscle to denervation arises mainly as a
result of an increase in the rate of protein degradation (7). Although protein synthesis rates decrease initially
at 3 and 7 days after denervation, the protein synthesis rate actually then increases (7, 9). Therefore, the
translocation of PKC- to the membrane upon denervation, together
with the translocation of a much greater amount with drug treatment,
correlates well with the levels of increase in protein synthesis rates
shown to occur with each of these treatments at 3 days (7,
22). Equally, the downregulation of PKC-
protein upon
denervation and the further loss of protein with clenbuterol treatment
may also correlate with increased protein synthesis, if PKC-
was a
negative regulator of this process. The fact that there was no
measurable effect on total membrane PKC activity in the 3-day
denervated muscle, despite translocation of PKC-
protein, may be
indicative of both the lesser amount of PKC-
translocated and the
concomitant reduction of PKC-
protein (and hence activity) in the membrane.
Localization of the regulatory domain of PKC- was consistent with
previous reports of detection at the neuromuscular junction (11). PKC-
was downregulated upon denervation, as
assessed by both Western and immunofluorescent analysis. PKC-
protein levels were further reduced in the presence of clenbuterol,
suggesting that the signaling function of this neuromuscular PKC
isoform may be altered by both denervation and clenbuterol treatment. The catalytic domain of PKC-
displayed fast-fiber specificity in
soleus muscle. This is consistent with previous Western analysis showing 2.5 times more PKC-
mass per unit of total protein in tensor
fascia latae (a fast-twitch muscle) than in soleus (a predominantly slow-twitch muscle) (6). Differences in localization of
the two PKC-
domains may be explained by the finding that PKC can undergo limited proteolysis by calpain proteases to form separate regulatory and catalytic domains (33). Such cleavage would
be consistent with the observed increase in calpain activity after clenbuterol treatment (1) and may account for the
decreased levels of full-length PKC-
protein detected in the Western
immunoblot. Clenbuterol-promoted muscle anabolism can involve both
increases in protein synthesis and decreases in protein degradation
rates, with the relative contribution of each depending on the nerve status as well as the muscle type (21, 22,
23). In innervated muscle, clenbuterol promotes fiber
hypertrophy predominantly through a reduction in protein degradation
(21, 31), whereas in denervated soleus
muscle, the amelioration of atrophy occurs mainly through an increase
in the rate of protein synthesis with a lesser effect on proteolysis
(22). If the observed effects of clenbuterol on PKC are
indeed related to the anti-atrophic response, then because the effects
are restricted solely to denervated muscle, this may suggest the
involvement of PKC-
and/or PKC-
isoforms in the control of
protein synthesis. Evidence for a link between PKC-
and the control
of protein synthesis in muscle has been documented previously. In L6
myoblasts, the stimulation of protein translation has been shown to
involve one or more of the PKC-
, -
, or -
isoforms
(34). Additionally, early work showing that the
arachidonic acid metabolite prostaglandin-F2
is involved in the stimulation of translation by insulin (32),
combined with the later observation that inhibition of PKC-
expression inhibits the release of arachidonic acid (8),
implies the involvement of this isoform in the control of protein
synthesis. Further studies also implicate PKC activity in the
regulation of catabolic pathways in skeletal muscle. The activation of
PKC with the DAG mimetic 12-O-tetradecanoylphorbol
13-acetate both stimulated and inhibited myofibrillar protein
degradation in muscle myotubes, implicating one or more classical or
novel PKC isoforms in this response (18, 35).
In conclusion, the present study demonstrates that the alleviation of
muscle atrophy induced by clenbuterol is associated with activation of
PKC- and downregulation of PKC-
. The known involvement of PKC in
the regulation of both anabolic and catabolic pathways in muscle is
consistent with alterations in PKC signaling being important for the
anti-atrophic action of the drug.
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ACKNOWLEDGEMENTS |
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The authors are grateful to the staff of the Small Animal Unit for the care of the animals.
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FOOTNOTES |
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The authors are also grateful for the support of this study by the Scottish Executive Rural Affairs Department.
Address for reprint requests and other correspondence: A. Sneddon, The Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen, Scotland AB21 9SB (E-mail: aas{at}rri.sari.ac.uk).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 8 November 1999; accepted in final form 22 February 2000.
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