Amelioration of denervation-induced atrophy by clenbuterol is associated with increased PKC-alpha activity

A. A. Sneddon, M. I. Delday, and C. A. Maltin

The Rowett Research Institute, Bucksburn, Aberdeen, Scotland AB21 9SB


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (alpha , varepsilon , theta , zeta , and µ) detected in soleus muscle by Western immunoblotting, clenbuterol treatment affected only the PKC-alpha and PKC-theta forms. PKC-alpha was translocated to the membrane fraction upon denervation, and the presence of clenbuterol increased membrane-bound PKC-alpha and active PKC-alpha as assayed by Ser657 phosphorylation. PKC-theta protein was downregulated upon denervation, and treatment with clenbuterol further decreased both cytosolic and membrane levels. Immunolocalization of PKC-theta showed differences for regulatory and catalytic domains, with the latter showing fast-fiber type specificity. The results suggest potential roles of PKC-alpha and PKC-theta in the mechanism of action of clenbuterol in alleviating denervation-induced atrophy.

skeletal muscle; beta -agonist; kinase activity


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DENERVATION-INDUCED ATROPHY has been used as a model to study the process of muscle wasting. Clenbuterol is one of a range of beta -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-alpha , -beta I, -beta II, and -gamma ) are Ca2+ dependent and diacylglycerol (DAG) activated; the novel PKC isoforms (nPKC-delta , -varepsilon , -eta , -theta , and -µ) are DAG activated but Ca2+ independent; and the atypical PKCs (aPKC-zeta and -lambda /iota ) 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-alpha , 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-alpha and nPKC-theta 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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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 beta -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-alpha , -beta , -gamma , -delta , -varepsilon , -µ, and -theta , Transduction Laboratories, Lexington KY; PKC-zeta and phospho-PKC-alpha , (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-alpha , Transduction Laboratories; PKC-theta , 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-theta , two primary antibodies were used, a rabbit polyclonal raised against amino acids 656-671 in the catalytic domain of mouse PKC-theta (Santa Cruz Biotechnology, Santa Cruz, CA) and a monoclonal antibody raised against amino acids 21-217 in the regulatory region of human PKC-theta (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-theta and the PKC-alpha 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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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta 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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Table 1.   Weight data from control and clenbuterol-treated animals with and without denervation

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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Fig. 1.   Total membrane protein kinase C (PKC) activity in innervated and denervated soleus muscle. Con, control diet; Clen, clenbuterol-containing diet; Den, control diet with unilateral denervation; Den Clen, Clen diet with Den; 3d, 3 day (groups 1-4); 7d, 7 day (groups 5-8). Values are presented as total membrane PKC activity as a percentage of the 3-day control values (means ± SE) and n = 4. * P < 0.05 and ** P < 0.01, Significance of the difference from the 3-day control value.

Detection of PKC Isoforms and Their Translocation in Soleus Muscle

Antibodies for eight PKC isoforms (alpha , beta , gamma , delta , varepsilon , theta , zeta , and µ) were used to determine the extent of PKC isoform expression in soleus muscle at the 3-day time point [nPKC-eta and aPKC-lambda /iota isoforms are not present in soleus muscle (29)]. Five PKC isoforms (alpha , varepsilon , theta , µ, and zeta ) were detected, whereas no signal was obtained with the anti-PKC-beta , -gamma , or -delta antibodies, despite a signal being obtained with the positive control.

PKC-alpha 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-alpha protein to the membrane, whereas clenbuterol treatment of denervated muscle resulted in the translocation of a consistently greater amount of PKC-alpha to the membrane fraction (Fig. 2). PKC-varepsilon 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-varepsilon 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-theta 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-theta protein in the cytosol (Fig. 2). Denervation resulted in a reduction of both the cytosolic and membrane PKC-theta protein, with a consistently greater reduction occurring in the cytosolic fraction. The level of PKC-theta protein in both fractions was further decreased in the clenbuterol-treated denervated muscle (Fig. 2). PKC-zeta 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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Fig. 2.   Western immunoblot analysis of PKC isoforms in soleus muscle. Co, control. Protein extracts from cytosol (C) and membrane (M) fractions from 3-day treatments were probed with antibodies to PKC-alpha , PKC-varepsilon , PKC-µ, PKC-theta , and PKC-zeta . The position of the 83-kDa molecular mass marker is indicated.

Detection of Phosphorylated PKC-alpha Protein in Soleus Membrane Fractions

An antibody that specifically recognizes PKC-alpha phosphorylated on Ser657 was used to detect the presence of phosphorylated PKC-alpha protein in membrane fractions at the 3-day time point. Equivalent amounts of phosphorylated PKC-alpha were detected in the membrane fraction of control and clenbuterol-treated innervated muscle (Fig. 3). Denervation of the muscle was associated with increased PKC-alpha phosphorylation, and drug treatment resulted in a higher level of phosphorylated PKC-alpha protein (Fig. 3).


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Fig. 3.   Western immunoblot analysis of phosphorylated PKC-alpha (PKC-alpha p) in soleus muscle. Inn, innervated. Protein extracts from membrane fractions from 3-day treatments were probed with anti-phospho-PKC-alpha Ser657 antibody. The position of the 83-kDa molecular mass marker is indicated.

Immunofluorescent Localization of PKC Isoforms in Soleus Muscle

Because both PKC-alpha and PKC-theta exhibited clenbuterol-induced alterations, immunofluorescent localization of these two isoforms was carried out in soleus muscle. PKC-alpha was located mainly at the sarcolemma of all fiber types, as described previously (11), and denervation was associated with an increased sarcolemmal staining of all fibers at 3 days (Fig. 4, A and B) and at 7 days (not shown). Treatment of denervated muscle with clenbuterol showed essentially the same staining pattern as in denervated muscle, with increased sarcolemmal staining compared with innervated muscle (Fig. 4C). The specificity of the PKC-alpha signal was demonstrated by the loss of staining when the antibody was preincubated with an excess of the immunogenic peptide (Fig. 4D). Two separate antibodies for PKC-theta localization were used, one against the amino-terminal regulatory domain and the other against the carboxy-terminal catalytic domain. Different distribution patterns were revealed for the different domains. The antibody against the regulatory region gave a strong signal only at certain sites on the muscle fiber membrane (Fig. 5, A-D) that were consistent with and presumed to be end plates (11). In addition, some fibers showed a more general sarcolemmal staining pattern (Fig. 5, A and B). Overall, there was generally stronger staining in the innervated muscle compared with denervated muscle (Fig. 5, A and B vs. C and D) but no obvious difference in levels of fluorescence or distribution pattern with clenbuterol treatment (Fig. 5, B and D vs. A and C). In contrast, the antibody raised against the catalytic domain of PKC-theta revealed a checkerboard pattern in soleus muscle (Fig. 6B). Examination of serial sections stained to demonstrate MHCf showed that fibers that gave a positive signal for the catalytic domain of PKC-theta were also positive for MHCf (Fig. 6, A and B). However, not all MHCf positive fibers gave a positive signal with the anticatalytic domain antibody. In particular, it was observed that intrafusal fibers in spindles, although staining positive with MHCf, did not show a positive reaction for the anticatalytic PKC-theta antibody (Fig. 6, A and B). Slow fibers were always negative for staining with the catalytic domain antibody. There was also inconsistent evidence of weak sarcolemmal staining with the PKC-theta catalytic domain antibody. In addition to muscle fibers, intramuscular nerves, motor end plates, and capillaries stained strongly with this antibody (not shown). The specificity of the signal was confirmed by the loss of signal when the antibody was preincubated with excess antigen (Fig. 6D).


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Fig. 4.   Immunolocalization of PKC-alpha in soleus muscle. A: transverse section of soleus muscle showing immunolocalization of PKC-alpha in 3-day innervated muscle. B: transverse section of soleus muscle showing immunolocalization of PKC-alpha in 3-day denervated muscle. C: transverse section of soleus muscle showing immunolocalization of PKC-alpha in 3-day denervated muscle treated with clenbuterol. D: a serial section to B reacted with PKC-alpha antibody that was preincubated with an excess of antigenic peptide.



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Fig. 5.   Immunolocalization of the regulatory subunit of PKC-theta in soleus muscle. A: transverse section of soleus muscle showing immunolocalization of the regulatory subunit of PKC-theta in 3-day innervated muscle. B: transverse section of soleus muscle showing immunolocalization of the regulatory subunit of PKC-theta in 3-day innervated muscle treated with clenbuterol. C: transverse section of soleus muscle showing immunolocalization of the regulatory subunit of PKC-theta in 3-day denervated muscle. D: transverse section of soleus muscle showing immunolocalization of the regulatory subunit of PKC-theta in 3-day denervated muscle treated with clenbuterol. E: a serial section to A reacted with antibody against the regulatory subunit of PKC-theta that was preincubated with an excess of antigen.



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Fig. 6.   Immunolocalization of the catalytic subunit of PKC-theta in soleus muscle. A: transverse section of 3-day innervated soleus muscle reacted to demonstrate the presence of the fast isoform of the myosin heavy chain (MHCf). B: immunochemical localization of PKC-theta in a section serial to A, with use of an antibody directed against the catalytic domain. C: a serial section to A reacted to demonstrate the activity of calcium-activated ATPase at pH 9.4 after methanol-free formalin fixation. D: a serial section to those in Figs. A-C reacted with antibody against the catalytic domain of PKC-theta that was preincubated with an excess of antigen.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (alpha , varepsilon , theta , zeta , and µ) in soleus muscle, in good agreement with the work of others (29). Three PKC isoforms (varepsilon , zeta , 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-alpha and PKC-theta exhibited clenbuterol-mediated changes, and these were restricted solely to denervated muscle. PKC-alpha 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-alpha and phosphorylation on Ser657. Both membrane translocation and phosphorylation of PKC correlate with activation (15, 16). In contrast, PKC-theta protein was downregulated upon denervation, as observed previously (12), and clenbuterol stimulated a further downregulation in PKC-theta protein levels. Therefore, activation of PKC-alpha 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-alpha 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-theta protein upon denervation and the further loss of protein with clenbuterol treatment may also correlate with increased protein synthesis, if PKC-theta 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-alpha protein, may be indicative of both the lesser amount of PKC-alpha translocated and the concomitant reduction of PKC-theta protein (and hence activity) in the membrane.

Localization of the regulatory domain of PKC-theta was consistent with previous reports of detection at the neuromuscular junction (11). PKC-theta was downregulated upon denervation, as assessed by both Western and immunofluorescent analysis. PKC-theta 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-theta displayed fast-fiber specificity in soleus muscle. This is consistent with previous Western analysis showing 2.5 times more PKC-theta 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-theta 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-theta 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-alpha and/or PKC-theta isoforms in the control of protein synthesis. Evidence for a link between PKC-alpha 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-alpha , -delta , or -varepsilon isoforms (34). Additionally, early work showing that the arachidonic acid metabolite prostaglandin-F2alpha is involved in the stimulation of translation by insulin (32), combined with the later observation that inhibition of PKC-alpha 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-alpha and downregulation of PKC-theta . 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.


    ACKNOWLEDGEMENTS

The authors are grateful to the staff of the Small Animal Unit for the care of the animals.


    FOOTNOTES

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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