Department of Physiology and Biochemistry, School of Medicine, University of São Paulo, 14049-900 Ribeirão Preto, São Paulo, Brazil
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ABSTRACT |
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The role of adrenoceptor
subtypes and of cAMP on rat skeletal muscle proteolysis was
investigated using a preparation that maintains tissue glycogen stores
and metabolic activity for several hours. In both soleus and extensor
digitorum longus (EDL) muscles, proteolysis decreased by 15-20%
in the presence of equimolar concentrations of epinephrine,
isoproterenol, a nonselective -agonist, or clenbuterol, a selective
2-agonist. Norepinephrine also reduced proteolysis but
less markedly than epinephrine. No change in proteolysis was observed
when muscles were incubated with phenylephrine, a nonselective
-agonist. The decrease in the rate of protein degradation induced by
10
4 M epinephrine was prevented by 10
5 M
propranolol, a nonselective
-antagonist, and by 10
5 M
ICI 118.551, a selective
2-antagonist. The
antiproteolytic effect of epinephrine was not inhibited by prazosin or
yohimbine (selective
1-and
2-antagonists,
respectively) or by atenolol, a selective
1-antagonist.
Dibutyryl cAMP and isobutylmethylxanthine reduced proteolysis in both
soleus and EDL muscles. The data suggest that catecholamines exert an
inhibitory control of skeletal muscle proteolysis, probably mediated by
2-adrenoceptors, with the participation of a
cAMP-dependent pathway.
epinephrine; clenbuterol; dibutyryl cAMP; isobutylmethylxanthine; protein degradation
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INTRODUCTION |
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IT IS WELL
ESTABLISHED from receptor-binding studies that the
2-adrenoceptor is the predominant
-adrenoceptor
subtype in skeletal muscle (10, 18), which may also
contain about 7-10%
1-adrenoceptors
(14) and a sparse population of
1-adrenoceptors (24). Recent studies
(4, 16) indicate that skeletal muscle may also be a target
for
3-adrenoceptor agonists, but no clear molecular
evidence has been provided for expression of this adrenoceptor in rat
skeletal muscle (9). Although the distribution of
1-,
1-, and
2-adrenoceptors in mammalian skeletal muscle has been well characterized, the physiological role of adrenoceptor subtypes on
protein metabolism remains unclear. Numerous studies, reviewed in Refs.
19, and 34, show that the administration of
2-adrenergic agonists, e.g., clenbuterol and cimaterol, increases skeletal muscle
mass in many species of animals. It has been reported that treatment
with clenbuterol induces a 10-20% increase in muscle weight in
normal rats (25) and reduces muscle waste in rats with
atrophy denervation (35), deprived of food
(5), and in hepatoma Yoshida AH-130-bearing rats
(8). Although the precise biochemical mechanism is not
known, the anabolic action of
2-adrenergic agonists in
vivo seems to be due, at least in part, to a reduction in the rate of
muscle protein breakdown. Indeed, epinephrine infusion into healthy
humans has been shown to induce a decrement in the rate of whole body
proteolysis that is still evident when endogenous insulin secretion is
blocked by somatostatin infusion (3). Also, we have
recently shown that guanethidine-induced adrenergic blockade increases
the rate of total protein degradation in rat soleus muscle after 2 days
of treatment (20). In contrast to the in vivo experiments,
which suggest that catecholamines exert an antiproteolytic action, the
experiments on the in vitro effect of adrenergic agonists on isolated
muscle preparations have produced contradictory results. It was found
that epinephrine can induce an increase in total protein degradation in
the rat epitrochlearis muscle during acute contractions in vitro
(21). On the other hand, a reduction in the release of
amino acids by epinephrine has been reported in perfused hemicorpus in
rats (15), but no effect of the catecholamine was detected
in perfused rat hindquarter (17).
It is well known that most of the catecholamine actions on glycogen and
lipid metabolism in several tissues, including muscle, are exerted
through a -adrenoceptor-mediated increase in intracellular cAMP and
subsequent activation of specific protein kinases. It has been recently
shown, using different
-adrenergic agonists in vitro, that, in rat
skeletal muscle, the increase in intracellular cAMP is mediated by
2- but not
3-adrenoceptors
(27). However, in contrast to glycogen and lipid
metabolism, the participation of cAMP in the regulation of skeletal
muscle protein metabolism has not been hitherto upheld by the
demonstration of direct effects of this cyclic nucleotide.
The present study examines the in vitro effects of different - and
-adrenergic agonists and antagonists on the rate of proteolysis, using a preparation of isolated rat skeletal muscles incubated at their
normal length under conditions that preserve the glycogen stores and
maintaining ATP and phosphocreatine levels and metabolic activity of
the incubated intact muscles for several hours, which has been shown to
be very adequate in estimating muscle proteolytic activity in several
situations (1, 12). The in vitro effects on muscle
proteolysis of dibutyryl cAMP (DBcAMP) and of isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, were also investigated.
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MATERIALS AND METHODS |
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Animals
Because the incubation procedure required intact muscles sufficiently thin to allow an adequate diffusion of metabolites and oxygen, young rats were used in all experiments. Male Wistar rats were housed in a room with a 12:12-h light-dark cycle and were given free access to water and normal lab chow diet forMuscle Proteolysis Studies
Incubation procedure. Rats were killed by cervical dislocation for muscle excision. The soleus and extensor digitorum longus (EDL) were rapidly dissected, care being taken to avoid damaging the muscles. Soleus muscles were maintained at approximately resting length by pinching their tendons in aluminum wire supports, and EDL muscles were maintained by pinning them on inert plastic supports. Tissues were incubated at 37°C in Krebs-Ringer bicarbonate buffer, pH 7.4, equilibrated with 95% O2-5% CO2, containing glucose (5 mM) and ascorbic acid (10 mM), and in the presence of cycloheximide (0.5 mM) to prevent protein synthesis and the reincorporation of tyrosine back into proteins. After a 1-h equilibration period, in the absence or presence of adrenergic agonists and/or antagonists, tissues were incubated for 2 h in fresh medium of identical composition.
Measurement of rates of protein degradation. The rate of proteolysis was determined by measuring the rate of tyrosine release in the incubation medium. Tyrosine was assayed as previously described (32). Muscle cannot synthesize or degrade tyrosine, and preliminary experiments showed that the intracellular pools of tyrosine of muscles incubated in the presence of adrenergic agonists or antagonists were not significantly affected by all the incubation conditions used here. Therefore, rates of amino acid release into the medium reflect rates of protein degradation.
Notwithstanding the absence of amino acids and the presence of an inhibitor of protein synthesis in the incubation medium, changes in proteolytic rates of this isolated muscle preparation have been shown to qualitatively reflect changes that occur in vivo in several situations, such as fasting (12) and diabetes (22). The preparation has also been found capable of reproducing in vitro, after addition of dexamethasone to the incubation medium, the proteolytic effect of the hormone in vivo (33).Drugs
(-)-Epinephrine; (-)-arterenol; (-)-isoproterenol; phenylephrine; clenbuterol; (-)-propranolol; prazosin; yohimbine; atenolol; (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol (ICI 118.551); DBcAMP; and IBMX were purchased from Sigma (St. Louis, MO).Statistical Methods
Data are means ± SE. Means from different groups were analyzed using Student's t-test. Multiple comparisons were made by using one- or two-way ANOVA followed by Bonferroni t-test. P < 0.05 was taken as the criterion of significance. ![]() |
RESULTS |
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Effect of Catecholamines on Muscle Proteolysis
As shown in Fig. 1A, the addition of 10
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In EDL muscles, a 22-25% decrease in proteolysis was obtained
with the two highest concentrations of epinephrine (106
and 10
4 M; Fig. 1A). Norepinephrine induced a
small (9%) but statistically significant reduction in the rate of
tyrosine release by EDL only at the highest concentration
(10
4 M; Fig. 1B).
Effect of - and
-Adrenergic Agonists on Muscle
Proteolysis
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Effect of Different Antagonists on the Antiproteolytic Adrenergic Action
In an attempt to determine the adrenoceptor subtypes involved in the inhibition of amino acid release by catecholamines, skeletal muscles were incubated with the highest dose of epinephrine used (10
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The selective 2-adrenoceptor antagonist ICI 118.551 (10
5 M) suppressed the antiproteolytic effect of
epinephrine (10
4 M; Fig.
4B) and clenbuterol
(10
5 M; Fig. 5) in both
muscles. Skeletal muscles incubated with epinephrine were also exposed
to different concentrations (10
8, 10
6, and
10
5 M) of atenolol, a selective
1-adrenoceptor antagonist. Unlike the
2-adrenoceptor antagonist, atenolol did not prevent the
reduction in protein degradation induced by epinephrine in soleus and
EDL muscles (Fig. 4A).
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Effect of DBcAMP and IBMX on Muscle Proteolysis.
The results of these experiments were strikingly similar in the two muscles (Fig. 6). Addition of DBcAMP at concentrations of 10
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DISCUSSION |
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The data of the present work clearly show that catecholamines exert an inhibitory action on proteolysis in rat isolated skeletal muscle. The epinephrine-induced reduction in the rate of protein breakdown in isolated muscles (~15-20%) was similar to that observed after epinephrine infusion in humans (30), in rat perfused hindlimb (11), and in muscle microdialysis experiments (28). An inhibitory action of catecholamines on skeletal muscle proteolysis was also suggested in a recent study from this laboratory (20) showing that rates of protein degradation in isolated muscles increase after acute chemical sympathectomy induced by guanethidine.
The findings that the antiproteolytic effect of epinephrine (Fig.
1A), which acts more selectively on -adrenoceptors, was more marked than that of norepinephrine (Fig. 1B) and that
isoproterenol (Fig. 2A) but not phenylephrine induced a
reduction in muscle proteolysis similar to that of epinephrine strongly
suggest that the inhibitory effect of catecholamines is mediated by
-adrenergic mechanisms. In consonance with these results,
isoproterenol has been shown to inhibit protein degradation in isolated
rat hemicorpus (15). The finding in this study that soleus
is more sensitive than EDL to the anticatabolic effect of epinephrine
(Fig. 1A) may be accounted for by differences in the density
of
-adrenoceptors, which has been shown to be twice as high in rat
skeletal muscles rich in type I fibers (e.g., soleus) than in muscles
containing mainly type II fibers (e.g., EDL; Refs. 10 and 14).
The present data provide strong evidence indicating that inhibitory
effects on skeletal muscle proteolysis are mediated by 2-adrenoceptors. Thus the antiproteolytic effect of
epinephrine in soleus and EDL was completely suppressed by propranolol
(Fig. 3A) and by the
2-adrenoceptor
antagonist ICI 118.55l (Fig. 4B). Clenbuterol, a selective
2-adrenergic agonist, induced a dose-dependent inhibition of proteolysis (Fig. 2B) that was also prevented
by ICI 118.551 in both muscles (Fig. 5). Another
2-adrenergic agonist, cimaterol, has also been found to
inhibit protein degradation in chick skeletal muscle (26)
and in rat myocardium in vitro (31). These data therefore
support the idea that the in vivo hypertrophic effect of clenbuterol
and cimaterol on skeletal muscle of different species (19)
is due, at least in part, to a reduction in muscle protein breakdown,
probably mediated by
2-adrenoceptors, as inferred from
the suppression by oral ICI 118.551 of the clenbuterol-induced hypertrophy of rat gastrocnemius muscles (6). Although in
vivo studies indicate that pale muscles, such as EDL, are more
responsive to the hypertrophic effect of
-adrenoceptor agonists than
red muscles (e.g., soleus; Ref. 34), we could not find any
statistically significant difference in the reduction of proteolysis
induced by clenbuterol in the two muscle types.
Recent studies (4, 16) suggest that skeletal muscle may be
also a target for 3-adrenergic agonists. BRL-37344
(BRL), a
3-adrenoceptor agonist, has been found to have
a biphasic effect on glucose utilization by isolated soleus and EDL,
increasing the uptake of the hexose at low concentrations
(10
11-10
9 M) and inhibiting this process
at higher concentrations (10
6-10
5 M)
(16). Preliminary results from our laboratory show that BRL does not affect muscle proteolysis at the concentrations of 10
11, 10
9, or 10
8 M but
induces a 15% decrease in tyrosine release in both soleus and EDL at
higher concentrations (10
6-10
5 M) that is
completely reversed by
2-adrenoceptor antagonist ICI
118.551 in both muscles (data not shown). These data suggest that the
inhibitory effect of BRL on muscle proteolysis is due to nonspecific
2-adrenoceptor-mediated effects of the higher doses of
the agonist and are in agreement with the findings that ICI 118.551 can
also reverse the inhibition of glucose utilization in soleus and EDL
induced by high concentrations of BRL (16). If this view
is correct, and if it assumed that the antiproteolytic effect of
catecholamines and other
-adrenergic agonists requires the mediation
of c-AMP, the lack of effect of the lower concentrations of BRL on
muscle proteolysis is to be expected, given the evidence that
3-adrenoceptors are not coupled to adenylate cyclase in rat skeletal muscle (27).
The present results clearly show that the proteolytic activity of rat
skeletal muscle is markedly decreased by DBcAMP or by IBMX (Fig. 6),
which may increase the intracellular concentration of cAMP in skeletal
muscle (27). No additive effects on proteolysis were
observed when clenbuterol and DBcAMP or IBMX were incubated together
(Fig. 6), suggesting that the inhibitory action of
2-adrenoceptor on skeletal muscle proteolysis is
mediated by cAMP. In agreement with this hypothesis, it has been shown
that daily administration of pentoxifylline, a xanthine derivative,
prevents muscle atrophy and suppresses increased muscle protein
breakdown in Yoshida sarcoma-bearing rats (7). These
results suggest that catecholamines inhibit skeletal muscle proteolysis
by activating cAMP-dependent protein kinases that in turn inactivate,
by phosphorylation, muscle proteolytic systems. Although the available
data do not allow any conclusion about the identity of these systems,
we have recently provided evidence for the existence of an inhibitory
adrenergic tonus in skeletal muscle that restrains proteolysis by
keeping the Ca2+-dependent pathway inhibited
(20). A close association between adrenergic activity and
Ca2+-dependent proteolysis has also been obtained in
numerous studies showing that the activity and gene expression of
µ-calpain and calpastatin, its endogenous inhibitor, are decreased
and increased, respectively, after
-adrenergic agonist treatment
(2, 13) and that the two calpains and calpastatin can be
phosphorylated by several kinases in different rat tissues, including
skeletal muscle (23, 29). That the ATP-ubiquitin-dependent
proteolytic system may also be involved in the action of catecholamines
is suggested by recent studies showing that the hyperactivation of this
system that occurs in skeletal muscle of tumor-bearing rats is
effectively reduced by clenbuterol (8) or pentoxifylline (7) treatment.
In summary, the present work shows that catecholamines and
-adrenoceptor agonists decrease in vitro the rate of proteolysis in
soleus and EDL muscles of rats, probably through an activation of
2-adrenoceptors. DBcAMP and IBMX also decreased
the rate of proteolysis in both muscles, suggesting the participation
of cAMP in the antiproteolytic action of catecholamines in rat skeletal muscle.
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ACKNOWLEDGEMENTS |
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The authors thank Elza Aparecida Filippin, Maria Antonieta R. Garófalo, José Roberto de Oliveira, and Victor Diaz Galbán for technical assistance, and Dr. Fernando Morgan de Aguiar Corrêa for careful reading and discussion of the manuscript.
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FOOTNOTES |
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This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 97/3950-5) and from the Conselho Nacional de Pesquisa (CNPq 501252/91-6). During this study, L.C.C. Navengantes received a fellowship from FAPESP (98/02591-4).
Address for reprint requests and other correspondence: Í. C. Kettelhut, Dept. of Biochemistry, School of Medicine, University of São Paulo, 14049-900 Ribeirão Preto, SP, Brazil (E-mail address:idckette{at}fmrp.usp.br).
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 18 January 2000; accepted in final form 25 April 2000.
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