Role of adrenoceptors and camp on the catecholamineinduced inhibition of proteolysis in rat skeletal muscle

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1 Am J Physiol Endocrinol Metab 280: E663 E668, Role of adrenoceptors and camp on the catecholamineinduced inhibition of proteolysis in rat skeletal muscle LUIZ CARLOS C. NAVEGANTES, NEUSA M. Z. RESANO, RENATO H. MIGLIORINI, AND ÍSIS C. KETTELHUT Department of Physiology and Biochemistry, School of Medicine, University of São Paulo, Ribeirão Preto, São Paulo, Brazil Received 18 January 2000; accepted in final form 25 April 2000 Navegantes, Luiz Carlos C., Neusa M. Z. Resano, Renato H. Migliorini, and Ísis C. Kettelhut. Role of adrenoceptors and camp on the catecholamine-induced inhibition of proteolysis in rat skeletal muscle. Am J Physiol Endocrinol Metab 280: E663 E668, 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 , 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 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 Address for reprint requests and other correspondence: Í. C. Kettelhut, Dept. of Biochemistry, School of Medicine, University of São Paulo, Ribeirão Preto, SP, Brazil ( address: idckette@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. Section 1734 solely to indicate this fact /01 $5.00 Copyright 2001 the American Physiological Society E663

2 E664 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. MATERIALS AND METHODS 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 for 1 day before the beginning of the experiments. Rats of similar body weight (65 70 g) were used in all experiments, which were performed at 8:00 AM. Drugs (-)-Epinephrine; (-)-arterenol; (-)-isoproterenol; phenylephrine; clenbuterol; (-)-propranolol; prazosin; yohimbine; atenolol; ( )-1-[2,3-(dihydro-7-methyl- 1 H-inden-4-yl)oxy]-3-[(1- methylethyl)amino]-2-butanol (ICI ); 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 Effect of Catecholamines on Muscle Proteolysis As shown in Fig. 1A, the addition of 10 8,10 6,or 10 4 M epinephrine to the incubation medium of soleus muscles reduced the rate of tyrosine release by 9, 12, and 15%, respectively. A similar antiproteolytic effect was observed when soleus muscles were incubated with norepinephrine, except that the lowest norepi- Muscle 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% O 2-5% CO 2, 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). Fig. 1. Effect of epinephrine (A) or norepinephrine (B) at different concentrations on rate of proteolysis of rat soleus and extensor digitorum longus (EDL) muscles. Data (means SE of 7 9 animals) are % of control rates, obtained in the absence of the agonists. Rates of tyrosine release of control values for soleus and EDL averaged, respectively (nmol tyrosine mg wet wt 1 2h 1 ): epinephrine experiments, and ; norepinephrine experiments, and * P 0.05 vs. controls.

3 E665 Fig. 3. Effect of propranolol (A) or prazosin yohimbine (B) at different concentrations on the inhibition of proteolysis induced by 10 4 M epinephrine in rat soleus and EDL muscles. Data (means SE of 7 9 animals) are % of control rates, obtained in absence of epinephrine or antagonists. Rates of tyrosine release of control values for soleus and EDL averaged, respectively (nmol tyrosine mg wet wt 1 2 h 1 ): propranolol experiments, and ; prazosin yohimbine experiments, and * P 0.05 vs. controls. Fig. 2. Effect of isoproterenol (A) or clenbuterol (B) at different concentrations on rate of proteolysis of rat soleus and EDL muscles. Data (means SE of 7 9 animals) are % of control rates, obtained in absence of the agonists. Rates of tyrosine release of control values for soleus and EDL averaged, respectively (nmol tyrosine mg wet wt 1 2h 1 ): isoproterenol experiments, and ; clenbuterol experiments, and * P 0.05 vs. controls. nephrine concentration (10 8 M) was ineffective (Fig. 1B). In EDL muscles, a 22 25% decrease in proteolysis was obtained with the two highest concentrations of epinephrine (10 6 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 An -adrenergic agonist (phenilephrine) and a -adrenergic agonist (isoproterenol) were used in these experiments. In both soleus and EDL muscles, a 12 18% decrease in the rate of total protein degradation was observed after addition of 10 6 and 10 4 M isoproterenol to the incubation medium (Fig. 2A). Phenylephrine (10 8 and 10 6 M) did not affect tyrosine release by soleus or EDL muscles (data not shown). Higher concentrations of phenylephrine interfered with the fluorometric method used for tyrosine determination. As shown in Fig. 2B, clenbuterol, a selective 2 -adrenoceptor agonist, caused a dose-dependent reduction of tyrosine release in soleus and EDL muscles. At the highest concentration used (10 4 M), clenbuterol induced a 27% decrease in proteolysis in both muscles. 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 4 M) in the absence or presence of different concentrations (10 8,10 6, and 10 5 M) of antagonists. Previous experiments showed that proteolysis was not affected by the isolated addition to the incubation medium of any of the antagonists, at the doses used. As shown in Fig. 3A, 10 5 M propranolol, a nonselective -antagonist, completely inhibited the fall in tyrosine release induced by epinephrine in both soleus and EDL muscles. No effect on epinephrine-induced reduction in proteolysis was observed when skeletal muscles were incu-

4 E666 bated with prazosin and yohimbine ( 1 - and 2 -adrenergic antagonists, respectively; Fig. 3B). The selective 2 -adrenoceptor antagonist ICI (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). 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 6 or 10 8 M had no effect on proteolysis (data not shown). However, the rate of tyrosine release into the incubation medium by soleus (Fig. 6A) and EDL muscles (Fig. 6B) was significantly reduced by higher concentrations of DBcAMP (10 3 M), as well as by the xanthine derivative, IBMX (10 3 M), which inhibits phosphodiesterase and increases Fig. 5. Effect of ICI at different concentrations on inhibition of proteolysis induced by 10 5 M clenbuterol in rat soleus and EDL muscles. Data (means SE of 7 9 animals) are % of control rates, obtained in the absence of clenbuterol or antagonist. Rates of tyrosine release of control values for soleus and EDL averaged, respectively (nmol tyrosine mg wet wt 1 2h 1 ): and * P 0.05 vs. controls. the intracellular concentration of camp in skeletal muscle (27). The antiproteolytic effect was especially marked in the experiments with IBMX, which induced a 40 48% decrease in the rate of proteolysis in both soleus and EDL muscles. The data in Fig. 6 also show that no further inhibition of tyrosine release by DBcAMP or IBMX was obtained when 10 5 M clenbuterol was added to the incubation medium of soleus (Fig. 6A) or EDL (Fig. 6B) muscles. Fig. 4. Effect of atenolol (A) or ICI (B) at different concentrations on inhibition of proteolysis induced by 10 4 M epinephrine in rat soleus and EDL muscles. Data (means SE of 7 9 animals) are % of control rates, obtained in the absence of epinephrine or antagonists. Rates of tyrosine release of control values for soleus and EDL averaged, respectively (nmol tyrosine mg wet wt 1 2 h 1 ): atenolol experiments, and ; ICI experiments, and * P 0.05 vs. controls. Fig. 6. Effect of 10 3 M IBMX, 10 3 M dibutyryl camp (DBcAMP), 10 5 M clenbuterol (Clenb), 10 5 M Clenb 10 3 M DBcAMP or 10 5 M Clenb 10 3 IBMX on rate of proteolysis in rat soleus (A) and EDL (B) muscles. Values are means SE of 7 8 muscles. * P 0.05 vs. controls.

5 E667 DISCUSSION 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 l (Fig. 4B). Clenbuterol, a selective 2 -adrenergic agonist, induced a dose-dependent inhibition of proteolysis (Fig. 2B) that was also prevented by ICI 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 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 (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 ( M) and inhibiting this process at higher concentrations ( M) (16). Preliminary results from our laboratory show that BRL does not affect muscle proteolysis at the concentrations of 10 11,10 9,or10 8 M but induces a 15% decrease in tyrosine release in both soleus and EDL at higher concentrations ( M) that is completely reversed by 2 -adrenoceptor antagonist ICI 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 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 Ca 2 -dependent pathway inhibited (20). A close association between adrenergic activity and Ca 2 -dependent proteolysis has also been obtained in numerous studies showing that the activity and gene expression of -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-ubiquitindependent 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.

6 E668 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. 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. 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 /91 6). During this study, L.C.C. Navengantes received a fellowship from FAPESP (98/ ). REFERENCES 1. Baracos V and Goldberg AL. 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