Overall proteolysis and the activity of skeletal muscle proteolytic systems were investigated in rats 1, 2, or 4 days after adrenodemedullation. Adrenodemedullation reduced plasma epinephrine by 95% and norepinephrine by 35% but did not affect muscle norepinephrine content. In soleus and extensor digitorum longus (EDL) muscles, rates of overall proteolysis increased by 15–20% by 2 days after surgery but returned to normal levels after 4 days. The rise in rates of protein degradation was accompanied by an increased activity of Ca2+-dependent proteolysis in both muscles, with no significant change in the activity of lysosomal and ATP-dependent proteolytic systems. In vitro rates of Ca2+-dependent proteolysis in soleus and EDL from normal rats decreased by ∼35% in the presence of either 10−5 M clenbuterol, a β2-adrenergic agonist, or epinephrine or norepinephrine. In the presence of dibutyryl cAMP, proteolysis was reduced by 62% in soleus and 34% in EDL. The data suggest that catecholamines secreted by the adrenal medulla exert an inhibitory control of Ca2+-dependent proteolysis in rat skeletal muscle, mediated by β2-adrenoceptors, with the participation of a cAMP-dependent pathway.
- dibutyryl cyclic adenosine monophosphate
- proteolytic systems
although the actions of catecholamines are usually associated with catabolic processes, promoting the breakdown of both glycogen and fat for use as fuel, a growing body of evidence suggests that they may have an antiproteolytic effect on muscle protein metabolism (13, 16, 23). However, the precise mechanism through which catecholamines produce this effect is not known.
Previous studies from this laboratory (18, 19) in rat skeletal muscles showed that short-term neuronal blockade with guanethidine induced time-dependent changes in skeletal muscle proteolysis, which increased after the first 2 days of treatment and reverted to levels below control values after 4 days (18). The early rise in overall proteolysis was accompanied by a rapid increase in the rate of Ca2+-dependent proteolysis, suggesting the existence of an inhibitory adrenergic tonus in skeletal muscle that restrains the activity of this proteolytic system (18). Because both plasma and muscle catecholamine levels were reduced by guanethidine treatment, we could not dissociate in these studies (18) the antiproteolytic effect of catecholamines secreted by the adrenal medulla from the effect of norepinephrine released directly by adrenergic innervation. One of the objectives of the present experiments was to assess overall proteolysis and the activity of the different proteolytic systems in skeletal muscle from rats adrenodemedullated a few days previously, a condition in which only plasma catecholamines are altered.
In our experiments with isolated rat muscles, we have also found that in vitro addition of epinephrine or norepinephrine induces a reduction in the rate of overall proteolysis (19) similar to that observed in human beings in vivo (8, 29) and in rat microdialysis studies (27). In agreement with these results, a close association between adrenergic activity and proteolysis has also been obtained in numerous other studies, showing that the activity and gene expression of Ca2+-dependent proteases are decreased after β2-adrenergic agonist treatment (2, 7, 12). Although these data have suggested that catecholamines inhibit Ca2+-dependent proteolysis by activating β2-adrenoceptors with the possible involvement of cAMP-dependent protein kinase (PKA), direct in vitro effects of β2-adrenergic agonists or of cAMP on the different muscle proteolytic systems have not yet been clearly demonstrated. In this respect, the only information available concerns in vitro effects of β-agonists on the activity of the lysosomal proteolytic system. Thus it has been reported that cimaterol decreases cathepsin B activity in myotubes (3) and in isolated muscles from the chicken (17) but that it has no effect on muscles from rats (15). On the other hand, other studies have shown that β-agonists may inhibit protein degradation in rat (31) and chicken muscles (25, 26), even in the presence of lysosomal activity inhibitors, suggesting that nonlysosomal pathways are responsible for the antiproteolytic effect.
The purpose of the present work was therefore twofold:1) to investigate the role of catecholamines released from the adrenal medulla in muscle protein metabolism by measuring the overall proteolysis and the activity of four proteolytic systems (lysosomal, Ca2+ dependent, ATP dependent, and ATP independent) in skeletal muscles obtained from rats adrenodemedullated 1–4 days before, and 2) to examine in skeletal muscles isolated from normal rats the in vitro effect of epinephrine, norepinephrine, clenbuterol, and dibutyryl cAMP (DBcAMP) on the activity of the proteolytic processes. The concentrations of muscle norepinephrine and of plasma catecholamines, corticosterone, insulin, and glucose in adrenodemedullated rats are also reported.
MATERIALS AND METHODS
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.
Adrenodemedullation and Muscle Proteolysis Studies
Adrenodemedullation was performed under ether anesthesia 1, 2, or 4 days before the animals were utilized in the experiments. The medulla of each adrenal was squeezed through a nick made on its capsula. The animals did not require saline as drinking water after surgery. Control rats were submitted to a sham operation in which the adrenals were visualized but not removed. Two days after adrenodemedullation, plasma levels (6 rats) of glucose (144 ± 3 mg/dl), insulin (1.1 ± 0.2 ng/ml), and corticosterone (2.2 ± 0.8 μg/dl) did not differ significantly from those of sham-operated animals (7 rats, 147 ± 3, 1.3 ± 0.1, and 2.4 ± 0.9, respectively). Also, no difference was observed in the body weight and the weight of soleus and extensor digitorum longus (EDL) muscles at any of the experimental periods (data not shown).
Rats were killed by cervical dislocation for muscle excision. The soleus and EDL were rapidly dissected, with 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 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 and containing glucose (5 mM) and in the presence of cycloheximide (0.5 mM) to prevent protein synthesis and the reincorporation of tyrosine back into proteins. Tissues were preincubated for 1 h in the buffer, and then incubated for 2 h in fresh medium of identical composition.
Measurement of rates of protein degradation.
The rates of overall proteolysis and of the different proteolytic systems were determined by measuring the rate of tyrosine release in the incubation medium. Because muscle cannot synthesize or degrade tyrosine, its release reflects the rate of protein breakdown. Tyrosine was assayed as previously described (32). Preliminary experiments showed that, as previously reported for normal animals (1), the intracellular pools of tyrosine of adrenodemedullated rats were not significantly affected by all of the incubation conditions used here. Therefore, rates of amino acid release into the medium reflect rates of protein degradation.
To measure the intralysosomal proteolysis, muscles from one limb were incubated in the absence of insulin and branched-chain amino acids, a condition in which the lysosomal process is activated. Contralateral muscles were incubated in the presence of insulin (1 U/ml), amino acids (leucine, 170 μM; isoleucine, 100 μM; valine, 200 μM), and methylamine (10 mM), a weak base that raises intralysosomal pH and inhibits lysosomal proteolysis (11). The difference in tyrosine release between the two muscles reflects the activity of the lysosomal proteolytic component.
To study the maximal activity of the Ca2+-dependent proteolysis, muscles from one limb were incubated in the presence of insulin and branched-chain amino acids (to block lysosomal process). Ca2+-dependent proteolysis was activated in the contralateral muscles by incubation in the presence of A-23187 (a Ca2+ ionophorum) or by allowing muscles to shorten during incubation (11). The difference in tyrosine release between the two muscles represents the Ca2+-dependent proteolytic process. Both methods were used in all experiments with similar results. Except for Fig. 4, values in the figures are those obtained with free shortening muscles.
In muscles maintained at resting length in the presence of insulin and amino acids, most protein breakdown occurs by a nonlysosomal Ca2+-independent process that requires ATP (11). To measure the ATP-dependent and energy-independent processes, muscles were first incubated under conditions that prevent activation of the lysosomal and Ca2+-dependent proteolytic systems by use of Ca2+-free medium and different inhibitors, including methylamine, insulin plus branched-chain amino acids, E-64, and leupeptin (11). The proteolytic activity measured in contralateral muscles incubated with dinitrophenol (DNP; 0.5 mM), 2-deoxyglucose (5 mM), and without glucose (to deplete them completely of intracellular ATP) must represent an ATP-independent proteolytic process. This residual process represents a distinct energy-independent degradative system and not just a failure to block completely the ATP-requiring process, because it varies in a distinct fashion (6, 11). The difference in tyrosine release between the two contralateral muscles (with and without ATP depletion) reflects the activity of the ATP-dependent proteolytic system.
Clenbuterol, Catecholamines, DBcAMP, and Muscle Proteolysis Studies
To investigate the in vitro effect of clenbuterol on the activity of the different proteolytic pathways, soleus and EDL muscles from normal rats were incubated in the presence of 10−5 M of this selective β2-adrenergic agonist by use of the same procedure already described. The in vitro effect of 10−4 M epinephrine, norepinephrine, and 10−3 M DBcAMP on the Ca2+-dependent proteolysis was also tested.
For the determination of catecholamine plasma levels, a group of rats was killed by decapitation 1, 2, and 4 days after adrenal medulla removal. Muscles (soleus and EDL) and plasma were stored at −70°C until assayed. Catecholamines were assayed as previously described (9) using HPLC (LC-7A, Shimadzu Instruments) with a Spherisorb ODS-2 (5 μm; Sigma-Aldrich) reversed-phase column.
Metabolites and hormone measurements.
In a group of adrenodemedullated and then decapitated rats, blood was collected over a 2-day period to determine the plasma concentrations of glucose, insulin, and corticosterone. Glucose concentration was determined with glucose oxidase by use of a glucose analyzer (Beckman). Hormone levels were determined by radioimmunoassay.
(−)-Epinephrine, (−)-arterenol, clenbuterol, (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)-amino]-2-butanol (ICI-118551), and DBcAMP were purchased from Sigma Chemical (St. Louis, MO).
Means of muscle samples from different groups of animals were analyzed using Student's nonpaired t-test. The pairedt-test was also used to compare the contribution of the proteolytic pathways. P < 0.05 was taken as the criterion of significance.
Plasma and Muscle Catecholamines
Adrenodemedullation induced a reduction of plasma epinephrine (95%) and norepinephrine (35%) concentration after 1, 2, or 4 days but did not affect the content of muscle norepinephrine during the same experimental period. Figure 1 shows the data for 2 days.
Proteolytic Activity and Adrenodemedullation
Skeletal muscle proteolysis in adrenodemedullated rats varied according to the time of surgery. In soleus and EDL muscles, a 15–20% increase in the rate of total protein degradation was observed 2 days after adrenal medulla removal (Fig.2). However, after 4 days, proteolysis in both muscles reverted to control values.
Lysosomal proteolytic activity in soleus and EDL at the three experimental intervals did not differ significantly in adrenodemedullated and sham-operated rats (Fig.3). Ca2+-dependent proteolytic activity in soleus and EDL muscles increased by 100% by 2 days after adrenal medulla removal (Fig. 3). However, by 4 days after surgery, the activity of this pathway returned to values similar to those of controls in both muscles. In both soleus and EDL, the activities of the ATP-dependent (Fig. 3) and of the ATP-independent (data not shown) proteolytic systems in adrenodemedullated rats did not differ significantly from those of control muscles at any of the experimental intervals.
In Vitro Effect of Clenbuterol, Catecholamines, and DBcAMP
Clenbuterol (10−5 M) added to the incubation medium of soleus or EDL muscles isolated from normal rats did not affect the activities of lysosomal, ATP-dependent, or ATP-independent proteolytic systems (Fig. 4) but reduced the rate of Ca2+-dependent proteolysis by 26 and 39%, respectively (Fig. 4). The decrease in proteolysis induced by clenbuterol in vitro was completely prevented by 10−5 M ICI-118551 (ICI), a selectice β2-adrenergic antagonist (Fig.5). As shown in Fig.6, the addition of 10−4epinephrine to the incubation medium of soleus and EDL muscles reduced the activity of the Ca2+-dependent proteolytic component by 39 and 40%, respectively. A similar antiproteolytic effect was observed when both muscles were incubated with norepinephrine (Fig. 6). DBcAMP (10−3 M) also reduced Ca2+-dependent proteolysis in both soleus (62%) and EDL (34%) (Fig. 6).
The present experiments show that the acute reduction in rat plasma catecholamines produced by adrenal medulla removal is accompanied by a transient 15–20% increase in the rate of skeletal muscle overall proteolysis (Fig. 2). That this early rise in proteolysis was probably a direct consequence of the reduction in plasma catecholamines is clearly indicated by the reiterated demonstration that these amines have an inhibitory effect on muscle proteolysis. Indeed, it has been shown that the infusion of epinephrine in humans (29) and in perfused rat hindquarters (10) induces a rapid 20% decrease in the rate of protein degradation, and we have recently (19) shown a similar effect of both epinephrine and norepinephrine on the proteolytic activity of isolated soleus and EDL muscles. We found in a previous work (18) that skeletal muscle overall proteolysis also increases 2 days after adrenergic blockade by guanethidine, a condition in which both plasma catecholamines and muscle norepinephrine are reduced. The verification in the present experiments that a similar increase in muscle proteolysis can be obtained in the presence of normal levels of tissue norepinephrine suggests that the main factor responsible for the acute increase in proteolysis induced by guanethidine treatment (18) was the reduction in plasma catecholamine concentration. Together, the experiments with adrenodemedullation and with chemical sympathectomy suggest that the sympathetic nervous system has an acute restraining effect on skeletal muscle proteolysis that is mediated mainly by catecholamines released from the adrenal gland. We have shown (19) that the rate of overall proteolysis in isolated skeletal muscles from normal rats is markedly reduced by clenbuterol, a selective agonist of β2-adrenoceptors, the predominant receptor in rat skeletal muscles (14). Furthermore, we have demonstrated (19) that the inhibitory effect of epinephrine on muscle protein degradation can be prevented by ICI, a selective β2-antagonist, suggesting that this adrenergic action is mediated by β2-adrenoceptors.
The data of the present work clearly show that the early rise in the rate of overall proteolysis after adrenal medulla removal was accompanied by a 100% increase in the activity of the muscle Ca2+-dependent proteolytic pathway (Fig. 3), with no changes in the activities of the lysosomal, ATP-dependent, and ATP-independent systems, which remained unaltered throughout the experimental period (Fig. 3). Also, the activities of these three proteolytic components in skeletal muscles from normal rats were not affected by 10−5 M clenbuterol in vitro (Fig. 4). In agreement with the results of adrenodemedullation, in the experiments with chemical sympathectomy, the increase in muscle total proteolysis observed after 2 days of guanethidine treatment was accompanied by a 45% increase in soleus Ca2+-dependent proteolysis (18). On the other hand, in entire agreement with these results, the inhibitory in vitro effects of epinephrine and norepineprine on the rate of overall proteolysis in isolated skeletal muscle from normal rats (19) were shown to be associated with a reduction in the activity of the Ca2+-dependent proteolytic system in both soleus and EDL (Fig. 6). The in vitro experiments also showed that the activity of Ca2+-dependent proteolysis in soleus and EDL is inhibited by the addition of clenbuterol to the incubation medium, an effect that was prevented by ICI (Fig. 5). Taken together, these data suggest that the catecholamines from adrenal medulla exert their acute restraining effect on skeletal muscle proteolysis by keeping the Ca2+-dependent pathway inhibited, probably through β2-adrenoceptor activation. Numerous studies have shown that the activity and gene expression of μ-calpain are decreased and those of calpastatin are increased after β2-agonist administration to different species (2, 7, 12), leading to the suggestion that this was the mechanism of the inhibitory effect of catecholamines on the Ca2+-dependent proteolytic process. This hypothesis has been reinforced by the recent finding that the infusion of epinephrine in pigs induces a 77 and 94% increase in calpastatin activity in skeletal and cardiac muscle, respectively (20). Because β2-agonists activate PKA in rat skeletal muscle (24), it has been proposed that calpain and/or calpastatin is a target for this kinase. In fact, calpastatin is phosphorylated by PKA (20, 28), and its inhibitory activity against μ-calpain is enhanced by phosphorylation in rat skeletal muscle in vitro (22). Moreover, recent evidence indicates that the calpastatin gene promoter is upregulated by DBcAMP, indicating that both the calpastatin gene promoter and protein are targets for PKA activity (4, 5). The inhibition of Ca2+-dependent proteolysis by DBcAMP observed in the present experiments in soleus and EDL muscles (Fig. 6) is consistent with the above studies.
The biphasic pattern of skeletal muscle proteolysis that has been found in catabolic states, including fasting (11), diabetes (21), and chemical sympathectomy (18), was also observed in adrenodemedullated rats (Fig. 2), further supporting the idea that the activation of regulatory mechanism(s) to prevent excessive breakdown of protein and spare muscle protein reserves is a characteristic feature of skeletal muscle (18). However, differently from the three conditions above mentioned, in which the initial rise in proteolysis was followed by a decrease in this process to levels significantly lower than controls (11, 18, 21), in adrenodemedullated rats after the initial increase, rates of muscle proteolysis only returned to normal levels (Fig. 2). It has been found that adrenodemedullation may induce an increase in the sympathetic activity in rat pancreas and adipose tissue (30). It seems reasonable to speculate that, if a similar compensatory increase occurred in muscle sympathetic activity in the adrenodemedullated rats, tissue norepinephrine would bring rates of proteolysis to normal levels, thus replacing the protein-sparing action of plasma catecholamines. Because of the suppression of skeletal muscle norepinephrine, this recourse is not available to rats submitted to chemical sympathectomy (18). Along this same line of reasoning, the decrease in muscle proteolysis to below control levels in these animals (18) could be explained by the intervention of other more potent, sympathetic-independent, antiproteolytic factors. Clearly, further experiments are needed to clarify the biochemical mechanisms involved in the biphasic pattern of muscle proteolysis in the different conditions.
In summary, the present work shows that a plasma catecholamine reduction 2 days after adrenodemedullation induces a transitory rise in the rate of overall proteolysis in soleus and EDL muscles that is accompanied by an increased activity of the Ca2+-dependent pathway. The reduction in the rate of Ca2+-dependent proteolysis in both muscles induced by catecholamines, clenbuterol, and DBcAMP in vitro suggests that catecholamines inhibit the activity of the Ca2+-dependent proteolytic process by binding to β2-adrenergic receptors in red and white skeletal muscles and activating intracellular pathways involving the cAMP-dependent protein kinase.
We are indebted to Elza Aparecida Filippin, Maria Antonieta R. Garófalo, and Victor Diaz Galbán for technical assistance.
This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, no. 97/3950–5) and the Conselho Nacional de Pesquisa (CNPq 501252/91–6). During this study L. C. C. Navegantes received a fellowship from the FAPESP (98/02591–4).
Address for reprint requests and other correspondence:Í. Kettelhut, Dept. of Biochemistry, School of Medicine, USP, Ribeirão Preto, 14049–900 SP, Brazil (E-mail:).
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- Copyright © 2001 the American Physiological Society