AJP - Endo AJP: Advances in Physiology Education
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 293: E932-E940, 2007. First published July 10, 2007; doi:10.1152/ajpendo.00175.2007
0193-1849/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/4/E932    most recent
00175.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Beitzel, F.
Right arrow Articles by Lynch, G. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Beitzel, F.
Right arrow Articles by Lynch, G. S.

beta-Adrenoceptor signaling in regenerating skeletal muscle after beta-agonist administration

Felice Beitzel,1 Martin N. Sillence,2 and Gordon S. Lynch1

1Basic and Clinical Myology Laboratory, Department of Physiology, The University of Melbourne, Victoria; and 2School of Agricultural and Veterinary Sciences, Charles Sturt University, Wagga Wagga, New South Wales, Australia

Submitted 18 March 2007 ; accepted in final form 4 July 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Stimulating the beta-adrenoceptor (beta-AR) signaling pathway can enhance the functional repair of skeletal muscle after injury, but long-term use of beta-AR agonists causes beta-AR downregulation, which may limit their therapeutic effectiveness. The aim was to examine beta-AR signaling during early regeneration in rat fast-twitch [extensor digitorum longus (EDL)] and slow-twitch (soleus) muscles after bupivacaine injury and test the hypothesis that, during regeneration, beta-agonist administration does not cause beta-AR desensitization. Rats received either the beta-AR agonist fenoterol (1.4 mg·kg–1·day–1 ip) or saline for 7 days postinjury. Fenoterol reduced beta-AR density in regenerating soleus muscles by 42%. Regenerating EDL muscles showed a threefold increase in beta-AR density, and, again, these values were 43% lower with fenoterol treatment. An amplified adenylate cyclase (AC) response to isoproterenol was observed in cell membrane fragments from EDL and soleus muscles 7 days postinjury. Fenoterol attenuated this increase in regenerating EDL muscles but not soleus muscles. beta-AR signaling mechanisms were assessed using AC stimulants (NaF, forskolin, and Mn2+). Although beta-agonist treatment reduces beta-AR density in regenerating muscles, these muscles can produce large cAMP responses relative to healthy (uninjured) muscles. Desensitization of beta-AR signaling in regenerating muscles is prevented by altered rates of beta-AR synthesis and/or degradation, changes in G protein populations and coupling efficiency, and altered AC activity. These mechanisms have important therapeutic implications for modulating beta-AR signaling to enhance muscle repair after injury.

muscle regeneration; beta-adrenoceptor; muscle function; muscle injury; adenylate cyclase; G protein

STUDIES IN RODENTS AND LIVESTOCK have shown that chronic administration of beta-adrenoceptor (beta-AR) agonists can lead to skeletal muscle hypertrophy and an increase in maximum force-producing capacity (5, 10, 15, 41, 50). The increase in muscle mass is the result of protein accretion via an increase in protein synthesis and a decrease in degradation, effects mediated via beta-AR activation, adenylate cyclase (AC) induction, and a consequent increase in intracellular cAMP concentration (15, 17, 23, 37). There is potential to exploit the anabolic effects of beta-agonists therapeutically, to promote muscle regeneration after injury, since beta-AR-mediated mechanisms are postulated to play a physiological role in successful muscle regeneration (34). Evidence comes from the fact that the beta-agonist fenoterol can hasten functional recovery of regenerating rat skeletal muscle after myotoxic injury. Previous studies have shown that daily fenoterol administration enhances the force-producing capacity of regenerating muscles by 19% compared with control by 14 days postinjury (5). The improvement in contractile function with fenoterol treatment was associated with increases in protein content and the cross-sectional area of regenerating muscle fibers (5). Additionally, fast-twitch muscles in the rat have a higher density of beta-AR when they are regenerating, suggesting that endogenous catecholamine stimulation could play a role in the regenerative process. However, a local increase in catecholamine activity, mediated by an increase in tissue sensitivity, would exploit the anabolic pathways linked to beta-AR, without systemic complications.

In addition to changes in beta-AR density, alterations in beta-AR signaling could influence muscle regenerative processes and specific responses to beta-AR stimulation after beta-agonist administration. In adult muscle, the hypertrophic response to beta-agonists may be limited by beta-AR downregulation (a reduction in the density of receptors at the cell membrane) and desensitization (uncoupling of the receptors from their response elements for cell signaling; see Refs. 26, 28, and 48). Downregulation occurs after prolonged exposure to high-dose beta-agonist administration, which renders beta-AR more susceptible to beta-AR kinase (beta-ARK), an enzyme that phosphorylates agonist-bound receptors and enhances their affinity for beta-arrestins. Further interaction between the beta-AR and G proteins is prevented, and endocytosis of the receptors is initiated, which leads to fewer receptors on the cell surface (18, 26, 28, 31). Studies in rat hindlimb muscles have demonstrated a reduction in beta-AR density with 7 days exposure to various beta-agonists, including clenbuterol, isoproterenol, and terbutaline (14, 46). Additionally, exposure of L6 myoblast membranes to isoproterenol can result in downregulation of beta-AR within 4 and 24 h of internalization of the beta-AR from the cell membrane (21).

beta-AR desensitization may be homologous or heterologous. As with downregulation, homologous desensitization also occurs when the beta-arrestins bind to the phosphorylated beta-AR and prevent further G protein interaction (22, 28, 33). However, unlike downregulation, homologous desensitization is reversible. In heterologous desensitization, elements downstream of the beta-AR, such as G proteins or AC itself, may be phosphorylated, or their expression and function altered to favor inhibition. This impairs signal efficacy and reduces AC activity, not only for beta-AR but for any receptor that shares this second messenger signaling pathway (16, 28, 52).

The attenuation of beta-AR signaling is important for cellular homeostasis, since continued overstimulation of the pathway can lead to cellular damage, changes in cell differentiation, and apoptosis (7, 13, 49). However, from a therapeutic standpoint, it has been suggested that beta-AR downregulation and desensitization may limit the effectiveness of chronic beta-agonist administration (11).

Several studies have examined the beta-AR signaling pathway during fetal or neonatal development. Experiments using heart, lung, brown fat, and liver suggest that immature, developing tissue is resistant to desensitization by chronic beta-agonist exposure (3, 4, 9, 29, 36). These observations indicate that the downregulation and desensitization responses may not be inherent but are acquired during development. Thus it is possible that changes in beta-AR signaling and an altered response to beta-agonists may occur in tissue that is regenerating after injury. If so, this could have important implications for the therapeutic use of beta-agonists to enhance muscle regeneration and promote functional restoration. Thus the aim was to examine the effect of beta-agonist administration on the beta-AR population and the downstream signaling pathway during early regeneration, in both fast-twitch and slow-twitch muscles, and test the hypothesis that, during regeneration, chronic beta-agonist administration does not cause beta-AR desensitization.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. The Animal Experimentation Ethics Committee of The University of Melbourne approved all experiments, which were conducted in accordance with the guidelines for Care and Use of Experimental Animals, as described by the National Health and Medical Research Council of Australia. Young adult (275–300 g) male Fischer 344 rats (total n = 116; Animal Resource Centre, Canning Vale, WA, Australia) were housed in the Biological Research Facility at The University of Melbourne. They were maintained under a 12:12-h light-dark cycle (light 0600–1800), with access to standard rat chow and water ad libitum.

Experimental muscle injury. All rats were anesthetized deeply (100 mg/kg ketamine and 10 mg/kg ip xylazine) such that they were unresponsive to tactile stimuli. The extensor digitorum longus (EDL) and soleus muscles of the right hindlimb were surgically exposed, with care taken to avoid damaging the nerve and blood supplies, and then injected to maximal holding capacity with 0.5% bupivacaine hydrochloride (Marcain, Astra, North Ryde, NSW, Australia), using techniques described previously (5, 19), to ensure degeneration of all muscle fibers (Fig. 1). The EDL and soleus muscles of the left hindlimb served as the uninjured controls. After surgery, the rats were assigned at random to receive daily treatment with either fenoterol, a full agonist of both beta2-AR and beta1-AR (1.4 mg·kg–1·day–1 ip dissolved in 1 ml isotonic saline; Sigma-Aldrich, Castle Hill, NSW, Australia; see Refs. 6 and 41), or an equal volume of saline for 2, 5, or 7 days. Previous studies using this protocol have shown that degeneration is maximal 2 days after bupivacaine, which is followed by spontaneous regeneration, such that immature but functional muscle fibers are present by 7 days postinjury (5, 20 and Fig. 1).


Figure 1
View larger version (121K):
[in this window]
[in a new window]

  		Fig. 1. Hematoxylin & eosin-stained cross sections of uninjured (top left) and regenerating skeletal muscle at 2 (top right), 5 (bottom left), or 7 (bottom right) days following maximal injection with bupivacaine hydrochloride. Note the maximal bupivacaine-induced degeneration at 2 days postinjury. By 5 days postinjury, small myotubes are evident, which mature to form the new muscle fibers seen at 7 days postinjury.

 
At the end of the treatment period, the rats were anesthetized deeply (100 mg/kg ketamine and xylazine 10 mg/kg ip xylazine), and the EDL and soleus muscles were surgically excised and trimmed of tendons and any adhering nonmuscle tissue. Muscles used for beta-AR density measurements, Western blotting, and PCR were frozen rapidly in isopentane cooled in liquid nitrogen and stored at –80°C for subsequent processing and analysis. Muscles used for AC activity assays were placed in ice-cold buffer for immediate preparation of cell membrane extracts from the fresh tissue. After the surgical procedures, the thoracic region was exposed, and the rats were killed by cardiac excision while still anesthetized.

Preparation of cell membranes. Procedures for the preparation of cell membrane fragments have been described previously (5, 41, 45, 47). Briefly, to obtain enough material for the assays, the EDL and soleus muscles were pooled (in pairs for radioligand-binding assays and in threes for cAMP) according to treatment group and injury state. The muscles were then homogenized in 2 ml ice-cold buffer [buffer A, in mM: 50 Tris (pH 7.0) 250 sucrose, and 1 EGTA; pH 7.4 at 4°C], and cell membrane fragments were prepared by centrifugation at 4°C. The centrifugation protocol ensures isolation of the sarcolemmal membrane fraction only. Therefore, the beta-AR examined are those present in the sarcolemma that are available for stimulation by both endogenous catecholamines and exogenously administered beta-agonists. The cell membrane pellets were resuspended in ice-cold buffer [for ligand binding 1 ml buffer B (in mM): 50 Tris (pH 7.6), 10 MgCl2·(6H2O), and 150 NaCl, pH 7.4 at 4°C, and, for cAMP assays, 550 µl buffer C (in mM): 50 Tris (pH 7.4), 5 MgCl2·(6H2O), and 1 EGTA, pH 7.4 at 4°C] using a hand-held glass homogenizer. The protein concentration of the membrane suspension was determined using a Bradford protein assay (Bio-Rad, Richmond, CA). Cell membrane samples for ligand binding assay were then stored at –80°C, whereas the preparations for AC assays were used immediately.

Radioligand binding assays. Radioligand binding assays were performed to measure the density of beta-AR in muscles from rats (n = 60; muscles pooled in pairs) at 2, 5, and 7 days postinjury using methods described previously (5). Single-point saturation assays were performed because of the limited amount of protein that could be obtained from the small muscles and to ensure that the sample protein concentration was within the concentration range of 0.05–0.3 mg/ml, in which the binding of the radioligand to the beta-AR sites has been shown to be linear. Measurement of beta-AR density in muscle membranes detects both beta1- and beta2-AR present. However, the beta-AR detected cannot be distinguished as beta1- or beta2-AR because of the limited amount of membrane protein that can be obtained from small muscles, since this procedure requires greater amounts of membrane. Therefore, the results for receptor density are expressed as total beta-AR. Briefly, the incubations were performed in 12 x 75-mm tubes containing 400 µl of the cell membrane suspension with 50 µl of the radioligand [125I]iodocyanopindolol (ICYP, 135 pM) and 50 µl of either buffer B (to determine the total binding of ICYP to beta-AR) or the nonselective beta-AR antagonist dl-propranolol (2 µM; to determine nonspecific binding of ICYP to the membrane). The receptor-bound radioligand was then collected by filtering the preparation through glass-fiber filter paper (Whatman GF-C filter paper, Maidstone, UK) and by rinsing each tube using a cell harvester (Brandel M-48R cell harvester; Biomedical Research and Development Laboratories, Gaithersburg, MD). Radioactivity remaining on the filters was determined using a gamma counter (1470 Wizard-automatic gamma counter; Wallac, Turku, Finland) at a counting efficiency of 78%. Results were expressed as {gamma}-radiation counts per minute, and a conversion was applied to determine the concentration of beta-AR (mol/mg of protein) based on the specific activity of the radioligand.

RNA analysis. Total RNA was isolated from small portions (~10 mg) taken from the midbelly region of the same EDL and soleus muscles at 2, 5, and 7 days postinjury. Samples were prepared using a commercially available kit and according to the manufacturer's instructions (kit no. 75742; Qiagen). Both beta1- and beta2-AR mRNA were examined because, although beta2-AR have been identified as the predominant subtype in skeletal muscle, some studies have shown that skeletal muscles may also contain ~7–10% beta1-AR, with this value slightly higher in slow-twitch muscles (25, 27). Levels of beta1-AR and beta2-AR mRNA were analyzed by semiquantitative RT-PCR based on techniques described previously, using 18S as a loading control (12), also using a commercially available kit (kit no. 74704; Qiagen, Valencia, CA).

Western blot analysis. Western immunoblot analysis was performed to measure Gs{alpha} splice variants in intact EDL and soleus muscles from rats (n = 8) at 7 days postinjury, utilizing the methodology of Schertzer and colleagues (42, 43). Primary antibodies were Gs{alpha} (K-20) and sc-823. A G protein-tagged fusion protein was used as positive control (Gs{alpha}-sc-4225 WB; Santa Cruz Biotechnology).

AC activity assays. AC activity was measured in muscles taken from rats (n = 48; pooled in triplicate) at 7 days postinjury based on methods described previously (45). Production of cAMP was measured in response to various stimulants that act at different points on the AC signaling pathway. The response to direct beta-adrenergic stimulation was determined using the nonselective beta-agonist isoproterenol (30). Maximal G protein activation was determined using NaF, which recruits all G proteins present (8, 53). Forskolin and MnCl2 were used to directly activate AC. Forskolin binds to a conserved region on the AC molecule and acts to "glue together" the two domains of the catalytic core, and Gs{alpha} association with AC enhances the effect of forskolin to increase AC activity (24, 44, 51). In contrast, Mn2+ activates AC by attaching to the Mg2+ binding site in the AC active site and interferes with G protein association such that Mn2+-stimulated AC activity is reduced (24, 32, 52). Use of various stimulants enables the identification of differences in the intracellular signaling pathway between uninjured and injured muscles, fast- vs. slow-twitch muscles, and whether aspects of the signaling pathway are altered by fenoterol treatment. Briefly, fresh L-isoproterenol (4 mM) and forskolin solutions (0.4 mM) were prepared, and an incubation buffer was made immediately before the incubation [in mM: 50 Tris (pH 7.7), 10 MgCl2·(6H20), 2 EGTA, 2 isobutyl methylxanthine, 0.4 mg/ml bacitracin, 6 mg/ml BSA 6, 40 phosphocreatine, 2 ATP, 0.2 GTP, and 0.6 mg/ml creatine phosphokinase, pH 7.4 at 30°C]. A small volume of incubation buffer was used to prepare NaF incubation buffer (20 mM) and Mn2+ incubation buffer (20 mM). All incubations were performed in triplicate. The final extraction of cAMP from samples was performed at 4°C by centrifugation with ethanol as described previously (45), and the samples were then assayed for cAMP concentration using a commercial assay kit (cAMP Biotrak EIA System, nonacetylation procedure; Amersham, GE Healthcare, Castle Hill, NSW, Australia; RPN 225 or 2255).

Statistical analysis. All values are expressed as means with bars denoting SE. Experimental groups from each time point were compared using a two-way ANOVA with main effects sought for treatment (saline vs. fenoterol) and injury (injured vs. uninjured). When significant differences were found, means were compared by using Fisher's least-significant difference post hoc multiple-comparison procedure. In all cases, differences between groups were considered significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
beta-AR density. Ligand binding assays showed that beta-AR density was ~2.5-fold greater in healthy slow-twitch soleus than in fast-twitch EDL muscles (Fig. 2). The comparatively low density of beta-AR in uninjured EDL was not reduced significantly by fenoterol treatment at any time tested (Fig. 2A). In regenerating EDL muscles, there was a marked increase in beta-AR density (~80%) that was evident at 2, 5, and 7 days postinjury. Fenoterol decreased the density of beta-AR in regenerating EDL muscles at 5 and 7 days postinjury compared with saline-treated regenerating muscles; however, beta-AR levels remained higher than in the contralateral fenoterol-treated uninjured muscles at those time points (Fig. 2A).


Figure 2
View larger version (30K):
[in this window]
[in a new window]

  		Fig. 2. beta-Adrenoceptor (beta-AR) density of uninjured and regenerating extensor digitorum longus (EDL; A) and soleus (B) muscles from rats treated with saline or fenoterol at 2, 5, or 7 days postinjury. In the EDL muscles (A), note the marked increase in beta-AR density during regeneration, and downregulation of beta-AR density in regenerating muscles occurred with fenoterol treatment. In the soleus muscles (B), downregulation of beta-AR density occurred with fenoterol treatment, with a greater effect in injured than uninjured muscles; n = 4–5 experiments. P < 0.05, different from saline-uninjured (control) muscles (*), difference between fenoterol-uninjured and fenoterol-injured muscles ({dagger}), and difference between saline-injured and fenoterol-injured muscles (#).

 
In uninjured soleus muscles, beta-AR density was not affected significantly after 2 and 5 days but was reduced by 20% compared with control levels after 7 days of fenoterol administration (Fig. 2B). In contrast to the EDL, in regenerating soleus muscles, beta-AR density was reduced by 47% compared with control muscles at 2 days but was restored to control levels by 5 days postinjury. However, similar to EDL muscles, fenoterol treatment prevented the restoration of beta-AR in regenerating soleus muscles such that levels were reduced compared with saline-treated regenerating muscles at 5 and 7 days postinjury. Thus, with fenoterol treatment, beta-AR levels were not restored to those found in the contralateral uninjured muscles (Fig. 2B).

beta-AR mRNA expression. Although fenoterol treatment did not cause downregulation of beta-AR in uninjured EDL muscles, after 5 days of fenoterol administration, there was a 51% and a 27% reduction in mRNA for beta1-AR and beta2-AR, respectively, that was restored to control levels by 7 days (Fig. 3, A and B). In regenerating EDL muscles, the marked increase in beta-AR density was accompanied by a 28 and a 37% increase in beta2-AR mRNA compared with control muscles at 5 and 7 days postinjury, respectively, whereas there was no change in beta1-AR mRNA levels at any time during muscle regeneration (Fig. 3, A and B). Although fenoterol attenuated the increase in beta-AR density in EDL muscles after injury, it did not prevent the increase in mRNA levels for beta2-AR at 5 days postinjury. beta1-AR mRNA was unchanged in regenerating muscles with fenoterol treatment compared with saline-treated regenerating muscles (Fig. 3, A and B).


Figure 3
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 3. Expression of beta2-AR and beta1-AR mRNA in uninjured and injured EDL (A and B) and soleus (C and D) muscles from rats treated with saline or fenoterol at 2, 5, or 7 days postinjury. In uninjured EDL muscles, 5 days of fenoterol treatment caused a reduction in beta2-AR mRNA (A) and beta1-AR mRNA (B). However, beta-AR mRNA levels in regenerating EDL muscles were not effected by fenoterol. In the soleus, beta2-AR mRNA (C) was reduced with fenoterol in both uninjured and regenerating muscles, and beta1-AR mRNA (D) levels were not affected by fenoterol treatment. (n = 4). P < 0.05, different from saline-uninjured (control) muscles (*), difference between fenoterol-uninjured and fenoterol-injured muscles ({dagger}), and difference between saline-injured and fenoterol-injured muscles (#).

 
In uninjured soleus muscles, beta1-AR mRNA was not affected by fenoterol treatment; however, beta2-AR mRNA was reduced (by 39 and 51%) after 5 and 7 days of administration, respectively. In regenerating soleus muscles, beta2-AR mRNA was reduced to 52%, beta1-mRNA increased by 90% compared with control muscles at 5 days, and mRNA levels for both beta-AR subtypes were returned to control values by 7 days postinjury (Fig. 3, C and D). With fenoterol treatment in regenerating soleus muscles, beta2-AR mRNA was reduced at 2, 5, and 7 days postinjury compared with control levels and was in fact reduced by 43% compared with levels in saline-treated injured muscles at 7 days postinjury (Fig. 3C). As for regenerating EDL muscles, beta1-AR mRNA was unchanged in regenerating soleus muscles after fenoterol treatment compared with saline-treated regenerating muscles (Fig. 3D).

G proteins. Similar to the observed differences in beta-AR density between fast- and slow- twitch muscles, the amount of total Gs{alpha} in control soleus muscles was ~2-fold greater than in control EDL muscles because of the ~2-fold increases in the amount of the Gs{alpha} splice variants, Gs{alpha}S and Gs{alpha}L, in the control soleus muscles (Fig. 4). Fenoterol treatment did not alter the amount of either splice variant of Gs{alpha} in uninjured EDL or soleus muscles (Fig. 4, C and D).


Figure 4
View larger version (23K):
[in this window]
[in a new window]

  		Fig. 4. Gs{alpha} proteins in regenerating EDL and soleus muscles from rats treated with saline or fenoterol at 7 days postinjury. Total Gs{alpha} (A) was determined by Western blotting (B). The sum of both the 45-kDa short (Gs{alpha}S; C) and the 52-kDa long (Gs{alpha}L; D) splice variants for EDL and soleus muscles. Note that fenoterol treatment did not alter the amount of either splice variant of Gs{alpha} in both uninjured and regenerating EDL and soleus muscles, however, the decrease in total Gs{alpha} in regenerating soleus muscles was only significant in control rats. (n = 4). P < 0.05, different from saline-uninjured (control) muscles (*) and difference between fenoterol-uninjured and fenoterol-injured muscles ({dagger}). UE, uninjured EDL; IE, injured EDL; US, uninjured soleus; IS, injured soleus.

 
In regenerating EDL muscles, Gs{alpha}S and Gs{alpha}L increased by 55 and 222%, respectively, so there was a 64% increase in total Gs{alpha} in regenerating EDL muscles. Fenoterol treatment did not alter the amount of either splice variant of Gs{alpha} in regenerating EDL muscles compared with saline treated regenerating muscles (Fig. 4, C and D).

In regenerating soleus muscles, levels of Gs{alpha}S decreased by 49%, whereas levels of Gs{alpha}L increased by 116% compared with control (Fig. 4, C and D). However, despite the marked increase in Gs{alpha}L because of the reduction in the predominant splice variant Gs{alpha}S, there was a net 40% reduction of total Gs{alpha} in regenerating soleus muscles compared with control (Fig. 4A). Although fenoterol treatment did not alter the amount or proportions of either isoform of Gs{alpha} in regenerating soleus muscles, the reduction of total Gs{alpha} was only significant in saline-treated rats, not in fenoterol-treated rats (Fig. 4A).

AC activity. Fenoterol treatment did not alter basal cAMP production or isoproterenol-, NaF-, Mn2+-, or forskolin-stimulated cAMP production in uninjured EDL muscles (Fig. 5). In regenerating EDL muscles, basal cAMP production was not different from controls and was not affected by fenoterol treatment (data not shown). beta-AR agonist (isoproterenol)-stimulated cAMP production was increased by 77% in regenerating EDL muscles; however, fenoterol treatment impaired the increase in isoproterenol-stimulated cAMP production (Fig. 5B). NaF-stimulated cAMP production during regeneration was not different from control but, with fenoterol treatment, was increased by 88% in regenerating muscles compared with control levels (Fig. 5C). Mn2+-stimulated cAMP production in regenerating muscles was not different from controls and was not affected by fenoterol treatment (Fig. 5D). Forskolin-stimulated cAMP production was increased by 64% in regenerating EDL muscles compared with controls but was also not affected by fenoterol treatment (Fig. 5E).


Figure 5
View larger version (34K):
[in this window]
[in a new window]

  		Fig. 5. Basal (A) and isoproterenol (B)-, NaF (C)-, Mn2+ (D)-, and forskolin (E)-stimulated cAMP production of uninjured and regenerating EDL and soleus muscles from rats treated with saline or fenoterol at 7 days postinjury. Note the marked increase in isoproterenol-stimulated AC activity during regeneration compared with control muscles, which was reduced with fenoterol treatment in regenerating EDL but not regenerating soleus muscles (A). NaF-stimulated cAMP production was enhanced during regeneration in the EDL with fenoterol treatment (B). Fenoterol did not affect basal, Mn2+-, or forskolin- stimulated cAMP production during regeneration. (n = 5–8). P < 0.05, different from saline-uninjured (control) muscles (*) and difference between fenoterol-uninjured and fenoterol-injured muscles ({dagger}).

 
In uninjured soleus muscles, fenoterol treatment did not alter basal cAMP production or the response to any of the stimulants tested (Fig. 5). In the soleus muscles, basal cAMP production during regeneration was increased by 77% compared with control, which was attenuated with fenoterol treatment (Fig. 5A). Isoproterenol-stimulated cAMP production during regeneration was 54% greater than control but, unlike basal AC activity, was not affected by fenoterol administration and remained 50% greater than control levels (Fig. 5B). Regenerating soleus muscles also showed marked increases in NaF-, Mn2+-, and forskolin-stimulated cAMP production by 135, 58, and 117%, respectively, compared with control. Fenoterol treatment did not attenuate these increases, which remained at 81, 40, and 145% greater than control levels for NaF-, Mn2+-, and forskolin-stimulated cAMP production, respectively (Fig. 5, C–E).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study identified unique characteristics of beta-adrenergic signaling in regenerating fast- and slow-twitch skeletal muscles that highlight this signaling pathway as a potential therapeutic target for promoting muscle repair after injury. In healthy adult muscle, beta-AR responses to beta-agonist stimulation are limited by downregulation and by desensitization of AC signaling (2, 26, 28). We found the capacity of fenoterol to downregulate beta-AR was influenced by the muscle phenotype, the basal level of beta-AR expression, and injury status. Greater downregulation of beta-AR was evident in regenerating muscles than in healthy uninjured muscles. The classical mechanism of beta-AR downregulation involves internalization of the receptor, and, consistent with this, the prolonged administration of a high-dose beta-agonist would make beta-AR more susceptible to phosphorylation by beta-ARK and downregulation from the cell surface (26, 28). However, we suggest that, although downregulation imposes limitations on beta-AR signaling in adult tissue, this does not necessarily occur in regenerating muscle, such as in soleus muscle where maximum signal transduction was not reduced by fenoterol treatment.

With chronic beta-agonist administration, beta-AR synthesis can also be impaired. In regenerating EDL muscles, the decrease of beta-AR density at the membrane at 5 and 7 days postinjury was not reflected by a similar reduction in beta1-AR or beta2-AR mRNA at these time points. This suggests that the reduction in beta-AR density at the membrane after fenoterol treatment was due primarily to their downregulation at the cell surface or in the posttranscriptional regulation of beta-AR.

In contrast, in the soleus muscles, the beta2-AR mRNA levels were reduced in both uninjured and injured muscles with 5 and 7 days of fenoterol treatment, respectively. These changes in mRNA levels did not match the time course of beta-AR density. These observations suggest that there is an even greater reduction in the rate of receptor degradation. In particular, fenoterol impaired the restoration of beta2-AR mRNA levels at 7 days postinjury, suggesting that treatment with the beta-agonist prevented the reduction in receptor degradation that would have otherwise occurred during regeneration. It should be noted that, during the inflammatory process that accompanies the muscle degeneration/regeneration process, it is possible that the isolated membrane fragment could contain macrophages and/or neutrophils that may themselves have disturbed beta-AR function. It has been shown previously in studies on airway smooth muscle that mediators released from inflammatory cells such as reactive oxygen species and fatty acid metabolites may directly or indirectly induce beta-AR dysfunction, which may have consequences for their immune function, mediator release, and effects on surrounding tissues (38). Thus, although the clear differences between the responses of fast and slow muscles in the present study suggest that the relative contribution of disturbed beta-AR function of macrophages and/or neutrophils is relatively small, nonetheless they should be taken into account when interpreting the findings.

Our results suggest that the proportion of beta1-AR in soleus muscles is likely to be higher than normal for a brief period during regeneration and higher still following fenoterol treatment because of a downregulation of beta2-AR mRNA at 7 days postinjury. However, the relative contribution of beta1-AR to muscle regeneration is unclear. Specifically, although fenoterol did not prevent the increase in beta1-AR expression at day 5, it reduced the total beta-AR density markedly. This suggests that, even after upregulation, the beta1-AR still represents a minor population relative to the beta2-AR. Nevertheless, the role of beta1-AR in maintaining beta-AR signaling during muscle repair is worthy of further investigation.

In regenerating EDL muscles at 7 days postinjury, the downregulation of beta-AR with fenoterol resulted in diminished beta-AR signaling, signified by a reduction in the isoproterenol-stimulated cAMP response. The maintained NaF response suggests that beta-AR downregulation caused a reduction in coupling between G proteins and the beta-AR; thus, the beta-AR-mediated cAMP response was reduced with fenoterol treatment at this time point. However, fenoterol did not impair elements of the downstream signaling pathway, since the responses to the downstream stimulants, NaF, Mn2+, and forskolin were not impaired, and amounts and proportions of G proteins were unaffected. In fact, this response was elevated in regenerating EDL muscles, suggesting that, at the level of postreceptor signaling, a heterologous mechanism occurs in regenerating muscles to enhance cAMP responses, despite fenoterol treatment.

A reduction in the cAMP response to isoproterenol was not observed in soleus muscles from fenoterol-treated rats at 7 days postinjury. This indicates that any downregulation caused by fenoterol was compensated for by alterations in downstream signaling such that the cAMP response was maintained. The coupling between G proteins and beta-AR was not impaired by fenoterol administration, the expression of beta1-AR remained increased at 5 days postinjury, and there was no significant reduction of total Gs{alpha} in regenerating muscles with 7 days of treatment. Additionally, the enhanced cAMP production observed in regenerating muscles at 7 days postinjury was maintained, since the responses of signaling elements downstream of receptor were unaffected by fenoterol and cAMP responses remained elevated.

Studies using similar AC activity measurements in developing rat fetal or neonatal heart and liver have demonstrated that, unlike in the adult, after chronic beta-agonist administration, the ability of beta-AR stimulation to elicit an increase in cAMP is not desensitized. Instead, signaling is maintained or even enhanced through heterologous sensitization of elements downstream of the receptor, at the levels of G protein coupling and AC activity (3, 4, 48, 53). For example, Auman and colleagues (3) showed that beta-AR/AC responses in the liver and heart were maintained in neonatal rats after chronic treatment with the beta2-agonist terbutaline despite beta-AR downregulation. In some cases, the neonatal beta-AR signaling pathway exhibited sensitization through mechanisms downstream of the receptor and possible deficiencies in the ability of immature cells to uncouple receptors from response elements. The observation that such processes enable beta-AR function to be maintained during heart and liver development highlights the potential value of the beta-AR signaling pathway as a useful therapeutic target for promoting growth and repair after muscle injury.

Our findings support the notion that the functional response to beta-agonists depends on the beta-AR-to-G protein-to-AC ratio rather than the absolute numbers of receptors and that the amount and isoforms of G proteins and AC are important (40). We have identified that these aspects of the signaling pathway vary depending on tissue type or state of growth, development, or repair.

Many studies have suggested that the number of beta-AR present exceeds that required to maximally activate the effector, since G proteins exist in great stoichiometric excess (1, 39). It has also been argued that maximal stimulation of cAMP production can be increased with an elevation in the total levels of AC (35). With an excess of receptors, a decrease in receptor density may decrease the sensitivity to an agonist but would only decrease the maximal effect if the drug was not a full agonist (4). Therefore, with the downregulation after 5 days of fenoterol treatment in regenerating muscles, an initial reduction in the potency of fenoterol may occur, although its ability to maximally stimulate AC activity would be unaffected. A further reduction of beta-AR density would lead to a proportional decrease in maximal stimulation because of a lack of spare receptors. Thus, in the EDL muscles, the reduction in beta-AR density would be expected to first decrease the potency of fenoterol to stimulate AC and subsequently to decrease the maximal effect of fenoterol. During regeneration of soleus muscles, there was no decrease in response with the decreased beta-AR, suggesting that the beta-AR density exceeds that required to maximally activate AC.

The findings suggest that cAMP output is elevated in regenerating muscles with fenoterol treatment because they have greater beta-AR stimulation than in untreated muscles. The alterations in beta-AR density and second messenger coupling are likely to alter tissue sensitivity and responsiveness to beta-agonist administration. Whereas the anabolic effect of a weak partial agonist drug may be attenuated over time (because of beta-AR downregulation), the response of a regenerating muscle to a full agonist is likely to be unaffected, even when the drug is given in high doses. Differences have been identified between liver and heart in the ability of their developing cells to desensitize following chronic beta-AR administration (4), and we have identified an enhanced responsiveness of the signaling pathway, shown by responses to NaF, forskolin, and Mn2+ may vary depending on tissue type or state of growth, development or repair. Thus it would be interesting to examine whether increased responsiveness of beta-AR signaling plays an important role in repair of other tissues, or attributes of signaling may be shared by other GPCRs, not only in skeletal muscle.

Our findings demonstrate that, despite the marked beta-AR downregulation within 5 days of beta-agonist administration, desensitization is prevented in regenerating skeletal muscle by alterations in the G protein population and the coupling efficiency and AC, which not only improve signaling and promote the physiological responses required for successful regeneration but have important implications when considering tissue sensitivity and responsiveness to beta-AR agonist therapies for promoting muscle repair.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported under the Australian Research Council's Discovery-Project funding scheme (project nos. DP0665071 and DP0772781) and the National Health and Medical Research Council of Australia (project nos. 350439 and 454561).


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. S. Lynch, Basic and Clinical Myology Laboratory, Dept. of Physiology, The Univ. of Melbourne, Victoria, 3010 Australia (e-mail: gsl{at}unimelb.edu.au)

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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Alousi AA, Jasper JR, Insel PA, Motulsky HJ. Stoichiometry of receptor-Gs-adenylate cyclase interactions. FASEB J 5: 2300–2303, 1991.[Abstract]
  2. Auman JT, Seidler FJ, Slotkin TA. Regulation of fetal cardiac and hepatic beta-adrenoceptors and adenylyl cyclase signaling:terbutaline effects. Am J Physiol Regul Integr Comp Physiol 281: R1079–R1089, 2001.[Abstract/Free Full Text]
  3. Auman JT, Seidler FJ, Tate CA, Slotkin TA. beta-Adrenoceptor-mediated cell signaling in the neonatal heart and liver: responses to terbutaline. Am J Physiol Regul Integr Comp Physiol 281: R1895–R1901, 2001.[Abstract/Free Full Text]
  4. Auman JT, Seidler FJ, Tate CA, Slotkin TA. Are developing beta-adrenoceptors able to densensitize? Acute and chronic effects of beta-agonists in neonatal heart and liver. Am J Physiol Regul Integr Comp Physiol 283: R205–R217, 2002.[Abstract/Free Full Text]
  5. Beitzel F, Gregorevic P, Ryall JG, Plant DR, Sillence MN, Lynch GS. beta2-Adrenoceptor agonist fenoterol enhances functional repair of regenerating rat skeletal muscle after injury. J Appl Physiol 96: 1385–1392, 2004.[Abstract/Free Full Text]
  6. Bremner P, Siebers R, Crane J, Beasley R, Burgess C. Partial vs. full beta-receptor agonism. A clinical study of inhaled albuterol and fenoterol. Chest 109: 957–962, 1996.[Web of Science][Medline]
  7. Burniston JG, Tan LB, Goldspink DF. beta2-Adrenergic receptor stimulation in vivo induces apoptosis in rat heart and soleus muscle. J Appl Physiol 98: 1379–1386, 2004.[Web of Science][Medline]
  8. Chaudhry A, Granneman JG. Developmental changes in adenylyl cyclase and GTP binding proteins in brown fat. Am J Physiol Regul Integr Comp Physiol 261: R403–R411, 1991.[Abstract/Free Full Text]
  9. Chaudhry A, Lahners KN, Granneman JG. Perinatal changes in coupling of beta1 and beta3 adrenergic receptors to brown fat adenylyl cyclase. J Pharmacol Exp Ther 261: 633–637, 1992.[Abstract/Free Full Text]
  10. Choo JJ, Horan MA, Little RA, Rothwell NJ. Anabolic effects of clenbuterol on skeletal muscle are mediated by beta2-adrenoceptor activation. Am J Physiol Endocrinol Metab 263: E50–E56, 1992.[Abstract/Free Full Text]
  11. Claing A, Laporte SA, Caron MG, Lefkowitz RJ. Endocytosis of G protein-coupled receptors:roles of G protein-coupled receptor kinases and beta-arrestin proteins. Prog Neurobiol 66: 61–79, 2002.[CrossRef][Web of Science][Medline]
  12. Collet F, Fève B, Frisdal E, Pavoine C, Pecker F, Atlan G. Pharmacological and molecular characterization of beta-adrenoceptors in adult rat diaphragm muscle. Respir Physiol 112: 1–12, 1998.[CrossRef][Web of Science][Medline]
  13. Communal C, Singh K, Pimental DR, Colucci WS. Norephinephrine stimulates apoptosis in adult rat ventricular myocytes by activation of the beta-adrenergic pathway. Circulation 98: 1328–1334, 1998.
  14. Elfellah MS, Reid JL. Regulation of beta-adrenoceptors in the guinea pig left ventricle and skeletal muscle following chronic agonist treatment. Eur J Pharmacol 182: 387–392, 1990.[CrossRef][Web of Science][Medline]
  15. Emery PW, Rothwell NJ, Stock MJ, Winter PD. Chronic effects of beta2-adrenergic agonists on body composition and protein synthesis in the rat. Biosci Rep 4: 83–91, 1984.[CrossRef][Web of Science][Medline]
  16. Giannuzzi CE, Seidler FJ, Slotkin TA. beta-Adrenoceptor control of cardiac adenylyl cyclase during development:agonist pretreatment in the neonate uniquely causes heterologous sensitization, not desensitization. Brain Res 694: 271–278, 1995.[CrossRef][Web of Science][Medline]
  17. Glass DJ. Molecular mechanisms modulating muscle mass. Trends Mol Med 9: 344–350, 2003.[CrossRef][Web of Science][Medline]
  18. Goodman Jnr OB, Krupnick JG, Santini F, Gurevich VV, Penn RB, Gagnon AW, Keen JH, Benovic JL. beta-Arrestin acts as a clatharin adaptor in endocytosis of the beta2-adrenergic receptor. Nature 383: 447–450, 1996.[CrossRef][Medline]
  19. Gregorevic P, Williams DA, Lynch GS. Hyperbaric oxygen increases the contractile function of regenerating rat slow muscles. Med Sci Sports Exerc 34: 630–636, 2002.
  20. Gregorevic P, Plant DR, Stupka N, Lynch GS. Changes in contractile activation characteristics of rat fast and slow skeletal muscle fibres during regeneration. J Physiol 558: 549–560, 2004.[Abstract/Free Full Text]
  21. Hardin AO, Lima JJ. beta2-adrenoceptor agonist induced down-regulation after short-term exposure. J Recept Signal Transduct Res 19: 835–852, 1999.[Web of Science][Medline]
  22. Hausdorf WP, Caron MG, Lefkowitz RJ. Turning off the signal: desensitization of beta-adrenergic receptor function. FASEB J 4: 2881–2889, 1990.[Abstract]
  23. Hinkle RT, Hodge KMB, Cody DB, Sheldon RJ, Kobilka BK, Isfort RJ. Skeletal muscle hypertrophy and anti-atrophy effects of clenbuterol are mediated by the beta2-adrenergic receptor. Muscle Nerve 25: 729–734 2002.
  24. Hurley JH. Structure, mechanism and regulation of mammalian adenylyl cyclase. J Biol Chem 274: 7599–7602, 1999.[Free Full Text]
  25. Jensen J, Brørs O, Dahl HA. Different beta-adrenergic receptor density in different rat skeletal muscle fibre types. Pharmacol Toxicol 76: 380–385, 1995.[Web of Science][Medline]
  26. Johnson M. Molecular mechanisms of beta2-adrenergic receptor function, response, and regulation. J Allergy Clin Immunol 117: 18–24, 2006.[CrossRef][Web of Science][Medline]
  27. Kim YS, Sainz RD, Molenaar P, Summers RJ. Characterisation of beta1- and beta2-adrenoceptors in rat skeletal muscles. Biochem Pharmacol 42: 1783–1789, 1991.[CrossRef][Web of Science][Medline]
  28. Kobilka B. Adrenergic receptors as models for G protein-coupled receptors. Ann Rev Neurosci 5: 87–114, 1992.
  29. Kudlacz EM, Navarro HA, Kavlock RJ, Slotkin TA. Regulation of postnatal beta-adrenergic receptor/adenylate cyclase development by prenatal agonist stimulation and steroids: alterations in rat kidney and lung after exposure to terbutaline or dexamethasone. J Dev Physiol 14: 273–281, 1990.[Web of Science][Medline]
  30. Lefkowitz RJ. Selectivity in beta-adrenergic responses: clinical implications. Circulation 49: 783–786, 1974.[Free Full Text]
  31. Lefkowitz RJ, Shenoy SK. Transduction of receptor signals by beta-arrestins. Science 308: 512–517, 2005.[Abstract/Free Full Text]
  32. Limbird LE, MacMillan ST. Mn2+-uncoupling of the catecholamine-sensitive adenylate cyclase system of rat reticulocytes. Parallel effects on cholera toxin-catalyzed ADP-ribosylation of the system. Biochim Biophys Acta 677: 408–416, 1981.[Medline]
  33. Lohse MJ, Benovic JL, Codina J, Caron MG, Lefkowitz RJ. beta-Arrestin: a protein that regulates beta-adrenergic receptor function. Science 248: 1547–1550, 1990.[Abstract/Free Full Text]
  34. Lynch GS, Schertzer JD, Ryall JG. Therapeutic approaches for muscle wasting disorders. Pharmacol Ther 113: 461–487, 2007.
  35. MacEwan DJ, Kim GD, Milligan G. Agonist regulation of adenylate cyclase activity in neuroblastoma x glioma hybrid NG 108-15 cells transfected to co-express adenylate cyclase type II and the beta2-adrenoceptor. Evidence that the adenylate cyclase is the limiting component for receptor-mediated stimulation of adenylate cyclase activity. Biochem J 318: 1033–1039, 1996.[Web of Science][Medline]
  36. Maier JA, Roberts JM, Jacobs MM. Ontogeny of fetal adenylate cyclase: mechanisms for regulation of beta-adrenergic receptors. J Dev Physiol 12: 249–261, 1989.[Web of Science][Medline]
  37. Navegantes LCC, Migliorini RH, Kettelhut IC. Adrenergic control of protein metabolism in skeletal muscle. Curr Opin Nutr Metab Care 5: 281–286, 2002.[CrossRef]
  38. Nijkamp FP, Henricks PA. Receptors in airway disease. beta-Adrenoceptors in lung inflammation. Am Rev Respir Dis 141: S145–S150, 1990.[Web of Science][Medline]
  39. Post SR, Hilal-Dandan R, Urasawa K, Brunton LL, Insel PA. Quantification of signaling components and amplification in the beta-adrenergic-receptor-adenylate cyclase pathway in isolated adult rat ventricular myocytes. Biochem J 311: 75–80, 1995.[Web of Science][Medline]
  40. Rousseau G, Guilbault N, Da Silva A, Mouillac B, Chidiac P, Bouvier M. Influence of receptor density on the patterns of beta2-adrenoceptor desensitization. Euro J Pharmacol 326: 75–84, 1997.[CrossRef][Web of Science][Medline]
  41. Ryall JG, Gregorevic P, Plant DR, Sillence MN, Lynch GS. beta2-Agonist fenoterol has greater effects on contractile function of rat skeletal muscles than clenbuterol. Am J Physiol Regul Integr Comp Physiol 283: R1386–R1394, 2002.[Abstract/Free Full Text]
  42. Schertzer JD, Green HJ, Duhamel TA, Tupling AR. Mechanisms underlying increases in SR Ca2+-ATPase activity after exercise in rat skeletal muscle. Am J Physiol Endocrinol Metab 284: E597–E610, 2003.[Abstract/Free Full Text]
  43. Schertzer JD, Ryall JG, Plant DR, Beitzel F, Stupka N, Lynch GS. beta2-Agonist administration increases sarcoplasmic reticulum Ca2+-ATPase activity in aged rat skeletal muscle. Am J Physiol Endocrinol Metab 288: E526–E533, 2005.[Abstract/Free Full Text]
  44. Seamon KB, Daly JW. Forskolin: its biological and chemical properties. Adv Cyclic Nucleotide Pro Phos Res 20: 1–150, 1986.
  45. Sillence MN, Matthews ML. Classical and atypical binding sites for beta-adrenoceptor ligands and activation of adenylyl cyclase in bovine skeletal muscle and adipose tissue membranes. Br J Pharmacol 111: 866–872, 1994.[Web of Science][Medline]
  46. Sillence MN, Matthews ML, Spiers WG, Pegg GG, Lindsay DB. Effects of clenbuterol, ICI118551 and solatol on the growth of cardiac and skeletal muscle and on beta2-adrenoceptor density in female rats. Naunyn-Schmiedeberg's Arch Pharmacol 344: 449–453, 1991.[Web of Science][Medline]
  47. Sillence MN, Moore NG, Pegg GG, Lindsay DB. Ligand binding properties of putative beta3-adrenoceptors compared in brown adipose tissue and in skeletal muscle membranes. Br J Pharmacol 109: 1157–1163, 1993.[Web of Science][Medline]
  48. Slotkin TA, Auman JT, Seidler FJ. Ontogenesis of beta-adrenoceptor signaling: implications for perinatal physiology and for fetal tocolytic drugs. J Pharmacol Exp Ther 306: 1–7, 2003.[Abstract/Free Full Text]
  49. Slotkin TA, Whitmore WL, Orbund-Miller L, Queen KL, Haim K. beta-Adrenergic control of macromolecular synthesis in neonatal rat heart, kidney and lung: relationship to sympathetic neuronal development. J Pharmacol Exp Ther 243: 101–109, 1987.[Abstract/Free Full Text]
  50. Smith WN, Dirks A, Sugiura T, Muller S, Scarpace P, Powers SK. Alteration of contractile force and mass in the senescent diaphragm with beta2-agonist treatment. J Appl Physiol 92: 941–948, 2002.[Abstract/Free Full Text]
  51. Tesmer JJG, Sprang SR. The structure, catalytic mechanism and regulation of adenylyl cyclase. Curr Opin Struct Biol 8: 713–719, 1998.[CrossRef][Web of Science][Medline]
  52. Zeiders JL, Seidler FJ, Slotkin TA. Agonist-induced sensitization of beta-adrenoceptor signalling in neonatal rat heart: expression and catalytic activity of adenylyl cyclase. J Pharmacol Exp Ther 291: 503–510, 1999.[Abstract/Free Full Text]
  53. Zeiders JL, Seidler FJ, Slotkin TA. Ontogeny of G-protein expression: control by beta-adrenoceptors. Dev Brain Res 120: 125–134, 2000.[Medline]



This article has been cited by other articles:


Home page
 J. Appl. Physiol.Home page
J. G. Ryall, J. D. Schertzer, T. M. Alabakis, S. M. Gehrig, D. R. Plant, and G. S. Lynch
Intramuscular {beta}2-agonist administration enhances early regeneration and functional repair in rat skeletal muscle after myotoxic injury
J Appl Physiol, July 1, 2008; 105(1): 165 - 172.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
G. S. Lynch and J. G. Ryall
Role of {beta}-Adrenoceptor Signaling in Skeletal Muscle: Implications for Muscle Wasting and Disease
Physiol Rev, April 1, 2008; 88(2): 729 - 767.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/4/E932    most recent
00175.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Beitzel, F.
Right arrow Articles by Lynch, G. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Beitzel, F.
Right arrow Articles by Lynch, G. S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.