Expression of adenylyl cyclase mRNAs in the denervated and in the developing mouse skeletal muscle

Yosuke Suzuki, Tiansheng Shen, Madeleine Poyard, Martin Best-Belpomme, Jacques Hanoune, and Nicole Defer

Institut National de la Santé et la Recherche Médicale Unité 99, Hôpital Henri Mondor, F-94010 Créteil, France

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Changes in the activity and in the expression of adenylyl cyclase (AC) were examined in mouse skeletal muscle after denervation and during development. Four isoforms of AC (AC2, AC6, AC7, and AC9) were detected by Northern blot analysis in gastrocnemius muscle, AC9 being the most abundant. After denervation, the levels of AC2 and AC9 mRNA decreased, whereas those of AC6 and AC7 increased. AC activity in response to several neurotransmitters was increased after denervation. During development, AC activity was high in fetus and neonate and declined in the adult; the sensitivity of AC activity to various neurotransmitters was the highest on the third postnatal day. The levels of AC6 and AC7 mRNAs were high on the third postnatal day and then decreased in adult, paralleling the decline in AC activity. All the characteristics of AC expression and activity in fetus and neonate resembled those observed in denervated adult muscle. These results indicate that changes in AC activity and AC mRNAs play an important role in the various physiopathological states of skeletal muscle, especially during muscle atrophy.

fast-twitch muscle; neurotransmitters; G proteins; Northern blot; in situ hybridization

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

ADENYLYL CYCLASES (ACs) catalyze the conversion of ATP to cAMP and play an important role in neurotransmission in the central nervous system as well as in the regulation of muscle contraction. Many neurotransmitters, including ACh and norepinephrine, transduce their signal into a cell by activating G protein-coupled receptors that modulate AC activity; changes in the intracellular cAMP, in turn, affect cAMP-dependent protein kinases (PKAs).

Genes for at least nine distinct mammalian ACs have been reported (reviewed in Refs. 29, 34, 62). The nine isoforms have been cloned, sequenced, and characterized. All of them are activated by forskolin, with the possible exception of AC9, and by the GTP-bound alpha -subunit of the stimulatory G protein (Gsalpha ). However, other regulatory properties vary among the different isoforms: for example, their susceptibility to protein kinase C (PKC) and PKA and to the GTP-bound alpha -subunit of the inhibitory G protein (Gialpha ) differ; PKC has been implicated in the stimulation of AC2 and AC5 but markedly inhibits the activity of AC6 (38, 41, 43, 75); PKA inhibits the activity of both AC5 and AC6 (33). AC1 and AC8 are specific for neural tissues and are activated by calmodulin in the presence of calcium (11, 40, 72). AC5, essentially expressed in heart and striatum, and AC6 can be inhibited by physiologically relevant concentrations of calcium (32, 37), whereas the calcium-mediated inhibition of AC3 activity in vivo is due to a direct phosphorylation of AC3 by a calmodulin kinase II (69, 70). AC2, AC4, and AC7 are insensitive to calcium but are activated by the beta gamma -subunits of the heterotrimeric G proteins (43, 61); moreover, they seem to be preferred substrates for PKC isoenzymes (35, 68). Finally, AC9 is not regulated by either calcium or beta gamma -subunits (55). It is likely that the diversity of these regulatory mechanisms accounts for the tissue-specific pattern of cAMP synthesis and, in particular, for the occurrence of subtle regulation by cross talk or convergence between independent signaling pathways.

Mammalian adult skeletal muscles are composed of heterogeneous populations of fibers that differ in their biochemical and structural characteristics due to the expression of an alternate subset of genes (reviewed in Refs. 15, 21). These fibers have been divided into four major classes on the basis of their expression of distinct isoforms of contractile proteins. Slow-twitch fibers (type I) express myosin heavy chain (MHC) type I and use mainly oxidative metabolism, whereas fast-twitch fibers (type II) express MHC IIa, IIb, or IIx and are mainly oxidative (type IIa) or glycolytic (types IIb and IIx) (reviewed in Ref. 52). The basis for such a muscle diversity is the existence of a heterogeneous population of myoblasts, differing in their gene expression, but evidence also exists that suggests gene expression in skeletal muscle is primarily determined by the stimulation pattern imposed on the fiber by its motoneuron.

Muscle denervation has been used as a model to study the molecular mechanisms by which muscle activity regulates synaptic protein expression. Dramatic changes occur in the morphological and biochemical properties of a skeletal muscle after section of its motor nerve (14, 24, 46). Some of these changes are thought to result from the immobility of the muscle after denervation, but deficiency of trophic factors resulting from the denervation may also be involved. This process is an important component of many human neurological diseases and has been extensively studied (26, 27). For example, after denervation there is a marked increase in extrajunctional ACh receptor (AChR) density, as well as an increase in the levels of mRNAs coding for the receptor subunits (4, 25, 49, 57), and denervation of skeletal muscle activates the expression of AChR genes (66). One candidate for a second messenger linking the action of nerve factors to AChR biosynthesis is cAMP. Analogs of this nucleotide, as well as activators of AC, stimulate AChR accumulation in cultured myotubes (6, 7), and cAMP is one of the second messengers that mediate the increase in AChR number elicited by calcitonin gene-related peptide (CGRP), a neuronal messenger coexisting with the classical transmitter ACh (42). As an early consequence of denervation, the expression of immediate early genes and the cAMP metabolism are affected, though differently in the different types of muscles. mRNA levels of the fos and jun protooncogenes are increased rapidly after denervation of the lower leg muscles of adult mice (1, 5). Moreover, the mRNAs for four muscle-specific regulatory factors, MyoD, myogenin, Myf-5, and muscle regulatory factor 4 (MRF4), which play an important role in controlling skeletal muscle development by regulating muscle-specific gene expression, rapidly increase following denervation of adult skeletal muscle. This increase can be suppressed by subsequent direct electrical stimulation (2, 9, 17, 67). In rat gastrocnemius muscle, AC activity was found to be increased by two- to fourfold from 14 to 28 days after sciatic nerve section (30, 51, 53). Moreover, chronic motor nerve pacing results in an increase in the AC activity; this increase is time dependent and appears to be consistent with the fiber type-specific differences in cAMP signaling (39, 65).

To date, little is known about the AC isoform expression and distribution in skeletal muscle. To explore the potential role of the ACs in gene regulation and signaling in muscle, we investigated 1) the expression of the different AC transcripts in normal adult gastrocnemius muscle on denervation of the sciatic nerve and during development, 2) the changes in AC activity in response to several neurotransmitters in these different conditions, and 3) the distribution of AC isoforms by in situ hybridization in mouse skeletal muscle before and after denervation.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animals and Tissue Preparation

C57BL/6 mice were purchased from Janvier. Male adult mice (aged 2 mo) weighing 20-25 g were used for the denervation study. Under pentobarbital anesthesia (60 mg/kg body wt, intraperitoneal injection), denervation was carried out by excision of a 5-mm piece of the left sciatic nerve at the midthigh region. Ten or thirty days after denervation, mice were decapitated and the gastrocnemius muscles were excised (D10 and D30, respectively). The contralateral muscle was used as a control (C10 or C30). No changes were found between C10 and C30 in AC activity assay and Northern blot analysis. Therefore, we denote the control muscle as simply C. For the fetal studies, time-mated females were decapitated after 19 days of gestation (E19). Mice at postnatal day 3 (PN3), PN21, and PN60 (adult) were decapitated, and their gastrocnemius muscles were rapidly removed. The muscles dissected on E19 or PN3 from the hindlimbs of fetal mice from a given litter were pooled and considered as a single preparation, respectively. These muscles may have contained minor amounts of bone residue that were difficult to remove during dissection.

AC Activity Assay

Isolated muscles were immediately homogenized in 10 volumes of ice-cold buffer containing (in mM) 50 Tris · HCl (pH 7.6), 2 EDTA (pH 7.6), 1 dithiothreitol, and 0.1 phenylmethylsulfonyl fluoride, and then they were centrifuged at 150 g for 10 min at 4°C. The pellet was discarded, and the supernatant was centrifuged at 17,000 g for 30 min at 4°C. The pellet that was obtained was resuspended in the same buffer and resubjected to the same centrifugation step. This procedure was repeated three times. The final pellet was resuspended in an appropriate volume of the same buffer and stored at -80°C until use.

Before the AC activity assay was started, the frozen membranes were thawed and centrifuged at 17,000 g for 30 min at 4°C. The pellet was resuspended in an ice-cold membrane buffer containing (in mM) 50 Tris · HCl (pH 7.6), 2 dithiothreitol, and 2 EGTA. The washing process was repeated twice. Finally, the pellet was resuspended with the membrane buffer at a concentration of ~1 mg protein/ml. AC activity was measured as described previously (29, 44), with some modifications. The standard reaction mixture contained (in mM) 50 Tris · HCl (pH 7.6), 1 EGTA, 5 MgCl2, 1 ATP containing [alpha -32P]ATP [1 × 106 counts/min (cpm)], 1 cAMP containing [8-3H]cAMP (10,000 cpm), and 5 phosphocreatine, with 250 µg/ml creatine phosphokinase, 10 µM GTP, and 30 µg of membrane protein in a final volume of 60 µl with or without test agents. Test agents were diluted with a 50 mM Tris · HCl buffer (pH 7.6) and added to the reaction mixture at the concentrations indicated in the text. Reactions were initiated by addition of membranes and run for 10 min at 37°C. The reactions were terminated by addition of 200 µl of 0.5 M HCl, followed by boiling for 7 min and neutralization by 200 µl of 1.5 N imidazole. cAMP formed during the incubation was separated by alumina column and corrected for recovery of added [3H]cAMP. Protein was measured by the method of Bradford (8), with BSA as the standard. CGRP (rat), salmon calcitonin (sCT), and amylin fragment 8-37 (human) were purchased from Sigma.

Probes

Probes specific for each of the AC subtypes were chosen from regions where the sequences are the most divergent. Probes specific for rat AC2, AC5, and AC6 were obtained by PCR, using mRNA from rat brain and the following specific oligonucleotides: the primers 5'-GGGAAGATTAGTACCACGGAT-3' (sense) and 5'-AGGAGAAGCCA AGGATGGACG-3' (antisense) for the 334-bp rat AC2 probe (20), 5'-CAGAGAACCAACTCCATTGGACACAATCCG-3' (sense) and 5'-CACACAGGCGTAGATCACAGATATAAACAC-3' (antisense) for the 459-bp rat AC5 probe (54), and 5'-TGCTGCTGGTCACCGTGCTCAT-3' (sense) and 5'-GGACGCTAAGCAGTAGATCATAGTGTCAA-3' (antisense) for the 493-bp rat AC6 probe (55). The amplified fragments were subcloned into the Sma I site of pGEM3 (Promega) for the rat AC2 or into the cloning site of pCRII (Invitrogen) for the rat AC5 and AC6 fragments. A 722-bp fragment cloned in pCRII and a 739-bp fragment cloned in pGEM3 were derived from human AC7 (nt 2453-3175; membrane-spanning domains 7-12) (50) and mouse AC9 (nt 1-739) (54), respectively. Various restriction fragments (1738, 1900, 1748, and 280 bp) of rat cDNAs coding for Gsalpha , Gi-1alpha , Gi-2alpha , and Gi-3alpha , respectively, were used as probes (36). A 1272-bp fragment inserted in the Pst I site of Bluescript was derived from rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (22). cDNA clones of the PKA regulatory subunit RIalpha and myogenin were generous gifts from M. C. Weiss [Unité de Génétique de la Différenciation, Unité de Recherche Associée (URA) 1149, Centre National de la Recherche Scientifique, Département de Biologie Moléculaire, Institut Pasteur] and D. Daegelen [Génétique et Pathologie Moléculaires de la Différenciation Musculaire, Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 129], respectively, and are detailed in Refs. 10 and 16, respectively.

cDNA probes were used for Northern blot analysis. The cDNA fragments were labeled with [alpha -32P]dCTP, using a standard random primer reaction (Megaprime, Amersham).

cRNA probes were used for in situ hybridization. Before in vitro transcription was started, the pGEM3 plasmids containing the 334-bp fragment of the rat AC2 and the 739-bp fragment of the mouse AC9 were linearized with EcoR I, the pCRII plasmids containing the 493-bp fragment of the rat AC6 and the 772-bp fragment of the human AC7 were linearized with Xho I and EcoR V, respectively, and the pSP65 plasmid containing the 295-bp fragment of PKA subunit RIalpha was linearized with EcoR I, to prepare antisense cRNA probe. The in vitro transcription was carried out at 42°C for 90 min in a 20-µl reaction mixture containing 1 µg of linearized plasmid templates and SP6 RNA polymerase in the presence of [alpha -33P]UTP (>3,000 Ci/mmol, 1 mCi = 37 MBq; ICN). The 33P-labeled probes were separated from free nucleotides by spin column (Sephadex G-50, fine) and then concentrated by ethanol precipitation.

Northern Blot Analysis

Total RNAs were prepared by the guanidinium thiocyanate-phenol extraction method (13). Poly(A)+ RNAs were isolated by the batch method with the use of the oligo(dT) cellulose. Ten micrograms of poly(A)+ RNA were electrophoresed on a 1% agarose gel containing 2.2 M formaldehyde as previously described (44) and transferred overnight onto Hybond N+ membrane (Amersham) by capillary blotting. The 0.24- to 9.5-kb RNA ladder from GIBCO BRL was used as the molecular mass standard. The membranes were hybridized for 2 h at 65°C (rapid hybridization buffer, Amersham) with 2 × 106 cpm/ml of probe and washed in 2× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate) containing 0.1% SDS at room temperature for 20 min, followed by 1× SSC-0.1% SDS, 0.5× SSC-0.1% SDS and 0.1× SSC-0.1% SDS at 65°C for 30 min, respectively, and were then exposed to Kodak XAR film with intensifying screens at -80°C. The membranes were subsequently rehybridized with the GAPDH cDNA probe. Messenger RNA levels were measured in each experiment. In each case, the hybridization signals were quantitated by densitometric scanning of the autoradiograms. mRNA values were expressed as a percent of the values from the control muscles (designated 100%) after normalization for GAPDH. Each experiment was performed at least three times with independent RNA preparations. Each time, similar results were obtained and the proportion of the different ACs was the same (see Figs. 4-7 for typical experiments).

In Situ Hybridization

In situ hybridization was performed essentially as described previously (44), with some modifications. Briefly, the excised gastrocnemius muscles were embedded immediately in Tissue-Tek OCT (Miles, Elkhart, IN) and quick frozen at -20°C. Tissue blocks were stored at -80°C. Eight-micrometer-thick cryostat sections were thaw mounted on gelatin-coated slides and stored in air-tight containers at -20°C. The frozen sections were fixed with 4% paraformaldehyde in PBS, treated with proteinase K (20 µg/ml) in 50 mM Tris · HCl (pH 8.0) and 5 mM EDTA for 7.5 min at room temperature, acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0), and delipidated in a graded series of ethanol and chloroform. Sections were then preincubated in a humid chamber at 52°C for 12 h with 30 µl of prehybridization buffer [50% formamide, 4× SSC, 1% sarkosyl, 1× Denhardt's reagent, 0.1 M phosphate buffer (pH 7.4), and 250 µg/ml denatured salmon sperm DNA]. Hybridization was performed overnight at 52°C in 30 µl of prehybridization buffer containing 10% dextran sulfate and 2.0 × 107 cpm/ml cRNA probe, under Parafilm coverslips. Slides were rinsed in 1× SSC and incubated for 30 min at 37°C with 20 µg/ml RNase A and 10 U/ml RNase T1 in 100 mM Tris · HCl (pH 8.0), 500 mM NaCl, and 1 mM EDTA. They were further washed in decreasing concentrations of SSC and finally washed with 0.1× SSC for 15 min at 37°C and then dehydrated in ethanol containing 300 mM ammonium acetate, dried, and exposed to BioMax film (Kodak) for 7 days to generate autoradiographic images. Nonspecific hybridization was determined by hybridization in a section pretreated with 20 µg/ml RNase A and 10 U/ml RNase T1 in 100 mM Tris · HCl (pH 8.0), 500 mM NaCl, and 1 mM EDTA at 37°C for 45 min. The signals observed with the antisense probes were almost totally eliminated in the control experimental conditions.

Data Analysis

Northern blots were performed at least three times for each AC type, G protein, and myogenin, and quantification was done by densitometric scanning and computer analysis with a laser densitometer (Ultroscan XL, LKB Instruments, Bromma, Sweden). For each AC type, G protein, and myogenin, the results were normalized using a GAPDH probe, and the values for the control and adult muscle were set arbitrarily as 100% for comparison after denervation and during development, respectively (see Figs. 4-7). To determine the relative levels of AC isoforms mRNAs, in each experiment, the length and the specific activity of the probes as well as the exposure time were taken into account (see Table 1). The values were expressed as arbitrary units per 100 bp of the probe per 24 h of exposure and normalized to GAPDH values, also corrected in the same way. Statistical comparisons were made by ANOVA followed by Student's two-tailed, nonpaired t-test. A value of P < 0.05 was considered to be significant. Data are presented as means ± SE.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

AC Activity Assay in Control and Denervated Muscle

After denervation, the weight of the gastrocnemius muscle decreased gradually, reaching 70 and 40% of that of the control, contralateral muscle 10 and 30 days after denervation (D10 and D30), respectively.

We first measured the basal and forskolin-, isoproterenol-, epinephrine-, and ACh-stimulated AC activities in the control and denervated muscles (Fig. 1). Basal AC activity in denervated muscle (D10 and D30) was higher than that in control muscle. The AC activity was 8.4 ± 0.34 pmol · min-1 · mg-1 in membranes from control animals and 11.97 ± 1.67 and 15.68 ± 1.56 pmol · min-1 · mg-1 in membranes from D10 and D30, respectively. Forskolin, isoproterenol, and epinephrine stimulated AC activity in a concentration-dependent manner. Compared with the basal AC activity, AC activity stimulated by forskolin (100 µM) was increased to 3.1-fold in the control and to 4.6- and 7.0-fold in D10 and D30, respectively. AC activity stimulated by isoproterenol (10 µM) was increased to 2.9-fold in the control and to 3.9- and 4.6-fold in D10 and D30, respectively, compared with the basal AC activity. AC activity stimulated by epinephrine (100 µM) was increased to 2.5-fold in the control and to 3.8- and 4.1-fold in D10 and D30, respectively, compared with the basal AC activity. On the other hand, ACh inhibited AC activity in a concentration-dependent manner. Compared with the basal AC activity, AC activity was decreased to 50% by ACh (100 µM) in the control and to 16 and 8% in D10 and D30, respectively.


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Fig. 1.   Effects of forskolin (FSK), isoproterenol (ISO), epinephrine (EPI), or ACh on adenylyl cyclase (AC) activity in control and denervated mouse gastrocnemius muscles. Left: AC activity. Right: percent of AC activity relative to basal activity. C, control muscle; D10 and D30, denervated muscle 10 and 30 days after surgery, respectively. Values are means ± SE of data from 3 experiments and triplicate determinations. #P < 0.05, * P < 0.01 compared with control muscle at same concentration of stimulus.

Furthermore, because CGRP, sCT, and amylin have been reported to specifically increase AC activity in skeletal muscle, we assessed their effects in control and denervated muscle (Fig. 2). These three peptides stimulated AC activity in a concentration-dependent manner. Compared with the basal AC activity, AC activity stimulated by CGRP (10 µM) was increased to 1.9-fold in the control and to 2.5- and 3.8-fold in D10 and D30, respectively. AC activity stimulated by sCT (10 µM) was increased to 1.6-fold in the control and to 2.2- and 3.5-fold in D10 and D30, respectively, compared with the basal AC activity. AC activity stimulated by amylin (10 µM) was increased to 1.7-fold in the control and to 2.0- and 2.9-fold in D10 and D30, respectively, compared with the basal AC activity.


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Fig. 2.   Effects of calcitonin gene-related peptide (CGRP), salmon calcitonin (sCT), or amylin (AMY) on AC activity in control and denervated mouse gastrocnemius muscle. Left: AC activity. Right: percent of AC activity relative to basal activity. Values are means ± SE of data from 3 experiments and triplicate determinations. #P < 0.05, * P < 0.01 compared with control muscle at same concentration of stimulus.

AC Activity Assay in Developing Muscle

We measured AC activity in the absence of stimuli (basal) or in the presence of 100 µM forskolin, 10 µM isoproterenol, 100 µM epinephrine, 100 µM ACh, 10 µM CGRP, 10 µM sCT, or 10 µM amylin at different ages (E19, PN3, PN21, and adult). The basal AC activities at these points were 12.4, 14.8, 10.6, and 8.4 pmol · min-1 · mg-1 protein, respectively. At E19, all the chemical agents (except ACh) already had significant stimulatory effects on AC activities, and ACh inhibited AC activity significantly. The basal and stimulated AC activities increased 1.2- to 1.8-fold to peak levels at PN3, followed by a decrease to the adult levels (Fig. 3). AC activity inhibited by ACh decreased 91% to the lowest level at PN3; the level in the adult was 1.9-fold greater than the E19 level (Fig. 3).


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Fig. 3.   AC activity in developing mouse gastrocnemius muscle. Membranes were prepared from 19-day-old fetus (E19), 3-day-old neonate (PN3), 21-day-old mouse (PN21) and 60-day-old adult (AD). A: AC activity was measured in absence or presence of forskolin (100 µM), isoproterenol (10 µM), epinephrine (100 µM), or ACh (100 µM) B: AC activity was measured in absence or presence of CGRP (10 µM), sCT (10 µM), or amylin (10 µM). Values are means ± SE of data from 3 experiments and triplicate determinations.

Northern Blot Analysis in Control and Denervated Muscle

Expression of AC2, AC6, AC7, and AC9 mRNAs. AC isoform mRNAs in mouse gastrocnemius muscle were resolved as single bands of 4.2, 6.5, 7.5, and 8.5 kb for AC2, AC6, AC7, and AC9, respectively, as described in other tissues (20, 54, 55, 68). We did not detect any AC5 mRNA. After 6 days of exposure, AC4 mRNA was barely detectable both in control and in denervated gastrocnemius muscle (not shown). Moreover, none of the calcium/calmodulin-stimulated AC mRNAs were observed either in rat or in mouse skeletal muscle. GAPDH mRNA, used as an internal control in all these experiments, migrated as a unique 1.5-kb band in mouse gastrocnemius muscle; its expression was constant after denervation, as observed in rat skeletal muscle (56). The levels of expression of AC2 and AC9 were decreased after denervation. Compared with control muscle, the levels of AC2 and AC9 mRNA were lower by 43.3% (D10) and 39.1% (D30) and by 35.9% (D10) and 30.6% (D30), respectively. In contrast, compared with control muscle, the levels of AC6 and AC7 mRNA were higher by 1.3-fold (D10) and 5.1-fold (D30) and by 4.4-fold (D10) and 10.8-fold (D30), respectively (Fig. 4). The relative levels of these four types of AC mRNA in control and D30 muscles were in the following orders: AC9 > AC2 > AC7 > AC6 in control muscles and AC7 > AC6 > AC9 > AC2 in D30 muscles (Table 1). The relative loss of AC9, compared with the other isoforms, may explain the higher sensitivity to forskolin of the total AC activity assayed in the denervated muscles.


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Fig. 4.   Northern blot analysis of AC mRNAs in control and denervated mouse gastrocnemius muscle. Left: 10 µg of poly(A)+ RNA were electrophoresed on 1% agarose-formaldehyde gel and transferred to a nylon membrane. Blots were hybridized with [alpha -32P]dCTP-labeled cDNA probes specific for each AC type (AC2, AC6, AC7, and AC9). After exposure for 1 day (AC9) or 2 days (AC2, AC6, and AC7) at -80°C, membranes were hybridized with 32P-labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probes. Exposure time for GAPDH was 3 h. Results are from 1 typical experiment of 3, with independent RNA preparations. Right: individual densitometric analysis for AC mRNA levels normalized to expression of GAPDH and expressed as percent of control muscle mRNA level. Values are means ± SE of data from 3 experiments. #P < 0.05, * P < 0.01 compared with control muscle.

                              
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Table 1.   Relative level of four types of AC mRNA in control muscle and muscles 10 and 30 days after denervation

Expression of Gsalpha , Gi-1alpha , Gi-2alpha , Gi-3alpha , and myogenin mRNAs. G protein and myogenin isoform mRNAs in mouse gastrocnemius muscle were resolved as single bands of 1.8, 2.4, 3.5, and 1.6 kb for Gsalpha , Gi-2alpha , Gi-3alpha , and myogenin, respectively, as described in previous reports (17, 36). We did not detect Gi-1alpha mRNA. The levels of expression of the four detectable mRNAs were increased after denervation. Compared with control muscle, the levels of Gsalpha , Gi-2alpha , Gi-3alpha , and myogenin mRNA were higher by 5.4-fold (D10) and 9.6-fold (D30), by 1.5-fold (D10) and 4.5-fold (D30), by 1.4-fold (D10) and 3.7-fold (D30), and by 30.9-fold (D10) and 61.1-fold (D30), respectively (Fig. 5). The increased level of expression of myogenin mRNA corresponded with a previous report (17).


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Fig. 5.   Northern blot analysis of Gsalpha , Gi-2alpha , Gi-3alpha , and myogenin mRNAs in control and denervated mouse gastrocnemius muscles. Left: for Northern blot analysis, same blots used for expression of AC (Fig. 4) were used for expression of G protein and myogenin. Autoradiograms were obtained after exposure for 2 h (Gsalpha ), 18 h (Gi-2alpha and Gi-3alpha ), 16 h (myogenin), or 3 h (GAPDH). Right: individual densitometric analysis for Gsalpha , Gi-2alpha , Gi-3alpha , and myogenin mRNA levels normalized to expression of GAPDH and expressed as percent of control muscle mRNA level. Values are means ± SE of data from 3 experiments. * P < 0.01 compared with control muscle.

Northern Blot Analysis in Developing Muscle

Expression of AC2, AC5, AC6, AC7, and AC9 mRNAs. Northern blot analysis of the relative levels of AC2, AC5, AC6, AC7, and AC9 mRNAs was performed to determine whether the levels of these messengers changed during development. The results are shown in Fig. 6, which depict the overall changes in the relative level of each RNA after correction for the GAPDH signal. The pattern of change in the RNA expression was different for the AC isoforms examined. The levels of expression of AC2 and AC9 were increased gradually during development. The relative ratios of AC2 and AC9 RNA to GAPDH messenger were calculated as 2.0-, 2.3-, and 2.6-fold increases and 2.2-, 3.3-, and 4.2-fold increases over the values for E19, PN21, and adult muscles, respectively. On the other hand, the levels of expression of AC6 and AC7 mRNAs were increased 1.1- and 1.4-fold to peak levels at PN3, respectively, followed by a decrease to adult levels. We did not detect AC5 mRNA at any stage during development.


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Fig. 6.   Expression of different ACs in mouse gastrocnemius muscle during development. Left: poly(A)+ RNA were isolated from mouse gastrocnemius muscles of a 19-day-old fetus (E19), 3-day-old neonate (PN3), 21-day-old mouse (PN21), and 60-day-old adult (AD); 10 µg of poly(A)+ RNA were loaded on 1% agarose-formaldehyde gel. Northern blot was performed as described in Fig. 4. Blots were hybridized with [alpha -32P]dCTP-labeled cDNA probes specific for each AC type (AC2, AC6, AC7, and AC9). After exposure for 1 day (AC9) or 2 days (AC2, AC6, and AC7) at -80°C, membranes were hybridized with 32P-labeled GAPDH cDNA probes. Exposure time for GAPDH was 3 h. Right: individual densitometric analysis for AC mRNA levels normalized to expression of GAPDH and expressed as percent of adult muscle mRNA level. Values are means ± SE of data from 3 experiments. #P < 0.05, * P < 0.01 compared with adult muscle.

Expression of Gsalpha , Gi-1alpha , Gi-2alpha , and Gi-3alpha mRNAs. The expression of G protein mRNAs was examined by Northern blot analysis during development. We did not detect Gi-1alpha mRNA during development. The levels of expression of the other three G protein mRNAs decreased gradually during development. The relative ratios of these mRNAs to GAPDH messenger in adult muscle were decreased by 22, 10, and 27% compared with the values at E19 for Gsalpha , Gi-2alpha , and Gi-3alpha , respectively (Fig. 7).


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Fig. 7.   Expression of Gsalpha , Gi-2alpha , and Gi-3alpha in mouse gastrocnemius muscle during development. Left: for Northern blot analysis, same blots used for expression of AC (Fig. 6) were used for expression of G protein subunits. Autoradiograms were obtained after exposure for 2 h (Gsalpha ), 18 h (Gi-2alpha and Gi-3alpha ), or 3 h (GAPDH). Right: individual densitometric analysis for Gsalpha , Gi-2alpha , and Gi-3alpha mRNA levels normalized to expression of GAPDH and expressed as percent of adult muscle mRNA level. Values are means ± SE of data from 3 experiments. #P < 0.05, * P < 0.01 compared with adult muscle.

In Situ Hybridization in Control and Denervated Muscle

Figure 8 shows X-ray film images of a series of control or denervated gastrocnemius muscle sections 10 days after operation, hybridized with the AC2, AC6, AC7, AC9, and PKA regulatory subunit RIalpha probes. As previously described by Imaizumi-Scherrer et al. (31), the arclike distribution of the neuromuscular junction (NMJ) was outlined by the RIalpha probe, and the number of the spots and the intensity of the RIalpha signal were enhanced after denervation. By contrast, the distribution of the four AC isoform mRNAs was uniform and diffuse. After denervation, the signal strength of AC2 and AC9 was slightly decreased, whereas that of AC6 and AC7 was slightly increased. These signals disappeared after pretreatment with RNase A and RNase T1. There is an apparent discrepancy between in situ hybridization and Northern blot concerning the level of expression of the ACs. The same situation exists in brain, where AC6, one of the major isoforms, has a weak and ubiquitous distribution, as opposed to the highly selective distribution of AC5 mRNA in striatum, nucleus accumbens, and olfactory tubercle (48).


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Fig. 8.   In situ hybridization analysis for AC2, AC6, AC7, AC9, and cAMP-dependent protein kinase subunit RIalpha mRNA expression in control and denervated mouse gastrocnemius muscle. Sections were hybridized with 33P-labeled antisense cRNA probes as described in MATERIALS AND METHODS. C10 and D10, control and denervated muscles 10 days after operation, respectively. RNase: sections were pretreated with 20 µg/ml RNase A and 10 U/ml RNase T1 at 37°C for 45 min before hybridization with RIalpha antisense cRNA probe. Autoradiograms were obtained after exposure for 7 days. Three separate experiments gave essentially same results. Bar, 5 mm.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The experiments presented in this study demonstrate that four AC isoforms (AC2, AC6, AC7, and AC9) are expressed in mouse gastrocnemius muscle, AC9 and AC2 being the most abundant. It is striking that skeletal muscle does not express a detectable amount of AC5, which is the predominant form in mature cardiac myocytes (19, 63). After denervation, the pattern of changes in the mRNA expression for these four ACs was markedly altered: the levels of AC2 and AC9 mRNA decreased, whereas those of AC6 and AC7 increased. Using in situ hybridization, we showed that the distribution of the four AC isoforms detected was uniform and diffuse both in normal and denervated muscle. In parallel with the changes in the AC expression program, we observed an increase in AC activity and a hypersensitivity to various effectors such as ACh, calcitonin, CGRP, amylin, isoproterenol and epinephrine, probably related to changes in the expression of G proteins. It is interesting to note that the characteristics of the AC isoform expression and activity in the fetus and the neonate resemble those observed in denervated adult skeletal muscle. Namely, compared with adult muscle, the levels of AC6 and AC7 mRNAs are high, those of AC2 and AC9 mRNAs are low, and the AC activities in response to various agents are high.

The patterns of AC isoform expression are different in skeletal, cardiac, and smooth muscles. We have shown that in the rat uterus five AC isoforms (AC2, AC4, AC6, AC7, and AC9) are expressed, with AC6 being the most abundant. The level of expression of these ACs increases during the course of pregnancy and diminishes near term and after delivery. The highest level of expression for each type of AC is consistently seen on the 17th day of pregnancy (59). In cardiac muscle, we (19), as well as Tobise et al. (63), reported an age-related change in the myocardial transduction pathway determined by a shift from a predominantly AC6 to a predominantly AC5 pattern. It is therefore obvious that the various AC genes are controlled by a unique combination of transcription factors in the different muscle cell types. The overall consequences of the changes in the AC pattern for muscle function are difficult to assess at present: AC2 and AC7 are closely related, and the only major difference between AC9 and AC6 is a potential weak inhibition by calcium of the latter.

Many studies have established that motoneuron activity influences gene expression in developing and adult skeletal muscle fibers. The nerve provides a trophic influence on the muscle by one or more specific transmitter substances and/or electrical activity. The neural dependence is further substantiated by the fact that neonatal denervation retards the development of fast-contracting muscle fibers (18). Gastrocnemius muscle contains 75-100% fast-twitch muscle fibers, depending on the species (28, 67). Denervation of fast-twitch muscles, such as extensor digitorum longus and tibialis anterior, results in a relative increase in the proportion of myofibers expressing slow myosin and slow troponin I (67, 68). A number of other phenotypic changes also follow denervation of fast-twitch muscles, including decreased myosin ATPase activity, increased levels of enzymes involved in oxidative pathways, prolonged contraction time, and increased proportion of slow-twitch fibers (27, 60, 64). Denervation results in muscle atrophy, and the denervated muscle becomes hypersensitive to the neurotransmitters present in the nerves that are destroyed, as the consequence of increases in neurotransmitter receptors. Although most skeletal muscle genes are expressed at similar levels in electrically active innervated muscle and in electrically inactive denervated muscle, a small number of genes, including those encoding AChR, the neural cell adhesion molecule (NCAM), and myogenin, are expressed at significantly higher levels in denervated than in innervated muscles (cf. references cited in Ref. 74). Denervation of adult muscle causes a dramatic increase in the sensitivity of the fibers to ACh, which is directly attributable to increases in the levels of alpha -, beta -, gamma -, and delta -subunit RNAs within 24-48 h and the expression of the embryonic type of nicotinic AChR (nAChR) (2). As an early event following denervation of adult rat hindlimb muscles, a rapid increase in the level of muscle regulatory factors (MyoD, myogenin, Myf-5, and MRF4) has been described (2, 67). The increase in myogenin transcripts in the denervated muscle is similar to the rapid increase in myogenin expression observed when myoblasts are committed to differentiate in culture. The increase in the myogenin mRNA expression is maintained even 30 days after denervation, when we observed an increase of AC6 and AC7 and of alpha -subunits of the heterotrimeric G proteins (Galpha s, Galpha i-2, and Galpha i-3). Genes similar to those that are active during muscle cell development may be switched on following a nerve injury, including AChRs, creatine kinase, and phosphoglyceromutase (3, 45). The return of these parameters to normal after reinnervation is variable and may be incomplete (23). It thus appears that, after denervation of skeletal muscle, a genetic program that includes myogenic differentiation and protooncogene expression is activated (1, 5, 71).

Before innervation, AChR are detected over the entire surface of the myotube. Once the motor nerve ending has contacted the muscle cells, AChR density increases rapidly at the NMJ while its biosynthesis declines in the extrajunctional regions (12). Outside the junction, the electrical activity evoked by the motor nerve suppresses the AChR gene expression primarily by a reduction of the transcription rates of AChR genes. At the NMJ, the degradation rate of AChRs is accelerated on denervation but can be stabilized by reinnervation or by cAMP. This effect is mediated through the PKA-dependent pathway (58, 73). The NMJ plays an important role in the course of development and maturation of skeletal muscle. Using in situ hybridization, we have shown that the distribution of the four AC isoforms detected was uniform and diffuse in the muscle and could not be detected preferentially in the NMJ, where the PKA RIalpha subunit and AChR mRNAs are distributed (12, 31). After denervation, RIalpha subunit mRNA increases specifically at the level of the NMJ and the program of AC expression is modified: AC2 and AC9 mRNAs decrease, whereas AC6 and AC7 mRNAs increase, without any apparent modification in their distribution. Imaizumi-Scherrer et al. (31) showed that PKA subunit RIalpha mRNA is localized at the NMJ and, in addition, demonstrated that RIalpha recruits a Calpha subunit to the junction, providing the potential for local responsiveness to cAMP at this site. Thus PKA is implicated in the establishment and/or maintenance of the unique pattern of gene expression occurring at the junction or in the modulation of synaptic activity via protein phosphorylation. Although the mRNA localization of the various ACs is diffuse, it is possible that a specific distribution of the different AC isoforms exists, playing an as yet unidentified but important role at the NMJ. However, this is still impossible to determine due to the absence of good antibodies specific for each isoform.

Because the mouse gastrocnemius muscle is composed of almost 100% type II fast-twitch fibers and because denervation results in an increased amount of slow-twitch fibers, we can assume that AC2 and AC9 are restricted to fast-twitch fibers and that AC7 and AC6 might be associated with the reprogramming after destruction of the motoneuron contacts. Torgan and Kraus (65) also showed that expression of AC2 in the muscle fiber significantly correlates with the percentage of fast-twitch muscle fibers: electrical motor nerve pacing, which induces fiber type transformation of fast-twitch to slow-twitch fibers, induces a decrease in AC2 mRNA preceding the fiber type conversion of metabolic and contracting proteins. This transformation is accompanied by an alteration in gene expression for proteins of energy metabolism and sarcoplasmic reticulum function and by switches in contractile protein isoform expression (39). Therefore, our results indicate that innervation is necessary to maintain the high levels of AC2 and AC9 transcripts and is also required to repress the AC6 and AC7 genes to the low levels found in the control muscle.

At the receptor level, denervation induces the appearance of new AChRs in the muscle membrane outside the endplate (47). cAMP plays a major role in this regulation, since it is one of the second messengers responsible for the increase in AChR number elicited by CGRP. More recently, it has been suggested that nerve regulation of the AChR degradation occurs also via a PKA-dependent pathway (73). In the present study, the basal and stimulated AC activity to various agents at D30 was higher than that at D10, a parallel change being observed in the expression levels of ACs (AC6 and AC7) as well as of G protein mRNAs. Taking these findings together, the changes in AC activity and sensitivity to various agents induced by denervation were due not only to an increase in the numbers of AC molecules as a consequence of increased levels of mRNAs coding for ACs (especially, AC6 and AC7) but also to changes in the receptor-G protein complex occurring together with muscle atrophy.

In conclusion, our studies illustrate the differential expression of AC2, AC6, AC7, and AC9 in skeletal muscle after denervation. Among these isoforms, AC9 and AC7 are the predominant subtypes in normal and denervated muscle, respectively. The expression of these AC isoforms is altered during development, but the pattern of their changes is different. A parallel decrease in mRNAs encoding for AC6 and AC7 and in AC activity during development might indicate that these two isoforms are the major factors contributing to the decline in the activity in the adult animals. Therefore, we suggest that changes in AC activity, determined by the content of specific isoforms, play an important role during muscle atrophy and possibly in other various physiopathological states of the skeletal muscle.

    ACKNOWLEDGEMENTS

We thank Dr. I. Espinasse [URA 1159, Centre National de la Recherche Scientifique (CNRS)] for the gift of all the G protein plasmids, Dr. M. C. Weiss (Unité de Génétique de la Différenciation, URA 1149, CNRS, Département de Biologie Moléculaire, Institut Pasteur) for the gift of PKA subunit RIalpha plasmid, and Dr. D. Daegelen (Génétique et Pathologie Moléculaires de la Différenciation Musculaire, INSERM Unité 129) for the gift of myogenin plasmid. We are also thankful to Drs. G. Guellaen, Y. Laperche, R. Barouki, J. F. Laycock and S. Lotersztajn for critical reading of the manuscript and to E. Grandvilliers for expert secretarial assistance.

    FOOTNOTES

This work was supported by INSERM and the Université Paris-Val de Marne.

Y. Suzuki is a recipient of a fellowship from the Fondation pour la Recherche Médicale. T. Shen is a recipient of a fellowship from the People's Republic of China.

Present addresses: Y. Suzuki, Department of Obstetrics and Gynecology, Fukushima Medical College, Fukushima, 960-12 Japan; T. Shen, c/o Dr. Rebecca Morris, Lankenau Medical Research Center, 100 Lancaster Ave. West, Wynnewood, PA 19096.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address reprint requests to J. Hanoune.

Received 15 January 1998; accepted in final form 5 March 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Abu-Shakra, S. R., A. J. Cole, and D. B. Drachman. Nerve stimulation and denervation induce differential patterns of immediate early gene mRNA expression in skeletal muscle. Brain Res. Mol. Brain Res. 18: 216-220, 1993[Medline].

2.   Adams, L., B. M. Carlson, L. Henderson, and D. Goldman. Adaptation of nicotinic acetylcholine receptor, myogenin, and MRF-4 gene expression to long-term muscle denervation. J. Cell Biol. 131: 1341-1349, 1995[Abstract].

3.   Andres, V., R. Cusso, and J. Carreras. Effect of denervation on the distribution and developmental transition of phosphoglycerate mutase and creatine phosphokinase isozymes in rat muscles of different fiber-type composition. Differentiation 43: 98-103, 1990[Medline].

4.   Axelsson, J., and S. Thesleff. A study of supersensitivity in denervated mammalian skeletal muscle. J. Physiol. (Lond.) 147: 178-193, 1959.

5.   Bessereau, J. L., B. Fontaine, and J. P. Changeux. Denervation of mouse skeletal muscle differentially affects the expression of the jun and fos proto-oncogenes. New Biol. 4: 375-383, 1990.

6.   Betz, H., and J. P. Changeux. Regulation of muscle acetylcholine receptor synthesis in vitro by cyclic nucleotide derivatives. Nature 278: 749-752, 1979[Medline].

7.   Blosser, J. C., and S. H. Appel. Regulation of acetylcholine receptor by cyclic AMP. J. Biol. Chem. 255: 1235-1238, 1980[Abstract/Free Full Text].

8.   Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254, 1976[Medline].

9.   Buonanno, A., L. Apone, M. I. Morasso, R. Beers, H. R. Brenner, and R. Eftimie. The MyoD family of myogenic factors is regulated by electrical activity: isolation and characterization of a mouse Myf-5 cDNA. Nucleic Acids Res. 20: 539-544, 1992[Abstract].

10.   Cadd, G., and G. S. McKnight. Distinct patterns of cAMP-dependent protein kinase gene expression in mouse brain. Neuron 3: 71-79, 1989[Medline].

11.   Cali, J. J., J. C. Zwaagstra, N. Mons, D. M. Cooper, and J. Krupinski. Type VIII adenylyl cyclase. A Ca2+/calmodulin-stimulated enzyme expressed in discrete regions of rat brain. J. Biol. Chem. 269: 12190-12195, 1994[Abstract/Free Full Text].

12.   Cartaud, J., and J. P. Changeux. Post-transcriptional compartmentalization of acetylcholine receptor biosynthesis in the subneural domain of muscle and electrolyte junction. Eur. J. Neurosci. 5: 191-202, 1993[Medline].

13.   Chomczynski, P., and N. Sacchi. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987[Medline].

14.   Coderre, L., M. M. Monfar, K. S. Chen, S. J. Heydrick, T. G. Kurowski, N. B. Ruderman, and P. F. Pilch. Alteration of the expression of GLUT-1 and GLUT-4 protein and messenger RNA levels in denervated rat muscles. Endocrinology 131: 1821-1825, 1992[Abstract].

15.   Donoghue, M. J., and J. R. Sanes. All muscles are not created equal. Trends Genet. 10: 396-401, 1994[Medline].

16.   Edmondson, D. G., and E. N. Olson. A gene with homology to the myc similarity region of MyoD1 is expressed during myogenesis and is sufficient to activate the muscle differentiation program. Genes Dev. 3: 628-640, 1989[Abstract].

17.   Eftimie, R., H. R. Brenneer, and A. Buonanno. Myogenin and MyoD join a family of skeletal muscle genes regulated by electrical activity. Proc. Natl. Acad. Sci. USA 88: 1349-1353, 1991[Abstract].

18.   Engel, W. K., and G. Karpati. Impaired skeletal muscle maturation following neonatal neurectomy. Dev. Biol. 17: 713-723, 1968[Medline].

19.   Espinasse, I., V. Iourgenko, N. Defer, F. Samson, J. Hanoune, and J. Mercadier. Type V, but not type VI, adenylyl cyclase mRNA accumulates in the rat heart during ontogenic development. Correlation with increased global adenylyl cyclase activity. J. Mol. Cell. Cardiol. 27: 1789-1795, 1995[Medline].

20.   Feinstein, P. G., K. A. Schrader, H. A. Bakalyar, W. J. Tang, J. Krupinski, A. G. Gilman, and R. R. Reed. Molecular cloning and characterization of a Ca2+/calmodulin-insensitive adenylyl cyclase from rat brain. Proc. Natl. Acad. Sci. USA 88: 10173-10177, 1991[Abstract].

21.   Firulli, A. B., and E. N. Olson. Modular regulation of muscle gene transcription: a mechanism for muscle cell diversity. Trends Genet. 13: 364-369, 1997[Medline].

22.   Fort, P., L. Marty, M. Piechaczyk, S. El Sabrouty, C. Dani, P. Jeanteur, and J. M. Blanchard. Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3-phosphate-dehydrogenase multigenic family. Nucleic Acids Res. 13: 1431-1442, 1985[Abstract].

23.   Frostick, S. P. The physiological and metabolic consequences of muscle denervation. Int. Angiol. 14: 278-287, 1995[Medline].

24.   Furuno, K., M. N. Goodman, and A. L. Goldberg. Role of different proteolytic system in the degradation of muscle proteins denervation atrophy. J. Biol. Chem. 265: 8550-8557, 1990[Abstract/Free Full Text].

25.   Goldman, D., J. Boulter, S. Heinemann, and J. Patrick. Muscle denervation increases the levels of two mRNAs coding for the acetylcholine receptor alpha -subunit. J. Neurosci. 5: 2553-2558, 1985[Abstract].

26.   Goldspink, D. F. The effects of denervation on protein turnover of rat skeletal muscle. Biochem. J. 156: 71-80, 1976[Medline].

27.   Guthmann, E., and J. Zelena. Morphological Changes in the Denervated Muscle. Prague: Czech. Acad. Sci., 1964.

28.   Hämäläinen, N., and D. Pette. The histochemical profile of fast fiber types IIB, IID, and IIA in skeletal muscle of mouse, rat, and rabbit. J. Histochem. Cytochem. 41: 733-743, 1993[Abstract/Free Full Text].

29.   Hanoune, J., Y. Pouille, E. Tzavara, T. Shen, L. Lipskaya, N. Miyamoto, Y. Suzuki, and N. Defer. Adenylyl cyclases: structure, regulation and function in an enzyme superfamily. Mol. Cell. Endocrinol. 128: 179-194, 1997[Medline].

30.   Hashimoto, K., Y. Watanabe, S. Uchida, and H. Yoshida. Increase in the amount of adenylate cyclase in rat gastrocnemius muscle after denervation. Life Sci. 44: 1887-1895, 1989[Medline].

31.   Imaizumi-Scherrer, T., D. M. Faust, J. C. Benichou, R. Hellio, and M. C. Weiss. Accumulation in fetal muscle and localization to the neuromuscular junction of cAMP-dependent protein kinase A regulatory and catalytic subunits RI alpha and C alpha. J. Cell Biol. 134: 1241-1254, 1996[Abstract].

32.   Ishikawa, Y., S. Katsushika, L. Chen, N. J. Halnon, J. Kawabe, and C. J. Homcy. Isolation and characterization of a novel cardiac adenylyl cyclase cDNA. J. Biol. Chem. 267: 13553-13557, 1992[Abstract/Free Full Text].

33.   Iwami, G., J. Kawabe, T. Ebina, P. J. Cannon, C. J. Homcy, and Y. Ishikawa. Regulation of adenylyl cyclase by protein kinase A. J. Biol. Chem. 270: 12481-12484, 1995[Abstract/Free Full Text].

34.   Iyengar, R. Molecular and functional diversity of mammalian Gs-stimulated adenylyl cyclases. FASEB J. 7: 768-775, 1993[Abstract/Free Full Text].

35.   Jacobowitz, O., J. Chen, R. T. Premont, and R. Iyengar. Stimulation of specific types of Gs-stimulated adenylyl cyclases by phorbol ester treatment. J. Biol. Chem. 268: 3829-3832, 1993[Abstract/Free Full Text].

36.   Jones, D. T., and R. R. Reed. Molecular cloning of five GTP-binding protein cDNA species from rat olfactory neuroepithelium. J. Biol. Chem. 262: 14241-14249, 1987[Abstract/Free Full Text].

37.   Katsushika, S., L. Chen, J. Kawabe, R. Nilakantan, N. J. Halnon, C. J. Homcy, and Y. Ishikawa. Cloning and characterization of a sixth adenylyl cyclase isoform: types V and VI constitute a subgroup within the mammalian adenylyl cyclase family. Proc. Natl. Acad. Sci. USA 89: 8774-8778, 1992[Abstract].

38.   Kawabe, J., G. Iwami, T. Ebina, S. Ohno, T. Katada, Y. Ueda, C. J. Homcy, and Y. Ishikawa. Differential activation of adenylyl cyclase by protein kinase C isoenzymes. J. Biol. Chem. 269: 16554-16558, 1994[Abstract/Free Full Text].

39.   Kraus, W. E., J. P. Longabaugh, and S. B. Liggett. Electrical pacing induces adenylyl cyclase in skeletal muscle independent of the beta -adrenergic receptor. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E226-E230, 1992[Abstract/Free Full Text].

40.   Krupinski, J., F. Coussen, H. A. Balkayar, W.-J. Tang, P. G. Feinstein, K. Orth, C. Slaughter, R. R. Reed, and A. G. Gilman. Adenylyl cyclase amino acid sequence: possible channel- or transporter-like structure. Science 244: 1558-1564, 1989[Medline].

41.   Lai, H. L., T. H. Yang, R. O. Messing, Y. H. Ching, S. C. Lin, and Y. Chern. Protein kinase C inhibits adenylyl cyclase type VI activity during desensitization of the A2a-adenosine receptor-mediated cAMP response. J. Biol. Chem. 272: 4970-4977, 1997[Abstract/Free Full Text].

42.   Laufer, R., and J. Changeux. Calcitonin gene-related peptide elevates cyclic AMP levels in chick skeletal muscle: possible neurotrophic role for a coexisting neuronal messenger. EMBO J. 6: 901-906, 1987[Abstract].

43.   Lustig, K. D., B. R. Conklin, P. Herzmark, R. Taussig, and H. R. Bourne. Type II adenylylcyclase integrates coincident signals from Gs, Gi, and Gq. J. Biol. Chem. 268: 13900-13905, 1993[Abstract/Free Full Text].

44.   Matsuoka, I., G. Giuili, M. Poyard, D. Stengel, J. Parma, G. Guellaen, and J. Hanoune. Localization of adenylyl and guanylyl cyclase in rat brain by in situ hybridization: comparison with calmodulin mRNA distribution. J. Neurosci. 12: 3350-3360, 1992[Abstract].

45.   Matsushita, H., S. Yamada, M. Adachi, T. Satoh, K. Kato, and H. Haimoto. Fetal-type creatine kinase in rat fast and slow muscles during denervation and reinnervation. Exp. Neurol. 97: 128-134, 1987[Medline].

46.   McLane, J. A., and I. R. Held. Effect of denervation on cyclic nucleotide metabolism in different types of skeletal muscle of the rat. J. Neurosci. Res. 6: 327-336, 1981[Medline].

47.   Miledi, R. The acetylcholine sensitivity of frog muscle fibers after complete or partial denervation. J. Physiol. (Lond.) 151: 1-23, 1960.

48.   Mons, N., and D. M. Cooper. Selective expression of one Ca2+-inhibitable adenylyl cyclase in dopaminergically innervated rat brain regions. Brain Res. Mol. Brain Res. 22: 236-244, 1993.

49.   Moss, S. J., D. M. Beeson, J. F. Jackson, M. G. Darlison, and E. A. Barnard. Differential expression of nicotinic acetylcholine receptor genes in innervated and denervated chicken muscle. EMBO J. 6: 3917-3921, 1987[Abstract].

50.   Nomura, N., N. Miyajima, T. Sazuka, A. Tanaka, Y. Kawarabayashi, S. Sato, T. Nagase, N. Seki, K. Ishikawa, and S. Tabata. Prediction of the coding sequences of unidentified human genes. The coding sequences of 40 new genes (KIAA0001-KIAA0040) deduced by analysis of randomly sampled cDNA clones from human immature myeloid cell line KG-1. DNA Res. 1: 27-35, 1994[Medline].

51.   Pacifici, G. M., C. Pellegrino, C. Maffei, and D. Beconcini. Effect of denervation on cyclic AMP regulating enzymes in rat gastrocnemius muscle. Ital. J. Biochem. 30: 20-29, 1981[Medline].

52.   Pette, D., and R. S. Staron. Cellular and molecular diversities of mammalian skeletal muscle fibers. Rev. Physiol. Biochem. Pharmacol. 116: 1-76, 1990[Medline].

53.   Piau, J. P., and G. Schapira. Adenyl cyclase in normal and denervated skeletal muscles. Enzymes 23: 36-45, 1978.[Medline]

54.   Premont, R. T., J. Chen, H. W. Ma, M. Ponnapalli, and R. Iyengar. Two members of a widely expressed subfamily of hormone-stimulated adenylyl cyclases. Proc. Natl. Acad. Sci. USA 89: 9809-9813, 1992[Abstract].

55.   Premont, R. T., I. Matsuoka, M. G. Mattei, Y. Pouille, N. Defer, and J. Hanoune. Identification and characterization of a novel and widely-expressed isoform of adenylyl cyclase. J. Biol. Chem. 271: 13900-13907, 1996[Abstract/Free Full Text].

56.   Ray, A., J. Kyselovic, J. J. Leddy, J. T. Wigle, B. J. Jasmin, and B. S. Tuana. Regulation of dihydropyridine and ryanodine receptor gene expression in skeletal muscle. Role of nerve, protein kinase C, and cAMP pathways. J. Biol. Chem. 270: 25837-25844, 1995[Abstract/Free Full Text].

57.   Shieh, B. H., M. Ballivet, and J. Schmidt. Quantitation of an alpha subunit splicing intermediate: evidence for transcriptional activation in the control of acetylcholine receptor expression in denervated chick skeletal muscle. J. Cell Biol. 104: 1337-1341, 1987[Abstract].

58.   Shyng, S. L., R. Xu, and M. M. Salpeter. Cyclic AMP stabilizes the degradation of original junctional acetylcholine receptors in denervated muscle. Neuron 6: 469-475, 1991[Medline].

59.   Suzuki, Y., T. Shen, N. Miyamoto, N. Defer, I. Matsuoka, and J. Hanoune. Changes in the expression of adenylyl cyclases in the rat uterus during the course of pregnancy. Biol. Reprod. 57: 778-782, 1997[Abstract].

60.   Syrovy, I., E. Gutmann, and J. Melichna. Differential response of myosin-ATPase activity and contraction properties of fast and slow rabbit muscles following denervation. Experientia 27: 1426-1427, 1971[Medline].

61.   Tang, W.-J., and A. G. Gilman. Type specific regulation of adenylyl cyclase by G protein beta gamma -subunits. Science 254: 1500-1503, 1991[Medline].

62.   Taussig, R., and A. G. Gilman. Mammalian membrane-bound adenylyl cyclases. J. Biol. Chem. 270: 1-4, 1995[Free Full Text].

63.   Tobise, K., Y. Ishikawa, S. R. Holmer, M. J. Im, J. B. Newell, H. Yoshie, M. Fujita, E. E. Susannie, and C. J. Homcy. Changes in type VI adenylyl cyclase isoform expression correlate with a decreased capacity for cAMP generation in the aging ventricle. Circ. Res. 74: 596-603, 1994[Abstract].

64.   Tomanek, R. J., and D. D. Lund. Degeneration of different types of muscle fibers. I. Denervation. J. Anat. 116: 395-407, 1973[Medline].

65.   Torgan, C. E., and W. E. Kraus. Regulation of type II adenylyl cyclase mRNA in rabbit skeletal muscle by chronic motor nerve pacing. Am. J. Physiol. 271 (Endocrinol. Metab. 34): E253-E260, 1996[Abstract/Free Full Text].

66.   Tsay, H. J., and J. Schmidt. Skeletal muscle denervation activates acetylcholine receptor genes. J. Cell Biol. 108: 1523-1526, 1989[Abstract].

67.   Voytik, S. L., M. Przyborski, S. F. Badylak, and S. F. Konieczny. Differential expression of muscle regulatory factor genes in normal and denervated adult rat hindlimb muscles. Dev. Dyn. 198: 214-224, 1993[Medline].

68.   Watson, P. A., J. Krupinski, A. M. Kempinski, and C. D. Frankenfield. Molecular cloning and characterization of the type VII isoform of mammalian adenylyl cyclase expressed widely in mouse tissues and in S49 mouse lymphoma cells. J. Biol. Chem. 269: 28893-28898, 1994[Abstract/Free Full Text].

69.   Wayman, G. A., S. Impey, and D. R. Storm. Ca2+ inhibition of type III adenylyl cyclase in vivo. J. Biol. Chem. 270: 21480-21486, 1995[Abstract/Free Full Text].

70.   Wei, J., G. Wayman, and D. R. Storm. Phosphorylation and inhibition of type III adenylyl cyclase by calmodulin-dependent protein kinase II in vivo. J. Biol. Chem. 271: 24231-24235, 1996[Abstract/Free Full Text].

71.   Weis, J. Jun, Fos, MyoD1, and myogenin proteins are increased in skeletal muscle fiber nuclei after denervation. Acta Neuropathol. (Berl.) 1: 63-70, 1994.

72.   Xia, Z., E.-J. Choi, F. Wang, C. Blazynski, and D. R. Storm. Type I calmoduline-sensitive adenylyl cyclase is neural specific. J. Neurochem. 60: 305-311, 1993[Medline].

73.   Xu, R., and M. M. Salpeter. Protein kinase A regulates the degradation rate of Rs acetylcholine receptors. J. Cell. Physiol. 165: 30-39, 1995[Medline].

74.   Zhu, X., J. E. Yeadon, and S. J. Burden. AML1 is expressed in skeletal muscle and is regulated by innervation. Mol. Cell. Biol. 14: 8051-8057, 1994[Abstract].

75.   Zimmermann, G., and R. Taussig. Protein kinase C alters the responsiveness of adenylyl cyclases to G protein alpha  and beta gamma subunits. J. Biol. Chem. 271: 27161-27166, 1996[Abstract/Free Full Text].


Am J Physiol Cell Physiol 274(6):C1674-C1685
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