Ultrastructural localization of cellular prion protein (PrPc) at the neuromuscular junction

We examined the localization of the normal cellular isoform of prion protein (PrPc) in mammalian skeletal muscle. Using two anti-PrP antibodies, the neuromuscular junction (NMJ) was preferentially stained after immunohistofluorescence. The mouse, hamster, and human NMJ displayed a fluorescent signal specific for PrPc. Postembedding immunoelectron microscopy analysis performed in the mouse muscle showed that the PrPc-specific colloidal gold immunolabelling was concentrated over the sarcoplasmic cytoplasm. The membrane of the postsynaptic domain was devoid of gold particles, while a weak signal was occasionally observed close to the presynaptic vesicles of the terminal axons. These results indicate that the PrP gene is expressed in mammalian muscle at the NMJ. The subsynaptic sarcoplasm of the NMJ appears to be the privileged site where PrPc presumably associated with endosome membrane may play a role in either physiological activity or maintenance of the morphological integrity of the synapse.     

Insulin-like growth factor-binding protein-5 is cleaved by physiological concentrations of thrombin

Insulin-like growth factor (IGF)-binding protein-5 (IGFBP-5) is cleaved by a serine protease that is secreted by fibroblasts and porcine smooth muscle cells (pSMC) in culture. To investigate whether other serine proteases could cleave this substrate at physiologically relevant concentrations, we determined the proteolytic effects of thrombin on IGFBP-5. Human alpha-thrombin (0.0008 NIH U/ml) cleaved IGFBP-5 into 24-, 23-, and 20-kDa non-IGF-I-binding fragments. Cleavage occurred at a physiologically relevant thrombin concentration. The effect was specific for IGFBP-5, as other forms of IGFBPs, e.g. IGFBP-1, IGFBP-2, and IGFBP-4 were not cleaved by thrombin. Although IGFBP-3 was cleaved by thrombin, this effect required a 50-fold greater thrombin concentration. [35S]Methionine labeling followed by immunoprecipitation confirmed that IGFBP-5 that was constitutively synthesized by pSMC cultures was also degraded by thrombin into 24-, 23-, and 20-kDa fragments. The binding of IGF-I to IGFBP-5 partially inhibited IGFBP-5 degradation by thrombin, and an IGF analog that does not bind to IGFBP-5 had no effect. Thrombin did not account for the serine protease activity that had been shown previously to be present in pSMC-conditioned medium. This was proven by showing that 1) no immunoreactive thrombin could be detected in the pSMC-conditioned medium; 2) the IGFBP-5 fragments that were generated by thrombin showed three cleavage sites (Arg192-Ala193, Arg156-Ile157, and Lys120-His121), whereas the serine protease in conditioned medium cleaves IGFBP-5 at a different site; and 3) hirudin had no effect on IGFBP-5 cleavage by the protease in pSMC medium; however, it inhibited IGFBP-5 degradation by thrombin. To determine the physiological significance of IGFBP-5 cleavage, the effect of an IGFBP-5 mutant that is resistant to cleavage by the pSMC protease and has been shown to inhibit IGF-I actions in pSMC was determined. This mutant inhibited IGF-I-stimulated DNA synthesis, but if thrombin was added simultaneously, IGF-I was fully active. In summary, physiological concentrations of thrombin degrade IGFBP-5. Degradation can be blocked by hirudin and is partially inhibited by IGF-I binding. Generation of active thrombin in vessel walls may be a physiologically relevant mechanism for controlling IGF-I bioactivity.     

Differential membrane localization and intermolecular associations of alpha-dystrobrevin isoforms in skeletal muscle

alpha-Dystrobrevin is both a dystrophin homologue and a component of the dystrophin protein complex. Alternative splicing yields five forms, of which two predominate in skeletal muscle: full-length alpha-dystrobrevin-1 (84 kD), and COOH-terminal truncated alpha-dystrobrevin-2 (65 kD). Using isoform-specific antibodies, we find that alpha-dystrobrevin-2 is localized on the sarcolemma and at the neuromuscular synapse, where, like dystrophin, it is most concentrated in the depths of the postjunctional folds. alpha-Dystrobrevin-2 preferentially copurifies with dystrophin from muscle extracts. In contrast, alpha-dystrobrevin-1 is more highly restricted to the synapse, like the dystrophin homologue utrophin, and preferentially copurifies with utrophin. In yeast two-hybrid experiments and coimmunoprecipitation of in vitro-translated proteins, alpha-dystrobrevin-2 binds dystrophin, whereas alpha-dystrobrevin-1 binds both dystrophin and utrophin. alpha-Dystrobrevin-2 was lost from the nonsynaptic sarcolemma of dystrophin-deficient mdx mice, but was retained on the perisynaptic sarcolemma even in mice lacking both utrophin and dystrophin. In contrast, alpha-dystrobrevin-1 remained synaptically localized in mdx and utrophin-negative muscle, but was absent in double mutants. Thus, the distinct distributions of alpha-dystrobrevin-1 and -2 can be partly explained by specific associations with utrophin and dystrophin, but other factors are also involved. These results show that alternative splicing confers distinct properties of association on the alpha-dystrobrevins.     

Postnatal development of Schwann cells at neuromuscular junctions, with special reference to synapse elimination

The neuromuscular junctions (NMJs) of postnatal rat soleus muscles were examined by immunohistochemical staining for S100, a marker of Schwann cells (SCs), and for protein gene product 9.5, a neuronal marker, to elucidate the involvement of SCs in synapse elimination. The morphological maturation of S100-immunoreactive terminal SCs at NMJs proceeded with the gradual increase in their number. The number of terminal SCs per NMJ was one or two at postnatal day (P) 7, reaching the adult number at P28, when it became three or four. Confocal laser scanning microscopic analysis of multi-innervated NMJs, whose number decreased between P7 and P14, revealed a change in the ratio between terminal SCs and axons with age. At P7, the ratio between axons and terminal SCs per NMJ was > or = 2:1, which was exactly the reverse of that in adults, while at P14 this had changed to 2:2. A structural change appeared to occur at the same time at the preterminal region, this being prior to the establishment of a 1:1 relationship between axon and SC sheath which was detected at P14, with the > or = 2:1 relationship seeming to occur at P7. Thus, synapse elimination seems to proceed, at least for one week, with the gradual loss of axons which are at different stages of maturation with respect to their spatial relationship with SCs. From our results it seems unlikely that SCs play an active role in selecting a single axon to survive.     

Formation of the neuromuscular junction: molecules and mechanisms

The vertebrate skeletal neuromuscular junction is the site at which motor neurons communicate with their target muscle fibers. At this synapse, as at synapses throughout the nervous system, efficient and appropriate communication requires the formation and precise alignment of specializations for transmitter release in the axon terminal with those for transmitter detection in the postsynaptic cell. Classical developmental studies demonstrate that synapse formation at the neuromuscular junction is a mutually inductive event; neurons induce postsynaptic differentiation in muscle cells and myofibers induce presynaptic differentiation in motor axon terminals. More recent experiments indicate that Schwann cells, which cap axon terminals, also play an active role in the formation and maintenance of the neuromuscular junction. Here, we review recent advances in the identification of molecules mediating such inductive interactions and the mechanisms by which they produce their effects. Although our discussion concerns events at developing neuromuscular junctions, it seems likely that similar molecules and mechanisms may act at neuron-neuron synapses in the peripheral as well as the central nervous system.     

An early stage in sodium channel clustering at developing rat neuromuscular junctions

Voltage-gated sodium channels (VGSCs) are concentrated in the postsynaptic membrane at the adult rat neuromuscular junction (NMJ). We have used immunolabelling to determine the pattern of initial VGSC accumulation during development. At birth, but not 3 days before, VGSC labelling is detectable at the NMJ and in the perijunctional (periJ) membrane but not elsewhere. A much higher density cluster of VGSCs forms at the NMJ itself 1-2 weeks later. If the nerve is cut 2 days after birth, VGSC labelling persists in the periJ region for at least 4 weeks but the clustering of VGSCs at the NMJ fails to develop. Thus an early, stable accumulation of VGSCs develops near the NMJ at least a week before high density postsynaptic VGSC clusters form.     

Acetylcholine receptor epsilon-subunit deletion causes muscle weakness and atrophy in juvenile and adult mice

In mammalian muscle a postnatal switch in functional properties of neuromuscular transmission occurs when miniature end plate currents become shorter and the conductance and Ca2+ permeability of end plate channels increases. These changes are due to replacement during early neonatal development of the gamma-subunit of the fetal acetylcholine receptor (AChR) by the epsilon-subunit. The long-term functional consequences of this switch for neuromuscular transmission and motor behavior of the animal remained elusive. We report that deletion of the epsilon-subunit gene caused in homozygous mutant mice the persistence of gamma-subunit gene expression in juvenile and adult animals. Neuromuscular transmission in these animals is based on fetal type AChRs present in the end plate at reduced density. Impaired neuromuscular transmission, progressive muscle weakness, and atrophy caused premature death 2 to 3 months after birth. The results demonstrate that postnatal incorporation into the end plate of epsilon-subunit containing AChRs is essential for normal development of skeletal muscle.     

Potent and selective thrombin inhibitors featuring hydrophobic, basic P3-P4-aminoalkyllactam moieties

Crystal structure and evolving SAR considerations of potent, selective benzylsulfonamide lactam thrombin inhibitors and related serine protease inhibitors have led to the design of novel thrombin inhibitors 1a-g, featuring hydrophobic, basic, P4-alkylaminolactam scaffolds that serve as novel types of P3-P4 dipeptide mimics. The design, synthesis, and biological activity of these targets is presented.     

Proteolysis of Saccharomyces cerevisiae RNase H1 in E coli

Expression of S cerevisiae RNase H1 in E coli leads to the formation of a proteolytic product with a molecular mass of 30 kDa that is derived from the 39-kDa full length protein. The 30-kDa form retains RNase H1 activity, as determined by renaturation gel assay. The amount of proteolysis observed depends on the procedure used in preparing the cell extracts for protein analysis. The cleavage site on the amino acid sequence of the 39-kDa RNase H1 was determined by N-terminal sequence analysis of the 30-kDa proteolytic form. The cut occurs between two arginines located at the amino terminus region of the protein. The pattern of proteolysis was examined for both the wild-type RNase H1 and a mutant RNase H1 that was constructed in this work. In the mutant the second arginine of the cleavage site was changed to a lysine. Comparisons of the expression of the wild-type and altered protein in two different E coli strains demonstrate that the protease responsible for the degradation has a specificity very similar to that of the OmpT protease. However, the proteolysis observed in an OmpT background in extracts, prepared by boiling the cells in SDS containing buffer, indicates that the protease may, unlike OH108.     

The peripheral nerve and the neuromuscular junction are affected in the tenascin-C-deficient mouse

A thorough examination of the structure and plasticity of the neuromuscular system was performed in tenascin-C mutant mice deficient in tenascin-C. The study of the peripheral nerve revealed a number of abnormal features. In the motor nerve, numerous unmyelinated and myelinated fibers with degraded myelin were present. Schwann cell processes often enclosed degenerative terminals. Transgene (beta-galactosidase) expression analyzed at the ultrastructural level was found to be unequally distributed in the mutant's neuromuscular tissues. At the NMJ, preterminal disorganization was prevalent. Some axon terminals exhibited abnormal overgrowth. A surprising lack of beta-galactosidase expression at some cellular sites known to possess tenascin-C in wild type mice correlated best with marked changes in the cytoarchitecture of the peripheral nerve and NMJ. In some other -but not all- cellular sites which normally express the molecule, immunofluorescence analysis suggested the presence of significant but low levels of tenascin-C-like immunoreactivity together with beta-galactosidase expression. Messenger RNA detection by RT-PCR confirmed the presence of low amounts of tenascin-C mRNA in skeletal muscle suggesting that the mice deficient in tenascin-C are not complete knock-outs of this gene, but low-expression mutants. Following in vivo injections of botulinum type-A toxin, we observed a greatly reduced sprouting response of the motor nerves in tenascin-C mutant mice. We also observed that N-CAM and beta-catenin were overexpressed in the mutant. Our results suggest that tenascin-C is involved both in stabilization and in plasticity of the NMJ.     

Synaptic development in the crayfish opener muscle

Nerve terminal regions in walking leg opener muscles of several crayfish of different ages (0 to 245 days after hatching) were examined by means of electron microscopy. This muscle is innervated by two axons (excitatory and inhibitory) and at maturity contains three classes of synapse: excitatory and inhibitory neuromuscular synapses, and inhibitory axo-axonal synapses. The muscle itself is initially a syncytium, which gradually becomes subdivided into distinct "muscle fibers" as the animal matures. Innervation was not found in the opener muscle just before or just after hatching, but was present in restricted locations on the inner side of the muscle within a few days of hatching. As the muscle enlarged and became subdivided, innervation appeared in various other locations. Synaptic contacts were located in young stages soon after hatching, and in later stages. Morphological differences characteristic of excitatory nerve terminals could be found even at the earliest stages of innervation. Both excitatory and inhibitory synapses, but particularly the former, showed evidence of progressive enlargement to a final size within the first two months, and no evidence for further enlargement of existing synapses thereafter. Synaptic maturation also involved the appearance of presynaptic "dense bodies" though to be regions at which transmitter substance is preferentially released. Nerve terminals at different levels of maturation were observed in opener muscles of young crayfish. Clear evidence for differential maturation of the three types of synapse present in this muscle was obtained. The inhibitory neuromuscular synapses attained their final average size and developed their dense bodies sooner than the excitatory neuromuscular synapses. The inhibitory axo-axonal synapses were the last to appear and to mature.     

The relationship between perlecan and dystroglycan and its implication in the formation of the neuromuscular junction

Perlecan is a major heparan-sulfate proteoglycan (HSPG) within the basement membrane surrounding skeletal muscle fibers. The C-terminus of its core protein contains three globular domain modules which are also found in laminin and agrin, two proteins that bind to dystroglycan (DG, cranin) on the muscle surface with these modules. In this study, we examined whether perlecan can also bind to DG and is involved in signaling the formation of the neuromuscular junction (NMJ). By labeling cultured muscle cells with a polyclonal anti-perlecan antibody, this protein is found both within the extracellular matrix in a fibrillar network and at the cell surface in a punctate pattern. In Xenopus muscle cells, the cell-surface perlecan is precisely colocalized with DG. Both perlecan and DG are clustered at ACh receptor clusters induced by spinal neurons or by beads coated with HB-GAM, a heparin-binding growth factor. Blot overlay assays have shown that perlecan binds alpha-DG in a calcium and heparin-sensitive manner. Furthermore, perlecan is present in muscle lysate immunoprecipitated with an anti-DG antibody. Immunolabeling also showed colocalization between HB-GAM and perlecan and between HB-GAM and DG. These data suggest that perlecan is anchored to muscle surface via DG-dystrophin complex. Since DG is also a site of agrin binding, the neural agrin secreted by motoneurons during NMJ formation may compete with the pre-existing perlecan for cell surface binding. This competition may result in the presentation of perlecan-bound growth factors such as HB-GAM to effect synaptic induction.     

The N-end rule pathway catalyzes a major fraction of the protein degradation in skeletal muscle

In skeletal muscle, overall protein degradation involves the ubiquitin-proteasome system. One property of a protein that leads to rapid ubiquitin-dependent degradation is the presence of a basic, acidic, or bulky hydrophobic residue at its N terminus. However, in normal cells, substrates for this N-end rule pathway, which involves ubiquitin carrier protein (E2) E214k and ubiquitin-protein ligase (E3) E3alpha, have remained unclear. Surprisingly, in soluble extracts of rabbit muscle, we found that competitive inhibitors of E3alpha markedly inhibited the 125I-ubiquitin conjugation and ATP-dependent degradation of endogenous proteins. These inhibitors appear to selectively inhibit E3alpha, since they blocked degradation of 125I-lysozyme, a model N-end rule substrate, but did not affect the degradation of proteins whose ubiquitination involved other E3s. The addition of several E2s or E3alpha to the muscle extracts stimulated overall proteolysis and ubiquitination, but only the stimulation by E3alpha or E214k was sensitive to these inhibitors. A similar general inhibition of ubiquitin conjugation to endogenous proteins was observed with a dominant negative inhibitor of E214k. Certain substrates of the N-end rule pathway are degraded after their tRNA-dependent arginylation. We found that adding RNase A to muscle extracts reduced the ATP-dependent proteolysis of endogenous proteins, and supplying tRNA partially restored this process. Finally, although in muscle extracts the N-end rule pathway catalyzes most ubiquitin conjugation, it makes only a minor contribution to overall protein ubiquitination in HeLa cell extracts.     

Clinical evaluation of technetium-99m-L,L-ethylenedicysteine in patients with chronic renal failure

1. Effects of feeding condition from birth were examined on the sensitivity of neuromuscular transmission to d-tubocurarine (dTc) in vitro in male mice of the ddY strain. 2. Mice were trained to climb two separated cylindrical steel-wire tubes for feeding and drinking, respectively, from 16 days of age. Some mice were conventionally fed, from 99 days of age. Nerve-muscle preparations were made from the left phrenic nerve diaphragm muscle (DPH), the sciatic nerve soleus muscle (SOL), and the sciatic nerve extensor digitorum longus muscle (EDL) of 99-day-old and 155-day-old mice. The nerve trunk was electrically activated with trains of four pulses and tetanic pulses. 3. The sensitivity to the effects of dTc decreased in the order EDL, SOL, and DPH. This result held true in all mice tested. 4. This sensitivity was significantly potentiated by the compulsory movement. 5. The supersensitivity remained even when mice were conventionally fed after 99 days of age. 6. The compulsion rendered EDL antifatigable on tetanic stimulation. This property was also retained after a return to conventional feeding. 7. These results suggest that the effects of feeding condition from birth might remain on neuromuscular functions after termination of the conditioning.     

Corticosteroid effects on diaphragm neuromuscular junctions

The effects of corticosteroid (CS) treatment (prednisolone continuously administered subcutaneously at a flow rate of 2.5 microl/h, daily dose 5.6 mg/kg, for 3 wk) on neuromuscular junction (NMJ) morphology and neuromuscular transmission in rat diaphragm muscle (Dimus) were compared with weight-matched (Sham) and ad libitum fed control (Ctl) groups. Fibers were classified on the basis of myosin heavy chain (MHC) isoform expression. CS treatment caused significant atrophy of fibers expressing MHC2X (type IIx), either alone or with MHC2B (type IIx/b). Fibers expressing MHCslow (type I) and MHC2A (type IIa) were unaffected by CS. The planar areas of nerve terminals and motor endplates at type IIx/b fibers were smaller in CS-treated Dimus compared with Sham and Ctl. However, CS-induced atrophy of type IIx/b fibers exceeded changes in NMJ morphology. Thus, when normalized for fiber diameter, NMJs were relatively larger in the CS-treated group compared with Ctl. Neuromuscular transmission failure, assessed in vitro by comparing force loss during repetitive (40 Hz) nerve vs. direct muscle stimulation, was less in CS-treated Dimus. These results indicate that alterations in NMJ morphology after CS treatment are dependent on fiber type and may contribute to improved neuromuscular transmission.     

Effects of prior exercise on exercise-induced arterial hypoxemia in young women

Insulin-like growth factor binding proteins (IGFBP) proteases have been proposed to be involved in changes of serum IGFBP pattern during pregnancy. IGFBP-4 and -5 are degraded specifically by proteases in pregnancy serum in vitro, whereas IGFBP-3 proteolytic activity was also detected in nonpregnancy serum. To identify and characterize IGFBP proteases, human pregnancy serum was fractionated by size exclusion chromatography revealing IGFBP-4 protease activities in fractions coeluting with proteins of approximately 600-kDa and 50- to 100-kDa molecular mass. In both fractions, a predominant 50-kDa gelatinase was found, suggesting that parts of the gelatinase activity might aggregate or are complexed with other proteins forming a higher molecular complex. Hydroxyapatite chromatography and chromatofocusing of the 50- to 100-kDa serum fraction showed that the IGFBP-4 protease and the 50-kDa gelatinase activity were copurified. When the 50-kDa gelatinase-containing band was excised from the polyacrylamide gel, it exhibited IGFBP-4 proteolytic activity, resulting in the formation of 17- and 10-kDa fragments. [125I] IGFBP substrate zymography combined with fragment blotting showed that the 1,10-phenanthroline-sensitive 50-kDa protease activity purified by chromatofocusing also cleaved IGFBP-3 and -5. Other proteases detected in pregnancy serum fractions with Mr estimates of 79-, 30-, and 22-kDa degraded IGFBP-3 and -5 but not IGFBP-4. [125I] IGFBP-5 substrate zymography revealed that the 30-kDa IGFBP protease was inhibited by serine protease inhibitors. Whereas 1,10-phenanthroline inhibited the IGFBP proteolytic activity in the solution assay, serine protease inhibitors failed to affect proteolysis, indicating the predominant contribution of the metalloproteinase to IGFBP proteolysis. Tissue inhibitors of matrix metalloproteinases-1 and -2 revealed weak or no inhibition of IGFBP-4 and -5 proteolytic activity, whereas a hydroxamic acid-based inhibitor, potentially inhibiting disintegrin metalloproteases, completely prevented the proteolysis of IGFBPs. Whereas no specific immunoreactivity of the 50-kDa protein with antimatrix metalloproteinase-1, -2, -3, -9, or -13 antibodies was observed, antidisintegrin domain-specific antibodies bound to the 50-kDa gelatinase. These studies provide the first direct biochemical evidence that human pregnancy serum contains a 50-kDa IGFBP protease with properties of a soluble disintegrin metalloproteinase that appears to be potentially involved in regulating IGF bioavailability for placental and fetal growth.     

Both basic fibroblast growth factor and ciliary neurotrophic factor promote the retention of polyneuronal innervation of developing skeletal muscle fibers

At birth, nearly all rat muscle fibers receive synaptic inputs from more than one motoneuron at a single end-plate site. By the end of the third postnatal week all but one of these inputs has been eliminated. During this loss of polyneuronal innervation, developing neuromuscular synapses compete with one another. Although the nature of this competition is not known, it is commonly assumed that it is mediated through differential activity of the competing inputs. One means by which such differential activity might be translated into a biological signal would be if the synapses compete in an activity-dependent manner for a scarce supply of neurotrophic molecules. A prediction of this hypothesis is that excess quantities of such trophic molecules will reduce competition and thereby slow down or abolish the normal loss of polyneuronal innervation. In newborn rats, the effects of injection of either basic fibroblast growth factor (bFGF) or ciliary neurotrophic factor (CNTF) on the outcome of neuromuscular synapse elimination were investigated. Daily injections of either bFGF or CNTF were made for 1 week into the lateral gastrocnemius muscle beginning at the postnatal age of 2 days. The amount of polyneuronal innervation of fibers in trophic molecule-injected muscles and saline-injected contralateral muscles was assayed using intracellular recording methods. For both bFGF- and CNTF-injected muscles, an increase in the percentage of polyneuronally innervated fibers relative to saline-injected muscles was noted. For bFGF-injected muscles, the amount of polyneuronal innervation remained at nearly 60% as late as the postnatal age of 14 days (P14). This is the amount of polyneuronal innervation found at age 6 days in normal animals. Nearly 40% of the fibers of CNTF-injected muscles remained polyneuronally innervated at age P14, the amount expected at age 9 days. These results indicate that both bFGF and CNTF exert powerful and long-lasting effects on developing neuromuscular synapses.     

Expression of the thrombin receptor in developing bone and associated tissues

Thrombin, a serine protease with a central role in thrombosis and hemostasis, is also a specific agonist for a variety of cellular responses in osteoblasts and stimulates bone resorption in organ culture. Cultured osteoblast-like cells express the proteolytically activated thrombin receptor, but the significance of this finding in vivo remains unknown. Immunohistochemistry was used to investigate the normal tissue distribution of the proteolytically activated thrombin receptor in developing rat bones and associated tissues. In hind limbs, the receptor was first observed on embryonic day 16 and became more abundant within the limb as gestation progressed. Thrombin receptor staining was detected on osteoblasts, macrophages, muscle cells, and endothelial cells, but not osteoclasts. Similarly, osteoblasts in developing calvariae stained positively for the thrombin receptor. The pattern of receptor expression by primary osteoblast cultures and freshly isolated macrophages and osteoclasts corresponded to that observed in vivo. The observed pattern of thrombin receptor expression in bone cells supports the hypothesis that cell-mediated thrombin-induced bone resorption is mediated by osteoblasts.     

Protein phosphorylation in fast and slow chicken skeletal muscles: effect of denervation

We have identified proteins in adult chicken skeletal muscle whose phosphorylation can be used as markers for the mature fast and slow muscle phenotype. These include phosphorylase, phosphorylase kinase, and a cyclic adenosine 3',5'-monophosphate (cAMP)-stimulated, calmodulin-inhibited 28-kDa band (markers for fast muscle), a calmodulin-stimulated 50-kDa band, and two cAMP-stimulated bands at 44 and 46 kDa (markers for slow muscle), and the relative concentrations of the regulatory subunits of cAMP-dependent protein kinase (RI and RII). After denervation the pattern of phosphorylation in fast muscle changed to resemble that of slow muscle: phosphorylation of the fast phenotype markers decreased; the slow phenotype markers, barely detectable in normal fast muscle, appeared as significant phosphoproteins; and the concentration of RII increased with no change in RI. This is consistent with denervation-induced changes observed using other phenotypic markers and indicates the potential for using these phosphoprotein markers in studies of muscle development and pathophysiology.