Lysosomes in skeletal muscle following denervation

Summary The in-vivo uptake of exogenously applied horseradish peroxidase and the activities of the lysosomal enzymes acid phosphatase and cathepsin D were studied histochemically and/or biochemically in innervated and 2–14 day-denervated tibialis anterior muscles of the mouse. The biochemically determined uptake of horseradish peroxidase showed a large increase already 4 days after denervation. The activities of the lysosomal enzymes increased in a more gradual fashion, and only cathepsin D showed an increase in activity when expressed as total activity per muscle. Histochemically horseradish peroxidase was found to be localized in muscle fibres in characteristic spindle-shaped segments after denervation. The main increase in the number of such segments per transverse section of the muscle occurred between 3 and 6 days after denervation. In serial sections these segments frequently showed positive staining also for acid phosphatase.It is concluded that exogenously applied horseradish peroxidase is taken up into the lysosomal system, which after denervation becomes organized into characteristic spindle-shaped segments in the muscle fibres. The endocytic activity of muscle fibres increases early after denervation. This is followed by a more gradual increase in activity of lysosomal enzymes and finally by an organization of the lysosomal system into characteristic spindle-shaped segments. The results are compatible with the working hypothesis that increased endocytosis may initiate lysosomal activation in denervated skeletal muscle.     

Effect of exercise on synthesis and degradation of muscle protein.

Several reports have shown that amino acid utilization via oxidation and gluconeogenesis is increased during exercise. The purpose of this study was to investigate whether these changes are accompanied by alterations in protein synthesis and degradation in the muscle of exercising rats. One group of rats was made in swim for 1h and then protein synthesis and protein degradation were measured in a perfused hemicorpus preparation. Protein synthesis was decreased and protein degradation was increased in exercised rats compared with sedentary control rats. Exercise also decreased amino acid incorporation by isolated polyribosomes from muscle. Measurement of several muscle proteinase activities demonstrated that exercise had no effect on alkaline proteinase or Ca2+-activated proteinase. However, the free (unbound) cathepsin D activity was elevated in muscle of exercised rats, whereas the total activity of catepsin D was unchanged. This increase in the proportion of free cathepsin D activity suggests that lysosomal enzymes may be involved in the increased protein degradation that was observed.     

Proteinase expression during differentiation of human osteoclasts in vitro

Osteoclasts are macrophage-derived polykaryons that degrade bone in an acidic extracellular space. This differentiation includes expression of proteinases and acid transport proteins, cell fusion, and bone attachment, but the sequence of events is unclear. We studied two proteins expressed at high levels only in the osteoclast, cathepsin K, a thiol proteinase, and tartrate-resistant acid phosphatase (TRAP), and compared this expression with acid transport and bone degradation. Osteoclastic differentiation was studied using human apheresis macrophages cocultured with MG63 osteosarcoma cells, which produce cytokines including RANKL and CSF-1 that mediate efficient osteoclast formation. Immunoreactive cathepsin K appeared at 3-5 days. Cathepsin K activity was seen on bone substrate but not within cells, and cathepsin K increased severalfold during further differentiation and multinucleation from 7 to 14 days. TRAP also appeared at 3-5 d, independently of cell fusion or bone attachment, and TRAP activity reached much higher levels in osteoclasts attached to bone fragments. Two proteinases that occur in the precursor macrophages, cathepsin B, a thiol proteinase related to cathepsin K, and an unrelated lysosomal aspartate proteinase, cathepsin D, were also studied to determine the specificity of the differentiation events. Cathepsin B occurred at all times, but increased two- to threefold in parallel with cathepsin K. Cathepsin D activity did not change with differentiation, and secreted activity was not significant. In situ acid transport measurements showed increased acid accumulation after 7 days either in cells on osteosarcoma matrix or attached to bone, but bone pit activity and maximal acid uptake required 10-14 days. We conclude that TRAP and thiol proteinase expression begin at essentially the same time, and precede cell fusion and bone attachment. However, major increases in acid secretion and proteinases expression continue during cell fusion and bone attachment from 7 to 14 days.     

Entamoeba histolytica: purification of cathepsin B

A cytotoxic cysteine proteinase with a molecular weight of 16,000 was isolated from axenically grown trophozoites of Entamoeba histolytica. The enzyme was purified from frozen-thawed strain HM-1 by ion-exchange chromatography on DEAE-cellulose, organomercurial agarose affinity chromatography, and size-exclusion chromatography. The purified enzyme had proteinase activity that could be demonstrated on azocasein (pH 5), hemoglobin (pH 5), or carbobenzoxy-L-arginyl--L-arginyl-7-amino-4-trifluoromethylcoumarin++ + (Z-arg-arg-AFC), a substrate specific for cathepsin B. Enzyme activity was stable to high pH, but not to 40 C for 1 hr or 56 C for 0.5 hr. As typical of cysteine proteinases, inhibition of activity on Z-arg-arg-AFC by p-chloromercuribenzoate or mercury was reversed by free sulfhydryl groups. Both the proteinase and cytotoxic activities of the purified amoebal cathepsin B were inhibited by leupeptin and serum and activated by free sulfhydryl groups, supporting the hypothesis that both activities are characteristics of amoebal cathepsin B. Virulent strains of E. histolytica (HM-1 and Rahman) had significantly more cathepsin B activity per milligram protein than less virulent strains (HK-9, Laredo, and Huff). The correlation between higher levels of cathepsin B activity in strains with greater virulence could indicate a role for amoebal cathepsin B in the pathogenesis of amoebiasis.     

Effects of chymostatin and other proteinase inhibitors on protein breakdown and proteolytic activities in muscle

To learn more about the enzymes involved in protein catabolism in skeletal and cardiac muscle and to identify selective inhibitors of this process, we studied the effects of proteinase inhibitors on protein turnover in isolated muscles and on proteolytic activities in muscle homogenates. Chymostatin (20μm) decreased protein breakdown by 20–40% in leg muscles from normal rodents and also in denervated and dystrophic muscles. These results are similar to our previous findings with leupeptin. The related inhibitors pepstatin, bestatin, and elastatinal did not decrease protein breakdown; antipain slowed this process in rat hind-limb muscles but not in diaphragm. Chymostatin did not decrease protein synthesis and thus probably retards proteolysis by a specific effect on cell proteinase(s). In homogenates of rat muscle, chymostatin, in common with leupeptin and antipain, inhibits the lysosomal proteinase cathepsin B, and the soluble Ca²⁺-activated proteinase. In addition, chymostatin, but not leupeptin, inhibits the chymotrypsin-like proteinase apparent in muscle homogenates. In muscles depleted of most of this activity by treatment with the mast-cell-degranulating agent 48/80, chymostatin still decreased protein breakdown. Therefore inhibition of this alkaline activity probably does not account for the decrease in protein breakdown. These results are consistent with a lysosomal site of action for chymostatin. Because of its lack of toxicity, chymostatin may be useful in maintaining tissues in vitro and perhaps in decreasing muscle atrophy in vivo.     

Neural influence on the expression of acetylcholinesterase molecular forms in fast and slow rabbit skeletal muscles

With the aim of investigating the roles of motor innervation and activity on muscle characteristics, we studied the molecular forms of acetylcholinesterase (AChE) in fast-twitch (semimembranosus accessorius; SMa) and slow-twitch (semimembranosus proprius; SMp) muscles of the rabbit. We have shown that SMa and SMp express different patterns and tissue distribution of AChE forms and that the effect of long denervation varies with age. Three principal findings concerning expression of AChE molecular forms emerge from these studies. (1) The activity of AChE and the pattern of its molecular forms are particularly altered in adult denervated SMa and SMp muscles. AChE activity increases by 10-fold in both muscles, but asymmetric forms disappear in SMa and increase by 20-fold in SMp muscles. A similar alteration of AChE is found after tenotomy of these muscles, showing that the effect of denervation may be partly due to suppression of muscle activity. (2) The different changes occurring in the composition of AChE molecular forms in adult denervated SMa and SMp muscles are consistent with fluorescent staining with anti-AChE monoclonal antibodies and with DBA or VVA lectins, which bind to AChE asymmetric, collagen-tailed forms. These lectins poorly stain denervated SMa muscle surfaces but intensely stain neuromuscular junctions and extrasynaptic areas in denervated SMp muscle. (3) In contrast with the adult, denervation of 1-day-old muscles does not markedly modify the total amount of AChE or the proportions of its molecular forms, despite dramatic effects on muscle structure. These results are supported by studies of labeling with fluorescent DBA: the lectin only slightly stains the muscle fiber surface of denervated 15-day-old SMp muscle. Taken together, these data show that denervated muscles escape physiological regulation, producing increased levels of AChE with highly variable cellular distribution and patterns of molecular forms, depending on the age of operation and on the type of muscle.     

Surgical Sympathetic Denervation Increases α1Adrenoceptor-Mediated Accumulation of myo-Inositol Trisphosphate and Muscle Contraction in Rabbit Iris Dilator Smooth Muscle

Sympathetic denervation of the iris muscle produces increases in both the breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) and in muscle contraction in response to norepinephrine (NE). To shed more light on the biochemical basis underlying this supersensitivity we investigated: the effects of NE on PIP2 breakdown, measured as myo-inositol trisphosphate (IP3) accumulation, and on muscle contraction in normal and denervated rabbit iris dilator; and the effects of denervation on selected biochemical properties of this muscle. The data obtained from these studies can be summarized as follows: The EC50 values (microM) for NE-induced IP3 accumulation in normal and denervated dilators were 14 and 3, respectively. This accumulation of IP3 was blocked by prazosin (1 microM). The EC50 values (microM) for NE-induced contraction for the normal and denervated muscles were 10 and 0.6, respectively. The NE-induced muscle contraction was blocked by prazosin (1 microM). The t1/2 values (s) for IP3 accumulation in normal and denervated muscles were 31 and 11, respectively, and for contraction the values were 19 and 9, respectively. Denervation increased significantly (15-18%) the basal labelling of phosphoinositides from myo-[3H]inositol, but not from 32P or [14C]arachidonic acid. Denervation had little effect on the activities of the enzymes involved in phosphoinositide metabolism. However, the activities of protein kinase C and Ca2+-ATPase increased in the denervated muscle. It is concluded that sympathetic denervation of the iris dilator renders the coupling between alpha1 receptors and PIP2 breakdown into IP3 and 1,2-diacylglycerol (DG) more efficient.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of different proteolytic systems in the degradation of muscle proteins during denervation atrophy

In order to clarify the cellular mechanisms of denervation atrophy of skeletal muscle, we have studied protein turnover in denervated and control rat soleus muscles in vitro under different conditions. By 24 h after cutting the sciatic nerve, overall protein breakdown was greater in the denervated soleus than in the contralateral control muscle, and by 3 days, net proteolysis had increased about 3-fold. Since protein synthesis increased slightly following denervation, the rise in proteolysis must be responsible for the muscle atrophy and the differential loss of contractile proteins. Like overall proteolysis, the breakdown of actin (as shown by 3-methyl-histidine production by the muscles) increased each day after denervation and by 3 days was 2.5 times faster than in controls. Treatments that block the lysosomal and Ca2(+)-dependent proteolytic systems did not reduce the increase in overall protein degradation and actin breakdown in the denervated muscles (maintained in complete medium at resting length). However, the content of the lysosomal protease, cathepsin B, increased about 2-fold by 3 days after denervation. Furthermore, conditions that activate intralysosomal proteolysis (incubation without insulin or amino acids) stimulated proteolysis 2-3-fold more in the denervated muscles than in controls. Also, incubation conditions that activate the Ca2(+)-dependent pathway (incubation with Ca2+ ionophores or allowing muscles to shorten) were 2-3 times more effective in enhancing overall proteolysis in the denervated muscle. None of these treatments affected 3-methylhistidine production. Thus, multiple proteolytic systems increase in parallel in the denervated muscle, but a nonlysosomal process (independent of Ca2+) appears mainly responsible for the rapid loss of cell proteins, especially of myofibrillar components.     

Increased endocytotic and lysosomal activities in denervated type I and type II muscle fibres.

Previous work has shown that increased endocytotic and lysosomal activities occur in the endplate region of denervated skeletal muscle fibres. This, however, does not engage all fibres of a muscle at a given time after denervation. The present study was carried out in order to determine if both type I (slow) and type II (fast) muscle fibres can react to denervation by increased endocytotic and lysosomal activities. Uptake of horseradish peroxidase as a marker for endocytosis was studied in conjunction with acid phosphatase staining for lysosomal activity in type I and type II fibres of the denervated mouse hemidiaphragm. Fibre typing was performed using a monoclonal antibody against fast skeletal myosin and by adenosine triphosphatase staining. The results show that increased endocytosis and lysosomal activation occur in both type I and type II fibres after denervation.     

Increased endocytotic and lysosomal activities in denervated type I and type II muscle fibres

Summary Previous work has shown that increased endocytotic and lysosomal activities occur in the endplate region of denervated skeletal muscle fibres. This, however, does not engage all fibres of a muscle at a given time after denervation. The present study was carried out in order to determine if both type I (slow) and type II (fast) muscle fibres can react to denervation by increased endocytotic and lysosomal activities. Uptake of horseradish peroxidase as a marker for endocytosis was studied in conjunction with acid phosphatase staining for lysosomal activity in type I and type II fibres of the denervated mouse hemidiaphragm. Fibre typing was performed using a monoclonal antibody against fast skeletal myosin and by adenosine triphosphatase staining. The results show that increased endocytosis and lysosomal activation occur in both type I and type II fibres after denervation.     

Sites of cleavage of rabbit muscle aldolase by purified cathepsin M from rabbit liver

Rabbit liver cathepsin M, a sulfhydryl proteinase similar in catalytic properties to cathepsin B, causes a decrease in the activity of rabbit muscle aldolase assayed with fructose 1,6-bisphosphate but not with fructose 1-phosphate. Proteolytic modification of aldolase by cathepsin M is limited to the removal of small peptides from the COOH-terminus, including the COOH-terminal hexapeptide NH2-Ile-Ser-Asn-His-Ala-TyrOH. Correlation of loss of aldolase activity with COOH-terminal modification indicates that only three of the four subunits of muscle aldolase contribute to the catalytic activity of the tetrameric enzyme.     

Differences in induction of lysosomal protease activity by protease inhibitors in B16 melanoma cell lines

1. The effects of potent protease inhibitors in vitro (leupeptin, pepstatin and E-64[N-[L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]agmatine]) on intracellular cathepsin B (EC 3.4.22.1), hemoglobin (Hb)-hydrolase and acid phosphatase (EC 3.1.3.2) from cultured B16 melanoma variants (B16-F1, F10 and BL6) were studied. 2. E-64 induced all the cultured B16 melanoma variants to decrease the activity of intracellular cathepsin B but did not have this effect with Hb-hydrolase or acid phosphatase. Furthermore, E-64 decreased the activity of cathepsin B in both the lysosomal and cytosol fractions. 3. Leupeptin induced all the cultured B16 melanoma variants to increase the activities of intracellular cathepsin B and Hb-hydrolase but not that of acid phosphatase. An increase in the level of cathepsin B activity was most significant in B16-BL6 followed by F10 and then F1 variants. 4. Leupeptin induced all the cultured B16 melanoma variants to increase the cathepsin B activity in the lysosomal fraction. Our data differed from the results of Tanaka et al. (1981) in that leupeptin induced rat cultured hepatocytes to inhibit the activity of intracellular cathepsin B and increase the Hb-hydrolase activity, especially in the cytosol fraction.     

Influence of denervation on the molecular forms of junctional and extrajunctional acetylcholinesterase in fast and slow muscles of the rat.

Acetylcholinesterase (AChE) molecular forms in denervated rat muscles, as revealed by velocity sedimentation in sucrose gradients, were examined from three aspects: possible differences between fast and slow muscles, response of junctional vs extrajunctional AChE, and early vs late effects of denervation. In the junctional region, the response of the asymmetric AChE forms to denervation is similar in fast extensor digitorum longus (EDL) and slow soleus (SOL) muscle: (a) specific activity of the A12 form decreases rapidly but some persists throughout and even increases after a few weeks; (b) an early and transient increase of the A4 AChE form lasting for a few weeks may be due to a block in the synthetic process of the A12 form. In the extrajunctional regions, major differences with regard to AChE regulation exist already between the normal EDL and SOL muscle. The extrajunctional asymmetric AChE forms are absent in the EDL because they became completely repressed during the first month after birth, but they persist in the SOL. Differences remain also after denervation and are, therefore, not directly due to different neural stimulation patterns in both muscles: (a) an early but transient increase of the G4 AChE occurs in the denervated EDL but not in the SOL; (b) no significant extrajunctional activity of the asymmetric AChE forms reappears in the EDL up till 7 wk after denervation. In the SOL, activity of the asymmetric AChE forms is decreased early after denervation but increases thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipoprotein lipase activity in skeletal muscles of the rat: effects of denervation and tenotomy.

The effects of denervation, tenotomy, or tenotomy with simultaneous denervation on the activity of heparin-releasable and intracellular, residual lipoprotein lipase (LPL) and triacylglycerol (TG) content were examined in rat skeletal muscles. An influence of muscle electrostimulation on denervated and tenotomized muscles was also evaluated. Activity of both LPL fractions was decreased in denervated and/or tenotomized soleus and red portion of gastrocnemius muscles. It was accompanied by a slight elevation of the intracellular TG content. Electrostimulation increased activities of both fractions of LPL in red muscles from intact hindlimbs. In stimulated denervated muscles without or with simultaneous tenotomy, activity of two LPL fractions was also enhanced, but control values were reached only in denervated soleus muscle. Electrical stimulation had no pronounced effect on LPL activity in tenotomized muscles. In conclusion, denervation and/or tenotomy decreases LPL activity in red muscles, indicating reduction of the muscle potential to utilize circulating TG. Electrostimulation only partly restores the diminished LPL activity in denervated muscles, without any effect in tenotomized ones. Thus, to maintain LPL activity in resting muscle, intact innervation and tension are needed.     

Lysosomal enzyme activities in muscle following starvation and refeeding in the saithe Pollachius virens L

Saithe (Pollachius virens L.) were starved for 66 days at 10 degrees C and activities of aryl sulfatase, acid proteinase, beta-glucuronidase, RNAase and acid phosphatase measured in homogenates prepared from fast and slow myotomal muscles. In fed fish, hydrolase activities were generally higher in slow than fast muscles. With the exception of acid proteinase activity in slow muscle, the activities of all the lysosomal enzymes increased by 70 to 100% during starvation. In general, there was a proportionally larger increase in the hydrolase activities in fast than in slow muscle. In a second experiment, fish were starved for 74 days, and refed for up to 52 days. The increases in aryl sulfatase and acid proteinase activity produced in fast muscle with starvation were found to be rapidly reversed by refeeding. Lysosomal enzyme activities in fish sampled after 10 days refeeding were not significantly different from fed controls. Membrane fractions enriched in aryl sulfatase activity were prepared from the fast muscle of 66-day starved fish. These were capable of degrading both myosin heavy chains and actin to lower molecular weight peptides at acid (pH 5.0), but not at neutral pH. The results suggest a role for lysosomal enzymes in the breakdown of myofibrillar proteins during starvation.     

Nucleo-cytoplasmic relationships of oestrogen receptors in rat liver during the oestrous cycle and in response to administered natural and synthetic oestrogen.

The mechanism of degradation of fructose-1,6-bisphosphate aldolase from rabbit muscle by the lysosomal proteinase cathepsin B was determined. Treatment of aldolase with cathepsin B destroys up to 90% of activity with fructose 1,6-bisphosphate as substrate, but activity with fructose 1-phosphate is slightly increased. Cathepsin L, another lysosomal thiol proteinase, and papain are also potent inactivators of aldolase, whereas inactivation is not caused by cathepsins D or H even at high concentrations, or by cathepsin B inhibited by leupeptin or iodoacetate. The cathepsin-B-treated aldolase shows no detectable change in subunit molecular weight, oligomer molecular weight or subunit interactions. Cathepsin B cleaves dipeptides from the C-terminus of th aldolase subunits. Four dipeptides are released sequentially: Ala-Tyr, Asn-His, Ile-Ser and Leu-Phe, and a maximum of five additional dipeptides may be released. There are indications that this peptidyldipeptidase activity of cathepsin B may be an important aspect of its action on protein substrates generally.     

Effect of maturation on activities of various proteases and protease inhibitors in the muscle of Ayu (Plecoglossus altivelis).

1. Increases in activities of muscle muticatalytic proteinase, modori-inducing proteinase (latent trypsin-like proteinase), cathepsin B and L-like proteases and cathepsin D were observed more markedly for male fish than female fish, in the spawning stage. 2. Decreases in inhibitory activities of muscle serine and cysteine protease inhibitors were observed more markedly for male fish than female fish in the spawning stage.     

Primary structure and expression of a sodium channel characteristic of denervated and immature rat skeletal muscle

The alpha subunit of a voltage-sensitive sodium channel characteristic of denervated rat skeletal muscle was cloned and characterized. The cDNA encodes a 2018 amino acid protein (SkM2) that is homologous to other recently cloned sodium channels, including a tetrodotoxin (TTX)-sensitive sodium channel from rat skeletal muscle (SkM1). The SkM2 protein is no more homologous to SkM1 than to the rat brain sodium channels and differs notably from SkM1 in having a longer cytoplasmic loop joining domains 1 and 2. Steady-state mRNA levels for SkM1 and SkM2 are regulated differently during development and following denervation: the SkM2 mRNA level is highest in early development, when TTX-insensitive channels predominate, but declines rapidly with age as SkM1 mRNA increases; SkM2 mRNA is not detectable in normally innervated adult skeletal muscle but increases greater than 100-fold after denervation; rat cardiac muscle has abundant SkM2 mRNA but no detectable SkM1 message. These findings suggest that SkM2 is a TTX-insensitive sodium channel expressed in both skeletal and cardiac muscle.     

Neural Control of Muscle Androgen Receptors

The number of cytosolic androgen receptors in rat skeletal muscle increases following denervation and disuse. This increase was postulated to represent altered intracellular distribution and consequent diminished sensitivity of skeletal muscle to androgens. To test this hypothesis, we measured total (homogenate) androgen receptor levels after denervation. Total (homogenate) androgen receptor binding did not change in response to denervation of leg muscles from adult male rats. An increase in cytosolic receptor number with no increase in total (homogenate) receptor levels supports the hypothesis of altered intracellular distribution of androgen receptors in denervated muscle. Cytosolic androgen receptor binding in muscle from male rats increased by 40% after denervation, whereas in females the increase was 17%. These increases could not be altered by endocrine manipulations of males or females.