Effect of prednisone on protease activities and structural protein levels in rat muscles in vivo

ELSEVIER

Clinica Chimica Acta 249 (1996) 47-58

Effect of prednisone on protease activities and structural protein levels in rat muscles in vivo J o h n W . H a y c o c k a, G a v i n F a l k o u s a, C h a r l o t t e A. M a l t i n b, M a r g a r e t I. D e l d a y b, D a v i d M a n t l e *a aMuscular Dystrophy Group Research Laboratories, Newcastle General Hospital, Newcastle upon Tyne, NE4 6BE, UK bRowett Research Institute, Greenburn Road, Bucksburn, Aberdeen, AB2 9SB, UK Received 1 August 1995; revision received 27 November 1995; accepted 4 December 1995

Abstract To further elucidate the biochemical mechanism by which the corticosteroid prednisone induces differential changes in muscle mass (via altered protein synthesis/degradation rates) in normal or degenerating muscle tissues, we have determined the activity of a range of proteolytic enzyme types, together with levels of muscle structural proteins, in five innervated and denervated muscle types from control and drug treated rats. In both normal and wasting muscles, the activity of many protease types was substantially down-regulated following treatment with prednisone; however, accompanying net decreases in muscle mass were observed (although the structural protein composition of muscles was unaltered following drug treatment). We conclude that whilst overall rates of protein degradation in both normal and degenerating muscle may be reduced (via protease down-regulation) following prednisone treatment, the effect of the latter in reducing protein synthesis rates must be proportionately greater (even in actively degenerating tissue). Thus, the data do not support the hypothesis that the beneficial effect of prednisone in maintaining muscle mass in pathological tissues (e.g., Duchenne muscular dystrophy (DMD)) operates principally via down-regulation of protease action/protein catabolism. Keywords: Muscle proteins; Prednisone; Muscular dystrophy; Protein metabolism; Proteases; Denervation * Corresponding author, Neurochemistry Department, Regional Neurosciences Centre, Newcastle General Hospital, Newcastle upon Tyne, NFA 6BE, UK. Tel.: 0191 2738811, ext. 22167; Fax: 0191 2260775. 0009-8981/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0009-8981 (95)06257-E

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1. Introduction The Xp-21 linked degenerative diseases of human skeletal muscle, Duchenne muscular dystrophy (DMD) and Becket muscular dystrophy (BMD) have been recognized to result from genetic mutations causing altered expression (quantity and/or molecular size) of the sarcolemmalassociated cytoskeletal protein, dystrophin [1,2]. As a result of these findings, much of the current research to develop therapeutic strategies for DMD/BMD patients has been directed towards cell therapy (via transfer of normal myoblasts [3]) or gene therapy (via vector insertion of normal DNA [4]). In the event of the above proving unsuccessful, it would be useful to develop alternative pharmacologically-based therapeutic strategies for treatment of DMD/BMD patients. A number of unsuccessful clinical trials, using a variety of drug compounds, have been reported previously (reviewed by Emery [5]). Only one compound, the corticosteroid prednisone (or its closely related derivatives) has consistently been reported to be of benefit in ameliorating the disease course in clinical drug trials in DMD patients (reviewed by Khan [6]); this is perhaps the more surprising since corticosteroids have been reported to induce myopathy when administered to patients who do not present with neuromuscular disorders [7-9]. The biochemical mechanism by which prednisone induces wasting of normal muscle tissues, whilst preventing wasting of pathological muscle tissues, is unknown, although it has been suggested that drug treatment primarily reduces overall protein synthesis in the former, whilst reducing overall protein degradation in the latter [10,11]. The effect of prednisone treatment on proteolytic enzyme activities in either normal or pathological muscle is unknown, although inhibition of muscle proteolysis in DMD patients has been suggested [12]. To determine a clearer understanding of the biochemical mechanism underlying the differential action of prednisone on protein turnover in normal and pathological tissues, the objectives of the work described in this paper were: (i) to determine the effect of prednisone treatment on the levels of activity of a range of proteolytic enzymes (responsible for the various stages of the protein degradation cascade) in normal innervated rat skeletal and cardiac muscles; (ii) to determine the effect of prednisone treatment on the activities of the above proteases in actively degenerating rat muscle tissues induced by denervation; (iii) to compare levels of structural proteins in innervated and denervated muscles from control and prednisone treated animals, correlating any changes in muscle composition with changes in protease activities in (i) and (ii) above. In the longer term, elucidation of the mechanism by which prednisone reduces wasting of pathological muscle tissue may facilitate development of more effective compounds, without the undesirable side effects currently associated with prednisone treatment, in treatment of DMD/BMD patients.

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2. Materials and methods

2.1. Animal protocols The experimental protocols for the animal feeding regimes, drug administration and skeletal muscle denervation were as described previously [13,14]. Briefly, at 19 days of age, 16 male weanling rats of the Rowett hooded Lister strain were all fed a control diet of standard chow feed for 3 days, followed by PW3 control semi-synthetic feed [15] for another 3 days. Animals were then organised into four groups of equal mean weight and two groups were unilaterally denervated; experimental groups were then fed as follows: (i) PW3 control diet for 7 days; (ii) prednisone-containing diet (in PW3) at 1.5 mg/kg body weight/day for 7 days; (iii) unilaterally denervated animals fed the PW3 control diet for 7 days; (iv) unilaterally denervated animals fed the prednisone-containing diet (in PW3) at 1.5 mg/kg body weight/day for 7 days. At the end of the experimental period, the animals were killed and the soleus, plantaris, gastrocnemius and extensor digitorum longus (EDL) muscles were removed bilaterally, together with the heart. All of the tissues were weighed and frozen in liquid nitrogen. 2.2. Analysis of structural muscle proteins following prednisone administration via SDS-PAGE Muscles were removed from control and prednisone treated animals and homogenised 1:9 (w/v) in 50 mmol/1 Tris/HCl buffer, pH 7.5, at 4°C in an Ultra Turrax homogeniser. The extracts were centrifuged at 2000 × g for 10 min and the pellet (structural proteins) retained for analysis. Pellets were reconstituted in the original volume of extraction buffer and boiled for 5 min after addition of 5% (w/v) sodium dodecyl sulphate (SDS), 5% (v/v) 2mercaptoethanol, 20% (v/v) glycerol, and 0.005% bromophenol blue tracking dye. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out using 14 × 10 cm slab gels following the method of Laemli [16], with a 4% stacking gel and a 5% to 20% linear resolving gel (sample loading 100 ~g protein per track). Gels were fixed and stained as described previously [17]. Protein stained gels were scanned using a Camag version III gel scanner. 2.3. Enzyme extraction and assay Whole muscle samples (40 to 100 mg, depending upon muscle type) were homogenised in an Ultra Turrax (3 x 15 s at 20 000 rev./min ). A 1:19 (w/v) tissue:buffer extract was prepared in a 50 mmol/l Tris/acetate buffer, pH 7.5, at 37°C for subsequent neutral protease measurement, and in a 50 mmol/1 CH3COOH/CH3COONa buffer, pH 5.5, for acidic protease measurement. The extracts were centrifuged at 2000 x g for 10 rain, and the supernatants retained for enzyme assay. Enzyme (0.1 ml supernatant) was incubated with appropriate assay medi-

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um (0.3 ml total volume) for 1 h at 37°C. Reactions were terminated by the addition of 0.6 ml of ethanol. The fluorescence of the liberated amino-4methylcoumarin (AMC) was measured by reference to a fluorescent standard tetraphenylbutadiene block (X,x 380 rim, X~m 440 nm). Assay blanks were run in which the enzyme was added to the medium immediately before the addition of ethanol. Assay conditions were modified for samples with high enzyme activity so that the extent of substrate utilization did not exceed 15%. Assays were carried out for the following enzyme types: alanyl aminopeptidase: 50 mmoF1 Tris/acetate buffer, pH 7.5, at 37°C, 5 mmol/1 CaCI2, 1 mmol/1 dithiotreitol (DTT), 0.25 mmoFl ala-AMC; arginyl aminopeptidase: 50 mmol/l K2HPOdKH2PO4 buffer, pH 6.5, 0.15 M NaC1, 1 mmol/l DTT, 0.25 mmol/l arg-AMC; leucyl aminopeptidase: 50 mmol/l giycine/NaOH buffer, pH 9.5, at 37°C, 5 mmoF1 MgC12, 1 mmol/l DTT, 2 mmol/l leu-AMC; pyroglutamyl aminopeptidase: 50 mmoFl glycine/NaOH buffer, pH 8.5, at 37°C, 2 mmol/l DTT, 0.25 mmol/1 pyroglutamyl AMC; dipeptidyl aminopeptidase I: 50 mmol/l CH3COOH/CH3CO ONa buffer pH5.5, 2 retool/1 DTT, 0.25 mmol/l gly-arg-AMC; dipeptidyl aminopeptidase II: 50 mmol/l CH3COOH/CH3COONa buffer, pH 5.5, 2 mmol/1 DTF, 0.25 mmoFl lysala-AMC; dipeptidyl aminopeptidase IV: 50 mmol/1 Tris/acetate buffer, pH 7.5, at 37°C, 2 mmoFl DTT, 0.25 mmol/1 gly-pro-AMC; tripeptidyl aminopeptidase: 50 mmol/1 Tris/acetate buffer, pH 7.5, at 37°C, 2 mmol/1 DTT, 0.25 mmol/l ala-ala-phe-AMC; proline endopeptidase: 50 mmoF1 Tris/ acetate buffer, pH 7.5, at 37°C, 2 mmoFl DTT, 0.25 mmoFl CBZ-gly-proAMC; cathepsin B or cathepsin B + L: 50 mmol/l CH3COOH/CH3COONa buffer, pH 5.5, 2 mmol/l DTI', 0.25 mmol/l CBZ-phe-arg-AMC (cathepsin B + L) or 0.25 mmoFl CBZ-arg-arg-AMC (cathepsin B only); cathepsin H: 50 mmol/l KH2POJK2HPO4 buffer, pH 6.0, 1 mmol/1 DTT, 0.5 mmol/l puromycin, 0.25 mmol/l arg-AMC. Assay of cathepsin D activity was based on the spectrophotometric procedure of Pluskal et al. [18]: 50 mmoFl CH3COOH/CH3COONa buffer, pH 3.5, 1 mmol/l DTI', 3 mg/ml acid-denatured haemoglobin substrate (total assay volume 0.5 ml). The reaction was terminated by addition of 0.5 ml 10% perchloric acid (PCA), the samples centrifuged at 2000 x g for 10 min, and the absorbance of acid-soluble peptides determined at 280 nm. Assay blanks were run as above. Non-collagen protein levels in muscle superuatants (for determination of specific activities of the above protease types) were determined by the method of Lowry et al. [19] with bovine serum albumin (BSA) as standard. 3. Results and discussion

Corticosteroids have been widely used as anti-inflammatory agents in the treatment of a wide range of surgical and medical disorders; however, the use

J.W. Haycock et al./ Clinica Chimica Acta 249 (1996) 47-58

51

of these agents in high dosage and/or long term treatment regimes has led to the clinical recognition of undesirable side effects, including gross wasting of the skeletal muscles. To date, it has not been possible to completely dissociate the beneficial action of these agents from such undesirable side effects. The precise biochemical mechanism by which corticosteroids induce wasting of skeletal muscle tissues is unknown; however, there is evidence from clinical studies with human patients and experimental studies using animal model systems that atrophic effects on skeletal muscle result primarily via suppression of protein synthesis [9,20,21], although increased protein degradation has also been suggested [22,23]. The effect of corticosteroid treatment on the levels of activity of various proteolytic enzyme types responsible for the various steps of the intracellular protein degradation cascade is unknown, although increased activity of muscle lysosomal proteases following corticosteroid treatment has been implicated [21]. The effect of prednisone treatment on subsequent levels of lysosomal and cytoplasmic specific protease activities (nmol/h/mg protein) is shown for different muscle types (fast, slow and intermediate) in Table 1 (as mean ± S.E.M.). It should be noted that levels of activity for the various protease types listed in Table 1 were determined using non-physiological substrates (i.e., fluorogenic aminoacyl 7amino-4-methyl-coumarin derivatives). Previous work [17] has shown that the characteristics of individual protease types determined using the latter type of substrates closely parallel enzyme characteristics determined (via high performance liquid chromatography (HPLC)-based assay methodologies) using oligopeptide substrates (which presumably represent the physiological substrates for these enzymes). Most importantly, it is possible to quantitate accurately the levels of activity for individual protease types in tissue extracts, without significant cross-assayinterference, using the assay procedures described under Materials and methods. Protease types not listed in Table 1 (e.g., carboxypeptidases, dicarboxypeptidases) were not included in the present investigation because of the low levels of activity of these enzymes in skeletal muscle tissue. Following prednisone treatment, mean muscle mass was reduced by 10%-20% (relative to control) depending on skeletal muscle type (significant for gastrocnemius and plantaris muscles). Of the lysosomal proteases, dipeptidyl aminopeptidase I, cathepsin B and cathepsin L showed significant reductions in activity (20%-50% of control activity, depending on muscle type) in all skeletal muscle-types following prednisone treatment; cathepsin H and cathepsin D activities showed significant activity reductions only in soleus and EDL muscles, respectively, whilst dipeptidyl aminopeptidase II activity was significantly increased in EDL muscle. Of the cytoplasmic proteases, leucyl-, pyroglutamyl- and tripeptidylaminopeptidase activities were significantly reduced (20OA-80%of control) in all skeletal muscle types following prednisone treatment; alanyl and arginyl aminopeptidase activities were reduced in EDL and soleus muscles, and

J.W. Haycock et aL / Clinica Chimica Acta 249 (1996) 47-58

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dipeptidyl aminopeptidase IV activity reduced in soleus muscle only. Surprisingly, proline endopeptidase showed significantly increased activity in all skeletal muscle types. Thus, it would be difficult to predict the effect of prednisone treatment on muscle proteolysis, since the effect of the latter on individual proteases differs in different muscle types. However, in general terms, the data show a significant and substantial overall reduction in muscle proteolysis following prednisone treatment. Thus, of the thirteen different protease types investigated (representative of the various stages comprising the protein catabolic cascade), 9 enzymes showed significantly reduced activity in EDL muscle, 10 enzymes in soleus muscle, 6 enzymes in gastrocnemius and 6 enzymes in plantaris, respectively. Quantification of changes in the levels of Ca 2÷ activated proteinases (calpains) was not attempted in the above experiments, since insufficient quantities of muscle tissue were available to permit chromatographic fractionation of these enzymes from their endogenous tissue inhibitor (calpastatin), which is required prior to assay [24]. The levels of activity of some lysosomal and cytoplasmic proteases in cardiac

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J. IV. Haycock et al. I Clinica Chimica Acta 249 (1996) 47-58

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55

muscle were significantly reduced following prednisone treatment (although cardiac muscle mass was unaltered); however, the reduction in mean activity levels for corresponding enzymes was in general substantially less than in skeletal muscles. These data suggest that proteolysis in cardiac muscle may be less susceptible to the influence of corticosteroid treatment. This finding is of interest since it has been reported previously that corticosteroids induce anabolic changes in cardiac muscle via a decreased rate of protein degradation [21,25]. The effect of prednisone treatment on the relative levels of muscle structural proteins (determined via SDS-PAGE analysis) is shown in Fig. 1 for soleus and EDL muscles, respectively. For innervated soleus (lanes 1 and 2) and innervated EDL (lanes 3 and 4), no differences were apparent in protein band staining intensities in the SDS-PAGE fractionation profiles following prednisone treatment; thus, the structural composition of these muscles was unchanged following administration of the latter. Similarly, the structural composition of denervated muscles (shown in lanes 5 and 6 for EDL muscle) was found to be unchanged following prednisone treatment. These results are in agreement with previous work which investigated the effects of corticosteroid treatment on muscle structure at the morphological level, via electron microscopic analysis [21], which concluded that administration of prednisone induced only minor changes in the ultrastructure of fast or slow muscle types. It should be noted, however, that the analytical approach outlined above does not exclude the possibility that changes in the levels of minor structural proteins may be induced in skeletal muscle tissue following administration of prednisone. This hyopthesis would require a more detailed analysis (e.g. 1 dimensional (1D) electrophoresis followed by immunoblotting using specific antibodies to individual muscle proteins, or via 2D electrophoresis) than that used in the present investigation; this in turn might provide useful data as to the site of action of corticosteroids at the molecular level. The beneficial action of prednisone in slowing the course of clinical disease (with respect to muscle function) in DMD patients was first reported by Drachman et al. [26]. This finding was somewhat surprising, since administration of corticosteroids (particularly in the longer term) in patients who do not present with neuromuscular disorders (e.g., as an anti-inflammatory agent for patients with rheumatoid arthritis) has been recognized to result in characteristic wasting of skeletal muscles. However, more detailed clinical trials have subsequently confirmed the beneficial effect of prednisone treatment on muscle function in DMD patients. Thus, Mendell et al. [27], in a double-blind, multicentre clinical trial, established that a daily dosage of 0.75 mg prednisone/kg body weight over a six month period resulted in improved muscle strength lasting over a subsequent two year period. Although the beneficial effect of prednisone treatment in DMD patients is now well

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J.W. Haycock et al. /Ciinica Chimica Acta 249 (1996) 47-58

established,the biochemical mechanism by which the former influencesmuscle function is poorly understood. In normal muscle tissue,it has been suggested that corticosteroid-induced myopathy results principally from a reduction in the fractional rate of protein synthesis (ks),with a lesser reduction in fractionalprotein degradation rate (kd) [8,28],whilst in pathological muscle tissue there is evidence that maintenance of muscle mass/function in DMD patients following prednisonc treatment results primarily from a reduction in kd [10,12,29].The effect of corticosteroid treatment on the activitiesof individual proteolyticenzymes (comprising the various steps of the intracellularprotein degradation cascade) in either normal or degenerating muscle is unknown, although a reduction in protease activity in muscle of DMD patients has been suggested [6].The effectof prednisone treatment on the activity levels of a range of proteolytic enzyme types in actively degenerating muscle tissues (induced following dcncrvation) was therefore determined (Table 2). Following denervation, a loss of muscle mass and increase in activity of many protcases (relativeto the corresponding innervated contralateralmuscle) was noted, as described previously [30]. In comparison to the effect of prednisone treatment on innervated muscles (where there was a general reduction in overall muscle proteolysis,Tablc I), the action of prednisonc on protcasc activitiesin denervated muscles was more variable. Thus, of the thirteen protcase types investigated, significant changes in activitylevelswere noted in EDL (alanyl- and Icucyl aminopeptidases reduced, proline endopcptidase and cathepsin L increased), in soleus (alanyl- and arginyl aminopeptidases, dipeptidyl aminopeptidasc IV, tripeptidyl aminopeptidase and cathcpsin D reduced, Icucyl aminopcptidasc and cathepsin L increased), in gastrocnemius (alanyl- and arginyl aminopeptidases,tripcptidylaminopeptidasc, proline endopeptidasc, cathepsins D and L increased), and in plantaris (arginyl and leucyl aminopeptidases, tripcptidyl aminopcptidase reduced, alanyl aminopeptidase, dipeptidyl aminopeptidasc I, proline endopeptidasc, cathcpsins D and L increased), respectively. There was no consistent pattern to these altered activity levels induced by prednisone, with individual cytoplasmic and lysosomal protcascs differently affected in different muscle types (Table 2). The data obtained above therefore do not support the hypothesis that ks is reduced in actively degenerating muscle following prcdnisonc trcatment, via a generalized down-regulation of muscle protcase activity. In summary, we conclude that whilst kd in normal innervated muscles may be reduced via protcase down-regulation following prednisonc treatment, the effect of the latter on ks must be proportionately greater to account for the net loss in muscle mass observed in the various muscle types investigated (in agreement with previous rcports on the rclativcinfluence of corticosteroid treatment on ks and kd, as outlined above). In actively degenerating muscle tissues (in this case induced by denervation), the data

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obtained do not support the hypothesis for a decrease in k d (relative to that of ks) via a generalized down-regulation of muscle proteolysis induced by prednisone (indeed a number of proteases show increased activity levels in denervated muscles following prednisone treatment). In view of the above, we therefore suggest that the beneficial action of prednisone on muscle function in DMD patients may be mediated via action on the inflammatory response mechanism [11], rather than by downregulation of muscle proteolysis; thus, development of improved pharmacologically-based therapeutic strategies (without the undesirable side effects associated with prednisone treatment) may be better focused upon the anti-inflammatory/immunosuppressant characteristics of corticosteroids.

Acknowledgements Dr. J. W. Haycock was supported by the Muscular Dystrophy Group of Great Britain and Northern Ireland. We thank Miss Carol Atkinson for the preparation of this manuscript.

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[12] Kawai H, Adachi K, Nishida Y, Invi T, Kimura C, Saito S. Decrease in urinary excretion of 3-methylhistidine by patients with Duchenne muscular dystrophy during glucocorticoid treatment. J Neurol 1993;140:181-186. [13] Maltin CA, Delday MI and Reeds PJ. The effect of a growth promoting drug, clenbuterol, on fibre frequency and area in hind limb muscles from young male rats. Biosci Rep 1986;6:193-299. [14] Maltin CA, Reeds PJ, Delday MI, Hay SM, Smith FG, Lobley GE. Inhibition and reversal of denervation induced atrophy by the B-agonist growth promoter clenbuterol. Biosci Rep 1986;6: 811-818. [15] Pullar JD, Webster AJ. The energy cost of fat and protein deposition in the rat. Br J Nutr 1977;37:355-363. [16] Laemii UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-685. [17] Mantle D, Lauffart B, McDermott JR, Smith AI, Pennington RJT. Purification and characterization of the major aminopeptidase from human skeletal muscle. Biochem J 1983;211:567-573. [18] Pluskal MG, Harris JB, Pennington RJT, Eaker D. Some biochemical responses of rat skeletal muscle to a single subcutaneous injection of a toxin isolated from the venom of the Australian tiger snake. Clin Exp Pharmacol Physiol 1978;5:131-141. [19] Lowry OH, Rosebrough N J, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193: 265-275. [20] McGrath JA, Goldspink DF. Ghicocorticoid action on protein synthesis and protein breakdown in isolated skeletal muscle. Biochem J 1982;206:641-645. [21] Kelly FJ, McGrath JA, Goldspink DF, Cullen MJ. A morphological/biochemical study on the actions of corticosteroids on rat skeletal muscle. Muscle, Nerve 1986;9:1-10. [22] Afifi AK, Bergman RA. A study of the evolution of the muscle lesion in rabbits. John Hopkins Med J 1969;123:158-174. [23] Walsh G, De Vivo D, Olsen W. Histochemical and ultrastructural changes in rat muscle: occurrence following adrenal corticotrophic hormone, glucocorticoids and starvation. Arch Neurol 1971;24: 83-93. [24] Mantle D, Perry EH. Comparison of Ca 2÷ activated proteinase enzyme and endogenous inhibitor activity in brain tissue from normal and Alzheimer's disease cases. J Neurol Sci 1991;102: 220-224. [25] Kelly F J, Goldspink DF. The differing responses of four muscle types to dexamethasone treatment in the rat. Biochem J 1982;208: 147-151. [26] Drachman DB, Toyka KV, Myer E. Prednisone in Duchenne muscular dystrophy. Lancet 1974;2:1409-1142. [27] Mendell JR, Moxley RT, Griggs RC et al. Randomised, double blind six month trial of prednisone in Duchenne muscular dystrophy. New Engl J Med 1989;320:1592-1597. [28] Tomas FM, Munro I-IN, Young VR. Effects of glucocorticoid administration on the rate of muscle protein breakdown in vivo in rats, as measured by urinary excretion of Nmethyl histidine. Biochem J 1979;178:139-146. [29] Rifai Z, Welle S, Moxley RT, Lorenson M, Griggs RC. Mechanism of action of prednisone in Duchenne dystrophy. Neurology 1992;42:(Suppl: Abstract 1428). [30] Mantle D, Delday MI, Maltin CA. Effect of clenbuterol on protease activities and prorein levels in rat muscle. Muscle, Nerve 1992;15:471-478.