J. Cell Sci. 83, 197-212 (1986) 197Printed in Great Britain © The Company of Biologists Limited 1986

BIOCHEMICAL ASPECTS OF BUPIVACAINE-INDUCED

ACUTE MUSCLE DEGRADATION

SHOICHI ISHIURA*, IKUYA NONAKA AND HIDEO SUGITANational Center for Nervous, Mental and Muscular Disorders, Kodaira, Tokyo 187,Japan

SUMMARY

A single injection of a local anaesthetic, bupivacaine, into the soleus muscle of adult rat has asevere mytoxic effect, i.e. rapid dissolution of myofilaments and degradation of myofibrillarproteins shortly after injection. Increased lysosomal enzymes were observed in homogenates ofaffected muscle. The activity of potent proteolytic enzyme, cathepsins B and L (assayed against anew synthetic substrate succinyl-Tyr-Met-naphthylamide), gradually increased and reached aplateau value that was 11-fold greater than the control 48 h after bupivacaine injection. Thechronological change in the activity of cathepsins B and L was reflected in the myofibrillar proteinpattern in bupivacaine-treated muscle. To determine whether the increase in lysosomal peptidehydrolases is due to activation of muscle lysosomes or not, mononuclear cells were separated fromboth injected and control muscles. The activity of cathepsins B and L in the lysate from injuredmuscle was 180-fold higher than the control. Affinity-purified antibody was used to study theintracellular localization of cathepsin B by immunohistochemical procedures. The results wereconsistent with the biochemical observation that the main source of cathepsin B in musclehomogenates was infiltrated mononuclear cells. Therefore, we conclude that the increasedlysosomal enzymes may be derived mainly from mononuclear cells (macrophages), not frommuscle lysosomes, in bupivacaine-induced acute muscle degeneration.

INTRODUCTION

Many proteolytic enzymes have been found and characterized in skeletal muscleand some of them seem to be involved in pathological protein turnover, as inmuscular dystrophy (Bird et al. 1980; Obinata et al. 1981). Lysosomal cathepsinsshow the most potent proteolytic activities in skeletal muscle and have beenimplicated in the normal turnover of myofibrillar components (Pennington, 1977).Earlier biochemical studies revealed that among many acid peptide hydrolases,cathepsins B, D and L had more potent endoproteolytic properties than otherexopeptidases, such as cathepsins C and H, and lysosomal carboxypeptidases(Barrett, 1977, 1980). A characteristic of cathepsins B and L is their sensitivity tothiol-blocking agents, and a characteristic of cathepsin D is its high sensitivity topepstatin (Barrett, 1980). These endopeptidases also degraded almost all of thestructural proteins in vitro. However, their significance in acute muscle degenerationis unknown. Non-lysosomal calcium-activated protease apparently initiates myo-fibrillar disorganization by removing Z-lines (Ishiura, 1981). Mast cells contain fairly

•Author for correspondence.

Key words: bupivacaine, muscle, protease, macrophages, cathepsin B.

198 S. Ishiura, I. Nonaka and H. Sugita

large amounts of alkaline proteolytic activities, but the physiological meaning of thisis still unclear (Edmunds & Pennington, 1981). The precise mechanism of proteinbreakdown in muscle remains to be elucidated.

Previous uncertainty as to the distribution of individual enzymes between musclecells and non-muscle cells prompted us to estimate the contribution of proteases ofnon-muscle origin. However, the number of these cells is too low in normal muscle todetermine the activities of these proteases of non-muscle origin. Previously, mono-nuclear cells were isolated from denervated rat muscle with a 4% yield (Maskleyet al. 1977). The preparation contained relatively high specific activities of lysosomalacid hydrolases. However, the total activities of these enzymes per muscle increasedas much as 4-fold 6 days after denervation. Animals that have hereditary dystrophyare other models for accelerated protein breakdown and various proteases have beenreported to be increased in skeletal muscle. In such cases, degeneration of musclefibres accompanies active muscle regeneration. So the calculated activity of a wholemuscle homogenate is the result of a mixture of both degenerating muscle fibres thathave large amounts of proteolytic enzymes and regenerating normal muscle fibres.

This consideration led to the present work in which we induced acute muscledegeneration in whole soleus muscle of rats, by administration of a local anaesthetic,bupivacaine (Libelius et al. 1970; Bradley, 1979), and measured the activity oflysosomal enzymes. The massive degeneration of injured soleus muscle continued forat least 72 h after drug treatment, and then the muscle began to regenerate. Completeregeneration was seen at 30 days after bupivacaine administration (Nonaka et al.1983). In studies of the action of bupivacaine, we found that a thiol-dependentsuccinyl-Tyr-Met-naphthylamide(Suc-Tyr-Met-NA)-hydrolytic activity increased11-fold in the muscle homogenate, whereas cathepsin D increased only threefold.Other lysosomal enzymes did not increase more than threefold. Accordingly, weconcluded that Suc-Tyr-Met-NA-degrading enzyme(s) was mainly involved in thebreakdown of muscle proteins in the myopathy. In this study, we investigated theexpression of cathepsin B in degenerating skeletal muscle by the indirectimmunofluorescence technique and found the limited occurrence of cathepsin B inmacrophages, which can at least partly account for the biochemical findings thatincreased lysosomal enzyme may originate from phagocytes, not from musclelysosomes, in bupivacaine-induced muscle degeneration.

MATERIALS AND METHODS

Bupivacaine-induced myopathyThe effect of 0 5 % bupivacaine • HC1 (Yoshotomi Pharm. Co., Tokyo) alone and in combination

with various agents was examined in male Wistar rats weighing about 250g each. Isotonicbupivacaine solution was injected in 0-2ml vol. intramuscularly into the soleus muscle in ratslightly anaesthetized with pentobarbital. The soleus muscle was exposed surgically and bu-pivacaine was injected directly into the muscle using a 26-5 gauge needle (Nonaka et al. 1983).Isotonic saline was injected into the contralateral soleus muscle as a control.

The rats were killed by cervical dislocation. Soleus muscles were removed and weighed. Themuscles were rapidly homogenized twice for 30 s in a glass homogenizer with 10 vol. of ice-cold5 mM-phosphate buffer (pH 7-0), containing 0-1 M-NaCl and 0-1 % Triton X-100. Approximately

Bupivacaine-induced muscle degradation 199

95 % of the lysosomal enzymes were solubilized by this procedure. After centrifugation at 10 000gfor 10 min, the resulting supernatant was used for each assay.

Biochemical assaysAcid phosphatase, a-glucosidase and ar-galactosidase were assayed as described (Ishiura et al.

1984). Cathepsins B and L, and cathepsin D were measured according to Barrett (1977). A newsynthetic substrate, Suc-Tyr-Met-NA, was used to determine the activity of thiol-dependentcathepsins. Previous studies in this laboratory indicated that purified calcium-activated proteasefrom muscle did not hydrolyse this substrate. Protein was determined by the Lowry method withbovine serum albumin as a standard (Lowry et al. 1951).

In vitro inhibition by bupivacaine and other compounds of lysosomal enzymesFor in vitro experiments, bupivacaine • HC1 was dissolved in deionized glass-distilled water. A

cysteine protease inhibitor, either Ep-475 (L-/raws-epoxysuccinyl leucylamide-(3-methyl)butane orE-64-c, kindly provided by Taisho Pharm. Co. Ltd, Ohmiya), was dissolved in 0-1 M-NaHCC>3.Pepstatin was first dissolved in ethanol and then diluted for each assay. Calcium-activated neutralprotease was purified from rat skeletal muscle as reported (Ishiura & Sugita, 1983). Crystallinecathepsin B from rat liver was a generous gift from Dr T. Noda, Tokushima University. CathepsinD from rat skeletal muscle was partly purified according to Weinstock & Iodice (1968).

Denervation and immobilizationIn four rats, the right sciatic nerve (5 mm in length) was removed at the level of the trochanter

major of the femur (Ishiura et al. 1981). Sciatic denervation caused degeneration of the right soleusmuscle 1 week after denervation, as judged from its weight.

Immobilization of the right soleus muscle fixed at the lengthened position (6 rats) or theshortened position (6 rats) was performed according to the method of Miyazawa et al. (1983),using rats of the same age. After 1 week of immobilization, the muscles were removed for theenzyme assay. In these experiments, contralateral muscles were used as a control.

Separation of mononuclear cells from bupivacaine-injected muscleSoleus muscles were removed and chopped up with scissors. The chopped material was weighed

and transferred to a small culture tube containing RPMI-1640 medium (Flow Labs, Virginia,USA, 10 ml/soleus). The tissue suspension was incubated for 1 h at 37°C with continuous shaking.After incubation, the suspension was filtered through lens paper to separate the cell suspensionfrom muscle residues. The cells were collected by centrifugation at 1500^ for 5 min, washed twicewith saline and suspended in phosphate-buffered saline containing 0-1% Triton X-100 for theenzyme assay. The residue was then homogenized with the same buffer as described above.

Gel electrophoresisOne-dimensional electrophoretic analysis of proteins was performed on high-resolution sodium

dodecyl sulphate (SDS)-polyacrylamide slab gels with the discontinuous Tris-glycine system(Laemmli, 1970). At appropriate intervals after bupivacaine administration, the soleus muscleswere removed and kept at —20°C for a week in 50% glycerol solution. This treatment isindispensable for visualizing structural proteins on gels because glycerination for a week clearlyremoves soluble proteins from the muscle. Preliminary experiments indicated that no proteolysisocurred in the above conditions, whereas prolonged preservation of muscle in glycerol led to theselective removal of a-actinin from Z-lines, even at —20°C. We have previously indicated that theremoval of tt-actinin might be catalysed by a Z-line-bound calcium-activated protease (Ishiuraet al. 1984). But the activity of this enzyme is negligible after preservation for a week. Afterglycerol treatment, the muscle was withdrawn and incubated in phosphate-buffered saline at roomtemperature for 5 min to remove excess glycerol. The muscles were teased, solubilized in buffercontaining 2% SDS and subjected to SDS-polyacrylamide gel electrophoresis. After electro-phoresis, gels were stained overnight in 45 % methanol, 7 % acetic acid, and 0-25 % CoomassieBlue R-250. Gels were destained with 7 % acetic acid, and photographed.

200 5. Ishiura, I. Nonaka and H. Sugita

Histochemical stainingAt the time of killing, the solei were removed immediately from each animal for histochemistry.

The specimen was frozen in isopentane cooled by liquid nitrogen. A routine battery of histo-chemical reactions was applied to 10 fim thick transverse sections as described (Nonaka et al.1983).

Indirect immunofluorescent microscopyAffinity-purified rabbit antibody against cathepsin B, provided by Professor N. Katunuma and

Dr E. Kominami (Tokushima University), was used to detect the reservoir of the enzyme. Thespecificity of the antibody has been reported (Kominami & Katunuma, 1982). Anti-vimentinantibody was obtained from Transformation Res. Inc., Framingham, USA. Fluorescein-conju-gated goat anti-rabbit IgG (immunoglobulin G) was purchased from Miles-Yeda, Japan.

Immunocytochemical reactions were applied to 3 fim thick transverse frozen sections. Prior tostaining the specimen with antibody, the sections on glass coverslips were fixed with cold acetonefor 5min, washed with saline, and transferred to a humidified chamber. A 20fd sample ofappropriately diluted antiserum was applied to each coverslip. The coverslips were incubated at37°C for lOmin, rinsed in saline and returned to the chamber. A 20//I sample of fluorescein-conjugated goat anti-rabbit IgG (1/800 dilution) was applied to each coverslip. They wereincubated at 37°C for lOmin, rinsed in saline, and mounted on glass slides. All specimens wereexamined with a Zeiss photomicroscope 18FL. Photomicrographs were made on Fuji Neopan 400film (ASA 400). Control experiments were carried out by incubation with preimmune serum oronly second antibody. No fluorescence was observed with these procedures.

RESULTS

Bupivacaine-induced myopathy

3 h after bupivacaine treatment the wet weight of the soleus muscle (n = 20)increased to 157% of that of the contralateral one as a result of swelling. At 48 hafter injection, the wet weight of the injected muscle had been reduced to 126%.Morphologically, 0-5 % or 16mM-bupivacaine produced muscle degeneration in thesoleus muscle, while injection of saline solution caused no observable effect on themuscle fibre, except for limited damage along the needle track 48 h after injection(Fig. 1); 24—48 h after injection, the necrotic areas were infiltrated by numerousacid-phosphatase-positive mononuclear cells. Electron microscopy showed thatalmost all of the necrotic fibres contained mononuclear cells with a number of Golgibodies, lysosomes and innumerable cytoplasmic processes (Nonaka et al. 1983;Ishiura et al. 1983a). Structural proteins decreased and their amounts were less than70 % of that of the control after 48 h. No regenerating fibres were observed until 48 hafter injection, indicating that all the muscle fibres in the soleus muscle underwentdegeneration simultaneously. The effect was confined to the injected soleus muscle.Bupivacaine at 1 % produced roughly similar lesions to those at 0-5%, but at 0-25 %they were less prominent and below 0-125 % there was no obvious effect.

Bupivacaine-induced increase in lysosomal enzyme activities

The increase in the lysosomal apparatus 48 h after injection of bupivacaine wascharacterized by assaying of typical marker enzymes. Table 1 shows that cathepsinsB and L are enriched approximately 11-fold in the affected muscles as compared tothe contralateral one. All the lysosomal enzymes tested increased as well. Up to 72 h

Bupivacaine-induced muscle degradation 201

after bupivacaine treatment, these enzymes increased in the muscle and thereafterdecreased gradually. The activity had returned to the normal level 30 days afteradministration, accompanied by complete regeneration of muscle fibres (Nonakaet al. 1983). Bupivacaine itself, when mixed into the assay medium in the concen-tration range of 1—30 mM, did not alter these enzyme activities.

So-called lysosomal activation has been found to be pronounced in denervatedmuscles or immobilized muscles fixed in the shortened position. We measured thelysosomal acid hydrolases 1 week after operation, when infiltration of mononuclearcells activated by surgical operations was not prominent, to observe the directcontribution of muscle lysosomes. As shown in Table 2, all of the lysosomal enzymesso far assayed increased in their specific activities per mg soluble proteins but thedegree of increase was rather low compared to that in bupivacaine-induced changes2 days after injection. The marked difference was that the specific activity ofcathepsins B and L was higher than that of other lysosomal enzymes in bupivacaine-treated muscles.

On the basis of the results presented above it was suggested that the activity oflysosomal enzymes of muscle origin increased twofold at most after denervation orimmobilization, even when the wet weight of the muscle decreased to 56% of thecontrol after 1 week, and that in bupivacaine-induced myopathy these lysosomal acidhydrolases had not originated from muscle lysosomes. To determine directly thecontribution of other cells that infiltrated into the muscle to the total enzyme activity

Fig. 1. Cross-section of the soleus muscle of rats 48 h after a single injection of 0-5%bupivacaine. Almost all of the muscle fibres have been damaged and broken down byphagocytes (B), while no significant lesion was observed in the contralateral saline-injected muscle (A). Haematoxylin and eosin. X170.

202 S. Ishiura, I. Nonaka and H. Sugita

Table 1. Effect of bupivacaine administration on lysosomal enzyme activities insoleus muscle 48 h after injection

Specific activity (unitsmg~')f

Enzymes

Cathepsins B and LJCathepsin D§Qf-GlucosidaseUar-GalactosidaseKAcid phosphataseH

Bupivacaine (B)

320 ± 9*0-467 ±0-021*2-48 ±0-193-95 ±0-18*

0-342 ±0-023*

Control (C)

30 ± 60-151 ±0-0152-54±0-14l-12±0-06

0-176 ±0-004

B/C

10-73-11-03-51-9

*P<0-01;n = 21.fMean±S.E.M.11 unit = nmol naphthylamineh"1.§ 1 unit = the amount of the enzyme that catalysed an increase of 1-0 unit of absorbance at

280 nm under our standard assay conditions (Ishiura et al. 1984).\ 1 unit = arbitrary unit.

Table 2. Effect of denervation and immobilization on lysosomal enzyme activities inrat soleus muscle

Experiment

Denervation (n = 4)Immobilization

Shortened (n = 6)Lengthened (w = 6)

Bupivacaine (n = 21)

CathepsinsB and Lf

127

131183

1070

Cathepsin Df

135

119123309

cr-Galactosidasef

128

123153353

NCPJ

56

7610668

•f The relative specific activities to the contralateral are given in this Table. Data (%) are theaverage of observations indicated. Bupivacaine-induced changes in lysosomal enzymes are moreprominent than those after other physical operations.

| NCP, non-collagen protein. NCP was extracted with 0-1 M-NaOH after removing the solubleproteins by repetitive washing.

in muscle homogenate, mononuclear cells were separated from affected muscles. Toavoid contamination by myofibrillar debris, we adopted the simple incubationmethod described by the Pennington group (Maskley et al. 1977). The recovery ofcathepsins B and L in cell lysates was approximately 12% of the whole muscleextract, which is higher than those reported by Etherington & Wardale (1982). Themononuclear cells retained 95 % viability as judged by Trypan Blue exclusion. Thecells obtained from bupivacaine-treated muscles adhered to the plastic plate andshowed macrophage-like morphology within 24 h. Therefore, we concluded thatmost of the harvested cells were macrophages.

The relative activity value for each enzyme is given in Table 3. The fraction frombupivacaine-treated muscles has 180-fold higher activity of cathepsins B and L, andthe lowest for cathepsin D, suggesting that the composition of acid hydrolase in

Bupivacaine-induced muscle degradation 203

Table 3. Recovery of lysosomal enzyme activity in the mononuclear cell fraction ofrat soleus muscle

Enzymes Bupivacaine/Controlf

Cathepsins B and L 181Cathepsin D 8-5ar-Glucosidase 23-4a-Galactosidase 15-4

f Mononuclear cells were harvested from both bupivacaine-injected and control muscles 48 hafter treatment. Data are averages of four animal experiments and presented as the portion of thosefor injured muscle relative to the control. All values were statistically significant (P< 0-001).

macrophages is different from that in the control muscle. Accordingly, we assumedthat most of the lysosomal enzymes are derived from macrophages in acutebupivacaine-induced myopathy.

Specificity of Suc-Tyr-Met-NA-hydrolysing enzyme(s)

Next, we investigated the specificity of Suc-Tyr-Met-NA-hydrolysing enzyme(s)in the bupivacaine-injected muscle homogenate. The various compounds thataffected proteolytic activities are listed in Table 4. The pH optimum for hydrolysis ofthis substrate is around 6. But non-thiol-dependent hydrolysis is rather high at pH6*0 compared to that at 5-0. Only a group of compounds whose action is accepted asbeing restricted to cysteine proteinases was effective in inhibiting the activity at morethan 10~sM, which is consistent with the previous in vitro data determined usingpurified enzymes, which shows that only cathepsin B and cathepsin L hydrolysedthis substrate, among various cathepsins (KatunumaeJ al. 1983). On the other hand,calcium ions had no effect in the concentration range from 10~s to 10~2M. Bestatinalso did not affect the activity.

In order to characterize the activity, the Suc-Tyr-Met-NA-degrading activity inthe crude muscle extract was quantitatively precipitated in cold acetone between45% and 75%, dissolved in lml of 20mM-sodium acetate (pH5-0), containing50mM-NaCl and 5 mM-2-mercaptoethanol (buffer A), and then applied to a columnof Sephadex G-100 (data not shown). A very minor amount of activity was found inthe flow-through ( = 5 % of the total activity). The peak was eluted at a molecularweight of 25 000 to 30000. The active enzyme was applied to a CM-Sephadex C-50column equilibrated with buffer A. The column was washed and then eluted step-wise with 0-1 M, 0-2M, 0-35 M, 0-5 M and 0-6M-NaCl in buffer A. The Suc-Tyr-Met-NA-degrading activity was eluted in 0-35—0-5M-NaCl, suggesting that cathepsin Hand Leu-naphthylamide-hydrolysing protease, hydrolase H (Okitani et al. 1981),were not involved in the preparation. Separate experiments indicated that a thiol-dependent Arg-MCA-degradating activity, supposed to be cathepsin H, was foundin the breakthrough fraction on CM-Sephadex column chromatography. We tookthese results to mean that, although many proteases may be mixed in the musclehomogenate, the activity of cathepsin B and that of cathepsin L (tentatively named as

204 5. Ishiura, I. Nonaka and H. Sugita

Table 4. Effects of activators and inhibitors on Suc-Tyr-Met-NA-hydrolysingenzyme(s)

(1) Conditions (pH)

344-555-566-578

(2) +Cysteine, +EDTA, pHSf

Effector

Trypsin inhibitor (soybean)IodoacetamideEp-475 (E-64-c)LeupeptinBestatinPepstatinZnCl2

FeCl3

CaCl2

MgCl2

MgCl2 + ATP

Activity (%)-Cysteine, - E D T A +Cysteine, +EDTAf

169

32333843180

Final concentration of effector

0-01 mg ml"1

10 mM0-01 mM0-01 mM0-1 mM0-1 mM

lmMlmM

0-01 mM or 10 mM10 mM2mM

1296691

1001008927

0

% Activity

100500

100100

40

10010099

The enzyme was preincubated with various concentrations of effectors for lOmin at 37°C andassayed as described in Materials and Methods; 100% activity is defined as maximum activityunder optimal conditions.

f Cysteine (2mM), EDTA (1 mM).

cathepsins B and L) is sufficiently determined by measuring the rate of hydrolysis ofSuc-Tyr-Met-NA.

The increase in activity of cathepsins B and L was examined in 0-5 % bupivacaine-treated soleus muscle (Fig. 2). The specific activity was almost the same as thecontrol, 6 h after the injection. The activity, however, began to increase and reacheda plateau value, which was 11 times higher than the control, after 48 h. Recovery 7days after bupivacaine treatment, however, was not as complete as recovery after 30days. The effect of a single injection of bupivacaine depends on its concentration.The increase in activity of cathepsins B and L at 48 h after bupivacaine injection at0-25 % is approximately 60 % of that at 0-5 %. As can be seen in Fig. 2 , the increasewithin 48 h is striking but has an apparent lag period. The change in activity seemedto be proportional to the number of invading cells detected by a histochemicaltechnique (Nonaka et al. 1983). Consequently, we believe that the chronologicalchanges in the activity of cathepsins B and L should be reflected in the proteinpattern in injected soleus muscle (see also Fig. 3).

Bupivacaine-induced muscle degradation-H 1 th

205

2 7Days after bupivacaine injection

Fig. 2. Graph (O) showing the cathepsins B and L activity of the individual soleusmuscles of the bupivacaine-treated legs expressed as units (nmol naphthylamine liber-ated h~') per mg soluble protein. ( • ) Saline-injected soleus muscle.

Change in structural proteins

In order to examine the degradation of structural proteins, individual proteinswere estimated by SDS—polyacrylamide gel electrophoresis. To rule out the possi-bility that bands in gels were due to the presence of contaminating serum, weremoved soluble proteins from the muscle by glycerination. Preliminary resultsindicated that serum albumin was clearly detected in the muscle without glycer-ination even 3 h after bupivacaine injection, which suggested an influx of the seruminto muscle cells. Accordingly, the glycerol treatment was necessary to exclude theseproteins. Identification of structural proteins was carried out by co-electrophoresiswith each protein purified from rat soleus muscle.

As shown in Figs 3 and 4, there was no sign of degeneration at 3 h except thespecific decrease in cr-actinin content. This decrease indicated the well-documenteddisappearance of the Z-line, which has been regarded as the initial change in musclefibres in this type of myopathy (Hall-Craggs, 1974; NonakaeJ al. 1983). At 6h afterbupivacaine injection, muscle showed a similar picture, with a slightly pronouncedloss of ar-actinin. At 24 h after bupivacaine application, other structural proteinssimultaneously began to decrease sharply. Almost all of the structural proteinsdisappeared and were degraded into smaller molecular weight products at 48 h. A43 000Afr protein, identified as actin by two-dimensional gel electrophoresis,however, is resistant to degradation (as indicated in Fig. 3), whereas the content ofmyosin heavy chain decreased with time. The above results suggested a close relationbetween the increase in the cathepsins B and L and the decrease in structural proteinsin bupivacaine-treated muscle.

206

Mr

x10"3

94

67

43

S. Ishiura, I. Nonaka and H. Sugita

1 2 3 4 5 6

MHC

30

20

Fig. 3. SDS-polyacrylamide (15%) gel electrophoresis of glycerinated myofibrilstreated with bupivacaine. Lane 1, marker proteins (in A/rXl0~3): phosphorylase b (94),bovine serum albumin (67), ovalbumin (43), carbonic anhydrase (30) and soybeantrypsin inhibitor (20); lane 2, non-treated soleus muscle; lane 3, 3 h; lane 4, 6h; lane 5,24h; lane 6, 48h after bupivacaine injection. MHC, myosin heavy chain; A, actin.Alpha-actinin is indicated by an arrow.

As shown in Fig. 4, the decrease in Q'-actinin at 3 h after bupivacaine injection wassuppressed by the co-injection of a protease inhibitor, Ep-475, suggesting thatcysteine-dependent protease(s) removed tf-actinin from the Z-lines.

Immunohistochemical localization of cathepsin B in degenerating muscle

In order to confirm the results described above, we investigated the localization ofone of these enzymes, cathepsin B, in bupivacaine-treated muscle using an immuno-fluorescent technique. A 3 [xm thick section was used to elucidate the location of theenzyme more clearly. However, we could hardly distinguish the muscle fibres inaffected muscle from the interstitial tissues by haematoxylin and eosin (H/E)staining because of the diffuse boundary of degenerating muscle fibres (see Fig. 1).Among various antibodies tested, anti-vimentin antibody gave a strong peripheralstaining pattern for indirect immunofluorescence of control and affected muscles

Bupivacaine-induced muscle degradation 207

(Fig. 5A,B). From this picture, the number of muscle fibres was calculated as lessthan 50% after 48 h of injection. On the other hand, comparable fluorescence-positive cells, stainable only with anti-cathepsin B antibody, were detected withinbupivacaine-treated muscle fibres (Fig. 5C,D) in addition to the small phagocytessurrounding degenerating muscle fibres. A high-magnification micrograph of thelocation of cathepsin B is shown in Fig. 6. Two serial sections were made frommuscle to identify each component, one of which was stained with H/E and the otherwith anti-cathepsin B. Notably, phagocytes in the muscles fibres were decorated withanti-cathepsin B, but the granulocytes (g) and degenerating muscle fibres were lessstainable. At this stage of necrosis, i.e. 48 h after bupivacaine injection, most of the

1 2 3 4 5 6 7

43

i* I30

Fig. 4. SDS-polyacrylamide 12 % gel electrophoresis of glycerinated myofibrils 3 h afterbupivacaine injection. Bupivacaine was injected as described in Materials and Methods.A protease inhibitor, Ep-475 (lmM), was coinjected with bupivacaine. Lane 1, markerenzymes (see legend to Fig. 3); lanes 2 and 3, control soleus muscles; lanes 4 and 5,bupivacaine-injected soleus muscles; lanes 6 and 7, 1 mM-Ep-475 administered to soleusmuscles with bupivacaine.

208 S. Ishiura, I. Nonaka and H. Sugita

Fig. 5. In normal muscle a bright peripheral vimentin-specific staining is seen in A;X140. After 48 h of treatment with bupivacaine a peripheral vimentin-specific fluor-escence is seen in only a few muscle fibres (B, X 140), amounting to less than 50 % of thecontrol. The bupivacaine-treated soleus muscle was indirectly stained by anti-cathepsin Bantibody (C,D, X280). C. Phase-contrast micrograph; D, the same muscle viewed withanti-cathepsin B fluorescence optics. Note the bright spotty fluorescence confined to thephagocytes.

infiltrated cells were identified as macrophages by electron microscopy (Ishiura et al.1983a). Basophilic mast cells were scarcely found by histochemical methods.

DISCUSSION

We have investigated the change in lysosomal enzymes in acute muscledegeneration induced by bupivacaine. It has previously been shown that localanaesthetics such as lidocaine, procaine, tetracaine, dibucaine and bupivacaine causedegeneration of muscle fibre (Foster & Carlson, 1980; Yagielae/ al. 1982). However,there have been few reports on the mechanism of myofibrillar degeneration by theselocal anaesthetics. One of the problems was the diversity of the conclusions, resulting

Bupivacaine-induced muscle degradation 209

Fig. 6. Transverse serial sections (3 ^m thick) of bupivacaine-injected soleus muscle.Haematoxylin and eosin; X350; B, anti-cathepsin B fluorescent micrograph; X350.Degenerating muscle fibre (asterisk); granulocytes (g).

from technical differences. In this experiment, only soleus muscle was used becauseuniform degenerative changes throughout the whole muscle were observed after asingle intramuscular injection of bupivacaine (Nonaka et al. 1983; Ishiura et al.1983a,6).

As shown in Table 3, the mononuclear cell fraction included large amounts ofcathepsins B and L, most of which might cause the increased activity of theseenzymes in the muscle homogenate after bupivacaine treatment. The acceleratedcatabolism of myofibrillar proteins following drug administration is certainly due tolysosomal proteases. Maskley et al. (1977) reported that 6 days after denervation ofrat hemidiaphragm there was a fourfold increase in cathepsin B in the muscle itself,but not in the mononuclear cells. Extracted mononuclear cells contained only a smallportion of these enzymes (1-4%). More recently, Etherington & Wardale (1982)showed that the contribution of mononuclear cells to the lysosomal enzyme activitiesof the whole muscle extract of normal rats is approximately 2-4—4-0 % for cathepsinsB and L. These results suggested that the population of mononuclear cells is so smallthat the quantitative extraction of these cells is difficult, apart from the seriousexperimental problem of the separation of these cells from fibrous muscle tissue. Ourfinding that 12% of cathepsins B and L is associated with the mononuclear cellfraction is a higher value than previous ones, but not conclusive because we do notknow the origin of the remaining 88 % of the activity. However, we demonstrated byantibody staining that cathepsin B was localized in invading cells. These two resultsled us to the conclusion that muscle fibres were digested by acid hydrolases derivedfrom mononuclear cells. An increased number of mononuclear cells in and aroundthe necrotic fibres after injection had histochemical and electron-microscopic charac-teristics of macrophages; they had high acid phosphatase activity and numerouscytoplasmic processes. We could hardly recognize fibroblasts in these fractions. Thecontribution of fibroblasts, even if present, can be ruled out because the content ofcathepsins B and L is relatively low in cultured fibroblasts (data not shown).

210 S. Ishiura, I. Nonaka and H. Sugita

Next, we must demonstrate that most of the induced enzyme is cathepsin B.According to Barrett (1977), lysosomal cathepsins are recognized to comprisedistinct groups, called exopeptidases and endopeptidases. The endopeptidases orproteinases, which are considered to be mainly involved in the degradation ofstructural proteins, are also classified into four groups depending on the catalyticmechanism of their active centre, which is easily revealed by the use of specificinhibitors. In our case, the inducible enzyme(s) can be blocked by iodoacetamide,leupeptin and Ep-475, suggesting that a cysteine residue should be involved in thecatalytic centre of the enzyme. Among many lysosomal enzymes, only cathepsins B,H and L are cysteine proteases, but cathepsin H can be excluded by the fact that thepurified enzyme did not hydrolyse Suc-Tyr-Met-NA at all (Katunuma et al. 1983).

The possibility that the thiol-dependent Suc-Tyr-Met-NA-hydrolysing activity isdue to contamination by other cellular cysteine proteases can be ruled out by thefollowing: (1) the pH optimum of the hydrolysis is below 6-0, and the addition ofTriton X-100 to the homogenizing buffer results in increased activity, suggestingthat the enzyme is of lysosomal origin. (2) The activity is not enhanced by calcium(10~s to 10~2M), indicating that two forms of calcium-activated proteases do notparticipate in the processes. (3) A high molecular weight thiol-activated protease,ingensin, found in mammalian tissues is not involved in the hydrolysis of Suc-Tyr-Met-NA, because the molecular weight of the enzyme is above 600000 on gelfiltration (Dahlmann et al. 1985; Ishiura et al. 1985), which is inconsistent with ourdata showing that 95 % of the Suc-Tyr-Met-NA-degrading enzyme is eluted from aSephadex G-100 column in a single peak of a molecular weight of 25 000 to 30000.The remaining 5 % of the activity is eluted near breakthrough. The nature of thelatter is still unknown. (4) Cathepsin C, known as a dipeptidylpeptidase, hydrolysedTyr-Met-NA (but not Suc-Tyr-Met-NA) and could be inhibited by a cysteineprotease inhibitor, Ep-475 (Sawada, 1982). However, the K{ of cathepsin C forEp-475 is 10~4M, which is 1000-fold higher than those of cathepsins B, H and L.Since the Suc-Tyr-Met-NA-hydrolysing activity is completely blocked by Ep-475 ata concentration of 10~6M, the involvement of cathepsin C can be ruled out. (5) Otherexopeptidases may not be able to hydrolyse this substrate, because the N-terminaltyrosine and C-terminal methionine are blocked by both succinyl and naphthylamidegroups. Taken together, the results and Table 4 strongly suggest that Suc-Tyr-Met-NA is digested by cathepsin B and cathepsin L.

Knowledge of the presence of cathepsin B in phagocytes has been obtained by twotechniques: biochemical assay and indirect fluorescent staining. Biochemically, atime lag was observed before both the burst of cathepsin B and L activity and thedegradation of myofibrillar components. The activity of cathepsins B and L inperitoneal macrophages drastically increased and reached a plateau value within 6 hafter the injection (Ishiura et al. 19836). In this respect, it is interesting that thedegradation rate of muscle is proportional to the activity of lysosomal enzymes ininjected muscle, not to the activity in the peritoneal cavity. This can be interpreted asshowing that infiltrating macrophages play an important role in the breakdown ofstructural proteins in this myopathy.

Bupivacaine-induced muscle degradation 211

The mechanism of bupivacaine-induced degeneration of skeletal muscle is stillobscure (Deshpande et al. 1982). Histological studies suggested that the earlydegenerative change is mediated by calcium ions released from the sarcoplasmicreticulum (SR) by bupivacaine treatment (Hall-Craggs, 1980). Verapamil, a calciumantagonist, prevented the muscle degeneration (Benoit et al. 1980). These resultssuggest that bupivacaine may either inhibit uptake of calcium in the SR or promotethe release of calcium from it. Increased intracellular calcium may activate a cellularprotease (Ishiura, 1981). In contrast to our expectations, however, degeneration ofskeletal muscle was not completed by calcium-activated protease in vitro. Theprotease digested some cytoskeletal proteins and induced disorganization of myo-filaments by removing Z-lines. The enzyme has never been found to digest all of thestructural proteins in vitro. Therefore, bupivacaine-induced calcium release cannotexplain the overall degeneration processes. Our results apparently explain thisdiscrepancy, as we showed that macrophage invasion is necessary for completedegeneration of myofibrillar components.

The authors thank Professor Nobuhiko Katunuma and Dr Eiki Kominami (TokushimaUniversity) for generous use of anti-cathepsin B and the synthetic substrate. We are indebted toMisses Satomi Okada and Harumi Anraku for their excellent technical assistance. This work wassupported by a Grant-in-Aid for New Drug Development from the Ministry of Health andWelfare, Japan.

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(Received 31 December 1985 -Accepted 11 February 1986)