CURRENTRESEARCHIN

MUSCULARDYSTROPHY，JAPAN（BIOMEDICALRESEARCHES）
Vol．3

TheProceedingsofthe

AnnualMeetingofMuscular
DystrophyResearchGroup，
1981，Tokyo1

CONTENTS

75

3Structural Changes in Striated Muscles of Dystrophic Hamster
Cell Adhesion Molecule of Cleavage Stage Mouse Embryos-Functions in "Compaction"
Phage Particle-MediatedGene Transfer into Cultured Mammalian Cells
A Study of Dystrophic Muscle Maintained in vitro
Mitotic Activities
of Mononucleate Cells Cultured in vitro Originated from Regenerating

9

Muscles ofNormal (+/ +) and Dystrophic(dy/dy) Mice (II)Requirement of Transferrin for Myogenic Cell Growth in vitro
Effects of Innervation on Muscle Regeneration
Postnatal Alterationsin the Number of Muscle Fibers in Normal and Dystrophic Chickens
Arrested Growth of Muscle Fibers in Muscular Dystrophic Mice
Comparison of the Alteration Patterns of Several Biochemical Markers in Mice with Some
Neuromuscular Mutations
Effect of Brain Extract on Development of Tetrodotoxin-Sensitive
Sodium Channel in L6
Myotubes

Developmental Studies of the Membrane Properties
Properties of Ca Channels during Differentiationof Striated Muscle Fibers-Changes
of Excitable Cells

Sensitivity
to Ca2+, Sr2+, and Ba2+ Ions of the Contractile
Amphibian Muscles

System of Slow and Fast Fibers

11
13

15
17
19

21

in the23ofAntibodies
to Acetylcholine
Receptor in Ocular Myasthenia Gravis
Neuronal Factor Stabilizing
Acetylcholine
Receptors on Cultured Muscle Cells
Synthesis and Differentiationof Acetylcholine
Receptors in Cultured Skeletal Muscle Cells
Studies on Acetylcholine Receptor of Dystrophic Chick Muscle in Culture
Freeze-Fracture Analysis of the Caveolae in Chick Skeletal Muscles
Freeze-Etch Replica Study of the Cytoskeleton in Skeletal Muscle Fibers
Different Rate of Change in Isoforms of Myosin Heavy and Light Chains during Developmentof Skeletal Muscle
Numberof Antitroponin I Striations
along the Thin Filament of Adult and Fetal Skeletal Musclesof Rabbit
Isoform Variants of Myofibrillar
Proteins during Muscle Regeneration after Cold Injury
Myosin Isozymes in Embryonic and Adult Chicken Skeletal Muscle
Native High Molecular Weight Protein from Chicken Breast Muscle
A Protein in Chicken Breast Muscle Which Inhibits
the ADP-Ribosylationof EF2 by Diphtheria
Toxin in vitro
A Trophic Interaction in Denervated Muscles from the Standpoint of Recovery of Ca-Uptake
Abilityof FSR

Changes in Protease Activities during Development of Chicken Muscles
Endogenous Thiol Proteinase Inhibitor
and Its Changes of Activity in Skeletal Muscle of
Dystrophic Hamsters
The Effects of Low Molecular Enzyme Inhibitors
on Enzyme Networks in Various Organs ofDystrophic Mice
The Role of Protease in the Development and Differentiationof Muscles
The Effect of Protease Inhibitoron the Calcium-Induced Degeneration of Myofilament in vitro
and in vivo
iii

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27
29
31
33

35
37

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43
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49
51
53

55
57
59

61

肋∫C〟brかγJJr（汐力γGro岬，JタβJ

STRUCTURAL CHANGESIN STRIATED MUSCLES OF DYSTROPHIC HAMSTER
YoshiakiNonomura
DepartmentofPharmacology，FacultyofMedicine，UniversityofTokyo，Tokyol13

SincethefirstintroductionofastrainofmyopathichamsterbyHomburger

sgroupl】in1962，attention

hasbeengiventothemyopathyofthisanimal，eSpeCiallyitscardiomyopathy．Thisstrainhasanautosomal
recessivelyinheritedpolymyOPathy，buthasno otherdefbctiveorgansortissuesexceptfbrthesestriatedmuscles．
Thepresentworkaimedatcheckingthedegreeofdamageinvariousskeletalmusclesatdifferentages．
BIO15．6malehamsterswereusedfbrthestudies．Afterbeingsacrificedbyablowontheheadandbleed−
ing，the animalwasfixed on a board and the various muscles were carefu11y removedincluding bothterminaltendons．Themuscles werefixed h7Sifulength on dentalwax and4％glutaraldehydebuffered
Withcacodylatewaspouredonthematerials．Theywerethenteasedintosmallbundlesoffibersbyforceps
30minlater and transfbrred to a smal1bottle containing a glutaraldehydefixative．The usualmethods
Werethenfbllowedofasecondfixation ofosmiumtetroxide，Stainingenbloc，dehydrationwith alcoholand acetone，and embeddingin epoxy resin．Arelatively thick section with toluidine blue stainingwas
prepared fbrlightmicroscopy and a thinsectionwithlead stainingfbr electronmicroscopy．The cross
SeCtionviewfbrlightmicroscopywasmainlyusedtodeterminethegradeofmuscledamageandthiswas
done by the criteria according to Bajusz etal．2），SlightlymodiGed asfbllows：士，less than4cells with
Centralnuclei（CN）amonglOOcrosssectionedcells；＋，lessthan8（CN）cellsinlOO；＋＋，mOrethan
8（CN）cellsinlOO；＋＋＋，abundanceofCNcellsandotherabnormalities，＋＋＋＋，SeVereabnormalities
Withnecroticdamage．

AsshowninTablel，12striatedmusclesofdi飴rentagesweretestedwithfixedmaferials．Allhad
SOme degree ofabnormalities，particularly severe onesin theshoulder muscle．When thecentralnuclei
WereSeeninalongitudinalsection，theywerearrangedinalongrow．Inthissamesectionanabnormal
arrangementofnucleiwasrecognizedinalongrowalthoughtheywerenotlocalizedatthecenterbutat
theperiphery（Fig．1）．Othernucleicabnormalitiessuchas perinuclearcavitiesorporesweresometimeS
present．Myofibri1susuallylookedgenerallynormaleveninagrade＋＋十muscle，eXCeptfbrthenuclear

anomaly．When the musclewas observedin detailunder highmagnincation，both free polysomes andmembrane−boundribosomes were distributed around the nucleus and submembraneous region，WhichCOuldnotbeseeninanormaladultmuscle．Inagrade＋＋＋＋musclesomevacuolesanddisarrangement
OfmyofibrilsappearedhereandtheretogetherwithabundantCN．
TABLEl．TheDegreeofMuscleDamage
77daysArmmuscles M．extensordigitrumcommunis
M．extensorcarpiradialisbrevis
Shouldermuscles M．omotransversarius
M．omobrachialis
Backmuscle M．lumbodorsalaponeurosis
Abdominalmuscle M．externaladbominaloblique
Esophagus M．esophagus
Diaphragma M．diaphragma
Legmuscles M．samitendinosus
M．soleus
M．extensordigitrumlongus
M．Sartorium＊AppearanCeOfnecroticcell．

97days llOdays
±

＋＋＋＊

＋＊

＋＋＋
＋＋
＋＋
一十＋＋
＋＋

＋＋＋＋＊

＋
＋
＋＋＊
＋

＋＋＋＋＊

130daysFIG．1．Electronmicrographoflongitudinalsectionoflegmuscleindystrophichamster．Nucleiarearrangedina
longrowattheperipheralreglOn．Nucleoliareclearlyobserved．

Theappearanceofdegenerativecellsmarked

byasterisk（＊）inTablel

seemedtobeinduced

byat

leastthreedi飴rentmechanisms．Thefirsttypewasaso−Calledfattydegeneration，butthisappearancewas
veryrareindystrophichamsterandonlyonecasewasfoundinthesartoriusmuscle．Thesecondtypewas
themostcorrunon，aCCOmpaniedbyvacuoledegenerationanddisarrangementofmyofibrils．Typicallyseen
inthelongitudinalsectionwasapanoramicviewwhichhadgonefromrelativelyslightdamagewithsmall
VaCuOles to severe damagewith disarrangement ofmyofibrils and the disappearance ofmyo丘Iaments．

ThethirdtyperarelyappearedbutitsimagewasverylmpreSSive；thistypewasmacroscoplCa11yrecognizedasbeinglikerefinedwhitewax．Themuscleswerereplacedwithnbroblastsorinterstitialce11sinacertain
areaandsomemusclecellsremainedlyingscatteredamongtheconcentratedfibroblasts．Whilesomemus−
Cleswerenormalexceptfbrtheircentralnuclei，Othersweredegeneratedwithvacuoles．Thesestructural
Changeswerenotprogressive，butsuchchangeswerefrequentlyobservedinthetissuefrom70to130days
afterbirth．
Inconclusion，allthestriatedmusclesofadystrophichamSterincludingtheesophagealmuscleshow
OVerallstruCturalabnormality，eSpeCially thecentralnuclei，desplte theapparent normalarrangement of
myofibrils．Furthermore，theprocessofoccurrenceofdegenerativecellsisverylikethegenesisofhumandystrophy．Anexperimentalapproachusingdystrophichamsterswouldmakegoodprogressinourstudies
onthis disease．

1）Homburger，F．，Baker，J．R．，Nixon，C．W．，andWhitney，R．：Med．Erp．，6，339−345，1962．
2）Bajusz，E．，Homburger，F．，Baker，J．，andOtie，LH．：Ann．N．Y．A（・ad．Sci．，138，213−231，1966．
2

．・lJ〃領・J′／ 汀D．l・∫J…J小・GJ・（川β．ノ9ぶJ

CELL ADHESION MOLECULE OF CLEAVAGE STAGE MOUSE EM【BRYOS−FUNCTIONSINCONIPACTIONM

T．S．Okada，Soh−ichiOgou，ChikakoYoshida−Noro，andMasatoshiTakeichi
DepartmentofBiophysics，FacultyofScience，UniversltyOfKyoto，Kyoto606

Compaction

isoneoftheverylmpOrtanteVentSinthepreimplantationstageofmammalianembryonicdevelopment．Inmouseembryosatthe8−Ce11stage，COntaCtSurfacesbetweeneachcell（blastomere）become
verywide，thereisamodificationofthece11shape，andthusanembryobecomesacompactmassl）．Itis
believedthatinteractiverelationshipsbetweenindividualembryoniccellsstarttobeestablishedonlyafter
thisstageof

compaction．

Inthisreport，WeShowthatacellsurfaceproteinofm．W．of140K（t−CDS）

functionsincompactionbymeansofimmunologlCaltechniques．
（、tイJ Jf〟Jで∫わJJJ川）んでJJ／（−∫
Cell−Cell contactin vertebrate cellsis established by the presence of hypotheticalmechanisms

dualadhesion2）．Wehavepreviouslyshownthateachmechanismisoperatedbythepresenceoftwodistinct

Cellsurfacesites．Oneofthem，CDS，afunctionofwhichisdependentonCa2＋，isparticularlyimportantfor

cellularrecognitionbetweendifferentcelltypesaswellasholdingadjoinlngCellsinstablecontact3）．CDS
wasclearlyidentifiedasaproteinofm．W．of150Kontheelectrophoresisofatotallysateofisotopicallylabelledcellsurfacecomponentsofavarietyofdi能rentiatedcellsaswellasofseveralestablishedcelllinesJ〃再汀（）、
Cellsofaparticularlineofmouseteratocarcinoma，F9，Whicharecharacterizedbytheirinabilityin
celldifferentiation，COntainanunusuallyhighcontentofCDS ontheircellsurfaces4）．However，them0−
1ecularweightofF9ce11S（t−CDS）isnot150Kask〉undinothercelltypes，but140K．Then，CDSinF9cells（andotherteratocarcinomacellsalso）isnotthesamemoleculeastheCDSpresentindif托rentiatedcells，thoughbothsharetheidenticalfunction5）．
5pec析Cαが路0め′（好α如才ceJJ−仇幼e∫わ〃mOJec〟Je
Anantibodyspecifictot−CDSwasraisedbyinjectingF9cellsintoarabbitandbyproperabsorption
toremovenon−SPeCificantibodies．Thisantibodyhasbeensuccessfullyutilizedasadiagnosticreagentto．三三二◆千二：て・宣．二．・二．・、．＿ト・一二千、二二二千二∵、、妻斗二・二三■しぎ■ごこ・さFIG．1．A：8−CellstagemouseembryosprlOrtO
compaction

COmpaCtion．

B：Embryosatthestageof

compaction：

ofoncecompactedembryosinducedbythetreatmentwiththeFabfragmentofanti−F9．
3

C：

De−

seewhethert−CDSispresentin earlymouseembryos befbreimplantation．This wascarried out byex−aminingwhethertheFabfragmentofanti−t−CDScanspecifiCallyinhibittheaggregationofembryosplaced
under aconstant gyration．Theresults clearly demonstrated thatmouseembryos ranglngfrom one−Cell

to8−Cellstagesareprovidedwitht−CDS，butnotwithCDSofm．W．of150K．Thus，WeCanCOnCludethat
Cleavage stage mouse embryos share a common celladhesion molecule（t−CDS）with teratocarcinoma
cellsbutnotwithdifftrentiatedcells6）．
カec（フナ叩αCJわ〃 qremふけ0∫わ′α〝J山一CDぶ

Whenmouseembryosatthestage ofcompaction（thelate8−Cellstage）Weretreated with theFab
fragmentofrabbitantibodiesagainstF9cells，theshapeofeachcellsoonbecameroundand thus thephenomenoncalled

decompaction

ofembryoswasinduced．SuchefftctoftheFabofnon−absorbedanti−

F9wascompletelyremovedbyabsorbingitwithF9cells，butnotwithseveraltypesofdifrtrentiatedmouse
cellsorwithseveral加vitrolinedce11SJtisknownthatthetreatmentofF9cellsbyproteaseandEDTA
underspecifiedconditionsremovesonlyt−CDS，1eavlngmOStSurfaceproteinsintact4）．Anabsorptionofthe
Fabofanti−F9withsuchtreatedF9cellsalsoabolisheditsefrtcttoinduce
becomesclearthat

decompaction

moleculefunctionsin

compaction

Itremainstobeinvestigatedwhy

decompaction．

isduetoaspecificeffectoftheantibody

Therefbre，lt

againstt−Cl）Sandthelatter

ofembryos．
compaction

occursatthe8−Cellstage，lnSpiteofthecontinuingpresenceoft−CDSsincetheone−Cellstage．Perhapsanaccumulationoft−CDSatthisparticularstagebe−
comeshighenoughtocause

compaction

ofembryos．

1）McLaren，A．：EarlyEventsinMammalianEmbryogenesis・hMechanismsofCellChange（Ebert，J・D・andOkada，
T．S．，eds．），pp．49−56，JohnWiley＆Sons，1979．
2）Takeiehi，M．：J．CelLBEol．，75，464474，1977．
3）Takeichi，M．，Ozaki，H．S．，Tokunaga，K・，andOkada，T・S・：Dev・Biol・，70，195−205，1979・
4）Takeichi，M．，Atsumi，T．，Yoshida，C．，Uno，K・，andOkada，T・S・：Dev・Biol．，87，340−350，1981．
5）Yoshida，C．andTakeichi，M．：Cbll，28，217−224．1982・
6）Ogou，S．−I．，Okada，T．S．，andTakeichi，M・：Dey・BioL・，inpress・4肋∫C〟br功JJr（やわ′Gr（）〟p，J夕βJ

PHAGE PARTICLE−MEDIATED GENE TRANSFERINTO CULTURED MAIJAN CELLS
YoshioOkadaandM．Ishiura＊

ResearchInstituteforMicrobialDiseases，OsakaUniversity，Osaka565＊NationalInstituteforBasicBiology，Okazaki444

Gene−tranSftrintoculturedcellsincludingcellsffomhumanhereditarydiseaseswillbecomeanessential
procedureforcellbiology．However，SeVeralimprovementsshouldbemadebecausethee瓜ciencyoftrans−
fbrmationbygenomicDNAhasnotbeenhighenoughtoovercomethespontaneousbackmutationofthemutant cells，the transfbrmants obtained by DNA transftrhave been reported to be unstable，and theDNAsequencesintroducedexogenouslysufferfromrearrangement．
Metaphasechromosomeshavealso been usedforthetransfbrofsingle−COpygeneSineukaryocytes
andhaveatleasttwoadvantagesasadonor，aSCOmparedtogenomicDNA：theefnciencyofgenetransftr
isfoundtobealwayslOtolOOtimeshigherforchromosomesthanforgenomicDNA，and thetrans−
fbrmantsobtainedfromchromosometransferexperimentsaremorestablethanthosefromgenomicDNAtransftr．Theseadvantages，Wethink，arepartial1yderivedfromtheorganizationofchromosomestructure
Where DNA sequences are covered with nuclear proteins，paCkaged compactinto chromatin structure，
andprotectedfromDNaseattacks．In spiteoftheseadvantages，however，metaphasechromosomesare
notachoiceasdonormaterialsfbrtheisolationoftheselectablemarkergenebecauseDNAsequencesin
Chromosomescannotbemanipulatedbefbreapplicationtotherecipientce11S．

Asadonorofgeneticmaterials，Wehaveselectedphageparticlesinwhichanexogenousgeneisen−
CapSulatedfbrthefollowlngreaSOnS：theyhaveastructuralorganizationanalogoustothatofchromosomes．

DNAsequencesarecoveredwithphagecoatproteinspackagedcompactintophageparticlesandprotected．
Fromthisstructuralanalogy，WeeXpeCtahighefnciencyofgenetransftrandgoodstabilityoftheresult−anttransfbrmantswhenphageparticlesareappliedtorecipientcells．Themethodfbrtheconstructionof
agenelibraryofmammalianDNAinlambdaphagevectorshasbeenalreadyestablished，andsequences
forgenomicDNAandlambdavectorscanbeeasilymanlpulatedbeforepackagingintophageparticles．
Inthisworkphageparticlescoprecipitatedwithcalciumphosphatewereappliedtotherecipientcells
forgenetransftrinplaceofeitherDNAorchromosomes．UsingCharon4Arecombiantphagecarrying
HSV−1tkgeneandLtk−cells，Wehaveestablishedtheconditionsofphage一mediatedgenetransfbr，attained

ahighefBciencyofgenetransftr，andalSohavefoundthattheresultanttransformantswereapparently
Stable．Furthermore，tO Our SurprlSe，theexogenouslyintroducedDNA sequencesin the transfbrmants
havebeenstablyconservedwithonlyslightrearrangement．
」占∫Jr〟CJげ班ere∫〝血
Recombinant phageparticlescarrylng the thimidine kinasegene ofherpes simplex typel andc0−
precipitatedwith

calcium

phosphate

efBciently

transfbrm

mouse

Ltk￣cells

tothetk＋phenotype．The

COnditions necessary to achievehighefnciency transfer ofthetkgene byphageparticle−mediated genetransftrwereinvestigated．Oftheparameters examined，pH ofabufferusedfbrthecoprecipitation ofphageparticleswithcalciumphosphate，thelengthoftimefbrthecoprecipitation，andthelengthofad−
SOrPtiontimewerefbundtosignificantlyalterthetransftre侃ciency．Theoptimalvaluesfortheseparame−
terswereasfollows：thebufftrofpH6．87at25OC；thecoprecipitationtimeof7to20min；theadsorp−
tion time of18to30hr．Treatmentwith dimethylsulfoxide，glycerol，OrSuCrOSedidnotenhancegene
transfbr．TheoptimalconditionsyieldedaboutonetransfbrmantPerlO5phageparticlesperlO6cellswith−
OutCarrierDNA．Increaslngthedosageofphageparticles，atleastupto5×107perlOOmmdish，reSulted

inalinearincreaseinthenumberoftransfbrmants．AdditionofcarrierphageuptolOlOphageparticles
5perlOOmmdish，SCarCelyaffectedthenumberoftransfbrmants・Thetransfbrmantswereapparentlystable
and the exogenouslyintroduced DNA sequencesin them were stably conservedwith only slight rear−
rangement．

Ishiura，M．，Hirose，S．，Uchida，T．，andOkada，Y．：Mbl．Cell．Biol．，inpress．

6肋∫ピ〟ねr功∫／rq〆γGr（フ〟p，J夕βJ

A STUDY OF DYSTROPHIC MUSCLE MAINTAINED ZN VtTRO
TakeshiYonezawa
DepartmentofPathology，KyotoPreftcturalUniversltyOfMedicine，Kyoto606
0urpreviOusworkonthealterationsofdystrophicmusclemaintained血Vitro（CalifbrniaDavisLine413）disclosedcharacteristicdegenerativechanges，SuChasabnormalfusingprocessesofmyoblasts，VaCu−
01ation ofsarcoplasm，fenestrationofsarcolemma，dilatation oftransversetubules，hypercontractionof
myofibrils，andsegmentalnecrosisofsarcoplasm・Hypercontractionofmyofibrilsandsegmentalnecrosis
werecloselyrelatedtothedilatedTsystem，WhichseemedtobecontinuoustothesarcolemmalopenlngS・ToclarifythecausativerelationshipsofthesealterationsandtheaccumulationofexternalfluidintheT
system，HRPtechniqueswereappliedtoculturesofdystrophicmuscle・UltrastruCturalstudieswerealso
performed・Fragmentsofsuperficialbreastmusclefromdystrophicchickandcrosssectionsofspinalcord

werecultivatedonco11agen−COatedcoverslipsandmaintainedwiththedoublecoverslipmethod．Cultures
wereobservedundertheordinarylightmicroscopeandNomarskyoptlCSwithhighmagnification・When
needed，Cultureswereimmersedinabalancedsaltsolution（BSS）containingHRP（2・5mg／ml）andmain−tainedforonehour．AfterrinsingwithBSS，Cultureswere丘xedwith2．5％glutaraldehydefor30minand
washedovernight，andthenstainedwithDABinthepresenceof0．01％ofH202．
Fenestrations ofmusclesurfaceexhibitedwithlightmicroscopicobservationswerefbundto corre−

spondtothewideningoforificesofthetransversetubularsystem・Inaddition，endocytoticvesiclesin−creasedinnumber，andweremainlylocatedunderneaththesarcolemma・Similarvesicularstructureswere
alsoseeninthedeepersarcoplasm．HRPreactionseemedtobecloselyrelatedtothesestructures，SuggeSt−
ingaccumulationofexternalnuidinsidethemusclefibers・Furthermore，withlightmiCroscopicobservation
itwasfbundthatHRPpositivereglOnSWereOftenassociatedwitheitherhypercontractionofmyofibrils
orsegmentalnecrosisofsarcoplasm．
NerveinnervationofmusclefibersmaintainedklVitromaypromotebettermaturationandmainte−
nancethanwouldotherwiseoccur．Developmentofdystrophicchangesinmuscleculturemaybeenhanced
intheinnervatedmusclefibers，SuggeStingthealterationsweremorefrequentlyproducedinmuscles matu−

rated both biochemically and structurally．Among thealterations described above，VaCuOlation ofthesarcoplasmseemedtobenonspecifiC，Whileothertypesofalterationwerecharacteristicforthisdisorder・Wideningand dilatationofthetubularsystemarecloselyrelatedtothenecroticfiberswhichexhibited
HRPdeposition．Furthermore，damageofthetubularsystemseemedtobeaninitialchangeleadingtothe
productionofnecroticinclusionandhypercontractionaswell・Thoughthesenecroticprocessesdifferedfrom the disturbanceoffusingprocesses ofmyotubes，theremaybeapossibilityofasinglecausative
factor，thatis，membraneabnormalitywhichisgenetical1yinherited・Iftrue，itisquitepossiblethatsome
othertypeofcellmaydevelopasimilarmembraneanomalyonageneticbasis・IntheDuchennetypeofmusculardystrophyithasbeensuggestedthatCa2＋depositioninthetubular

systemactivatesthecalciumdependentproteinase，reSultinginsegmentalnecrosisofmusclefibersand
productionofopaquefibers・Thesechangesinpatientsareidenticaltothoseobservedinchickdystrophic
muscles正目正価仇7肋∫C〟ねr勒∫／rqpわ，Gro岬，JpgJ

MITOTIC ACTIVITIES OF MONONUCLEATE CELLS CULTURED ZN VtTRO ORIGINATED
FROMREGENERATINGMUSCLESOFNORMAL（＋／＋）ANDDYSTROPHIC（dy／dy）MICE（II）
TsutomuKagawa
LaboratoryofCellBiology，ToneyamaNationalHospital，Toyonaka560

Wehavereportedthat duringthesamecultureperiodthenumberofcelldivisions（NCDs）ofdys−
trophic myogenic cells（dy／dy）was signifiCantly smaller than that ofnormal（＋／＋）onesl）．To clarify
WhetherthedifferenceintheNCDswasduetotheprolongationofgenerationtimeofdystrophicmyogenic
Cellsortotheirearlierdeprivationofmitoticactivities，WeCarriedoutclonalculturesofeithernormalor
dystrophicmyogeniccellsandobtainedtheirgrowthcurves．
Adult normal（＋／＋）and dystrophic（dy／dy）mice were used throughout．The procedures fbr the
preparationofregeneratingmuSCles2，3）andtheliberationofmononucleatecellsfromeithersource4）were
describedpreviouslybyKagawaetal．Cultureswereestablishedbyplating500cellsfromeithersourcein
5mlofthefreshrnedium2）onto a60−mm Falcon plastic petridishcoatedwithO．2％gelatin5）．Tncu−

bationwascontinuedat370Cinahumidifiedatmospheregasedwith5％CO2and95％air．Eachculture
WaSftdwith5mlofthe

growthmedium2〉onedayafterplating．Threeseriesof．culturesofthesametype（cultures1，2，and3）werecarriedout．EachofthemconsistedoflOnormalandlOdystrophiccul−
tures．In each series，30−45colonies ofmyogenic ce11s from either origin were randomiy selected andmarkedwithnumbers2daysafterplating．Thecellnumberofeachwasenumeratedeverydaythereafter
andgrowthcurveswereobtained．Colonieswhichlostthepotencyofce11growthwereexcludedand，in
COnSequenCe，thenumberofcoloniesusedforthecountingofNCDsdecreasedwiththeadvancement of
thedaysofcultures．
Theaveragenumberofcelldivisionsoccurringduringthecultureperiodofdystrophicmyogeniccells（7days）wassmallerthanthatofnormalinallcultures and the differences weresignificantinculturesland2（Tablel）．AsshowninFig．1，thedifferencesintheNCDsbetweennormalanddystrophicmyogenic
Cellswhich occurred during thefirst two culture days were also significantin culturesl and2．To
excludethepossibilitythatthedifferenceintheNCDswasduetotheincreaseofcellnumberbythefusion
Oftwoormorecoloniesinnormalcultures，NCDs occurringafter2through7daysofincubationwere
TABLEl．AverageNumberofCellDivisions（0−7days）
Culturel Culture2

Culture3

6．21土2．08

5．24士2．18（43）

5．02士2．04（31）

Dystrophy

3．80土1．73

2．37士1．11

4．25士1．86

トStat．（P＜）

（29）
0．01

（16）
0．01

Nomal

（23）
（26）

NS

1−Stat・：1−Statistics．（）：numberofcoloniesusedforthecountofnumberofcelldivisions．NS：nOtSignlficant．
TABLE2．AverageNumberofCellDivisions（2−7days）
Culturel Culture2
NormalCulture3

3．06土1．89

2．99±1．86

（43）

（31）

（23）

Dystrophy

1．45土1．47

1．32土0．92

2．51士1．50

（29）
0．01

（16）
0．01

（26）

トStat．（P＜）

2．93士1．63

NS

t−Stat・：1−Statistics．（）：numberofcoloniesusedforthecountofnumberofcelldivisions．NS：nOtSigni丘cant．
9

Culturel

Culture3

Culture2

エ言OJu ござ盲○ S Sだu〇一〇こOL36コN訳

SUO叫∽て竃 qU hO Jむ一∈nN
3

4

5

6

FIG．1．Growthcurvesandthecurvesshowingthenumberofcolonieswithmitoticactivity．−aVeragenumber
ofcelldivisions（0−7days）．，一一aVeragenumberofcelldivisions（2−7days）．一一−％numberofcoloniesshowing

cellgrowth．Thet−Statisticsfortwomeanswasusedtocomparethenumberofcelldivisionsofnormalmyogenic
cellsandthatofdystrophicones：＊＊＊P＜0．01，■＊P＜0．05，＊NS．

alsoobtained．Theywerealsosmallerindystrophicculturesthaninnormalonesinallseriesofculturesandthedifftrencesweresignificantinculturesland2（Table2）．Thesefindingsagreedwiththeresultswepre−
Viouslyreportedl）．
Thegradeofeachgrowthcurve（Fig．1）showsthevelocityofcelldivisions．Thegradesofthegrowth
curvesofdystrophicmyogeniccellswereapproximatelyparalleltothoseofnormalinallcultures（Fig・1）
indicatingthatthegenerationtime，WhichisthereclprOCalofthevelocityofcelldivision，Ofdystrophic
myogeniccellswasapproximatelythesameasthatofnormalcells．
Coloniesshowingcellgrowthdecreasedinnumbermuchmorerapidlyindystrophicculturesthaninnormalonesinculturesland2，inbothofthesethenumberofcelldivisionsofdystrophicmyogeniccells
wassignifiCantlysmallerthanthatofnormalmyogeniccells（Fig．1）．
Thepresentstudysuggestedthat thedecreasein the numberofcelldivisions foundindystrophic
cultureswas not dueto theprolongationofgenerationtimeofdystrophicmyogeniccells but to theirearlierdeprivationofmitoticactivity．
1）Kagawa，T．，Chikata，E．，andHamano，R．：CurrentResearchinMuscularDystrophy，Japan，2，11，1980・
2）Kagawa，T．，Chikata，EりandTani，J．：Dev．BioL55，402，1977．

3）Kagawa，T．，Chikata，E．，Tani，J．，andTsutamune，T．：Dev．Biol．65，526，1978・
4）Kagawa，T．，Chikata，ETani，J．，andTsutamune，T．：30thAnnu．MeetingofJapanSoc・OfCellBioLAbst・（A−28），

1977．

5）Richler，C．andYa能，D．：Dev．BioL．，23，1，1970・

10肋∫C〝ねr勒∫什qp々γGrp〟p，J夕βJREQUIREMENT OF TRANSFERRIN FOR MYOGENIC CELL GROWTH m T7ZTt10
EijiroOzawa，IchiroKimura，TakayukiHasegawa，IchioIi，YasukoHagiwara，andKqjiSaito
DivisionofCellBiology，NationalCenterfbrNervOuS，MentalandMuscularDisorders，Kodaira187
Chick myogenic cells do not growin a basalculture medium（BCM）composed of85％Eagle

S

minimumessentialmediumand15％horseserumbutgrowwellwhenasmallamountofmuscletrophic
factor（MTF）is addedtothemediuml）．MTFis aproteinprepared丘omchickadultserum21whichis
Salmonpinkincolorandhasamolecularweightof80Kdalton．
InordertodeterminethenatureofMTF，itwascomparedwithchickserumtransftrrin（STf）preparedfromchickserum，andovotransftrrin（OTf）ffomchickeggwhite3・4）．
ThemolecularweightofsTfwasals080KdaltonandthatofoTfwasalittlesmallerwhendetemined
bySDSacrylamidegelelectrophoresis．ProteolyticdigestsofMTF，STf，andoTfbyα−Chymotrypslnand
papainshowedindistinguishablepatterns．

Asingleandidenticalprecipitinlinewasfbrmedinadoubleimmunodiffusionprecipitationtestwhenanti−STfandoTflrabbitserareactedinagargelwithMTF，STfioTfiorchickserum．
Thesethreeproteins boundftrricion．Absorptionat465nm，Whichischaracteristicofiron−boundtransftrrin，increasedastheamountofftrricionboundtotheapo−tyPeOfMTF，STf，andoTfincreased．
Thesetworeachedaplateauwhenthemolarratioof80Kdaltonproteinagainstironwasapproximately
l：2．Thisstoichiometricratioisalsocharacteristicoftransferrin，indicatingthatthemainproteinsbelong
tothetransftrrinfami1y．
Iron−free proteins showed no myotrophicactivity．Myotrophicactivityappearedwhen theprotein
boundiron．Itincreasedalmostproportionallywiththeamountoftheboundiron，andreachedaplateauat

05︵X︶︵Lモ S叫P＼E SqlOEヱ﹂言三一Ud鍼0︵○︶？OtXEU＼苫＼空くMoIar ratio of Fe3＋added：Tf

FIG・1・RelationofmolarratioofFe3＋vs・TftomolarratioofboundFe3＋vs・Tf，Opticalabsorptionat465miLm，andcreatinekinase（CK）activityasanindexofmyotrophicactivity．
11thesamemolarratio（Fig．1）．Complexesoftransftrrinwithmetalsotherthanironsofarexaminedwere
withoutactivlty；therefore，theactivltyCanbesolelyascribedtoiron−boundtransftrrin．Allofthesedata
alsoindicatethatMTFisidenticaltotransftrrin．
Ferricorftrrousion，nOtboundtotransferrin，WaSalsoefrtctiveinpromotingmyogeniccellgrowth，althoughmorethan200timestheamountofironasacomplexwithtransftrrinwasrequired．Othermetals，
SuChasMn，Cr，Cu，Co，Ni，Cd，andZn，WerenOteffective．
We have also prepared transftrrin from chick embryo extract（EE）．The preparation method was
similar to that usedfor MTF preparation，With somemodifications which willbe detai1edelsewhere5）．
Thistransftrrin（EETf）andsTfwereindistinguishablefromeachotherphysicochemically，immunologi−
Callyandinmyotrophicactivitysofarexamined，eXCeptthatEETfwasmainlycomposedoftransftrrin
specieswithOorlsialicacidresidue，WhereassTfhadmainly20rlsialicacidresidues．Myotrophicactivity
didnotdependonthenumberofsialicacidresidues．
When transftrrinwas removed from EE byimmunoprecipitation oriron bound to transftrrinwas
removedfromEEbydialysisatpH4．5，myOtrOPhicactivitywaslostbutwasrestoredbytheadditionof
transftrrin oriron to both kinds ofinactivated EE．Thisindicates that transfbrrinis a verylmpOrtantcomponent ofEEformyogenesis．Further，OneOfthemostimportantroleofEEistoglVeirontothe
Cells．

HorsetransftrrinpresentinBCM didnotshowmyotrophicactivityonchick cells．Thiscanbeex−
plainedonthebasisoftheclass−SPeCificnatureoftransftrrin；mammaliantransftrrin，eXCePtbovinetrans−

ftmin，WaSnOteffectivebutaviantransftrrinwaseffectiveinpromotingchickmyogeniccellgrowth・Chick transferrin，irrespectiveofthesources ormethods ofpreparation，PrOmOtedchickmyogenic
cellgrowthsofarasitboundferricion．Itisdeducedthatironisabsolutelyrequiredeitherfbrmultiplica−

tionofmyoblastorgrowthofmyotubes．TransftrrinservesasanefBcientiron−donnertocells．Withoutit，
grownmyotubesdegenerateandaredestroyed6）．
Someoftheresultsonthissubjecthavebeenpreviouslypublished7●10）

1）Hagiwara，Y．，Kimura，I．，andOzawa，E．：Dev．GrowthD脾r．23，249，1981．
2）Ozawa，E．andKohama，K．：Proc．J（PanAcad．，49，852，1973．
3）Leibman，A．J．andEisen，P．：Arch．Biochem．Biqphys．，121，717，1967．
4）Kimura，I．，Hasegawa，TりMiura，T．，andOzawa，E．：A■OC．JqpanAcad．，57B，200，1981．

5）Ii，I．，Kimura．I．，andOzawa．E．：Submitted．
6）Ozawa，E．andHagiwara，Y．：Biomed．Res・，3，16，1982・
7）Hasegawa，TりSaito，K．，Kimura，I．，andOzawa，E．：Proc．JqpanAcad．，57B，206，1981．

8）Ii，I．，Kimura，LHasegawa．T．，aIldOzawa，E．：Proc．JLPanAcad．，57B，211，1981・
9）Ozawa，E．andHagiwara，Y．：Proc．JEPanAcad．，57B，406，1981．

10）Hagiwara，Y．andOzawa，E．：Dev．GrowthD脾r．，24，115，1982．

12

〟〟∫C〟ねr上）γ∫什qpわ，Gro岬，ノタβノ

EFFECTS OFINNERVATION ON MUSCLE REGENERATION
ToshioTerao，KazukoMashino，andNakaakiOhsawa＊

TheFirstDepartmentofInternalMedicine，TeikyoUniversitySchoolofMedicine，Tokyo173＊TheThirdDepartmentofInternalMedicine，FacultyofMedicine，UniversltyOfTokyo，Tokyol13

In transplanted muscles myotubes are formed by satellite cells followed by striate formation，and
Subsequentlydevelopintonewmaturemusclefibers．Am如Ordifferenceinthemusclestructurebeforeandaftertransplantationisthatthediameteroftheregeneratedmusclefiberissmaller．
Majorfactorscontributingtotheincreaseinthediameteroftheseregeneratedmusclesaretherecovery
Ofbloodsupplyandmuscletensionaswellasthereinnervationtothemuscles，Whichincludestheregenera−tionofnervesfromthehostbodyandtheirconnectiontotheregeneratedmuscle．
The present studies were undertakenin order to elucidate theinfluence ofreinnervation on the re−generationoftransplantedmuscles．
Undernembutalanesthesia，OrthotoplCalautotransplantationofbothsidesoftheextensordigitorum
longusmusclewasperformedinrats．Ishiadicnerveofonesidewasdenervatedandtheintactsideserved
as acontrol．

Thetransplantedmuscleswereextlrpatedsuccessivelyafterthetransplantationandwereweighedand
SeCtionedwithacryostatt．ThehistologicaldifferenceswereanalyzedbetweenthecontrolanddenervatedmusclesbothbyordinarystainlngSSuChasHematoxylin−EosinandTrichrome，andhistochemicalstainings
SuChasATPase，DPNH，andSDH．
Fourdaystooneweekfollowingthemuscletransplantations，numbersoffibroblasts，myOblasts，andmacrophagesappearedintheperipheralreglOnSOfthetransplantedmusclesfollowedbythedevelopment

FIG・1・Regeneratedmusclesthreeweeksaftertransplantation・C：COntrOIside，d：denervatedside．
13

FIG．2．Histologyoftheregeneratedmusclesthreeweeksaftertransplantation・ATPasestaining・left：denervated
Side，right：COntrOIside．

ofnewregeneratedmuscles．Aftertwoweeks，theregeneratedmusclesgrewtooccupythewholeareaof
thegrafts．Ananalysisofthesizeoftheregeneratedmusclefibersrevealedlittledifferencebetweenthe
denervatedandtheintactmuscles，althoughthediametersofdenervatedmusclefiberstendedtobesmaller
thanthose ofthecontrol．

Thereafter，thedi鮎rencesbecameclearer．FigurelshowsregeneratedmusclesofthedenervatedandthecontroIsidesthreeweeksaftertransplantation．Asshown，thesizeoftheregeneratedmusclefromthe
denervatedsideisapparentlysmallerthanthatofthecentral．Thedifferenceincreasedgraduallyandsuc−cessively．Atfourweeks aftertransplantationtheaverageweights oftheregeneratedmuscles from thecontroIsidewereapproximately50mg，Whereasthose ofthe denervatedmuscles were usuallylessthan
halfthatofcontrol．
Figure2showsthehistologyoftheregeneratedmuscles ofthedenervatedandcontroIsidesthree
weeksfollowlngtranSplantation．Herethedenervatedmusclerevealspoorerregenerationwithplentyof
connectivetissues，Whichleadtothefibrosisfrequentlyobservedinthe centralportion・Thenumberof
regeneratedmusclefibersdecreasedinthedenervatedside・Further，thecontroIswereapparentlyuniform
insize，Whereasthedenervatedmuscleswerevariableandtendedtobesmaller・
Theaboveresultsindicate

that

theformation

ofthemyoblastandthemyotubeduringmusclere一

generationwasnotinnuencedbytheinnervation・However，thedevelopmentoftheregeneratedmuscles
wasprogressivelyimpairedwithtime．Furtherstudiesareneededtoanalyzethesephenomena・
1）Gutmann，E．：Thedenervatedmuscle，p．121，PublishingHouseoftheCzechoslovakAcademyofScience，Prague，
1962．

2）Saunders，J．H．andSissons，A．：J．BoneJt．SLLrg．，35B，113，1953・
3）Denny−Brown，D：J．Neurppathol．，10，94，1951・

14

施JJC〃血r勒∫什qpわ′Gr（，岬，J987

POSTNATAL ALTERATIONSIN THE NUMBER OF MUSCLE FIBERSIN NORMAL AND
DYSTROPHIC CHICKENS
TetsujiHironaka，YoshioIkari，YuheiMiyata＊，andMasanoriOtsuka＋

DepartmentofPharmacology，TeikyoUniversitySchoolofMedicine，Tokyo173＊DepartmentofPharmacology，FacultyofMedicine，TokyoMedicalandDentalUniversity，Tokyo113

Previous results obtained from cross−tranSplantation experiments on extensor carpiradialislongus（ECRL）muSCles suggested that someextramuscularfactor（S）playsanimportant roleinmusclegrowth
innormalanddystrophicchickensll．Ontheotherhand，abnormaldischargesoriginatingftomthecentral
nervoussystemhavebeendemonstratedinthemusclesofthedystrophicchicken2）．
ThenormaldevelopmentofskeletalmuscleinvoIvesaseriesofinteractionsbetweenmotorneurons
and musclefibers．Forinstance，muSClefibers areinnervated polyneurona11yin neonatal animals andSOme Ofthesesynapticinputsareeliminated duringsubsequentdevelopmentresultingln a Slngleinner−
Vationby2−3Weeksofage3，7）．Therearealsoseveralreportsofanincreaseinthenumberofmusclefibers
arterbirth6・8￣lUItis，therefbre，interestingtoexaminewhetherornotthereexistanydisordersinmusclegrowthandmaturationinthedystrophicchicken．Inthepresentstudy，Wehaveexploredthepostnatalchangesinthe
numberofmusclefibersinnormalanddystrophicchickens．Itwasfbundthatthepostnatalmusclepr0−
1iftrationwasgreatlysuppressedinthedystrophicchicken．

Normal（1ine412）ordystrophic（line413）ChickenswereanesthetizedwithetherandtheECRLmuscle
WaSeXpOSed．Theexcisedmusclewasfixedinaphosphate−bufrtredfbrmalinsolution，andtransversesec−
tionsweretakenffomthewidestportionofthemusclebelly．Thetotalnumberofmusclefiberswascounted
OnapreParationstainedwithhaematoxylinandeosin．
TheresultsaresummarizedinTablel．TheECRLmusclesinnormalanddystrophicchickensat2−3
daysexovocontainedaboutthesamenumberofmusclefibers．From2−3to12−14dayspostpartumthere
WaSa77％increaseinthenumberofmusclefibersinthenormalchicken，Whereasonlya15％increase
WaSObservedinthedystrophicchicken．Thenumberofmusclefibersthengraduallydecreasedinboththe
dystrophicandthenormalmuscles，reaChingasteady−Statein about60days；however，thedecreasewas
muchmorepronouncedinthedystrophicchickenthanin thenormalchicken．Akineticanalysis ofthe
decreasingphaseusingthe

steady−Statetheoryofmutationrates12）gavetherateconstantsofO．128／day

andO．134／dayinnormalanddystrophicchickens，reSpeCtively，SuggeStingthatthereisnoessentialdifl
ftrenceintherateofdegradationbetweenthetwo．
The present studyshowed that a postnatalincreaseand a decreasein thenumberofmusclefibers
OCCurintheECRLmuscleofbothnormalanddystrophicchickens．Withthelightmicroscope，therewere
SOmeunCertaintiesinidentifyingindividualmusclefibers，eSpeCiallyinyounganimals．Althoughtheelectron
TABLEl．NumberofFibersinNormalandDystrophicECRLMusclesoftheChickens
Age

2−3days12−14days

25−26days

25，382
士1，777

20，664

Normal

14，375
土1，699士1，472

18，077
土2，028

7才＝5

〝＝3

JJ＝5

17，173
士869

13，316
土660

土1，013

〝＝4

〝＝4

〝＝4

Dystrophic

14，912
士1，311〟＝4

Sign摘cance＊

no

〃＝4

f＜0．0142−43days

60−65days

7，462

アく0．01

Mean士S．E．＊t−teStforthedはbrencebetweennormalanddystrophjc．
15

microscopiccountgave higher values by20％than those obtained bylight microscopic countinrat
lumbricalmuscle6㌧thiswouldgivesimilarbiasesto thevaluesin both normalanddystrophicchickens．
Therefbre，itislikelythatnosignifiCantabnormalitiesinmuscleproliftrationexistedattheembryonicstage，butthisprocesswasgreatlya斤bctedindystrophic musclesin the early postnatal period of2−14days．
IntermSOfthenerve−muSCleinteractions，itisconceivablethatthedystrophicnerveshavesomedeftctin
theirabilityto promotepostnatalproliftrationofthemusclefibers．Alternatively，thedystrophicmuscle
fibersthemselvesmaybeunabletorespondproperlyto theneuralinfluence．Furtherstudyisneededto
elucidatethemechanismsofthesuppressedproliferationduringtheearlypostnatalstageinthedystrophic
Chicken．

1）Hironaka，T．，Ikari，Y．，Miyata，Y．，andOtsuka，M．：CurrentResearchinMuscularDystrophy，Japan，pp．5−6，
1980．

2）Miyata，Y．，Morimoto，S．，Ikari，Y．，andHironaka，T．：Biomed．Res．，2，664−672，1981．
3）Red托rn，P．A．：エ勒わブリ209，701−709，1970．
4）Bennett，M．R．andPettigrew，A．G．：J．PhysEol．，241，515−545，1974．
5）Brown，M．C．，Jansen，J．K．S．，andVanEsser），D．：J．Physiol．，261，387−122，1976．
6）Betz，W．J．，Caldwe11，J．H．，andRibchester，R．R．：J．PhysEoZ．，297，463−478，1979．
7）Miyata，Y．andYoshioka，K：J．Physlol．，309，631−646，1980．
8）Chiakulus，J．J．andPauly，J．E．：Anat．Rec．，152，55−61，1965．
9）Bridge，D．T．andAl1brock，D．：J．Ana1．，106，285−295，1970．
10）Rayne，J．andCrawfbrd，G．N．C．：J．Ana1．，119，347−357，1975．
11）Ontell，M．andDunn，R．F．：dJ乱J．d〝加．，152，539−556，1978．
12）Johnson，F．H．，Eyring，H．，andStover，B．J．：TheTheoryofRateProcessesinBiologyand Medicine，pp．549−
598，1974．

16

施J∫C〟br勒∫／r叩わ′Gr伽p，J夕gJ

ARRESTED GROWTH OF MUSCLE FIBERSIN MUSCULAR DYSTROPHIC MICE
TsuyoshiTotsuka，KimiWatanabe，IsaoUramoto，SigehiroKiyono，AkikoOhshima，AkiraMizutani，andKatsuhiko
Shimada＊InstituteforDevelopmentalResearch，AichiPrefecturalColony，Kasugai480−03＊NagoyaMunicipalWomensJuniorCollege，Nagoya464

We have proposed a bone−muSCleimbalance hypothesis for the pathogenesisofmurine muscular
dystrophyandpossiblesymptomatictreatments（Fig・1）・Ourrecentfindingthatamajordystrophicsymp−
tomindystrophicmicecouldbemaskedbytheintroductionofgenedwstronglysupportsthishypothesisl）．
RecentlyasimilarhumancasehasbeenreportedbyZatzetal・2），andanimprovedrightingability（flipnumber）hasbeenreportedindystrophicchickenstreatedwithananti−thyroiddrug（propylthiouracil）byKinggJd．8）．
Inthehindlimbsofthedystrophicmouse，lmpairedgrowthofskeIetalmusclesisduetomaturational
deftctsofrnusclefibers4）．Thus，inthepresentstudythreetypesofexperimentswere carriedoutinordertoascertainthematurationaldefbctsofmusclefibersinboththefbre−andhindlimbsofthedystrophic
mouse（C57BL／6Jdy／dy）．
腑′∫C〟ねr即戊edpoJe〃ぬた

Evokedpotentialsofgastrocnemiusmusclesofnormalanddystrophicmicewererecordedbyasciatic
nervestimulation（0・05msecpulse）・Theydisplayedabiphasicwavewithhighamplitudes（Fig．2A，upPer
half）・WhenthefrequencyofstimulationwasraisedfromO．5to5Hz，amplitudesofthemuscularpotentials
innormaladultmicewererapidlyandseverely reduced，Whereasthoseinnormalyounganddystrophic（bothyoungandadult）micechangedonlyslightly（Fig．2A，lowerhalfandFig．2B）．Thus，muSClesof
dystrophicmiceappeartobefatigue−reSistantcomparedwiththoseofnormaladultmice，afindingsimilar
tothatofSandowandBrust（tensionanalysis）5）．Musclesofdystrophicmicecantherefbrebesaidtobe
VerySimilartothoseofnormalyoungmice．

A BONE−MUSCLEIMBALANCE HYPOTHESISGene dy

The growth ofskeletalmusclesis arrested：
Maturational defects of muscle fibers．

The growthofbonesis almost normaL

Age−relatedincreaseinbone−muSCleimbalance：Progress ofthe disease？
POSSIBLESYMPTOMATIC

TREATMENTS？

I．Hypophysectomy＊or−anti−GH
lI．Thyroidectomyordrugs・

anti−thyroid

drugs■t

FIG・1・A bone−muSCleimbalancehypothesisfbrthepathogenesis ofmurinemuscular dystrophyandpossible
SymptOmatictreatments・＊Latent dystrophic symptomin genotypically dystrophic・dwarfmicel），and asimilar
humancase2）・＊＊ImprovedrightingabiJityofpropylthiouracil−treateddystrophicchickens3J．

17

4︳﹄面■

3膚■■■

2Ⅷ■¶﹄

∵−運■浬■

Control

H膚﹄﹃選一

A

B
0

4

8

Ageofmouse（week）

FIG・2（1eft）・Evokedpotentialsofm・gaStrOCnemiusbysciaticnervestimulatior）innormalanddystrophicmice・A）RepresentativepatternsbyO．5（upper）and5Hz−Stimu）ation（loWer）．1−Iand1−1l：2−n：Onth・01ddystrGPhicm（e．
2，3，and4：2−mOnth，7−mOnth，and16−day−Oldnormalmice．Calibration：40mVand4msec．B）Time−COurSeS Of
changesinmuscularpotentialsduriIlg5Hz−Stimulation．Meanarnplitudes（％oftheinitiaHel、el）areplotted versus
time．Arrowindicatesvaluesobtainedimmediatelyafterthestartof5Hz−Stimulation．●：2−mOnth−01ddystrophic
mice（n＝7and7）．△，O，and口：16−day（n＝8），2−mOnth（n＝13），and7，mOnth−Old（n＝7）normaJmice．
FIG．3（right）．Age−relatedchangesin［12511α−BGTbindingbyforelimb tricepsofdystrophicandnormalmice．

肋∫Cわガムerd由me相和
HistologiCalobservationswereperformedonmusclespecimens（7FLm−thickcrosssectionsofpara庁in−embedded forelimb triceps and hindlimb biceps）．Increaseinfiber−SPlitting，丘ber necrosis，CentralandClusterednucleation，atrOPhicfiber，COnneCtivetissue，andfiber−diametervariationwaspronouncedthrough−Outthefore−andhindlimbmusclesofdystrophicadultmice6）．Inthepresentstudy，thediametersofmuscle
fibersweremeasured．Thoseinhindlimbsofnormalyounganddystrophic（bothyoungandadult）miceaveragedonlyabout25FLm，Whereasthoseofnormaladultmice wereverylarge（around40FLm）．Fore−limbmusclefibersofadultmicehadalargediameter（37iLminaverage）innormalmiceandasmallone（23FLminaverage）indystrophicmice．Theseresultssuggestan age−relatedincreasein thediameterof
musclefibersinfbrelimbsaswellasinhindlimbsinnormalmice，butnotindystrophicmice．
α一月〟JぽαrOわズ血ム加晦Theforelimbtricepswasquicklyremoved，Weighedandhomogenizedinabu飴redsalinewithPoly−tron PT−10．After centrifugation at28．000×g fbr30min，the pellet was suspendedin the same saline，
SOnicated andused forl125I］α一bungarotoxin（α−BGT）binding experiments．Musclesfrom normalyoung
anddystrophic（bothyoungandadult）micewerefbundtobindsimilaramountsofα−BGTonawetweightbasis（Fig．3）．An age−related change（decrease）inα−BGT binding was observedonlyin normalmice．
This may be atleast partly attributable to an age−related decreasein thesurface area−VOlume ratio ofmusclefibersresultingfromthematuration（enlargement）ofthesefibers．
Inconclusion，the present丘ndings demonstrate thatmaturationalprocessesofmusclefibersin thefore−andhindlimbsmaybeseverelyinhibitedinmusculardystrophicmice．
1）Totsuka，T．，Watanabe，K．，andKiyono，S．：Proc．JqpalZAcad．，57Jl，109−113，1981．
2）Zatz，M．，Betti，R．T．B．，andLevy，J．A．：Am．J．Med．Gene1．，10，30lr304，1981．
3）King，D．B．，King，C．R．，andJacaruso，R．B．：L的Sc1．，28，577−585，1981．

4）Totsuka，T．，Watanabe，K．，andKiyono，S．：Cof好．AflOm．，21，253−259，1981．
5）Sandow，A・andBrust，M・：Am・J・Pb）Siol・，202，815−820，1962・
6）Totsuka，T．andWatanabe，K．：Ehp．AnEm．，30，465−470，1981．
18

肋∫C〟kr勒∫汀叩妙Gro〟p，J夕βJ

COMPARISON OF THE ALTERATION PATTERNS OF SEVERAI．BIOCHEMICAL MARKERS
IN〜ⅢCE WITH SOME NEUROMUSCULAR MUTATIONS
ShigekatsuTsujiandHiroshiMatsushita
DepartmentofPhysiology，WakayamaMedicalCoIJege，Wakayama640
Thepurposeofthisworkwastocompareabnormalchangesofcertainenzymeactivitiesinseveral
tissuesandofcreatineconcentrationsinurineffommicewiththreedi飴rentmutationsofmuscular・dys−
trophy，SuChasDystrophiamuscularis（dy），Myodystrophy（myd），andnewlydiscoveredmutation（mut），
andtoelucidatethedi能rencesofintrinsicorgenetica11ycontrolledchangesinthedevelopmentofdisease
amongthesemutations∴

Theexperimentswerecarriedoutwith6−12weekolda能ctedhomozygotesanduna能ctedlittermate
animalsfromstrainsofC57BL／6J−dy，MYD／J−myd，andSM／J−mut．
Hindlegmusclesandbrainwereremovedffomthemiceimmediatelyaftercervicaldislocationand
WerehomogenizedwithlOvolumesoficecoldO．32MSuCrOSeinateaonhomogenizer．Themusclehomo一

genateswerethenfhctionatedbytheSchneidermethodandthecytosolfractionswereusedasenzyme
SOurCeS・Thepurifiedmyelin丘actionwasisolatedn■Omthebrainhomogenatewithaconventionalsub−

CellularLhctionationprocedure，eSSentiallyaccordingtoNortonandPoduslol）・Thebloodsampleobtained
ffomthecarotidarterywasallowedtocoagulateandtheserumwasseparatedinthecentrifugeandthen
usedfbrenzymeassay・Urinesamplescollectedfromfivemiceofeachexperimentaltrialwereappropr1−
atelydilutedandfi1teredtoremoveimpurities．

Forthedeterminationofcreatinekinase（CPK）activityinmuscleandserum，thespectrophotometricmethodbyHessetal・2）wasused．Pyruvatekinase（PK）andlactatedehydrogenase（LDH）activitiesin
muscle and serum were also measured by the spectrophotometric methods ofTanaka et al．3）and of
WroblewskiandLaDue4），reSpeCtively．2′，3′−Cyclicnucleotide3′−phosphohydrolase（CNP）andcholestero1−

esterhydrolase（CEH）activitiesinthepurifiedmyelin鉦actionofbrainweredeterminedbythemethods
PreViouslydescribed5）・Estimationofcreatineandcreatinineinurinewasperformedbythemethodof
JaffereactionaccordingtoTaussky6）．
InTablelareshownthecalculatedresultsofthealterationoftheenzymeactivitiesinserum，muSCle，

andbrain，andofcreatineconcentrationsinurineofthea能ctedmice．Eachvalueinthetableshowsthe
PerCentOfchangeofafftctedmicetothatofuna恥ctedcontrolmice・Itisnotedthatinmydmice，SerumlevelsofallthoseenzymeSrepOrtedtobehighinhumanmusculardystrophyarealsosigni負cantlyelevated．
TABLEl．AlterationsofEnzymeActivitiesin SeveralTissuesand ofCreatine Contentsin Urine from ALrected
MiceMediatedbyThreeDi庁brentNeuromuscularMutations．

Dystrophy Myodystrophy Newmutant
Serum CPK
PK
LDH
Muscle CPKPK
LDHBrain CNP
CEH
Urine Creatine
Creatinine
Creatine／Creatinineratio

128．1…
329．3＊

107．4
54．9＊
25．6＊
72．9＊

66．7♯
52．3＊
235．5＊
88．5
276．5＊＊ア＜0．01，＊＊タ＜0．05．

19

TheactivitylevelsforcorrespondingmuscleenzymeSShowsignificantdecreasewhichisanalogoustothe
resultobtainedindymice．LDHactivitiesinserumofdystrophicmiceandinmuscleofnewmutantmice
arenormal．AllserumenzymelevelsexceptCPKlevelinnewmutantmicetendtodecrease．Thereisslg−
nificantdecreaseofmyelinCNPactivitiesinthebrainofbothdystrophicandmyodystrophicmice，but
thisisunchangedinthebrainofnewmutantmice．However，SignificantdecreaseofmyelinCEHisob−
servedinthebrainofdystrophicandnewmutantmice．Ontheotherhand，myelinCEHactivityinbrain
ofmyodystrophicmiceshowsaslightbutnotsignificantincrease．
Signi丘cantincrease ofcreatineconcentrations andslight but not signi丘cantdecreaseofcreatinine

concentrationsareobservedinallafrtctedmiceascomparedwiththoseofuna馳ctedlittermates．There−fbre，thecreatine／creatinineratioinurineofdystrophic，myOdystrophic，andnewmutantmiceismuch
higherthanthatoftheirnormallittermates．
Theresultsobtainedinthisexperimentindicatethatmanysimilaritiesinthealterationpatternsofsome
biochemicalmarkersofmusclediseasecouldlinknewmutantmicewithdystrophicmice．Itisofinterest
thatthedecreaseofmyelinCEHactivitywhichinvolvesincholesterolmetabolismofthemembranestruc−tureinthenervetissueisalsoobservedinthebrainofbothdystrophicmiceandnewmutantmice．The

alterationpatternofsomebiochemicalmarkersinthisexperimentresultedinmyodystrophicmicecom−parabletothoseinapseudohypertrophictypeofhumanmusculardystrophy・
1）Norton，W．T．andPoduslo，S．E．：J．Neurochem・，21，749−757，1973・
2）Hess，J．W．，Murdock，K．J．，andNatho，G・J・W・：Am・J・Clin・劫thoL，50（1），89−97，1968・
3）Tanaka，T．，Harano，Y．，Sue，F．，andMorimura，H・：J・Biochem・，62，71−91，1967・
4）Wroblewski，F．andLaDue，J．S．：Proc．Soc．Eh，・Biol・Med・，90，210−213，1955・
5）Tsuji，S．andMatsushita，H．：CurrentResearchinMuscularDystrophy，Japan，1，pp・47−48，1979・
6）Taussky，H．H．：J．Biol．Chem．，208，853−861，1954・

20

肋∫C〟ねr勒∫／rqpわ′Gr（）叩，J夕gJ

EFFECT OFBRAINEXTRACT ON DEVELOPMENT OF TETRODOTOXIN−SENSITIVE SODIUMCHANNEI，IN L6Ⅳ【YOTUBES
MasaakiraKano，SuteoYamazaki，TeikichiSatoh，andMitsueYamamoto
DepartmentofPhysiology，SchoolofMedieine，KitasatoUniversity，Sagamihara228
Inadultinnervatedvertebrateskeletalmuscletheactionpotentialisgenerallyblockedbytetrodotoxin（TTXlO￣6M），indicatingthat

theactionpotentialis

operatedbyaTTX−SenSitive

sodiumchannel．Im−

mediatelyafterbirth，however，ratSkeletalmusclefibersarecapableofgeneratinganactionpotentialthat
isnotblockedbyTTX、andby20daysofagetheactionpotentialbecomeTTX−SenSitiveinrelationto
theestablishmentofnormalinnervationl）．Conversely，afterdenervationofadultskeletalmuscletheaction
potentiallosesitsnormalsensitivitytoTTX，butisstillsodium−dependent2）．Theseobservationsindicate
thattherearetwo di斤brent sodiumchannels：Oneis sensitiveto TTXwhereas the otheris not．Mainly
TTX−reSistant sodium channels are presentin rat skeletalmuscle at birth and，in contrast，the sodium
channelsinadultratinnervatedskeletalmuscleareTTX−SenSitivebutrevertbacktoTTX−reSistantsodium
channelsfbllowingdenervation．ThedevelopmentandmaintenanceofTTX−SenSitivesodiumchannelsin
theskeletalmusclecellmembranecouldbeconsidered，then，aSOneOfthemembranepropertiesregulated
byinnervation．

Usingskeletalmusclecultures，itwasshownthatce11−freenerveextractscanduplicateatleastapartoftheeffectofinnervationonthedevelopmentandmaintenanceoftheTTX−SenSitivesodiumchannelsin
uninnervated skeletalmuscle cells3・4）．Thus，the resultsindicate that the trophic controlofthe sodium
channelsofmusclecellsmightwellbemediatedbyatrophicsubstanceproducedbynervetissues．
TheL6myotubes5）Sharemanycharacteristicsofembryonicanddenervated skeletalmusclecells，in−cludingresistivityoftheactionpotentialtoTTX6・7）．Thismaybecorrelatedwithalackoftheneurotrophic
effect．Thepresentstudydealswiththetrophice鮎ctofbrainextractonthesodiumchannelsinL6myo−tubes．WehavefoundthatbrainextractiscapableofinducingthedevelopmentofTTX−SenSitivesodium
ChannelsinuninnervatedL6myotubes．

Stockcultures ofL6myoblasts（giftofDr．T．Amano，Mitsubishi−KaseiInstituteofLift Sciences）
weregrowninDulbecco

smodifiedEagle

smediumsupplementedwith10％fbtalcalfserum・Experimental

disheswerepreparedbyplating3×106cellsper60mmtissueculturedishcontaining3mlofculturemedium，
towhichhorseserumwasaddedinplaceofthefttalcalfseruminordertoacceleratemyotubefbrmation．
Brainextractwaspreparedfrom brain of20−dayoldratfttusesbycentrifugingal：2homogenatein

Tyrode，ssolutionat20，000×gfor30min・Culturesweremaintainedintheabsenceorthepresenceof
brainextractfbr180r19days．
Amyotubein18−Or19−dayoldcultureswaspenetratedwithcloselyadjacentconventionalrecording
andcurrent−paSSlngmicroelectrodes．Tominimizetheefrectofvariationintherestingmembranepotential
andtoensuremaximalactionpotentialgeneration，themembrane potentialwas presetineachcaseat−80mVbypassingasteadyhyperpolarizingcurrent，andanactionpotentialwasevokedbyasuperimposed
depolarizingcurrentpulse．
Theactionpotentialwascomposedofaspikeoperatedbysodiumchannels，fbllowedbyaplateau
TAtiLEl．MaximumRateofRiseofSodiumSpikes（V／sec）inCulturedL6Myotubes
Normalsaline TTXcontainingsaline
Contro1
Brainextract

132．50士10．72（14）153．34土10．23（16）122．23士15．54（13）78．75土9．04（16）＊Valuesaremeanj＝S．E．Numbersinparenthesesaremyotubesexamined．事Indicatesasignificantdecrease．

21

operatedbycalciumchannels，aSrePOrtedbyothers6・7）・Thernaximumrateofriseofthespikewasmeasured
withadifferentiatingcircuitandusedasanindexofthedensityofthesodiumchannels・
AsshowninTablel，themeanValueofthemaximumrateofriseofthesodiumspikesincultures
growninthepresenceofbrainextractdoesnotsignificantlydifftrfromthatincontroIculturesgrowninthe
absenceofbrainextract．However，thevalueincultureswithbrainextractisreducedontheaverageby
about50％byTTX（10−6M），althoughthevalueincontroIculturesisnotaffectedbyTTX・
TheseresultsindicatethatthereisalossofTTX−reSistantsodiumchannelsandagainofTTX−SenSitive
sodiumchannelsincultureswithbrainextract，t．e．，theL6myotubescandifftrentiatewithrespecttothe
sodiumchannelswhengrowninthepresenceofbrainextract，eVenintheabsenceofinnervation・1）HarriS，J．RandMarshall，M．W・‥NatureNewBlol・，243，19ト192，1973・
2）Redftrn，P．andThesleff，S．：AcfaPhysiol・Scafld・，82，70−78，1971・
3）Kuromi，H．andHasegawa，S．：BrainRes・，100，178−181，1975・
4）Kano，MりSuzuki，N．，andqjima，H．：J．Cell・Physiol・，99，327−332，1979・

5）Ya仔岳，D∴fr（）C．凡JJJ．dcαd∫cf．払ぶ．A，61，477−483，1968・
6）Kidokoro，Y．：Nat〟reNewBiol．，241，158−159，1973・
7）Land，B．RリSastre，A．，andPodleski，T・R・：J・Cell・Pjzysiol・，82，497−510，1973・22

肋∫C〟ねr勒∫／rqp力yGr（）岬，J夕βJ

DEVELOPMENTAL STUDIES OF THE MEMBRANE PROPERTIES OF STRIATED MUSCLEFIBERS−CHANGESIN THE PROPERTIES OF Ca CHANNELS DURING DIFFERENTIATION
OF EXCITABLE CELLSTomooHiranoandKunitaroTakahashi
LaboratoryofNeurobioIogy，Instituteofl3rainResearch，FacultyofMedicine，UniversityofTokyo，Tokyol13
Themechanism thatinduces the Cainfluxfrom outsideby depolarization ofthemembraneisone
oftheessentialcomponentsintheexcitablemembraneandiscalledtheCachannel．ThisCachannelshowswide distribution among excitable ce11s ofvarious adult animalsl）．The existenceofCachannelsin theheart musclemembrane and the smooth musclemembrane ofthe vertebrates has been well established．

AlthoughthestriatedmusclefibersinthevertebrateshavebeenconsideredtoevokeonlyNaspikesand
inducenoCacurrent，therecentpreciseanalysisofthemembranecurrentwith TTXandTEApresence
revealsCa−dependentinwardcurrentwhichisclearlydistinguishedfrom Nacurrentbythetimecourse
undervoltage−Clamp．Furtherduringontogenesis，theCachannelsarecharacteristic components ofdif−
ftrentiatingexcitablemembranesintheearlystagesofdevelopment．Forexample，thepresenceofaCa−
dependentplateaufollowlngafterafastNa−dependentspikehasbeen reportedinthemembraneofthe
myotubesinvertebrateembryos2）．Therefbre，ChangesinthequalityandquantityofCachannelscanusual1y
beexpectedduringthedifferentiationofexcitablemembranes．
TheCachannelwhichwasfirstdescribedinthecrustaceanstriatedmusclefiberandthenfoundinthe
neuronsofthemolluscangangliahasbeenknowntoshowcharacteristichighpermeabilitytoBaionsand
along−1astinginwardcurrentwithmuchlessinactivationthantheNachannel．FurtherintheCachannel
theCacurrentismoreinactivatedthantheSrorBacurrent，indicatingthattheinactivationisdependent
Ontheionspeciescarryingthecurrent．ThisisassumedtobeduetoCa−inducedinhibitionofCachannels
by theaccumulationofCaionscarried bythecurrentitself．On theotherhand，reCent Studiesinour
laboratoryhaverevealedanothertypeofCachannelinvariousoocytesofthemouse，thetunicateand
seaurchin3）．ThisCachannelshowsapotential−dependentinactivationwhichisveryslmilartotheHodgkin
andHuxleytypefoundintheNachannel．Thetimecourseandtheinactivationarenotdependentupon
theionspeciescarrylngthecurrent．Further，theCachannelisthemostpermeabletoSrions．Thus，theeggCachannelisclearlydifferentftomtheCachannelfoundinotherexcitablemembranes．Sincetheegg
cellmembraneis actually at thestartingpoint ofthe diffbrentiation ofthe membranefunction，the Ca
Channelmaychangeitspropertiesfromeggcelltypetoexcitablecelltypeinassociationwiththedifrbrenti−
ationoftheembryonicmembrane．
Inthetunicateembryooftheprotochordateithasbeenreportedthattheblastomeresinthecleavage−arrested8−Or16−Cellembryocanshow difrtrentiationoftheexcitablemembranebyinducingNaorCa
actionpotentials4）．Eveninlargeblastomeresofthecleavage−arreSted1−，2−，and4−Cellembryodi能renti−
ation ofthemembranealso occurs．In thosecases，aSingleblastomeremaybeexpectedtohavemosaic
properties ofvarious types ofexcitable ce11S，because those blastomeres are not unlquely determined．
However，inreality，the di舵rentiated unicellular embryo shows the characteristiclong−1asting action
potentialswhichareidentifiedastheepidermalcelltype．Themembranecurrentinthedifrtrentiateduni−
Ce11ularembryoisshowntobepureCa−Channelcurrent．Thus，uSlngaunicellularembryo，analysisofthe
ChangesinthepropertiesofCachannelsareattemptedduringthedifrtrentiationofthemembranen

Om

thatoftheorlglnaleggcell．
肋才力αた
Adultascidian，Hah7g〃thiaroretzioratIranthLm，Whichhadmaturedeggsandsperm，Werekeptina
23

COldaquariumOf4Oc．Theeggsandspermwereobtainedfromspawnlnganimalsafter24hrinawarm
bath of120C．The eggs were fertilized by the sperm ffom heteronymous animals．Developmentaltime
WaSmeaSuredfromthisfertilizationintheincubationbathataconstanttemperatureoflOOC．Beforefirst
Cleavage the embryos were transftrred toanother bath containing cytochalasin BIpg／mlin order toarresttheircell−Cleavageandwerefurthercultureduntilthecontrolintact tadpolelarvaehatched．Afterremovingthechorionmembraneby digestionwithpronaselOmg／ml，thecleavage−arreStedembryowas
keptinthehighproteinousexternalsolutionandwaspenetratedbytwomicroelectrodes．Theexperiments
Weredonebyeitherconstantcurrentstimulationorvoltage−Clamp，aSdescribedpreviously5）．Thestandard
externalsolutionwaslOOmMSrC12，400mMNaCl，10mMKCl，5mMPIPES−Na（PH7．0），10mg／mlBSA，
and2FLg／mlcytochalasinB．Whenitwasnecessary，SOdiumwasreplacedbyequimolarcholine orstron−
tiumwithequlmOlarcalciumorbarium．InordertoanalyzetheinactivationprocessoftheCachannel，thetailcurrentoftheCacurrentatvariousperiodsffomthevoltage−StepWaSrr）eaSuredand thetimecourse
oftheinwardCacurrentwasdecidedwithoutcontaminationoftheoutwardcurrent．
Re∫〟JJ∫

1）AfterculturinginseawatercontainingcytochalasinBuntilthecontrolintactlarvaehatched，therestingpotentialofthedi仔brentiatedunicellularembryowasabout r70mV，beingatKequilibriumpo−
tentialinthestandard external solution．The action potentialwas evoked bydepolarizationabove−30
mV anditsduration was more than two seconds．TheactionpotentialwasabolishedbyeliminatingSr
ions，but not changed by the replacement of Nawith choline．The overshoot was about50mV．No
evidenceofthedevelopeddelayedKrectificationwasnotobtained．Thistypeofactionpotentialhasbeen
reportedastheepidermaltype．

2）Under a voltage−Clamp condition，thelessinactivatinginward current was observedwith de−
POlarizationabove r30mVandthecurrent showed themaximumvalueat15mVin standardexternal
solution．In Mnexternalsolution theinwardcurrentwaseliminated butno outwardcurrentwasobserved
except theleakagecurrentwithouttime−dependentkinetics．Thus，themembranecurrentinthedi飴enti−
atedunicellularembryowasidentifiedasalmost pureCa−Channelcurrent．Thetimecourseofthedecay
PhaseinstandardsolutionwasmuchslowerthanthatoftheeggCa−Channelcurrent．
3）In Ca external solution the membrane current showed the outward component，but this wasabolishedbyirjectingEGTAintheunicellularembryo．Thus，theoutwardcurrentwasconsideredtobe
Ca−dependentKcurrentandnotdelayedKrectification．
4）TheionselectivityoftheCachannelinthedi舵rentiatedunicellularembryowasCa＜Sr＜Ba，
being

difftrent

h■Om

that

oftheegg

Cachannel，Ca≒Ba＜Sr．TheinactivationoftheCa−Channelcur−

rentwasonlyfoundinCaexternalsolutioninthecaseofthedifferentiatedunicellularembryo，Whilethe
inactivationoftheeggCa−Channelcurrentwaspotential−dependentandobservedineitherCaorSrorBa
externalsolution．
上）血C〟∫∫わ〃

AlltheaboveresultsindicatethattheCachannelinthedifrtrentiatedunicellularembryowasexactly
identicalwiththosefbundin otherdif托rentiatedexcitablecellsanddefinitelydifftrentfromthosefbund
invariousoocytes．Thus，itissuggestedthatthedifftrenceofthepropertiesinCachannelsispartlydue
tothechangesofchannelstructureduringthedifrtrentiationofexcitablemembranes．
1）Hagiwara，S．andByerly，L．：Annu．Rev，Ne〟rOSCi．，4，69−125，1981．

2）Kano，M．：J．CeJJ．Cb〝岬．アわ′∫わ7．，86，503−510，1975．
3）Okamoto，H．，Takahashi，K．，andYamashita，N．：J．PhysEol．，267，465−195，1977．
4）Takahashi，K．andYoshii，M．：J．Physiol．，315，515−529，1981．
5）Okamoto，H．，Takahashi，K．，andYoshii，M．：J．P々ysiol．，254，607−638，1976．

24

肋∫C〟ねr勒∫什qpわ′Gr（フ岬一J夕βJSENSITIVITYTOCa2＋，Sr2＋，ANDBa2＋IONSOFTHECONTRACTILESYSTEMOFSLOWAND

FAST FIBERS OF AWHIBIAN MUSCLES
MakotoEndoandKeisukeHoriuti
DepartmentofPharmacology，TohokuUniversitySchoolofMedicine，Sendai980
Itiswellknownthattherearetwotypesofmusclefibers，Slowandfast，inamphibianskeletalmuscles．
Theslowfibersofamphibianmusclesaredifferentfromslowtwitchfibersofmammalian musclesatleast
inthatactionpotentialscannotbeevokedintheformerbutcanbeinthelatter．Ithasbeenreportedthat
inmammalianmuscles，WhereasCa2＋−SenSitivitiesofslowandfasttwitchfibersarerathersimilar，Sr2十一
SenSitivitieSarequitedifftrent，theSr2＋−SenSitivityofslowtwitchfibersbeingsignificantlyhigherthanthat

Offast twitch丘bersl，2）．Itisinteresting，therefbre，tOeXaminewhetherornotasimilardifftrenceexists
betweenfastandslowfibersofamphibianmuscles．WehaveexaminedsensitivitiestoCa2＋andSr2＋ions
aswellastoBa2＋ionsofthecontractilesystemoffastandslowfibersofamphibianmusclesasthefirst

StepOfourstudiesondifrtrentiationanddevelopmentofmuscletissues．
IliofibularismuscleswereisolatedfromtheadultAfricanclawedtoad，Xenppush7eVLy，andsmallbun−
dlesweredissectedoutoftheminafrogRingersolution．Thebundleswereimmersedinarelaxingsolution
andsinglefiberswereisolatedandthenskinnedeitherbytearingthemlongitudinallywith fbrcepsorby
immersingtheminadetergent（0．5W／V％Brij58）−COntainingrelaxingsolutionfbr30min．Isometrictension
WaSreCOrdedwithastrain−gaugetranSducer（UC−20r−20，Shinkoh）anddisplayedonapen−reCOrder．Maxi−
mumshorteningvelocitywasmeasuredbytheslacktesttechniqueas reportedpreviously3）anddenoted
asFh・ExperimentswereperfbrmedinthepresenceoflmMfreeMg2＋，4mMMgATP2−，10mMtOtalEGTA，
and20mMPIPSE（pH7．0）atanionicstrengthofO．15Mandat2OC．ActivatingsolutionscontainedCa2＋，
Sr2＋，Orl3a2＋andthefreedivalentcationconcentrationsinthesesolutionswerecalculatedbyassuming

apparentassociationconstantsforCa−，Sr−，andBa−EGTAto belO6・20，103・71，andlO3・62，reSpeCtively4，5）．

亡○叫的uヱむ＞コdlむだ

巴む∈00誌SJ鼠ujugJOJS
EもtJ虐pだ叫nbとむ月↑

已○叫的亡39＞コdl申出
10￣7

10嶋6

10￣5

10−4

10−3hI

ConcentratiorL Ofthe free metalion

Low High
Sr2十一SenSitivity

FIG・1（left）・Therelationbetweenfreedivaldntcationconcentrationsandtensiondevelpoedbyfhst（upper）andSlow（lower）fibersofamphibianskeletalmuscles．
FIG・2（right）・CorrelationbetweenshorteningvelocityandSr2＋−SenSitivityintwotypesofBbers．

25

Histochemically，theamphibianskeletalmusclefibershavebeendividedintofivetypeswhichwere
namedfromtheirappearancesaslargepale，1argedark，Smalldark，Smal1pale，andsmallclear6）・Thefirst
threetypesareconsideredtobethefastfibers，thelasttheslowfibers，andthefburththeintermediate
fibers6−9）．Fibersofthelargepaletypeweredissectedasatypicalfastfiberandtheirdivalentcation−SenSi−
tivitiesweredetermined．AsshownintheupperpartofFig．1，Sr2＋−SenSitivityofthesefiberswassome30
timeslowerthantheirCa2＋−SenSitivity．WithBa2＋ions，thesensitivitywasstilllowerandmaximaltension

couldnotbereachedevenattheconcentrationoflmM．Skinnedfibersfromthelargedarkandprobablythesmalldarkfibertypesshowedsimilardivalentcation−SenSitivitiestothoseofthelargepalefibers・However，
skinnedfibersfromfibersoftheprobablysmallcleartypeexhibitedquitedi能rentproperties・AsshowninthelowerpartofFig・1，WhiletheirCa2＋−SenSitivitywasnotmuchdifferentfromthatofthelargepale
fibers，theirSr2＋−SenSitivitywassomuchhigherthattheSr2＋requlrementOfthecontractilesystemofthis
fibertypewasalmostthesameasitsCa2＋requlrement・Ba2十一SenSitivityofthisfibertypewasalsomuch

higherthanthatofthelargepalefibersandalmostmaximaltensioncouldbeobtainedwithO・1mMOfthe
Ba2＋ions．Wecouldnotrecognizethreefibertypesamongthesmallfibersand，therefore，Smallpalefibers

describedintheliteraturemightbeincludedeitherinoursmalldarkfibersoroursmallclearfibers・
Theresultsdescribedaboveclearlyshowthattherearetwodistincttypesoffibersinamphibianmuscles
showingeitherhighorlowsensitivitiestoSr2＋andBa2＋ions・FromfiberappearanceandpreviOusreports，

fibersshowingthelower sensitivitiesmay befastfibersandthoseshowingthehighersensitivitiesslow

fibers．Indeed，meaSurementSOfVoofthesefibersindicatedthatthisisthecase・AsShowninFig・2，allthehighlySr2十一SenSitivefibersshortenedveryslowlyandrequiredaperiodoftimeseveraltimeslonger
forshorteningofthelpmdistancepersarcomerethandidfibersoflowerSr2＋−SenSitivity・ItwasthusshownthatSr2＋−SenSltlVltyOffastandslowfibersoftheamphibianskeletalmusclesissimi1artothoseoffastandslowtwitchfibersofmarnmalianmuscles．ThedifferencebetweenSr2＋−andBa2十一SenSi−

tivitiesofthesefibertypesmightreflectthedifferencebetweenthea侃nitiesoftroponintothesedivalentcations．Infact，inmammalianandavianmusclesithasbeenreportedthattroponinfromredorslow
muscleshashigherafnnitytoSr2＋ionsthanthatfromwhiteorfastmusclesl・10）・lfthisisthecase，trOPOnin

ofslowandfastfibersofamphibianskeletalmusclesaredifferentfromeachother・Whetherthisdifftr−
enceintroponinstructureshasaphysioIogicalsignifiCanceinperformingcontractionswithdifrtrentspeeds，
andhowthedi仔もrentiationoftroponinstructuresisdevelopedareamongtheproblemstobeinvestigatednext．
1）Ebashi，S．，Endo，M．，andOhtsuki，I．：Q．Rev．Bipphysり2，351−384，1969・

2）Takagi，A．andEndo，M．：旬．Ne〟rOl．，55，95−101，1977・
3）Endo，M．etal．：CurrentResearchinMuscularDystrophy，Japan，2，27−28，1980・
4）Harafuji，H．andOgawa，Y．：J．Bioche肌，87，1305−1312，1980・
5）Schwarzenbach，GリSenn，H．，andAnderegg，G．：Helv．Cum．Acfa，40，1886L1900，1957，6）Smith，R．S．andOvalle，Jr．，W．K．：J．Afl01．，116，1r24，1973・
7）Lannergren，J．andSmith，R．S．：ActaPhysEol．Scand．，68，263−274，1966．
8）L且nnergren，J．：J．PJlySiol．，283，501−521，1978．
9）Lannergren，J．：Nafure，279，254−256，1979・
10）Kohama，K．：J．βわCお肌．，86，811−820，1979．

26

肋∫C〟ねr勒∫什qPわ′Gr∂岬，J夕gJ

ANTIBODIES TO ACETYLCHOLINE RECEPTORIN OCULAR MYASTHENIA GRAVIS
Hiroshi，Kuriyama，Yushi，Ito，andKenichiro，Oda
DepartmentofPharmacology，FacultyofMedicine，KyushuUniversity，Fukuoka812
Anti−aCetylcholinereceptor（ACh−R）−antibodiesaredetectedintheseraofmostpatientswithmyas−
theniagravisl・2）．However，theseraofpatientswithocularrnyastheniaoftenshoweda negativetitreora
Signi丘CantlylowertitrethanthoseobservedinCasesofgeneralizedmyastheniagravisl・3）．Toimprovethe
SenSitivityoftheradioimmunoassaymethodforanti−ACh−R−antibody，largeamountsofseraftompatients
With myasthenia gravIS，and higher concentrations of antigens and rabbit anti−hurnan−IgG−antiserum，WereuSed．TheseproceduresenabledmeasurementofthetitrevalueofoverO．04pmol／mlserumandthis
ValuerevealedasensitivityaboutlOtimeshigherthanthatpredictedusingthepreviousmethod．
Usingthemoresensitiveradioimmunoassaymethod，WeCOmparedthetitrevaluesoftheantibodyto
ACh−Rinthesera ofocularandgeneralizedmyastheniagravisinrelationto theclinicalstatusofthesepatients．
Serawereobtainedftom17Japanesepatientswithmyastheniagravisrestrictedtotheeyelidsandocular
muscles，37patientswithgeneralizedmyastheniagraviS，andlOnormalcontroIs．Allthepatients（10men

and24womenaged21−63andwithdefinitegeneralizedmyastheniagravis）hadclassichistories，Signsand
SymPtOmS，and the diagnosis was supported byclinicalimprovement fbllowinganticholinesterasemedi−
Cation．

Aspatientswithdefiniteocularmyasthenia，llmalesand6ftmales（aged9−65years）wereselected．
Theseindividualshadbeenafftctedfortwomonthsto18years．Allpatientshadbeenonanticholinesterase
medicationbuthadnotundergonethymeCtOmynOrbeengivensteroids．
Radioimmunoassayofanti−ACh−R−antibodywasfundamental1ythesameasthatdescribedelsewhere
forhumanantigens2J．
Theamountofanti−ACh−R−antibodywasmeasuredbyincubatinglnCreaSingamountsofhumanserum
withafixedquantityof125Ⅰ−α−BuTx−ACh−Randsubsequentlyprecipitatingthe125I−α−BuTx−ACh−Rcom−

plexed to anti−ACh−R−antibodies byrabbitanti−human−IgG serum．Intheroutine assay，thetest serum
WaSdilutedlO−fbldwithphosphate−bufreredsaline−0．5％TritonX−100，andlO，20，and50FLlaliquots ofthedilutedserumwereincubatedwithaconstantvolume（500FLl）of1251−α−BuTx−ACh−R（0．4pmol／ml），at

40C．AfterlOhr ofincubation，450FLlofrabbit antiserum（which contained sumcient anti−human−IgG
antibodiestoprecipitatealltheIgGinthetestserum）wasaddedandthemixturewasfurtherincubatedat
40Cfor16hr．ThefinalvolumewaslmlfbllowingadditionofPBS．Theinsolubleimmunecomplexfbrmed
WaSpreCipitatedbycentrifugationat2，500rpmfbr5minat20C，WaShedthreetimeswithicecoldPBS，
andtheradioactivityintheprecipitatewasmeasuredbyagammascintillationcounter．
When the sera showedless thanlpmol／mltitre values（1ess than800cpm／5fd serum），these sera
WerereaSSayedusingthemoresensitiveprocedures．Whenanincreasedamountofdilutedsera（250→500FLl）wasused，andinordertoobtainthesamefinaltubevolumeasintheroutineassay（1ml），COnCentrated
125Ⅰ−α−BuTx−ACh−R（2．Opmol／ml）and rabbit anti−human−IgG were used toprecipitatetheanti−ACh−R−

antibodies．Namely，thelabelled ACh−Randrabbit anti−human−IgG wereconcentratedabout5−OrlO−
fbld usingamacromolecularconcentrator（Amicon，PM30）or33％Na2SO4，reSpeCtively．
Thirty−fiveoutof37patientswith generalizedmyastheniagravisshowedmeasurableamountsofthe
antibodytitresinroutineradioimmunoassays．Thetwopatientswithanegativetitrevaluewereinaremis−
Sion．TheantibodyconcentrationsinthesepatientsrangedfromO．44pmol／mlto252pmol／ml（themean
Valuewas25．42土43．30（土S．D．）pmol／ml）．TherangeofnormalcontroIwasOpmoltoO．4pmolwitha
27

meanvalueofO．18±0．10pmol／ml（土S．D．）．Ontheotherhand，intheocular group，anti−ACh−R−anti−
bodiesweredetectedinonly5of17patientsandthetitrevaluesoftheocularpatientsrangedfromOpmol
toll．24pmol．
Thustheincidenceofanti−ACh−R−antibodyintheoculargroup（30％）waslowerthaninthegeneralized
myastheniagravis（90％），aSdeterminedusingaroutineassay・However，ifpatientswithocularmyasthenia
gravishaveantibodiesoflessthanO．4pmol／ml，theincidenceofanti−ACh−R−antibodyintheoculargroup
Withthesensitiveradioimmunoassaymethod，anti−ACh−R−antibodiesweredetectedin80f120Cular
patientswithanegativetitre，aSmeaSuredbyroutineassay・Theantibodyconcentrationsofthesepatients
rangedfromO・042toO・16pmoLIntheoculargroup，therefore，130f17patientshadmeasurabletitre
values．ThesevaluesrangedbetweenO．042andll．2pmol／ml，andthemeantitrevalue（1・79士3・18（土S・D・））
wassignifiCantlylowerthanthevaluesobtainedinthegeneralizedgroup（P＜0・01）・
Withthisimprovedassayprocedure（10−fbldincreaseinthesensitivity），theincidenceofantibody
againstACh−Rincasesofocularmyastheniawaselucidatedinabout70％andthatofgeneralizedmyasthenia
gravisinabout90％ofthepatients．Theincidenceofanti−ACh−R−antibodyinthetwogroupsdidnotdiffer，
yetabsolutetitrevaluesweresignificantlylowerintheoculargroupthaninthegeneralizedgroup・Fur−thermore，anti−ACh−R−antibodywasnotdetectableinfburpatientswithocularsymptomsofseveralmonths
duration．ThesensitivityofthepresentassaymaynotbesufRcienttodetectminuteamountsofantibodies
intheseraofthesepatients，and／ortheantibodiesagainstACh−Rmaynotevenbepresentintheirsera・
AnotherpossibilityisthatthereissomedifferentantlgenicdeterminantbetweenACh−Roftheocular
musclesandthatofthepectoralmuscles．
1）Lindstrom，J．M．，Seybold，M．E．，1．ennon，V．A．，Whittingham，S・，andDuane，D・D・：Ne〟rOlqgy，26，1054−1059，
1976．

2）Oda，K．，Goto，L．，Kuroiwa，Y．，Onoue，K・，andIto，Y・：NeEIrOkw，30，543−546，1980・
3）Lefvert，A．K．，llergstr6m，K．，Matell，GりOsterman，P．0．，andPirskanen，R・：J・Ne〟rOl・肋urosurg・PWchiatr・，
41，394−403，1978．

28

〃〟∫C〟血r上）γ∫Jrqp力γGr（）叩，Jタβノ

NEURONALFACTORSTABILIZINGACETYI・CHOLINERECEPTORSONCULTUREDMUSCLECELLSYashiroHagiharaandShl再iHasegawa
DepartmentofNeuropharmaco】ogy，73rainResearchInstitute，SchoolofMedicine，ChibaUniversity，Chiba280
Thedistributionandmetabolicstabilityoftheacetylcholinereceptors（ACh−R）ontheskeletalmuscle
Ofvertebratesarein且uencedbytheinnervatingnerve・TheACh−RofaduItmusclefibersarelocalizedat
neuromuscularjunctionsanddegradedslowly，WhereastheACh−Rofembryonicmusclefibersaredis＿

tributeddiffuselyanddegradedrapidly・TheACh−Ronculturedmyotubesaremetabolizedasrapidlyas
thoseonembryonicmusclefibers．
TheneurotrophicmechanismbywhichtheACh−Ronthemusclemembranearestabilizedinetab。Ii＿

Callyduringdevelopmentisunclear・Herewereportthatafactorproducedandreleasedbyneuronalcells
regulatestheturnoveroftheACh−Ronculturedmyotubes．
Primaryculturesofembryonicskeletalmusclecellswerepreparedfromhindlimbsofratembryosas

describedbyChristianetal・1）・Fourorfivedaysafterplatingculturesweretreatedfbronedaywithl0−5M
fluorodeoxyuridineandmaintainedinDulbecco，smodifiedEagle，smedium（DMEM）containinglO％
horseserumandO・5FLg／mltetrodotoxin・Onthedaybefbretreatmentwithneuronalconditionedmedium，
horseserumwasreplacedby2mg／mlbovineserumalbumin．Neuronalconditionedmedium（NCM）was
preparedusingtheneurobIastomaxgliomahybridcelllineNGlO8−152）・NGlO8−15cellswere grownin
DMEMcontaininghypoxanthine，thymidine，andlO％fttalcalfserum．Serumfreemediumwasexposed
tocon伽entculturesofNGlO8−15cellsforoneday，COnCentratedbyultrafiltration，dialyzedagainstdei−
Onizedwater，andstoredat−20OC・ThelyophilizedmaterialwasredissoIvedataconcentrationofO．4
mg／mlinDMEMcontainingbovineserumalbuminandtetrodotoxinandaddedtomyotubecultures．
MediumconditionedbythegliomacellIineC6BU−1（GCM）wasalsopreparedbythesameprocedure．

Time（hr）

FIG・1・RateofdegradationofACh−R・Twoninedayo】dcultureswerepre−treatedfor17hrwithNCMorcontrol
medium・Cultureswerethenlabelledwith125I−ABTandtherateofdegradationof125I・ABT−ACh−Rcomplexwas
measured．

29

TheratesofdegradationoftheACh−Ronthesurfacemembraneofmyotubesweredeterminedac−
cordingtotheprocedureofDevreotesandFambrough3）・Seventotendayculturedmyotubesweretreated
with0．4mg／mlNCMorGCMfor16to45hrandincubatedwithlOnM125Ⅰ−Cr−bungarotoxin（ABT）fbr
60min．Unbound125Ⅰ−ABTwasremoved．Thenthelossof125Ⅰ一ABT−ACh−Rcomplexonmyotubeswas

monitoredinNCM，GCM，OrCOntrOlmediumover40hrbymeasuringradioactivityreleasedbymyotubes
into themedium．
ThesizeoftheintracellularpoolofinternalizedACh−Randitsdegradationproductswascalculatedby
subtractingtheamountoflabelboundtosurfaceACh−Rduringl−hrincubationwith125Ⅰ−ABTfromthe
amountoflabelboundtosurfaceACh−RandinternalizedACh−Rduring20−24hrincubationwith125Ⅰ−ABT・ThedegradationcurveSOftheACh−Ronmyotubesfollowedasingleexponentialprocessover40hr（Fig．1）．Thehalf−time（Tl／2）ofthedegradationof125Ⅰ−ABT−ACh−RcomplexonmyotubesincontroI
cultureswascalculatedtobe22．6j＝0．9hr（mean土S．E．）．Tl／20fthedegradationof125Ⅰ−ABT−ACh−Ron

myotubesinNCM−treatedcultureswas132％ofthatincontroIcultures・SinceTl／20ftheACh−Rdegra−
dationinGCM＿treatedcultureswassimilartothatincontroIcultures，theem㍍tOfconditionalmedium

onstabilizingACh−Ronculturedmyotubesisspecifictoneuronalcells・ThedecreaseintherateofdegradationofsurfaceACh−Rthatwasobservedbymeasurlngthedegra−
dationproductsiscausedbyslowingoneofthefbllowingprocesses‥1）internalization

ofthesurface

ACh−R，2）intracellulardegradation，Or3）releaseofdegradationproductsintotheextrace11ularmedium・
IfthetreatmentofmyotubeswithNCMslowsthesecondorthethirdprocess，theincreasedaccumulation
ofinternalized125Ⅰ−ACh−Risexpected・Therefbre，theeffectofNCMontheintracellularaccumulationof

125I−ABT−ACh−Rcomplexanditsdegradedproductswasexamined・Thearnountofintracellularlyac−
cumulated125I−ABT−ACh−RanditsdegradedproductsinNCM−treatedmyotubeswassimilartothatin
controIcultures．InmyotubestreatedwithlOFLMChloroqulneinsteadofNCM・theamountofinternalized
125I−ABT・ACh−RcomplexincreasedtothreetimesthatofcontroIculturesandTl／20ftheACh−Rdegra−
dationincreasedtoeighttimesthatofcontrol．
InourexperimentsbothNCMandchloroquinedecreasedtherateofdegradationofACh−R・Whereas
chloroqulneinhibitedthereleaseofinternalized125Iintotheextracellularmedium4），NCMhadnoeffectonthisrelease．NCM，therefore，aCtSbydecreasingtherateofACh−Rinternalization・Ourresultssuggest
thatadi飢siblefactorwhichisproducedandreleasedbyneuronalcellsisinvoIvedintheneurotrophic
mechanismfbrthestabilizationofACh−Ronthesurfacemembraneofmusclecells．
1）Christian，C．N．，Daniels，M．P．，Sugiyama，H．，Voge】，Z・，Jacques，L，andNelson，P・G・：Proc・Natl・Acad・Sct・
扶且」り75，4011−1015，1975．
2）Bauer，H．C．，Daniels，M．P．，Pudimat，P．A．，Jacques，L，Sugiyama，H・，andChristian，C・N・：BrainRes・，209・
395−358，1981．

3）Devreotes，P．N．andFambrough，D・M・：J・CelLBiol・，65，335−358，1975・
4）Libby，P．，Bursztajn，S．，andGoldberg，A・L・：Cell，19，481−491，1980・

30

〟〝∫C〟ねr旦yJJrqpわ′Gro岬，J夕βJSYNTHESIS AND
DIFFERENTIATION OF ACETYLCHOLINE RECEPTORSIN CULTUREDSKELETAL MUSCLE CELLS
HiroyukiSugiyamaandYukikoYamashita
LaboratoryofMembraneBioIogy，NationalInstituteforPhysiologicalSciences，Okazaki444
Twotypesofacetylcholinereceptors（AChRs）havebeenfoundinthemembraneofvertebrateskeletalmuscZe，OnelocalizedatahighdensityattheadultneuromuscularJunCtionsandtheotherdistributedat
alowerdensityovertheextrajunctionalreglOnSOfdenervatedmuscles．Thesetwotypesofreceptors，JunC−
tionalandextrajunctional，difftrinanumberofpropertiesinadditiontotheirlocationanddensities．Bio＿

Chemica11y，thejunctionalreceptorhasaslightlylowerisoelectricpointthandoestheextrajunctionalre−
CePtOr・Embryonicmusc】eshavetheextrajunctionaltypeofreceptor，andthejunctionaltypeofreceptor
appears，atleasthttu，Onlyaftermotorinnervationofthemusclesoccurs．Althoughthedi貌rentiationof

AChRsisconsideredtobeunderthein月uenceoftheinnervatingnerves，theunderlyingmechanismremains
largelyunknown．

Inthepresentstudy，WeeXaminedthebiochemiCalpropertiesofthereceptorsonthesurfacemem−
branesandintracellularα−bungarotoxin（αBT）bindingcomponentsofculturedmusclece11sfromnewborn

rats，Chickembryos，andmouseclonalmusclecellsG8−1preparedasdescribedpreviouslyl，．Thesu血ce
receptors orintracellularαBT−binding components were differentiallylabeledwith125Ⅰ−Or3H−1abeled

αBT2，，eXtraCtedwith2％TritonX−100，andanalyzedbygelfi1tration，SuCrOSedensitygradientcentrifuga−tion，Orisoelectricfocusing・Thedetailedexperimentalprocedureshavebeendescribedpreviously3・4）．
TheresultsshowedthatintheabsenceofneuronalinfluencesculturedmusclecellsoftheratandthemousecanproducetwofbrmsofreceptorsonthecellsurLbcemembranes，aSdemonstratedbyisoelectric
focuslng，withpIvaluesindistinguishablefromthoseofjunctionalandextrajunctionalreceptors．Incontrast，
OnlyaslnglemaJOrCOmpOnentwithapIvalueclosetothatoftheextrajunctionalreceptorswasfbundon
thesurfacemembranesofculturedchickmusclecells（Tablel）．
TheculturedcellshadtwoclassesofintracellularαBT−bindingcomponents：Onehadthesamesedi−mentationcoe侃cientasthatofsurfacereceptors（9S），andtheotherhadmuchsmallerapparentmolecular
TABLEl・IsoelectricPointsofAChRsofDifrtrentFormsfrom VariousSourcesComplexedwithRadiolabeled
αBTs

pre。arati。nS。。rCe

霊認諾崇慧霊霊諾S
AcidicChickerlembryomusclecells
SurhceInsideMouse−bllasicDegraded

5．39土0．09（7）
5．60
（1）

6．02土0．22（10）
n．d．e

n．d．e

Diaphragm
G8−l cellsurhce
Rat

5．15土0．10（2）5．09土0．11（9）5．45±0．08（9）5．72土0．11（2）6．01±0．16（8）Diaphragm
Newbornratmusclecells
SurhceInside5．18（1）5．41±0．02（2）5．75土0．12（2）5．14土0．08（8）5．44土0．10（8）5．56土0．10（9）5．94土0．19（7）5．88土0．12（5）＆Valuesdeterminedusing3H−αBT（monoPH】propionyl−bderivative）1）and125I−aBT（mono−anddi【1251】iododerivatives）

WerePOOledwithoutdiscriminatioや，andexpressedinpHunit；mean士StandarddeJiation（numb＆0ldeterminations）．
bNotdetectableunderourconditions．
C Notdeterrr）ined．
31

weights．OnlyaslnglemaJOrCOmpOnentWaSdetectedbyisoelectricfocusinganalysisofthe9Sintrace1−
1ularαBT−binding component，With a pIvalue close to that of the extrqjunctionalreceptor（Tablel，
Inside）．

Theseresultssuggest：1）thatthejunctionalandextr毎unctional typesof AChRsofratormousemusclesmaybesynthesizedthroughacommonprecursoranddifrtrentiatedintothetwoformswithdifJ
ferentpIsbysomepost−tranSlationalmodi丘Cations，and2）thatinthecaseofthechickmuscles，thejunc−
tionalandextrajunctionalAChRsmayhaveanidenticalpIvalue・
1）Sugiyama，H．：EEBSLet1．，84，257−260，1977．
2）Sugiyama，H．andYamashita，Y．：J．Biochem．，90，729−735，1981・
3）Sugiyama，H．：Proc．JqpalZAcad．，55B，75−78，1979・
4）Sugiyama，H．：Biomed．Res．，1，91−95，1980・

32↓〃′∫川／ ∫′■J）．lI∫什（）両．l・GJ刷岬．／9朗

STUDIESONACETYLCHOLINERECEPTOROFDYSTROPHICCHICKMUSCLEINCULTURE
KenHottaandNobukoTomita
DepartmentofPhysiology，NagoyaCityUniversityMedicalSchool，Nagoya467
Wereportedpreviouslythattheelectricalpropertiesofchickdystrophicmusclecellsweresigni丘cantly
difrbrentfromnormalcontroIseveninveryearlystagesofgenesisl）・Inthisstudytheturnoverofacety1−

Cholinereceptor（ACh−R）ofdystrophicmusclecellswasinvestigatedtolearniftherewereanydi騰rencesfromnormalmuscleuslngthecellculturetechnique．
Thestartingmaterialwaschickembryo（N．H．412，413）whichhadbeenincubatedat37OCf。r12
days・The breastmusclewas dissectedandexplanted oncollagencoatedpetridishescontainlngthe

medium（MEM，10％HS，3％FCS，and2％CEE）・Cultivationwasperformedunder5％CO2−airaImos−
phereat370cfor5tolldays・ThemyoblastsinculturegrewrapidlyandfuSedwitheachotherfbrmlng
myotubules，aSShowninFig・1（6days）・Striationsappearedapproximately8daysaftertheexplantation．

Therewasnorecognizable di能rence between dystrophicmuscles and normalonesbyphasecontrast
microscopicobservationsuptolldays．
3H−labe11ederabutoxin（30nM）orα−bungarotoxin（2nM），WhichbindsACh−Rspecifically，WaSapplied

Onthesampleinculture・Afterwashingexcesstoxininthemedium，thesamplewasincubatedinthenew
medium・Theappearanceoftoxinand／oritsdegradatedproductsintothemediumwasfbllowedfor30hr．
TypicalexamplesoftoxinbindinganditsdegradationareshowninFig・2（erabutoxin）andFig．3（α−bungarotoxin）・Theinitialbindingofthetoxintodystrophicmusclewasonly50−80％ofthattonormal
muscleinbotherabutoxinandα−bungarotoxin（Fig．2a，3a）．Thisprobablyindicatesthenumberofthebindingsites（ACh−R）islessindystrophicmusclesthaninnormalones・Thehalfllifttimeofthereceptor（丁）wasobtainedbyplottingthelogarithmofremainingtoxinversusincubationtime（Fig．2b，3b）．The
Valueobtainedfromerabutoxinbindingwasconsiderablyshorterthanthatn・Omα−bungarotoxinbinding；apparently，dystrophicmusclealwaysexhibitedfasterturnoverofACh−R・Therelativetoxinbindingof
dystrophictonormalmuscles（D／N）andtheapparenthalf−lifもtime（T）ofACh−Robtainedfromdifferent
SamplesarelistedinTab】eI・TObtainedfromα−bungarotoxinbindingisinagreementwiththatofrat
Skeletalmuscle2）．

FIG・1・Phasecontrastmicroscopicobservationofcultureddystrophicchickmyotubules（6−dayoldinculture）（×160）．
33

％ Totalcount

0

10

20

30（hr）

20

20

b％100

10

0

30（hr）

FIG．2．ErabutoxinbindingtoACh−Randitsdegradation．
0normal，●dystrophy．a：relativebinding，initialvalueof

1030（hr）

20

30（hr）

FTG．3．α−BungarotoxinbindingtoACh−R．

NotationsandsymboIsaresameasFig・2・

normalmuscleis takenasl00％．b：logofrelativebindingvers〟Stime，eaChjnitialvalueistakenaslO0％．

TABLEl．NumbersofBindingSitesandHalfLiftTime（N，nOrmalmuscle；D，dystrophicmuscle；D／N，ratioof
toxinbinding（100％，initialvalueofnormalmuscle）；T，halfllifetime（hr））・
8

9

11

TotalD／N O．65
N
9．3
r（hr）D8
D／N O．86

0．40
9
8．3
0．92

0・40
7．7
6．1
0．78

Erabutoxin B Exp．No．

12

17

18

0．85

0．72

0．74

7．7
6・5
0・84

7．7
5・7
0・74

7．7
6・7
0．87

N：臥3±0．8．D：7．1土1．0．
α・Bungarotoxin Exp．No．

21

22

TotalD／N O．62
N
39．2

0．61
34．4

抽）・呂′N

雪・4

雪・2

24

0．36
31．8

31・8

27

28

29

1・0
41．5

0．78

0．68

31．0

34．2

43二g6

23：…4

2昌：言。

N：35．5土4．0．D：33．2土5．7．
ThefastturnoverofACh−Rmeasuredbyerabutoxinbindingmaybeduetotheweakbindingofthe
toxintothereceptorsites．Someofthebounderabutoxincouldbereleaseddirectlyintothemediumwith−outdegradation．Nevertheless，thereseemsto besignificant differencein theseturnoverrates between
dystrophicandnormalmuscles．
TheresultsobtainedintheseexperimentssuggestthattheACh−Rinadevelopingdystrophicmuscle
hasdistinctpropertiescomparedwiththatinanormalcontrol：1）densityoftheACh−Ronthemembrane
isless，2）ithasweakafnnitytoerabutoxin，and3）itexhibitsLasterturnovertThesefindingsareconsistent
withthepreviouslyknownfactthattheturnoverofmuscleproteinsIS，1ngeneral，enhancedindystrophic
muscle．
、

1）Hotta，K．，Tomita，N．，andSugiyama，H．：CurrentResearchinMuscularDystrophy，Japan，2，Pp・29−30，1981・
2）Steinbach，J．H．，Merline，J．，Heinemann，S．，andBloch，R．：ルoc．NatL．Acad・Sci・U・S・A・，76，3547−3551，1979・
34

九九∫C〟ねr恥什qpカγGrO叫），JタβJ

FREEZE−FRACTUREANALYSIS OFTHE CAVEOLAEIN CHICKSKEI．ETALMUSCLESYuzoIwasakiandHirokoIshii
NationalCenterforNervous，MentalandMuscularDisorders，Kodaira187
Lastyearwereportedthatincreaseinnumberandlossoforderlyalignmentofcaveolaeontheplasma

membranemightprecedethedevelopmentoffiberdegenerationindystrophicchickskeletalmuscles．For
thefurtherunderstandingofthesignificanceofthesecaveolarchangesindystrophicmuscles，Weanalyzed
thesizedistributionofcaveolaeinskeletalmusclesofdeveloplngnOrmalcontroIchickensandcompared
theresultswiththoseobtainedffomage一matCheddystrophicchickens・Asreportedpreviously，SuPerfiCial

PeCtOralisandadductorlateralmusclesofnewlyhatched，2and5weekoldchickensoflines412（COntrOl）
and413（dystrophic）wereobtainedunderpentobarbitalanesthesia．Smallpiecesofmuscletissuewere
immediatelyimmersedin2・5％glutaraldehydeinphosphatebufferedsaline（pH7．4，285mOsm）．After
fiXationinglutaraldehydefbrafbwhoursat40C，thetissueswereglycerinatedin25％glycerininPBSand
quenchedinliquidnitrogencooled Freon12．Thetissuewasffacturedat−1150cunderavacuum of
1−2×10，7TorrwithanEikofreezeetchdevice・Theffacturesurfacewasimmediatelycoatedwithplatinum

andcarbon．Thereplicas wereretrieved after digestionwith acommercialbleachsolution andwashedwithseveralchangesofdistilledwater・PhotographsweretakenwithaHitachiH−600electronmicroscope
atamagn抗cationof40，000．Thesizeofcaveolaewasdeterminedbymeasuringthesmallestdiameterandenlarglngittoafinalmagnificationof120，000withtheaidofaKontronImageAnalyzer．
Basedontheirdiametersthecaveolaeofchickskeletalmusclesseemedtobedividedintotwogroups；
thosewithadiameteroflessthan50nmandothersmorethan50nmindiameter．Thesizedistribution
OfthoseineachmusclegrouplSSummarizedinTablel・Exceptfbrthesuperficialpectoralismuscleof
newlyhatchedchickens，thecaveolaeareapparentlyheterogeneousinsize；Smallandlargearemixedin
Variousratios・Themoststrikingfindingindystrophicmusclesistheincreaseddensityofcaveolaeinsuper−
ficialpectoralismusclesof2and5weekoldchickens；inthelateraladductormuscleof5weekoldchickens
thisislargelyduetotheincreasednumberofsmallcaveolaelessthan50nmindiameter．ThisobservationagreeswiththereportonhumanmusculardystrophybyBonilla，Fischbeck，andSchotlandl）．Eventhough
theunderlyingmechanismsofmuscledegenerationmayd騰rinhumanandchickmusculardystrophy，itisofinterestthattheincreasednumberofsmal1Caveolaedevelopedasanearlychangeofmuscledegen−erationinboth．Wearestilluncertainabout thephysioIogiCalroleofcaveolaeinskeletalmuscles．The
PreSentObservationmaysuggestthatacertainpopulationofsmallcaveolaecanbeassociatedwithab＿normaltransmembranetransportsonlymanifestedunderpathologicalconditions，ifsuchexist．Itisalso
interestingthattheplasmamembraneofthesuperBcialpectoralismuscleofnewlyhatchedchickenswas

dominatedbysmallcaveolaeevenincontroIchickens・Sinceitisknownthatthesemusclesareverylm−
matureatthetimeofhatchingwithasmall丘berdiameterandpoorlydevelopedT−SyStem，itcanbeassumed

TABLEl．SizeDistributionofCaveolaeTime

MuscleSuperficialpectoralis
Lateraladductor
SuperfiCialpectoralis
Lateraladductor
SuperfiCialpectoralis
Lateraladductor＜25nm

25−19nm50−74nm0＊（40）

97（102）4（15）138（37）34（81）
206（87）155（11）15（2）62（85）
60（71）
86（56）
39（55）
102（108）

0（0）
0（0）
0（0）
2（0）
26（0）

＊Dystrophicchickens，OcontroIchickens，PerlOFLm2．

35

75nm＜

thesesmallcaveolaerepresentincreasedpinocytoticactivitytocompensateforthefunctionofthisimmature
T−SyStem．

1）Bonilla，E．，Fischbeck，K．，andSchotland，D・L・：Am・J・hthol・，104，167−173，1981・

36

肋∫C〟ねr勒∫汀叩力γGro岬，JタβJ

FREEZE．ETCH REPLTCA STUDY OF THE CYTOSKELETONIN SKELETAL MUSCLE FIBERS
HarunoriIshikawa，JiroUsukura，ShoichiroTsukita，andSachikoTsukita
DepartmentofAnatomy，FacultyofMedicine，UniversityofTokyo，Tokyol13
Thecytoskeletonmaybedefinedasacytoplasmicstructurewhichmechanicallysupportsthece11and

ismainlycomprisedofmiCrotubules，intermediatefilamentsandmicrofilaments．Inmusclecellsthecyt0−
SkeletonseemstobeinvoIvedinthecontractileactivitiesandoverallorganizationofmyofibrilsandmem−
branoussystems．Wehavebeenstudyjngelectronmicroscopicallytherolesofthecytoskeletalcomponents
inthedifftrentiationanddevelopmentofskeletalmusclecellsinculture．Thereisatransientincreaseof
microtubulesandintermediatefilamentsduringtheearlystageofcellelongationandmyoabri1formation．
Inmatureskeletalmusclesmicrotubules andintermediatefilamentsaresmal1in number，but oftenshow
moreorlessregulardistributionwithrespecttothesarcomerepatternofmyofibrils．
MorphologlCalstudies on the cytoskeleton have been carried out mostly by thin−SeCtion electron
microscopy．Recentprogressinafreeze−etChreplicamethodhaspromptedustousethismethodtoultra−
struCtural1yanalyzethecytoskeletalcomponentsinmusclecellswithspecialrefbrencetomyogenesis．In
particular，thedeep−etChreplicamethodcombinedwith rapid freezlnglSmOSt pertinenttothree−dimen−
sionallyobservetheultrastructuresofunfixed，freshtissues．However，OneSeriousprobleminthismethodarisesffomthefactthatonlythesuper丘Ciallayeroftissueisrnorphologically well−preServedandfreeofanydetectableicecrystals，eVenbyrapidfteezlng．Itisdifhculttoexposetheinterioroftissuewithinthis
superficiallayerforfreeze−etChreplicationincommerciallyavailableffeeze−etChdevices・Toovercomethis

dimcultywehavemadesometechnicalimprovements，themaJOrpOintofwhichistoapplycryomicrotomy
tothe丘eeze−etChreplicamethodl）．
Asmal1pieceoftissueismountedonaspecimenholder（Fig．la）andrapidlyfrozenbybringingthe
tissuesampleintocontactwithacopperblockcooledbyliquidhelium2）．Thesamplethusfrozenisbrought
intoacryokit（FTC／LTS−2）fittedtoaSorval1MT−2ultramicrotomeandcutwithaglasskniftwitha
settingoflFLmthicknessat−1100Ct0−1200ctoremovetheoutermost，meta1−COntaCtedsurfaceofthe
sample（Fig．lb−1）．Thepreftrabledepthofremovalisusually3to15fLmforsamples鉦ozenatthetem−
peratureofiiquidhelium．Sampleswithanexposedsurfacearetransfbrredtoaconventionalfreeze−etChdevice（EikoFD−2S）inwhichtheexposedsurfaceisdeeplyetched（Fig．lb−2）andthenrotary−Shadowed苧︸

withplatinumandcarbon（Fig．lb−3）．Replicasareobtainedbycleaninginhouseholdbleach，＿arepicked

uponformvar−fi1medcoppergridsandthenexaminedunderaHitachiHU−12electronmicroscope．

■■

1

2

FIG．1．Improvedrapid−freeze，deep−etCh replicamethod．a：un触ed，fresh tissuesamplesonspeCimenholders・b：（1）cuttingasampleinacryokittoexposethetissuewithinthesuper鮎iallayer，（2）deeplyetchinginan

etchreplicadevice，and（3）rotary−Shadowingwithplatinumandcarbonforreplication・
37

eeZe−

FIG．2．Freeze−etChreplicafeatureofunfixed，freshskeletalmuscle．Thickandthinmyofilamentsareclearlyvisible
withinthemyofibrils．A：Aband，I：Iband，Z：Zdisc．Frogsartoriusmuscle・

Forpreparingfreeze−etChreplicasofskeletalmuscles，Smallstripsofmuscletissueswerefreshlydis−

Sectedandfastenedinanextendedstateontospecimenholders（Fig．1a）and，Withoutchemicalfixation，
were丘Ozenrapidlyatthetemperatureofliquidhelium（40K）．Theultrastructureofmyofibrilsasseenin
replicaswasbasicallythesameasthatinthinsections（Fig．2）．Thethickandthinfilamentswithinmyo−
fibrilswereclearlyvisualizedinthree−dimensionandathighresolution3）．
In summary，Weimproved therapid−freeze，deep−etCh replicamethod by combiningcryomicrotomy
andshowedthefeasibilityofusingthisimprovedmethodforanultrastructuralexaminationofthecyto−skeleton．

1）Ishikawa，H．，Usukura，J．，andYamada，E．：J．ElectronMicrosc．，30，215，1981．
2）Usukura，J．andYamada，E．：Biomed．Res．，2，177，1981．
3）Usukura，J．，Tshikawa，H．，andYamada，E．：J．ElectronMicroscり30，237，1981．

38

肋∫C〟ねr砂∫汁qpわ′Gro岬，J987

I）IFFERENT RATE OF CHANGEINISOFORMS OF MYOSIN HEAVY ANDLIGHT CHAINSDURING DEVELOPMENT OF SKELETAL MUSCI．E
TakashiMikawa，SetsuroEbashi，TakashiObinata，＊TomohMasaki，＊＊・＊＊＊andHiromiOhmuro−Takano＊＊＊DepartmentofPharmacology，FacultyofMedicine，UniversltyOfTokyo，Tokyol13＊DepartmentofBiology，ChibaUniversity，Chiba280＊＊DepartmentofPharmacology，InstituteofBasicMedicalSciences，TheUniversltyOfTsukuba，1baraki300−31＊＊＊TheTokyoMetropolitanInstituteofMedicalScience，Tokyol13

1tiswellknownthatsevqralcontractileproteins，SuChasmyosln，aCtin，trOPOmyOSln，trOpOnln，and
α−aCtinin，havemultipleisofbrms．ImmunOChemiCalandelectrophoreticstudieshaveshownthatthedifl
ftrentfbrmsofeachcontractileproteinareusuallylocatedindifferentce11types，reSPeCtivelyl￣6）．Thus，aspecifiC

kit

ofproteinscharacteristicforeachofthecellulartypesisshowninthedifferentiatedcelLThehypothesisproposedforthemechanismofthisspecificexpressionisthatthegenesofeach

kit

areundercoordinatecontrolduringdifftrentiationofthece11S6−8）．
Inthepresentworkwehaveattemptedtostudywhattypeofcontrolisoperatinginthesynthesisof
myosinmoleculesinthedeveloplngmuSCle・Onemoleofmyosinmoleculeiscomposedofsixmolesofsub−

unitswhichareclassifiedintothreetypesoftwomoleseach：heavychains（HC），phosphorylatablelightchain（PLC），andunphosphorylatablelightchains（UPLC）．Sofar，SixisofbrmSOfHC，threeofPLCandfburofUPLC，WhichwouldbecodedindifrtrentstruCturalgenes，havebeendetectedinthesameanimal．
Althoughabout270typesofmyosinareexpectedfrom therandomassernblyofisofbrmSSubunit，difl
ferentiatedcellscontainonlyafbwtypesofmyosin2−5）．Asmyosiniscomposedofthreetypesofsubunits，
thepresenceofonespecificformofmyosinrequlreStheregulationofthreestructuralgenestobeexpressed
ffomthegenepooIs．

Weusedanteriorlatissimusdorsi（ALD）muscleofchicken，becausethismuscleinanadultanimalis
predominantlycomposedofonecellulartype，theslowtwitchmusclefibers，andcontainsonlyoneisofbrm
OfHC，PLC，andUPLC5）．
Totalcellularproteinsextractedfromembryonicmuscleatavarietyofstagesarefractionatedbytwo

dimensional（IEF／SDS）polyacrylamide gelelectrophoresis（2D−PAGE）．Asreported previously，SeVen
isofbrmsoftheLCsubunitshavedi能rentmolecularweightsand／ordi飴rentisoelectricpoints5）andhence
eachofthemismappedatdi能rentpositionson2D−PAGE．TheaccumulatedLC

sinembryonicmuscle

wereidentifiedbythreecriteria：1）tobecomigratedwithauthenticLCmarkers，2）tohaveLC−antigenicity，
3）toshowidenticalpeptidepatternSWithLCmarkers bylimitedproteolysis．Resultsindicatedthatin
embryonicALDmuscle，theLCisoformscorrespondingtothefastmuscleform（fPLCandfUPLC）were
expressedinadditiontotheslowmusclefbrmS（sPLCandsUPLC）whicharespecificfbrthedi能rentiated
ALDmuscle．Ataveryearlystageofdevelopment（10daysembryonicstage），fPLCandsUPLCcouldbe
faintlyseenincombinationwithpredominantlyfUPLCandsPLC．Thiscombinationof theLCpatterns
ofmyosinwasalsoseeninembryonicALDmuscle．ThespotoffPLCfirstbecamefainterandthenwasno
longer detectableinmuscle ofthe17−day embryo．The sUPLC／fUPLC ratio graduallyincreasedwith
development，andfUPLCwasstilldetectableataftwdayspost−hatching．Thus，fPLCandfUPLCdis−
appearatdifferentratesfromtheALDmuscleasitdevelops．Theseresultssuggestthatatsomestageof
ALDmuscledevelopment，ahybridmyosinmoleculemightexistwhichiscomposedoffastfbrmLCin
combinationwithslowfbrmLC．Toconfirmthisfurther，WeeXaminedtheassemblyofHCandLCby
non−denaturantpyrophosphategelelectrophoresis（PPi−PAGE）9）．Resultsindicatedthatmyosininadult
ALDmusclemlgratedasoneband，WhereasembryonicmyosinofALDmuscleshowedadditionalbands
39

（emb・M），besidestheadulttype（ad．M）．Theemb．M／ad．Mratiowasgraduallydecreasedwithdevelopment，
butemb．MwasstilldetectableinALDmuscleat16weekspost−hatching．AstheLCsexpressedatthis
StageWereOnlyslowmuscleform，thedif托rentmobilityofemb．Mfromthatofad．Mmight beduetothedifftrenceofHC．ThisislikelybecausethelimitedproteolyticpeptidemapsofHCfromemb．Mand
ad・Maredistinctlydifferent．TheHCpeptidemapsofad．Minembryonicmusclebycleaningwithpro−
teolyticenzymewasidenticalwithad．Minadultmuscle．Thead．Mofembryonicmuscle，however，COn−tainsafastformofLC．ltislikelythatinembryonicALDmuscle，hybridmoleculesmightexist．Inaddi−tion，thechangeinHCexpressionfromtheembryonictotheadulttypetakesalongertimecoursethanthe
CaSeOfLCsubunits．Theseresultsclearlyindicatethedifferentratesofexpressionamongthethreesub−
unitsofmyosinmoleculesduringdevelopment．Theaccumulation ofeach subunitcan becontrolled ata
numberofstepssuchasreplication，SPlicing，tranSlation，pOSt−tranSlationalmodification，aSSembly，andfi1amentformation・Theresultspresentedheresuggestthatthegenesofeachsubunitarenotundersimple
COOrdinatecontrol；SOmeStePS beforeassemblymaydeterminetherateofaccumulationofthesubunits．
1）Dhoot，G．K．，Frearson，NりandPerry，S．V．：Exp．CellResり112，339，1979．

2）Rubinstein，N．A．，Mabuchi，K．，Pepe，F．A．，Salmons，S．，Gergeley，J．，andSreter，F．：J．CellBiol．，79，252，1978．
3）Gauthier，G．F．andLowey，S．：J．CellBiol．，74，760，1977．
4）Pette，D，andSchnez，U．：Histochemisfry，54，97，1977．
5）Mikawa，T．，Takeda，S．，Shimizu，T．，andKitazawa，T．：J．Biochem．，89，1951，1981．
6）Dhoot，G．K．andPerry，S．V．：Nat〟re，278，714，1979．

7）A恥ra，N．A．，Daubas，P．，Weydent，A．，andGros，F．：J．Mol．Biol．，140，459，1980．
8）Dhoot，G．K．andPerry，S．V．：Exp．CellRes．，127，75，1980．
9）Hoh，J．F．Y．：fEβ∫上eJJり98，267，1979．

40

肋∫CIJねr旦γ∫汀叩砂GJ℃岬，JタβJ

NUMllER OF ANTITROPONINI STRIATIONS ALONG THE THIN FILANlENTOF ADULTANDFETAI．SKELETAL MUSCLES OF RABBIT
IwaoOhtsukiandYoshimichiOnoyama
DepartmentofPharmacology，FacultyofMedicine，KyushuUniversity，Fukuoka812
ImmunoelectronmiCroscopICStudyon thechicken skeletalmuscleshowedthattroponindistributes
alongthewholelengthofthethinfilamentatregularintervalsof38nm．Thenumberofantibodystriations
alongeach bundleofthinfilamentswasalwaystwenty−fbur．It wasalsoshownthat，in the developlng
myo丘brils，antitroponinfbmSmOrethantwenty−fourstriations．Thisfindingindicatesthatthefinalmatu−
ration ofthinfi1amentin chicken skeletalmuscleconsists ofa shortening ofthelongerfi1amentSinto a
constantlength after hatching．This shorteningofthinfilament occurred muchlaterinthe dystrophic
muscleofchickenthaninintactmuscle．
Thepresentstudywasundertakentoinvestigatewhethertheshorteningofthinfilamentisalsofbund
duringthedevelopmentofrabbitskeletalmuscle．Glycerinatedmusclefibrersfromtheiliopsoasmuscle
ofan adult and a29−day fttus were usedin the experiments．AntitroponinI antiserum from goat was
purifiedbythemethodofethanolfractionation．Twomethodswereusedfbrantibodystaining・Thefirstwasasaponin techniqueinwhich musclefiberwastreatedwithlO，4g／mlsaponin andthenwithanti−
troponinIinarelaxingsolutioncontainingO．lMKCl，10mMMgC12，10mMK−phosphate（pH7．0），3mM
ATP，andO．4mMEGTA．Themusclefiberwasfixedandembeddedinepoxyresinfbrthinsectioning．In
thesecondmethod，thinfilamentsweredissociatedfromthickfilamentsbyhomogenlZlngin therelaxlng
SOlution，treatedwith antibody，and stainedwith uranylacetate．Thesespecimens were observed byan
electronmicroscope（HitachiHS−8）．
AntitroponinTantibodyalwaysformedtwenty−Sixstriationsalongthethinfilamentofadultmuscle
atintervalsof38nm．Ontheotherhand，theantibody formedmorethantwenty−SixstriationsalOngthe
thinfi1amentofthe29−dayfetusasshowninFig．1．Uptothirty−fourstriationscouldbecountedalongthe
bundleofthefilaments．Thelengthorthethinfilamentsineach bundleseemedto be variablebutwas
longerthanthefi1amentoftheadultmuscle．Thesefindingsclearlyindicatethattheshorteningofthefila−rr）entalsooccursduringthedeveloprnentofskeletalmuscleinrabbit．
1）Ohtsuki，Ⅰ．：J．βわCカe肌，．75，755−765，1974．
2）Ohtsuki，Ⅰ∴J∴及bdkm．．85，1377−1378，1979．
3）Ohtsuki，I．：CurrentResearchinMuscularDystrophy，Japan，2，pp．23−24，198l．

41

．︵○暮︷ON︻×︶gH・NEOとpOJGn00uO叫義とS吉讃JOuO叫︶叫SOd呈︼∽OJe葛uェ＜︶U一切udて︺0空き．︻uモOdOと叫lud上美声pUu叫雲SJ叫qq空Snぶて言p・系dE〇七〇︻USn∈一票0︻OJSJ〇一も巴叫OJU叫∈uOJJUO芯．﹁営山肋∫C〟血r旦γ∫Jr（岬わ′Gr（）岬，JタgJ

ISOFORM VARIANTS OF MYOFIBRILLAR PROTEINS DURING MUSCI．E REGENERATIONAFTER COLDINJURY
YutakaShimada，NaqiiToyota，NorikoShimizu，andDonaldA．Fischman＊

DepartmentofAnatomy，SchoolofMedicine，ChibaUniversity，Chiba280＊DepartmentofAnatomyandCellBiology，SUNY，DownstateMedicalCenter，Brooklyn，N．Y．11203，U・S・A．

Skeletalmusclefibers ofadult chickens were fbcallyinjured by aliquid−nitrogen cooled brass rod，and the regeneration ofthe frozen zones were examined byimmunofluorescencemicroscopy and two−
dimensionalelectrophoresis．Transversesectionsofinjuredfiberswerestainedwithantibodiesspecificfor
troponin（TN）components（T，Ⅰ，andC）fromadultchickenbreastandcardiacmuscles，andwithmono−clonalantibodiesMF−21andElOID6raisedagainstadultandembryonicbreastC−prOtein，reSpeCtively．
Anteriorlatissimusdorsi（ALD）muscleofnormalanimalsboundonlyanti−CardiacTN−C，Whilethe
regeneratingmuscle stained positivelyfor breast TN−T，−I，and−CandcardiacTN−T and−C．Normal
posteriorlatissimus dorsi（PLD）musclestainedpositivelywithantibodiesto allbreast muscleTNsub−
units，WhiletheregeneratingmusclereactedstronglywithantibodiestoallbreastTNsubunitsandtocardiac
TN−TanJ−C（Fig．1）．Wehavereportedpreviouslythatal1embryonicskeletalmusclesarestainedwith
antibodiesagainstbreastTNcomponentsaswe11ascardiacTN−Tand−Cl）．Thus，OurpreSentreSultsshowed
thatreactivitiesofregeneratingmyotubesinALDandPLDmusclestoantibodiesagainstTNcomponents
arethesameasthoseseeninembryonicskeletalmuscles．
MonoclonalantibodyMF−21didnotstaincontroIALDmuscle，yetreaCted stronglywithregenerat−
ingALDmyotubes．ElOID60nlystainedregeneratingALDandPLD；uninjuredfiberswereunreactive
fbrthisantibody．
Two−dimensionalelectrophoresisconfirmedthepresenceofisoformvariantsofTNandmyosinduring
regenerationofeachmuscle．Inadditiontoproteinsofeachtype，1nregeneratingALDcardiactypeTN−T
and−CandPLDtypemyosinlightchainSwerefound，andinregeneratingPLD cardiactypeTN−Cwasdetected．TheresultsaresummarizedinTablel．
Fromtheresultsobtainedinthepresentstudy，itisconcludedthatembryonic−likeTN，myOSin，and
C−PrOteinisofbrmsaresynthesizedwithinadultmuscleduringtheregeneration丘omcoldinjury．
TABLEl．RegenerationofMyofibrillarProteins

＊
＊
＊

＋
＋
＋＋＋

一

一＋＋＋

一
＋
＋

43

＋＋＋＋＋

一

＊NotconBrmedbytwo−dimensionalelectrophoresis．

＋

一

PLDLCI
Myosin Two−dimensionalPLDLC2
1ightchain electrophoresis PLDLC3
E ALD−CardiacLCI
ALD−CardiacLC2

＋

十

一

C−prOtein Immuno−（Adultbreast）
貝uorescence ElOI D6（Embryobreast）

＊

＋

＋

4weeks
＋十＋一一

一

MF−21

PLD

lweek

l一一

一

Breast TN−T
Immuno−Breast TN−I
餌orescenceand Breast TN−C
two−dimensional Cardiac TN−T
electrophoresis Cardiac TN−I
Cardiac TN−C

Normal十＋＋＋一＋

4weeks＋＋＋Troponin

ALD

lweek

＋＋＋＋一十Myofibrillar Methods
proteins Normal

Regeneration

ALD

P L D

（1weekafterinjury）

AntiLbreast TN−T

Anti−breastTN−I

Anti−breast TN−C

Anti−Cardiac TN−T

AntiLCardiac TN−I

Anti−Cardiac TN−C

FJG・1・RegeneratingALDandPLD，7daysaftercoldinjury．ALD（a−f）andPLD（g−1）werestainedwithanti−
bodiestoalIskeletalTNcomponents（a−＜andg−i）andtocardiacTN−TandC（d，f，j，andl）（Bar，10FLm）．
1）Toyota，N．andShimada，Y．：J．CellBiol．，91，497−504．1981．

肋∫r〟／dr勒∫Jrqpわ′Gro叩，ノ9βノ

MYOSINISOZYMESINEMBRYONIC

AND

ADULT

CHICKEN

SKl三LETALMUSCLE

TakashiObinata，＊，＊＊HiromiOhmuro，＊＊andTomoh Masaki＊＊・＊＊＊＊DepartmentofBiology，FacultyofScience，ChibaUniverslty，Chiba280＊＊TheTokyoMetropolitanInstituteofMedicalScience，Tokyol13＊＊＊DepartmentofPharmacology，InstituteofBasicMedicalSciences，TheUniversityofTsukuba，lbaraki300−31

Myofibrillarproteinsareknowntoexistinseveraldi能rentisoforms，andmanyrecentstudieshave
demonstratedthattheproteinsinembryonicmusclearediffbrentfromthoseinthenormaL adult tissue．
Changesinmyosinisofbrmsduringmuscledevelopmenthaveparticularlybeenamatterofwideinterest
andanincreasingbodyofliteraturenowexistswhichstronglyindicatesthatthetypeofmyosinpresentin
embryonicskeletalmusclediffersfromthatinhomologousmaturemuscles1−5）・However，theprecisechemi−
Calnatureoftheembryonicvariationsremainscontroversial・Inthepresentinvestigation，WeObserved
thatmyosinisozymesfromchickenembryonicandadultpectoralismuscleare distinguishablefromeach
Other by pyrophosphate（PPi）−aCrylamide gelelectrophoresis，and obtained additional evidences which
SuggeStSthatthelightchaincompositionandpeptidefragmentsofheavychainsdiffersinembryonicand
adultmyosinofthechicken6）．
MyosinwaspreparedfrompeCtOralismuscleofvariousdevelopmentalagesofchickenembryoorposト

hatchedchickenasdescribedearlier2），andembryonicandadultmyosinisozymeinamonomericformwas
examinedbyPPi−aCrylamidegelelectrophoresisaccordingtoHoh6）・AsshowninFig・1，threefastmyosin
isozymes，FMl，FM2，andFM3，Weredetectedinadultchickenpectoralismuscleaspreviouslydemon−
Strated7）（Fig・1e）・Incontrast，WhenthemyosinfromthepectoralismuscleoflO−tO13−dayoldchickembryowasexamined，Onlytwobands，Onemajor（EM3）andonetrace（EM2），WereObserved（Fig．la，1b），
After15daysofincubationanothermyosinisozyme（EMl）appearedinthepectoralismuscleinaddition
toEM8andEM2（Fig・1C）・EMBWaSClearlydistinguishedfromtheadultmyosinisozymesbythediffbrence
inelectrophoreticmobilityonPPi−aCrylamidegel；itmlgratedmoreslowlythantheadultthreeisozymes
andthereforewhenitwasmixedwithadultmyosinisozymes，fourbandswereobtainedonthePPiacryl−amidegel（Fig・1g）・Theelectrophoreticmobilitiesoftheotherembryonicmyosinisozymes，EM2andEMl，
WereClosetothoseofadultmyosinisozymesFM8andFM2，reSpeCtively，butnotexactlyidentical．The
mobilityofEM2WaSalsoveryclosetothatofcardiacmyosinisozyme（CM）．EM2COuldbeanectopic
Cardiac−typemyOSinl）whichappearedintheembryonicpectoralismuscle・Slowmyosinisozymesfrom
theanteriorlatissimusdorsi（ALD）didnotcomigratewithembryonicpectoralis myosinisozymes．The
myosinn

OmthemuscleofalO−dayoldpost−hatchedchickencontainedthreemyosinisozymes（Fig．1d）andthemobilitiesoftheseisozymesonPPi−aCrylamidegelwerenotdistinguishablefromthoseofthethree
adultmyosinisozymeS（Fig・lf）・Ithasbeenpointedoutthatmyosinfromembryonictissueisoccasionally

EM3磯野

EM3

＼EM了
d

e

f

FIG・1・PPi−aCrylamidegelelectrophoresisofmyosinfromthebreastmuscleof10−dayembryo（a），13−dayembryo（b），15−dayFmbryo（C），10−daypost−hatchedchicken（d），andadultchicken（e）・（f）MixtureoflO−dayembryoand
adultmyosln・（g）MixtureoflO−daychickenandadultchickenmyosin．OthersymboIs：SeeteXt．
45

3
2
1
3
2

F二とE F F F

MMMMMM

TAl】LEl．SubunitsinChickenEmbryoandAdultChickenBreastMyosin
Myosinisozymes Heavychains Lightchains
EMHC
Lrl，Lf2，1紬182（L。1，1。2）
EMHC
Lfl，Lf2，151，Ls2（Lel，Le2）
EMHC
しわ，Lf3FMHC
Lfl，Lf2
FMHC
Lfl，Lr2，Lf3
FMHC
Li2，Lf3
1

EMHC，embryonicmyosinheavychain；FMHC，adultfast−typemyOSinheavychain・OthersymboIs：SeeteXt・
contaminated with RNA8），but the diffbrencein electrophoreticmobility between embryonicand adult
myosinisozymeswasnotduetothebindingofRNAtoembryonicmyosinbecausethemobilitiesofem−
bryonicmyosinisozymeswerenotafrtctedbythetreatmentofembryonicmyosinwithRNase・Wecannot
excludethepossibilitythatsomemyosin−bindingproteinsareassociatedwithembryonicmyosinandthere−
forethemobilitiesofembryonicmyosinisozymesdifferedformthoseofadultmyosinisozymes．However，
C−prOtein，OneOfthetypicalmyosin−bindingproteins，COuldnotbedetectedonthemyosinbandonPPi−
acrylamidegel．WeassumedthatthedifrtrenceinthemobilityofembryonicandadultmyosinonPPi−aCryl−amidegelwasmainlyduetothestructuraldifrerenceinmyosinsubunits，andweexaminedbothheavyand
lightchainsubunitsofembryonicandadultmyosin．

Inordertoanalyzethetypeoflightchainspresentineachmyosinisozyme，PPiacrylamidegelswere
stainedfaintlywithCoomassiebrilliantblueandeachbandofmyosinisozymewascutout．Eachgelslice
wasthenappliedfbrSDS−POlyacrylamidegelelectrophoresisafter treatmentwithanSDS−SOlutioncon−
taining2％SDS，2％2−merCaPtOethano15）．Thepatternoflightchainswasvisualizedbymeansofahighly
sensitivesilveraccordingtoOakleyetal．9）．Confirmingthepreviousreport7），themyosinisozymesfrom
adultchickenpectoralismusclecontainedonlyfastlightchains（L仁，Lfも，andLfも）but eachisozymeex−
hibitedaspecificcombinationoflightchains（seeTablel）．Thelightchaincombinationinembryonicmyo−sinisozymeS，EM3andEM2，WaSquitedifferentfromthoseofadultmyosinisozymes：bothembryonicmyosinisozymescontainedtwoslow（Lsl，Ls2）and／ortwocardiaclightchains（Lcl，Lc2）inadditionto
twofastlightchains（Lfl，Lら）（Tablel）．CoexistenceoffastandsIow（Orcardiac）lightchainsinEM3WaS
furtherconfirmedbyanimmunologicalmethod．
Inordertoexaminethestructuraldifferencesintheheavychainsofpectoralismyosininembryonicandadultchickens，myOSinwaspartiallydigestedwithα−Chymotrypsin，andthentheresultantpeptidefrag一

mentswereanalyzedbymeansoftwo−dimensionalgelelectrophoresis6）．Thepatternofthecleavageproductofembryonicmyosinwasdifftrentfromthatofadultmyosin．Mostofthepeptidefragmentsfromem−
bryonicmyosincorrespondedtothoseffomadultmyosin，butembryo−SPeCificpeptidesalsoexisted・Some
ofthepeptidefragmentSfromadultmyosinweremissinginthecleavageproductsofembryonicmyosin・

Thepatternofthepeptidefragmentsfromembryonicpectoralismyosinwasquitedi能rentfromthoseof
slowskeletalorcardiacmyosin．Theseresultsindicate that the peptidestructure ofembryonicmyosin
heavychainresemblesthatofadultbreast（fast）myosin．Inthepreliminaryexperiment，Wedidnotfindany

remarkabledifftrenceinheavychainstructurebetweenembryonicmyosinisozymeS．
Fromthepresentobservations，WeCOnCludethatthemyosinisozymeinchickenbreastmusclecontains
heavychainwhichisdistinctfromthoseinadulttissueasinthecaseofmammalianfetalmyosin5），butwe
furtherconcludethatlightchaincompositionsalsodi能rbetweenembryonicandadultmyosinisozymeS・
1）Masaki，T．andYoshizaki，C．：J．Biochem．，76，123−131，1974．2）Obinata，T．，Masaki，T・，andTakano，H・：J・Biochem．，87，81−88，1980，3）Hoh，J．F．Y．：托βSLelt．，98，267−270，1979．4）Rushbrook，J・Ⅰ・andStracher，A・：
丹oc．Nall．AcadSci．tLS．A．，76，4331L4334，1979．5）Whalen，R．G．，Sell，S．M．，Butler−Browne，G・SリSchwartz，K．，Bouveret，PりandPinset−Harstr6m，I．：NbtLLre，292，805−809，1981．

6）Takan0−Ohmuro，H・，Obinata，T・，

Masaki，T．，andMikawa，T．：J．Biochem．，91，1305−1311．1982． 7）Hoh，J・F・Y・：EEBSLett・，90，297−300，1978・
8）Bari1，E．F．andHerrmann，H．：Dev．Biol．，15，318−333，1967．9）Oakley，B・R・，Kirsch，D・R・，andMorris，N・R・：」〃αJ．βわdk研．，105，1951−1962，1980．
46

肋∫C〟hr砂∫Jr呼わ・Gro岬，J夕βJ

NATIVE HIGH MOLECULAR WEIGHT PROTEIN FROM CHICKEN BREAST MUSCLE
KoscakMaruyamaandSumikoKimura
DepartmentofBiology，FacultyofScience，ChibaUniversity，Chiba280
WehaveshownthatanelasticproteindesignatedconnectinHisresponsiblefbrelasticityandmechani−

Calcontinuityofskeletalmuscle，andthatconnectionconsistsofproteinveryhighinmolecularweight（Onemi11ion）andfbrmingverythinfilaments（2nmindiameter）1）．RecentlyWangandhisassociatesreported
thatthehighmolecularweightproteinthattheycalledtitincanbeisolatedbygelfiltrationffomadirect
SDSextractofintactmusclefibers2）．WeconfirmedWnag

swork，butreachedtheconclusionthattheirtitin

isidenticalwiththeconnectinwepreviouslydescribed3）．Previously connectinwasextractedwithanSDS
SOlutionfromthoroughlyextractedmuscleresiduesU．Inanycase，COnneCtinwasisolatedasadenatured
rom．
Inthepresentstudywehavetriedtoisolateconnectionasanativeformwithoutuseofadenaturing
agent．

Theindirectimmunofluorescent technique revealed that connectinislocatedin the ATIjunction
areaandinI−bandsbutnotinZ−linesl）．Observationsthatsomeconnectiniscoextractedwithmyosin3）andthatbothends ofisolatedAsegmentsarecoveredwithconnectinfilaments4）suggestthat someofthesefilamentsplayaroleinlocatingtheA−bandinthecenterofasarcomere．Baseduponthisview，Wepartially
SuCCeededinreleaslngCOnneCtinfi1amentsintoasaltsolutionbysuddenlycontractingswollenmyofibrils
withtheadditionofsalt．
Chickenbreastmusclewasfreshlycutout，andth占myofibri1Swerepreparedbywashingthemmore

thantentimeswith50・mMKCIcontaininglmMNaHCO3．Theywerethenwashedexhaustivelywith5−
10mM NaHCO3tO remOVe Water−eXtraCtable proteins such as alpha−aCtinin．Followlng this，the myo−
fibrilswerewashedwithpurewatertoreducetheionicstrengthtoapproximatelyO．001，andtheybecame
SWOllen．NaCIwasrapidlyaddedtoafinalconcentrationofO．2M，WhilebeingvlgOrOuSlymixed．Afterone
hourextraction，thesuspensionwasfiIteredthroughasheetoffiIterpaper．Thefiltratecontainedthehighmolecularweightprotein（HMWprotein）togetherwithsomemyosinandactin．
TABLEl．AminoAcidComposition
Connectinftom

HSext3J

′ b
1
l
′ b 7
7
7
1
5 7

3 ′ b 7 0 0 7 ′ b 5 ／ b 0 0 ′ 0 ′ b ′ b

0 ノ ′ D

O
3
2 7
1
5

O ノ q ノ 0 0 094776008739
71
41
糾6
10
26
865
21
95
25
85
8
1

HMW Whole3J Residue3）9575691674716211851059673027821555
102716822∬1872
乃7
刀刃68ガ罰771447
1197726116朋81824ガ47乃75〃31月1850

AS︒mr監伽豊野豊Leu監禁毎

Actin3）

ThewayofextractingexclusivelyHMWproteinhasnotyetbeenestablished，butoccasionallymore
than90％purityhasbeenobtainedusingSDSgelelectrophoresiscriteria．Usingthissample，SOmepre−
liminarycharacterizationhasbeenperfbrmed．
Fromthemobilityin3％acrylamide gelelectrophoresis，the apparentmolecularweight ofHMW
PrOteinwasmorethanonemillion，Whichisingoodagreementwith the values ofconnectin prepara−tionsl，3）．UVabsorptionspectraweretyplCalofproteinnaturewithamaximumat280nm．Theaminoacid
COmPOSitionwasalmostidenticalwiththatofisolatedconnectininadenaturedstate（Tablel）．Althoughtheaminoacidcompositionofthenativeconnectinwassimi1artothatofactin，SeVeralaminoacidcom−
ponentsweredistinctly difrtrent（Tablel）．Theviscosity ofnative HMWprotein was verysmallwhen
measuredbytheusualOstwaldtypeofviscometer：1essthanl．lcpforO．2mg／mlat avelocitygradient
Ofl，000ec．1．However，ataVerylowvelocitygradient，e．g．0．0008sec．1，meaSuredinaLow−ShearRheo一

meter，itwasaslargeas30，000cp．Thissuggeststhatthreedimensionalnetsarefbrmedinsolutionandthe
StruCtureiseasilybrokendownbyaweakexternalfbrce．Thisisnotthecasewithdenaturedconnectin
SOlutionin1％SDS．Infact，thefrozenreplicamethodrevealedthatthethreadformedbyconcentration
COnSistedofverythin丘laments．Furthermore，negativelystainedHMWproteinsolutionexhibitedafila−

mentousimageunderanelectronmicroscope．Moreworkisinprogresstoelucidatethestructureandfunc−
tionofnativehighmolecularproteinaswellasitspossibleinteractionwithmyosinfi1amentS．
1）Maruyama，K．，Matsubara，S．，Natori，R．，Nonomura，Y．，andKimura，S．：J．Biochem．，82，317−337，1977．
2）Wang，K．，McClure，J．，andTu，A．：ルoc．Natl．Acad．U．S・A．，76，3698−3702，1979・
3）Maruyama，K．，Kimura，S．，Kuwano，Y．，andOhashi，K．：J．Biocke〝肌，89，701−709，1981．

4）Ohashi，K．，Fischman，D．A．，Obinata，T．，andMaruyama，K．：Biomed．Res．，2，330−333，1981．

48

肋∫C〝ねr勒∫汁q〆γGrO〟p，JタβJ

APROTEININCHICKENBREASTMUSCLEWHICHINHIBITSTHEADP−RIBOSYLATIONOF
EF2BY DIPHTHERIA TOXINムV17TRO
TomohMasaki書，＊＊andAkiraYoshikawa＊＊＊DepartmentofPharmaco】ogy，InstituteofBasicMedicalSciences，TheUniversityofTsukuba，1baraki300−31＊＊TheTokyoMetropolitanInstituteofMedicalSciences，Tokyol13

Wereportedontheactivationofproteinsynthesisdirectedbypoly（U）inthepresenceofproteinfactors
inthelOO，000×gSupernatantOfdystrophicchickenmuscleinapreviouspaperl，2）．Toclarifythemecha−
nismofactivationofproteinsynthesisindystrophicchickenmuscle，WemeaSuredtheamountofelonga−
tionfactor2（EF2）inthelOO，000×gSupernatant・TheEF2contentcanbeconvenientlydeterminedby
measuringtheamountofl14C］ADP−ribosylEF2fbrmedbydiphtheriatoxincatalysisbecauseEF2isthe

Onlyproteinwhichisknowntoberibosylatedbydiphtheriatoxin3・4）．
InsomepreparationwefoundthatEF2wasnotalwaysribosylatedbydiphtheriatoxinandthatin
thesesamplesaproteinwhichinhibitedtheADP−ribosylationofEF2bythistoxin（INH）wascontained．
To furtheridentifythisprotein，purifiCationandcharacterizationofINHwas performed．Sofaraswe
know，INHisnotasubstancewhichhaspreviouslybeenknown．
Wepuri丘edINH as fbllows：Chicken breastmusclewashomogenizedwith2volumes ofbufftrA（0・lMKCl，10mMMg（OAc）2，20mMTriS−HCl（pI17．4），6mMβ−merCaptOethanol，andO．25MSuCrOSe）
inaWaringblender．The・homogenatewascentrifugedatlOO，000×gfor15min．Thesupernatantwascen−
trifugedat40，000rpmfbr4hrinaBeckman60Tirotor．Thesupernatant（100，000×gSup）wasfhctionated

▲■

一

mIl−■

byammoniumsulfateprecipitationandtheffactionbetween50−75％saturationwascollected．Thepre−

8︵TOtX邑U︶Nh甲田dq三U ﹈0
6
4︵TOlX︶．き．∑

0

0．2

0．4

0．6

0．8

1．O

0．03

0．1

0．3

1．O

EF2（mg／ml）

Relative mobility

FIG・l（left）・Molecularweight ofINH determinedbySDS−pOlyacrylamide gel（10％）electrophoresis by themethodofWeberandOsborn5㌧Acolumn：INH・Bcolumn‥phosphorylaseB；94，000，13SA；67，000，OValbumin；

43，000，Carbonicanhydrase；30，000，lactoalbumin；14，400，andlNH．
FIG・2（right）・Inhibitionofformationofl14C］ADP−ribosylEF2byIPH・ThereactionmixtureinO・lmlcontained

4pmoIDTT，5FEmOITriS−HCl（pH7・4），0・lmgBSA，2FLmOlhistamlne，56pmol【14CJNAD（534mCi／mmol），3jLg
diphtheria toxin，40FLgINH，and various amounts ofpigliver EF2．Theactivity ofINHisdecreasedwiththeincreasingamountofEF2．
49

cipitatewasdissoIvedinbufferB（0・1MKCl，20mMTris−HCl（pH7・4），6mMβ−merCaptOethanol，lmM
EDTA，andO．25MSuCrOSe）andappliedonaSephacrylS−200Column（2・6×95cm）・ThepeakwhichshowedinhibitingactivitywasfractionatedagainbyammOniumsulfateandthe丘actionbetween66・5−

75％saturationwascollected．TheprecipitatewasdissoIvedanddialyzedagainstbu飴rBandappliedon
ahydroxyapatitecolumn（2×10cm）・TheeluatebetweenO・1andO・2Mphosphatebufferwasco11ected，tO

whicharrunoniumsulfatewasaddedto60％saturation．INHwaselutedwithlOmMTriS−HCl（pH7・4）・
TomeasureEF2content，thereactionmixturecontained4pmoIDTT，5pmoITris−HCl（pH7・4），
0．1mgBSA，2FLmOlhistamine，56pmoll14C］NAD（534mCi／mm01），3FLgdiphtheriatoxin，21FLgpigliver
EF2，andvariousamountsofINHorlOO，000×gSupinO・lml・Incubationwascarriedoutat37OCfbr

15min．AnaliquotofthereactionmixturewasspottedonaWhatman3MMfi1terpaperdiskandwashed3timeswithcold5％TCAandoncewith99％ethanol．Afterdryingthepaperdisk，【14C］ADP−ribosyl
EF2wasmeasuredbyaliquidscintillationcounter・
ThemolecularweightofINHis40，000daltonsasdeterminedbySDS−POlyacrylamidegelelectro−
phoresisbythemethodofWeberandOsborn5，（Fig・1）・INHisalsopresentinliveraswellasinmuscle，
howevertheamountseemstobelessthanthatinmuscle．SDS−gelelectrophoresisshowsthatchickenem−
bryonicbreastmusclecontainsonlyasmallamountofINHandtheamountincreaseswiththestageof
development・ThisisconfirmedbythefactthattherateofADP−ribosylationofEF2bydiphtheriatoxin
inthelOO，000×gSupfromembryonicchickenishigherthaninthatfromadultchicken・ItisalsoprobablethatthecontentofEF2inachickenbreastmuscleatayoungerstageofdevelopmentmightbelargerthan
thatofadultchickensbecausetheinhibitingactivityofINHisblockedbyincreaslngamOuntSOfEF2（Fig．2）andnotbyanincreasingamountofdiphtheriatoxinorl14C］NAD・INHhasnoADP−ribosylEF2hydrolaseactivity・ThissuggeststhatthemechanismofinhibitionbyINHmaylnVOIvebindingatthe
specifiCsiteofEF2wherediphtheriatoxinbindsandINHshowscompetitiveinhibition・Innormalchicken
breastmuscle，theinhibitingactivityofINHappearsat20r3weeksafterhatching，Whileindystrophic
chickenbreastmuscleitappearsat40r5weeksafterhatching・RibosylationofEF2inthe3−Weekold
dystrophiclOO，000×gSupisinhibitedbyadditionof3−Weekoldnorma1100，000×gSupWhichshows
INHactivity．
Infuturewemustclarifythequestionofwhetherornotthisphenomenonisdueonlytodevelopmental
delayofdystrophicmuscleasshownbyNonakaeEal・，Takedaetal・，andObinataelal・6．8）
Withtheexceptionofmono−ADPribosylation，manyrePOrtSaboutpoly−ADPribosylationhavebeen
published．Thoughthephysiologicalsignificanceofpoly−ADPribosylationisstillunclear，SOmerepOrtS
havesuggestedthatpoly−ADPribosylationofhistonesisrelatedtothedifferentiationofcells9）・
1）Masaki，T．andYoshikawa，A．：CurrentResearchinMuscularDystrophy，Japan，pp・22−23，1977・
2）Yoshikawa，A．andMasaki，T．：J．Biochem・，90，1775−1786，1981・
3）Hopjo，T．，Nishizuka，Y．，Hayaishi，0・，andKato，T・：J・Biol・Chem・，243，3553−3555，1968・
4）Hayaishi，0．andUeda，K．：Ann・Rev・Biochem・，46，95−116，1977・

5）Weber，K．andOsborn，M．：J．Biol・Chem・，244，44064412，1969・
6）Nonaka，Ⅰ．andSugita，H．：SaLshEfZなaku，5，907−915，1980（ilZJqpanese）・
7）Takeda，S．andNonomura，Y．：Biomed・Res・，1，176−179，1980・
8）Obinata，T．，Ohmuro，T．H．，andMatsuka，R．：EEBSLett・，120，195−198，1980・
9）Caplan，A．I．andRosenberg，M．J．：丹oc．Natl・AcadlSci・tLS・A・，72，1852−1857，1975・

50

ルh∫CJ血r勒∫／r叩わ′Gr（）岬，JタβJ

A TROPHICINTERACTIONINI）ENERVATED MUSCLES FROM THE STANDPOINT OFRECOVERY OF Ca−UPTAKE ABILITY OF FSR
ToshioSakai，HiroakiNisllijima，andTakayukiImai
DepartmentofPhysiology，TheJikeiUniversitySchoolofMedicine，TokyolO5
In previOuspapersl・望），Wehavereported acharacteristics ofCa−uPtake ability of丘agmented sarco−

plasmicreticulum（FSR）inM．pectoralis，fastposteriorlatismus（PLD）andanteriorlatismusdorsi（ALD）
OfdystrophicchickenandalsohavedescribedamodifiCationofthepreparationmethod．

ItisknownthattheM．SoleusandM．extensordigitorumlongues（EDL）ofratlegmuscleareequiva−
lenttothesIow andfastmuscle，reSPeCtively，Whichexactlycorrespond to ALD and PLDin chicken．
Therefore，in the present experiment，We meaSured the Ca−uPtake ability of fragmented sarcoplasmic
reticulumfromEDLand M．soleusin atrophyinduced by denervation orin recovery by reinnervation．
FromtheresultsobtaineditwasconsideredwhetherornotthechangesofCa−uPtakeabilityofFSRffom
dystrophicmusclehaveananalogousconnectionwiththoseffomdenervatedmuscle．Further，aPrOCedureof denervationandreinnervation on nervesinnervated tothe EDL and soleusmusclewere done at sites
differentdistancesfromthemuscle，t．e．，attheterminalbranchtoeachmuscle，（EDL−N．tibialis，M．soleus−
N．fibralis）and at the N．ichiadicin which several sensory and motor nerveS areinvoIved．Usingour
modifiedmethod，thedifferencein Ca−uptake abilityofFSRfrom denervated and reinnervatedmuscles
WaSCOmpared3・4）．
IntheseparationofFSRffomthedenervatedmuscleinthelowerlegofrats，incontrasttothatofdys−
trophicmusclesnochangesofpHintheprocessofhomogenizationorofnormalmuscleweredone．The
yieldofH−fhction（8，500−36，500×g）andL−fraction（above36，500×g）ofFSRfrommuscleunderdenerva−

tionfor4weekswasmuchthesameaSthatfromnormalmuscle．Therefore，WeCOnjecturedthatintheSR
Ofdenervatedmuscletherewerenomembranedistractionswhichcouldbeseensuchasthoseindystrophic
muscle．

In the present experiments themaximumamOunt OfCa−uPtakeonFSRfromthesoleusmusclewas
about180×10．9mol／45sec；什omEDL，itwas240×10●9mol／mgprotein／30sec．TheamountofCa−uPtake

OnFSRfromthesoleusmusclewas，therefore，lowerthanthatfromEDL．ThislowervalueofCa−uPtakeon

100

90

0
0

︵訳︶む駕言ヲdU

80

70

60

0

1

2

3

4

5

6

7

8

0

9101632

Weeks afterinnervation

1

2

3

4

5

6

7

8

91016

32

Weeks afterinnervation

FIG．1．ChangesofCa−uptakeabilityofFSRfromEDLorsoleusmusclesbydenervationofN．tibialisorN．fibralis（一〇一）andN．ichiadics（−●−）．
51

nU︵訳︶良三dn・帽U

0

1

2

3

4

0

1

2

3

4

Weeks ofdenervation

Weeks ofdenervation

FIG．2．ChangesofCa−uPtakeabilityonFSRfromEDLorsoleusmusclesbyreinnervationofN．tibialisorN．fibralis（−O J andN．ichiadics（−●−）．

FSRfromthesoleusmusclecouldalsobeseenonFSRfromALDofthedystrophicchicken．
ThedegreeofchangeinCa−uPtakeabilityinthedenervationprocesswasthesameinFSRfromEDL
andfromthesoleusmuscle，Whenonlytheterminalbranchtoeachmusclewascut（Fig．1）．Butwhenthe
ichiadicnerveWaSCut，the Ca−uPtakeability ofFSRffomEDL decreasedmoremarkedlythanthat of
FSRfromthesoleusmuscle．AIso，theCa−uPtakerateintheH−fractionandthatintheL−fractionofFSR
fromPLDandM．pectoralisofdystrophicchickenwasreducedmorethaninALD．Fromtheseresults，lt
WaSaSSumedthatthefunctionofCa−uptakeonFSRfromfasttypemusclewasinhibitedmorethanthat
ffomslowtypemuscles．
In orderto observeareinnervationeffect，thechanges ofCa−uptakeability ofFSRfrom therein−
nervatedmusclewerestudied．Evenifthe terminalbranch ofthenerve to thesoleusandEDLmuscles orN．ichiadicwasreinnervatedsoonafterdenervation，theCa−uPtakeabilityofFSRfrombothmuscleswas
SupPreSSedandwasminima13weeksafterreinnervation（Fig．2）．
When theN．ichiadics werecut，SuPpreSSion ofCa−uPtakeability ofFSRfrom bothmuscles，EDL
andM．soleus，WaSmOreremarkablethanthatofthefbrmerexperimentalresultmentioned．Butthesup−
pressedvaluesofCa−uPtakeabilityonFSRasshowninFig．2increasedslowlyupotthatofnormalmuscles
aftershowingtheminimumat3weeksofreinnervation．
TheseresultsseemtosuggestthattheCa−uPtakeabilityofFSRrecoveredwithoutregardtodistance
betweencuttingpointofthenerveandmusclesandthenumberoffuniculesinthenerve．Insummary，We
WOuld suggest that trophicinteraction plays aprominent role between themuscle and nerve on thosemuscleswhichwereexperimentallytestedforprocedureofdenervationandinnervation．
1）Sakai，T．，Nishijima，H．，Iwagaki，S．，andHattori，A．：CurrentResearchinMuscularDystrophy，Japan，pp．45−16，
1979．

2）Sakai，T．，Nishijima，H．，Iwagaki，S．，Imai，T．，andWatanabe，M．：Current Researchin MuscularDystrophy，Japan，pP．41−−42，1980．
3）Nishijima，H．，Ito，Y．，andKuriyama，H．：froc．J（Pa〝Acad．，49，367−371，1973．

4）Nishijima，H．，Tani，Y．，andOgino，U．：JikeikaiMed．J．，25，287−290，1978．

52

肋∫C〟ねr功JJr（やわ′Gro叩，J夕βJ

CHANGESIN PROTEASE ACTIVITIES DURING DEVELOPⅣlENT OF CHICKEN MUSCLES
KenjiTakahashiandYoshikazuIchihara
Departmentof13iochemistry，PrimateResearchTnstitute，KyotoUniverslty，Inuyama484
Therehavebeenftwreportssofaronthe developmentalchangesinthelevelsofproteaseactivities
inmusculartissues．Inordertogainsomeknowledgeaboutsuchchanges，Weinvestigatedthechangesin
thelevelsofcertainproteaseactivitiesduringtheearlystageofdevelopmentofchickenmuscles．
Muscles were obtained from14−day and19−day（i．e．，1−day befbre hatching）embryos and7−day
ChickensofWhiteLeghorn．Eachmusclewas homogenizedin5volumes of5mMHepes buffer，pH7．5，
COntainingO．25MSuCrOSeandlOmM2−merCaPtOethanolwithaPotter−EIvehjemtypeglass−Teflonhom0−
genizer．ThehomogenatewascentrifugedatlO，000×gfbrlOminandthesupernatantwasfurthercentri−
fugedat78，000×gfor60min．Thesupernatantfractionthusobtainedwasusedforproteaseassay．The

precipitate（microsomalfraction）waswashedwithlMKCltoobtainlMKC1−insolublemicrosomalfrac−tionwhichwasthoughttocontainfirmlymembrane−boundproteases，andthesefractionswerealsoused
forproteaseassay．CathepsinDactivitywasmeasuredatpH3．Owithhemoglobinasasubstrate．Dipep−
tidylaminopeptidaseIVactivitywasdeterminedatpH7．5withglycy1−L−prOline−4−methyl−COumary1−7−amideasasubstrate．Aminopeptidaseactivitieswere determinedatpH7．0with2−naphthylamidesofL−alanine，
L−leucine，andL−arglnlneaSSubstrates．

Thespecificactivities（activity／proteinweight）ofcathepsinDofthesupernatantfractionsoflegand
breastmusclesdidnotchangesignificantlyduringtheperiOdexamined，Whereasthoseofheartandgizzard
musclesshowedamarkedincrease：thoseofthe7−daychickensweremorethanfivetimeshigherthanthe
14−dayembryos．Similarbutlessmarkedincreasesintheactivity（aboutl．5−2−fold）werealsoobservedfbr

themicrosomefractionsofheartandgizzardmuscles，andwiththelMKC1−WaShedmicrosomefraction
Ofgizzardmuscle．ThespecificactivityofthelMKCl−WaShedmicrosomefractionofheartmuscle，however，
decreasedsomewhattowardhatchingandthenmaintainedaconstantlevel．Ontheotherhand，thespecificactivitiesofthemicrosomeandlMKCl−WaShedmicrosome丘actionsoflegandbreastmusclesshoweda
gradualdecreaseduringdevelopment．
Thespecificactivities ofdipeptidylaminopeptidaseIVofmostmusclefractionsshowedageneraltend−
encytoincrease toward hatching，andafterhatchingto keepaconstantlevelortograduallydecrease．A
notableincrease（about4−fbld）inspecificactivitytowardhatchingwasobservedfbrthelMKCl−WaShed
microsomefractionofgizzardmuscle．ThismayreflectthefactthatdipeptidylaminopeptidaseIVisthought
tobeoneofthemicrosomalmembrane−boundenzymes．
Thechangesinthelevelofalanineaminopeptidaseactivitywereroughlysimilartothoseofdipeptidyl−
aminopeptidaseIV．InmostcasesthespecifiCactivityoftheaminopeptidaseincreasedtowardhatchingand
afterhatchingitwasalmostconstantordecreasedgradua11y．Simi1arpatternsofactivitychangesduring
developmentwerealsoobservedforleucineaminopeptidaseandarginineaminopeptidase．
Sofarasexamined，thespecificactivities ofproteasesin1egandbreastmuscleswereverycIoseand
Changedalmostinparallelwitheachother，Whereasthoseofheartandgizzardmusclesweredif托rentand
Changedseparatelybothffomeachotherandfromlegandbreastmuscles．ThismayreflectthedifRrencein thetypeofmuscles andsuggeststhat theremaybesignificantdifftrenceinproteinCatabolisminthe
early stage ofdevelopment among these muscles．In order to obtain more conclusive results，however，
furtherexperimentaldataisnecessary，lnCludingthoseonseveralotherproteases，eSPeCial1yendopeptidases
SuChascathepsinB，Calcium−aCtivatedneutralproteinase，alkalineproteinase，andmembrane−boundneu−
tralproteinasel）．Changesinthelevelofproteaseactivitiesinthelaterstageofdevelopmentmustalsobe
53

investigated．1nthepreviousstudies2，3）theactivitiesofvariousproteaseswerefoundtobemarkedlyele−
Vatedinthebreastmuscle ofdystrophicchickens．Thereforeitwi11bealsoimportant toinvestigate thedevelopmental changesin protease activitiesin the muscles ofdystrophic chickensin comparison withnormalones．
1）Ichihara，Y．，Sogawa，K．，andTakahashi，K．：J．Biochem．，91，87，1982．
2）Takahashi，K．，Ichihara，Y．，and Sogawa，K．：Current ResearchinMuscular Dystrophy，Japan，2，pp．49−50，
1981．

3）Aoyagi，T．andWada，T．：CurrentResearchinMuscularDystrophy，Japan，2，Pp．47−18，1981．

54

肋∫C〟わr勒Jか叩わ，Gro岬，ノタgJ

ENDOGENOUS THIOL PROTEINASEINHIBITOR ANDITS CHANGES OF ACTIVITYIN
SKELETAL MUSCLE OF DYSTROPHIC HAMSTERS
NobuhikoKatunuma，EikiKominami，NobuakiWakamatsu，andKazuhideTsogai
Department ofEnzyme Chemistry，Institute for Enzyme Research，Schoolof Medicine，Tokushima University，Tokushima770

Endogenous thiolproteinaseinhibitoris foundin various organs of many animalsl）．We have re−Centlypurifiedtheinhibitorfromthecytosol丘actionofratliveruslnga庁initychromatographyonpapain−
boundSepharose2）．Puri丘edinhibitorismonomericproteinandhasamolecularweightof12，500．Itinhibits
VariousthioIproteinases（CathepsinB，H，L，Candpapain）but not serine proteinases orcarboxylpro−
teinase．CalciumdependentproteinaseisathioIproteinasebutisnotinhibited bythisinhibitor．Anim−munodouble diffusion test using antibody against thioIproteinaseinhibitor from ratliver showed thatliverandmuscleinhibitorareindistinguishableimmunologically．
Activitiesoflysosomalthiolproteinases（CathepsinBandH）andoftheirendogenousinhibitorwere
assayedwiththighmuscleofnormal（FIB）anddystrophic（BIO14．6）harnsters．Musclehomogenatefrom
15weekoldhamsterswascentrifugedat12，000×gfbr20minandthecentrifugedprecipitateswereusedfbrassay oftheinhibitor．AsShowninFig．1，amarkedincreaseofcathepsinB（ZrArg−Arg−βNA hydrol−ase）and cathepsinH＋B（BZ−Arg−βNAhydrolase）were observed．But anotherlysosomalmarker，aCid
phosphate，WaSnOtincreased，SuggeStingspecifiCincreaseofproteasesinmusclelysosomesofdystrophic
hamsters．ThiolproteinaseinhibitorisalsoincreasedinmuscleofdystrophicanimalS，butothercytosolic
enzymes，aldolaseandlacticdehydrogenaseareratherdecreasedindystrophicanimalS．Itisunknownat
presentwhethertheincreaseofthiolproteaseinhibitorinmusclesofdystrophichamstershasanybiologicalSigniGcance．Nosignificantincreasein thiolproteinaseinhibitorinliversofdystrophicanimals suggests
thatincreaseoftheinhibitorislimitedtotargetorgansofdystrophy．

00

茎SSコで⊃uFIG・1・Variousenzymeactivitiesin skeletalmuscleofnormaland dystrophic hamsters．Values areexpressedasmeans土StandarddeviationoflOhamsters．⊂コ；nOrmal，筐喜≡ヨ；dystrophic．
1）Lenl−ey，J・F・：CurrentTopicsinCe11ularRegulation（Horecker，B．LandStadtman，E．R．，eds．）Vol．17，p．25，
AcademicPress，NewYork，1981．
2）Kominami，E・，Wakamatsu，N．，andKatunuma．N．：Biochem．Biqphys．Res．Cbmmtm．，99，568，1981．
55

肋∫C〃ねr砂∫Jrqpわ′Gr0叩，J夕βノ

THE EFFECTS OF LOW MOLECULAR ENZYMEINHIBITORS ON ENZYME NETWORKSINVARIOUS ORGANS OF DYSTROPHIC MICE
TakaakiAoyagiandTakaoWada
InstituteofMicrobialChemistry，Tokyo141
1npreviouspaperswesuggestedthepossibilitythatnotonlyabnormalitiesinendopeptidasesbutalso
abnormalitiesinexopeptidasesmightplayaroleinpathogenesisofmusculardystrophyl・2）．Thisprompted
thetrialofbestatinfortreatmentofmousemusculardystrophy．Itactuallyshowedane爪：Ctinsuppresslng
theoccurrenceofdystrophy3）．Wedoubtedwhetherornotthise鮎ctwasduetotheinhibitionofelevatedaminopeptidasesindystrophicmuscle．ItisalSopossiblethatbestatinwhichbindstoce11sinducesachange
inproteasepatternsindystrophicmuscleandhasthee仔bctofsuppressingtheprogressofdystrophy．Thus

We decided to compare thechanges ofenzyme activitiesin various organs ofdystrophicmiceaftertheadministration ofleupeptin4），bestatin5），and forphenicino16），Whichhave difrerent spectra ofenzymein−
hibitingactionsinvitro7）．

Three−Week−Old dystrophicmice（C57BL／6J dy／dy）were obtained from the CentralInstitute forExperimentalAnimals，Japan．Micewerekilled bycervicaldislocation3hra丘er thefinaliqjection，and

Organhomogenateswerepreparedinphosphate−bu能redsaline（PBS）usinganUltra−turraXatmaXimum
SPeedforlmin．Thehomogenatewascentrifuged（5，000rpmfbr20min）andthesupernatantnuidwas
Withdrawntomeasureenzymeactivities．TheinhibitorsweredissoIvedinO．2mlofsaline，andwereglVen
toexperimentalanimalsdailyintraperitoneal1yfor8daysinthefollowingdoses：leupeptin500FEg，bestatin
200FLg，andfbrphenicino1500FLg．ControlanimalsweregivenO．2mlsalinedaily for8days．Thesuper−
natantsoforganhomogenatesweredispensedintotesttubes（1．5×10cm）withPBScontainingtherespec−tivesubstrates．Thetesttubeswereincubatedforlhrat370C．AssaysweredoneatpH7．21，27．
Tablel showstheenzymaticchangesinthefbrelimbmuscleofdystrophicmiceinducedbythelow
molecularinhibitors．Ascanbeseen，leupeptintendedtoinducethedecreaseofallaminopeptidaseactivi−
tiesexceptfMet−AP．Bestatinandforphenicinoltendedtodecreasenotonlyaminopeptidaseactivitiesbut
TAl】LEl．EnzymaticActivityChangesinForelimbMuscleofDystrophicMiceInducedbyLowMolecularInhibitors
EllZymeAP−A
AP−B
Pro−Al）
Leu−APrMet−AP
Phe−AP
Gly−Pro・Leu−AP
Trypsin−like
Chy−try−1ike
EIastase−like
CathepsinC
α一D−Glucosidase
α−D−Mannosidase
GIc−NH2aSe＊＊

CPK
Phosphatase
Esterase

Specificactivity＊±SD

None

1．29土0．22
10．28土1．61
2．97土1．75
8．13土 0．90
5．33土0．40
13．17土0．91
1．00土0．02
86．62土 5．20
1（泊．97土17．47
56．35土 7．78
1．19土1．01
1．04土0．42
0．16土0．28
2．67土 0．91
22．51土4．76
6．27土1．58
206．20土79．30

Leupeptln

Bestatin

0．95土0．60
8．39土 2．29
2．42土0．49
5．68土 3．45
6．34±1．41
10．87土 5．32
0．94土0．48
102．07±47．93
134．26士25．32
48．74土18．50
2．37土0．49
0．74土0．16
0．31士0．28
2．22土1．07
19．75土10．43
1．67土1．23
67．62±16．80＊nmol／min／rngprotein．＊＊N−aCetyl−P−D−glucosaminidase．

57

Forphenicinol

1．26±0．22
9．22土1．14
1．45±0．43
7．67土0．74
5．17士0．67
10，93土2．31
0．97±0．18
54．42±21．40
63．53±23．28
45．76土18．82
0．99土0．19
0．40±0．04
0．14士0．03
1．22±0．25
16．05土2．74
1．46士0．20
62．57±2．85

1．28±0．06
9．08圭1．42
2．54±0．85
7．58±0．12
4．60土0．07
13．59±0．58
1．03士0．15
55．11士13．43
90．06土 5．96
54．11土 7．22
1．38±0．11
0．43土0．00
0．19土0．05
1．13±0．14
19．27± 3．48
1．42土0．19
57．31±4．81

TABLE2．Correlations Among Enzymatic ChangesInduced by Low Molecular WeightInhibitorsinVarious
Organs
Leupeptln

Organs；Bestatin

MuscleForelimb
Hindlimb
Heart
SpleenLiverForphemicinol

0．12

0．05

0．69＊＊

0．69＊＊

0．55＊

0．31

0．66＊＊

0．22

0．43く＊）

0．71＊＊

0．81＊＊＊

0．75＊＊＊

0．49（＊〉

0．13
0．21

−0．04
0．37

KidneyBestatin

ForphenicinoI

0．59＊（＊）：P＜0．1，＊：P＜0．05，＊＊：タ＜0．01，＊＊＊：ア＜0．001．
Spearmansrankcorrelations（r8Values）Werecalculatedbytheequationsbelow．

rs＝1−

62（Tix−Tiy）2〝＝16，′＝1，2，3…………16718−〃

alsotheactivitiesofendopeptidasesandglycosidases．Simi1arstudiesweredoneon50therkindsoforgans
SuChashindlimbmuscle，heart，Spleen，liver，andkidney．Thedegreeofenzymaticchangesin60rganSare
expressedasStudent

s t−Values．Asjudgedfromthese1−Values，theinhibitorssignifiCantlya斤bctedmany

enzymeactivitiesinal160rganSteSted．However，thereweredif7brencesamongtheresponsepatternsofthe
Variousorgans．InordertoquantitativelycomparetheenzymaticresponsepatternsamOngthe3inhibitors
andthe60rganS，Spearmansrankcorrelations（rsvalues）wereadopted．Eachofthet−Valuesfbrthe16

enzymaticactivitieswasgivenarankaccordingtoitssize．Theseriesofrankswereevaluatedfbrthedegree
Ofcorrelation amongvariousmousegroupswhich wereglVen theinhibitors．Theobtainedrsvalues areShowninTable2．Therewerehighcorrelationsamongtheeffectsofthe3inhibitorsinseveralorgans．
Theb2Vivoefrtctsofenzymeinhibitorsrevealedinthepresentstudywerehardlyimaginablefromthe
Viewpointofinvitroactionsofenzymeinhibitors．First，themovementsofthevariousenzymeactivities

WereSeeminglyindependentofthepurelybiochemicalactionsoftheinhibitors．Second，theresponsesof
thevariousorganswerenotuniform；eVen between themusculartissuesofforelimbandhindlimbthere
Were discrepanciesin enzymaticresponses．Third，there were frequent correlations among theresponse
patternsoforganstodifftrentenzymeinhibitors．Significantchangesofenzymaticactivityinmuscleand
Various organsinduced byleupeptin，bestatin，and fbrphenicinoIsuggest that the therapeutic effect ofleupeptinandbestatinonmousemusculardystrophymaynotbeduetotheinhibitionofenzymessensitive
totheseinhibitorsbutduetotheirindirectefrtcts3・8，9J．ItmoreoversuggeststhatfbrphenicinoIwhichbinds
tocellsurfacesmaybeacompoundworthyoffurtherstudyagainstmuscledystrophy．
1）Aoyagi，T．，Wada，T．，KQjima，F．，Nagai，MりandUmezawa，H．：J．Clin．1nve丑，67，51−59，1981．

2）Wada，T・，Kqjima，F．，Nagai，M．，Aoyagi，T．，andUmezawa，H．：J．PJlarm．LD・11．，4，184−190，1981．
3）Matsushita，H．：Studyonthe ClinicalApplicationsofSecondary Metabolites fromMicrobes（AnnualReport
OfMuscularDystrophyCommittee，SuPPOrtedbyMinistryofHealthandWelfare），45−50，1980rinJapanese）．
4）Aoyagi，T．，Miyata，S．，Nambo，M．，Kojima，F．，Matsuzaki，M．，Tshizuka，M．，Takeuchi，T．，andUmezawa，H．：
J，」〃7品加．，22，558−568，1969．
5）Umezawa，HリAoyagi，T．，Suda，HりHamada，M．，andTakeuchi，T．：J．Afltibiot．，29，97−99，1976．
6）Aoyagi，T．，Yamamoto，T，，Kqjiri，K．，Kqiima，F．，Hamada，M．，Takeuchi，T．，andUmezawa，H．：J．Antlblotり

31，2率ト246，1978．
7）Aoyagi，TリWada，T．，Iwabuchi，M．，KQjima，F．，Nagai，M．，andUmezawa，H．：Biockem．ht．，1982，inpress．

8）Stracher，A．，McGowan，E．B．，andShafiq，S．A∴Scie17Ce，200，50r51，1979．
9）McGowan，E．B．，Sha頁q，S．A．，andStracher，A．：Erp．NeLIrO1．，50，649−657，1976．

58

肋∫C〟hr上）γ∫汁qp力γGr（フ岬，ノタβJ

THE ROLE OF PROTEASEIN THE DEVELOPMENT ANDDIFFERENTIATION OF MUSCLES
KazutomoImahori，KoichiSuzuki，＊ShuichiTsuji，＊Shun−ichiroKubota，＊YoshioKimura，＊andMegumuTakahashi−

Nakamura

TokyoMetropolitanInstituteofGerontology，Tokyo173＊DepartmentofBiochemistry，FacultyofMedicine，TheUniversityofTckyo，Tokyol13

Some proteases are thought to beinvoIvedin the development and di能rentiation ofmuscles，eS−
PeCiallyintheturnoverofmuscleproteinsandtheactivationofproteinkinases．Asacandidateofsucha
protease we selected calcium activated neutralprotease（CANP）and purifieditl）．However，Since thisCANPrequiredanunphysiologicallyhighconcentrationofCa2＋itmaynotoperateunderphysiological
COnditions．RecentlyanotherCANPwhichrequiredmicr0−mOlarCa2＋（jL−CANP）wasfound2）besidesthe
onewhichnecessitatesmilli−mOlarCa2＋（m−CANP）．Thuswehaveundertakenworktoclarifytherelation

betweenthesetwoCANPsandhavelearnedthatFL−CANPisderivedfromm−CANPbyitsautolysis．This
COnVerSion may trigger someimportant stepin the differentiation ofmuscle cells．Anotherimportant
regulatorysystemorCANPactivitywouldbetheendogenousinhibitor．Thuswepurifiedandcharacterized
itindetail．ThisreportwilldealwiththeconversionofCANPandthecharacterizationoftheinhibitor．
Re∫〟JJ∫

Theconversionofm−CANPtoFL−CANPwasfoundbychance3）．Wetriedtopuri付CANPbyamnity
chromatography．Partiallypurifiedm−CANPofchickenmusclenwasappliedtoacolumnofcasein−Sepha−rosejnthepresenceof20mMCa2＋．AfterwashingouttheunabsorbedmaterialCANPwaselutedwitha
SOlutioncontaining10mMEGTA．Theeluted

CANPwashighlysensitivetoCa2＋withKd30FLMWhiletheorlglnalCANPwasfarlesssensitivetoCa2＋withKdO．8mM．Thuswecalledtheformerandthelatter

FL−CANPandm−CANP，reSpeCtively．Themolecularweightsofm−andFL−CANPswereidentifiedas82K
and79K，reSpeCtively，aSjudged by SDS−gelelectrophoresis．Thissuggested that FL−CANP wasderived
bytheautolysisofm−CANPinthecolumnwhereenoughCa2＋existedtoactivatem−CANP．

In orderto prove this suggestion weconducted thefollowlngeXPeriments4I．Purifiedm−CANPwas
incubatedwithlOmMCa2＋atOOC．Atintervalsanampleamountoftheincubationmixturewaswithdrawn
andassayedinthepresenceof50FLM

Ca2＋．In

parallel，apart

OfthesamplewasanalyzedonSDS−gelelectrophoresis．TheactivityofFL−CANPappearedveryrapidly，reaChedthemaximumat3minandthen
graduallydecreased．Inthesampletakenat3min，the82Kbandwasreplacedbythe79Kbandasjudged
bySDS−gelelectrophoresis．Afterwardsthe60Kbandalsoappearedandtheywerefinallyreducedtothe
30r33K band．We have separated components of79K，60K，and30Kfrom the digestionmixture．The
COmpOnentWiththemolecularweightof79KgavethesameCa2＋sensitivityasthatofFL−CANPobtained

bya餌nitychromatography．Thecomponentof60Khadpropertiessimilartothe79Kcomponent，While
the30Kcomponentwastota11yinactive．Theseresultsindicatedthatp−CANPisderivedfromm−CANP
throughautolysisofthelatter．
Itisinterestingthattheautolysism−CANPstopsatthestageof79Kandnofurtherdegradationtakes
placewhenitiscarriedoutinafBnitychromatography．Thismaysuggestthatinthepresenceofthesub−
Strate，aSinthatcellularcytoplasm，theconversiontoFL−CANPwillproceedinaproperway・
In orderto clarifyanother regulatorymechanism ofCANP activity we purifieditsendogenousin−
hibitor5）．Fromtheextractsofthemusclece11CANPwasprecipitatedatitsisoelectricpH．Throughseveral
Purification steps，including DEAE−Cellulose，QAE−Sephadex chromatography，isoelectric focusing，and
phenylSepharosechromatographywesucceededinpurifiCationoftheendogenousinhibitorinhomogenity．
Themolecularweightwasestimatedas70Kfromgelfiltrationbut35KfromSDS−gelelectrophoresis．Thus59theinhibitorwasidentifiedasadimericprotein，judgedbyitssusceptibilitytoseveralhydrolases．When
CANPactivitywasassayedinthepresenceofseveralamount oftheinhibitoritwasclarified thateachSubunitoftheinhibitorinhibitsonemolecule ofCANP byfbrmlngatightcomplex．Theinhibitorwas
effectiveagainstbothm−and FL−CANPsbut thecomplexformationtookplaceonlywhenCANPswere
activatedbyCa2＋．Theinhibitorwasine能ctiveagainstanyotherproteasessofartested．
AlthoughithasnotyetbeenclarifiedhowCANPisinVoIvedinthedifferentiationofmusclecell，thepresentresults do providea breakthroughfbrthe problem．Underthephysiologicalconditionm−CANP
WOuldbehaveasaprecursorofp−CANP．WhenitisnecessarytransientorlocalinfluxofCa2十ionswi11

takeplaceandtheconversionofm−CANPtoFL−CANPwillbetriggered．TheresultingFL−CANPwouldful−fillitsphysiologlCalrolebutitsunfavorableactionmaybepreventedbytheendogenousinhibitor．Onthe
Otherhand，WhenanuncontrolledinmlXOfCa2＋takesplacem−CANPwilldigestseveralmuscleproteins

andresultinadiseasesuchasmusculardystrophy．

1）lshiura，S．，Murofushi，H．，Suzuki，K．，andImahori，K：J．Biochem．，84，225，1978．
2）Mellgren，R．L．：fEβ∫上eJJ．，109，129，1980．
3）Kubota，S．，Suzuki，K．，andImahori，K．：Biochem．Biqphys．Res．CbmmLJl7．，100，1189，1981．
4）Suzuki，K．，Tsttji，S，，Kubota，S．，Kimura，Y．，andImahori，K．：J．Biochem．，90，275，】981．
5）Takahashi−Nakamura，M．，Ts可i，S，，andImahori，K．：J．Biochem．，90，1583，1981．

60

肋C〟hr勒JJr叩わ′Gr（）岬，79βJ

THE EFFECT OF PROTEASEINHIBITOR ON THE CALCIUM−INDUCED DEGENERATION
OF］M【YOFILAMENT．〃Ⅴγ′mO AND麒VI′JyO
HideoSugita，IkuyaNonaka，andShoichiIshiura
DivisionofNeuromuscularResearch，NationalCenterforNervous，MentalandMuscularDisorders，Kodaira187
A non−lysosomalcalcium−aCtivatedneutralprotease（CANP）is suggested to beinvoIvedinmuscle
PrOteinturnOVer，butitsintracellularroleisstillobscure．ToclarifytheroleofCANPかyivo，Weemployedanewtechniqueofmuscleincubationwithcalciumhzvitro．CalCiumionswereintroducedintointactrat
SOleusmusclebyA23187andthee恥ctoftheproteaseinhibitorwasstudiedmorphologlCallyandbio−
Chemically．

Intactratmusclewasremovedwithtendonsandincubatedinasolutioncontaining4mlofKrebs−
Ringerbufrtr，pH7．4，andcycloheximide．WhencalciumionswereinvoIved，Z−linelossandconcomitantreleaseofα−aCtinininto themediumwereobserved・Thereleasedα−aCtininwasdeterminedbySDS−gelelectrophoresis・Athiolproteaseinhibitor，E−64−C，addedexogenouslyintotheincubationmixture，Sup−
PreSSedthecalcium−inducedreleaseofα−aCtininby45％（Tablel）．
TAllLEl．TheReleaseofα−ActininfromIntactRatSoleusMuscle
Experiments

ControI

E−64−Ci可ected

Additions

Releasedα−aCtinin（g／3hr）

EGTA
CaC12
E−64−C，CaC12
EGTA
CaC12

9．1土2．0（〃＝5）
55．3土5．2（〝＝5）
33．1士7．4（〝＝5）
12．1土3．2（〝＝5）56．1土5．5（〃＝5）Normalmalerats （100g）weresacri貢cedbypentobarbitaloverdoseadministration．Thepairedsoleusmuscleswere

Carefu11yremovedwithtendonsintactandweighed．MuscleswereimmediatelyincubatedinKrebs−Ringerbu能r（4ml）
COntaining25FLg／mlcycloheximideunder95％02−5％CO2at370C．A23187wasdissoIvedinDMSO rfinalcon−
Centration，0・5％）・ThecontroIwastreatedasmentionedaboveexceptthatKrebs・Ringerbufrtrwaspreparedwith−
OutCalciumbutcontainedO・lmMEGTA．After3hrincubation，aliquotsofthemediumweresubjectedtoSDS−gelelectrophoresis・Thereleasedα一aCtinin，identified byimmunoreplica，WaSdeterminedspeetrophotometrically．In

SeParategrOupSOfrats，E−64−CWaSinjectedsubcutaneously（10mg／kg）inavolumeofO．2mlofsaline．Animals
WereSaCrificedafter24hrandthesoleusmusclewasrapidlyremovedasdescribedabovc．Calcium−inducedrelease
Ofα・aCtininwasdeterminedinthesamemanner．Tntramusculardistributionofl3H】E−64−CWaSdeterminedbyprepar−

ingthreefractions，myO重Iaments，mitochondrial，andlysosomal丘actions（ML）andsupernatant．Almost70％ofthe
injectedE−64−CWaSlocalizedinthecytosol，nOtinthelysosome．

However，SubcutaneousinjectionofE−64−C（10mg／kg，24hrlater）hadnoefftctonthecalcium−induced
Z−lineloss（SeeTablel）．Theintracellularconcentration ofitjectedl3H］E−64−CWaSalso studied．The

radioactivityin thepostmitochondrialsupematantindicatedthatapproximatelyO・23FLgOfE−64−CWaS
takenupintolgofmuscle．Therefbre，WeCOnCludedthatCANPwasnotinhibitedbyE−64−CininjectedmusclebecauseOfthelowcontentofE−64−Cinthemusclece11．
1）Kameyama，T．andEtlinger，J．D．：Nature，279，344−346，1979．
2）Sugita，H・，Ishiura，S・，Suzuki，K．，andImahori，K．：Muscte＆Nerve，3，335−339，1980．
3）Tshiura，S・，Nonaka，I．，andSugita，H．：J．Biochem．，90，2837285，1981．
4）Ishiura，S・，Hanada，K・，Tamai，M．，Kashiwagi，K．，andSugita，H．：J．Biochem．，90，1567−1570，1981．
5）Ishiura，S．：上的∫C7．，29，1079−1087，1981．
6）Obinata，T・，Maruyama，K・，Sugita，HりKohama，K．，andEbashi，S．：肋scle＆Nerve，4，456188，1981．

61

MEMBERS OF THE RESEARCH GROUP ON
MUSCULARI）YSTROPHYINJAPAN（BiomedicalSciences）

Chairman
Ebashi，Setsuro

DepartmentofPharmacology，FacultyofMedicine，UniversityofTokyo，Tokyo
Members
Aoyagi，Takaaki

InstituteofMicrobialChemistry，Tokyo
Endo，Makoto
DepartmentofPharmacology，TohokuUniversity，SchoolofMedicine，Sendai
Hagihara，Yashiro
Department ofNeuropharmacology，Brain ResearchInstitute，SchoolofMedicine，ChibaUniversity，
Chiba
Hotta，Ken
DepartmentofPhysiology，NagoyaCityUniversity，MedicalSchool，Nagoya
Imahorj，Kazutomo
TokyoMetropolitanInstituteofGerontology，Tokyo
Ishikawa，Harunori
DepartmentofAnatomy，FacultyofMedicine，UniversityofTokyo，Tokyo
Iwasaki，Yuzo
DivisionofUltrastructureResearch，NationalCenterforNervOuS，MentalandMuscularDisorders，Tokyo
Kagawa，Ts止Omu

LaboratoryofCellBiology，ToneyamaNationalHospital，OsakaKano，Ma弧akira

DepartmentofPhysiology，SchoolofMedicine，KitasatoUniversity，Sagamihara
Xatunum，NobubikoDepartmentofEnzymeChemistry，InstitutefbrEnzymeResearch，SchoolofMedicine，TokushimaUmi−
VerSity，Tokushlma
Xwiyama，Hiroshi
DepartmentofPharmaCOlogy，FacultyofMedicine，KyushuUniversity，Fukuoka−63−

Mbruyama，Koscak
DepartmentofBiology，FacultyofScience，ChibaUniversity，Chiba
Mb鼠aM，Tomoh

DepartmentofPharmacology，InstituteofBasicMedicalSciences，TheUniversityofTsukuba，Ibaraki，andTheTokyoMetropolitanInstituteofMedicalScience，Tokyo
Matsu5hita，Hiroshi
DepartmentofPhysioIogy，WakayamaMedicalCollege，Wakayama
Nomomum，Yoshiaki
DepartmentofPharmacology，FacultyofMedicine，UniversityofTokyo，Tokyo
Obinah，Takasbi
Departmentofliiology，FacultyofScience，ChinaUniversity，Chiba
Ohts山d，Iwao
DepartmentofPharmacology，FacultyofMedicine，KyushuUniversity，Fukuoka
Okada，T．S．
DepartmentofBiophysics，FacultyofScience，UniversityofKyoto，Kyoto
Okada，Yosuo
ResearchInstituteforMicrobialDiseases，OsakaUniversity，Osaka
Ots血，Ma￡anOri
DepartmentofPharmacology，FacultyofMedicine，TokyoMedicalandDentalUniversity，Tokyo
OZaWa，Eijiro
DivisionofCellBiology，NationalCenterfbrNervous，MentalandMuscularDisorders，Tokyo
Sakai，Tostlio
DepartmentofPhysiology，TheJikeiUniversity，SchoolofMedicine，Tokyo

Shima血，Yutaka
DepartmentofAnatomyandBiology，FacultyofMedicine，ChibaUniversity，Chiba
Sugita，Hideo
Division ofNeuromuscular Research，National Center fbr Nervous，Mentaland Muscular Disorders，Tokyo，andDepartmentofNeuroIogy，InstituteofBrainResearch，FacultyofMedicinC，Universityof
Tokyo，Tokyo
Sugiyama，Hiroyu最LaboratoryofMembraneBiology，NationalInstituteforPhysiologicalSciences，Okazaki
TakaIlaSbi，Kenji
DepartmentofBiochemistry，PrimateResearchInstitute，KyotoUniversity，Inuyama−64−Takahashi，Kumitaro
DepartmentofNeurophysioIogy，InstituteofBrainResearch，FacultyofMedicine，UniversityofTokyo，Tokyo
Terao，Toshio
TheFirstDepartmentofInternalMedicine，FacultyofMedicine，TeikyoUniversity，Tokyo
Totsuka，Tsuyoshi
DepartmentofPhysiology，InstitutefbrDevelopmentalResearch，AichiPreftcturalColony，Aichi
Yomezawa，Takeshi
DepartmentofPathology，KyotoPreftcturalUniversltyOfMedicine，Kyoto−65−