Muscle contractions induce interleukin6 mRNA production in rat skeletal muscles.docx
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Muscle contractions induce interleukin6 mRNA production in rat skeletal muscles.docx
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Musclecontractionsinduceinterleukin6mRNAproductioninratskeletalmuscles
JPhysiol. 2000October1; 528(Pt1):
157–163.
doi:
10.1111/j.1469-7793.2000.00157.x
PMCID:
PMC2270126
Musclecontractionsinduceinterleukin-6mRNAproductioninratskeletalmuscles
IHJonsdottir, PSchjerling, KOstrowski, SAsp,* EARichter,* and BKPedersen†
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Abstract
1.Thepresentstudyexploredthehypothesisthatinterleukin-6(IL-6)mightbelocallyproducedinresponsetoskeletalmusclecontractionsandwhethertheproductionmightreflectthetypeofmusclecontractionperformed.Ratswereanaesthetizedandthecalfmusclesofonelimbwerestimulatedelectricallyforconcentricoreccentriccontractions(4×10contractionswith1minofrestbetweenthe4series,100Hz).Thecontralateralmusclesservedasunstimulatedcontrols.ThemRNAlevelsforIL-6,theglucosetransportproteinGLUT-4andβ-actinintheratmuscles(whiteandredgastrocnemiusandsoleus)werequantifiedbyquantitativecompetitiveRT-PCR.
2.TheIL-6mRNAlevel,measured30minafterthestimulation,increasedafterbotheccentricandconcentriccontractionsandtherewerenosignificantdifferencesinIL-6mRNAlevelsbetweenthedifferentmusclefibretypes.NosignificantincreaseinIL-6mRNAlevelwasseenintheunstimulatedcontralateralmusclefibres.
3.NoincreaseinGLUT-4mRNAlevelwasdetected,indicatingthattheincreaseinIL-6mRNAlevelwasnotduetogeneralchangesintranscription.
4.WeconcludethatIL-6islocallyproducedaftermusclecontraction,withnosignificantdifferencesbetweendifferentmusclefibretypes.ThislocalproductionofIL-6isnotduetogeneralchangesintranscription,sincenochangesinthelevelofGLUT-4mRNAwerefound.ThefactthatincreasedIL-6mRNAlevelswereseenafterbothconcentricandeccentriccontractionsindicatesthattheproductionofIL-6isnotsolelyduetomuscledamage,seenprimarilyaftereccentricexercise.
Itiswelldocumentedthatunaccustomedexercise,particularlyinvolvingeccentricmusclecontractions,resultsindisruptionofcontractiletissueandcytoskeletalcomponents(Smith,1991; Friden&Lieber,1992).Furthermore,severalinvestigatorsagreethatsomeaspectsofaninflammatoryresponseareseenfollowingeccentricexercise,involvingproductionofcytokinesthatarereleasedatthesiteofinflammation(Smith,1991).Increasedplasmalevelsofinflammatorycytokines,primarilyIL-6,havebeenfoundinresponsetoexercise(Pedersen etal. 1997).IL-6hasbeenshowntobeapotentinducerofacutephaseproteinsynthesisinbothratsandhumans(Heinrich etal. 1990)andithasbeensuggestedthatIL-6mightbeinvolvedinthegenerationofacutephaseresponsesseenafterexercise(Northoff&Berg,1991).
IncreasedplasmalevelsofIL-6haveprimarilybeendescribedinrelationtoeccentricexercisebutIL-6hasalsobeenshowntobeelevatedafterconcentricexercise. Ullum etal. (1994) investigatedtheeffectofconcentricexercise(cycling)oncytokineplasmalevelsandfounda2-foldincreaseinIL-6inplasmawhereasIL-1α,IL-1βandTNF-αremainedbelowdetectionlimits.
Bruunsgaard etal. (1997) showedthathigh-intensityeccentricexercisecausedamorepronouncedincreaseintheplasmalevelofIL-6,thanconcentricexercise,suggestingthattheelevationofIL-6couldberelatedtomuscledamageseenpredominantlyaftereccentricexercise.Wehaverecentlyshown,usingaqualitativePCRtechnique,thatmRNAforIL-6wasdetectableinhumanskeletalmuscleafterintenseprolongedexercise.IL-6mRNAwasnotconcomitantlydetectableincirculatingbloodmononuclearcells(BMNCs),suggestingthatIL-6couldbelocallyproducedintheskeletalmuscle(Ostrowski etal. 1998).
TheaimofthepresentstudywastoexplorefurtherthehypothesisthatIL-6couldbelocallyproducedinskeletalmusclefollowingmusclecontractions.Byusingthequantitativecompetitivereversetranscriptionpolymerasechainreaction(QCRT-PCR)techniqueIL-6mRNAlevelsweremeasuredinmuscleafterconcentricoreccentriccontractionsandinthecontralateralnon-contractingmuscle.ThestudywasalsodesignedtoexamineiftherewereanydifferencesinIL-6levelsbetweendifferenttypesofmusclefibres.
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METHODS
Animals
MaleWistarratsweighing200–250gwereusedinthisstudy.Allanimalswerekeptona12–12hlight-darkcycleandfedonan adlibitum standardchowdiet.
Theanimalsweredividedrandomlyintothefollowinggroups:
(a)non-stimulatedcontrols(n =5);(b)concentricstimulationgroup(n =6);(c)eccentricstimulationgroup(n =6).Intheeccentricstimulationgroup,boththestimulatedmusclesandthecontralateralunstimulatedmuscleswereincludedinthestudy.
AllexperimentswereapprovedbytheDanishAnimalInspectorate.
Eccentric/concentriccontractionmodel
Thecalfmusclesononesidewerestimulatedforconcentricoreccentriccontraction.Thecontralateralmusclesoftheratsstimulatedwitheccentriccontractionsservedasunstimulatedcontrols.
TheratswereanaesthetizedbyanintraperitonealinjectionofDormicum(midazolam,0.5mg(kgbodywt)−1,Roche,Switzerland)andHypnorm(fentanyl,20mg(kgbodywt)−1 andfluoanison,1mg(kgbodywt)−1,Janssen,HighWycombe,Bucks,UK),laidontheirbacks,andbothhindlimbswerefixedoveraplasticblockbytheuseofVelcrostrips.Supplementarydoses(Dormicum,0.5mgkg−1;Hypnorm,20mgkg−1)ofanaestheticwereadministeredasindicatedbythewithdrawalreflexresponsetotoeand/ortailpinch.Thelimbswereplacedsothehipjointhadanapproximately60degangle,thekneejointanapproximately60degangleandtheanklejointhada90degangle.HookswereplacedundertheAchilles’tendonandconnectedtoastraingaugeandanairpressuresystem.OnelowerlimbwasstimulateddirectlyandexternallyforcontractionthroughaneedleplacedinthemostdistalpartofthequadricepsmuscleandtheipsilateralhookundertheAchilles’tendon.Thestimulusconsistedoffoursessionsoftentrains(durationof1000ms,pulseduration1ms,100Hz).Inordertogainapproximatelythesameforcedevelopmentduringthesessionsthestimulusamplitudewasincreasedgraduallyfrom10Vduringthefirstsessionto15Vduringthesecondsession,20Vduringthethirdsessionand25Vduringthefinalsession.Thetrainsineachsessionwereseparatedby4s,andthesessionswereseparatedbyacoupleofminutes.Theelectricalstimulus perse madethecalfcontractconcentrically,andthisprocedurewasusedintheconcentricgroup.Intheeccentricgroupthehindlimbwasfirststimulatedforconcentriccontractionasdescribedaboveandwitha400msdelaytheactivecalfwasstretchedusingtheairpressuresystem.Aspringwasinsertedinseriestosmooththemusclestretch,andastraingaugewasalsoinsertedtomeasurethepoweroutputandinput.Allexercisesessionswerefinishedbefore12am.
Bilateralcalfmuscleswereobtained30minafterthestimulation.Thesuperficialpartofthegastrocnemiusmuscle,whichconsistsmainlyoffast-twitchwhitefibreswascutoutandclamped.Thesoleusmuscle,whichconsistsmainlyofslow-twitchfibreswasreflectedandclamped.Finally,aportionofthedeeppartofthemedialheadofthegastrocnemius,consistingmainlyoffast-twitchredfibres,wascutoutandclamped.Theanimalswerekilledwithanoverdoseofbarbiturate.Allsampleswerefrozenimmediatelyinliquidnitrogenandstoredat−80°C.
Wehavepreviouslyshown,usingthismodel,thatnomuscledamagewasinflictedbyconcentriccontractionsassuggestedbylackofhistologicalchangesandnormalglycogenresynthesis.Apriorstudyrevealednohistologicalsignsofmuscledamageandthemuscleglycogenconcentrationreturnedtothepre-stimulationlevellessthan24haftertheconcentricstimulation.Incontrast,eccentriccontractionscausedhistologicalsignsofmuscledamageandthemuscleglycogenremainedsubnormalmorethan48hafterthestimulation(Asp etal. 1995).
RNAextraction
TotalRNAwasisolatedessentiallyasdescribedby Chomczynski&Sacchi(1987).Firsta100–300mgmuscletissuesamplewashomogenizedin3mlGITsolution(4Mguanidinethiocyanate,25mMsodiumcitrate,100mMβ-mercaptoethanol,pH7)usingapolytron(Ultra-TurraxT8,IkaLabortechnik,Staufen,Germany).Then50μlSarkosyl,300μlsodiumacetate(pH4.0)and3mlphenolwereaddedandthesamplewasmixedthoroughly.Afterthis,600μlchloroform-isoamylalcohol(49:
1)wasaddedandthesamplemixedthoroughly,followedby15minincubationonice.Aftercentrifugationat5000 g at4°Cfor20min,theaqueousphasewasprecipitatedonicewithonevolumeofisopropanolfor1h.Afterafurthercentrifugationat5000 g at4°Cfor30min,thepelletwasresuspendedin600μlGITsolutionandprecipitatedonicewith600μlisopropanolfor1h.Finally,aftercentrifugationat13000 g at4°Cfor10min,thepelletwaswashedoncewith1ml80%ethanol.Thedrypelletwasresuspendedin100μlRNase-freewaterandstoredat−80°Cforlateruse.
Competitorpreparation
CompetitorsforQCRT-PCRwereconstructedusingPCRtointroduceasmalldeletioninthewild-typePCRproduct(Celi etal. 1993).The Pfu polymerase(Stratagene,LaJolla,USA)wasusedtoamplifyboththecompetitorandthewild-typePCRproductsfromratmusclecDNAusingtheprimerslistedin Table1.ThePCRproductswereclonedintothe Sma IsiteofthepBlueScriptIISK(+)vector(Alting-Mees&Short,1989),withorientationparalleltothe lac Zgene,resultingintheplasmidslistedin Table1.Theplasmidswerecutwith Pvu
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