Kuyera zvinokonzerwa nemethylene blue mumitochondria: okisijeni, ATP, nepotenshiyari yemembrane

Ngatitii rabhoritari inowedzera methylene blue kumitochondria dzine kukanganisika kwekufema uye yoona kushandiswa kweokisijeni kuchikwira. Mutsara unongoerekana wauya mupfungwa ndewekuti dhayi yadzorera kushanda kwemitochondria. Mutsara iwoyo ungave usiri iwo. Kuyerera kweokisijeni, kugadzirwa kweATP, nepotenshiyari yemembrane zvinhu zvitatu zvakasiyana. Zvinogoverana organelle imwe chete sezvinoita kuchengetedza kopi, kuenzanisa mafaera, nekugoverana mafaera muforodha imwe chete iri mugore: zvakabatana, asi zvinotongwa nemitemo yakasiyana. Kana ukavhiringidza chimwe nechimwe, dudziro inokanganisika, kunyange kana chiyero chose chaitwa nemazvo.

Methylene blue inoita kuti kuvhiringidzika uku kuve nyore nokuti inopinda mukushanda kwehurongwa hwairi kuyera. Inotenderera muredox, ichigamuchira maerekitironi kumatanho ekutanga yoapa kumatanho anotevera; ication inofarira mafuta inoungana mumitochondria dzine simba; uye ikemikari ine ruvara rwebhuruu rwakasimba inotora nekuburitsa chiedza mumabhendi mamwe chete nemaprobu anowanzoshandiswa. Chiyero chimwe chete hachigoni kutakura mutoro iwoyo. Nzira yakavimbika, yakaratidzwa muzvidzidzo zvemitochondria zveBudapest, ndeyekuyera kuyerera kweokisijeni pamwe nemwero wekugadzirwa kweATP pamwe nepotenshiyari yemembrane pamwe nekuburitswa kwehydrogen peroxide, paine bvunzo dzekudzora dzakanangana nedhayi padanho rimwe nerimwe.

Respirometry inoyera kuyerera kweokisijeni, kwete simba rinobuda

Respirometry ine kunyatsoyera kwakakwirira, ingaitwa neOroboros Oxygraph kana Seahorse extracellular flux analyzer, inoratidza chinhu chimwe chete: kumhanya kuri kuita kugamuchira maerekitironi kweokisijeni. Hairatidzi kuti mapurotoni mangani akapombwa paerekitironi imwe neimwe, kuti musiyano wekugoverana kwawo wakachengetedzwa zvakadini, kana kuti ATP synthase yakaushandisa here.

Mashoko anoshandiswa ane basa. Mumitochondria dzakaparadzaniswa nemasero, substrate isina ADP inopa kufema kunobva pakuvuza kwemapurotoni, substrate ine ADP yakawanda inokwana kuzadza zvinodiwa inopa kugona kweoxidative phosphorylation, uye kuwedzera uncoupler zvishoma nezvishoma kunopa kugona kukuru kwekutamisa maerekitironi. Mumasero asina kuvhurwa, zviyero zvinoenderana nazvo zveSeahorse ndezvekufema kwepakutanga, kufema kwakabatana neATP kunodzorwa neoligomycin, kuvuza kwemapurotoni, kufema kukuru kunotsanangurwa neFCCP, uye kushandiswa kweokisijeni kusiri kwemitochondria kusingamiswa nerotenone pamwe neantimycin. Kudaidza zviyero zveSeahorse kuti State 3 neState 4 kunopa pfungwa yekunyatsotsanangura zvinhu isingatsigirwi nemagadzirirwo emuenzaniso wacho, sezvinojekeswa nehurongwa hweAgilent Mito Stress Test.

Methylene blue inoputsa fungidziro yokuti okisijeni yakawanda inoreva simba rakawanda. Mumitochondria dzakaparadzaniswa kubva muuropi hweguinea pig, dhayi iri pa100 nM kusvika pa1 uM yakawedzera kushandiswa kweokisijeni panguva yekuzorora paishandiswa masubstrate eglutamate pamwe nemalate, succinate, uye alpha-glycerophosphate, paine kukwira kweinenge 64 muzana paalpha-glycerophosphate. Asi kufema kunokurudzirwa neADP mumitochondria dzine utano hakuna kunyatsochinja, maererano nechidzidzo chemitochondria dzakaparadzaniswa kubva muuropi. Nokuti kufema kunobva pakuvuza kwakakwira asi kuyerera kunokurudzirwa neADP kwakaramba kwakafanana, reshiyo inoverengwa yekudzora kufema inoderera. Kuderera ikoko hakurevi kuti dhayi yakakuvadza mitochondria. Kunoreva kuti dhayi yakawedzera kutenderera kweredox kunoshandisa okisijeni pamusoro pekufema kwemazuva ose. Bvunzo yekushandiswa kweokisijeni nemethylene blue inongoratidza kukwira ikoko inenge yayera kemisitiri chaiyo asi isina kutaura chinhu pamusoro pekuchengetedzwa kwesimba.

Zviedzo zvekudzoreredza kushanda zvinoratidza mukaha mumwe chete pamwero mukuru. Pakavharwa Complex I, kuyerera kweokisijeni kwakadzoka kusvika kunenge katatu asi kugadzirwa kweATP kwakadzoka kusvika kungangoita ka1.5 chete. Okisijeni yakawanda yakasvika paerekiturodhi. Chikamu chidiki chayo ndicho chakashandiswa kugadzira ATP. Chero chirevo chekuti oxidative phosphorylation yavandudzwa chinoda rutivi rweATP rwekuverenga uku, zviri nani ruchiratidzwa sereshiyo yeATP paokisijeni, pachitsanangurwa nzira inoshandiswa kuverenga nhamba iri pasi pereshiyo.

Bvunzo dzeATP dzinoyera mwero wekugadzirwa, usingatsiviwi nehuwandu huripo

Bvunzo yeATP inopindura mumwe mubvunzo: ATP yakawanda sei inogadzirwa pachikamu chenguva. Chiyero chinotevera shanduko nekufamba kwenguva, kungave kupenya kwefirefly luciferase kunoteverwa kwemaminitsi kana bvunzo yakabatanidzwa yehexokinase pamwe neglucose-6-phosphate dehydrogenase inoverengwa pa340 nm, chinopa mwero wekugadzirwa. Kuyera ATP yose kamwe chete panoperera bvunzo kunopa huwandu huripo, hunoratidza kugadzirwa kubvisa kushandiswa uye husingatauri zvakawanda pamusoro peoxidative phosphorylation kana huri hwega.

Boka reBudapest rakashandisa nzira yemaenzayimu akabatanidzwa, adenylate kinase yakadziviswa, rikaona kuti methylene blue iri pa1 kusvika pa2 uM yakawedzera kugadzirwa kweATP zvine mwero asi zvine kukosha kwenhamba mumitochondria dzakaiswa rotenone nedzakaiswa antimycin. Uku kudzoreredzwa kwechokwadi, asi kune chikamu chete. Chidzidzo chehupfu hweTEGDMA chakawana mhinduro ine chimiro chimwe chete mune mumwe muenzaniso: 5 mM yedental monomer TEGDMA yakaderedza zvikuru kufema kwakabatana neComplex I kubva paanenge 286 kusvika pa28 emayuniti ekuyerera, uye 2 uM yedhayi yakakudzorera kusvika paanenge 86.

Bvunzo mbiri dzekudzora hadzifaniri kusiyiwa kana uchishandisa kemikari ine ruvara inoshanda muredox. Kutanga, ita bvunzo neoligomycin kuti uone kuti chikamu chakadii chekugadzirwa kweATP kwakayerwa chinotsamira paATP synthase. Chechipiri, ita bvunzo isina masero yekuwana zvakare ATP yakawedzerwa, uchishandisa ATP ine huwandu hunozivikanwa pamwe nemushonga webvunzo wakakwana pamwe nekuwanda kwedhayi chaiko kwakashandiswa, kusanganisira kanenge 0, 100 nM, 300 nM, 1 uM, uye 2 uM. Dhayi inogona kutora chiedza chinoburitswa, kudzivisa enzayimu inopa chiratidzo, kana kushandura kemisitiri yeredox yemushonga webvunzo. Nhungamiro yebvunzo dzeluciferase inokurudzira bvunzo dzebiochemical dzekuongorora kukanganiswa kwakadai. Usafungidzira kuti 1 uM haina kukanganisa chiyero nokuti kudzima kupenya kunowanzova kwakanyanya pakuwanda kwakakwirira.

Maprobu epotenshiyari yemembrane anoyera kugoverana kwedhayi, kwete kuyerera

Safranine O, TMRM neTMRE, rhodamine 123, uye JC-1 ose macation anogona kupfuura nemumembrane. Anoungana mumatrix ine chaji isina kunaka maererano nekuenzana kwemagetsi nekemikari, saka fluorescence yose inoratidza potenshiyari nenzira isina kunanga kuburikidza nenzvimbo iri probu. Hapana rimwe rawo rinoyera ATP kana kuyerera kwemaerekitironi.

Probu imwe neimwe ine magadzirirwo emuenzaniso ainonyanya kukodzera. Safranine O inokodzera mitochondria dzakaparadzaniswa nemasero akaitwa kuti membrane yawo ipindike, kazhinji iri pedyo ne2 uM, ichikurudzirwa nechiedza chiri pedyo ne495 nm uye ichiburitsa chiedza chiri pedyo ne585 nm. Inoshanda mumamiriro ekudzima kupenya, umo kupinda kwayo kunoshandura fluorescence yose. TMRM neTMRE zvinokodzera masero asina kuvhurwa pakuiswa muhuwandu hushoma hwenanomolar, mumamiriro asina kudzima kupenya, umo kuderera kwepotenshiyari kunoburitsa dhayi uye fluorescence yemitochondria inodzikira; kana dhayi yakaiswa yakawanda, mafambiro echiratidzo anogona kupinduka nokuda kwekudzima kupenya kwayo pachayo. JC-1 inopa reshiyo iri nyore yegirinhi kune tsvuku asi inotsamira pahuwandu hwayakaiswa uye pahuwandu hwemitochondria, zvichiita kuti ive isina kunyatsokodzera kuyerwa kwenhamba seTMRM yakanyatsodzorwa. Zvakanakira nezvisina kunakira izvi zvinopfupikiswa muongororo yemaprobu epotenshiyari yemembrane yemitochondria inoshandiswa zvakanyanya.

Methylene blue inokanganisa kuyerwa kwechiedza pahuwandu chaihwo hunoshandiswa pabasa. Mabhendi ayo ekutora chiedza ari pedyo ne600 kusvika ku665 nm anopindirana nekuburitswa kwechiedza chitsvuku nemaprobu, saka kukanganisa kweinner-filter, kudzima kupenya, uye kutorwa kwechiedza chinokurudzira probu zvose zvinogona kuitika. Dhayi iyi zvakare imwe cation inofarira mafuta, ine kugoverana kwayo kunochinja nepotenshiyari iri kuyerwa. Kudzivirira izvi kunoda zviyero zvinotsigirana pamwe nemienzaniso isina chinoyerwa: probu yoga, dhayi yoga, probu pamwe nehuwandu humwe nohumwe hwedhayi, kubvisa potenshiyari zvachose neFCCP sechiyero chezero, uye kana zvichibvira probu yechipiri ine hunhu hwechiedza hwakasiyana. Chidzidzo chekupfuura Complex III neimwe nzira chakaratidza nzira iyi nokudzokorora kuyerwa kwekudzoreredzwa kwepotenshiyari yemembrane chichishandisa zvose safranine O neTMRM mushure mekudziviswa neantimycin nemyxothiazol, chikawana kudzoka kwakafanana pahutano hwechiratidzo. Chiratidzo chimwe chete chinobva pachiedza chinopa fungidziro. Zviratidzo zviviri zvakazvimirira zvinopa humbowo.

Magadzirirwo matatu emienzaniso, mibvunzo mitatu yakasiyana

Mitochondria dzakaparadzaniswa, masero akaitwa kuti membrane yawo ipindike, uye masero asina kuvhurwa hazvisi zvigadziriso zvinotsiviwa zvekunzwa kwebvunzo imwe chete. Pfungwa dzakasiyana dzine misimboti yekushanda yakasiyana.

Mitochondria dzakaparadzaniswa dzinochengetedza cheni yekutamisa maerekitironi, membrane yemukati, uye hurongwa hwematrix, asi dzichibvisa plasma membrane, cytosol, nechimiro chemambure emitochondria. Kubvisa ikoko ndiko chinangwa: masubstrate, ADP, zvinodzivisa, uye kuwanda kwedhayi zvinotsanangurwa chaizvo, saka nzira iri kushanda inogona kuzivikanwa. Zvinorasika ndezvekupiwa kwemasubstrate kwechisikigo, kutumirwa kwezviratidzo, uye kudiwa kwesimba.

Masero akaitwa kuti membrane yawo ipindike anochengetedza mitochondria mukati mechikamu chikuru chechimiro chesero, achibvumira masubstrate neADP zvinobva kunze kupfuura nemuplasma membrane yakavhurwa. Ndiwo mudziyo wakakodzera pakuziva kukanganisika kwenzira yesubstrate mukati memamiriro esero. Kuvhura membrane zvakanyanyisa kunokuvadza membrane yekunze, uye kudzora kunoitwa necytosol kunosukwa kuchibuda.

Masero asina kuvhurwa anochengetedza zvose: chipingamupinyi cheplasma membrane, zvinhu zvinopa maerekitironi zviri mucytosol, kutakurwa kwemasubstrate anobva mukati, mambure emitochondria, uye kudiwa kweATP. Ndiwo ari pedyo zvikuru nekushanda kwemuviri asi ari kure zvikuru nekutsanangura mashandiro chaiwo, nokuti kupinda kwedhayi, kugamuchira kwayo maerekitironi, uye kubatwa kwayo munzvimbo dzakasiyana zvose zvinoumba huwandu hunosvika pamitochondria huchinyatsoshanda.

Chidzidzo cheTEGDMA chakashandisa matanho ose matatu nemaune: mitochondria dzakaparadzaniswa kubva muuropi pakudzoreredzwa kweoxidation nepotenshiyari nedhayi, stem cells dzepulp yemazino dzakaitwa kuti membrane yadzo ipindike kunyanya kutsanangura kuti kukanganisika kwaiva paoxidation yesubstrate yakabatana neComplex I, uye stem cells dzisina kuvhurwa pakuona mhedzisiro yose ye2 uM yedhayi pakushandiswa kweokisijeni kwakanga kwadzvinyirirwa. Kuvaverengi vanoenzanisa zviyero zvemitochondria mumagadzirirwo akasiyana, chinyorwa chinofambidzana neichi chinotsanangura mafambisirwo anoitwa maerekitironi nemethylene blue mucheni yekufema chinopa hwaro hwenzira yacho, uye gwaro rekuverenga zvidzidzo zvekufema zvemethylene blue rinotsanangura kufunga kunoshandiswa pakusarudza huwandu nezvinodzivisa.

Bvunzo dzekudzora dzinodiwa nedhayi inoshanda muredox

Chinhu chinotenderera muredox chinoda runyorwa rurefu rwebvunzo dzekudzora kupfuura chinodzivisa chenguva dzose, nokuti kemisitiri yedhayi yoga inogona kutevedzera zvinoitwa nezvipenyu.

Pa respirometry, shandisa kamuri ine buffer yoga kuti uone kutsauka kwechiyero, kamuri ine buffer pamwe nedhayi pasina zvinhu zvezvipenyu, dhayi pamwe nechero chinhu chinopa maerekitironi chiri kunze kwemasero mubvunzo, uye rotenone pamwe neantimycin pakupera kuti utsanangure kushandiswa kweokisijeni kusiri kwemitochondria mumasero asina kuvhurwa. Cherechedza kuti dhayi yakagamuchira maerekitironi inoita oxidation zvakare nokutamisa maerekitironi kuokisijeni ichigadzira hydrogen peroxide. Saka muenzaniso wedhayi yoga usina chinopa maerekitironi unofungidzira pasi kushandiswa kweokisijeni kusina masero kunooneka kana NADH kana NADPH yavepo.

Pahydrogen peroxide, sarudza Amplex UltraRed yakaiswa pachiyero pamwe nehorseradish peroxidase panzvimbo pedhayi dzeDCFDA dzisingasarudzi chinhu chimwe chete, simbisa necatalase, uye ongorora kutsamira pachiedza, nokuti dhayi inoita kuti maitiro akurudzirwe nechiedza. Chidzidzo chemitochondria dzeuropi chakawana kuti mutoro weperoxide wakakwira kupfuura kanokwana kana pakungova ne100 nM yedhayi, uku kubviswa kwayo kuchinonoka. Peroxide yaigadzirwa zvakananga nedhayi yakagamuchira maerekitironi pasina superoxide yepakati. Tsanangudzo dzekusarudza probu nebvunzo dzekudzora chiedza dziri muchinyorwa chinofambidzana neichi pamusoro pekuyera marudzi eokisijeni anoshanda zvakanyanya muzviedzo zvedhayi, uye mubvunzo wakakura wekuti peroxide inoshanda rini sechiratidzo kana semutoro unotsanangurwa pamwe nekuenzana kweredox mumasero nezvirevo zveantioxidant.

Pazvirevo zvine chekuita nenzira yekushanda, edza nzvimbo dzose dziri mbiri dzinodziviswa paComplex III: antimycin A panzvimbo yeQi uye myxothiazol panzvimbo yeQo, pamwe nekuongorora danho rekupedzisira reComplex IV necyanide. Chidzidzo chepakutanga cheuropi hwemakonzo chakati dhayi yakadzorera kufema kwakavharwa nerotenone asi kwete kwakavharwa neantimycin, asi chidzidzo chakazotevera chemhando nhatu dzemhuka chakawana kudzoreredzwa kwechikamu nezvinodzivisa zvose zviri zviviri uye humbowo hwekupiwa maerekitironi kwakananga kucytochrome c. Bata pfungwa yekupfuura neimwe nzira seinoenderana nedata idzva, kwete seyakatosimbiswa, nokuti kuwanda kwedhayi, nzvimbo inodziviswa, mamiriro esubstrate, uye kugoverana kwedhayi zvose zvinochinja nzira inoonekwa.

Tafura yekusarudza bvunzo

Shandisa tafura iyi kusarudza chiyero kubva pamubvunzo, kwete kusarudza mubvunzo kubva pachiyero.

Chirevo chidiki chinogutsa chinoda kuti huwandu humwe chete hwedhayi, 100 nM, 300 nM, 1 uM, uye 2 uM, huongororwe pakuyerera kweokisijeni mumamiriro ekuvuza, ADP, oligomycin, uye uncoupler; pamwero wekugadzirwa kweATP; papotenshiyari yemembrane, zvichinyanya kunaka kushandisa maprobu maviri; uye pakuburitswa kweperoxide kwakaiswa pachiyero, paine catalase nebvunzo dzekudzora dzisina masero. Ratidza boka rezviyero zvakabatana: kuyerera kweokisijeni pamwe nemwero weATP pamwe neATP paokisijeni pamwe nepotenshiyari pamwe neperoxide. Chero imwe yenhamba idzodzo iri yoga chiyero chiri kutsvaga zvachinoreva.