Ukulinganisa imiphumela ye-methylene blue kuma-mitochondria: i-oxygen, i-ATP, namandla kagesi olwelwesi

Ake sithi ilabhorethri ifaka i-methylene blue kuma-mitochondria anokuphefumula okuphazamisekile bese ibona ukusetshenziswa kwe-oxygen kukhuphuka. Umusho obonakala ufanele ukubhalwa uthi udayi ubuyisele ukusebenza kwama-mitochondria. Lowo musho ungase ube yiphutha. Ukugeleza kwe-oxygen, ukukhiqizwa kwe-ATP, namandla kagesi olwelwesi kuyimiqondo emithathu ehlukene. Kwenzeka esakhiweni esisodwa seseli ngendlela ukufakwa kwekhophi eyisipele, ukuvumelanisa, nokwabelana okusebenzisa ngayo ifolda eyodwa esefwini: kuyahlobana, kodwa kulawulwa yimithetho ehlukene. Uma okunye kudidaniswa nokunye, incazelo iba yiphutha, ngisho nalapho zonke izilinganiso zenziwe kahle ngokobuchwepheshe.

I-methylene blue yenza lokhu kudideka kube lula ngoba ibamba iqhaza ohlelweni elulinganisayo. Ingumjikelezi we-redox owamukela ama-electron engxenyeni engaphambili yomzila bese uwanikela engxenyeni elandelayo, iyi-cation ethanda amafutha enqwabelana kuma-mitochondria anikwe amandla, futhi iyinhlanganisela enombala oluhlaza okwesibhakabhaka oqinile emunca futhi ikhiphe ukukhanya emabhendini afanayo nalawo ezihlonzi ezivamile. Isilinganiso esisodwa asinakuthwala wonke lowo mthwalo. Indlela ethembekile, eboniswe ocwaningweni lwama-mitochondria lwaseBudapest, iwukulinganisa ukugeleza kwe-oxygen kanye nezinga lokukhiqizwa kwe-ATP kanye namandla kagesi olwelwesi kanye nokukhishwa kwe-hydrogen peroxide, kusetshenziswa izivivinyo zokulawula eziqondene nodayi esinyathelweni ngasinye.

Ukulinganisa ukuphefumula kulinganisa ukugeleza kwe-oxygen, hhayi amandla akhiqizwayo

Ukulinganisa ukuphefumula okunokunemba okuphezulu, kungaba nge-Oroboros Oxygraph noma nge-Seahorse ehlaziya ukugeleza ngaphandle kwamaseli, kubika into eyodwa: ukuthi i-oxygen yamukela ama-electron ngokushesha kangakanani. Akubiki ukuthi mangaki ama-proton aphonswe ngaphesheya kolwelwesi nge-electron ngayinye, ukuthi ungakanani umehluko ogciniwe phakathi kwezinhlangothi zolwelwesi, noma ukuthi i-ATP synthase iwusebenzisile yini.

Amagama asetshenziswayo abalulekile. Kuma-mitochondria ahlukanisiwe, i-substrate engenayo i-ADP iveza ukuphefumula okuhambisana nokuvuza kwama-proton, i-substrate ene-ADP eningi ngokwanele ukuba igcwalise isidingo iveza umthamo we-oxidative phosphorylation, kanti ukwengezwa kancane kancane kwesihlukanisi sokuhlanganiswa kokuphefumula nokukhiqizwa kwe-ATP kuveza umthamo omkhulu wokudluliselwa kwama-electron. Emaselini aphelele, izilinganiso ze-Seahorse ezihambisana nalokhu ukuphefumula okuyisisekelo, ukuphefumula okuhlobene ne-ATP okuvinjwa yi-oligomycin, ukuvuza kwama-proton, ukuphefumula okuphezulu okuchazwa yi-FCCP, nokusetshenziswa kwe-oxygen okungaveli kuma-mitochondria okungavinjelwa yi-rotenone kanye ne-antimycin. Ukubiza imiphumela ye-Seahorse ngokuthi i-State 3 ne-State 4 kuboleka ukunemba okungasekelwa yisampula elilungiselelwe, njengoba uhlaka lwe-Agilent Mito Stress Test lucacisa.

I-methylene blue iphula ukuxhumana okulindelekile phakathi kwe-oxygen eyengeziwe namandla engeziwe. Kuma-mitochondria ahlukaniswe ebuchosheni bezingulube zaseGuinea, udayi ongu-100 nM kuya ku-1 uM wakhuphula ukusetshenziswa kwe-oxygen ngesikhathi sokuphumula lapho kusetshenziswa ama-substrate e-glutamate kanye ne-malate, i-succinate, ne-alpha-glycerophosphate, ngokukhuphuka okucishe kube ngamaphesenti angu-64 ku-alpha-glycerophosphate. Nokho, ukuphefumula okukhuthazwa yi-ADP kuma-mitochondria aphilile cishe akuzange kushintshe, ngokusho kocwaningo lwama-mitochondria obuchopho ahlukanisiwe. Njengoba ukuphefumula okuhambisana nokuvuza kwakhuphuka kodwa ukugeleza okukhuthazwa yi-ADP kwahlala kungashintshi, isilinganiso sokulawulwa kokuphefumula esibaliwe siyehla. Lokho kwehla akusho ukuthi udayi ulimaze ama-mitochondria. Kusho ukuthi udayi wengeze umjikelezo we-redox osebenzisa i-oxygen phezu kokuphefumula okuvamile. Ukuhlolwa kokusetshenziswa kwe-oxygen nge-methylene blue okubika ukukhuphuka kuphela kulinganise ukusabela kwamakhemikhali kwangempela kodwa akushongo lutho ngokulondolozwa kwamandla.

Izivivinyo zokubuyisela ukusebenza zibonisa igebe elifanayo ngezinga elikhulu. Lapho i-Complex I ivinjiwe, ukugeleza kwe-oxygen kwabuyela ngokuphindwe cishe kathathu, kuyilapho ukukhiqizwa kwe-ATP kwabuyela ngokuphindwe cishe ngo-1.5 kuphela. I-oxygen eningi yafinyelela ku-electrode. Ingxenye encane yayo yasiza ukukhiqiza i-ATP. Noma yisiphi isimangalo sokuthuthuka kwe-oxidative phosphorylation sidinga nohlangothi lwe-ATP lwaleso silinganiso, okungcono luvezwe njengesilinganiso se-ATP nge-oxygen ngayinye, kuchazwe nendlela esetshenziswe entweni ehlukanisa isilinganiso.

Ukuhlolwa kwe-ATP kulinganisa izinga lokukhiqizwa, elingenakuthathelwa indawo yinani elikhona

Ukuhlolwa kwe-ATP kuphendula umbuzo ohlukile: ingakanani i-ATP ekhiqizwa ngeyunithi ngayinye yesikhathi. Ukufunda izinguquko ngokuhamba kwesikhathi, kungaba ngokulandelela ukukhanya kwe-luciferase kakhanyikhanyi imizuzu ethile noma ngokuhlolwa okuhlanganisa i-hexokinase kanye ne-glucose-6-phosphate dehydrogenase okufundwa ku-340 nm, kuveza izinga lokukhiqizwa. Isilinganiso esisodwa se-ATP ephelele ekugcineni siveza inani elikhona, elimelela ukukhiqizwa kususwe ukusetshenziswa, futhi sisodwa sikutshela okuncane nge-oxidative phosphorylation.

Iqembu laseBudapest lasebenzisa indlela yama-enzyme ahlanganisiwe licindezele ukusebenza kwe-adenylate kinase, lathola ukuthi i-methylene blue engu-1 kuya ku-2 uM yakhuphula ukukhiqizwa kwe-ATP kancane kodwa ngokuphawulekayo kuma-mitochondria ayefakwe i-rotenone kanye nalawo ayefakwe i-antimycin. Lokho kuwukubuyiselwa kokusebenza kwangempela, futhi kuyingxenye kuphela. Ucwaningo lobuthi be-TEGDMA lwafinyelela emphumeleni ofanayo ohlelweni oluhlukile: i-monomer yamazinyo i-TEGDMA engu-5 mM yehlisa kakhulu ukuphefumula okuhlobene ne-Complex I kusuka cishe kumayunithi okugeleza angu-286 kuya ku-28, futhi udayi ongu-2 uM wakubuyisela cishe ku-86.

Izivivinyo zokulawula ezimbili azinakushiywa uma kusetshenziswa inhlanganisela enombala esebenza ku-redox. Okokuqala, sebenzisa i-oligomycin ukuthola ukuthi kungakanani ukukhiqizwa kwe-ATP okulinganisiwe okuncike ku-ATP synthase. Okwesibili, yenza ukuhlolwa kokutholwa kabusha kwe-ATP efakiwe endaweni engenamaseli, usebenzisa inani le-ATP elaziwayo kanye nayo yonke i-reagent kanye namanani okugxila odayi afana ncamashi nalawo asetshenzisiwe, okungenani u-0, 100 nM, 300 nM, 1 uM, no-2 uM. Udayi ungamunca ukukhanya okukhishwayo, uvimbe i-enzyme ekhiqiza isignali, noma uguqule amakhemikhali e-redox e-reagent, futhi isiqondiso esijwayelekile sokuhlolwa nge-luciferase sincoma ukuhlolwa okungeziwe kwamakhemikhali ezinto eziphilayo ukuze kubonakale lolu hlobo lokuphazamiseka. Ungacabangi ukuthi ku-1 uM akukho miphumela mbumbulu ngoba ukucishwa kwesignali kuvame ukuba kubi kakhulu emazingeni okugxila aphezulu.

Izihlonzi zamandla kagesi olwelwesi zilinganisa ukusabalala kodayi, hhayi ukugeleza

I-Safranine O, i-TMRM ne-TMRE, i-rhodamine 123, ne-JC-1 konke kungama-cation akwazi ukudlula olwelwesini. Kunqwabelana ku-matrix eneshaji engaphansi kuka-zero ngokuvumelana nokulingana kwamakhemikhali nogesi, ngakho i-fluorescence yesampula lonke iveza amandla kagesi ngokungaqondile, ngokuthi isihlonzi sitholakala kuphi. Akukho kulokhu okulinganisa i-ATP noma ukugeleza kwama-electron.

Isihlonzi ngasinye sinohlobo lwesampula olusifanele kakhulu. I-Safranine O ifanele ama-mitochondria ahlukanisiwe namaseli enziwe avumele izinto zidlule olwelwesini, ngokuvamile iseduze no-2 uM, nokukhanya okuyivusayo kuseduze no-495 nm nokukhanya ekukhiphayo kuseduze no-585 nm. Isebenza esimweni sokucishwa kwe-fluorescence lapho ukungena kwayo kushintsha i-fluorescence yesampula lonke. I-TMRM ne-TMRE zifanele amaseli aphelele lapho zifakwa ngamazinga aphansi e-nanomolar esimweni esingacishi i-fluorescence, lapho ukulahleka kwamandla kagesi kukhipha udayi bese i-fluorescence yama-mitochondria yehla; lapho kufakwa inani eliphezulu, indlela isignali eshintsha ngayo ingaphenduka ngenxa yokuzicisha kwe-fluorescence. I-JC-1 iveza isilinganiso esilula sokukhanya okuluhlaza kuya kokubomvu, kodwa incike enanini elifakiwe nasesisindweni sama-mitochondria, okuyenza ingabi namandla njengendlela ye-TMRM elawulwa ngokucophelela emsebenzini wokulinganisa amanani. Lezi zinzuzo nokulinganiselwa kufingqiwe ekubuyekezweni kwezihlonzi zamandla kagesi olwelwesi lwama-mitochondria okusetshenziswa kabanzi.

I-methylene blue iphazamisa izilinganiso zokukhanya kuwo kanye amazinga okugxila asetshenziswayo. Amabhendi ayo okumunca ukukhanya aseduze no-600 kuya ku-665 nm ahlangana nokukhanya okubomvu okukhishwa yizihlonzi, ngakho imiphumela yokuhlunga ukukhanya ngaphakathi kwesampula, ukucishwa kwe-fluorescence, nokumuncwa kokukhanya okuvusa isihlonzi konke kungenzeka. Udayi ubuye ube yi-cation yesibili ethanda amafutha, ukusabalala kwayo okushintsha ngokwamandla kagesi alinganiswayo. Indlela yokuzivikela ukuphinda ngezindlela ezizimele kanye nokusebenzisa amasampula ayizikhombo: isihlonzi sodwa, udayi wodwa, isihlonzi kanye nezinga ngalinye lokugxila kodayi, ukulahleka okuphelele kwamandla kagesi nge-FCCP njengesikhombo sika-zero, futhi uma kungenzeka, isihlonzi sesibili esisebenzisa ukukhanya okuhlukile. Ucwaningo lokudlula i-Complex III lwabeka leso sibonelo ngokuphinda ukuhlolwa kokubuyiselwa kwamandla kagesi olwelwesi nge-safranine O nange-TMRM ngemva kokuvinjelwa nge-antimycin nange-myxothiazol, lwathola ukubuyiselwa kokusebenza okufanayo ngokohlobo loshintsho. Isihlonzi esisodwa sokukhanya siyinkomba. Izihlonzi ezimbili ezizimele ziwubufakazi.

Izinhlobo ezintathu zamasampula alungiselelwe, imibuzo emithathu ehlukene

Ama-mitochondria ahlukanisiwe, amaseli enziwe avumele izinto zidlule olwelwesini, namaseli aphelele akuzona izilungiselelo zokuzwela ezishintshaniswayo ekuhlolweni okukodwa. Kuyimiqondo ehlukene esebenza ngezimiso ezihlukene.

Ama-mitochondria ahlukanisiwe agcina iketango lokudluliswa kwama-electron, ulwelwesi lwangaphakathi, nezinqubo ezisebenza ku-matrix, kodwa asuse ulwelwesi lweseli, i-cytosol, nokwakheka kwenethiwekhi. Lokho kususa kuyinhloso: ama-substrate, i-ADP, izivimbeli, namazinga okugxila kodayi kunqunywa ngokunembile, ngakho kungakhonjwa umzila othintekayo. Okulahlekayo ukunikezwa kwama-substrate ngokwemvelo, ukuthunyelwa kwezimpawu, nesidingo samandla.

Amaseli enziwe avumele izinto zidlule olwelwesini agcina ama-mitochondria engaphakathi kwengxenye enkulu yokwakheka kweseli, kodwa avumele ama-substrate avela ngaphandle ne-ADP ukuba kudlule olwelwesini lweseli oluvuliwe. Ayithuluzi elifanele lokukhomba ukuphazamiseka komzila we-substrate esimweni seseli. Ukuvumela ukudlula ngokweqile kulimaza ulwelwesi lwangaphandle, futhi ukulawula okuvela ku-cytosol kuyagezeka kuphume.

Amaseli aphelele agcina konke: isithiyo solwelwesi lweseli, izinto ezinikela ngama-electron ku-cytosol, ukuthuthwa kwama-substrate angaphakathi, inethiwekhi yama-mitochondria, nesidingo se-ATP. Asondele kakhulu ekusebenzeni kwemvelo kodwa akude kakhulu nokucacisa indlela yokusebenza, ngoba ukungena kodayi, ukwamukela kwawo ama-electron, nokuvaleleka kwawo ezindaweni ezithile konke kunquma ukuthi ama-mitochondria ahlangana nenani elingakanani elisebenzayo lodayi.

Ucwaningo lwe-TEGDMA lwasebenzisa wonke la mazinga amathathu ngenhloso: ama-mitochondria obuchopho ahlukanisiwe ukuhlola ukubuyiselwa kwe-oxidation namandla kagesi ngodayi, amaseli esiqu omgogodla wezinyo enziwe avumele izinto zidlule olwelwesini ikakhulukazi ukukhomba ukuthi ukuphazamiseka kuseku-oxidation yama-substrate ehlobene ne-Complex I, namaseli esiqu aphelele ukuhlola umphumela wokugcina ngokwemvelo wodayi ongu-2 uM ekusetshenzisweni kwe-oxygen okucindezelwe. Kubafundi abaqhathanisa izilinganiso zama-mitochondria ezinhlotsheni ezahlukene zamasampula alungiselelwe, incazelo ehambisana nalesi sihloko yokuthi i-methylene blue ihambisa kanjani ama-electron eketangweni lokuphefumula inikeza isizinda semizila, kanti umhlahlandlela wokufunda ucwaningo lokuphefumula nge-methylene blue uchaza umqondo wenani elisetshenziswayo nezivimbeli.

Izivivinyo zokulawula ezidingwa udayi osebenzayo ku-redox

Umjikelezi we-redox udinga uhlu olude lwezivivinyo zokulawula kunesivimbeli esivamile, ngoba ukusabela kwamakhemikhali okwenziwa udayi wodwa kungalingisa okwenzeka ezintweni eziphilayo.

Ekulinganiseni ukuphefumula, sebenzisa igumbi elinesixazululo esigcina i-pH kuphela ukuze uhlole ukunyakaza kwesilinganiso ngokuhamba kwesikhathi, igumbi elinaleso sixazululo kanye nodayi kodwa elingenazo izinto eziphilayo, udayi kanye nanoma iyiphi into enikela ngama-electron engaphandle kwamaseli esetshenziswayo ekuhlolweni, bese ekugcineni usebenzisa i-rotenone kanye ne-antimycin ukuchaza ukusetshenziswa kwe-oxygen okungaveli kuma-mitochondria emaselini aphelele. Qaphela ukuthi udayi owamukele ama-electron ubuye uwalahle ngokuwadlulisela ku-oxygen ukuze kwakheke i-hydrogen peroxide, ngakho isampula eliyisikhombo elinodayi wodwa elingenayo into enikela ngama-electron libukela phansi ukusetshenziswa kwe-oxygen okungenamaseli okwenzeka lapho i-NADH noma i-NADPH isikhona.

Nge-hydrogen peroxide, khetha i-Amplex UltraRed elungiswe ngezilinganiso ezaziwayo kanye ne-horseradish peroxidase kunodayi be-DCFDA abangakhethi into ethile kuphela, qinisekisa nge-catalase, futhi uhlole ukuthi umphumela uncike yini ekukhanyisweni, ngoba udayi ukhuthaza ukusabela okubangelwa ukukhanya. Ucwaningo lwama-mitochondria obuchopho lwathola ukuthi umthwalo we-peroxide wakhuphuka ngokuphindwe ngaphezu kokune ngodayi ongu-100 nM kuphela, kuyilapho ukususwa kwayo kwehla. I-peroxide yakheka ngokuqondile ngodayi owamukele ama-electron, ngaphandle kwe-superoxide njengesinyathelo esiphakathi. Imininingwane yokukhethwa kwezihlonzi nezivivinyo zokulawula ukukhanya itholakala esihlokweni esihambisanayo sokulinganisa izinhlobo ze-oxygen ezisabela kalula ezivivinyweni zodayi, kanti umbuzo obanzi wokuthi i-peroxide isebenza nini njengophawu futhi nini njengomthwalo uchazwa kanye nokulingana kwe-redox emaselini nezimangalo zama-antioxidant.

Ngezimangalo zemizila, hlola zombili izindawo zezivimbeli ze-Complex III: i-antimycin A endaweni ye-Qi ne-myxothiazol endaweni ye-Qo, kanye nokuhlolwa kwe-Complex IV ekugcineni nge-cyanide. Ucwaningo lwangaphambili lobuchopho begundane lwabika ukubuyiselwa ngodayi kokuphefumula okuvinjwe yi-rotenone kodwa hhayi okuvinjwe yi-antimycin, kuyilapho ucwaningo lwakamuva lwezinhlobo ezintathu zezilwane lwathola ukubuyiselwa okuyingxenye ngazo zombili izivimbeli nobufakazi bokuthi i-cytochrome c yamukela ama-electron ngokuqondile. Phatha ukudlulwa komzila njengokuhambisana nedatha entsha kunokuba kube yinto esiqinisekisiwe, ngoba izinga lokugxila kodayi, indawo yesivimbeli, isimo se-substrate, nokusabalala kodayi konke kushintsha umzila obonakalayo.

Ithebula lokukhetha ukuhlolwa

Sebenzisa leli thebula ukukhetha isilinganiso ngokombuzo, hhayi ukwenza okuphambene nalokho.

Isimangalo esikholisayo esidinga okungenani ubufakazi obanele sisebenzisa amazinga okugxila kodayi afanayo, u-100 nM, 300 nM, 1 uM, no-2 uM, ukuhlola ukugeleza kwe-oxygen ngaphansi kwezimo zokuvuza kwama-proton, ze-ADP, ze-oligomycin, nezokuhlukaniswa kokuhlanganiswa kokuphefumula nokukhiqizwa kwe-ATP, izinga lokukhiqizwa kwe-ATP, amandla kagesi olwelwesi okungcono ngezihlonzi ezimbili, nokukhishwa kwe-peroxide okulungiswe ngezilinganiso ezaziwayo, kusetshenziswa i-catalase nezivivinyo zokulawula ezingenamaseli. Bika iqoqo elixhumene: ukugeleza kwe-oxygen kanye nezinga le-ATP kanye ne-ATP nge-oxygen ngayinye kanye namandla kagesi olwelwesi kanye ne-peroxide. Noma iyiphi kulezi zinombolo iyodwa iyisilinganiso esisafuna incazelo.