Gupima ingaruka z'ubururu bwa metilene kuri mitokondiriya: ogisijeni, ATP na potansiyeli y'agahu

Tekereza laboratwari yongeye ubururu bwa metilene kuri mitokondiriya zifite ikibazo cyo guhumeka, maze ikabona ikoreshwa rya ogisijeni ryiyongera. Interuro umuntu yahita yandika ni uko irangi ryasubije mitokondiriya ubushobozi bwo gukora. Iyo nteruro ishobora kuba itari yo. Urujya n'uruza rwa ogisijeni, ikorwa rya ATP na potansiyeli y'agahu ni ibintu bitatu bitandukanye. Bihuriye kuri kamwe mu duce tugize ingirabuzimafatizo nk'uko kubika kopi y'inyongera, guhuza amakuru no kuyasangiza bihurira mu bubiko bumwe bwo kuri interineti: birafitanye isano, ariko bigengwa n'amategeko atandukanye. Iyo kimwe gifashwe nk'ikindi, ibisobanuro by'ibisubizo biba bitari byo, nubwo buri gipimo cyaba cyafashwe neza mu buryo bwa tekiniki.

Ubururu bwa metilene butuma uko kwitiranya gushoboka cyane kuko bugira uruhare muri sisitemu bupimirwamo. Ni ikinyabutabire cyisubiramo mu kwakira no gutanga elegitoroni muri redox, kizakira mu ntambwe zibanza kikazitanga mu zikurikiraho; ni iyoni ifite umuriro mwiza w'amashanyarazi kandi ikunda ibinure, yirundanyiriza muri mitokondiriya zifite ingufu; kandi ni ikinyabutabire cy'ubururu bukomeye gikurura kikanohereza urumuri mu byiciro by'uburebure bw'imiraba bikoreshwa n'ibipimisho bisanzwe. Igipimo kimwe ntigishobora gutanga ibisobanuro by'ibyo byose. Uburyo bwizewe, bwagaragajwe mu bushakashatsi bwa mitokondiriya bwakorewe i Budapest, ni ugupima urujya n'uruza rwa ogisijeni hamwe n'umuvuduko w'ikorwa rya ATP, potansiyeli y'agahu n'isohorwa rya perogiside ya hidurojeni, hakoreshejwe igerageza ry'igenzura ryihariye kuri iryo rangi kuri buri ntambwe.

Gupima uguhumeka bipima urujya n'uruza rwa ogisijeni, si ingufu zikomokaho

Gupima uguhumeka ku rwego rwo hejuru rw'ubusobanuro, haba hakoreshejwe Oroboros Oxygraph cyangwa igikoresho cya Seahorse gipima urujya n'uruza rw'ibinyabutabire hanze y'ingirabuzimafatizo, bitanga igipimo kimwe: umuvuduko ogisijeni yakiraho elegitoroni. Ntibigaragaza umubare wa porotoni zisunikwa kuri buri elegitoroni, urugero rw'itandukaniro ryabitswe hagati y'impande z'agahu, cyangwa niba ATP synthase yarikoresheje.

Amagambo akoreshwa ni ingenzi. Muri mitokondiriya zatandukanyijwe n'ingirabuzimafatizo, ikinyabutabire gikoreshwa mu guhumeka kidafite ADP gitanga uguhumeka kujyanye no kunyura kwa porotoni mu gahu bidakoze ATP; icyo kinyabutabire hamwe na ADP ihagije kugeza ku rugero rwuzuye bitanga ubushobozi bwo gukora ATP hakoreshejwe fosiforilasiyo ishingiye kuri ogisidasiyo; naho kongera buhoro buhoro ikinyabutabire gitandukanya uguhumeka n'ikorwa rya ATP bitanga ubushobozi ntarengwa bwo guhererekanya elegitoroni. Mu ngirabuzimafatizo zitangiritse, ibipimo bihwanye n'ibyo bya Seahorse ni uguhumeka kw'ibanze, uguhumeka kujyanye n'ikorwa rya ATP guhagarikwa na oligomycin, kunyura kwa porotoni mu gahu bidakoze ATP, uguhumeka ntarengwa kugenwa na FCCP, n'ikoreshwa rya ogisijeni ritari irya mitokondiriya ritahagarikwa na rotenone hamwe na antimycin. Kwita ibisubizo bya Seahorse State 3 na State 4 biha ibyo bipimo ubusobanuro ubwo buryo bwo gutegura icyitegererezo butemerera, nk'uko urwego rw'imikorere rwa Agilent Mito Stress Test rubisobanura.

Ubururu bwa metilene busenya igitekerezo cyoroshye cy'uko ogisijeni nyinshi ikoreshwa isobanura ingufu nyinshi. Muri mitokondiriya zatandukanyijwe n'ubwonko bw'ingurube zo muri Gineya, irangi riri ku rugero rwa 100 nM kugeza kuri 1 uM ryongereye ikoreshwa rya ogisijeni mu gihe cyo kuruhuka hakoreshejwe glutamate hamwe na malate, succinate na alpha-glycerophosphate nk'ibinyabutabire byo guhumeka. Iryo zamuka ryari hafi 64 ku ijana kuri alpha-glycerophosphate, nyamara uguhumeka gukangurwa na ADP muri mitokondiriya zimeze neza kwabaye nk'ukudahindutse, nk'uko ubushakashatsi kuri mitokondiriya zatandukanyijwe n'ubwonko bubigaragaza. Kubera ko uguhumeka kujyanye no kunyura kwa porotoni mu gahu kwiyongereye ariko urujya n'uruza rukangurwa na ADP ntiruhinduke, igipimo kibarwa cy'igereranya ry'igenzura ry'uguhumeka kiragabanuka. Iryo gabanuka ntirisobanura ko irangi ryangije mitokondiriya. Risobanura ko irangi ryongeye uruziga rwa redox rukoresha ogisijeni ku guhumeka gusanzwe. Ikizamini cy'ikoreshwa rya ogisijeni gikoresha ubururu bwa metilene kigatanga gusa iryo zamuka kiba cyapimye impinduka nyakuri z'ubutabire, ariko ntikigire icyo kivuga ku ibikwa ry'ingufu.

Amagerageza yo gusubizaho imikorere agaragaza icyo cyuho ku rugero runini kurushaho. Iyo Complex I yahagaritswe, urujya n'uruza rwa ogisijeni rwagarutse ku rugero rwikubye hafi gatatu, mu gihe ikorwa rya ATP ryagarutse ku rugero rwikubye hafi 1.5 gusa. Ogisijeni nyinshi yageze kuri elegitorode. Ariko umugabane muto kurushaho wayo ni wo watanze ingufu zo gukora ATP. Kuvuga ko fosiforilasiyo ishingiye kuri ogisidasiyo yarushijeho gukora neza bisaba no kugaragaza ibipimo bya ATP, byaba byiza bigatangwa nk'igipimo cya ATP kuri ogisijeni, hagasobanurwa uburyo bwakoreshejwe mu kugena igabanyabare.

Ibizamini bya ATP bipima umuvuduko w'ikorwa ryayo, udashobora gusimbuzwa ingano ihari

Ikizamini cya ATP gisubiza ikindi kibazo: ingano ya ATP ikorwa mu gihe runaka. Igipimo gikurikirana impinduka uko igihe gihita, cyaba ari urumuri rutangwa na luciferase y'agacurama rukurikiranwa mu minota cyangwa ikizamini cya hexokinase ifatanyijwe na glucose-6-phosphate dehydrogenase gipimwa kuri 340 nm, gitanga umuvuduko. Gupima ATP yose rimwe gusa ku musozo bitanga ingano ihari, igaragaza ikorwa ukuyemo ikoreshwa, kandi yonyine ntitanga amakuru menshi kuri fosiforilasiyo ishingiye kuri ogisidasiyo.

Itsinda ry'i Budapest ryakoresheje uburyo bwa enzime zifatanijwe, ribuza adenylate kinase gukora, maze ribona ko ubururu bwa metilene kuri 1 kugeza kuri 2 uM bwongereye ikorwa rya ATP ku rugero ruto ariko rufite agaciro mu ibarurishamibare, muri mitokondiriya zavuwe na rotenone n'izavuwe na antimycin. Uko ni ugusubizaho imikorere nyakuri, kandi ni igice cyayo gusa. Ubushakashatsi ku burozi bwa TEGDMA bwabonye ibisubizo bisa muri sisitemu itandukanye: 5 mM ya monomeri TEGDMA ikoreshwa mu buvuzi bw'amenyo yagabanyije cyane uguhumeka gufitanye isano na Complex I, kuva hafi 286 kugeza kuri 28 by'ibipimo by'urujya n'uruza, naho irangi rya 2 uM ririsubiza kuri hafi 86.

Hari amagerageza abiri y'igenzura adashobora gusimbukwa ku kinyabutabire gifite ibara kandi kigira uruhare muri redox. Icya mbere, koresha oligomycin kugira ngo umenye umugabane w'ikorwa rya ATP ripimwa ushingiye kuri ATP synthase. Icya kabiri, kora igerageza ryo kongeramo ATP izwi ingano no kugenzura ingano yongeye kuboneka, nta ngirabuzimafatizo zirimo, ukoreshe ATP izwi ingano hamwe n'imvange yuzuye y'ibinyabutabire by'ikizamini n'ingano nyazo z'irangi zakoreshejwe, nibura 0, 100 nM, 300 nM, 1 uM na 2 uM. Irangi rishobora gukurura urumuri rusohoka, kubuza enzime itanga ikimenyetso gukora, cyangwa guhindura ubutabire bwa redox bw'ibinyabutabire by'ikizamini. Amabwiriza rusange y'ibizamini bya luciferase asaba amagerageza y'inyongera y'ubutabire bw'ibinyabuzima agenzura neza ubwo bwoko bw'ibangamira. Ntugatekereze ko 1 uM itagira ibisubizo biyobya kubera gusa ko kugabanuka kw'urumuri akenshi gukomera ku rugero rw'irangi ruri hejuru.

Ibipimisho bya potansiyeli y'agahu bipima uko irangi ryikwirakwiza, si urujya n'uruza

Safranine O, TMRM na TMRE, rhodamine 123 na JC-1 byose ni iyoni zifite umuriro mwiza w'amashanyarazi kandi zishobora kwambuka agahu. Zirundanyiriza mu gice cy'imbere cya mitokondiriya gifite umuriro mubi w'amashanyarazi hakurikijwe iringaniza ry'ubutabire n'amashanyarazi. Bityo, floresansi rusange igaragaza potansiyeli mu buryo buziguye, binyuze aho igipimisho giherereye. Nta na kimwe muri ibyo gipima ATP cyangwa urujya n'uruza rwa elegitoroni.

Buri gipimisho gifite uburyo bwo gutegura icyitegererezo bukibereye. Safranine O ibereye mitokondiriya zatandukanyijwe n'ingirabuzimafatizo hamwe n'ingirabuzimafatizo zagizwe izemerera ibinyabutabire kwinjira. Akenshi ikoreshwa hafi ya 2 uM, ikabyutswa n'urumuri ruri hafi ya 495 nm kandi igasohora urumuri ruri hafi ya 585 nm, ikora mu buryo bugabanya urumuri aho kwinjira kwayo guhindura floresansi rusange. TMRM na TMRE bibereye ingirabuzimafatizo zitangiritse, bigashyirwamo ku rugero ruto rwa nanomolar mu buryo butagabanya urumuri. Muri ubwo buryo, gutakaza potansiyeli bituma irangi risohoka maze floresansi ya mitokondiriya ikagabanuka; iyo hashyizwemo irangi ryinshi, icyerekezo cy'ikimenyetso gishobora guhinduka kubera ko irangi ubwaryo rigabanya urumuri rwaryo. JC-1 itanga igipimo cyoroshye cy'igereranya ry'icyatsi n'umutuku, ariko gishingira ku rugero rw'irangi ryashyizwemo n'ingano ya mitokondiriya, bigatuma idakwiriye cyane umurimo wo gupima ingano kurusha TMRM igenzuwe neza. Ibyiza n'imbogamizi by'ubwo buryo byavuzwe mu ncamake mu isesengura ry'ibipimisho bya potansiyeli y'agahu ka mitokondiriya rikoreshwa cyane.

Ubururu bwa metilene bubangamira ibipimo by'urumuri kuri za ngano nyine zikoreshwa mu kazi. Ibyiciro by'uburebure bw'imiraba bukururiraho urumuri, hafi ya 600 kugeza kuri 665 nm, bihurirana n'urumuri rutukura rusohorwa n'ibipimisho. Bityo, ingaruka zo kuyungurura urumuri imbere mu cyitegererezo, kugabanuka kw'urumuri no gukurura urumuri rubyutsa igipimisho byose bishobora kubaho. Iryo rangi kandi ni indi iyoni ifite umuriro mwiza w'amashanyarazi ikunda ibinure, na yo ihindura uko yikwirakwiza bitewe na potansiyeli iri gupimwa. Uburyo bwo kwirinda ibyo ni ibipimo byuzuzanya hamwe n'amagerageza atarimo ibipimwa: igipimisho cyonyine, irangi ryonyine, igipimisho hamwe na buri rugero rw'irangi, gukuraho potansiyeli yose hakoreshejwe FCCP nk'igipimo fatizo cya zeru, ndetse byaba byiza hakoreshejwe n'ikindi gipimisho gifite imiterere y'urumuri itandukanye. Ubushakashatsi ku kunyura ahandi hatari muri Complex III bwatanze urugero rw'ubwo buryo: bwongeye gupima isubiraho rya potansiyeli y'agahu bukoresheje safranine O na TMRM nyuma yo guhagarika imikorere hakoreshejwe antimycin na myxothiazol, maze bubona isubiraho risa mu miterere yaryo. Igipimisho kimwe cy'urumuri gitanga ikimenyetso cy'ibishoboka. Ibipimisho bibiri byigenga bitanga ibimenyetso bifatika.

Uburyo butatu bwo gutegura ibyitegererezo, ibibazo bitatu bitandukanye

Mitokondiriya zatandukanyijwe n'ingirabuzimafatizo, ingirabuzimafatizo zagizwe izemerera ibinyabutabire kwinjira, n'ingirabuzimafatizo zitangiritse si amahitamo asimburana y'urwego rw'ubushobozi bw'ikizamini kimwe bwo kumva impinduka. Ni uburyo butandukanye bufite amahame y'imikorere atandukanye.

Mitokondiriya zatandukanyijwe n'ingirabuzimafatizo zigumana uruhererekane rwo gutwara elegitoroni, agahu k'imbere n'ibice bikora byo mu gice cy'imbere, ariko zigakurwaho agahu gakikije ingirabuzimafatizo, sitozole n'imiterere y'urusobe rwazo. Uko gukuraho ibyo bice ni yo ntego: ibinyabutabire byo guhumeka, ADP, ibibuza imikorere n'ingano z'irangi bigenwa neza, bityo hakamenyekana inzira ikoreshwa. Igihombo ni ukubura itangwa risanzwe ry'ibinyabutabire byo guhumeka, itumanaho ry'ibimenyetso n'ubukene bw'ingufu.

Ingirabuzimafatizo zagizwe izemerera ibinyabutabire kwinjira zigumana mitokondiriya mu gice kinini cy'imiterere yazo yo mu ngirabuzimafatizo, ariko zikemerera ibinyabutabire byo guhumeka biturutse hanze na ADP kunyura mu gahu gakikije ingirabuzimafatizo kafunguwe. Ni igikoresho gikwiye cyo kumenya ikibazo cy'inzira ikoresha ikinyabutabire runaka mu miterere y'ingirabuzimafatizo. Gufungura inzira zo kwinjira birenze urugero byangiza agahu k'inyuma, kandi uburyo bwo kugenzura bukomoka muri sitozole bukavamo.

Ingirabuzimafatizo zitangiritse zigumana byose: inzitizi y'agahu gakikije ingirabuzimafatizo, ibinyabutabire byo muri sitozole bitanga elegitoroni, itwarwa ry'ibinyabutabire byo guhumeka bikomoka imbere, urusobe rwa mitokondiriya n'ubukene bwa ATP. Ni zo zegereye cyane imikorere isanzwe y'ibinyabuzima, ariko zikaba kure cyane yo gusobanura uburyo nyabwo bukora, kuko kwinjira kw'irangi, kwakira elegitoroni kwaryo no kwigizwayo kwaryo byose bigena urugero nyakuri rugera kuri mitokondiriya.

Ubushakashatsi bwa TEGDMA bwakoresheje izo nzego zose uko ari eshatu ku bushake: mitokondiriya zatandukanyijwe n'ubwonko kugira ngo harebwe isubiraho rya ogisidasiyo na potansiyeli bitewe n'irangi; ingirabuzimafatizo shingiro zo mu mwinjiro w'iryinyo zagizwe izemerera ibinyabutabire kwinjira, cyane cyane kugira ngo hamenyekane ko ikibazo kiri muri ogisidasiyo y'ibinyabutabire ifitanye isano na Complex I; n'ingirabuzimafatizo shingiro zitangiritse kugira ngo harebwe ingaruka rusange za 2 uM y'irangi ku ikoreshwa rya ogisijeni ryari ryaragabanutse. Ku basomyi bagereranya ibipimo bya mitokondiriya mu buryo butandukanye bwo gutegura ibyitegererezo, inyandiko ijyana n'iyi isobanura uko ubururu bwa metilene bwimura elegitoroni mu ruhererekane rw'uguhumeka itanga amakuru y'ibanze ku nzira ikoreshwa, naho amabwiriza yo gusoma ubushakashatsi ku guhumeka gukoresha ubururu bwa metilene asobanura impamvu zo guhitamo ingano n'ibibuza imikorere.

Igerageza ry'igenzura rikenewe ku irangi rigira uruhare muri redox

Ikinyabutabire cyisubiramo muri redox gikenera urutonde rurerure rw'amagerageza y'igenzura kurusha ikinyabutabire gisanzwe kibuza imikorere, kuko ubutabire bw'irangi ryonyine bushobora kwigana ibibera mu binyabuzima.

Mu gupima uguhumeka, koresha icyumba kirimo gusa umuti ukomeza pH kugira ngo ugenzure guhindagurika kw'igipimo; icyumba kirimo uwo muti n'irangi, nta bintu bikomoka ku binyabuzima; irangi hamwe n'ikinyabutabire cyose gitanga elegitoroni kiri hanze y'ingirabuzimafatizo kandi kiri mu kizamini; ndetse na rotenone hamwe na antimycin ku musozo, kugira ngo ugenere ikoreshwa rya ogisijeni ritari irya mitokondiriya mu ngirabuzimafatizo zitangiritse. Zirikana ko irangi ryakiriye elegitoroni ryongera kuzitanga kuri ogisijeni, rigakora perogiside ya hidurojeni. Bityo, igerageza ririmo irangi ryonyine ariko ridafite utanga elegitoroni rigaragaza ikoreshwa rya ogisijeni ridafite ingirabuzimafatizo nk'aho ari rito kurusha iribaho iyo NADH cyangwa NADPH ibonetse.

Kuri perogiside ya hidurojeni, hitamo Amplex UltraRed yagenwe neza ibipimo hamwe na peroxidase yo mu gihingwa cya horseradish, aho gukoresha amarangi ya DCFDA adatoranya neza icyo apima; genzura ukoresheje catalase, kandi ugerageze niba ibisubizo bishingira ku kumurikirwa, kuko irangi rituma urumuri rutera impinduka z'ubutabire. Ubushakashatsi kuri mitokondiriya z'ubwonko bwabonye ko umutwaro wa perogiside wiyongereye ukarenga inshuro enye ku rugero rw'irangi rwa 100 nM gusa, mu gihe kuyikuraho byatinze. Perogiside yakorwaga mu buryo butaziguye n'irangi ryakiriye elegitoroni, nta ntambwe yo hagati ya superoxide. Ibisobanuro birambuye ku guhitamo igipimisho no kugenzura urumuri biri mu nyandiko ijyana n'iyi ku gupima ubwoko bwa ogisijeni bukora cyane mu magerageza y'amarangi. Ikibazo cyagutse cyo kumenya igihe perogiside ikora nk'ikimenyetso n'igihe iba umutwaro gisobanurwa hamwe n'iringaniza rya redox mu ngirabuzimafatizo n'ibivugwa ku birwanya ogisidasiyo.

Ku bivugwa ku nzira ikoreshwa, gerageza ahantu hombi ibibuza Complex III gukorera: antimycin A ku mwanya wa Qi na myxothiazol ku mwanya wa Qo, wongereho n'ikizamini cya nyuma cya Complex IV ukoresheje cyanide. Ubushakashatsi bwa mbere ku bwonko bw'imbeba bwagaragaje ko irangi ryasubijeho uguhumeka kwahagaritswe na rotenone, ariko ntirisubizeho ukwahagaritswe na antimycin. Ubushakashatsi bwakurikiyeho ku moko atatu bwabonye isubiraho ry'igice cy'imikorere ku bibuza byombi, n'ibimenyetso by'uko cytochrome c yakiraga elegitoroni mu buryo butaziguye. Fata iyo nzira inyura ahandi nk'ihuje n'amakuru mashya aho kuyifata nk'iyamaze kwemezwa burundu, kuko ingano y'irangi, umwanya ikibuza imikorere gikoreraho, uko ibinyabutabire byo guhumeka bimeze n'uko irangi ryikwirakwiza byose bihindura inzira igaragara.

Imbonerahamwe yo guhitamo ibizamini

Koresha iyi mbonerahamwe uhitemo igipimo ukurikije ikibazo, aho guhitamo ikibazo ukurikije igipimo.

Kugira ngo icyemezo cyemeze nibura, ingano zimwe z'irangi, 100 nM, 300 nM, 1 uM na 2 uM, zigomba gukoreshwa mu gupima urujya n'uruza rwa ogisijeni mu gihe cyo kunyura kwa porotoni mu gahu bidakoze ATP, no mu buryo bukoresha ADP, oligomycin n'ikinyabutabire gitandukanya uguhumeka n'ikorwa rya ATP; umuvuduko w'ikorwa rya ATP; potansiyeli y'agahu, byaba byiza hakoreshejwe ibipimisho bibiri; n'isohorwa rya perogiside ryagenwe neza ibipimo, hakoreshejwe catalase n'amagerageza y'igenzura adafite ingirabuzimafatizo. Tanga urusobe rw'ibipimo bifitanye isano: urujya n'uruza rwa ogisijeni hamwe n'umuvuduko w'ikorwa rya ATP, ATP kuri ogisijeni, potansiyeli na perogiside. Kimwe muri ibyo bipimo cyonyine ni igipimo kikiri gushakirwa icyo gisobanura.