Te ine i ngā pānga o te kikorangi metilini ki ngā mitokōria: te hāora, te ATP, me te pūmanawa kiriuhi

Me whakaaro ka tāpirihia e tētahi taiwhanga te kikorangi metilini ki ngā mitokōria kua ngoikore te whakahā, ā, ka kitea te pikinga o te whakapau hāora. Ko te rerenga māori hei tuhi, kua whakahokia e te waikano te mahi a ngā mitokōria. Tērā pea kei te hē taua rerenga. He ariā motuhake te rere hāora, te hanga ATP, me te pūmanawa kiriuhi. Kei te okana pūtau kotahi ēnei, pēnei i te tārua pūrua, te tukutahi, me te tiritiri e whakamahi ana i te kōpaki kapua kotahi: he hononga tō rātou, engari he ture rerekē e whakahaere ana i ia mea. Ki te rangiruatia tētahi ki tētahi, ka hē te whakamāramatanga, ahakoa he tika ā-hangarau ia inenga.

Ka whakanuia e te kikorangi metilini te tūpono ki tēnei rangirua, nā te mea ka whai wāhi ia ki te pūnaha e inea ana e ia. He matū huringa hāora-whakaheke e tango ana i ngā irahiko i te taha whakarunga o te ara, ā, e tuku ana ki te taha whakararo; he katote tōrunga rata-ngako ka whakaemi ki ngā mitokōria whai pūngao; he pūhui kikorangi kaha hoki e ngongo ana, e tuku ana i te aho i ngā awhe kotahi ki ērā o ngā pūoko noa. Kāore e taea e te inenga kotahi te kawe i taua taumahatanga. Ko te huarahi pono, kua kitea puta noa i ngā rangahau mitokōria o Budapest, ko te ine i te rere hāora me te tere hanga ATP me te pūmanawa kiriuhi me te tukunga hauwai perōkai, me ngā mana whakahaere motuhake mō te waikano i ia wāhanga.

Ka ine te ine whakahā i te rere hāora, ehara i te putanga pūngao

Kotahi noa te mea e pūrongotia ana e te ine whakahā taumira-teitei, mā te Oroboros Oxygraph rānei, mā te kaitātari rere ā-waho-pūtau Seahorse rānei: te tere o te whakahekenga o te hāora. Kāore e pūrongo i te maha o ngā iraoho i papua mō ia irahiko, i te nui o te rōnaki i tiakina, i te whakamahinga rānei o taua rōnaki e te whākōkī ATP synthase.

He mea nui ngā kupu. I ngā mitokōria kua wehea, ka puta te whakahā turuturu i te matū whāngai kāore he ADP; ka puta te āheinga phosphorylation hāora i te matū whāngai me te ADP whakakī rawa; ā, mā te tāpiri ā-taumata i tētahi kaiwetewete hononga ka kitea te āheinga mōrahi whakawhiti irahiko. I ngā pūtau mau tonu, ko ngā inenga taurite a Seahorse ko te whakahā taketake, te whakahā hono-ATP ka tairongo ki te oligomycin, te turuturu iraoho, te whakahā mōrahi kua tautuhia mā te FCCP, me te whakapau hāora ehara i te mea nō ngā mitokōria, e ātete ana ki te rotenone me te antimycin. Ko te tapa i ngā putanga a Seahorse ko State 3 me State 4 he whakatau i tētahi tōtika kāore e tautokona e taua whakaritenga, e whakamārama ana te anga Mito Stress Test a Agilent.

Ka whati te kikorangi metilini i te hononga e whakaarohia noatia ana i waenga i te nui ake o te hāora me te nui ake o te pūngao. I ngā mitokōria kua wehea mai i te roro poaka kini, nā te waikano 100 nM ki te 1 uM i whakapiki te whakapau hāora i te wā whakatā, me ngā matū whāngai glutamate me te malate, succinate, me te alpha-glycerophosphate. Tata ki te 64 ōrau te pikinga i runga i te alpha-glycerophosphate, engari kāore i tino rerekē te whakahā i whakaohongia e te ADP i ngā mitokōria hauora, e ai ki te rangahau mō ngā mitokōria roro kua wehea. Nā te pikinga o te turuturu me te noho pūmau o te rere i whakaohongia e te ADP, ka heke te ōwehenga whakahaere whakahā kua tātaihia. Ehara taua hekenga i te tohu kua tūkinotia ngā mitokōria e te waikano. Ko tōna tikanga, kua tāpirihia e te waikano tētahi huringa hāora-whakaheke whakapau hāora ki runga i te whakahā noa. Ki te pūrongo tētahi whakamātautau whakapau hāora mō te kikorangi metilini i te pikinga anake, kua ine ia i tētahi tukanga matū tūturu, engari kāore anō kia kōrero mō te tiaki pūngao.

Ka whakaatu ngā whakamātautau whakaora i taua āputa anō, i tētahi tauine nui ake. I te aukatinga o te Matatini I, tata ki te toru whakareatanga te whakaoranga o te rere hāora, engari tata ki te 1.5 whakareatanga noa te whakaoranga o te hanga ATP. He nui ake te hāora i tae ki te kawehiko. He iti ake te wāhanga i whakamahia hei hanga ATP. Me whai inenga ATP ngā whakapae katoa kua pai ake te phosphorylation hāora; ko te mea pai, me whakaatu hei ōwehenga ATP mō ia hāora, me te whakamārama i te tikanga o te tauraro.

Ka ine ngā whakamātautau ATP i te tere hanga; kāore te rahinga e taea te whakakapi i tērā

He pātai kē tā te whakamātautau ATP e whakautu ai: e hia te ATP ka hangaia mō ia wāhanga wā. Mā tētahi inenga ā-wā, arā, te aroturuki i te whitinga aho o te luciferase namu ahi mō ētahi meneti, mā tētahi whakamātautau hono o te hexokinase me te glucose-6-phosphate dehydrogenase rānei, ka pānuitia i te 340 nm, ka whiwhi i tētahi tere. Mā te inenga kotahi o te ATP katoa i te pito whakamutunga ka whiwhi i te rahinga. E whakaata ana tēnei i te hanga tangohia te whakapau, ā, he iti noa tāna e whakaatu ai mō te phosphorylation hāora ina tū takitahi.

I whakamahia e te rōpū Budapest te tikanga whākōkī hono, me te pēhi i te adenylate kinase. I kitea e rātou he pikinga iti, engari he pikinga whai hiranga ā-tatauranga, o te hanga ATP i ngā mitokōria kua tukuna ki te rotenone me te antimycin, i te 1 ki te 2 uM kikorangi metilini. He whakaoranga tūturu tērā, ā, he whakaoranga wāhanga noa. I puta he whakautu pēnei i tētahi tauira rerekē i te rangahau tāoke TEGDMA: nā te waetahi niho TEGDMA 5 mM i turaki te whakahā hono ki te Matatini I mai i te āhua 286 ki te 28 waeine rere, ā, nā te waikano 2 uM i whakahoki ki te āhua 86.

E rua ngā mana whakahaere e kore e taea te waiho atu mō tētahi pūhui tae e whai tauhohenga hāora-whakaheke ana. Tuatahi, whakamahia te oligomycin hei whakatau i te wāhanga o te hanga ATP kua inea e whakawhirinaki ana ki te ATP synthase. Tuarua, whakamātauria te whakaoranga o tētahi tāpiritanga mōhiotia, kāore he pūtau, me te ATP mōhiotia, te huinga tauhohe katoa, me ngā kukūtanga waikano tonu i whakamahia; ko ngā kukūtanga iti rawa hei whakamātau ko te 0, 100 nM, 300 nM, 1 uM, me te 2 uM. Ka taea e te waikano te ngongo i te aho ka tukuna, te aukati i te whākōkī tohu, te whakarerekē rānei i ngā tauhohenga hāora-whakaheke o ngā matū tauhohe. E taunaki ana ngā aratohu whānui mō ngā whakamātautau luciferase kia whakamahia ngā whakamātautau whakaū ā-koiora-matū mō tēnei momo whakararuraru tonu. Kaua e whakapae kāore he whakapohehe ine i te 1 uM nā te mea he kaha ake te tāmitanga aho i ngā kukūtanga teitei ake.

Ka ine ngā pūoko pūmanawa kiriuhi i te tohanga waikano, ehara i te rere

Ko te Safranine O, te TMRM me te TMRE, te rhodamine 123, me te JC-1 he katote tōrunga katoa ka taea te whakawhiti i te kiriuhi. Ka whakaemi ki te kōpū mitokōria whai utanga tōraro, e ai ki te taurite matū-hiko. Nō reira, mā te wāhi e noho ai te pūoko ka tohu autaki te kōrekoreko tapeke i te pūmanawa. Kāore tētahi o ēnei e ine i te ATP, i te rere irahiko rānei.

He whakaritenga e tino hāngai ana ki ia pūoko. E pai ana te Safranine O mō ngā mitokōria kua wehea me ngā pūtau kua whakapuarehia, ko te tikanga tata ki te 2 uM, me te aho whakaongaonga tata ki te 495 nm me te aho tuku tata ki te 585 nm. Ka mahi i tētahi āhua tāmitanga aho, e whakarerekē ai te urunga ki roto i te kōrekoreko tapeke. E pai ana te TMRM me te TMRE mō ngā pūtau mau tonu, me te utanga iti kei te taumata nanomolar, i te āhua kore-tāmitanga. I tēnei āhua, ka tuku te whakakorenga pūmanawa i te waikano, ā, ka heke te kōrekoreko mitokōria; ina nui te utanga, ka taea te huri o te ahunga tohu nā te tāmitanga whaiaro. Ka whakarato te JC-1 i tētahi ōwehenga kākāriki ki te whero e ngāwari ana te whakamahi, engari ka whakawhirinaki ki te utanga me te papatipu mitokōria. Nā reira, he ngoikore ake mō ngā mahi ine rahinga i te TMRM e āta whakahaerehia ana. Kua whakarāpopototia ēnei painga me ngā here i tētahi arotake e whakamahia whānuitia ana mō ngā pūoko pūmanawa kiriuhi mitokōria.

Ka whakararuraru te kikorangi metilini i ngā inenga ā-aho i ngā kukūtanga e whakamahia ana. Ko ōna awhe ngongo tata ki te 600 ki te 665 nm e īnaki ana ki te aho whero ka tukuna e ngā pūoko, nō reira he take tūturu pea ngā pānga tātari ā-roto, te tāmitanga aho, me te ngongo i te aho whakaongaonga. He katote tōrunga rata-ngako tuarua hoki te waikano, ā, ka rerekē tōna ake tohanga i runga i te pūmanawa e inea ana. Ko te ārai ko ngā inenga whakaū maha me ngā tauira pātea: ko te pūoko anake, ko te waikano anake, ko te pūoko me ia kukūtanga waikano, ko te whakakorenga katoa o te pūmanawa mā te FCCP hei tohutoro kore, ā, ki te taea, ko tētahi pūoko tuarua he rerekē ōna āhuatanga aho. I whakatakotoria taua tauira e te rangahau karo i te Matatini III, mā te tāruarua i te whakaoranga pūmanawa kiriuhi ki te safranine O me te TMRM i muri i te aukatinga mā te antimycin me te myxothiazol, ā, i kitea te whakaoranga kounga ōrite. He tīwhiri te pūoko aho kotahi. He taunakitanga ngā pūoko motuhake e rua.

E toru ngā whakaritenga, e toru ngā pātai rerekē

Ehara ngā mitokōria kua wehea, ngā pūtau kua whakapuarehia, me ngā pūtau mau tonu i ngā tautuhinga tairongo ka taea te whakawhitiwhiti i te whakamātautau kotahi. He ariā rerekē ēnei, he rerekē hoki ngā mātāpono whakahaere.

Ka mau tonu i ngā mitokōria kua wehea te mekameka kawe irahiko, te kiriuhi o roto, me ngā pūnaha o te kōpū mitokōria, engari kua tangohia te kiriuhi pūtau, te wē pūtau, me te hanganga whatunga. Ko taua tangohanga tonu te whāinga: ka tino tautuhia ngā matū whāngai, te ADP, ngā kaiārai, me ngā kukūtanga waikano, kia taea ai te tautuhi i tētahi ara. Ko te utu, ka ngaro te tuku matū whāngai māori, te tuku tohu, me te hiahia pūngao.

Ka pupuri ngā pūtau kua whakapuarehia i ngā mitokōria ki roto i te nuinga o tō rātou hanganga pūtau, me te tuku kia uru mai ngā matū whāngai o waho me te ADP mā te kiriuhi pūtau kua whakatuwheratia. Ko ēnei te taputapu tika hei tautuhi i tētahi hapa o te ara matū whāngai i roto i te horopaki pūtau. Mā te whakapuare nui rawa ka tūkinotia te kiriuhi o waho, ā, ka horoia atu te whakahaere a te wē pūtau.

Ka pupuri ngā pūtau mau tonu i ngā mea katoa: te ārai kiriuhi pūtau, ngā matū whakaheke i te wē pūtau, te kawe matū whāngai ā-roto, te whatunga mitokōria, me te hiahia ATP. Ko ēnei te mea tata rawa ki ngā mahi noa o te pūtau, engari ko te mea tawhiti rawa i te tikanga mahi motuhake, nā te mea mā te urunga, te whakahekenga, me te pupuritanga motuhake o te waikano e whakatau te pānga tūturu ki ngā mitokōria.

I āta whakamahia e te rangahau TEGDMA ngā taumata e toru: ngā mitokōria roro kua wehea hei tirotiro i te whakaoranga o te hāoratanga me te pūmanawa mā te waikano; ngā pūtau kakau penupenu niho kua whakapuarehia hei tautuhi matua i te wāhi o te hapa ki te hāoratanga matū whāngai hono ki te Matatini I; me ngā pūtau kakau mau tonu hei ine i te pānga kupenga ā-pūtau o te waikano 2 uM ki te whakapau hāora kua pēhia. Mō ngā kaipānui e whakataurite ana i ngā inenga mitokōria puta noa i ngā whakaritenga, ka whakarato te whakamārama hoa mō te kawe irahiko a te kikorangi metilini i roto i te mekameka whakahā i te papamuri o ngā ara, ā, ka whakamārama te aratohu pānui rangahau whakahā kikorangi metilini i te arorau o te rahinga me ngā kaiārai.

Ngā mana whakahaere e hiahiatia ana mō tētahi waikano hohe ki ngā tauhohenga hāora-whakaheke

He roa ake te rārangi mana whakahaere e hiahiatia ana mō tētahi matū huringa hāora-whakaheke i tērā mō tētahi kaiārai noa, nā te mea ka taea e ngā tauhohenga o te waikano anake te whakatauira i ngā mahi koiora.

Mō te ine whakahā, whakamahia he rūma whai wairewa pūreirei anake hei ine i te paheke o te tohu; he rūma whai wairewa pūreirei me te waikano, kāore he matū koiora; te waikano me ngā matū whakaheke ā-waho-pūtau e noho ana i te whakamātautau; me te rotenone me te antimycin i te mutunga hei tautuhi i te whakapau hāora ehara nō ngā mitokōria i ngā pūtau mau tonu. Kia mōhio, ka hāora anō te waikano kua whakahekea mā te whakawhiti irahiko ki te hāora hei hanga hauwai perōkai. Nō reira, mā tētahi tauira pātea waikano-anake kāore he kaituku irahiko e whakatau iti rawa te whakapau hāora kore-pūtau ka puta ina wātea te NADH, te NADPH rānei.

Mō te hauwai perōkai, me pai kē ki te Amplex UltraRed kua whakatauritehia me te horseradish peroxidase, kaua ki ngā waikano DCFDA kāore e whiriwhiri motuhake. Whakaūngia mā te catalase, ā, whakamātauria te whakawhirinaki ki te aho, nā te mea he matū whakapiki tauhohenga mā te aho te waikano. I kitea e te rangahau mitokōria roro, neke atu i te whā whakareatanga te pikinga o te kawenga perōkai i te waikano 100 nM noa, me te pōturi haere o te whakakorenga. I hanga hāngaitia te perōkai e te waikano kua whakahekea, kāore he matū takawaenga superoxide. Kei te tuhinga hoa mō te ine i ngā momo hāora tauhohe i ngā whakamātautau waikano ngā taipitopito mō te kōwhiri pūoko me ngā mana whakahaere aho. Kei te taha o te taurite hāora-whakaheke ā-pūtau me ngā whakapae ārai-hāora te kōrerorero whānui mō te wā e mahi ai te perōkai hei tohu, hei kawenga rānei.

Mō ngā whakapae mō te ara, whakamātauria ngā wāhi kaiārai e rua o te Matatini III: te antimycin A i te wāhi Qi me te myxothiazol i te wāhi Qo, me tētahi whakamātautau whakamutunga o te Matatini IV mā te cyanide. I pūrongo tētahi rangahau roro kiore tōmua i te whakaoranga mā te waikano o te whakahā i aukatihia e te rotenone, engari kāore i whakaorangia te whakahā i aukatihia e te antimycin. Heoi, i kitea e te rangahau momo-kararehe e toru o muri mai he whakaoranga wāhanga me ngā kaiārai e rua, me ngā taunakitanga mō te whakahekenga hāngai o te cytochrome c. Me whakaaro he hāngai te ara karo ki ngā raraunga hou, kaua hei mea kua whakatauria pū, nā te mea ka whakarerekē te kukūtanga waikano, te wāhi kaiārai, te āhua matū whāngai, me te tohanga waikano i te ara ka kitea.

Ripanga kōwhiri whakamātautau

Whakamahia tēnei ripanga hei kōwhiri i te inenga i runga i te pātai, kaua ko te pātai i runga i te inenga.

Ko te huinga iti rawa mō tētahi whakapae whakaponohia, me whakamahi ngā kukūtanga waikano ōrite, 100 nM, 300 nM, 1 uM, me te 2 uM, mō te rere hāora i raro i ngā tikanga turuturu, ADP, oligomycin, me te kaiwetewete hononga; mō te tere hanga ATP; mō te pūmanawa kiriuhi, me ngā pūoko e rua ki te taea; me te tukunga perōkai kua whakatauritehia, me ngā mana whakahaere catalase me te kore-pūtau. Pūrongotia te huinga hono: te rere hāora me te tere ATP me te ATP mō ia hāora me te pūmanawa me te perōkai. Ki te tū takitahi tētahi o aua tau, he inenga noa e rapu ana i tōna tikanga.