Maƙala
Shuɗin methylene da nitric oxide: NOS, cGMP, da isar da sigina a jijiyoyin jini
Tambayar tana kama da mai sauƙi: shin shuɗin methylene yana hana nitric oxide aiki? A dakin gwaje-gwaje, amsar ta kasu zuwa tambayoyi huɗu daban-daban, kuma kowace tana da nata gwajin. Haɗa su wuri ɗaya shi ne babban dalilin da ya sa rubuce-rubuce game da shuɗin methylene da nitric oxide suke nuna tabbaci amma ba su fayyace abin da suke nufi ba.
Ka ɗauki siginar nitric oxide a matsayin sarka mai gaɓoɓi huɗu. Ana kwafar bayanin wata kwayar gado domin samar da nitric oxide synthase. Enzayim ɗin yana mayar da arginine zuwa nitric oxide. Gas ɗin yana yaɗuwa zuwa abin da zai yi wa aiki kafin ya yi martani da wani abu dabam. Guanylate cyclase mai narkewa yana gano shi, sannan ya samar da GMP mai zagaye. An wallafa tasirin shuɗin methylene a kowace gaɓa, amma shaidun sun fito ne daga shirye-shiryen gwaji daban-daban, da matakan taro daban-daban, da nau'o'in enzayim daban-daban. Sakamako a wata gaɓa ba ya tabbatar da tasiri a wata.
Sarka ta asali, a bayyane
A bangon jijiyar jini, tsarin da aka sani yana gudana haka. Wani abin motsawa yana aiki a kan ƙwayar endothelium. Nitric oxide synthase na endothelium, wanda ake kira eNOS ko NOS3, yana samar da ɗan gajeren zango na nitric oxide. Gas ɗin yana yaɗuwa zuwa ƙwayar tsoka mai santsi da ke kusa, ya ɗaure da heme mai baƙin ƙarfe a yanayin raguwa na guanylate cyclase mai narkewa, sannan ya hanzarta mayar da GTP zuwa GMP mai zagaye. GMP mai zagaye yana kunna protein kinase G, yadda ake sarrafa calcium ya sauya, sannan tsokar ta sake.
Akwai wata sarka ta biyu da ya dace a ware. Yayin kamuwa da cuta ko fallasa ga cytokine, ƙwayoyin jiki suna kwafar bayanin wani enzayim dabam, nitric oxide synthase da ake iya jawo samarwarsa, ko iNOS, wanda kuma ake kira NOS2. Yana samar da kwararar nitric oxide mafi yawa kuma mafi ɗorewa fiye da eNOS. Nau'i na uku, NOS na ƙwayoyin jijiyoyin sadarwa ko nNOS, yana da nasa yanayin dabam. Idan wata takardar bincike ta ambaci nitric oxide synthase ba tare da bayyana nau'insa ba, ka tsaya ka duba wanne aka auna. Bayanin yadda shuɗin methylene ke motsa electrons yana yin wannan gargaɗin game da abubuwan da martanin redox ke shafa gaba ɗaya: mai isarwar ba ya yin komai shi kaɗai, kuma abokin hulɗar da ke kowane ƙarshen ne ke tantance sakamakon.
GMP mai zagaye da aka auna shi ma sakamako ne da ya haɗa tasirin abubuwa da dama, ba gwajin enzayim kai tsaye ba. Matsayinsa yana nuna abin da cyclase ya samar bayan an cire abin da phosphodiesterases suka rushe, musamman PDE5 a tsokar jijiyoyin jini mai santsi. Saboda haka, raguwar GMP mai zagaye tana nufin cewa sigina kaɗan ne ya isa ga mai gano ta. Wannan kaɗai ba ya nuna gaɓar da ta gaza.
Gaɓa ta 1: ana samar da enzayim na iNOS kaɗan
A cikin macrophages na ƙananan beraye da aka motsa da lipopolysaccharide na ƙwayoyin bacteria, interferon gamma, ko duka biyun, shuɗin methylene ya hana ƙaruwar furotin na iNOS da ake tsammani, kuma ya rage RNA mai ɗaukar saƙon iNOS sosai a gwaje-gwajen kwafar bayanin kwayar gado a macrophages. Abin da ya fi bayar da haske shi ne abin da bai sauya ba. Rushewar mai hanawa I kappa B alpha, haɗa phosphate da NF kappa B da STAT1, da motsawar waɗannan abubuwan zuwa cikin tsakiya sun ci gaba da gudana kusan yadda suke. Abin da ya ragu shi ne ɗaurewarsu da DNA, kamar yadda gwajin chromatin immunoprecipitation ya tabbatar ta hanyar nuna raguwar zamansu a yankin fara kwafar bayanin iNOS.
Wannan tasiri ne kan sarrafa kwayar gado a matakin da yake kafin yawan enzayim, ba toshe enzayim da ya riga ya kasance nan take ba. Yana cikin isar da sigina ta kumburi, inda abubuwan da ke sarrafa kwafar bayanin kwayar gado dole su ɗaure da yankin fara kwafar kafin furotin ya bayyana. Ba ya faɗin wani abu kai tsaye game da eNOS da yake aiki kullum a jijiyar jini da ke hutawa, kuma ya kamata a bayyana shi haka.
Gaɓa ta 2: enzayim na NOS da ya riga ya kasance yana aiki a hankali
Wani bincike a kan beraye ya gwada wani mataki dabam. An bai wa beraye maza da suke farke lipopolysaccharide ta jijiya, sannan bayan sa'o'i uku aka ba su ko dai ruwan gishiri ko shuɗin methylene a 3 mg a kowace kg ta jijiya. An auna aikin nitric oxide synthase a ɓangaren tsakiya na ƙasan hypothalamus ta hanyar mayar da arginine mai alamar ganowa zuwa citrulline a binciken aikin NOS a hypothalamus. Damuwa tare da ruwan gishiri kaɗai sun jawo wani aiki da bai dogara da calcium ba, sannan lipopolysaccharide ya ɗaga shi zuwa kusan ninki goma na matakin rukunin kwatantawa. Shuɗin methylene ya kawar da dukkan ɓangarorin aikin biyu da aka jawo. Aikin da yake faruwa kullum kuma ya dogara da calcium bai sauya a wannan nama ba, a wannan adadin da wannan lokacin.
Ka karanta wannan sakamakon daidai yadda aka rubuta shi: an rage aikin irin iNOS da aka auna a ƙarƙashin waɗannan yanayi. Ba auna yadda ake bayyana kwayar gado ba ne, kuma ba hujjar cewa koyaushe yana fifita hana iNOS aiki fiye da eNOS ko nNOS ba ce. Aikin gwaji da tsarkakakken enzayim yana ƙara bayyana dalilin yin taka-tsantsan. A gwaje-gwajen kai tsaye na tsarkakakken nitric oxide synthase, shuɗin methylene ya toshe samuwar citrulline gaba ɗaya a 30 micromolar, tare da matakin taron da ke hana rabin aiki kusa da 5.3 micromolar ba tare da superoxide dismutase ba, da 9.2 micromolar tare da shi, kamar yadda aka ruwaito a kwatancen tsarkakakken NOS da guanylate cyclase. Wani bincike dabam na tsarkakakken nNOS ya nuna cewa oxidation na heme da ke ɗaure da enzayim yana da hannu. Darasin da za a ɗauka shi ne cewa hana NOS aiki kai tsaye gaskiya ne a tsarin da aka ware, amma ba za a faɗaɗa sakamakon hypothalamus zuwa da'awar gama-gari cewa shuɗin methylene ba ya shafar dukkan NOS da suke aiki kullum a ko'ina ba.
Gaɓa ta 3: nitric oxide yana ɓacewa kafin ya isa
Mafi bayyanannen gwajin da ya nuna cewa wani abu dabam da toshe enzayim na iya bayyana raguwar GMP mai zagaye ya fito ne daga ƙwayoyin tsoka masu santsi na jijiyar huhu ta zomo da aka reno. Tsarin gwajin ya kauce wa NOS na cikin ƙwayoyin gaba ɗaya ta hanyar ƙara masu bayar da nitric oxide kai tsaye, sannan ya bincika ko shuɗin methylene har yanzu yana rage martanin GMP mai zagaye a binciken superoxide a tsokar jijiyoyin jini mai santsi.
An gwada masu bayarwa uku daga 1 zuwa 100 micromolar: S nitroso L cysteine, sodium nitroprusside, da wani tabbataccen sinadari mai kama da su wanda aka cire wa rukunin amine, mai suna MPANO. Dukkansu sun ƙara GMP mai zagaye tsakanin kusan ninki 1.5 da ninki 12. Shuɗin methylene a 10 micromolar ya rage tasirin biyun farko da kashi 51 zuwa 100, amma bai sauya martanin MPANO ba. Da a ce shuɗin methylene yana toshe wurin aikin cyclase ne kawai, zai yi wuya a bayyana wannan bambancin tsakanin masu bayarwar, domin dukkansu ukun suna haɗuwa a enzayim ɗaya da ke matakin gaba.
Sai gwaje-gwajen tabbatarwa guda uku suka nuna cewa superoxide ne sanadin wannan tasirin. Na farko, samar da superoxide a wajen ƙwayoyin ta hanyar xanthine tare da xanthine oxidase ya maimaita irin wannan tsari: an toshe tasirin CYSNO da nitroprusside, amma ba a shafi MPANO ba. Na biyu, shuɗin methylene ya sa ƙwayoyin tsoka masu santsi suka samar da superoxide gwargwadon adadi da lokaci, wanda aka auna ta hanyar reduction na cytochrome c. Na uku, superoxide dismutase ya hana wannan ragewar aiki gaba ɗaya, amma catalase bai yi haka ba. Wannan yana nuna superoxide, maimakon hydrogen peroxide, a matsayin sinadarin da ya haifar da tasirin kai tsaye a wannan shirin gwaji.
Ya dace a bayyana nau'in gwajin da aka reno ƙwayoyi tare, domin yana nuna wurin da tasirin ya faru. An reno ƙwayoyin endothelium da ke kan ƙananan gilashin rufe samfur tare da ƙwayoyin tsoka masu santsi na ɗan lokaci, don haka abin da endothelium yake fitarwa a yanayin asali ya ƙara GMP mai zagaye na tsoka mai santsi ninki uku zuwa takwas. Yi wa kowane sashe maganin shuɗin methylene kafin gwajin ya rage wannan sigina, kuma superoxide dismutase ya dawo da wani ɓangare na siginar da ta ragu bayan an yi wa endothelium maganin. Wato, ba lallai ne shuɗin methylene ya yi aiki a cikin ƙwayar tsoka mai santsi da ke gano siginar ba. Zai iya rage siginar ta hanyar kawar da mai ɗaukar saƙon yayin tafiyarsa. Jagorar karanta binciken shuɗin methylene tana ɗaukar wannan a matsayin ƙa'ida ta gama-gari: a bayyana sashen da aka yi shisshigi kafin a bayyana sinadarin da aka yi wa aiki.
Kiran wannan hana NOS aiki zai zama kuskure. Masu bayarwar ba su buƙaci wani mataki na synthase domin su yi aiki ba, amma duk da haka siginar ta ragu.
Gaɓa ta 4: mai gano siginar kansa yana ba da martani kaɗan
Akwai tasiri kai tsaye a kan guanylate cyclase mai narkewa, amma matakan taro suna da muhimmanci. A kwatancen tsarkakakkun enzayim da aka ambata a sama, cyclase da nitric oxide ya kunna ya buƙaci shuɗin methylene kusan 60 micromolar domin hana rabin aikinsa, idan aka kwatanta da hana rabin aikin NOS a kusan 5 zuwa 9 micromolar. Ko da 1 millimolar ya samar da toshewar da ba ta kai cikakkiyar toshewa ba. Wannan bambanci ne na kusan ninki shida zuwa goma sha ɗaya. Saboda haka, gwajin ƙwayoyin da ke amfani da ƙaramin matakin micromolar kuma yake rage GMP mai zagaye ba za a kira shi hana cyclase aiki musamman ba saboda ƙimar GMP mai zagaye kaɗai.
Wani binciken redox da aka yi daga baya ya ƙara gargaɗi na biyu: shuɗin methylene ya kashe aikin cyclase da nitric oxide ya kunna ta wata hanya da ta bambanta da oxidation na baƙin ƙarfen heme ta iska kawai. Don haka, ana iya raunana mai gano siginar, amma har yanzu ba a warware cikakken tsarin sinadaran ba. Bambancin matakan taron yana nufin cewa ya dace a fara kawar da yiwuwar tasirin superoxide da NOS a ƙaramin matakin micromolar. A nan ne kuma ya dace a yi gwajin sarrafa phosphodiesterase. Sai dai idan an auna ko an toshe rushewar, raguwar taruwar GMP mai zagaye na iya dacewa da ƙarancin samarwa, saurin rushewa, ko duka biyun.
Dalilin da ya sa ba za a taƙaita wannan zuwa da'awa ɗaya game da hawan jini ba
An tabbatar da kowace gaɓa a wani tsari dabam. Kwafar bayanin kwayar gado a macrophages ba aikin enzayim a hypothalamus ba ne. Aikin hypothalamus bayan lipopolysaccharide ba yanayin matsewar endothelium a hutawa ba ne. Abin da endothelium ke fitarwa a yanayin asali yayin renon ƙwayoyi tare ba faɗaɗawar jijiyoyin jini da sinadarin motsawa ya jawo ba ce. Ƙwayoyin jijiyar huhu da aka reno a 10 micromolar na shuɗin methylene ba tsarin zagayawar jinin mutum ba ne. Haɗa su zuwa magana irin ta cewa shuɗin methylene yana ɗaga matsin jini ta hanyar toshe nitric oxide yana watsar da ainihin bambance-bambancen da aka tsara gwaje-gwajen tabbatarwa domin su kiyaye.
Abubuwan dubawa uku suna sa bayanin ya tsaya kan gaskiya. Na farko, a bayyana nau'in enzayim: eNOS don yanayin matsewar jijiyoyin jini a hutawa, iNOS don kwararar da aka jawo ta siginar kumburi, nNOS don yanayin ƙwayoyin jijiyoyin sadarwa da wasu gwaje-gwajen tsarkakakken enzayim. Na biyu, a bayyana yadda siginar nitric oxide ta samu: samarwa daga ciki, fitarwar endothelium a yanayin asali, ko wani takamaiman mai bayarwa kamar CYSNO, nitroprusside, ko MPANO, domin tabbataccen mai bayarwar ya jure tasirin shuɗin methylene, amma masu saurin sauyawa ba su jure ba. Na uku, a bayyana matakin taro da abin da ya dawo da aiki: dawo da aiki ta superoxide dismutase yana nuna hannun superoxide, gazawar catalase tana saɓa wa ra'ayin cewa hydrogen peroxide ne sanadi a wannan shirin gwaji, maimaitawar tasiri ta xanthine oxidase tana nuna cewa wannan hanya kaɗai ta isa, kuma gwaje-gwajen tsarkakakken enzayim suna ware tasirin kai tsaye kan aikin enzayim daga asarar da ke faruwa yayin tafiyar saƙo.
Ga taƙaitaccen haɗin bayanin. Ya fi dacewa a ɗauki shuɗin methylene a matsayin abin da ke sauya wurare da dama a isar da sigina daga nitric oxide zuwa GMP mai zagaye, maimakon mai toshe cyclase musamman. Dangane da shirin gwaji da matakin taro, zai iya rage ɗaurewar abubuwan da ke sarrafa kwafar bayanin iNOS, hana enzayim na NOS aiki kai tsaye, rage samuwar nitric oxide ta hanyar samar da superoxide, da raunana cyclase da nitric oxide ya kunna. Rawar isar da sigina ta GMP mai zagaye a binciken jijiyoyin jini da kwatancen masu bayar da nitric oxide da ake amfani da su a gwaje-gwajen ƙwayoyin jiki kowannensu yana tattauna ɗaya daga cikin waɗannan rassan dalla-dalla, kuma hanyoyin da ake amfani da su wajen ware tasirin redox daga tasirin enzayim suna bayyana dalilin da ya sa superoxide dismutase tare da catalase da kuma tabbataccen mai bayarwa a matsayin gwajin tabbatarwa suke bayyana akai-akai a waɗannan wallafe-wallafen bincike.
Ƙarancin ƙimar GMP mai zagaye bayan shuɗin methylene yana nuna cewa sarkar ta karye. Gwaje-gwajen tabbatarwa kaɗai ne suke nuna gaɓar da ta karye.