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ID naloge: 3 Letnik: 1997 Predmet: patofiziologija
Vpliv glukokortikoidov na ekspresijo
acetilholinesteraze in butirilholinesteraze
v možganih in jetrih sesalca
Avtor: Urša Weber
Mentor: prof. dr. Zoran Grubic
IZHODIŠCA. V organizmu sesalca obstojata dve obliki holinesteraznih encimov - acetil- (AChE) in butiril- (BuChE) holinesteraza. V holinergicnih sinapsah AChE razgrajuje živcni prenašalec acetilholin (ACh) in tako prekine sinapticni prenos. ACh v veliko manjši meri razgrajuje tudi BuChE, vendar njen fiziološki pomen še ni znan. Pomen socasnega obstoja teh dveh katalitsko zelo sorodnih encimov, kot tudi njun pomen v številnih neholinergicnih tkivih, sta prav tako še nepojasnjena. Encima sta verjetno vpletena tudi v patogenezo Alzheimerjeve bolezni in nekaterih malignomov, s cimer tovrstne študije pridobijo tudi klinicni pomen. V mnogih raziskavah skušajo osvetliti vlogo teh encimov s proucevanjem njunega odziva v specificnih fizioloških ali farmakoloških stanjih. Tako je bilo ugotovljeno, da so med aktivnostmi BuChE razlicnih organov podgane statisticno znacilne korelacije, kar pa ne velja za aktivnosti AChE. Opazovali so tudi vpliv hipofizektomije na aktivnost teh encimov in ugotovili, da se je v vecini organov aktivnost BuChE spremenila veliko bolj kot pa aktivnost AChE, obenem pa je prišlo tudi do porušenja omenjenih korelacij aktivnosti BuChE. To kaže, da je uravnavanje aktivnosti AChE bolj tkivno specificno, uravnavanje aktivnosti BuChE pa bolj pod vplivom sistemsko delujocih dejavnikov, hkrati pa nakazuje razlicen pomen teh encimov v organizmu sesalca. Vendar pa je interpretacija omenjenih opažanj po hipofizektomiji težka, saj pride v takih razmerah do prekinitve v številnih hormonskih zankah, obenem pa so spremembe encimskih aktivnosti lahko posledica tako spremenjene sinteze, kot tudi razgradnje opazovanih encimov.
NAMEN. Namenili smo se raziskati vpliv glukokortikoidov (GK) na presnovo AChE in BuChE v možganih in jetrih podgane ter s tem tudi ozadje omenjenih sprememb po hipofizektomiji. GK smo izbrali zato, ker pripadajo s hipofizo uravnavani hormonski zanki, so znani regulatorji beljakovinske sinteze, nekateri njihovi ucinki na aktivnost holinesteraz pa so tudi že bili opisani. Preverjali smo naslednje hipoteze:
1.) pod vplivom GK se aktivnost BuChE v opazovanih organih bolj spremeni kot pa aktivnost AChE;
2.) pod vplivom GK pride do porušenja korelacije med možganskimi in jetrnimi aktivnostmi BuChE;
3.) spremembe aktivnosti AChE in BuChE pod vplivom GK so predvsem posledica sprememb na ravni sinteze teh beljakovin.
METODE. Poskuse smo izvedli na samicah podgan soja Wistar. Desetim poskusnim živalim smo vsakodnevno vbrizgavali deksametazon (4,5 mg/kg t.t.), dokler nismo ugotovili 15 % padca telesne teže, kar je merilo za polno izražene ucinke deksametazona v organizmu. Kontrolna skupina živali (n = 10) je prejemala enako kolicino fiziološke raztopine. Živali smo žrtvovali, izolirali opazovane organe in iz njih pripravili tkivne homogenate in polisomske frakcije. V njih smo z radiometricno metodo na osnovi tankoplastne kromatografije dolocili encimske aktivnosti AChE in BuChE. S primerjavo encimskih aktivnosti v polisomski frakciji in celokupnem tkivnem homogenatu smo ocenjevali ucinke GK na ravni sinteze opazovanih encimov. Spremembe encimskih aktivnosti smo statisticno ovrednotili z Wilcoxonovim testom z vsoto rangov, za oceno korelacij encimslcih aktivnosti med opazovanimi organi pa smo izracunali koeficiente korelacije ranga po Spearmanu.
REZULTATI. Pri poskusni skupini je aktivnost BuChE statisticno znacilno padla tako v možganih (- 30,3 %; p < 0,01), kot tudi v jetrih (- 60,7 %; p < 0,01). Aktivnost AChE se v možganih ni statisticno znacilno spremenila (p > 0,05), v jetrih pa je statisticno znacilno padla (- 18,8 %; p < 0,01). Med možganskimi in jetrnimi aktivnostmi BuChE smo v kontrolni skupini opazili statisticno znacilno korelacijo (p = 0,89; p < 0,01), medtem ko je v poskusni skupini ni bilo (p = 0,06; p > 0,05). V primeru BuChE so se padci aktivnosti v celokupnih tkivnih homogenatih dobro ujemali s padci aktivnosti v polisomskih frakcijah (možgani: - 27,3 %; p < 0,05; jetra: - 59,5 %; p < 0,05). V primeru AChE pa niti v možganih niti v jetrih nismo ugotovili statisticno znacilnih padcev aktivnosti v polisomski frakciji (p > 0,05).
ZALJUCKI.
1.) Opaženi ucinki deksametazona podpirajo teorijo, da je aktivnost BuChE bolj pod vplivom sistemsko
delujocih dejavnikov, aktivnost AChE pa bolj pod nadzorom tkivno specificnih dejavnikov.
2.) Ker do porušenja korelacije med možganskimi in jetrnimi aktivnostmi BuChE pride tako v razmerah hipofizektomije kot tudi po tretiranju podgan z deksametazonom, je možno, da je vzrok za to porušenje odsotnost ucinkov adrenokortikotropnega hormona (ACTH). ACTH je namrec dejavnik, katerega plazemska koncentracija je znižana v obeh omenjenih poskusnih razmerah, poleg tega pa ima že dokazane neposredne ucinke na presnovo nekaterih beljakovin.
3.) Aktivnost možganske in jetrne BuChE uravnavajo GK predvsem na ravni njene sinteze, medtem ko so za padec aktivnosti AChE v jetrih odgovorni drugi mehanizmi, verjetno pospešena razgradnja tega encima. Na aktivnost možganske AChE, kot tudi na njeno sintezo, GK najverjetneje nimajo vpliva.
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[Abstract / English version]
Glucocorticoid influence on acetylcholinesterase
and butyrylcholinesterase expression
in mammalian brain and liver
Author: Urša Weber
Mentor: prof. dr. Zoran Grubic
BACKGROUND. In mammalian organism there are two cholinesterase enzymes - acetyl- (AChE) and butyryl(BuChE) cholinesterase. In cholinergic synapses AChE degrades the neurotransmitter acetylcholine (ACh) and in this way terminates synaptic transmission. To a much lesser degree, ACh is also degraded by BuChE, of which physiological function is still unknown. The role of the coexistence of both enzymes with very similar catalytic properiies and the role of these enzymes in many noncholinergic tissues, are also not clear. These enzymes possibly have some patogenetic role in Alzheimer's disease and some cancers, which gives a certain clinical background to such investigations. In many researches the approach to investigate the function of these enzymes is to study their response to specific physiological or pharmacological conditions. It was previously shown that in rats there are strong cross-tissue correlations of BuChE activity, but not of AChE. The effect of hypophysectomy was also investigated and it was found, that in most organs the BuChE activity was far more affected than that of AChE and beside that, complete disruption of cross-tissue correlations of BuChE activity was observed. This implies, that the regulation of AChE is tissue-specific, while BuChE depends on a regulation by general systemic factors. It also may reflect different roles of these enzymes in mammalian organism. However, the changes observed after hypophysectomy are difficult to interpret, because they may reflect disturbances of several different hormonal axes and at the same time, the changes of enzyme activities could be the result of either influences on the enzyme synthesis level or changed enzyme degradation.
OBJECTIVE. We aimed to elucidate the glucocorticoid (GC) influence on the metabolism of AChE and BuChE in rat brain and liver and at the same time, to investigate the background of changes observed after hypophysectomy. GCs were applied, because they are pituitary regulated hormones, they represent well known protein synthesis regulatory factors and because some of their effects on cholinesterase metabolism were already shown. Following hypotheses were tested:
1.) BuChE activity is more influenced by GCs than AChE activity;
2.) GCs eliminate the correlation between brain and liver BuChE activity;
3.) the effects of GCs on the activities of AChE and BuChE are primarily exerted on the level of enzyme synthesis.
METHODS. Female Wistar rats were used in our experiments. Ten tested animals were daily injected with dexamethasone (4,5 mg/kg t.t.) until 15 % loss of body weight, indicative for fully expressed effects of dexamethasone, was observed. Animals of control group (n = 10), on the other hand, received the same amount of physiologic solution. Animals were sacrificed, organs were isolated and their tissue homogenates and polysomic fractions were prepared. Corresponding enzyme activities were measured by radiometric assay on the basis of thin layer chromatography. The effects of GCs on the enzyme synthesis level were evaluated by comparition between enzyme activities in polysomic fractions and activities in whole tissue homogenates. Alterations in enzyme activities were statistically evaluated by the rank-sum technique of Wilcoxon. Spearman's rank correlation coefficients (p) were calculated to evaluate the correlations between brain and liver enzyme activities.
RESULTS. Under the influence of dexamethasone BuChE activity was significantly afected in both, brain (- 30,3 %; p < 0,01) and liver (- 60,7 %; p < 0,01). While AChE activity in brain remained practically unchanged (p > 0,05), liver AChE activity decreased significantly (- 18,8 %; p < 0,01) after dexamethasone treatment. Significant correlation between brain and liver BuChE activity was observed in the control group (p = 0,89; p < 0,01). This correlation disappeared (p = 0,06; p > 0,05) after dexamethasone treatment. In the case of BuChE, dexamethasone effects on activities in total tissue homogenates matched well with those in polysomic fraction (brain: - 27,3 %; p < 0,05; liver: - 59,5 %; p < 0,05). On the other hand, AChE activities in the brain and liver polysomic fractions remained unchanged (p > 0,05) after dexamethasone treatment.
CONCLUSIONS.
1.) Observed effects of GCs support the theory, that BuChE activity is primarily regulated by general systemic
factors, while the regulation of AChE activity is mainly tissue-specific.
2.) Because the elimination of the correlation between brain and liver BuChE activities is observed both, after hypophysectomy and after dexamethasone treatment, it is possible that this effect is the result of reduced plasma adrenocorticotropin (ACTH) level. ACTH is namely a factor, of which plasma concentration is reduced in both experimental conditions and beside that, it also exerts direct effects on metabolism of some proteins.
3.) In the case of brain and liver BuChE, GCs exert their effects on the level of enzyme synthesis. On the other hand, reduced AChE activity in rat liver must be the result of other effects of GCs, possibly increased degradation of this enzyme. Most probably there is no effect of GCs on the brain AChE activity nor its
synthesis.
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