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ID naloge: 28 Letnik: 1998 Predmet: patofiziologija
Vpliv elektricnega draženja
na presnovo acetilholinesteraze v skeletni mišici
v razmerah glukokortikoidne miopatije
Avtor: Vid Zgonc, Janez Vodiškar
Mentor: Prof. dr. Zoran Grubic
Somentor: Doc. dr. Samo Ribaric
IZHODIŠCE. Med kljucne komponente živcnomišicnega stika sodi tudi encim acetilholinesteraza (AChE), ki prek hidrolize živcnega prenašalca acetilholina prekinja živcnomišicni prenos. Poznavanje dejavnikov, ki uravnavajo ekspresijo tega encima v skeletni mišici, in poznavanje medsebojnih vplivov teh dejavnikov je kljucnega pomena za razumevanje procesov, ki so odgovorni za uravnavanje AChE aktivnosti in njene razporeditve v mišicnih vlaknih. Poleg elektromehanicne aktivnosti in raznih lokalno delujocih živcnih faktorjev, uravnavajo ekspresijo AChE v sesalskih skeletnih mišicah tudi hormonski dejavniki. Med slednje sodijo glukokortikoidi (GK), ki v odrasli hitri mišici upocasnijo sintezo AChE. GK upocasnijo tudi sintezo vrste drugih mišicnih beljakovin in s tem povzrocijo glukokortikoidno miopatijo (GM) z znacilno mišicno usahlostjo in izgubo moci.
NAMEN IN HIPOTEZE. V tej nalogi smo preucevali medsebojne vplive elektricnega draženja in GK na ekspresijo AChE v hitri podganji mišici. Uporabili smo živalski model GM in ugotavljali, kako v takšnih razmerah elektricno draženje po vzorcu pocasne mišice vpliva na ekspresijo AChE v hitri mišici. Ucinke na ravni AChE smo primerjali z ucinki na ravni celokupnih beljakovin, ki jih lahko spremljamo prek merjenja mišicne teže. Ucinke na hitri mišici (EDL) smo primerjali z ucinki na pocasni mišici (SOL) in preverjali dve hipotezi:
Hipoteza 1. Elektricno draženje hitre podganje mišice EDL po vzorcu, ki je znacilen za pocasna skeletnomišicna vlakna, preprecuje z GK povzroceno usahlost te mišice.
Ker je znano, da GM prizadene le hitra glikoliticna mišicna vlakna, ne pa tudi pocasnih oksidativnih, pricakujemo, da bo v takih razmerah draženje hitre mišice EDL po vzorcu, ki je znacilen za pocasna mišicna vlakna, preprecevalo usahlost in torej izgubo teže te mišice.
Hipoteza 2. Ucinki, ki jih imajo na aktivnost AChE v hitri podganji mišici GK in elektricno draženje po vzorcu pocasne mišice, so aditivni.
Znano je, da elektromehanicna aktivnost zavira sintezo AChE predvsem na ravni njene mRNA, medtem ko GK upocasnjujejo sintezo tega encima na translacijski in/ali zgodnji posttranslacijski ravni. Ker imata torej elektromehanicni in hormonski regulator prijemališci na razlicnih ravneh biosinteze AChE, lahko pricakujemo, da bosta njuna ucinka aditivna. Potrditev hipoteze bi utrdila prepricanje, da mehanizmi delovanja obeh regulatorjev nimajo skupnih poti.
METODE. Podganje samice soja Wistar smo razdelili v štiri skupine: v prvi so živali prejemale samo fluorirani glukokortikoidni preparat deksametazon (5mg/kg t.t, i.p), v drugi smo posredno, prek ishiadicnega živca, elektricno dražili mišici EDL in SOL z vzorcem, ki je znacilen za pocasno mišico (10 Hz, 1mA, 200µs, 12 h/dan), v tretji pa smo istocasno izvajali obe opisani tretiranji. V cetrti, kontrolni skupini, so živali namesto raztopine deksametazona prejemale enako kolicino fiziološke raztopine. Po petih dneh smo živali žrtvovali in izmerili teži mišic EDL in SOL. V tkivnih homogenatih teh mišic smo z radiometricno metodo na osnovi tankoplastne kromatografije izmerili aktivnost AChE. S primerjavo mišicnih tež in aktivnosti AChE glede na kontrolo smo ocenjevali ucinke GK in elektricnega draženja na presnovo beljakovin in AChE v skeletni mišici. Rezultate smo statisticno ovrednotili z neparametricnim Wilcoxonovim testom z vsoto rangov.
REZULTATI. Z deksametazonom tretirana mišica EDL je bila za 9 % lažja (p<0,05) od kontrolne mišice. Pri mišici EDL, ki je bila elektricno dražena po vzorcu pocasne mišice, je prišlo do porasta teže za 15 % (p<0,05) v primerjavi s kontrolo. Po hkratnem tretiranju z deksametazonom in elektricnim draženjem, se teža mišice EDL ni statisticno znacilno razlikovala (p>0,05) od kontrole. V mišici EDL je aktivnost AChE po dajanju deksametazona padla za 12 % (p<0,05) glede na kontrolo, medtem ko je bila v elektricno draženi mišici EDL za 34 % manjša (p<0,01) od kontrolne aktivnosti. Po hkratnem tretiranju z deksametazonom in elektricnim draženjem je bila aktivnost AChE v mišici EDL za 32 % manjša (p<0,01) od kontrole, a se ni statisticno znacilno razlikovala od aktivnosti v mišici EDL, ki je bila le elektricno dražena. Pri mišicah SOL nismo opazili statisticno znacilnih razlik v primerjavi s kontrolno mišico.
ZAKLJUCKI. Potrdili smo našo prvo hipotezo in pokazali, da elektricno draženje hitre podganje mišice EDL po vzorcu, ki je znacilen za pocasna skeletnomišicna vlakna, preprecuje usahlost te mišice v razmerah GM. Pokazali smo torej, da je sama elektromehanicna aktivnost po opisanem vzorcu dovolj za preprecevanje omenjene miopatije. Dobljeni rezultati pa niso potrdili naše druge hipoteze, saj v elektricno draženi mišici ucinkov GK na AChE aktivnost nismo zaznali. Pravilneje bi bilo torej sklepati, da elektricno draženje, poleg zaviranja sinteze AChE, zavira tudi ucinke GK na sintezo AChE. Naši rezultati napeljujejo k razmišljanju, da mehanizmi, prek katerih uravnava presnovo mišicnih beljakovin elektromehanicna aktivnost, prepoznavajo in locujejo sinapticne beljakovine od izvensinapticnih, medtem ko mehanizmi, prek katerih delujejo GK takega prepoznavanja niso zmožni.
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[Abstract / English version]
Effects of electrical stimulation
on acetylcholinesterase expression in skeletal muscle
under conditions of glucocorticoid myopathy
Author: Vid Zgonc, Janez Vodiškar
Mentor: Prof. dr. Zoran Grubic
Co-mentor: Doc. dr. Samo Ribaric
BACKGROUND. Enzyme acetylcholinesterse (AChE) is one of the key components of the neuromuscular junction: it hydrolyzes acetylcholine and thus terminates neuromuscular transmission. Identification of factors controlling AChE expression, and elucidation of their mutual interactions, is essential for understanding regulation of AChE activity and its distribution along the skeletal muscle fiber. Beside electromechanical activity and some locally acting factors, derived by motor nerve, hormones also regulate AChE expression in the mammalian skeletal muscles. Glucocorticoids (GC) were demonstrated to downregulate AChE synthesis in the fast rat skeletal muscle. GCs also impair synthesis of other muscular proteins causing in this way glucocorticoid myopathy (GM), which is characterized by muscle atrophy and loss of strength.
AIM AND HYPOTHESES. We studied the effects of electrical stimulation, GCs and their mutual interactions on the AChE expression in the fast rat muscle. The effects on AChE were compared to the effects observed at the general muscular protein level roughly determined by measuring muscle weights. The effects on fast (EDL) and slow (SOL) muscles were compared and the following two hypotheses were tested:
Hypothesis 1. Muscle weight loss of fast EDL muscle due to GC treatment can be prevented by electrical stimulation with a pattern characteristic for slow muscle.
It is well known that GM is restricted to fast-glycolytic muscle fibers while slow-oxidative fibers are relatively spared. Therefore, one could hypothesize that electromechanical activity of the slow muscle pattern will prevent fast muscle to develop glucocorticoid-mediated weight loss and atrophy.
Hypothesis 2. The effects of slow pattern electrical stimulation and of GCs on AChE activity of fast rat EDL muscle are not mutually exclusive but aditive.
According to previous studies, electromechanical activity slows down AChE synthesis primarily at the mRNA level, while GCs downregulate syntesis of this enzyme at the translational and/or early posttranslational level. Since both regulators act at different levels of AChE biosyntesis one could hypothesize that their individual effects will be aditive rather than mutually exclusive.
METHODS. Female Wistar strain rats were divided into four groups: the first one was receiving dexametasone (5mg/kg m.w., i.p.); in the second group EDL and SOL muscles of one leg were indirectly stimulated with a slow muscle pattern (10 Hz, 1 mA, 200µs, 12 h/day), while in the third group both treatments were applied simultaneously. Animals of the control group were receiving saline instead of dexamethasone. After five days animals were sacrificed and EDL and SOL muscle weights were determined. AChE activities were measured in homogenates of EDL and SOL by radiometric assay based on thin layer chromatography. The effects of GC and electrical stimulation on the protein and AChE metabolism in skeletal muscle were evaluated by comparison of muscle weights and AChE activities in treated animals and controls. Results were statistically evaluated by the Wilcoxon's rank-sum technique.
RESULTS. In the dexametasone treated-only EDL muscles we observed a significant (p<0,05) loss of weight of 9 % in comparison to control. Weights of EDL muscles electrically stimulated with the slow muscle pattern were significantly (p<0,05) increased (+15 %) in comparison to control. Weight of EDL muscle treated with dexametasone and simultaneously electrically stimulated with the slow muscle pattern did not differ from control weight (p>0,05). AChE activity in EDL muscle treated solely with dexamethasone was decreased for 12 % in comparison to control (p<0,05), while AChE activity of electrically stimulated-only EDL muscle was lower for 34 % in comparison to control (p<0,01). AChE activity of dexamethasone treated and simultaneously electrically stimulated EDL muscle was decreased for 32 % in comparison to control (p<0,01), but was not significantly different from the activity of electrically stimulated-only EDL muscle. In the case of SOL muscles no significant effects could be observed (p>0,05).
CONCLUSIONS. We confirmed our first hypothesis by demonstrating that electrical stimulation of fast rat EDL muscle with the slow muscle pattern prevents GC-mediated atrophy of this muscle. In this way we demonstrated that electromechanical activity of slow pattern is sufficient to prevent GM. However, we could not detect any GC effect on AChE activity in the electrically stimulated muscle and therefore failed to confirm our second hypothesis. According to our results, electromechanical activity of slow muscle pattern not only downregulates AChE activity in fast muscle but also blocks GC effects on the AChE activity in this muscle. Our results suggest that the mechanisms controlling muscle protein metabolism through electromechanical activity recognize and differentiate between synaptic and extrasynaptic proteins, while GC control of this metabolism has no capacity of differentiation on this basis.
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