20.500.12556/RUL-178890 Hormonski in presnovni mehanizmi uravnavanja Na+,K+-ATPaze v skeletni mišici ​Hormonal and metabolic mechanisms of regulation of Na+,K+-ATPase in skeletal muscle Na+,K+-ATPaza (NKA) je heterodimerna ionska črpalka, ki igra ključno vlogo pri uravnavanju ionske homeostaze, hkrati pa je pomemben porabnik celičnega ATP. Natančna usklajenost ionskega transporta in presnove energije je potrebna med mišičnimi kontrakcijami. Signalne poti, ki povezujejo NKA in celično presnovo energije, večinoma niso bile raziskane. Regulacija majhnega transmembranskega regulatorja FXYD1 je v veliki meri neznana. Z uporabo celičnih modelov mišice in ledvic smo raziskali molekularne mehanizme regulacije NKA/FXYD1 preko presnovnih dražljajev. Pokazali smo, da od AMP odvisna protein kinaza (AMPK) progresivno zavira fosforilacijo Tyr10 v človeških in podganjih mišičnih cevčicah, medtem ko v ledvičnih celicah prepreči z epidermalnim rastnim faktorjem (EGF) spodbujeno fosforilacijo Tyr10. Učinki na NKA so rezultat zaviranja EGF receptorja (EGFR), kar prikazuje ključno vlogo AMPK pri uravnavanju Tyr fosforilacije. Nasprotno pa inhibitor NKA ouabain poveča fosforilacijo Tyr10 ter tako nakazuje, da ima od AMPK odvisna defosforilacija Tyr10 stimulatorni učinek na NKA. Razjasnili smo mehanizem fosforilacije Tyr10. Rastni faktorji, ne pa inzulin, močno spodbudijo fosforilacijo Tyr10. Aktivacija EGFR je nujna, vendar ne zadostna za od EGF odvisno fosforilacijo Eyr10. Kinaze družine Src so glavne kinaze Tyr10, saj občutno znižajo bazalno in popolnoma preprečijo od EGF odvisno fosforilacijo Tyr10 brez učinka na EGFR. Utišanje Src kinaze zniža bazalno, vendar ne od EGF odvisno fosforilacijo Tyr10, kar prikazuje, da Src ni edini regulator Tyr10. Ščitnični hormon (T3) ni imel vpliva na izražanje NKA/FXYD1 mRNA in/ali vsebnost proteinov. Slaba odzivnost naših celic opozarja na omejitve celičnih modelov. T3 je povečal oksidativno in glikolitično proizvodnjo ATP, neodvisno od NKA. Kljub temu smo opazili donorsko specifičen učinek T3 na izražanje FXYD1. Zaključimo lahko, da smo vzpostavili nov mehanizem, s katerim AMPK uravnava NKA, in predlagali, da je izražanje FXYD1 lahko odvisno od presnovnih signalov, s čimer smo opisali povezavo med presnovo in od NKA odvisnim ionskim transportom. Na+,K+-ATPase (NKA), a heterodimeric ion pump, is essential for ion homeostasis and important sink of cellular ATP. In contracting skeletal muscles, tight coordination of ion transport and energy metabolism is required. Signaling pathways that link NKA and cell energy metabolism are not well understood. Regulation of small transmembrane NKA regulator FXYD1 in muscle is largely unknown. We have dissected molecular mechanisms of NKA/FXYD1 regulation via important metabolic stimuli, using muscle and kidney cell models. Our results demonstrate AMP-activated protein kinase (AMPK) progressively suppressed the phosphorylation of Tyr10 in cultured human and rat myotubes, while preventing the epidermal growth factor (EGF)-mediated Tyr10 phosphorylation in kidney. AMPK exerts its effects on NKA via suppression of EGF receptor (EGFR), highlighting its role in tyrosine phosphorylation. Conversely, NKA inhibitor ouabain increased Tyr10 phosphorylation, suggesting AMPK-mediated NKA dephosphorylation has stimulatory effect on NKA. We have elucidated the mechanism of Tyr10 phosphorylation. Growth factors, but not insulin, potently stimulated Tyr10 phosphorylation. EGFR activity is necessary but not sufficient for EGF-mediated Tyr10 phosphorylation. Src-family kinases are the main kinases of Tyr10. Src family kinase inhibition markedly suppressed basal Tyr10 phosphorylation and completely prevented the EGF-stimulated Tyr10 phosphorylation without affecting EGFR. Gene silencing of Src decreased basal, but not EGF-stimulated Tyr10 phosphorylation, indicating Src is not a sole regulator of Tyr10. We failed to observe significant effects of thyroid hormones (T3) on the NKA/FXYD1 gene expression and/or protein levels. Our cells exhibit poor responsiveness to T3 stimulation, indicating limitations of cell models. T3 increased oxidative and glycolytic ATP production, irrespective of NKA. Nevertheless, we have observed a donor-specific effect of T3 on the expression of FXYD1. Collectively, we have established a novel mechanism by which AMPK regulates NKA and suggested FXYD1 expression may be dependent on metabolic signals, providing new links between energy metabolism and NKA-mediated ion transport. NKA Na+ K+ -ATPaza Tyr10 fosforilacija AMPK presnova Src EGF inzulin T3 ščitnični hormon FXYD1 skeletna mišica človeške cevčice HK-2 NKA Na+ K+ -ATPase Tyr10 phosphorylation AMPK metabolism Src EGF insulin T3 thyroid hormone FXYD1 skeletal muscle human myotubes HK-2 true false false Slovenski jezik Angleški jezik Doktorsko delo/naloga 2026-01-31 07:15:09 2026-01-31 07:15:30 2026-02-01 04:37:59 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 38504 8518.pdf 8518.pdf 1 363EF88823138818C2EB3A20A51B22E0 2ff9db0abc998afafd1e184e3742262411ebfb99f59d7f5ca2c13ace5170f4f3 3293c00e-fe6c-11f0-a1ba-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=226920 Medicinska fakulteta 0 0 0