Author/Editor		Bezjak, Katja; Pirkmajer, Sergej; Matkovič, Urška
Title		Vpliv ouabaina na izražanje natrij-kalijeve črpalke in znotrajcelično signaliziranje v kulturi človeških in podganjih skeletnomišičnih celic
Translated title		The effect of ouabain on the expression of the sodium-potassium pump and intracellular signaling in cultured human and rat skeletal muscle cells
Type		monografija
Place		Ljubljana
Publisher		[K. Bezjak]
Publication year		2021
Volume		str. 60 f., I-VI, 1-23 f. pril.
Language		slv
Abstract		Uvod: Ouabain je kardiotonični glikozid, ki svoje učinke sproži preko natrij-kalijeve črpalke (NKA), ki je doslej njegov edini znan receptor. Skeletne mišice so največji organ v človeškem telesu in zato vsebujejo največjo količino NKA ter tako predstavljajo pomembno tarčno tkivo za ouabain. Ouabain s svojo vezavo na NKA ne vpliva samo na njeno transportno funkcijo, ampak tudi na znotrajcelično signalizacijo, kar bi lahko vplivalo tudi na endokrino funkcijo skeletnih mišic (npr. izločanje interlevkinov). Skeletnomišične celice izločajo interlevkin-6 (IL-6) med telesno aktivnostjo, med katero se najverjetneje iz nadledvičnih žlez izloča tudi endogeni ouabain. Namen: Ugotoviti, ali bo ouabain v skeletnomišičnih celicah vplival na izražanje NKA in ali bo aktiviral signalne poti, ki so lahko prožene preko NKA in so za odziv mišice na telesno aktivnost še posebej pomembne. Hipoteze: 1) Ouabain zniža izražanje proteina NKA[alfa]1 v človeških skeletnomišičnih celicah; 2) Ouabain aktivira signalni poti Src/ERK1/2 in Akt/mTOR v človeških skeletnomišičnih celicah; 3) Ouabain zavira signalno pot IL-6/JAK/STAT3 v človeških skeletnomišičnih celicah; 4) Ouabain ne vpliva na izražanje NKA[alfa]1 in ne proži signalnih poti v podganjih skeletnomišičnih celicah. Metode: Kot model smo uporabili človeške skeletnomišične celice in podganje skeletnomišične celice L6 ter določili izražanje proteinov z metodo prenosa western, izražanje genov pa z metodo RT-qPCR. Rezultati: V človeških skeletnomišičnih celicah je ouabain znižal izražanje proteina NKA[alfa]1, aktiviral signalni poti Src-ERK1/2 in Akt/mTOR in zavrl je signalno pot IL-6/JAK/STAT3. V podganjih skeletnomišičnih celicah ouabain ni vplival na izražanje NKA[alfa]1 in ni imel pomembnejših učinkov na aktivacijo znotrajceličnih signalnih poti. Zaključki: Ugotovili smo, da je mehanizem znižanja izražanja proteina NKA[alfa]1 v človeških skeletnomišičnih celicah posttranskripcijski. Odzivnost signalne poti Src-ERK1/2 je relativno majhna, aktivacija signalne poti Akt/mTOR pa je najverjetneje neodvisna od Src. Pri z ouabainom povzročenim zavrtjem signalne poti IL-6/JAK/STAT3 je udeleženih več mehanizmov, za razjasnitev katerih bi bili potrebni dodatni poskusi. Na podlagi rezultatov na podganjih skeletnomišičnih celicah smo ugotovili, da so signalni učinki ouabaina odvisni od prisotnosti na ouabain občutljive NKA[alfa]1.Background: Ouabain, cardiotonic steroid, trigggers its effects via the sodium-potassium pump (NKA), which is the only known receptor for ouabain so far. Skeletal muscles, which are the largest organ in human body, contain the largest pool of NKA and consequently represent the major target tissue for ouabain. In addition to being an inhibitor of ion transport, ouabain is also capable of inducing signals, which may influence the endocrine function of skeletal muscle. Skeletal muscle cells secrete Interleukin-6 during exercise, when endogenous ouabain is also secreted, probably from the adrenal gland. Aim: To determine whether ouabain has an effect on the expression of NKA and whether it activates any of the signalling pathways known to be activated through NKA and which are especially important for muscle response during exercise. Hypotheses: 1) Ouabain lowers the expression of protein NKA[alpha]1 in human skeletal muscle cells, 2) Ouabain activates the Src-ERK1/2 and Akt/mTOR signaling pathway in human skeletal muscle cells, 3) Ouabain inhibits the IL-6/JAK/STAT3 signalling pathway in human skeletal muscle cells, 4) Ouabain has no effect on the expression of NKA[alpha]1 and on signalling pathways in rat skeletal muscle cells. Methods: As an experimental model, we used human skeletal muscle cells and L6 rat skeletal muscle cells. We determined the expression of proteins with Western Blot and gene expression with RT-qPCR. Results: Ouabain reduced the expression of NKA[alpha]1 protein and activated the Src-ERK1/2 and PI3K/Akt/mTOR signalling pahways in human skeletal muscle cells. The IL-6/JAK/STAT3 signalling pathway in human skeletal muscle cells was inhibited by ouabain. In contrast, in rat skeletal muscle cells ouabain had no effect on the expression of protein NKA[alpha]1 and did not display major effects on intracellular signalling. Conclusions: We concluded ouabain reduces the expression of NKA[alpha]1 protein in human skeletal muscle cells in post-trancriptional manner. Src-ERK1/2 signalling pathway is barely responsive on treatment with ouabain, activation of Akt/mTOR signalling pathway is probably Src independent. Ouabain inhibits IL-6/JAK/STAT3 signalling pathway in human skeletal muscle cells through more than one mechanism, which has to be addressed by future studies. Based on results in rat skeletal muscle cells, we concluded that ouabain's effect on intracellular signalling is dependent on the presence of ouabain-sensitive NKA[alpha]1.
Descriptors		Skeletne mišice
Keywords		ouabain natrij-kalijeva črpalka znotrajcelična signalizacija interlevkin-6