title: Ouabain-induced changes in MAP kinase phosphorylation in primary culture of rat cerebellar cells.
abstract: Cardiotonic steroid (CTS) ouabain is a well-established inhibitor of Na,K-ATPase capable of inducing signalling processes including changes in the activity of the mitogen activated protein kinases (MAPK) in various cell types. With increasing evidence of endogenous CTS in the blood and cerebrospinal fluid, it is of particular interest to study ouabain-induced signalling in neurons, especially the activation of MAPK, because they are the key kinases activated in response to extracellular signals and regulating cell survival, proliferation and apoptosis. In this study we investigated the effect of ouabain on the level of phosphorylation of three MAPK (ERK1/2, JNK and p38) and on cell survival in the primary culture of rat cerebellar cells. Using Western blotting we described the time course and concentration dependence of phosphorylation for ERK1/2, JNK and p38 in response to ouabain. We discovered that ouabain at a concentration of 1 μM does not cause cell death in cultured neurons while it changes the phosphorylation level of the three MAPK: ERK1/2 is phosphorylated transiently, p38 shows sustained phosphorylation, and JNK is dephosphorylated after a long-term incubation. We showed that ERK1/2 phosphorylation increase does not depend on ouabain-induced calcium increase and p38 activation. Changes in p38 phosphorylation, which is independent from ERK1/2 activation, are calcium dependent. Changes in JNK phosphorylation are calcium dependent and also depend on ERK1/2 and p38 activation. Ten-micromolar ouabain leads to cell death, and we conclude that different effects of 1-μM and 10-μM ouabain depend on different ERK1/2 and p38 phosphorylation profiles. Copyright © 2016 John Wiley & Sons, Ltd.
authors.affiliation: Research Center of Neurology, Moscow, Russia.|Bach Institute of Biochemistry, Research Center of Biotechnology, Russian Academy of Sciences, Moscow, Russia.
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Lopachev
authors.affiliation: Research Center of Neurology, Moscow, Russia.|Lomonosov Moscow State University, International Biotechnological Center, Moscow, Russia.
authors.forename: Olga M
authors.initials: OM
authors.lastname: Lopacheva
authors.affiliation: Lomonosov Moscow State University, International Biotechnological Center, Moscow, Russia.|Lomonosov Moscow State University, Faculty of Chemistry, Department of Chemical Enzymology, Moscow, Russia.
authors.forename: Ekaterina A
authors.initials: EA
authors.lastname: Osipova
authors.affiliation: Lomonosov Moscow State University, Faculty of Biology, Department of Biochemistry, Moscow, Russia.
authors.forename: Elizaveta A
authors.initials: EA
authors.lastname: Vladychenskaya
authors.affiliation: Lomonosov Moscow State University, International Biotechnological Center, Moscow, Russia.|Lomonosov Moscow State University, Faculty of Biology, Department of Biochemistry, Moscow, Russia.
authors.forename: Larisa V
authors.initials: LV
authors.lastname: Smolyaninova
authors.affiliation: Research Center of Neurology, Moscow, Russia.
authors.forename: Tatiana N
authors.initials: TN
authors.lastname: Fedorova
authors.affiliation: Bach Institute of Biochemistry, Research Center of Biotechnology, Russian Academy of Sciences, Moscow, Russia.
authors.forename: Olga V
authors.initials: OV
authors.lastname: Koroleva
authors.affiliation: St. Petersburg State University, Institute of Translational Biomedicine, St. Petersburg, Russia.
authors.forename: Evgeny E
authors.identifier: http://orcid.org/0000-0002-2552-9512
authors.initials: EE
authors.lastname: Akkuratov
mesh_terms: D000818:Animals; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology*; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008954:Models, Biological; D010042:Ouabain / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D013997:Time Factors; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D010042:Ouabain; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases
keywords: ERK1/2; JNK; MAP kinases; Na,K-ATPase; ouabain; p38