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.

Transcriptomic changes triggered by ouabain in rat cerebellum granule cells: Role of α3- and α1-Na+,K+-ATPase-mediated signaling

“…The animal experiments were approved by the Bioethics Committee of the Faculty of Biology, Lomonosov Moscow State University (#82-O). Cerebellum was isolated from 7- to 8-day-old rat pups under sterile conditions as described previously [33]. Cerebellum cells were dissociated with 0.25% Trypsin-EDTA (Paneco) for 15 min at 37°C.…”

Endogenous Ouabain and Related Genes in the Translation from Hypertension to Renal Diseases

“…It was demonstrated that it is an integrated component of a hypothalamic renin-angiotensin-aldosterone system (RAAS) and also plays an important role in regulation of systemic Blood Pressure (BP) [ 37 , 40 , 56 ]. The relation between the central and peripheral RAASes with central and peripheral EO is still not completely understood [ 55 ], but it was discovered that EO-induced signaling in neurons had positive and direct consequences on rat brain in terms of brain cells survival [ 57 ]. Similarly, in mouse models, the reduction of endogenous steroids seems to have a protective effect on oxidative stress for CNS [ 58 ].…”

Intracerebroventricular injection of ouabain causes mania-like behavior in mice through D2 receptor activation

“…Recent studies have demonstrated that α-synuclein 12 and β-amyloid 13 oligomers can inhibit Na,K-ATPase function, which may be one of the mechanisms underlying the relationship between Na,K-ATPase function and neurodegenerative processes. CTS ouabain is known to activate several signaling cascades in neurons, such as MAPK 14,15 , Akt 16 , PKC 17 , etc. Several studies have demonstrated that ouabain can modulate the function of the dopaminergic system by binding to Na,K-ATPase that functionally interacts with dopamine receptors 18 .…”