Ouabain activates NFκB through an NMDA signaling pathway in cultured cerebellar cells.

Na,K-ATPase, an ion pump, has been shown to interact with other proteins in signaling complexes in cardiac myocytes, renal and glial cells, and several other cell types. Our previous in vivo studies indicated that intrahippocampal administration of ouabain (OUA), an inhibitor of Na,K-ATPase, induces NFκB activation, leading to an increase in mRNA levels of target genes of this transcription factor in the rat hippocampus. The present work investigated whether OUA can regulate NF-κB in primary cultured rat cerebellar cells. Cells were treated with different concentrations of OUA (1, 10 or 100 µM) for different periods of time (1, 2 and 4 h). OUA induced a time- and concentration-dependent activation of NFκB (peak of activation: 10 µM, 2 h), involving both p50/p65 and p50/p50 NFκB dimers. OUA (10 µM, 2 h) induced upregulation of tumor necrosis factor α (Tnf-α), interleukin-1ß (Il-1ß), and brain derived neurotrophic factor (Bdnf) mRNA levels. Both NFκB activation and gene expression activation induced by OUA (10 µM) were abolished when cells were pre-treated for 20 min with MK-801 (N-Methyl-D-Aspartate (NMDA) receptor antagonist), manumycin A (farnesyltransferase inhibitor), PP-1(Src-family tyrosine kinase inhibitor) and PD98059 (mitogen-activated protein kinase (MAPK) inhibitor). OUA (10 µM) alone or in the presence of MK-801, PP-1, PD98059 did not cause cell death or DNA fragmentation. These findings suggest that OUA activates NFκB by NMDA-Src-Ras-like protein through MAPK pathways in cultured cerebellar cells. This pathway may mediate an adaptive response in the central nervous system.

Influence of N-methyl-D-aspartate receptors on ouabain activation of nuclear factor-κB in the rat hippocampus.

It has been shown that ouabain (OUA) can activate the Na,K-ATPase complex and mediate intracellular signaling in the central nervous system (CNS). Inflammatory stimulus increases glutamatergic transmission, especially at N-methyl-D-aspartate (NMDA) receptors, which are usually coupled to the activation of nitric oxide synthase (NOS). Nuclear factor-κB (NF-κB) activation modulates the expression of genes involved in development, plasticity, and inflammation. The present work investigated the effects of OUA on NF-κB binding activity in rat hippocampus and the influence of this OUA-Na,K-ATPase signaling cascade in NMDA-mediated NF-κB activation. The findings presented here are the first report indicating that intrahippocampal administration of OUA, in a concentration that did not alter Na,K-ATPase or NOS activity, induced an activation of NF-κB, leading to increases in brain-derived neurotrophic factor (Bdnf), inducible NOS (iNos), tumor necrosis factor-α (Tnf-α), and B-cell leukemia/lymphoma 2 (Bcl2) mRNA levels. This response was not linked to any significant signs of neurodegeneration as showed via Fluoro-Jade B and Nissl stain. Intrahippocampal administration of NMDA induced NF-κB activation and increased NOS and α(2/3) -Na,K-ATPase activities. NMDA treatment further increased OUA-induced NF-κB activation, which was partially blocked by MK-801, an antagonist of NMDA receptor. These results suggest that OUA-induced NF-κB activation is at least in part dependent on Na,K-ATPase modulatory action of NMDA receptor in hippocampus. The interaction of these signaling pathways could be associated with biological mechanisms that may underlie the basal homeostatic state linked to the inflammatory signaling cascade in the brain.