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

Abstract

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.

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“…Protein concentration was determined using the BioRad protein reagent. EMSA to NF-κB was performed using a gel shift assay kit from Promega [27-29]. NF-κB double-stranded consensus oligonucleotide (5'-AGTTGAGGGGACTTTCCCAGGC- 3') was end-labeled with γ- 32 P-ATP using T4 polynucleotide kinase.…”

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“…However, this story does not end here. In addition to maintaining plasma membrane electrochemical gradients, Na,K-ATPases are now established as an unusual class of transmembrane receptors that mediate signaling elicited by ouabain and related compounds (Kawamoto et al, 2012;Lima et al, 2013). Through interactions with Src tyrosine kinases and IP3-receptors, Na,K-ATPases mediate the activation of Src tyrosine kinase-and intracellular Ca +2 -dependent signaling cascades, respectively (Aperia et al, 2016;Madan et al, 2016).…”