Signaling function of Na,K-ATPase induced by ouabain against LPS as an inflammation model in hippocampus

Signaling function of Na,K-ATPase induced by ouabain against LPS as an inflammation model in hippocampus Kinoshita et al. Kinoshita et al. Journal of Neuroinflammation 2014, 11:218 http://www.jneuroinflammation.com/content/11/1/218 Kinoshita et al. Journal of Neuroinflammation (2014) 11:218 DOI 10.1186/s12974-014-0218-z RESEARCH JOURNAL OF NEUROINFLAMMATION Open Access Signaling function of Na,K-ATPase induced by ouabain against LPS as an inflammation model in hippocampus Paula Fernanda Kinoshita1, Lidia Mitiko Yshii1, Andrea Rodrigues Vasconcelos1, Ana Maria Marques Orellana1, Larissa de Sá Lima1, Ana Paula Couto Davel2, Luciana Venturini Rossoni2, Elisa Mitiko Kawamoto1 and Cristoforo Scavone1* Abstract Background: Ouabain (OUA) is a newly recognized hormone that is synthesized in the adrenal cortex and hypothalamus. Low doses of OUA can activate a signaling pathway by interaction with Na,K-ATPase, which is protective against a number of insults. OUA has central and peripheral anti-inflammatory effects. Lipopolysaccharide (LPS), via toll-like receptor 4 activation, is a widely used model to induce systemic inflammation. This study used a low OUA dose to evaluate its effects on inflammation induced by LPS injection in rats. Methods: Adult male Wistar rats received acute intraperitoneal (ip) OUA (1.8 μg/kg) or saline 20 minutes before LPS (200 μg/kg, ip) or saline injection. Some of the animals had their femoral artery catheterized in order to assess arterial blood pressure values before and after OUA administration. Na,K-ATPase activity, cytokine mRNA levels, apoptosis-related proteins, NF-κB activation brain-derived neurotrophic factor BDNF, corticosterone and TNF-α levels were measured. Results: OUA pretreatment decreased mRNA levels of the pro-inflammatory cytokines, inducible nitric oxide synthase (iNOS) and IL-1β, which are activated by LPS in the hippocampus, but with no effect on serum measures of these factors. None of these OUA effects were linked to Na,K-ATPase activity. The involvement of the inflammatory transcription factor NF-κB in the OUA effect was indicated by its prevention of LPS-induced nuclear translocation of the NF-κB subunit, RELA (p65), as well as the decreased cytosol levels of the NF-κB inhibitor, IKB, in the hippocampus. OUA pretreatment reversed the LPS-induced glial fibrillary acidic protein (GFAP) activation and associated inflammation in the dentate gyrus. OUA also prevented LPS-induced increases in the hippocampal Bax/Bcl2 ratio suggesting an anti-apoptotic action in the brain. Conclusion: Our results suggest that a low dose of OUA has an important anti-inflammatory effect in the rat hippocampus. This effect was associated with decreased GFAP induction by LPS in the dentate gyrus, a brain area linked to adult neurogenesis. Keywords: Ouabain, Na,K-ATPase, TNF-α, NF-κB, Inflammation, LPS * Correspondence: 1 Molecular Neuropharmacology Laboratory, Department of Pharmacology, Institute of Biomedical Science, University of São Paulo, 05508-900 São Paulo, Brazil Full list of author information is available at the end of the article © 2014 Kinoshita et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kinoshita et al. Journal of Neuroinflammation (2014) 11:218 Introduction Na,K-ATPase (NKA) is a membrane protein that is essential for the survival of the organism. This enzyme is expressed in all the cells of the human body, having many functions including the maintenance of osmotic balance, cell volume, pH and membrane potential. This occurs by the hydrolysis of an adenosine triphosphate (ATP) molecule leading to the export of three sodium ions and the import of two potassium ions into the cell, which is fundamental for neuronal excitability and cell maintenance [1,2]. NKA is constituted of three subunits: α, β and γ [3], with each subunit having a number of isoforms that provide functional versatility across different cell types, in turn highlighting the different roles and responses produced by NKA activation across cell types [4-8]. However, the γ (gamma) subunit, is not present in all the cells, with the other subunits being required for NKA to be functional [9]. In the adult brain, α1 is expressed in all cells, with α2 being expressed primarily in astrocytes and α3 in neurons [10,11]. Mutations in the α2- and α3-isoform genes are involved in neurological disorders, such as familial hemiplegic migraine type-2 [12], rapid-onset dystonia [13], alternating hemiplegia of childhood [14] and cerebellar ataxia, areflexia, pes cavus, optic atrophy and sensorineural hearing loss (CAPOS) [15], with genetic variations in NKA also associating with bipolar disorder, suggesting a role for this enzyme in the etiology of this disease [16]. The NKA α-isoform plays a critical role in the modulation of learning and memory, in turn regulating susceptibility to Alzheimer’s disease [17]. Several works show NKA to operate as a receptor and not only as a pump, with a number of intracellular pathway activations driving its effects [18,19]. Ouabain (OUA) is synthesized by the adrenal gland and hypothalamus [20,21] and is likely to have important physiological roles in both the central and peripheral nervous systems [22,23]. OUA binds to NKA in hippocampal astrocytes, activating inositol trisphosphate receptor (InsP3R), which generates calcium oscillations, thereby activating NF-κB [23]. Xie and Askari [24] also showed OUA to act as a signal transducer, by binding to NKA and thereby activating the Ras-Raf-MAPK signaling cascade by the epidermal growth factor receptor (EGFR). OUA has a dual role, given its dose-dependent response curve effects. A high concentration of OUA can cause cell death, driving neuronal necrosis via NKA inhibition, leading to potassium ion depletion and thereby increasing intracellular sodium and calcium ions [25]. Conversely, low concentrations of OUA (0.01 nM) are protective against kainic acid-induced lesions in the rat striatum, where it reduces apoptosis by increasing Bcl-2 [26]. Similarly, OUA affords protection in rat kidney primary Page 2 of 12 cultures against Shiga toxin [27]. As such, OUA can afford protection both peripherally and centrally. NF-κB is a nuclear transcription factor, which is commonly induced following danger or inflammatory signaling, including by lipopolysaccharide (LPS) [28]. NF-κB comprises homo- and heterodimers via the combination of the subunits p65 (RELA), p50, p52, c-REL and REL of the REL/NF-κB family of proteins [28]. Different dimer combinations can activate or (...truncated)