Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA263

Poster Communications

Effect of therapeutic concentration of lithium on live HEK293 cells results in increase of Na+/K+-ATPase, change of protein composition and induction of oxidative stress.

M. Vošahlíková1, L. Roubalova1, H. Ujcikova1, S. Musil2, M. Alda3,4, P. Svoboda1

1. Institute of Physiology of the Czech Academy of Sciences, Praha, Czechia. 2. Institute of Analytical Chemistry of the Czech Academy of Sciences, Brno, Czechia. 3. Dalhousie University, Halifax, Nova Scotia, Canada. 4. National Institute of Mental Health, Klecany, Czechia.

Lithium (Li) represents the standard for the treatment of bipolar disorder. Mechanism of therapeutic Li action at the cellular and molecular level remains not fully elucidated. Among many different hypotheses, induction of oxidative stress (OS) and alteration of expression level and/or activity of membrane ionic channels/transporters received much attention. In this work, we tested whether the long-term exposure of live HEK293 cells to therapeutic Li concentration results in change of plasma membrane (PM) content of Na+/K+-ATPase. Na+/K+-ATPase level was determined in parallel with estimation of general OS and products of lipid peroxidation. HEK293 cells were cultivated in the presence or absence of 1 mM LiCl for 7 (L7 Li-treated, C7 control) or 21 days (L21, C21). Three independent PM and post-nuclear supernatant (PNS) preparations were isolated from all cell types. Na+/K+-ATPase level was determined by [3H]ouabain binding and immunoblot assays in PM. General OS was monitored by fluorescent dye 2′,7′-dichlorofluorescein diacetate (DCF) in whole cells, PNS and PM. 4-hydroxy-2-nonenal (4-HNE)-protein adducts (immunoblot assay) and malondialdehyde (MDA, HPLC analysis) were determined as products of lipid peroxidation in PNS. Overall protein composition was determined in PNS by 2D electrophoresis followed by MALDI-TOF MS/MS. Values are means±SEM expressed as % of control values (100%); the significance of difference between control and Li samples was determined by unpaired Student's t-test (triplicate for each sample preparation). Na+/K+-ATPase was increased in PM isolated from cells exposed to Li (L7 153±15% p<0.05, L21 115±5%, p<0.05 immunoblot; L7 121±8% p<0.05, L21 120±3% p<0.05 [3H]ouabain binding). In Li-treated cells, the highly increased (p<0.05) DCF fluorescence intensity was noticed in whole cells, PNS and PM. Accordingly, production of 4-HNE-protein adducts (p<0.05) and MDA (p<0.01) was clearly increased in Li-treated cells. The analysis of CCB-stained gels revealed 16 altered proteins in L7 compared with C7 and 25 in L21 versus C21. The intensity of staining of these protein spots was decreased (>2-fold). All proteins were functionally related to glycolysis, oxidative stress or calcium homeostasis. Our findings bring the evidence for adaptation of HEK293 cell metabolism to prolonged exposure to therapeutic Li concentration. This adaptation proceeds as up-regulation of Na+/K+-ATPase and limited reorganization of cellular protein composition. Metabolism of HEK293 cells growing in culture in the presence of a therapeutic Li concentration may be characterized as the state of discomfort induced by mild OS which does not prevent cells from fast growth.

Where applicable, experiments conform with Society ethical requirements