Effect of ouabain on calcium signaling in rodent brain: A systematic review of in vitro studies

Abstract

The Na⁺/K⁺-ATPase is an integral membrane ion pump, essential to maintaining osmotic balance in cells in the presence of cardiotonic steroids; more specifically, ouabain can be an endogenous modulator of the Na⁺/K⁺-ATPase. Here, we conducted a systematic review of the in vitro effects of cardiotonic steroids on Ca²⁺ in the brain of rats and mice. Methods: The review was carried out using the PubMed, Virtual Health Library, and EMBASE databases (between 12 June 2020 and 30 June 2020) and followed the guidelines described in the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA). Results: in total, 829 references were identified in the electronic databases; however, only 20 articles were considered, on the basis of the inclusion criteria. The studies demonstrated the effects of ouabain on Ca²⁺ signaling in synaptosomes, brain slices, and cultures of rat and mouse cells. In addition to the well-known cytotoxic effects of high doses of ouabain, resulting from indirect stimulation of the reverse mode of the Na⁺/Ca²⁺ exchanger and increased intracellular Ca²⁺, other effects have been reported. Ouabain-mediated Ca²⁺ signaling was able to act increasing cholinergic, noradrenergic and glutamatergic neurotransmission. Furthermore, ouabain significantly increased intracellular signaling molecules such as InsPs, IP3 and cAMP. Moreover treatment with low doses of ouabain stimulated myelin basic protein synthesis. Ouabain-induced intracellular Ca²⁺ increase may promote the activation of important cell signaling pathways involved in cellular homeostasis and function. Thus, the study of the application of ouabain in low doses being promising for application in neurological diseases. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42020204498, identifier CRD42020204498.

Keywords: Na+/K+-ATPase; calcium; nervous system; ouabain; signaling.

FIGURE 1

Protein representation of rat Na⁺/K⁺-ATPase α1β1γ isoform subunits. (A) In green, α1 subunit, in cyan, β1 subunit, and in pink, γ (FXYD2) subunit. The residues displaying the substitutions present in the rat compared to the human Na⁺/K⁺-ATPase protein. In orange, magnesium ions. The gray dots represent the Na⁺/K⁺-ATPase position at the plasma membrane, and the arrow points to the pocket where ouabain (in yellow) interacts. (B) Magnification of the binding pocket with ouabain (inside the red circle) seen from the bottom. The protein was constructed by homology using as reference the human crystallographic structure (pdb id:4RET) using the Swiss-model webserver, and for the representation the software PyMOL 7 was used.

FIGURE 2

Flow diagram of study selection. The search process using the PRISMA flow diagram.

FIGURE 3

Representative schedule of the effects of ouabain on Ca²⁺ signaling. Ouabain in high concentrations selectively binds to Na⁺/K⁺-ATPase, inhibiting its pump activity, leading to increased intracellular Na⁺ and Ca²⁺ concentrations. The latter by inducing a lower/reverse action of the Na⁺–Ca²⁺ exchanger (NCX) colocalizes with the Na⁺/K⁺-ATPase. Ouabain effects on intracellular Ca²⁺ influence the release of acetylcholine (Ach) and norepinephrine (NE3H) and glutamate (Glu). Binding of ouabain to Na⁺/K⁺-ATPase triggers intracellular signaling networks in the glutamate signaling cascade through protein-protein interactions, generating many effects independent of the impairment of electrochemical gradients. In this case, ouabain (and other cardiotonic steroids) would act as an agonist, stimulating these pathways, acting in a different way observed as Na⁺/K⁺-ATPase inhibitor. In low concentrations ouabain can stimulate changes in Na⁺/K⁺-ATPase that triggers signaling complexes such PI3K-AKT pathways, increasing cAMP. Also, ouabain modulates Ca²⁺ intracellular oscillation through activation of ryanodine receptor (RyR) and IP3 receptor (IP3R) that provoke the increasing of NF-κB and activation of PKC. LD- Low Dose and HD- High dose (Figure were created with BioRender.com).