Intracerebroventricular administration of ouabain, a Na/K-ATPase inhibitor, activates mTOR signal pathways and protein translation in the rat frontal cortex

Abstract

Intracerebroventricular (ICV) injection of ouabain, a specific Na/K-ATPase inhibitor, induces behavioral changes in rats in a putative animal model of mania. The binding of ouabain to Na/K-ATPase affects signaling molecules in vitro, including ERK1/2 and Akt, which promote protein translation. We have also reported that ERK1/2 and Akt in the brain are involved in the ouabain-induced hyperactivity of rats. In this study, rats were given an ICV injection of ouabain, and then their frontal cortices were examined to determine the effects of ouabain on the mTOR/p70S6K/S6 signaling pathway and protein translation, which are important in modifications of neural circuits and behavior. Rats showed ouabain-induced hyperactivity up to 8h following injection, and increased phosphorylation levels of mTOR, p70S6K, S6, eIF4B, and 4E-BP at 1, 2, 4, and 8h following ouabain injection. Immunohistochemical analyses revealed that increased p-S6 immunoreactivity in the cytoplasm of neurons by ouabain was evident in the prefrontal, cingulate, and orbital cortex. These findings suggested increased translation initiation in response to ouabain. The rate of protein synthesis was measured as the amount of [(3)H]-leucine incorporation in the cell-free extracts of frontal cortical tissues, and showed a significant increase at 8h after ouabain injection. These results suggest that ICV injection of ouabain induced activation of the protein translation initiation pathway regulated by ERK1/2 and Akt, and prolonged hyperactivity in rats. In conclusion, protein translation pathway could play an important role in ouabain-induced hyperactivity in a rodent model of mania.

Keywords: 4E-BP; 4′,6′-diamidino-2-phenylindole; ANOVA; BDNF; Bipolar disorder; DAPI; EGFR; ERK; ICV; MAPK; NMDA; Na-pump; Na/K-ATPase; OD; PKC; PLC; PMS; Protein synthesis; S6; aCSF; analysis of variance; artificial cerebrospinal fluid; brain-derived neurotrophic factor; eIF4B; epidermal growth factor receptor; eukaryotic translation initiation factor 4B; eukaryotic translation initiation factor 4E binding protein; extracellular signal regulated kinase; intracerebroventricular; mTOR; mammalian target rapamycin; mitogen-activated protein kinase; n-methyl-d-aspartate; optical density; p70 ribosomal S6 kinase; p70S6K; phospholipase C; postmitochondrial supernatant; protein kinase C; small ribosomal protein 6; sodium and potassium-activated adenosine triphosphatase.