Receptor-mediated Oxidative Stress in Murine Cerebellar Neurons is Accompanied by Phosphorylation of MAP (ERK 1/2) Kinase

Yulia      Rybakova; Evgeny      Akkuratov; Konstantin      Kulebyakin; Olga      Brodskaya; Antonina      Dizhevskaya; Alexander      Boldyrev

Abstract

A primary culture of murine cerebellar neurons was used to induce oxidative stress resulting in the accumulation of reactive oxygen species (ROS) and activation of ERK 1/2 kinase. Short-term incubation (15 min) of cerebellar neurons with homocysteine (HC) or N-methyl-D-aspartate (NMDA) induced partial ERK 1/2 phosphorylation thus providing the activation of the enzyme. Inhibitors of NMDA receptors, MK-801 or D-AP5, both prevented the activation of cells by HC or NMDA. Another receptor-dependent means of oxidative stress stimulation is exposure of cells to the cardiac glycoside ouabain, a specific inhibitor of Na/K-ATPase. Ouabain induces ROS accumulation and substantial ERK1/2 activation in neuronal cells at concentrations as low as 1 nM – 1 M, which corresponds to participation of Na/K-ATPase in intracellular signalling. Neuropeptide carnosine added to the cells 2 hours before oxidative stress prevented both ROS accumulation and ERK1/2 activation. As ERK1/2 kinase plays a key role in gene expression responsible for either cell adaptation or cell death, the model used gives a useful tool to characterize the effect of natural and synthetic anti-cancer drugs on cellular life. The data presented show that carnosine is a natural modulator of oxidative stress in neuronal cells, providing regulation of ERK1/2 activity via buffering intracellular ROS levels.

Keywords: Carnosine, ERK 1/2, homocysteine, Na/K-ATPase, neurons, NMDA, ouabain, oxidative stress, murine cerebellar neurons, reactive oxygen species