Nitric Oxide Synthase Inhibitors Modulate Nerve-Growth-Factor Mediated Activation of Akt

Cheryl L Cragg,Janet C Mackinnon,Bettina E Kalisch

doi:10.5402/2012/847974

Abstract

Nitric oxide (NO) modulates nerve-growth-factor- (NGF-) mediated signaling and gene expression. In the present paper, the role of NO in NGF-mediated Akt activation in PC12 and IMR32 cells was investigated. Cells were treated with NGF (50 ng/mL) in the presence or absence of NO synthase (NOS) inhibitors and Akt phosphorylation assessed by western blot analysis. In both cell lines, Akt was phosphorylated within 15 min of NGF treatment. In PC12 cells, this level of phosphorylation was sustained for 60 min, while in IMR32 cells, the activation decreased after 30 min of NGF treatment. The nonselective NOS inhibitor Nω-nitro-L-arginine methylester (L-NAME; 20 mM) had no effect on NGF-mediated Akt phosphorylation in PC12 cells but in combination with NGF, the iNOS selective inhibitor s-methylisothiourea (S-MIU; 2.0 mM) maintained Akt phosphorylation up to 2 h. In IMR32 cells, both L-NAME and S-MIU prolonged the activation of Akt. Pretreatment with 50 μM U0126, a MAP kinase pathway inhibitor, also increased the activation of Akt in both cell lines. These data suggest that NO modulates the duration of phosphorylation of Akt in response to NGF and that this effect may, in part, be mediated by the effects of NO on the Ras-MAP kinase pathway.

Highlights

Binding of nerve-growth-factor (NGF) to its high affinity receptor (TrkA) elicits a range of cellular responses such as neuronal survival, cell cycle arrest, and phenotypic and morphological differentiation
We provide evidence that nitric oxide (NO) modulates the NGF-induced activation of Akt
We demonstrated that the inducible NOS (iNOS)-selective inhibitor S-MIU did not result in an alteration in the level of initial NGF-mediated phosphorylation of Akt, but did increase the level or duration of activation of Akt in both PC12 and IMR32 cells

Summary

Introduction

Binding of nerve-growth-factor (NGF) to its high affinity receptor (TrkA) elicits a range of cellular responses such as neuronal survival, cell cycle arrest, and phenotypic and morphological differentiation. Stimulation of TrkA receptors activates protein kinases and adaptor proteins in multiple signaling pathways regulating the expression and activation of transcription factors and other cellular proteins [1]. Blockade of Akt activation by PI3 kinase inhibitors reveals that activation of this important signaling pathway is required for NGFmediated increases in the levels of the endothelial isoform of the enzyme nitric oxide synthase (eNOS or NOS III) [2]. Nitric oxide (NO) plays a role in NGF-mediated neurotrophic response [3,4,5,6] Activation of both Akt [7,8,9] and NO [3,4,5,6] appears to be required for many of the neurotrophic actions of NGF, the exact link between NO production and the phosphorylation of Akt is not clear

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