Abstract
Purpose: :
Oxidative damage to the Retinal Pigment Epithelium (RPE) is thought to play a major role in the development and progression of Age-related Macular Degeneration (AMD). The purpose of this in vitro study was to evaluate the ability of the selective 5-HT1A agonist, AL-8309A, to protect RPE cells against oxidative damage and to define the mechanism of the cytoprotective action.
Methods: :
Using the human RPE cell line ARPE-19, we have developed an in vitro survival assay using the cytotoxin 7-ketocholesterol (7kCh). Oxidative damage and apoptosis is induced by the addition of 7kCh, and the tetrazolium salt, WST-1, is used to assess cell viability following treatment. AL-8309A was evaluated in this model at concentrations from 0.01 nM to 100 µM. Western analysis was performed to assess changes in protein concentration and activation state in response to treatment with AL-8309A. The selective 5-HT1A antagonists, NAN-190 and WAY 100635, and specific Mek inhibitor, U0126, were used to examine the mechanism of action of AL-8309A.
Results: :
Treatment with AL-8309A caused a significant, dose dependent increase in survival of RPE cells exposed to oxidative damage, with an EC50 of 4.8 nM. 12 hours pre-treatment was required for full protection and this protection decayed with a half-life of approximately 12 hours following the removal of drug. Concurrent treatment with cycloheximide and DRB caused a blockade of protection with AL-8309 demonstrating a requirement for both transcription and translation. Treatment with AL-8309A led to an increase of Mek 1/2 and Erk 1/2 phosphorylation and to subsequent upregulation of anti-oxidant and anti-apoptotic proteins, including SOD-1, SOD-2, Bcl-2 and Bcl-XL. Protection from oxidative damage with AL-8309A was completely blocked by NAN-190, WAY 100635 and U0126.
Conclusions: :
The serotonin agonist AL-8309A protects RPE cells from oxidative damage induced by 7kCh. This protection is mediated by 5-HT1A receptor activation and signal transduction via the Mek/Erk pathway. This alters gene expression, upregulating anti-oxidant and anti-apoptotic proteins. AL-8309 may be useful in the treatment of AMD.
Keywords: retinal pigment epithelium • oxidation/oxidative or free radical damage • age-related macular degeneration