Protective mechanism of agmatine pretreatment on RGC-5 cells injured by oxidative stress

Braz J Med Biol Res, April 2010, Volume 43(4) 356-358

Protective mechanism of agmatine pretreatment on RGC-5 cells injured by oxidative stress

Y. Iizuka^1,2, S. Hong², C.Y. Kim², W.I. Yang^1,3, J.E. Lee⁴ and G.J. Seong²

¹Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea

²Department of Ophthalmology, Institute of Vision Research, Yonsei University College of Medicine, Seoul, Korea

³Department of Pathology, Yonsei University College of Medicine, Seoul, Korea

⁴Department of Anatomy, Yonsei University College of Medicine, Seoul, Korea

^{Correspondence and Footnotes}

Abstract

Agmatine has neuroprotective effects on retinal ganglion cells (RGCs) as well as cortical and spinal neurons. It protects RGCs from oxidative stress even when it is not present at the time of injury. As agmatine has high affinity for various cellular receptors, we assessed protective mechanisms of agmatine using transformed RGCs (RGC-5 cell line). Differentiated RGC-5 cells were pretreated with 100 μM agmatine and consecutively exposed to 1.0 mM hydrogen peroxide (H₂O₂). Cell viability was determined by measuring lactate dehydrogenase (LDH), and the effects of selective alpha 2-adrenergic receptor antagonist yohimbine (0-500 nM) and N-methyl-D-aspartic acid (NMDA) receptor agonist NMDA (0-100 µM) were evaluated. Agmatine’s protective effect was compared to a selective NMDA receptor antagonist MK-801. After a 16-h exposure to H₂O₂, the LDH assay showed cell loss greater than 50%, which was reduced to about 30% when agmatine was pretreated before injury. Yohimbine almost completely inhibited agmatine’s protective effect, but NMDA did not. In addition, MK-801 (0-100 µM) did not significantly attenuate the H₂O₂-induced cytotoxicity. Our results suggest that neuroprotective effects of agmatine on RGCs under oxidative stress may be mainly attributed to the alpha 2-adrenergic receptor signaling pathway.

Key words: Agmatine; Neuroprotection; Oxidative stress; Retinal ganglion cell

Introduction

Glaucoma is the leading cause of irreversible blindness worldwide. Although elevated intraocular pressure has been known as a major risk factor for the development and progression of glaucoma, oxidative stress is also associated with glaucomatous visual field deterioration (1-3). Retinal ganglion cells (RGCs) are susceptible to oxidative stress (3-5). As glaucomatous damages are mostly irreversible, many glaucoma researchers are searching for a neuroprotective strategy for RGCs.

Agmatine is a primary amine, which is formed by decarboxylation of L-arginine in the mammalian brain. Agmatine has high affinity for various cellular receptors. It is an agonist for alpha 2-adrenergic and imidazoline receptors and an antagonist for N-methyl-D-aspartic acid (NMDA) receptors (6-8). Agmatine has neuroprotective effects against cortical and spinal neuronal injuries (9-11). It also protects RGCs from hypoxic damage (12). Even when agmatine is not present at the time of injury, it seems to protect RGCs by later blocking cellular death induced by oxidative stress (13). However, the precise protective mechanisms of agmatine pretreatment have not been well established.

We investigated the mechanisms of action of agmatine pretreatment on RGCs under oxidative stress using transformed RGCs (RGC-5 cell line).

Material and Methods

Cell culture and agmatine pretreatment

RGC-5 cells, which belong to a transformed RGC line developed from postnatal Sprague-Dawley rats, were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) plus 10% heat-inactivated fetal bovine serum (FBS; Gibco), 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco). For differentiation, the cells were incubated with 1.0 μM staurosporine (Sigma-Aldrich, USA) for 6 h; and then they were cultured in 10% FBS-DMEM for another 3 days (13).

After differentiation, the cells were pretreated with 100 μM agmatine (Sigma-Aldrich) in serum-free DMEM for 2 h (13), and then rinsed with phosphate-buffered saline (PBS; Gibco). Control cells were incubated in serum-free DMEM.

Oxidative stress with agonist/antagonist

After agmatine (100 μM) pretreatment for 2 h, the cells were exposed to 1.0 mM hydrogen peroxide (H₂O₂; Sigma-Aldrich) for 16 h (13). The mechanisms of action of agmatine pretreatment were investigated using the specific alpha 2-adrenergic receptor antagonist yohimbine (0-500 nM; Sigma-Aldrich), and NMDA receptor agonist NMDA (0-100 μM; Calbiochem, USA). In addition, the effect of agmatine was compared to that of MK-801 (0-100 µM; Sigma-Aldrich), a selective NMDA receptor antagonist.

Cytotoxicity assay

Cell viability was determined by measuring lactate dehydrogenase (LDH; CytoTox 96^® Non-Radioactive Cytotoxicity Assay Kit; Promega, USA) according to manufacturer instruction. Briefly, the LDH released from injured cells into the culture medium was reacted with tetrazolium salt. The red formazan product was measured at 490 nm using an ELISA plate reader (Vmax; Molecular Device, USA). Cytotoxicity was calculated as the proportion of injured cells to the total cell population (14). Data are reported as means ± SEM.

Statistical analysis

Data were analyzed by one-way ANOVA, followed by post hoc comparisons (Student-Newman-Keuls) using the Statistical Package for Social Sciences (SPSS) program for Windows, version 12.0 (SPSS Inc., USA). P values below 0.05 were considered to be significant.

Results

Differentiated RGC-5 cells were pretreated with 100 μM agmatine for 2 h, and then exposed to 1.0 mM H₂O₂ for 16 h for induction of oxidative stress. After agmatine pretreatment, the cytotoxicity of H₂O₂ declined from above 50 to approximately 30% (Figure 1A,B).

Neither yohimbine nor NMDA, at any concentration, had any significant effect on oxidative stress-induced cell death (data not shown); 10 nM yohimbine and 100 μM NMDA were used for further experiments. While yohimbine almost completely blocked the protective effects of agmatine pretreatment (Figure 1A), NMDA did not (Figure 1B).

To evaluate the contribution of NMDA receptor signaling, the protective effect of agmatine was compared to that of MK-801. MK-801 did not significantly affect the H₂O₂-induced cytotoxicity of RGC-5 cells (Figure 1C).

Discussion

Agmatine is known to be an agonist for alpha 2-adrenergic and imidazoline receptors as well as an antagonist of NMDA receptors (6-8). Wheeler and WoldeMussie (15) and Kalapesi et al. (16) studied the alpha 2-adrenergic receptors of human ganglion cells and the RGC-5 cell line. They demonstrated the presence of alpha 2A-adrenergic receptors on both undifferentiated and succinyl concanavalin-A differentiated RGC-5s. They also showed a higher expression of alpha 2A-adrenergic receptors on 7-day differentiated RGC-5 cells compared to the undifferentiated and early differentiated cells. In addition, RGCs also expressed NMDA glutamate receptors.

In the present study, the protective effects of agmatine pretreatment on RGC-5s under oxidative stress were completely abolished by 10 nM yohimbine but only partially decreased by 100 µM NMDA. Even the powerful NMDA antagonist MK-801 did not reduce the RGC-5 cell death induced by H₂O₂. These results suggest that agmatine pretreatment may rescue RGCs from oxidative stress mainly through alpha 2-adrenergic signaling rather than through the NMDA receptor pathway.

We have demonstrated that agmatine pretreatment has anti-apoptotic effects on differentiated RGC-5s under oxidative stress (13). In the present study, its protective mechanisms were investigated. Considering the many biological functions of agmatine, this study is not sufficient since its focus was limited to alpha 2-adrenergic and NMDA receptor pathways. Further experiments are needed to fully understand the neuroprotective mechanisms of agmatine pretreatment.

The neuroprotective mechanisms of agmatine pretreatment on RGCs under oxidative stress in vitro may be mainly associated with the alpha 2-adrenergic receptor signaling pathway.

References

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Acknowledgments

The authors thank Alcon Research, Ltd., for providing the RGC-5 cell line. Research supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD; KRF-2007-314-E00134).

Address for correspondence: G.J. Seong, Department of Ophthalmology, Institute of Vision Research, Yonsei University College of Medicine, 712 Eonjuro, Gangnam-gu, Seoul 135-720, Korea. Fax: [phone]. E-mail: [e-mail]

Received August 22, 2009. Accepted February 23, 2010. Available online March 19, 2010. Published April 12, 2010.

The Brazilian Journal of Medical and Biological Research is partially financed by

Figure 1. Determination of H₂O₂ cytotoxicity by the lactate dehydrogenase assay. Differentiated RGC-5 cells were pretreated with 100 μM agmatine and then exposed to oxidative stress with yohimbine (A) or N-methyl-D-aspartic acid (NMDA) (B). The cells were also treated with MK-801 (C). Panels A and B, *P < 0.05 (one-way ANOVA followed by post hoc comparisons).

Correspondence and Footnotes

Publication Dates

• Publication in this collection
  05 Apr 2010
• Date of issue
  Apr 2010

History

• Received
  22 Aug 2009
• Accepted
  23 Feb 2010