Agmatine protects retinal ganglion cells from hypoxia-induced apoptosis in transformed rat retinal ganglion cell line

Abstract

Background: Agmatine is an endogenous polyamine formed by the decarboxylation of L-arginine. We investigated the protective effects of agmatine against hypoxia-induced apoptosis of immortalized rat retinal ganglion cells (RGC-5). RGC-5 cells were cultured in a closed hypoxic chamber (5% O2) with or without agmatine. Cell viability was determined by lactate dehydrogenase (LDH) assay and apoptosis was examined by annexin V and caspase-3 assays. Expression and phosphorylation of mitogen-activated protein kinases (MAPKs; JNK, ERK p44/42, and p38) and nuclear factor-kappa B (NF-kappaB) were investigated by Western immunoblot analysis. The effects of agmatine were compared to those of brain-derived neurotrophic factor (BDNF), a well-known protective neurotrophin for retinal ganglion cells.

Results: After 48 hours of hypoxic culture, the LDH assay showed 52.3% cell loss, which was reduced to 25.6% and 30.1% when agmatine and BDNF were administered, respectively. This observed cell loss was due to apoptotic cell death, as established by annexin V and caspase-3 assays. Although total expression of MAPKs and NF-kappaB was not influenced by hypoxic injury, phosphorylation of these two proteins was increased. Agmatine reduced phosphorylation of JNK and NF-kappaB, while BDNF suppressed phosphorylation of ERK and p38.

Conclusion: Our results show that agmatine has neuroprotective effects against hypoxia-induced retinal ganglion cell damage in RGC-5 cells and that its effects may act through the JNK and NF-kappaB signaling pathways. Our data suggest that agmatine may lead to a novel therapeutic strategy to reduce retinal ganglion cell injury related to hypoxia.

Figure 1

LDH release in RGC-5 cells. LDH release in RGC-5 cells, illustrating the neuroprotective effects of agmatine and BDNF against hypoxia for (A) 12 hours, (B) 24 hours, and (C) 48 hours. Data are shown as mean ± S.E.M. of 32 measurements. *P < 0.001.

Figure 2

Hoechst 33342 and propidium iodide double staining in RGC-5 cells. Agmatine and BDNF reduce the hypoxia-induced cell death in RGC-5. RGC-5 cells were exposed to hypoxia for 48 hours either alone (B) or in the presence of 100 μM agmatine (C) or 10 ng/mL BDNF (D). A control normoxic culture is shown in (A). The cultures were stained with Hoechst 33342 and propidium iodide. The magnification is × 400.

Figure 3

Annexin V assay in RGC-5 cells. Flow cytometric analysis of effects of agmatine and BDNF on the hypoxia-induced apoptosis of RGC-5 cells. Cells were exposed to hypoxia for 24 hours either alone (B) or in the presence of 100 μM agmatine (C) or 10 ng/mL BDNF (D). A control normoxic culture is shown in (A). Cultures were stained with annexin V-FITC and propidium iodide. Cells of high reactivity with FITC and low reactivity with propidium iodide (right lower area) are the apoptotic cells.

Figure 4

Caspase-3 assay in RGC-5 cells. Colorimetric analysis of the effects of agmatine on the caspase-3 activity induced by hypoxic injury in RGC-5 cells. Cells were exposed to hypoxia for 24 hours with or without 100 μM agmatine or caspase-3 inhibitor Z-VAD-FMK (50 μM). Specific activity of caspase-3 was measured by cleavage of the caspase-3 substrate Ac-DEVD-pNA.

Figure 5

Western blot analysis of MAPKs in RGC-5 cells. Western blot analysis showing effects of agmatine and BDNF on mitogen-activated protein kinases (MAPKs). Western immunoblots probed with antibodies against JNK and phospho-JNK (A), ERK and phospho-ERK (B), p38 and phospho-p38 (C), and β-actin (D).

Figure 6

Western blot analysis of NF-κB in RGC-5 cells. Western blot analysis showing the effect of agmatine and BDNF on nuclear factor-kappa B (NF-κB). Western immunoblots probed with antibodies against NF-κB and phospho-NF-κB from nuclear (A) and cytosolic (B) proteins. Histone 3 (A) and β-actin (B) were used as internal controls.