The D₁ dopamine receptor agonist, SKF83959, attenuates hydrogen peroxide-induced injury in RGC-5 cells involving the extracellular signal-regulated kinase/p38 pathways

Abstract

Purpose: Oxidative stress is widely implicated in the death of retinal ganglion cells associated with various optic neuropathies. Agonists of the dopamine D(1) receptor have recently been found to be potentially neuroprotective against oxidative stress-induced injury. The goal of this study was to investigate whether SKF83959, a next-generation high-affinity D(1) receptor agonist, could protect retinal ganglion cell 5 (RGC-5) cells from H(2)O(2)-induced damage and the molecular mechanism involved.

Methods: We examined expression of the D(1) receptor in RGC-5 cells with reverse-transcription-PCR and immunoblotting and assessed neuroprotection using propidium iodide staining and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In addition, we monitored the activation and involvement of members of mitogen-activated protein kinase family, extracellular signal-regulated kinase (ERK), p38 and c-Jun NH(2)-terminal kinase, with western blot and specific inhibitors.

Results: We found that the D(1) receptor was expressed in RGC-5 cells, but the sequence analysis suggested this cell line is from mouse and not rat origin. SKF83959 exhibited a remarkable neuroprotective effect on H(2)O(2)-damaged RGC-5 cells, which was blocked by the specific D(1) receptor antagonist, SCH23390. ERK and p38 were activated by SKF83959, and pretreatment with their inhibitors U0126 and SB203580, respectively, significantly blunted the SKF83959-induced cytoprotection. However, the specific c-Jun NH(2)-terminal kinase inhibitor, SP600125, had no effect on the SKF83959-induced protection.

Conclusions: We conclude that SKF83959 attenuates hydrogen peroxide-induced injury in RGC-5 cells via a mechanism involving activation of the ERK and p38 pathways and the D(1) receptor is a potential molecular target for developing neuroprotective drugs.

Figure 1

The dopamine D₁ receptor was expressed in RGC-5 cells. A: Total mRNA was extracted from RGC-5 cells, and an amplicon of about 215 bp was obtained by RT–PCR using specific primers designed according to the cDNA sequence of the D₁ receptor. B: D₁ receptor protein from RGC-5 cell lysate was analyzed with western blot. Con: control, SKF: treated with 30 μM SKF83959, positive: positive control (lysate from mouse brain homogenate). A specific D₁ receptor protein band with a molecular weight of approximately 50 kDa was detected. Treatment with SKF83959 had no significant influence on the expression of the D₁ receptor related to the internal control, β-actin. A representative blot is shown from at least three independent experiments. C: Analysis and comparison of D₁ receptor amplicons were performed using the BLAST tool (NCBI online). The maximum identification was highlighted with a pink rectangle.

Figure 2

Pre-treatment with SKF-83959 attenuated H₂O₂-induced death in RGC-5 cells. RGC-5 cells were pre-treated with either various concentrations of SKF-83959 or vehicle for 30 min prior to and during H₂O₂ treatment for an additional 5 h. A-F: Cell death was detected with PI staining. The dead cells were positively tracked with PI shown in red fluorescence. Left panel: fluorescence images (PI). Right panel: merged fluorescent images with binocular convert images. A, B: RGC-5 cells were treated with 500 μM H₂O₂; C, D, pretreated with 30 μM SKF-83959 before 500 μM H₂O₂; E, F, or vehicle. Scale bar = 50 μm. G: Cell viability was determined via the MTT assay. Data are expressed as percentage of relative cell viability (mean±SEM, from at least three independent experiments) in relation to control treatment. ***p<0.001, compared with 500 μM H₂O₂ treatment. H: Antagonist of the D1 receptor, SCH23390, abolished the SKF-83959-induced neuroprotection. Again, viability of RGC-5 cells was determined by MTT assay. Data were obtained from at least three independent experiments and expressed as the mean±SEM. ***p<0.001, compared to the SKF-83959 treated cells.

Figure 3

H₂O₂ treatment temporally decreased active levels of ERK in RGC-5 cells. RGC-5 cells were treated with various agents for designated times. Cells were then harvested and lysed for immunoblot assay A: 500 μM H₂O₂-treatment caused a gradual reduction in p-ERK levels in a time-dependent manner (1–5 h), but had little influence on total ERK compared to controls. Con: control. Each experiment was repeated at least three times, and a representative blot is shown. B: The reduction in p-ERK was quantitatively measured, and statistical significance was analyzed. Data were obtained from at least three independent experiments and expressed as the mean±SEM ***p<0.001, compared to controls. C: Total ERK levels were quantitatively measured, and statistical differences were analyzed. Data were obtained from at least three independent experiments and expressed as the mean±SEM ***p<0.001, compared to controls.

Figure 4

SKF83959 treatment preserved active levels of ERK in H₂O₂-treated RGC-5 cells. A: Pretreatment with 30 μM SKF83959 prevented the H₂O₂-induced reduction of p-ERK levels. Treatment with the specific inhibitor of MEK/ERK, 20 μM U0126, abolished the SKF83959-induced increase of p-ERK. SKF: SKF83959. Each experiment was repeated at least three times, and a representative blot is shown. B: The reduction of p-ERK was quantitatively measured, and statistical significance was analyzed. Data were obtained from at least three independent experiments and expressed as the mean±SEM ***p<0.001, compared to control. C: U0126 attenuated SKF83959-mediated neuroprotection. RGC-5 cells were pretreated with 20 μM U0126 30 min before 30 μM SKF83959 was added in the presence of 500 μM H₂O₂ for 5 h. Cell viability was determined with MTT assay. Data were obtained from at least three independent experiments and expressed as the mean±SEM ***p<0.001, compared to SKF83959-pretreated cells.

Figure 5

H₂O₂ treatment reduced levels of p-p38. RGC-5 cells were treated with 500 μM H₂O₂ for the designated times. Cells were then harvested and lysed for immunoblot assay. A: H₂O₂ treatment caused the gradual reduction of p-p38 in a time-dependent manner (1–5 h) but had little influence on total p38 levels compared with controls. Con: control. Each experiment was repeated at least three times, and a representative blot is shown. B: The reduction of p-p38 levels was quantitated and statistically analyzed. Data were obtained from at least three independent experiments and expressed as the mean±SEM ***p<0.001, compared with controls. C: Total p38 levels were quantitated and statistically analyzed. Data were obtained from at least three independent experiments and expressed as the mean±SEM ***p<0.001, compared with controls.

Figure 6

SKF83959 treatment preserved levels of active, p-p38 in H₂O₂-treated RGC-5 cells. RGC-5 cells were treated with various agents for the designated times. Cells were then harvested and lysed for immunoblot assay. A: Pretreatment with 30 μM SKF83959 prevented the H₂O₂-induced reduction of p-p38 levels. The specific inhibitor of p38, 200 μM SB203580, abolished the SKF83959-induced preservation of p-p38 levels. SKF: SKF83959. Each experiment was repeated at least three times, and a representative blot is shown. B: Levels of p-p38 were quantitated and statistically analyzed. Data were obtained from at least three independent experiments and expressed as the mean±SEM ***p<0.001, compared with controls. C: SB203580 attenuated SKF83959-mediated neuroprotection. RGC-5 cells were pretreated with 200 μM SB203580 30 min before 30 μM SKF83959 was added in the presence of 500 μM H₂O₂ for 5 h. Cell viability was determined with MTT assay. Data were obtained from at least three independent experiments and expressed as the mean±SEM ***p<0.001, compared with SKF83959-pretreated cells.

Figure 7

p-JNK levels were not affected by H₂O₂ treatment. RGC-5 cells were treated with 500 μM H₂O₂ for the designated times. Cells were then harvested and lysed for immunoblot assay. A: Treatment with 500 μM H₂O₂ had no significant effect on p-JNK or total JNK levels detected with western blot from 1 h to 5 h relative to the controls. Con: control. Each experiment was repeated at least three times, and a representative blot is shown. B: p-JNK levels were quantitated and statistically analyzed compared to controls. Data were obtained from at least three independent experiments and expressed as the mean±SEM C: Total JNK levels were quantitated and statistically analyzed compared to controls. Data were obtained from at least three independent experiments and expressed as the mean±SEM.

Figure 8

JNK activity was not associated with SKF83959-induced neuroprotection in H₂O₂-treated cells. A: The JNK inhibitor was unable to attenuate SKF83959-mediated neuroprotection. RGC-5 cells were pretreated with 100 μM SP600125 30 min before addition of 30 μM SKF83959 in the presence of 500 μM H₂O₂ for 5 h. Cell viability was determined with MTT assay. Data were obtained from at least three independent experiments and expressed as the mean±SEM B: Cotreatment with ERK and 38 inhibitors completely abolished SKF83959-mediated neuroprotection. RGC-5 cells were pretreated with 20 μM U0126 and 200 μM SB203580 30 min before 30 μM SKF83959 was added in the presence of 500 μM H₂O₂ for 5 h. Cell viability was determined with MTT assay. Data were obtained from at least three independent experiments and expressed as the mean±SEM ***p<0.001, compared with SKF83959-pretreated cells.