Melanocortin receptor agonists MCR1-5 protect photoreceptors from high-glucose damage and restore antioxidant enzymes in primary retinal cell culture

Abstract

Retinal photoreceptors are particularly vulnerable to local high-glucose concentrations. Oxidative stress is a risk factor for diabetic retinopathy development. Melanocortin receptors represent a family of G-protein-coupled receptors classified in five subtypes and are expressed in retina. Our previous data indicate that subtypes 1 and 5 receptor agonists exert a protective role on experimental diabetic retinopathy. This study focuses on their role in primary retinal cell cultures in high-glucose concentrations. After eye enucleation from wild-type male C57BL/6 mice, retinal cells were isolated, plated in high-glucose concentration and treated with melanocortin receptors 1 and 5 agonists and antagonists. Immunocytochemical and biochemical analysis showed that treatment with melanocortin receptors 1 and 5 agonists reduced anti-inflammatory cytokines and chemokines and enhanced manganese superoxide dismutase and glutathione peroxidase levels, preserving photoreceptor integrity. According with these evidences, we propose a major role of melanocortin receptors 1 and 5 on primary retinal cell response against high glucose or oxidative insults.

Keywords: hyperglycaemia; melanocortin receptor agonists; oxidative stress; photoreceptors; primary retinal cell cultures.

Figure 1

RT‐PCR analysis showed (A) MCR ₁ (melanocortin receptors 1) and (B) MCR ₅ (melanocortin receptor 5) gene expression in retinal cells cultured in high‐glucose (25 mM) concentration, and in the presence or absence of MCR ₁ agonist BMS‐470539 and MCR ₅ agonist PG‐ 901. The results are reported as the mean ± S.E.M. of n = 3 treatments and the significant results expressed as °P < 0.01 versus control and **P < 0.01 versus high glucose.

Figure 2

ELISA assay showed high levels of MCR _1,5 protein in retinal cells cultured in high‐glucose concentration. These were significantly decreased by the treatment with BMS‐470539 and PG‐ 901 (MCR ₁ and MCR ₅ agonists). The results are reported as the mean ± S.E.M. of n = 3 treatments and the significant results expressed as °P < 0.01 versus control and **P < 0.0 versus high glucose.

Figure 3

Western blotting analysis showed that high glucose (25 mM) decreases MnSOD and GPx enzyme levels; (A, B). Treatment with the compounds BMS‐470539 (MCR ₁ agonist) and P‐901 (MCR ₅ agonist) restored the MnSOD and GPx enzyme levels. (A, B). MnSOD: manganese superoxide dismutase; GPx: glutathione peroxidase. The results are reported as the mean ± S.E.M. of n = 3 treatments and the significant results expressed as *P < 0.05 versus high glucose, **P < 0.01 versus high glucose and °P < 0.01 versus control.

Figure 4

Depicted are representative immunocytochemistries of retinal cells cultured in 5 mM or 25 mM glucose and labelled with opsin, recoverin and 4′,6‐diamidino‐2‐phenylindole (DAPI) antibodies. Cells were treated with BMS‐470539 and PG‐901, and representative microscopic fields for each treatment are shown. Accordingly, the percentage of recoverin and opsin‐positive cells is represented in the graph. The results are expressed as mean ± S.E.M. of the percentages of positive cell/total cell counted in each analysed field for each treatment. The statistical significance was reported as °P < 0.01 versus control; **P < 0.01 versus high glucose. 40× magnification.