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Supplementary Material for: 7-Methylxanthine Influences the Behavior of ADORA2A-DRD2 Heterodimers in Human Retinal Pigment Epithelial Cells

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posted on 20.06.2022, 10:01 authored by Fan Y., Li J., Huang L., Wang K., Zhao M.
Introduction: The goal of this study was to investigate the presence of ADORA2A-DRD2 heterodimers in human RPE cells; determine if 7-methylxanthine (7-MX), a non-selective adenosine receptor antagonist which was used to control myopia progression can influence the behavior of retinal pigment epithelial (RPE) cells through the ADORA2A-DRD2 receptor pathway, and assess the changes in the expression of signaling molecules during cellular signal transduction. Methods: Human RPE cells were cultured in vitro in the presence or absence of 7-MX. Cell proliferation was evaluated with the CCK-8 assay. Apoptosis and necrosis rates were determined by annexin V–FITC/propidium iodide (PI) staining and flow cytometry. Immunofluorescence and coimmunoprecipitation were used to examine the protein expression and colocalization of ADORA2A and DRD2 in RPE cells. ADORA2A and DRD2 were knocked down with small interfering RNAs (siRNAs). Changes in the protein expression of ERK1/2 and phospho-ERK1/2 (pERK 1/2), which are signaling molecules downstream of dopamine (DA) receptors, were evaluated by western blot analysis. Results: Immunofluorescence and coimmunoprecipitation showed that ADORA2A and DRD2 were colocalized in RPE cells. The expression of ADORA2A in RPE cells was inhibited by treatment with 50 µmol/l 7-MX for 48 h, and the expression of DRD2, ERK1/2, and pERK1/2 was increased after treatment with 50 µmol/l 7-MX for 48 h. After siRNA-mediated knockdown of DRD2 in RPE cells and further treatment with 50 µmol/l 7-MX for 48 h, the expression of DRD2 was nearly restored to the level observed in the native control. At the experimental concentrations, 7-MX and siRNAs did not affect the proliferation or apoptosis of human RPE cells. Conclusions: ADORA2A and DRD2 heterodimers were present in RPE cells. 7-MX may affect the behaviors of RPE cells through the ADORA2A-DRD2 receptor pathway. 7-MX is an inhibitor of ADORA2A receptors that can prevent inhibition of the D2 receptor pathway and increase D2 receptor pathway activity. This phenomenon may explain the mechanism of action through which 7-MX can control myopia progression.

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