Presence and regulation of cannabinoid receptors in human retinal pigment epithelial cells.
OBJECTIVE
Cannabinoid receptors have been detected in neuron cells and proposed as potential therapeutic agents in neurodegenerative disorders because of their involvement in controlling neural cell survival and death. However, their presence and role in human retinal pigment epithelial (RPE) cells, which play a key role in initiating and developing age related macular degeneration (ARMD), have never been investigated. Here we analyzed the expression of and changes in cannabinoid receptors (CB1 and CB2) and one enzyme responsible for endocannabinoid hydrolysis, fatty acid amide hydrolase (FAAH), in RPE cell oxidative damage process, a cellular model of ARMD.


METHODS
Primary human RPE cells and cells from the ARPE-19 cell line were cultured and exposed to H2O2 for 24 h to induce oxidative damage. Real time RT-PCR, immunofluorescent staining, and western blot methods were performed to study the expression of and changes in CB1 and CB2 receptors, and FAAH. Cell viability and reactive oxygen species (ROS) production were measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and a dichlorofluorescein (DCF) assay, respectively. PI3K/Akt and ERK1/2 protein expression and activation of signaling molecules were assessed by western blot analysis.


RESULTS
By using real time RT-PCR, immunofluorescent staining and western blot methods, we showed that human RPE cells express CB1, CB2, and FAAH. Meanwhile, oxidative stress can upregulate CB1 and CB2 receptor expression, and downregulate FAAH expression. The CB1/CB2 receptor agonist, CP55,940, and the CB2 receptor agonist, JWH015 significantly protected RPE cells from oxidative damage. In addition, CP55,940 significantly reduced the levels of intracellular ROS, strengthened oxidative stress-induced activation of PI3K/Akt and reduced activation of the ERK1/2 signal pathway.


CONCLUSIONS
The results demonstrate the expression and regulation of CB1 and CB2 receptors and FAAH in human RPE cells. The modulation of cannabinoid receptor tone warrants consideration for future therapeutic strategies of ARMD.
Wei
Yan
Y
Department of Ophthalmology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China.
Wang
Xu
X
Wang
Ling
L
D000328:Adult; D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D002070:Butadienes / Q000494:pharmacology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003511:Cyclohexanols / Q000494:pharmacology; D005786:Gene Expression Regulation; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008297:Male; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D043884:Receptor, Cannabinoid, CB1 / Q000235:genetics / Q000378:metabolism*; D043885:Receptor, Cannabinoid, CB2 / Q000235:genetics / Q000378:metabolism*; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000099252:Fatty Acid Amide Hydrolases
D000581:Amidohydrolases; D002070:Butadienes; D003511:Cyclohexanols; D006861:Hydrogen Peroxide; D020928:Mitogen-Activated Protein Kinases; D009570:Nitriles; D019869:Phosphatidylinositol 3-Kinases; D017382:Reactive Oxygen Species; D043884:Receptor, Cannabinoid, CB1; D043885:Receptor, Cannabinoid, CB2; D000099252:Fatty Acid Amide Hydrolases; C113580:U 0126; C054649:3-(2-hydroxy-4-(1,1-dimethylheptyl)phenyl)-4-(3-hydroxypropyl)cyclohexanol