u-0126 and 3-(2-hydroxy-4-(1-1-dimethylheptyl)phenyl)-4-(3-hydroxypropyl)cyclohexanol

Classically, G protein-coupled receptor activation by a ligand has been viewed as producing a defined response such as activation of a G protein, activation or inhibition of adenylyl cyclase, or stimulation of phospholipase C and/or alteration in calcium flux. Newer concepts of ligand-directed signaling recognize that different ligands, ostensibly acting at the same receptors, may induce different downstream effects, complicating the selection of a screening assay. Dynamic mass redistribution (DMR), a label-free technology that uses light to measure ligand-induced changes in the mass of cells proximate to the biosensor, provides an integrated cellular response comprising multiple pathways and cellular events. Using DMR, signals induced by opioid or cannabinoid agonists in cells transfected with these receptors were blocked by pharmacologically appropriate receptor antagonists as well as by pertussis toxin. Differences among compounds in relative potencies at DMR versus ligand-stimulated GTPγS or receptor binding endpoints, suggesting functional selectivity, were observed. Preliminary evidence indicates that inhibitors of intermediate steps in the cell signaling cascade, such as receptor recycling inhibitors, mitogen-activated protein kinase kinase/p38 mitogen-activated protein kinase inhibitors, or cytoskeletal disruptors, altered or attenuated the cannabinoid-induced response. Notable is the finding that mitogen-activated protein kinase kinase 1/2 inhibitors attenuated signaling induced by the cannabinoid type 2 receptor inverse agonist AM630 but not that stimulated by the agonist CP 55,940. Thus, DMR has the potential to not only identify ligands that activate a given G protein-coupled receptor, but also ascertain the signaling pathways engaged by a specific ligand, making DMR a useful tool in the identification of biased ligands, which may ultimately exhibit improved therapeutic profiles.

Topics: Analgesics, Opioid; Animals; Butadienes; Cannabinoids; Carrier Proteins; Chemistry Techniques, Analytical; CHO Cells; Cricetinae; Cyclohexanols; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GTP-Binding Protein alpha Subunits; Indoles; Male; Morphine; Nitriles; Optical Phenomena; Pertussis Toxin; Protein Serine-Threonine Kinases; Rats; Rats, Wistar; Receptor, Cannabinoid, CB2; Receptors, G-Protein-Coupled; Receptors, Opioid; Signal Transduction

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.. 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.. 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.. 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.

Topics: Adult; Amidohydrolases; Blotting, Western; Butadienes; Cell Line; Cell Survival; Cyclohexanols; Gene Expression Regulation; Humans; Hydrogen Peroxide; Male; Microscopy, Fluorescence; Middle Aged; Mitogen-Activated Protein Kinases; Nitriles; Phosphatidylinositol 3-Kinases; Reactive Oxygen Species; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Retinal Pigment Epithelium; Reverse Transcriptase Polymerase Chain Reaction