As shown in Physique 7, in control virus (ad.shLacZ)Ctreated porcine TM cells, the PEA induced an increase in the phosphorylation of p42/44 MAPK. of the p42/44 MAPK pathway, blocked both PEA-induced enhancement of aqueous humor outflow facility and PEA-induced phosphorylation of p42/44 MAPK. Conclusions. Our results demonstrate that PEA increases aqueous humor outflow through the TM pathway and these effects are mediated by GPR55 and PPAR receptors through activation of p42/44 MAPK. Introduction Elevated intraocular pressure (IOP) is one of the major risk factors for glaucoma, a leading cause of human blindness.1C3 Marijuana smoking and administration of cannabinoids are known to reduce IOP.4,5 The maintenance of IOP depends on the dynamic sense of balance between the secretion of aqueous humor by the ciliary body and the outflow of aqueous humor via the trabecular meshwork (TM) and uveoscleral routes.1,3 Both cannabinoid 1 (CB1) and cannabinoid 2 (CB2) cannabinoid receptors are known to be expressed in the TM, an important site for modulating the aqueous humor outflow.6C8 Our previous studies have demonstrated that this IOP-lowering effects of cannabinoids are at least partly mediated by increasing aqueous humor outflow via the TM pathway.7C10 In addition, previously we have demonstrated that both CB1 and CB2 Angiotensin (1-7) receptors are involved in the aqueous humor outflow-enhancing effects of various synthetic and endogenous cannabinoids.7C10 Palmitoylethanolamide (PEA) was first discovered to be an endogenous ligand in mammalian tissues in the 1960s.11 It is a saturated fatty acid amide analog of the endocannabinoid arachidonylethanolamide (AEA), also named anandamide. 12 PEA is usually synthesized from palmitic acid and ethanolamine in various tissues by actions of two enzymes, for 10 minutes, and the supernatant obtained was incubated with 4 Laemmli sample buffer at Angiotensin (1-7) room temperature for 20 minutes. Subsequently, proteins were resolved on a 10% SDSCpolyacrylamide gel using a minigel electrophoresis system (Invitrogen) and protein bands were transferred onto a nitrocellulose membrane. The nitrocellulose membranes were blocked with 5% nonfat dried milk in TBS-T buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.3% Tween 20) for 1 hour and then incubated overnight at 4C with the anti-GPR55 antibody (Cayman Chemical, Ann Arbor, MI) or the anti-PPAR antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Subsequently, the membranes were washed twice for 5 minutes each time with TBS-T buffer and incubated with horseradish peroxidaseClabeled secondary antibody for 1 hour at room temperature. The membranes were then washed three times with TBS-T buffer for 5 minutes each time and the antibody-recognized protein bands were visualized by an enhanced chemiluminescence detection kit (Thermo Scientific, Rockford, IL). p42/44-MAPK Activity Assay The TM cells were maintained in serum-free medium for 18 hours before various treatments. To activate the p42/44 MAPK, the cells were pretreated with URB597 for 15 minutes, followed by treatment with PEA plus URB597 for 10 minutes. To study the effects of various antagonists or inhibitors, the cells were pretreated with URB597 plus these antagonists or inhibitors (SR141716A, SR144528, GW6471, or PD98059) for 15 minutes, followed by PEA plus URB597 treatment for 10 minutes. At the end of the incubation period, 150 L of ice-cold lysis buffer made up of 50 mM -glycerophosphate, 20 mM EGTA, 15 mM MgCl2, 1 mM NaVO4, 1 mM dithiothreitol (DTT), and 1 g/mL of a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN) were added. The cell lysates were incubated on ice for 5 minutes and then transferred to microcentrifuge Egfr tubes. The lysates were clarified by centrifugation at 14,000for 10 minutes, the supernatants were collected, and protein concentrations were measured (Bradford protein assay reagent; Bio-Rad, Hercules, CA). After boiling with 2 Laemmli sample buffer for 5 minutes, 40 L of cell lysate (made up of 25 g of protein) was run on a 10% SDSCpolyacrylamide gel. Subsequently, the proteins were transferred onto a nitrocellulose Angiotensin (1-7) membrane, and the total p42/44 MAP kinase bands were detected by Western blot analysis by using a rabbit polyclonal anti-p42/44 MAP kinase antibody (Cell Signaling Technology, Beverly, MA). The levels of phosphorylated p42/44 MAPK were determined using a mouse monoclonal antibody against phospho-p44/42 MAPK (Thr202/Tyr204; Angiotensin (1-7) Cell Signaling Technology), according to procedures described by the manufacturer. Data Analyses For the anterior segment perfusion studies, outflow facility was calculated as the ratio of the rate of flow of perfusate (L/min) to the steady state perfusion pressure (mm Hg). Drug effects were evaluated in each eye as the percentage change of outflow facility.