Evidence for a GPR18 Role in Diurnal Regulation of Intraocular Pressure

Purpose The diurnal cycling of intraocular pressure (IOP) was first described in humans more than a century ago. This cycling is preserved in other species. The physiologic underpinning of this diurnal variation in IOP remains a mystery, even though elevated pressure is indicated in most forms of glaucoma, a common cause of blindness. Once identified, the system that underlies diurnal variation would represent a natural target for therapeutic intervention. Methods Using normotensive mice, we measured the regulation of ocular lipid species by the enzymes fatty acid amide hydrolase (FAAH) and N-arachidonoyl phosphatidylethanolamine phospholipase (NAPE-PLD), mRNA expression of these enzymes, and their functional role in diurnal regulation of IOP. Results We now report that NAPE-PLD and FAAH mice do not exhibit a diurnal cycling of IOP. These enzymes produce and break down acylethanolamines, including the endogenous cannabinoid anandamide. The diurnal lipid profile in mice shows that levels of most N-acyl ethanolamines and, intriguingly, N-arachidonoyl glycine (NAGly), decline at night: NAGly is a metabolite of arachidonoyl ethanolamine and a potent agonist at GPR18 that lowers intraocular pressure. The GPR18 blocker O1918 raises IOP during the day when pressure is low, but not at night. Quantitative PCR analysis shows that FAAH mRNA levels rise with pressure, suggesting that FAAH mediates the changes in pressure. Conclusions Our results support FAAH-dependent NAGly action at GPR18 as the physiologic basis of the diurnal variation of intraocular pressure in mice.

both eyes were immediately removed and placed in an Eppendorf tube on dry ice. The eyes were then stored at -80 °C. To begin the lipid extraction, samples were shock frozen in liquid nitrogen, which allowed them to be easily removed from the Eppendorf tube and weighed before being transferred to a 15 mL centrifuge tube. The mass of the largest sample was multiplied by 50 to determine how many milliliters of HPLCgrade methanol (Avantor Performance Materials, Inc., Center Valley PA) was to be added to the centrifuge tube. Then, 5 µL of vortexed 1 µM deuterium-labeled N-arachidonoyl glycine (d8NAGly) (Cayman Chemical, Ann Arbor, MI) was added to each test tube to serve as an internal standard. The spiked tubes were covered with Parafilm and were allowed to sit in the covered ice bucket for 2 hours. The eyes were then briefly homogenized using a sonicator (VirTis, Gardiner NY).
Then, samples were spun in a centrifuge at 19,000g for 20 minutes at 20°C.
After centrifugation, supernatant was poured from the centrifuge tubes into 15 mL polypropylene tubes. Enough HPLC H2O (EMD Millipore Corporation, Billerica, MA) to make a 75:25 water to organic solution was added to the supernatant. To partially purify the supernatant/water solution, solid phase extraction columns were used. One solid-phase 500mg C18 extraction cartridge (Agilent Technologies, Lake Forest CA) for each tube of extract was inserted into a Preppy vacuum manifold apparatus located in a fume hood. To activate the hydrophobic carbon chains in the column, 5 mL of HPLC methanol was added to each column. When the methanol almost reached the bottom of the columns, 2.5 mL of HPLC H2O was added to the columns to activate the polar silica in the columns. When the water had almost run through the column, the supernatant/water solution was added and allowed to slowly drip through the column. After the solution had eluted, another 2.5 mL of HPLC H2O was added to the columns to wash off impurities.
Then, 1.5 mL of 40% methanol was added to the column to wash off more impurities. The 40% methanol was allowed to completely elute and any eluate in the collector vials was discarded. The collector vials were then replaced with labeled autosampler vials (Perkin Elmer, Waltham, MA) that corresponded to each sample. A series of 4 elutions with 1.5 mL of 60%, 75%, 85%, and 100% methanol as the eluting solvent was performed to partially purify the lipids being measured.
More polar lipids, such as PGE2 or PGF2α, were purified in the 60% and 75% elutions. On the other hand, lipids such as 2-AG and AEA were purified in the 100% elution and lipids such as NAGly were purified in the 85% elution. Vials of eluants were stored in the -80°C freezer until they were ready for analysis.

Quantitative Polymerase Chain Reaction Analysis
Primers for selected components of the endocannabinoid system were designed Tissue samples were extracted and immediately stored at -80C. RNA was extracted using a Trizol reagent (Ambion, Austin, TX) and genomic DNA was removed with DNase (NEB, Bethesda, MD) following the manufacturer's instructions. RT-PCR was performed using a one-step, Sybr Green amplification process (PwrSybr, Applied Biosystems, Carlsbad, CA). Quantitative PCR was performed using an Eppendorf RealPlex2 Mastercycler thermocycler.
A primer for b-actin was used as an internal control for each experimental condition with the threshold cycle set within the linear range (10 fold above baseline). Once the standard critical threshold (Ct) was set, the relative expression levels for genes were determined. Data analysis and statistics were performed using Excel (Microsoft Corp., Redmond, WA) and Prism (GraphPad Software Inc., San Diego, CA) software. Values were compared using an unpaired t test.