Endocannabinoids in the intact retina:³H-anandamide uptake, fatty acid amide hydrolase immunoreactivity and hydrolysis of anandamide

Abstract

There is much evidence for an endocannabinoid system in the retina. However, neither the distribution of endocannabinoid uptake, the regulation of endocannabinoid levels, nor the role of endocannabinoid metabolism have been investigated in the retina. Here we focused on one endocannabinoid, anandamide (AEA), and its major hydrolyzing enzyme, fatty acid amide hydrolase (FAAH), in the goldfish retina. Immunoblots of FAAH immunoreactivity (IR) in goldfish retina, brain and rat retina, and brain homogenates showed a single band at 61 kDa that was blocked by preadsorption with peptide antigen. Specific FAAH IR (blocked by preadsorption) was most prominent over Müller cells and cone inner segments. Weaker label was observed over some amacrine cells, rare cell bodies in the ganglion cell layer, and in four lamina in the inner plexiform layer. FAAH activity assays showed that goldfish-retinal and brain homogenates hydrolyzed AEA at rates comparable to rat brain homogenate, and the hydrolysis was inhibited by methyl arachidonyl fluorophosphonate (MAFP) and N-(4 hydroxyphenyl)-arachidonamide (AM404), with IC(50)s of 21 nM and 1.5 microM, respectively. Cellular 3H-AEA uptake in the intact retina was determined by in vitro autoradiography. Silver-grain accumulation at 20 degrees C was most prominent over cone photoreceptors and Müller cells. Uptake was significantly reduced when retinas were incubated at 4 degrees C, or preincubated with 100 nM MAFP or 10 microM AM404. There was no differential effect of blocking conditions on the distribution of silver grains over cones or Müller cells. The codistribution of FAAH IR and 3H-AEA uptake in cones and Müller cells suggests that the bulk clearance of AEA in the retina occurs as a consequence of a concentration gradient created by FAAH activity. We conclude that endocannabinoids are present in the goldfish retina and underlay the electrophysiological effects of cannabinoid ligands previously shown on goldfish cones and bipolar cells.

Involvement of the Endocannabinoid System in Retinal Damage after High Intraocular Pressure–Induced Ischemia in Rats

“…This unprecedented evidence gives biochemical support to previous functional and immunocytochemical data. 18,20,21 We also showed that high IOP-induced ischemic insult, followed by reperfusion, results in a significant decrease of the endogenous tone of AEA in the retina of rat. This effect was associated with altered endocannabinoid metabolism, because ischemia-reperfusion also resulted in enhanced activity of the AEA-hydrolase FAAH, whereas the AEA-transporter AMT or the AEA-synthase NAPE-PLD were not significantly affected.…”

“…42 To discriminate non-carrier-mediated from carrier-mediated transport of AEA through synaptosome-like vesicles, control experiments were performed at 4°C, and the results were subtracted from those at 37°C. 18,42 AMT activity was expressed as picomoles AEA taken up per minute per milligram protein.…”

“…15 Since then, the presence of a functional endocannabinoid system in the eye has been widely documented. In fact, porcine ocular tissues synthesize and degrade AEA, 16 as do bovine 17 and rat retina 18 and various human eye tissues. 19 In addition, CB1 receptors, FAAH, and TRPV1 have been shown to be widespread in the retina of rats and other mammals by immunocytochemical methods.…”

Evidence for a GPR18 Role in Diurnal Regulation of Intraocular Pressure

“…22,23 The role of FAAH in breaking down anandamide was established relatively early, 24 and anandamide metabolism, presumed to occur via FAAH activity, has been studied in the eye. 25,26 Importantly in our case, FAAH and anandamide are implicated in the production of N -arachidonoyl glycine (NAGly), a candidate GPR18 agonist. 27,28 NAPE-PLD was proposed early on as the enzymatic source of anandamide, 29,30 but the first knockout study was discouraging, setting off a search for other candidate pathways.…”

Aplicaciones de los cannabinoides en glaucoma

“…Se ha evidenciado la presencia, síntesis y degradación de AEA en distintas estructuras oculares de diferentes mamíferos en modelos porcino 14 , bovino 15 , murino 16 y en humano 17 . Por otro lado, se ha demostrado la presencia de los principales subtipos de receptores CB1 y CB2 en retina de rata, así como receptores vaniloides por los que ciertos compuestos de cannabinoides presentan afinidad 18 .…”