Endocannabinoids in the intact retina: 3 H-anandamide uptake, fatty acid amide hydrolase immunoreactivity and hydrolysis of anandamide.
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.
Department of Biochemistry and Cell Biology, Stony Brook University, New York 11794-5230, USA.
Sherrye T
ST
Glaser
Dale G
DG
Deutsch
Keith M
KM
Studholme
Sarah
S
Zimov
Stephen
S
Yazulla
D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism* / Q000494:pharmacology; D001345:Autoradiography; D015153:Blotting, Western; D063385:Cannabinoid Receptor Modulators / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D063388:Endocannabinoids; D006054:Goldfish / Q000502:physiology*; D006868:Hydrolysis; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D053284:Polyunsaturated Alkamides; D012160:Retina / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D017949:Retinal Cone Photoreceptor Cells / Q000378:metabolism; D016622:Silver Staining; D000099252:Fatty Acid Amide Hydrolases
D000581:Amidohydrolases; D001095:Arachidonic Acids; D063385:Cannabinoid Receptor Modulators; D063388:Endocannabinoids; D053284:Polyunsaturated Alkamides; D000099252:Fatty Acid Amide Hydrolases; C078814:anandamide; C107594:N-(4-hydroxyphenyl)arachidonylamide