Glaser Sherrye T, Deutsch Dale G, Studholme Keith M, Zimov Sarah, Yazulla Stephen
Department of Biochemistry and Cell Biology, Stony Brook University, New York 11794-5230, USA.
Vis Neurosci. 2005 Nov-Dec;22(6):693-705. doi: 10.1017/S0952523805226020.
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.
有大量证据表明视网膜中存在内源性大麻素系统。然而，内源性大麻素摄取的分布、内源性大麻素水平的调节以及内源性大麻素代谢的作用在视网膜中均未得到研究。在此，我们聚焦于金鱼视网膜中的一种内源性大麻素——花生四烯乙醇胺（AEA）及其主要水解酶——脂肪酸酰胺水解酶（FAAH）。金鱼视网膜、脑以及大鼠视网膜和脑匀浆中FAAH免疫反应性（IR）的免疫印迹显示，在61 kDa处有一条单一的条带，该条带可被肽抗原预吸附阻断。特异性FAAH IR（被预吸附阻断）在 Müller 细胞和视锥细胞内节最为显著。在一些无长突细胞、神经节细胞层中罕见的细胞体以及内网状层的四个板层中观察到较弱的标记。FAAH活性测定表明，金鱼视网膜和脑匀浆水解AEA的速率与大鼠脑匀浆相当，且水解受到甲基花生四烯酰氟磷酸酯（MAFP）和N-(4-羟基苯基)-花生四烯酸酰胺（AM404）的抑制，其半数抑制浓度（IC50）分别为21 nM和1.5 μM。通过体外放射自显影测定完整视网膜中细胞对3H-AEA的摄取。在20℃时，银粒积累在视锥光感受器和 Müller 细胞上最为显著。当视网膜在4℃孵育或用100 nM MAFP或10 μM AM404预孵育时，摄取显著降低。阻断条件对视锥细胞或 Müller 细胞上银粒分布没有差异影响。视锥细胞和 Müller 细胞中FAAH IR与3H-AEA摄取的共分布表明，视网膜中AEA的大量清除是由FAAH活性产生的浓度梯度所致。我们得出结论，内源性大麻素存在于金鱼视网膜中，并构成了先前显示的大麻素配体对金鱼视锥细胞和双极细胞的电生理效应的基础。
