The Metabolism of 2-arachidonoylglycerol in Rod Outer Segments Is Modulated by Proteins Involved in the Phototransduction Process.

We previously reported that 2-arachidonoylglycerol (2-AG) synthesis by diacylglycerol lipase (DAGL) and lysophosphatidate phosphohydrolase (LPAP) and hydrolysis by monoacylglycerol lipase (MAGL) in rod outer segments (ROS) from bovine retina were differently modified by light applied to the retina. Based on these findings, the aim of the present research was to evaluate whether 2-AG metabolism could be modulated by proteins involved in the visual process. To this end, ROS kept in darkness (DROS) or obtained in darkness and then subjected to light (BROS) were treated with GTPγS and GDPßS, or with low and moderate ionic strength buffers for detaching soluble and peripheral proteins, or soluble proteins, respectively. Only DAGL activity was stimulated by the application of light to the ROS. GTPγS-stimulated DAGL activity in DROS reached similar values to that observed in BROS. The studies using different ionic strength show that (1) the highest decrease in DROS DAGL activity was observed when both phosphodiesterase (PDE) and transducin α (Tα) are totally membrane-associated; (2) the decrease in BROS DAGL activity does not depend on PDE association to membrane, and that (3) MAGL activity decreases, both in DROS and BROS, when PDE is not associated to the membrane. Our results indicate that the bioavailability of 2-AG under light conditions is favored by G protein-stimulated increase in DAGL activity and hindered principally by Tα/PDE association with the ROS membrane, which decreases DAGL activity.

Binding of rhodopsin and rhodopsin analogues to transducin, rhodopsin kinase and arrestin-1.

AIM: To investigate the interaction of reconstituted rhodopsin, 9-cis-retinal-rhodopsin and 13-cis-retinal-rhodopsin with transducin, rhodopsin kinase and arrestin-1. METHODS: Rod outer segments (ROS) were isolated from bovine retinas. Following bleaching of ROS membranes with hydroxylamine, rhodopsin and rhodopsin analogues were generated with the different retinal isomers and the concentration of the reconstituted pigments was calculated from their UV/visible absorption spectra. Transducin and arrestin-1 were purified to homogeneity by column chromatography, and an enriched-fraction of rhodopsin kinase was obtained by extracting freshly prepared ROS in the dark. The guanine nucleotide binding activity of transducin was determined by Millipore filtration using ß,γ-imido-((3)H)-guanosine 5'-triphosphate. Recognition of the reconstituted pigments by rhodopsin kinase was determined by autoradiography following incubation of ROS membranes containing the various regenerated pigments with partially purified rhodopsin kinase in the presence of (γ-(32)P) ATP. Binding of arrestin-1 to the various pigments in ROS membranes was determined by a sedimentation assay analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. RESULTS: Reconstituted rhodopsin and rhodopsin analogues containing 9-cis-retinal and 13-cis-retinal rendered an absorption spectrum showing a maximum peak at 498 nm, 486 nm and about 467 nm, respectively, in the dark; which was shifted to 380 nm, 404 nm and about 425 nm, respectively, after illumination. The percentage of reconstitution of rhodopsin and the rhodopsin analogues containing 9-cis-retinal and 13-cis-retinal was estimated to be 88%, 81% and 24%, respectively. Although only residual activation of transducin was observed in the dark when reconstituted rhodopsin and 9-cis-retinal-rhodopsin was used, the rhodopsin analogue containing the 13-cis isomer of retinal was capable of activating transducin independently of light. Moreover, only a basal amount of the reconstituted rhodopsin and 9-cis-retinal-rhodopsin was phosphorylated by rhodopsin kinase in the dark, whereas the pigment containing the 13-cis-retinal was highly phosphorylated by rhodopsin kinase even in the dark. In addition, arrestin-1 was incubated with rhodopsin, 9-cis-retinal-rhodopsin or 13-cis-retinal-rhodopsin. Experiments were performed using both phosphorylated and non-phosphorylated regenerated pigments. Basal amounts of arrestin-1 interacted with rhodopsin, 9-cis-retinal-rhodopsin and 13-cis-retinal-rhodopsin under dark and light conditions. Residual arrestin-1 was also recognized by the phosphorylated rhodopsin and phosphorylated 9-cis-retinal-rhodopsin in the dark. However, arrestin-1 was recognized by phosphorylated 13-cis-retinal-rhodopsin in the dark. As expected, all reformed pigments were capable of activating transducin and being phosphorylated by rhodopsin kinase in a light-dependent manner. Additionally, all reconstituted photolyzed and phosphorylated pigments were capable of interacting with arrestin-1. CONCLUSION: In the dark, the rhodopsin analogue containing the 13-cis isomer of retinal appears to fold in a pseudo-active conformation that mimics the active photointermediate of rhodopsin.