Diacylglyceride lipase activity in rod outer segments depends on the illumination state of the retina

We have demonstrated that the competition between phosphatidic acid (PA) and lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P) for lipid phosphate phosphatases (LPP) generates different levels of diacylglycerol (DAG) depending on the illumination state of the retina. The aim of the present research was to determine the diacylglyceride lipase (DAGL) activity in purified rod outer segments (ROS) obtained from dark-adapted retinas (DROS) or light-adapted retinas (BLROS) as well as in ROS membrane preparations depleted of soluble and peripheral proteins. [2-3H]monoacylglycerol (MAG), the product of DAGL, was evaluated from [2-3H]DAG generated by LPP action on [2-3H]PA in the presence of either LPA, S1P or C1P. MAG production was inhibited by 55% in BLROS and by 25% when the enzymatic assay was carried out in ROS obtained from dark-adapted retinas and incubated under room light (LROS). The most important events occurred in DROS where co-incubation of [2-3H]PA with LPA, S1P or C1P diminished MAG production. A higher level of DAGL activity was observed in LROS than in BLROS, though this difference was not apparent in the presence of LPA, S1P or C1P. DAGL activity in depleted DROS was diminished with respect to that in entire DROS. LPA, S1P and C1P produced a similar decrease in MAG production in depleted DROS whereas only C1P significantly diminished MAG generation in depleted BLROS. Sphingosine and ceramide inhibited MAG production in entire DROS and stimulated its generation in BLROS. Sphingosine and ceramide stimulated MAG generation in both depleted DROS and BLROS. Under our experimental conditions the degree of MAG production depended on the illumination state of the retina. We therefore suggest that proteins related to phototransduction phenomena are involved in the effects observed in the presence of S1P/sphingosine or C1P/ceramide.