Abstract
Arachidonic acid is an essential constituent of cell membranes that is esterified to the sn-2-position of glycerophospholipids and is released from selected lipid pools by phospholipase cleavage. The released arachidonic acid can be metabolized by three enzymatic pathways: the cyclooxygenase pathway forming prostaglandins and thromboxanes, the lipoxygenase pathway generating leukotrienes and lipoxins, and the cytochrome P450 (cP450) pathway producing epoxyeicosatrienoic acids and hydroxyeicosatetraenoic acids. The present study describes a novel group of cP450 epoxygenase-dependent metabolites of arachidonic acid, termed 2-epoxyeicosatrienoylglycerols (2-EG), including two regioisomers, 2-(11,12-epoxyeicosatrienoyl)glycerol (2-11,12-EG) and 2-(14,15-epoxyeicosatrienoyl)glycerol (2-14,15-EG), which are both produced in the kidney and spleen, whereas 2-11,12-EG is also detected in the brain. Both 2-11,12-EG and 2-14,15-EG activated the two cannabinoid (CB) receptor subtypes, CB1 and CB2, with high affinity and elicited biological responses in cultured cells expressing CB receptors and in intact animals. In contrast, the parental arachidonic acid and epoxyeicosatrienoic acids failed to activate CB1 or CB2 receptors. Thus, these cP450 epoxygenase-dependent metabolites are a novel class of endogenously produced, biologically active lipid mediators with the characteristics of endocannabinoids. This is the first evidence of a cytochrome P450-dependent arachidonate metabolite that can activate G-protein-coupled cell membrane receptors and suggests a functional link between the cytochrome P450 enzyme system and the endocannabinoid system.
Highlights
When cells are stimulated by relevant growth factors or hormones, the essential constituent of cell membrane, arachidonic acid, is hydrolyzed from the sn-2-position of selected glycero
The present study describes a novel group of cytochrome P450 enzyme system (cP450) epoxygenase-dependent metabolites of arachidonic acid, termed 2-epoxyeicosatrienoylglycerols (2-EG), including two regioisomers, 2-(11,12-epoxyeicosatrienoyl)glycerol (2-11,12-EG) and 2-(14,15-epoxyeicosatrienoyl)glycerol (2-14,15-EG), which are both produced in the kidney and spleen, whereas 2-11,12-EG is detected in the brain
CP450 enzymes were first recognized to metabolize arachidonic acid in 1981 [2,3,4], and the demonstration of such cP450 arachidonate metabolites in various tissues, plasma, and urine unequivocally established the cP450-dependent metabolism of arachidonic acid as a third metabolic pathway of arachidonic acid in vivo [14, 16, 46]
Summary
Chemicals and Antibodies—Arachidonic acid was purchased from Nu Chek Prep (Elysian, MN). [3H]CP55940 (specific activity 168 Ci/mmol) was from PerkinElmer Life Sciences. 2-Arachidonylglycerol (2-AG), WIN55212-2, AM251, and AM630 were from Tocris Cookson Inc. (Ellisville, MO). Analysis of Endogenous 2-EG by ESI/LC/MS/MS Analysis— Synthetic mixtures of glycerol 1- and 2-positional isomers of 11,12- and 14,15-EG were injected onto a reversed phase HPLC column (Kromasil 100 C18, 3 m, 150 ϫ 2.1 mm) and eluted using a solvent gradient from initially 30% H2O, 70% CH3OH containing 25 M AgBF4 to 100% CH3OH containing 25 M AgBF4 in 10 min and at a flow of 0.2 ml/min. To compare the levels of endogenous 2-11,12- and 2-14,15-EG with that of 2-AG in the fresh tissues, including kidney, spleen, and brain, both 2-EGs and 2-AG were measured in the same batch of lipid extract samples by ESI/LC/MS/MS analysis and calculated based on their internal standards, respectively. Binding of [3H]CP55940 (specific activity 168 Ci/mmol; PerkinElmer Life Sciences) to plasma membranes (50 g of membrane protein/150-l reaction) was performed essentially as described previously [25]. Mice were placed individually in the center of each open field activity chamber equipped with three vectors of infrared photo beam emitters
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