Anandamide (N-arachidonoylethanolamine) is known to be an endogenous ligand of cannabinoid and vanilloid receptors. Its congeners (collectively referred to as Nacylethanolamines) also show a variety of biological activities. These compounds are principally formed from their corresponding N-acyl-phosphatidylethanolamines by a phosphodiesterase of the phospholipase D-type in animal tissues. We purified the enzyme from rat heart, and by the use of the sequences of its internal peptides cloned its complementary DNAs from mouse, rat, and human. The deduced amino acid sequences were composed of 393-396 residues, and showed that the enzyme has no homology with the known phospholipase D enzymes but is classified as a member of the zinc metallohydrolase family of the ␤-lactamase fold. As was overexpressed in COS-7 cells, the recombinant enzyme generated anandamide and other N-acylethanolamines from their corresponding N-acyl-phosphatidylethanolamines at comparable rates. In contrast, the enzyme was inactive with phosphatidylcholine and phosphatidylethanolamine. Assays of the enzyme activity and the messenger RNA and protein levels revealed its wide distribution in murine organs with higher contents in the brain, kidney, and testis. These results confirm that a specific phospholipase D is responsible for the generation of N-acylethanolamines including anandamide, strongly suggesting the physiological importance of lipid molecules of this class.

Transmembrane signals initiated by a broad range of extracellular stimuli converge on nodes that regulate phospholipase C (PLC)-dependent inositol lipid hydrolysis for signal propagation. We describe how heterotrimeric guanine nucleotide-binding proteins (G proteins) activate PLC-βs and in turn are deactivated by these downstream effectors. The 2.7-angstrom structure of PLC-β3 bound to activated Gα q reveals a conserved module found within PLC-βs and other effectors optimized for rapid engagement of activated G proteins. The active site of PLC-β3 in the complex is occluded by an intramolecular plug that is likely removed upon G protein-dependent anchoring and orientation of the lipase at membrane surfaces. A second domain of PLC-β3 subsequently accelerates guanosine triphosphate hydrolysis by Gα q , causing the complex to dissociate and terminate signal propagation. Mutations within this domain dramatically delay signal termination in vitro and in vivo. Consequently, this work suggests a dynamic catch-and-release mechanism used to sharpen spatiotemporal signals mediated by diverse sensory inputs.Phospholipase C (PLC) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] to the second messengers inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3 ] and diacylglycerol in an essential step for the physiological action of many hormones, neurotransmitters, growth factors, and other extracellular stimuli (1-3). These cascades use

Mice lacking the gene encoding the receptor for prostaglandin F2alpha (FP) developed normally but were unable to deliver normal fetuses at term. Although these FP-deficient mice showed no abnormality in the estrous cycle, ovulation, fertilization, or implantation, they did not respond to exogenous oxytocin because of the lack of induction of oxytocin receptor (a proposed triggering event in parturition), and they did not show the normal decline of serum progesterone concentrations that precedes parturition. Ovariectomy at day 19 of pregnancy restored induction of the oxytocin receptor and permitted successful delivery in the FP-deficient mice. These results indicate that parturition is initiated when prostaglandin F2alpha interacts with FP in ovarian luteal cells of the pregnant mice to induce luteolysis.

Bioactive N-acylethanolamines, including anandamide (an endocannabinoid) and N-palmitoylethanolamine (an anti-inflammatory and neuroprotective substance), are hydrolyzed to fatty acids and ethanolamine by fatty acid amide hydrolase. Moreover, we found another amidohydrolase catalyzing the same reaction only at acidic pH, and we purified it from rat lung (Ueda, N., Yamanaka, K., and Yamamoto, S. (2001) J. Biol. Chem. 276, 35552-35557). Here we report complementary DNA cloning and functional expression of the enzyme termed "N-acylethanolamine-hydrolyzing acid amidase (NAAA)" from human, rat, and mouse. The deduced primary structures revealed that NAAA had no homology to fatty acid amide hydrolase but belonged to the choloylglycine hydrolase family. Human NAAA was essentially identical to a gene product that had been noted to resemble acid ceramidase but lacked ceramide hydrolyzing activity. The recombinant human NAAA overexpressed in HEK293 cells hydrolyzed various N-acylethanolamines with N-palmitoylethanolamine as the most reactive substrate. Most interestingly, a very low ceramide hydrolyzing activity was also detected with NAAA, and N-lauroylethanolamine hydrolyzing activity was observed with acid ceramidase. By the use of tunicamycin and endoglycosidase, NAAA was found to be a glycoprotein. Furthermore, the enzyme was proteolytically processed to a shorter form at pH 4.5 but not at pH 7.4. Expression analysis of a green fluorescent protein-NAAA fusion protein showed a lysosome-like distribution in HEK293 cells. The organ distribution of the messenger RNA in rats revealed its wide distribution with the highest expression in lung. These results demonstrated that NAAA is a novel N-acylethanolamine-hydrolyzing enzyme that shows structural and functional similarity to acid ceramidase.

We used mice deficient in each of the eight types and subtypes of prostanoid receptors and examined the roles of prostanoids in dextran sodium sulfate-induced (DSS-induced) colitis. Among the prostanoid receptor-deficient mice, only EP4-deficient mice and not mice deficient in either DP, EP1, EP2, EP3, FP, IP, or TP developed severe colitis with 3% DSS treatment, which induced only marginal colitis in wild-type mice. This phenotype was mimicked in wild-type mice by administration of an EP4-selective antagonist (AE3-208). The EP4 deficiency impaired mucosal barrier function and induced epithelial loss, crypt damage, and aggregation of neutrophils and lymphocytes in the colon. Conversely, administration of an EP4-selective agonist (AE1-734) to wild-type mice ameliorated severe colitis normally induced with 7% DSS, while that of AE3-208 suppressed recovery from colitis and induced significant proliferation of CD4 + T cells. In vitro AE3-208 enhanced and AE1-734 suppressed the proliferation and Th1 cytokine production of lamina propria mononuclear cells from the colon. DNA microarray analysis revealed elevated expression of genes associated with immune response and reduced expression of genes with mucosal repair and remodeling in the colon of EP4-deficient mice. We conclude that EP4 maintains intestinal homeostasis by keeping mucosal integrity and downregulating immune response.

Endocannabinoids are endogenous ligands of the cannabinoid receptors CB1 and CB2. Two arachidonic acid derivatives, arachidonoylethanolamide (anandamide) and 2-arachidonoylglycerol, are considered to be physiologically important endocannabinoids. In the known metabolic pathway in mammals, anandamide and other bioactive N-acylethanolamines, such as palmitoylethanolamide and oleoylethanolamide, are biosynthesized from glycerophospholipids by a combination of Ca 2+ -dependent N-acyltransferase and N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D, and are degraded by fatty acid amide hydrolase. However, recent studies have shown the involvement of other enzymes and pathways, which include the members of the tumor suppressor HRASLS family (the phospholipase A/acyltransferase family) functioning as Ca 2+ -independent N-acyltransferases, N-acyl-phosphatidylethanolamine-hydrolyzing phospholipaseDindependent multistep pathways via N-acylated lysophospholipid, and N-acylethanolamine-hydrolyzing acid amidase, a lysosomal enzyme that preferentially hydrolyzes palmitoylethanolamide. Although their physiological significance is poorly understood, these new enzymes/pathways may serve as novel targets for the development of therapeutic drugs. For example, selective N-acylethanolamine-hydrolyzing acid amidase inhibitors are expected to be new anti-inflammatory and analgesic drugs. In this minireview, we focus on advances in the understanding of these enzymes/pathways. In addition, recent findings on 2-arachidonoylglycerol metabolism are described.

Anandamide (an endocannabinoid) and other bioactive long-chain NAEs (N-acylethanolamines) are formed by direct release from N-acyl-PE (N-acyl-phosphatidylethanolamine) by a PLD (phospholipase D). However, the possible presence of a two-step pathway from N-acyl-PE has also been suggested previously, which comprises (1) the hydrolysis of N-acyl-PE to N-acyl-lysoPE by PLA1/PLA2 enzyme(s) and (2) the release of NAEs from N-acyllysoPE by lysoPLD (lysophospholipase D) enzyme(s). In the present study we report for the first time the characterization of enzymes responsible for this pathway. The PLA1/PLA2 activity for N-palmitoyl-PE was found in various rat tissues, with the highest activity in the stomach. This stomach enzyme was identified as group IB sPLA2 (secretory PLA2), and its product was determined as N-acyl-1-acyl-lysoPE. Recombinant group IB, IIA and V of sPLA2s were also active with N-palmitoyl-PE, whereas group X sPLA2 and cytosolic PLA2a were inactive. In addition, we found wide distribution of lysoPLD activity generating N-palmitoylethanolamine from N-palmitoyl-lysoPE in rat tissues, with higher activities in the brain and testis. Based on several lines of enzymological evidence, the lysoPLD enzyme could be distinct from the known N-acyl-PE-hydrolysing PLD. sPLA2-IB dose dependently enhanced the production of N-palmitoylethanolamine from N-palmitoyl-PE in the brain homogenate showing the lysoPLD activity. N-Arachidonoyl-PE and N-arachidonoyl-lysoPE as anandamide precursors were also good substrates of sPLA2-IB and the lysoPLD respectively. These results suggest that the sequential actions of PLA2 and lysoPLD may constitute another biosynthetic pathway for NAEs, including anandamide.

We used mice deficient in each of the eight types and subtypes of prostanoid receptors and examined the roles of prostanoids in dextran sodium sulfate–induced (DSS-induced) colitis. Among the prostanoid receptor–deficient mice, only EP4-deficient mice and not mice deficient in either DP, EP1, EP2, EP3, FP, IP, or TP developed severe colitis with 3% DSS treatment, which induced only marginal colitis in wild-type mice. This phenotype was mimicked in wild-type mice by administration of an EP4-selective antagonist (AE3-208). The EP4 deficiency impaired mucosal barrier function and induced epithelial loss, crypt damage, and aggregation of neutrophils and lymphocytes in the colon. Conversely, administration of an EP4-selective agonist (AE1-734) to wild-type mice ameliorated severe colitis normally induced with 7% DSS, while that of AE3-208 suppressed recovery from colitis and induced significant proliferation of CD4+ T cells. In vitro AE3-208 enhanced and AE1-734 suppressed the proliferation and Th1 cytokine production of lamina propria mononuclear cells from the colon. DNA microarray analysis revealed elevated expression of genes associated with immune response and reduced expression of genes with mucosal repair and remodeling in the colon of EP4-deficient mice. We conclude that EP4 maintains intestinal homeostasis by keeping mucosal integrity and downregulating immune response