Assay of DAGLα/β Activity

Bisogno, Tiziana

doi:10.1007/978-1-4939-3539-0_16

Cited by 3 publications

(4 citation statements)

References 13 publications

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“…DAGL-α enzyme activity was assessed as previously reported [40, 41] by using membrane preparations (50 μg of protein) obtained from COS-7 cells overexpressing the human recombinant DAGL-α enzyme, and 1-[ 14 C]oleoyl-2-arachidonoylglycerol (1.0 mCi/mmol, 25 μM, synthesized as previously reported [40, 41], as substrate in the presence of vehicle or increasing concentrations of PEA (0.1–25 μM) in Tris-HCl 50 mM pH 7.4. After the incubation (20 min at 37 °C), lipids were extracted with two volumes of CHCl 3 /CH 3 OH (2:1, v/v).…”

Section: Methodsmentioning

confidence: 99%

“…DAGL-β enzyme activity was assessed by using membrane preparations (100 μg of protein) obtained from RBL-2H3 cells, and 1-[ 14 C]oleoyl-2-arachidonoylglycerol (1.0 mCi/mmol, 50 μM, [40, 41]), as substrate in the presence of vehicle or increasing concentrations of PEA (1–25 μM) in Tris-HCl 50 mM pH 7.4 or in Tris-HCl 50 mM pH 7.4 and CaCl 2 10 mM. After the incubation (20 min at 37 °C), the protocol followed the same procedures as above reported for DAGL-α enzyme activity assay.…”

Section: Methodsmentioning

confidence: 99%

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Palmitoylethanolamide counteracts substance P-induced mast cell activation in vitro by stimulating diacylglycerol lipase activity

Petrosino

Moriello

Verde

et al. 2019

J Neuroinflammation

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BackgroundPalmitoylethanolamide (PEA) is a pleiotropic endogenous lipid mediator currently used as a “dietary food for special medical purposes” against neuropathic pain and neuro-inflammatory conditions. Several mechanisms underlie PEA actions, among which the “entourage” effect, consisting of PEA potentiation of endocannabinoid signaling at either cannabinoid receptors or transient receptor potential vanilloid type-1 (TRPV1) channels. Here, we report novel molecular mechanisms through which PEA controls mast cell degranulation and substance P (SP)-induced histamine release in rat basophilic leukemia (RBL-2H3) cells, a mast cell model.MethodsRBL-2H3 cells stimulated with SP were treated with PEA in the presence and absence of a cannabinoid type-2 (CB2) receptor antagonist (AM630), or a diacylglycerol lipase (DAGL) enzyme inhibitor (OMDM188) to inhibit the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). The release of histamine was measured by ELISA and β-hexosaminidase release and toluidine blue staining were used as indices of degranulation. 2-AG levels were measured by LC-MS. The mRNA expression of proposed PEA targets (Cnr1, Cnr2, Trpv1, Ppara and Gpr55), and of PEA and endocannabinoid biosynthetic (Napepld, Dagla and Daglb) and catabolic (Faah, Naaa and Mgl) enzymes were also measured. The effects of PEA on the activity of DAGL-α or -β enzymes were assessed in COS-7 cells overexpressing the human recombinant enzyme or in RBL-2H3 cells, respectively.ResultsSP increased the number of degranulated RBL-2H3 cells and triggered the release of histamine. PEA counteracted these effects in a manner antagonized by AM630. PEA concomitantly increased the levels of 2-AG in SP-stimulated RBL-2H3 cells, and this effect was reversed by OMDM188. PEA significantly stimulated DAGL-α and -β activity and, consequently, 2-AG biosynthesis in cell-free systems. Co-treatment with PEA and 2-AG at per se ineffective concentrations downmodulated SP-induced release of histamine and degranulation, and this effect was reversed by OMDM188.ConclusionsActivation of CB2 underlies the inhibitory effects on SP-induced RBL-2H3 cell degranulation by PEA alone. We demonstrate for the first time that the effects in RBL-2H3 cells of PEA are due to the stimulation of 2-AG biosynthesis by DAGLs.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Palmitoylethanolamide counteracts substance P-induced mast cell activation in vitro by stimulating diacylglycerol lipase activity

Petrosino

Moriello

Verde

et al. 2019

J Neuroinflammation

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“…In the case of DAGL, responsible for the formation of 2-AG, a highly sensitive radiometric assay using 1-oleoyl[1-14C]-2-AG as a substrate can measure its activity. This is based on the use of methods allowing for lipid extraction, fractionation by thin-layer chromatography (TLC) and quantification of radiolabeled [14C]-oleic acid by scintillation counting [127].…”

Section: Alterations In Protein Activitymentioning

confidence: 99%

Molecular Alterations of the Endocannabinoid System in Psychiatric Disorders

Navarro

Gasparyan

Navarrete

et al. 2022

IJMS

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The therapeutic benefits of the current medications for patients with psychiatric disorders contrast with a great variety of adverse effects. The endocannabinoid system (ECS) components have gained high interest as potential new targets for treating psychiatry diseases because of their neuromodulator role, which is essential to understanding the regulation of many brain functions. This article reviewed the molecular alterations in ECS occurring in different psychiatric conditions. The methods used to identify alterations in the ECS were also described. We used a translational approach. The animal models reproducing some behavioral and/or neurochemical aspects of psychiatric disorders and the molecular alterations in clinical studies in post-mortem brain tissue or peripheral tissues were analyzed. This article reviewed the most relevant ECS changes in prevalent psychiatric diseases such as mood disorders, schizophrenia, autism, attentional deficit, eating disorders (ED), and addiction. The review concludes that clinical research studies are urgently needed for two different purposes: (1) To identify alterations of the ECS components potentially useful as new biomarkers relating to a specific disease or condition, and (2) to design new therapeutic targets based on the specific alterations found to improve the pharmacological treatment in psychiatry.

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“…2‐AG synthetase and catabolic enzymes were diacylglycerol lipas (DAGL) and monoacylglycerol lipase (MAGL), respectively. The former has two isoforms, DAGLα and DAGLβ, that shared 33% sequence identity (Bisogno, 2016). DAGLα appears to be the major 2‐AG synthetase and plays a key role in regulating retrograde synaptic plasticity in the nervous system (Gao et al, 2010).…”

Section: The Ecb Systemmentioning

confidence: 99%

Role of the endocannabinoid system in neurological disorders

Zou

et al. 2019

Intl J of Devlp Neuroscience

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Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder that begins in infancy. Although the etiology and pathogenesis are poorly understood, many studies have shown that ASD is closely related to structural and functional defects in the nervous system, especially synaptic transmission. The endocannabinoid (eCB) system is an important regulatory system of the central nervous system that regulates neurotransmission and synaptic plasticity and plays an important role in emotional and social responses and cognitive function. The relationship between eCB system and ASD has attracted increasing attention from scholars. In this review, we discuss the complex lipid signaling network of the eCB system, intracellular transport pathways, abnormal expression and association with various neurological diseases, and direct and indirect evidence for the link between eCB and ASD. Collectively, the findings to date indicate that the eCB system plays a key role in the pathophysiology of ASD and can provide new insights into potential interventions and rehabilitation strategies for ASD.

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Assay of DAGLα/β Activity

Cited by 3 publications

References 13 publications

Palmitoylethanolamide counteracts substance P-induced mast cell activation in vitro by stimulating diacylglycerol lipase activity

Palmitoylethanolamide counteracts substance P-induced mast cell activation in vitro by stimulating diacylglycerol lipase activity

Molecular Alterations of the Endocannabinoid System in Psychiatric Disorders

Role of the endocannabinoid system in neurological disorders

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