Abstract
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.
Highlights
Palmitoylethanolamide (PEA) is a pleiotropic endogenous lipid mediator currently used as a “dietary food for special medical purposes” against neuropathic pain and neuro-inflammatory conditions
The maximum effect was observed at the highest concentration tested of PEA (10 μM) (Fig. 1a), which inhibited the release of histamine from SPstimulated RBL-2H3 cells, as compared to substance P (SP)-stimulated RBL-2H3 cells treated with the vehicle of PEA (Fig. 1b)
When SP-stimulated RBL-2H3 cells were co-treated with PEA (10 μM) and AM630 (0.1 μM), histamine release was comparable to that observed in SP-stimulated RBL-2H3 cells treated with the vehicle, or with AM630 (0.1 μM) (Fig. 3a)
Summary
Palmitoylethanolamide (PEA) is a pleiotropic endogenous lipid mediator currently used as a “dietary food for special medical purposes” against neuropathic pain and neuro-inflammatory conditions. PEA is considered an endogenous lipid mediator produced on demand in several mammalian cell types and tissues to counteract inflammatory and other noxious responses [2]. On the other hand, decreased PEA levels were reported in granuloma in rats, a model of chronic inflammation sustained by neoangiogenesis [7], and in spinal and supraspinal brain regions involved in nociception in mice with neuropathic pain [8]. While the increase of endogenous PEA levels in some disorders might be a compensatory response aiming at counteracting inflammatory processes, their decrease in other pathological conditions could contribute to the etiology of the disease
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