dinoprost and glyceryl-2-arachidonate

A leading hypothesis of N-acyl ethanolamine (NAE) biosynthesis, including the endogenous cannabinoid anandamide (AEA), is that it depends on hydrolysis of N-acyl-phosphatidylethanolamines (NAPE) by a NAPE-specific phospholipase D (NAPE-PLD). Thus, deletion of NAPE-PLD should attenuate NAE levels. Previous analyses of two different NAPE-PLD knockout (KO) strains produced contradictory data on the importance of NAPE-PLD to AEA biosynthesis. Here, we examine this hypothesis with a strain of NAPE-PLD KO mice whose lipidome is uncharacterized. Using HPLC/MS/MS, over 70 lipids, including the AEA metabolite, N-arachidonoyl glycine (NAGly), the endocannabinoid 2-arachidonyl glycerol (2-AG) and prostaglandins (PGE(2) and PGF(2α)), and over 60 lipoamines were analyzed in 8 brain regions of KO and wild-type (WT) mice. Lipidomics analysis of this third NAPE-PLD KO strain shows a broad range of lipids that were differentially affected by lipid species and brain region. Importantly, all 6 NAEs measured were significantly reduced, though the magnitude of the effect varied by fatty acid saturation length and brain region. 2-AG levels were only impacted in the brainstem, where levels were significantly increased in KO mice. Correspondingly, levels of arachidonic acid were significantly decreased exclusively in brainstem. NAGly levels were significantly increased in 4 brain regions and levels of PGE(2) increased in 6 of 8 brain regions in KO mice. These data indicate that deletion of NAPE-PLD has far broader effects on the lipidome than previously recognized. Therefore, behavioral characteristics of suppressing NAPE-PLD activity may be due to a myriad of effects on lipids and not simply due to reduced AEA biosynthesis.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Brain; Brain Stem; Cerebellum; Cerebral Cortex; Chromatography, High Pressure Liquid; Corpus Striatum; Dinoprost; Dinoprostone; Endocannabinoids; Ethanolamines; Glycerides; Glycine; Hippocampus; Hypothalamus; Lipid Metabolism; Lipids; Mesencephalon; Mice, Knockout; Phosphatidylethanolamines; Phospholipase D; Polyunsaturated Alkamides; Tandem Mass Spectrometry; Thalamus

Activation of G protein-coupled receptors (GPCRs) can induce vasoconstriction via calcium signal-mediated and Rho-dependent pathways. Earlier reports have shown that diacylglycerol produced during calcium signal generation can be converted to an endocannabinoid, 2-arachidonoylglycerol (2-AG). Our aim was to provide evidence that GPCR signaling-induced 2-AG production and activation of vascular type1 cannabinoid receptors (CB1R) is capable of reducing agonist-induced vasoconstriction and hypertension. Rat and mouse aortic rings were examined by myography. Vascular expression of CB1R was demonstrated with immunohistochemistry. Rat aortic vascular smooth muscle cells (VSMCs) were cultured for calcium measurements and 2-AG-determination. Inhibition or genetic loss of CB1Rs enhanced vasoconstriction induced by angiotensin II (AngII) or phenylephrine (Phe), but not by prostaglandin(PG)F2α. AngII-induced vasoconstriction was augmented by inhibition of diacylglycerol lipase (tetrahydrolipstatin) and was attenuated by inhibition of monoacylglycerol lipase (JZL184) suggesting a functionally relevant role for endogenously produced 2-AG. In Gαq/11-deficient mice vasoconstriction was absent to AngII or Phe, which activate Gq/11-coupled receptors, but was maintained in response to PGF2α. In VSMCs, AngII-stimulated 2-AG-formation was inhibited by tetrahydrolipstatin and potentiated by JZL184. CB1R inhibition increased the sustained phase of AngII-induced calcium signal. Pharmacological or genetic loss of CB1R function augmented AngII-induced blood pressure rise in mice. These data demonstrate that vasoconstrictor effect of GPCR agonists is attenuated via Gq/11-mediated vascular endocannabinoid formation. Agonist-induced endocannabinoid-mediated CB1R activation is a significant physiological modulator of vascular tone. Thus, the selective modulation of GPCR signaling-induced endocannabinoid release has a therapeutic potential in case of increased vascular tone and hypertension.

Topics: Angiotensin II; Animals; Aorta; Arachidonic Acids; Benzodioxoles; Calcium; Calcium Signaling; Dinoprost; Endocannabinoids; Gene Expression Regulation; Glycerides; GTP-Binding Protein alpha Subunits, Gq-G11; Hypertension; Lactones; Lipoprotein Lipase; Male; Mice; Mice, Knockout; Monoacylglycerol Lipases; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Orlistat; Phenylephrine; Piperidines; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Tissue Culture Techniques; Vasoconstriction

Endocannabinoids have been shown to play a role in the regulation of vascular tone. The effects of 2-arachidonoyl glycerol (2-AG) on induced-tone were examined in rat aortic rings in vitro.. Aortic rings from Wistar Kyoto (WKY) rats were suspended in organ chambers for recording isometric tension development in response to 2-AG. The production of TXA2 in response to 2-AG was also assessed by enzyme immunoassay.. In endothelium-intact rings pre-contracted to PGF(2alpha), 2-AG (10 nM-30 microM) induced a biphasic effect: a weak relaxation from 10 nM to 0.1 microM, which turned into a concentration-dependent contraction from 3 to 30 microM. Endothelium-denudation did not change 2-AG-mediated vascular effects. 2-AG-induced contraction was unaffected by both the cannabinoid CB1 receptor antagonist SR141716A (3 microM) and the CB2 receptor antagonist SR144528 (1 microM). In contrast, the anandamine transport inhibitor (AM404, 100 microM) and the amino hydrolase inhibitor (PMSF, 30 microM) attenuated (P<0.05) the contractile response evoked by 2-AG in endothelium-intact and rubbed aortic rings. In addition, the cyclooxygenase inhibitor (indomethacin, 10 microM) and the thromboxane A2 (TXA2) receptor (TP receptor) antagonist GR32191 (0.3 microM) totally abolished the contraction elicited by 2-AG in endothelium-intact and rubbed aortic rings. Challenge of isolated aortic rings with 2-AG (10 microM) evoked a significant increase in TXA2 level (measured as TXB2 level) in endothelium-intact and rubbed aortic rings.. These data suggested that the contraction elicited by 2-AG resulted from the vascular smooth muscle cell uptake and conversion of 2-AG to constrictor prostanoid TXA2, which in turn caused vasoconstriction through the stimulation of TP receptor.

Topics: Animals; Aorta; Arachidonic Acids; Cannabinoid Receptor Modulators; Dinoprost; Dinoprostone; Endocannabinoids; Glycerides; In Vitro Techniques; Male; Muscle, Smooth, Vascular; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Inbred WKY; Thromboxane A2; Vasoconstrictor Agents