N-arachidonoyl Glycine Research Articles

Lipid fingerprinting and geographical origin identification of large yellow croaker (Larimichthys crocea) using UHPLC-QTOF-MS

The Large Yellow Croaker (LYC) with rich lipids offers numerous health benefits, yet its lipid profile remains underexplored. Therefore, the lipids of LYC were comprehensively profiled and compared based lipidomics. Higher nutritional value of LYC was identified based on lipid and fatty acid assessment, particularly ovary and brain. A total of 963 lipid species belonging to 47 lipid subclasses were identified, exhibiting higher levels of glycerophospholipids (GPs) and sphingolipids (SPs) in the brain, as well as high levels of glycolipids (GLs) in the muscle. Furthermore, unique lipid subclasses were detected in the brain (Acylcarnitine (CAR), Alpha-hydroxy-N-stearoyl phytosphingosine (Cer-AP)) and ovary (N-arachidonoyl glycine (NAGly)). Notably, 8 lipid subclasses were selected as potential contributors for four tissues differentiation. Additionally, identification of LYC from various origins was achieved through lipidomics for the first time. LYC from Zhejiang and Fujian provinces could be distinguished by 100 lipid biomarkers. Most different lipids significantly negatively correlated with seawater pH and dissolved oxygen, but positively correlated with netting density, depth, environmental temperature and salinity. This comprehensive analysis provided valuable insights into LYC’s nutritional values and origin identified.

Molecular Insights into Single Chain Lipid Modulation of Acid-Sensing Ion Channel 3.

Polyunsaturated fatty acids (PUFAs) and their analogs play a significant role in modulating the activity of diverse ion channels, and recent studies show that these lipids potentiate acid-sensing ion channels (ASICs), leading to increased activity. The potentiation of the channel stems from multiple gating changes, but the exact mechanism of these effects remains uncertain. We posit a mechanistic explanation for one of these changes in channel function, the increase in the maximal current, by applying a combination of electrophysiology and all-atom molecular dynamics simulations on the open-state hASIC3. Microsecond-scale simulations were performed on open-state hASIC3 in the absence and presence of a PUFA, docosahexaenoic acid (DHA), and a PUFA analog, N-arachidonyl glycine (AG). Intriguingly, our simulations in the absence of PUFA or PUFA analogs reveal that a tail from the membrane phospholipid POPC inserts itself into the pore of the channel through lateral fenestrations on the sides of the transmembrane segments, obstructing ion permeation through the channel. The binding of either DHA or AG prevented POPC from accessing the pore in our simulations, relieving the block of ionic conduction by phospholipids. Finally, we use the single-channel recording to show that DHA increases the amplitude of the single-channel currents in ASIC3, which is consistent with our hypothesis that PUFAs relieve the pore block of the channel induced by POPCs. Together, these findings offer a potential mechanistic explanation of how PUFAs modulate ASIC maximal current, revealing a novel mechanism of action for PUFA-induced modulation of ion channels.

N-arachidonylglycine is a caloric state-dependent circulating metabolite which regulates human CD4+T cell responsiveness

Caloric deprivation interventions such as intermittent fasting and caloric restriction ameliorate metabolic and inflammatory disease. As a human model of caloric deprivation, a 24-h fast blunts innate and adaptive immune cell responsiveness relative to the refed state. Isolated serum at these time points confers these same immunomodulatory effects on transformed cell lines. To identify serum mediators orchestrating this, metabolomic and lipidomic analysis was performed on serum extracted after a 24-h fast and re-feeding. Bioinformatic integration with concurrent peripheral blood mononuclear cells RNA-seq analysis implicated key metabolite-sensing GPCRs in fasting-mediated immunomodulation. The putative GPR18 ligand N-arachidonylglycine (NAGly) was elevated during fasting and attenuated CD4+T cell responsiveness via GPR18 MTORC1 signaling. In parallel, NAGly reduced inflammatory Th1 and Th17 cytokines levels in CD4+T cells isolated from obese subjects, identifying a fasting-responsive metabolic intermediate that may contribute to the regulation of nutrient-level dependent inflammation associated with metabolic disease.

Identification and characterisation of lipids that are positive allosteric modulators of glycine receptors.

Glycine receptors are ligand-gated ion channels that mediate fast inhibitory neurotransmission within the nervous system. They have recently emerged as targets for chronic pain therapies due to their role in nociceptive signalling. N-arachidonyl glycine is an endogenous lipid that modulates glycine receptors, however its mechanism of action remains unclear. We have identified a series of N-acyl amino acids that positively modulate glycine receptors. From this, a second series of lipids containing a phenylene-moiety within the lipid tail were synthesised. This improved their efficacy by up to 6-fold, and increased the potency of receptor activation by over 10-fold. (8-2-octylphneyl)octanoyl)glycine (OPOG) had the greatest activity - potentiating the glycine EC5 by 1500% with an EC50 of 664 nM at the α1 receptor. OPOG was also found to be analgesic and produces a dose-dependent reversal of allodynia in rodent models of neuropathic pain. The second aim of this study was to identify lipid binding sites on the glycine receptor. Molecular dynamic simulations of OPOG at the α1 receptor were conducted and indicated consistent binding within a cavity at the intracellular portion of transmembrane domains (TM) 1 and 4. This site has previously been suggested to bind neurosteroids. A cryoEM structure of OPOG bound to the zebrafish glycine receptor was also obtained and identified binding to an inter-subunit cavity between the extracellular portions of TM1(-), TM2(+) and TM3(+). This cavity has previously been shown to bind the positive allosteric modulator ivermectin. Mutagenesis of the residues identified within the two sites was indicated potentiation occurs primarily through the neurosteroid binding site involving aromatic and positively charged residues. Further studies are required to explore binding within the ivermectin cavity and its relative contribution to lipid modulation.

Abstract 15584: Metabolomic Signature of Fontan Circulation

Introduction: Patients born with a range of cardiac defects, not amenable to biventricular repair are palliated with establishment of Fontan circulation. Although majority of patients with Fontan circulation survive childhood, eventually they experience multi-system dysfunction, mainly frailty, impaired exercise capacity, blunted exercise-augmented hemodynamics, progressive liver fibrosis, and shortened survival. Fontan circulation associated biochemical abnormalities have not been well described. Materials and Methods: Twenty adult patients with Fontan circulation and 20 age- and sex-matched healthy subjects underwent a non-targeted metabolomics assessment using an LC-QTOF-MS system to detect potential Fontan-related biomarkers. These study participants were also investigated for markers of frailty, cardiopulmonary exercise testing, and resting as well as exercise-augmented hemodynamic evaluation. Results: A total of 1679 known plasma metabolites were detected in electrospray positive and negative modes among which 37 metabolites were significantly different (T-Test Unpaired (p<0.05) followed by a multiple testing correction of p values (Benjamini-Hochberg)). These metabolites belonged to several classes of compounds including lipids, bile acids, amino acids, and derivatives. Several bile acids such as chenodeoxycholic acid glycine conjugate, glycoursodeoxycholic acid, and deoxycholic acid glycine conjugate were upregulated in patients with Fontan while 8-hydroxyguanine, N-arachidonoyl glycine, uracil, and cysteine were down-regulated in comparison to healthy subjects. Conclusion: In-depth plasma metabolomic analysis demonstrated metabolic pathways activation involved in bile acid formation, markers of oxidative stress, damage of DNA, whereas lower concentrations of several amino acids in patients with Fontan.

Putative role of N-Arachidonoyl glycine (NAGly) Acute hyperphagia in BALB/c mice

Introduction: Obesity is a pandemic problem associated to development of chronic degenerative diseases. N-arachidonylglycine (NAGly) is speculated to participate in pain regulation, immunity and insulin secretion; some N-acyl amino acids induce hyperphagia; in light of this, NAGly metabolic functions related to energy homeostasis has not been clarified yet. We aim to elucidate the effect of NAGly administration on weight gain, food intake, diet preferences and inflammatory profile changes in a murine model. Methods: 8-week-old BALB/c mice (n=78) were allocated into 4 groups for either sex: control, vector, NAGly-LD (1nM) and NAGly-HD (10nM). NAGly was subcutaneous administered 5 consecutive days for 3 weeks. Mice were exposed to standard diet (SD), high-fat diet (HFD) and high-sugar diet (HSD) simultaneously; weight gain, food intake and diet preferences were evaluated for 21-days. Finally, cytokines were analyzed by enzyme-linked immunosorbent assay (ELISA). Results: final weight gain was found higher in those following NAGly [LD: 21.6±1.1 g and HD: 21.4±2.1 g, (p=0.001)]. NAGly groups consumed more food in the first days; NAGly groups consumed more HFD [5.06±1.60; (p=0.001)] and HSD [1.10±0.69; (p=0.001)]. Conclusion: Our data propose that subcutaneous NAGly promotes acute hyperphagia events. We propose that NAGly might play a role related to the hunger-satiety circuit.

Rescue of two trafficking-defective variants of the neuronal glycine transporter GlyT2 associated to hyperekplexia

Hyperekplexia is a rare sensorimotor syndrome characterized by pathological startle reflex in response to unexpected trivial stimuli for which there is no specific treatment. Neonates suffer from hypertonia and are at high risk of sudden death due to apnea episodes. Mutations in the human SLC6A5 gene encoding the neuronal glycine transporter GlyT2 may disrupt the inhibitory glycinergic neurotransmission and cause a presynaptic form of the disease. The phenotype of missense mutations giving rise to protein misfolding but maintaining residual activity could be rescued by facilitating folding or intracellular trafficking. In this report, we characterized the trafficking properties of two mutants associated with hyperekplexia (A277T and Y707C, rat numbering). Transporter molecules were partially retained in the endoplasmic reticulum showing increased interaction with the endoplasmic reticulum chaperone calnexin. One transporter variant had export difficulties and increased ubiquitination levels, suggestive of enhanced endoplasmic reticulum-associated degradation. However, the two mutant transporters were amenable to correction by calnexin overexpression. Within the search for compounds capable of rescuing mutant phenotypes, we found that the arachidonic acid derivative N-arachidonoyl glycine can rescue the trafficking defects of the two variants in heterologous cells and rat brain cortical neurons. N-arachidonoyl glycine improves the endoplasmic reticulum output by reducing the interaction transporter/calnexin, increasing membrane expression and improving transport activity in a comparable way as the well-established chemical chaperone 4-phenyl-butyrate. This work identifies N-arachidonoyl glycine as a promising compound with potential for hyperekplexia therapy.

Cannabidiol Ameliorates Monocrotaline-Induced Pulmonary Hypertension in Rats.

Cannabidiol (CBD) is known for its vasorelaxant (including in the human pulmonary artery), anti-proliferative and anti-inflammatory properties. The aim of our study was to examine the potential preventive effect of chronic CBD administration (10 mg/kg/day for three weeks) on monocrotaline (MCT)-induced pulmonary hypertension (PH) rats. PH was connected with elevation of right ventricular systolic pressure; right ventricle hypertrophy; lung edema; pulmonary artery remodeling; enhancement of the vasoconstrictor and decreasing vasodilatory responses; increases in plasma concentrations of tissue plasminogen activator, plasminogen activator inhibitor type 1 and leukocyte count; and a decrease in blood oxygen saturation. CBD improved all abovementioned changes induced by PH except right ventricle hypertrophy and lung edema. In addition, CBD increased lung levels of some endocannabinoids (anandamide, N-arachidonoyl glycine, linolenoyl ethanolamide, palmitoleoyl ethanolamide and eicosapentaenoyl ethanolamide but not 2-arachidonoylglycerol). CBD did not affect the cardiopulmonary system of control rats or other parameters of blood morphology in PH. Our data suggest that CBD ameliorates MCT-induced PH in rats by improving endothelial efficiency and function, normalization of hemostatic alterations and reduction of enhanced leukocyte count determined in PH. In conclusion, CBD may be a safe, promising therapeutic or adjuvant therapy agent for the treatment of human pulmonary artery hypertension.

Inhibition of store-operated calcium channels by N-arachidonoyl glycine (NAGly): no evidence for the involvement of lipid-sensing G protein coupled receptors

N-arachidonoyl glycine (NAGly) is an endogenous lipid deriving from the endocannabinoid anandamide (AEA). Identified as a ligand of several G-protein coupled receptors (GPCRs), it can however exert biological responses independently of GPCRs. NAGly was recently shown to depress store-operated Ca2+ entry (SOCE) but its mechanism of action remains elusive. The major aim of this study was to gain a better knowledge on the NAGly-dependent impairment of SOCE in neurons of the central nervous system (CNS) from mice. First, we examined the expression of genes encoding for putative lipid sensing GPCRs using transcriptomic data publicly available. This analysis showed that the most abundant GPCRs transcripts present in the cerebral cortices of embryonic brains were coding for lysophosphatidic acid (LPA) and sphingosine-1 phosphate (S1P) receptors. Next, the presence of functional receptors was assessed with live-cell calcium imaging experiments. In primary cortical cells S1P and LPA mobilize Ca2+ from internal stores via a mechanism sensitive to the S1P and LPA receptor antagonists Ex26, H2L5186303, or Ki16425. However, none of these compounds prevented or attenuated the NAGly-dependent impairment of SOCE. We found no evidence for the requirement of lipid sensing GPCRs in this inhibitory process, indicating that NAGly is an endogenous modulator interfering with the core machinery of SOCE. Moreover, these data also raise the intriguing possibility that the depression of SOCE could play a role in the central effects of NAGly.

Molecular Modeling of an Orphan GPR18 Receptor

Background: : One of the best known to date GPCR class A (Rhodopsin) includes more than 100 orphan receptors for which the endogenous ligand is not known or is unclear. One of them is N-arachidonyl glycine receptor, named GPR18, a receptor that has been reported to be activated by Δ9-THC, endogenous cannabinoid receptors agonist anandamide and other cannabinoid receptor ligands suggesting it could be considered as third cannabinoid receptor. GPR18 activity, as well as its distribution might suggest usage of GPR18 ligands in treatment of endometriosis, cancer, and neurodegenerative disorders. Yet, so far only few GPR18 antagonists have been described, thus only ligand-based design approaches appear to be most useful to identify new ligands for this orphan receptor. Methods: : Main goal of this study, GPR18 inactive form homology model was built on the basis of the evolutionary closest homologous template: Human P2Y1 Receptor crystal structure. Results: : Obtained model was further evaluated and showed active/nonactive ligands differentiating properties with acceptable confidence. Moreover, it allowed for preliminary assessment of proteinligand interactions for a set of previously described ligands. Conclusion:: Thus collected data might serve as a starting point for a discovery of novel, active GPR18 blocking ligands.

Protective Effect of N-Arachidonoyl Glycine-GPR18 Signaling after Excitotoxical Lesion in Murine Organotypic Hippocampal Slice Cultures.

N-arachidonoyl glycine (NAGly) is an endocannabinoid involved in the regulation of different immune cells. It was shown to activate the GPR18 receptor, which was postulated to switch macrophages from cytotoxic to reparative. To study GPR18 expression and neuroprotection after NAGly treatment we used excitotoxically lesioned organotypic hippocampal slice cultures (OHSC). The effect of NAGly was also tested in isolated microglia and astrocytes as these cells play a crucial role during neuronal injury. In the present study, the GPR18 receptor was found in OHSC at mRNA level and was downregulated after N-Methyl-D-aspartate (NMDA) treatment at a single time point. Furthermore, treatment with NAGly reduced neuronal damage and this effect was abolished by GPR18 and cannabinoid receptor (CB)2 receptor antagonists. The activation but not motility of primary microglia and astrocytes was influenced when incubated with NAGly. However, NAGly alone reduced the phosphorylation of Akt but no changes in activation of the p44/42 and p38 MAPK and CREB pathways in BV2 cells could be observed. Given NAGly mediated actions we speculate that GPR18 and its ligand NAGly are modulators of glial and neuronal cells during neuronal damage.

Bioactive Lipids in Cancer, Inflammation and Related Diseases : Acute and Chronic Mild Traumatic Brain Injury Differentially Changes Levels of Bioactive Lipids in the CNS Associated with Headache.

Headache is a common complaint after mild traumatic brain injury (mTBI). Changes in the CNS lipidome were previously associated with acrolein-induced headache in rodents. mTBI caused similar headache-like symptoms in rats; therefore, we tested the hypothesis that mTBI might likewise alter the lipidome. Using a stereotaxic impactor, rats were given either a single mTBI or a series of 4 mTBIs 48 h apart. 72 h later for single mTBI and 7 days later for repeated mTBI, the trigeminal ganglia (TG), trigeminal nucleus (TNC), and cerebellum (CER) were isolated. Using HPLC/MS/MS, ~80 lipids were measured in each tissue and compared to sham controls. mTBI drove widespread alterations in lipid levels. Single mTBI increased arachidonic acid and repeated mTBI increased prostaglandins in all 3 tissue types. mTBI affected multiple TRPV agonists, including N-arachidonoyl ethanolamine (AEA), which increased in the TNC and CER after single mTBI. After repeated mTBI, AEA increased in the TG, but decreased in the TNC. Common to all tissue types in single and repeated mTBI was an increase the AEA metabolite, N-arachidonoyl glycine, a potent activator of microglial migration. Changes in the CNS lipidome associated with mTBI likely play a role in headache and in long-term neurodegenerative effects of repeated mTBI.

Phyto and endocannabinoids exert complex actions on calcium and zinc signaling in mouse cortical neurons

Live-cell imaging experiments were performed with the fluorescent Ca2+ and Zn2+ probes Fluo-4 and FluoZin-3 on cultured cortical neurons dissociated from embryonic mice to investigate the effects of the cannabinoids anandamide (AEA), cannabidiol (CBD), and N-arachidonoyl glycine (NAGly) on neuronal store-operated Ca2+ entry (SOCE). When tested individually AEA, CBD or NAGly inhibited SOCE. CBD and NAGly also released Ca2+ from the endoplasmic reticulum. Furthermore, NAGly mobilized Zn2+ from a store distinct from the endoplasmic reticulum and mitochondria, and up-regulated the thapsigargin-evoked Ca2+ release. All these effects developed in a cannabinoid receptor CB1/2 independent manner via an intracellular pathway sensitive to the GPR55 antagonist ML193. Evidence is presented that cannabinoids influence Ca2+ and Zn2+ signaling in central nervous system neurons. The lipid sensing receptor GPR55 seems to be a central actor governing these responses. In addition, the alteration of the cytosolic Zn2+ levels produced by NAGly provides support for the existence of a connection between endocannabinoids and Zn2+ signaling in the brain.

The quest for endothelial atypical cannabinoid receptor: BKCa channels act as cellular sensors for cannabinoids in in vitro and in situ endothelial cells

Endothelium-dependent component of cannabinoid-induced vasodilation has been postulated to require G-protein-coupled non-CB1/CB2 endothelial cannabinoid (eCB) receptor. GPR18 was proposed as a candidate for eCBR. To address the hypothesis that the effects attributed to eCBR are mediated by G-protein-coupled receptor (GPCR)-independent targets, we studied the electrical responses in endothelial cells, focusing on BKCa channels. In patches excised from endothelial-derived EA.hy926 cells, N-arachidonoyl glycine (NAGly) and abnormal cannabidiol (abn-cbd), prototypical agonists for eCB receptor, stimulate single BKCa activity in a concentration- and Ca2+-dependent manner. The postulated eCB receptor inhibitors rimonabant and AM251 were found to inhibit basal and stimulated by NAGly- and abn-cbd BKCa activity in cell-free patches. In isolated mice aortas, abn-cbd and NAGly produced endothelial cell hyperpolarization that was sensitive to paxilline, a selective BKCa inhibitor, but not to GPR18 antibody, and mimicked by NS1619, a direct BKCa opener. In excised patches from mice aortic endothelium, single channel activity with characteristics similar to BKCa was established by the addition of abn-cbd and NAGly. We conclude that the two cannabinoids abn-cbd and NAGly initiate a GPR18-independent activation of BKCa channels in mice aortic endothelial cells that might contribute to vasodilation to cannabinoids.

Ca2 +-dependent potassium channels and cannabinoid signaling in the endothelium of apolipoprotein E knockout mice before plaque formation

Endothelial Ca2+-dependent K+ channels (KCa) regulate endothelial function. We also know that stimulation of type 2 cannabinoid (CB2) receptors ameliorates atherosclerosis. However, whether atherosclerosis is accompanied by altered endothelial KCa- and CB2 receptor-dependent signaling is unknown. By utilizing an in situ patch-clamp approach, we directly evaluated the KCa channel function and the CB2 receptor-dependent electrical responses in the endothelium of aortic strips from young ApoE−/− and C57Bl/6 mice. In the ApoE−/− group, the resting membrane potential (−30.1±1.1mV) was less negative (p<0.05) compared to WT (−38.9±1.4mV) and voltage ramps generated an overall KCa current of reduced amplitude. The peak hyperpolarization to 2μM Ach was not different between the groups. However, the sustained component was significantly reduced in ApoE−/− strips. In contrast, the peak hyperpolarization to 0.2μM Ach was increased in the ApoE−/− group, and SKA-31, a direct IKCa/SKCa channel opener, produced a hyperpolarization and whole-cell current of greater amplitude. The BKCa opener NS1619 produced hyperpolarization that was enhanced in ApoE−/− group. N-arachidonoyl glycine, a BKCa opener, produced a hyperpolarization of enhanced amplitude in ApoE−/− arteries. Selective CB2 receptor agonist AM1241 (5μM) had no effect on endothelial membrane potential in WT group; however, in ApoE−/− group, it elicited hyperpolarization that was inhibited by a selective CB2 receptor antagonist AM630. Conclusively, our data point to functional down-regulation of basal IKCa activity in unstimulated endothelium of ApoE−/− mice. Direct and indirect IKCa stimulation resulted in increased recruitment of the channels. In addition, our data point to up-regulation of endothelial BKCa channels and CB2 receptors in ApoE−/− arteries.

N-Acyl Amino Acids (Elmiric Acids): Endogenous Signaling Molecules with Therapeutic Potential.

The subject of N-acyl amino acid conjugates has been rapidly growing in recent years, especially with regard to their analgesic and anti-inflammatory actions. The field comprises a large family of lipid signaling molecules whose importance is only now being fully realized. The most widely studied member is N-arachidonoyl glycine (NAGly), which differs structurally from the endocannabinoid anandamide (N-arachidonoyl ethanolamide) by a single oxygen atom even as the two are metabolically related. Topics that are covered in this minireview are: biosynthetic pathways for N-acyl amino acids, receptors for N-acyl amino acids, physiologic actions of N-acyl amino acids, pharmacological effects of N-acyl amino acids, and molecular mechanisms believed to be responsible for their effects. On the subject of mechanisms, we propose several possibilities whose basis is the currently available information. Four putative pathways can be suggested: 1) inhibition of fatty acid amide hydrolase-induced increases in anandamide or 2-arachidonoyl glycerol (2-AG) levels, resulting in analgesic activity; 2) binding to GPR18, initiating the production of anti-inflammatory eicosanoids (specifically, the data suggest roles for 15-deoxy-Δ12,14-prostaglandin-J2 and lipoxin A4, both of which are potent inflammation-resolving molecules); 3) inactivation of T-type Cav3 channels; and 4) inhibition of the GLYT2 glycine transporter. Each pathway would produce analgesic effects. Also, the N-acyl amino acids do not bind to either cannabinoid or opioid receptors, thus reducing adverse actions and making them good templates for novel drug candidate molecules.

N-arachidonoyl glycine, another endogenous agonist of GPR55

Interest in lipoamino acids as endogenous modulators of G-protein coupled receptors has escalated due to their involvement in a variety of physiologic processes. In particular, a role for these amino acid conjugates has emerged in the endocannabinoid system. The study presented herein investigated the effects of N-arachidonoyl glycine (NAGly) on a candidate endocannabinoid receptor, GPR55. Our novel findings reveal that NAGly induces concentration dependent increases in calcium mobilization and mitogen-activated protein kinase activities in HAGPR55/CHO cells. These increases were attenuated by the selective GPR55 antagonist ML193 (N-[4-[[(3,4-Dimethyl-5-isoxazolyl)amino]sulfonyl]phenyl]-6,8-dimethyl-2-(2-pyridinyl)-4-quinolinecarboxamide), supporting receptor mediated signaling. To our knowledge this is the first report identifying GPR55 as a target of the endogenous lipoamino acid, NAGly.

N-Oleoylglycine-Induced Hyperphagia Is Associated with the Activation of Agouti-Related Protein (AgRP) Neuron by Cannabinoid Receptor Type 1 (CB1R).

N-Acyl amino acids (NAAAs) are conjugate products of fatty acids and amino acids, which are available in animal-derived food. We compared the effects of N-arachidonoylglycine (NAGly), N-arachidonoylserine (NASer), and N-oleoylglycine (OLGly) on in vivo food intake and in vitro [Ca2+]i of Agouti-related protein (AgRP) neurons to identify the role of these compounds in energy homeostasis. Hypothalamic neuropeptide expression and anxiety behavior in response to OLGly were also tested. To further identify the underlying mechanism of OLGly on food intake, we first detected the expression level of potential OLGly receptors. The cannabinoid receptor type 1 (CB1R) antagonist was cotreated with OLGly to analyze the activation of AgRP neuron, including [Ca2+]i, expression levels of PKA, CREB, and c-Fos, and neuropeptide secretion. Results demonstrated that only OLGly (intrapertioneal injection of 6 mg/kg) can induce hyperphagia without changing the expression of hypothalamic neuropeptides and anxiety-like behavior. Moreover, 20 μM OLGly robustly enhances [Ca2+]i, c-Fos protein expression in AgRP neuron, and AgRP content in the culture medium. OLGly-induced activation of AgRP neuron was completely abolished by the CB1R-specific antagonist, AM251. In summary, this study is the first to demonstrate the association of OLGly-induced hyperphagia with activation of the AgRP neuron by CB1R. These findings open avenues for investigation and application of OLGly to modulate energy homeostasis.

Calcium signaling involved in bovine herpesvirus 1 replication in MDBK cells.

Calcium is one of the most prominent second messengers in eukaryotic cells. The involvement of calcium signaling in bovine herpesvirus 1 (BoHV-1) replication was not yet reported. In this study, we revealed that the L-type Ca2+ calcium channel blocker, Verapamil and store-operated calcium channel blocker, 2-aminoethyl diphenylborinate (2-APB) inhibited BoHV-1 replication in MDBK cells at the post-entry stages, and the Na+/Ca²+ exchanger inhibitor, N-arachidonoyl glycine exchanger (NAGly) interfered with the viral entry process. NAGly also effected the phosphorylation of PLCγ-1 at Ser1248, which corroborated our previous findings, that PLCγ-1 is important for BoHV-1 entry. Collectively, these results suggest that diverse calcium channels are employed by BoHV-1 for efficient replication.

The endogenous lipid N-arachidonoyl glycine is hypotensive and nitric oxide-cGMP-dependent vasorelaxant

N-arachidonoyl glycine (NAGLY), is the endogenous lipid that activates the G protein-couple receptor 18 (GPR18) with vasodilatory activity in resistance arteries. This study investigates its hemodynamic effects and mechanisms of vasorelaxation. Hemodynamic effects of NAGLY in rats were assessed using a Biopac system and its vascular responses were assessed using a wire myograph. NAGLY (1mg/kg) decreased blood pressure by 69.4±5.5% and reduced renal blood flow by 88±12% and the effects were not sensitive to inhibition by O-1918 (3mg/kg). In resistant vessels, NAGLY (1–30µM) induced concentration- and endothelium-dependent vasorelaxation and the effect was inhibited by the nitric oxide synthase inhibitor, L-NAME (300µM), a cGMP synthase inhibitor, ODQ (10µM), the antagonists of “endothelial anandamide” receptor, rimonabant (3µM) and O-1918 (10µM) and the inhibitor of Na+/Ca2+ exchanger (NCX), KB-R7943 (10µM). On the other hand, NAGLY-induced vasorelaxation was not affected by CID 16020046 (GPR55 antagonist), AM 251 (cannabinoid CB1 receptor antagonist), AM 630 (cannabinoid CB2 receptor antagonist), capsazepine (TRPV1 antagonist), indomethacin (cyclooxygenase inhibitor), TRAM34 (IKCa channel blocker), iberiotoxin (BKCa channel blocker) and GW9662 (PPARɤ antagonist). At low concentrations of carbachol, NAGLY potentiated carbachol-induced vasorelaxation. NAGLY is an endothelium-dependent vasodilator and hypotensive lipid. The vasorelaxation is predominantly via activation of nitric oxide-cGMP pathway and NCX and probably mediated by the "endothelial anandamide” receptor, while the hypotensive effect of NAGLY appears not to involve the anandamide receptor. NAGLY also potentiates carbachol-induced vasorelaxation, the mechanism of which might involve stimulation of NO release.