jwh-133 and glyceryl-2-arachidonate

The inflammatory sequence is the first phase of wound healing. Macrophages (MPhs) and mesenchymal stromal cells (MSCs) respond to an inflammatory microenvironment by adapting their functional activity, which polarizes them into the pro-inflammatory phenotypes M1 and MSC1. Prolongation of the inflammatory phase results in the formation of chronic wounds. The endocannabinoid system (ECS) possesses immunomodulatory properties that may impede this cellular phenotypic switch.. We investigated the immunosuppressive influence of the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) on the M1 and MSC1 cytokine secretion. Lipopolysaccharides (LPS) were used as inflammagen to stimulate MPhs and MSCs. Both inflammatory phenotypes were co-exposed to AEA or 2-AG, the specific cannabinoid receptor CB2 agonist JWH-133 served as reference. The inflammatory responses were detected by CD80/163 immuno-labelling and by ELISA measures of secreted IL-6, IL-8, MIF, TNF-α, TGF-β, and VEGF.. M1 cells were found positive for CD80 expression and secreted less IL-6 and IL-8 than MSC1 cells, while both cell types produced similar amounts of MIF. TNF-α release was increased by M1, and growth factors were secreted by MSC1, only. Cannabinoid receptor ligands efficiently decreased the inflammatory response of M1, while their impact was less pronounced in MSC1.. The ECS down-regulated the inflammatory responses of MPhs and MSCs by decreasing the cytokine release upon LPS treatment, while CB2 appeared to be of particular importance. Hence, stimulating the ECS by manipulation of endo- or use of exogenous cannabinoids in vivo may constitute a potent therapeutic option against inflammatory disorders.

Topics: Arachidonic Acids; B7-1 Antigen; Cannabinoids; Cells, Cultured; Cytokines; Endocannabinoids; Glycerides; Humans; Immunosuppression Therapy; Inflammation; Lipopolysaccharides; Macrophages; Mesenchymal Stem Cells; Phenotype; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB2

G-protein coupled cannabinoid CB2 receptor signaling and function is primarily mediated by its inhibitory effect on adenylate cyclase. The visualization and monitoring of agonist dependent dynamic 3',5'-cyclic adenosine monophosphate (cAMP) signaling at the single cell level is still missing for CB2 receptors. This paper presents an application of a live cell imaging while using a Förster resonance energy transfer (FRET)-based biosensor, Epac1-camps, for quantification of cAMP. We established HEK293 cells stably co-expressing human CB2 and Epac1-camps and quantified cAMP responses upon Forskolin pre-stimulation, followed by treatment with the CB2 ligands JWH-133, HU308, β-caryophyllene, or 2-arachidonoylglycerol. We could identify cells showing either an agonist dependent CB2-response as expected, cells displaying no response, and cells with constitutive receptor activity. In Epac1-CB2-HEK293 responder cells, the terpenoid β-caryophyllene significantly modified the cAMP response through CB2. For all of the tested ligands, a relatively high proportion of cells with constitutively active CB2 receptors was identified. Our method enabled the visualization of intracellular dynamic cAMP responses to the stimuli at single cell level, providing insights into the nature of heterologous CB2 expression systems that contributes to the understanding of Gαi-mediated G-Protein coupled receptor (GPCR) signaling in living cells and opens up possibilities for future investigations of endogenous CB2 responses.

Topics: Arachidonic Acids; Cannabinoids; Colforsin; Cyclic AMP; Endocannabinoids; Fluorescence Resonance Energy Transfer; Glycerides; Guanine Nucleotide Exchange Factors; HEK293 Cells; Humans; Polycyclic Sesquiterpenes; Receptor, Cannabinoid, CB2; Signal Transduction; Single-Cell Analysis

The exact role of the endocannabinoid system (ECS) during spermatogenesis has not been clarified. We used purified germ cell fractions representative of all phases of spermatogenesis and primary cultures of spermatogonia. This approach allowed the precise quantification of the cannabinoid receptor ligands, anandamide and 2-arachidonoylglycerol, and of the expression at transcriptional and transductional levels of their metabolic enzymes and receptors. Our data indicate that male mouse germ cells possess an active and complete ECS, which is modulated during meiosis, and suggest the presence of an autocrine endocannabinoid signal during spermatogenesis. Mitotic cells possess higher levels of 2-arachidonoylglycerol, which decrease in spermatocytes and spermatids. Accordingly, spermatogonia express higher and lower levels of 2-arachidonoylglycerol biosynthetic and degrading enzymes, respectively, as compared to meiotic and postmeiotic cells. This endocannabinoid likely plays a pivotal role in promoting the meiotic progression of germ cells by activating CB(2) receptors. In fact, we found that the selective CB(2) receptor agonist, JWH133, induced the Erk 1/2 MAPK phosphorylation cascade in spermatogonia and their progression toward meiosis, because it increased the number of cells positive for SCP3, a marker of meiotic prophase, and the expression of early meiotic prophase genes.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Cannabinoids; Cell Differentiation; Cells, Cultured; Endocannabinoids; Fluorescent Antibody Technique; Glycerides; Male; MAP Kinase Signaling System; Meiotic Prophase I; Mice; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; RNA, Messenger; Spermatogenesis; Spermatogonia; TRPV Cation Channels

To date, it has been thought that cannabinoid receptors in CNS are primarily of the CB1R subtype, with CB2R expressed only in glia and peripheral tissues. However, evidence for the expression of CB2 type cannabinoid receptors at neuronal sites in the CNS is building through anatomical localization of receptors and mRNA in neurons and behavioural studies of central effects of CB2R agonists. In the medial entorhinal area of the rat, we found that blockade of CB1R did not occlude suppression of GABAergic inhibition by the non-specific endogenous cannabinoid 2-AG, suggesting that CB1R could not account fully for the effects of 2-AG. Suppression could be mimicked using the CB2R agonist JWH-133 and reversed by the CB2R inverse agonist AM-630, indicating the presence of functional CB2R. When we reversed the order of drug application AM-630 blocked the effects of the CB2R agonist JWH-133, but not the CB1R inverse agonist LY320135. JTE-907, a CB2R inverse agonist structurally unrelated to AM-630 elicited increased GABAergic neurotransmission at picomolar concentrations. Analysis of mIPSCs revealed that CB2R effects were restricted to action potential dependent, but not action potential independent GABA release. These data provide pharmacological evidence for functional CB2R at CNS synapses.

Topics: Animals; Arachidonic Acids; Benzofurans; Cannabinoid Receptor Modulators; Cannabinoids; Central Nervous System Agents; Dioxoles; Endocannabinoids; Entorhinal Cortex; gamma-Aminobutyric Acid; Glycerides; Hippocampus; In Vitro Techniques; Indoles; Male; Neural Inhibition; Neurons; Quinolones; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Synapses; Synaptic Transmission

Cannabinoids have long been proposed to affect the immune system, especially as one of the cannabinoid receptors, the cannabinoid receptor-2 (CB(2)R) has been found almost exclusively on immune cells. Here, using human in vitro activated peripheral blood-derived T lymphocytes we investigated the long-term changes in cannabinoid receptor protein expression following cellular activation and the effects of cannabinoids on migration. We report that resting T lymphocytes do not detectably express either the cannabinoid receptor-1 (CB(1)R) or CB(2)R at the protein level. However, CB(2)R protein expression is upregulated in a biphasic manner in T lymphocytes following activation by superantigen. The cannabinoids 2-AG and JWH-133 were found to elicit activation of downstream biochemical effectors (as assessed by the phosphorylation of the ERK1/2 MAP kinases). Neither 2-AG nor JWH-133 induced chemotaxis in day 5 activated T lymphocytes, when receptor expression was at its highest. Interestingly, both 2-AG and JWH-133 inhibited CXCL12-induced chemotaxis, suggesting a modulatory role for cannabinoids in activated T lymphocytes.

Topics: Adult; Amidohydrolases; Arachidonic Acids; Cannabinoids; Chemokine CXCL12; Chemokines, CXC; Chemotaxis, Leukocyte; Endocannabinoids; Extracellular Signal-Regulated MAP Kinases; Glycerides; HT29 Cells; Humans; Monoacylglycerol Lipases; Phosphorylation; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB2; RNA, Messenger; T-Lymphocytes