The cannabinoid acids, analogs and endogenous counterparts.

The cannabinoid acids are a structurally heterogeneous group of compounds some of which are endogenous molecules and others that are metabolites of phytocannabinoids. The prototypic endogenous substance is N-arachidonoyl glycine (NAgly) that is closely related in structure to the cannabinoid agonist anandamide. The most studied phytocannabinoid is Δ(9)-THC-11-oic acid, the principal metabolite of Δ(9)-THC. Both types of acids have in common several biological actions such as low affinity for CB1 anti-inflammatory activity and analgesic properties. This suggests that there may be similarities in their mechanism of action, a point that is discussed in this review. Also presented are reports on analogs of the acids that provide opportunities for the development of novel therapeutic agents, such as ajulemic acid.

Effect of endocannabinoids on soybean lipoxygenase-1 activity.

Endocannabinoids appear to be involved in a variety of physiological processes. Lipoxygenase activity has been known to be affected by unsaturated fatty acids or phenolic compounds. In this study, we examined whether endocannabinoids containing both N-acyl group and phenolic group can affect the activity of soybean lipoxygenase (LOX)-1, similar to mammalian 15-lipoxygenase in physicochemical properties. First, N-arachidonoyl dopamine and N-oleoyl dopamine were found to inhibit soybean LOX-1-catalyzed oxygenation of linoleic acid in a non-competitive manner with a Ki value of 3.7 µM and 6.2 µM, respectively. Meanwhile, other endocannabinoids failed to show a remarkable inhibition of soybean LOX-1. Separately, N-arachidonoyl dopamine and N-arachidonoyl serotonin were observed to inactivate soybean LOX-1 with Kin value of 27 µM and 24 µM, respectively, and k3 value of 0.12 min(-1) and 0.35 min(-1), respectively. Furthermore, such an inactivation was enhanced by ascorbic acid, but suppressed by 13(S)-hydroperoxy-9,11-octadecadienoic acid. Taken together, it is proposed that endocannabinoids containing polyunsaturated acyl moiety and phenolic group may be efficient for the inhibition as well as inactivation of 15-lipoxygenase.

GPR18 undergoes a high degree of constitutive trafficking but is unresponsive to N-Arachidonoyl Glycine.

The orphan receptor GPR18 has become a research target following the discovery of a putative endogenous agonist, N-arachidonoyl glycine (NAGly). Chemical similarity between NAGly and the endocannabinoid anandamide suggested the hypothesis that GPR18 is a third cannabinoid receptor. GPR18-mediated cellular signalling through inhibition of cyclic adenosine monophosphate (cAMP) and phosphorylation of extracellular signal-regulated kinase (ERK), in addition to physiological consequences such as regulation of cellular migration and proliferation/apoptosis have been described in response to both NAGly and anandamide. However, discordant findings have also been reported. Here we sought to describe the functional consequences of GPR18 activation in heterologously-expressing HEK cells. GPR18 expression was predominantly intracellular in stably transfected cell lines, but moderate cell surface expression could be achieved in transiently transfected cells which also had higher overall expression. Assays were employed to characterise the ability of NAGly or anandamide to inhibit cAMP or induce ERK phosphorylation through GPR18, or induce receptor trafficking. Positive control experiments, which utilised cells expressing hCB1 receptors (hCB1R), were performed to validate assay design and performance. While these functional pathways in GPR18-expressing cells were not modified on treatment with a panel of putative GPR18 ligands, a constitutive phenotype was discovered for this receptor. Our data reveal that GPR18 undergoes rapid constitutive receptor membrane trafficking-several-fold faster than hCB1R, a highly constitutively active receptor. To enhance the likelihood of detecting agonist-mediated receptor signalling responses, we increased GPR18 protein expression (by tagging with a preprolactin signal sequence) and generated a putative constitutively inactive receptor by mutating the hGPR18 gene at amino acid site 108 (alanine to asparagine). This A108N mutant did cause an increase in surface receptor expression (which may argue for reduced constitutive activity), but no ligand-mediated effects were detected. Two glioblastoma multiforme cell lines (which endogenously express GPR18) were assayed for NAGly-induced pERK phosphorylation, with negative results. Despite a lack of ligand-mediated responses in all assays, the constitutive trafficking of GPR18 remains an interesting facet of receptor function and will have consequences for understanding the role of GPR18 in physiology.

Δ(9)-THC and N-arachidonoyl glycine regulate BV-2 microglial morphology and cytokine release plasticity: implications for signaling at GPR18.

Microglial cells are extremely plastic and undergo a variety of CNS-prompted shape changes relative to their location and current role. Signaling molecules from neurons also regulate microglial cytokine production. Neurons are known to employ the endogenous cannabinoid system to communicate with other cells of the CNS. N-arachidonoyl glycine (NAGly) and Δ(9)-tetrahydrocannabinol (Δ(9)-THC) signaling via GPR18 has been introduced as an important new target in microglial-neuronal communication. Our hypothesis is that endogenous NAGly-GPR18 signaling regulates phenotypic shape and cytokine production in microglia, and is mimicked by Δ(9)-THC in the BV-2 microglia model system. BV-2 microglia were exposed to NAGly and Δ(9)-THC or Vh for 12 h, which resulted in significant differences in the cell morphologies expressed. Cannabidiol (CBD) was effective at antagonizing the effects of both NAGly and Δ(9)-THC. Using ELISA-based microarrays, BV-2 microglia were exposed to NAGly and Δ(9)-THC or Vh for 3 h and the presence of 40 cytokines in the culture media quantified. Production of Axl, CD40, IGF-I, OPN, and Pro-MMP-9 were significantly altered by NAGly and Δ(9)-THC, and antagonized by CBD. These data add to an emerging profile that emphasizes NAGly as a component of an endogenous system present in the CNS that tightly integrates microglial proliferation, recruitment, and adhesion with neuron-glia interactivity and tissue remodeling.

Effect of endocannabinoids on IgE-mediated allergic response in RBL-2H3 cells.

Recently, some endocannabinoids were reported to show anti-inflammatory and anti-allergic activities. In this respect, various arachidonoyl endocannabinoids were screened for the inhibition of allergic response in IgE-activated RBL-2H3 cells. Among arachidonoyl endocannabinoids with a low cytotoxicity, only NA-5HT remarkably inhibited the release of ß-hexosaminidase (IC(50), 13.58 µM), a marker of degranulation, and tumor necrosis factor-α (IC(50), 12.52 µM), a pro-inflammatory cytokine, in IgE-activated RBL-2H3 cells. Additionally, NA-5HT markedly suppressed the formation of prostaglandin D(2) (PGD(2)) with IC(50) value of 1.27 µM and leukotriene B(4) (LTB(4)) with IC(50) value of 1.20 µM, and slightly LTC4. When effect of NA-5HT on early stage of FcεRI cascade was investigated, it significantly inhibited phosphorylation of Syk, but not Lyn. Furthermore, NA-5HT suppressed phosphorylation of PLCγ1/2 and PKCδ, related to degranulation process, as well as phosphorylation of LAT, ERK1/2, p38, JNK, Gab2, PI3K and Akt, implicated in the expression of pro-inflammatory cytokines. Relative to its effect on the late stage, NA-5HT slightly reduced phosphorylation of 5-lipoxygenase (5-LO) and cyclooxygenase-2 (COX-2). Additionally, NA-5HT significantly reduced the level of p40(phox), and partially inhibited the expression of p47(phox) and p67(phox). From these results, it is suggested that NA-5HT expresses anti-allergic action by suppressing the activation of Syk, LAT, p38, JNK, PI3K and Akt, as well as the expression of ERK1/2 and NADPH oxidase subunits. Further, a strong inhibition of PGD(2) or LTB(4) biosynthesis by NA-5HT may be an additional mechanism for its anti-allergic action. Such anti-allergic actions of NA-5HT may contribute to further information about its biological functions.