Sachiko Kondo

Evidence That 2-Arachidonoylglycerol but Not N-Palmitoylethanolamine or Anandamide Is the Physiological Ligand for the Cannabinoid CB2 Receptor COMPARISON OF THE AGONISTIC ACTIVITIES OF VARIOUS CANNABINOID RECEPTOR LIGANDS IN HL-60 CELLS

We examined the effect of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, on the intracellular free Ca2+ concentrations in HL-60 cells that express the cannabinoid CB2 receptor. We found that 2-arachidonoylglycerol induces a rapid transient increase in intracellular free Ca2+concentrations in HL-60 cells. The response was affected by neither cyclooxygenase inhibitors nor lipoxygenase inhibitors, suggesting that arachidonic acid metabolites are not involved. Consistent with this notion, free arachidonic acid was devoid of any agonistic activity. Importantly, the Ca2+ transient induced by 2-arachidonoylglycerol was blocked by pretreatment of the cells with SR144528, a CB2 receptor-specific antagonist, but not with SR141716A, a CB1 receptor-specific antagonist, indicating the involvement of the CB2 receptor but not the CB1 receptor in this cellular response. Gi or Go is also assumed to be involved, because pertussis toxin treatment of the cells abolished the response. We further examined the structure-activity relationship. We found that 2-arachidonoylglycerol is the most potent compound among a number of naturally occurring cannabimimetic molecules. Interestingly, anandamide and N-palmitoylethanolamine, other putative endogenous ligands, were found to be a weak partial agonist and an inactive ligand, respectively. These results strongly suggest that the CB2 receptor is originally a 2-arachidonoylglycerol receptor, and 2-arachidonoylglycerol is the intrinsic natural ligand for the CB2 receptor that is abundant in the immune system.

Evidence That the Cannabinoid CB1 Receptor Is a 2-Arachidonoylglycerol Receptor STRUCTURE-ACTIVITY RELATIONSHIP OF 2-ARACHIDONOYLGLYCEROL, ETHER-LINKED ANALOGUES, AND RELATED COMPOUNDS

An endogenous cannabimimetic molecule, 2-arachidonoylglycerol, induces a rapid, transient increase in intracellular free Ca2+ concentrations in NG108–15 cells through a cannabinoid CB1 receptor-dependent mechanism. We examined the activities of 24 relevant compounds (2-arachidonoylglycerol, its structural analogues, and several synthetic cannabinoids). We found that 2-arachidonoylglycerol is the most potent compound examined so far: its activity was detectable from as low as 0.3 nm, and the maximal response induced by 2-arachidonoylglycerol exceeded the responses induced by others. Activities of HU-210 and CP55940, potent cannabinoid receptor agonists, were also detectable from as low as 0.3 nm, whereas the maximal responses induced by these compounds were low compared with 2-arachidonoylglycerol. Anandamide was also found to act as a partial agonist in this assay system. We confirmed that free arachidonic acid failed to elicit a response. Furthermore, we found that a metabolically stable ether-linked analogue of 2-arachidonoylglycerol possesses appreciable agonistic activity, although its activity was apparently lower than that of 2-arachidonoylglycerol. We also confirmed that pretreating cells with various cannabinoid receptor agonists nullified the response induced by 2-arachidonoylglycerol, whereas pretreating cells with other neurotransmitters or neuromodulators did not affect the response. These results strongly suggested that the cannabinoid CB1 receptor is originally a 2-arachidonoylglycerol receptor, and 2-arachidonoylglycerol is the intrinsic physiological ligand for the cannabinoid CB1 receptor.

2‐Arachidonoylglycerol, an endogenous cannabinoid receptor agonist: identification as one of the major species of monoacylglycerols in various rat tissues, and evidence for its generation through Ca2+‐dependent and ‐independent mechanisms

The molecular species compositions of monoacylglycerols obtained from various rat tissues were examined by reverse‐phase high‐performance liquid chromatography (HPLC) and gas chromatography‐mass spectrometry (GC‐MS) analyses. We confirmed that 2‐arachidonoylglycerol, an endogenous cannabinoid receptor agonist, is one of the most abundant molecular species of monoacylglycerols in the brain. Substantial amounts of 2‐arachidonoylglycerol were also found in the liver, spleen, lung and kidney, but the levels were considerably lower than that in the brain. We found that a small amount of 2‐arachidonoylglycerol was generated in a brain homogenate during incubation in the absence of Ca2+. Importantly, the generation of 2‐arachidonoylglycerol was markedly augmented in the presence of Ca2+, suggesting that Ca2+ plays a key role in regulation of the generation of 2‐arachidonoylglycerol in this tissue.

Enzymatic synthesis of oleamide (cis‐9,10‐octadecenoamide), an endogenous sleep‐inducing lipid, by rat brain microsomes

The enzymatic synthesis of a novel sleep‐inducing lipid, oleamide (cis‐9,10‐octadecenoamide), was studied using rat brain subcellular fractions as enzyme sources. We found that oleamide was formed from oleic acid and ammonia on incubation with a brain homogenate. The enzyme activity catalyzing the formation of oleamide from oleic acid and ammonia was highest in the microsomal fraction among the subcellular fractions. Boiled microsomes did not exhibit appreciable enzyme activity. These results strongly suggest that oleamide can be synthesized enzymatically in the brains of stimulated animals.

Mice Deficient in N-Acetylgalactosamine 4-Sulfate 6-O-Sulfotransferase Are Unable to Synthesize Chondroitin/Dermatan Sulfate containing N-Acetylgalactosamine 4,6-Bissulfate Residues and Exhibit Decreased Protease Activity in Bone Marrow-derived Mast Cells

Chondroitin sulfate (CS) and dermatan sulfate (DS) containing N-acetylgalactosamine 4,6-bissulfate (GalNAc(4,6-SO4)) show various physiological activities through interacting with numerous functional proteins. N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate from 3′-phosphoadenosine 5′-phosphosulfate to position 6 of N-acetylgalactosamine 4-sulfate in CS or DS to yield GalNAc(4,6-SO4) residues. We here report generation of transgenic mice that lack GalNAc4S-6ST. GalNAc4S-6ST-null mice were born normally and fertile. In GalNAc4S-6ST-null mice, GalNAc(4,6-SO4) residues in CS and DS disappeared completely, indicating that GalNAc4S-6ST should be a sole enzyme responsible for the synthesis of GalNAc(4,6-SO4) residues in both CS and DS. IdoA-GalNAc(4,6-SO4) units that account for ∼40% of total disaccharide units of DS in the liver of the wild-type mice disappeared in the liver DS of GalNAc4S-6ST-null mice without reduction of IdoA content. Bone marrow-derived mast cells (BMMCs) derived from GalNAc4S-6ST-null mice contained CS without GlcA-GalNAc(4,6-SO4) units. Tryptase and carboxypeptidase A activities of BMMCs derived from GalNAc4S-6ST-null mice were lower than those activities of BMMCs derived from wild-type mice, although mRNA expression of these mast cell proteases was not altered. Disaccharide compositions of heparan sulfate/heparin contained in the mast cells derived from BMMCs in the presence of stem cell factor were much different from those of heparan sulfate/heparin in BMMCs but did not differ significantly between wild-type mice and GalNAc4S-6ST-null mice. These observations suggest that CS containing GalNAc(4,6-SO4) residues in BMMCs may contribute to retain the active proteases in the granules of BMMCs but not for the maturation of BMMCs into connective tissue-type mast cells.

N-arachidonoylethanolamine (anandamide), an endogenous cannabinoid receptor ligand, and related lipid molecules in the nervous tissues.

The effects of N-arachidonoylethanolamine (anandamide) and related compounds on the binding of [3H]CP55940 to rat brain synaptosomes were examined. Anandamide was shown to inhibit competitively the specific binding of [3H]CP55940 to synaptosomal membranes. The Ki value was 89 nM. In contrast, N-acylethanolamines containing saturated or monoenoic fatty acids did not exhibit high binding affinity. Several structural analogues of anandamide showed some binding activity. Among them, 2-arachidonoylglycerol is noteworthy because of its occurrence in mammalian tissues. A biosynthetic study indicated that anandamide can be synthesized via two separate synthetic pathways. The first is synthesis from free arachidonic acid and ethanolamine, and the second is the formation of N-arachidonoyl phosphatidylethanolamine (PE) from diarachidonoyl phospholipids and PE and the subsequent enzymatic release of N-arachidonoylethanolamine. The latter pathway appears to explain very well the fatty acid composition of N-acylethanolamines present in mammalian tissues.