Shinji Nakane

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.

Identification of 9-hydroxyoctadecadienoic acid and other oxidized free fatty acids as ligands of the G protein-coupled receptor G2A.

G2A is a G protein-coupled receptor that is predominantly expressed in lymphoid tissues and macrophages. G2A can be induced by diverse stimuli to cause cell cycle arrest in the G2/M phase in pro-B and T cells. G2A is also expressed in macrophages within atherosclerotic lesions, suggesting G2A involvement in atherosclerosis. Recently, G2A was discovered to possess proton-sensing ability. In this paper, we report another function of G2A, that is, as a receptor for 9-hydroxyoctadecadienoic acid (9-HODE) and other oxidized free fatty acids. G2A, expressed in CHO-K1 or HEK293 cells, showed 9-HODE-induced intracellular calcium mobilization, inositol phosphate accumulation, inhibition of cAMP accumulation, [35S]guanosine 5′-3-O-(thio)triphosphate binding, and MAP kinase activation. Furthermore, G2A was activated by various oxidized derivatives of linoleic and arachidonic acids, but it was weakly activated by cholesteryl-9-HODE. Oxidized phosphatidylcholine (1-palmitoyl-2-linoleoyl) when hydrolyzed with phospholipase A2 also evoked intracellular calcium mobilization in G2A-expressing cells. These results indicate that G2A is activated by oxidized free fatty acids produced by oxidation and subsequent hydrolysis of phosphatidylcholine or cholesteryl linoleate. Thus, G2A might have a biological role in diverse pathological conditions including atherosclerosis.

Occurrence of lysophosphatidic acid and its alkyl ether-linked analog in rat brain and comparison of their biological activities toward cultured neural cells.

Rat brain was found to contain substantial amounts of potent bioactive lipids lysophosphatidic acid (acyl LPA) (3.73 nmol/g tissue) and lysoplasmanic acid (alkyl LPA) (0.44 nmol/g tissue). The presence of alkyl LPA was confirmed by mild alkaline hydrolysis analysis and by gas chromatography/mass spectrometry analysis of the trimethylsilyl derivative. This is the first clear evidence of the occurrence of an alkyl LPA in nature. The predominant molecular species of acyl LPA are 18:1-, 18:0- and 16:0-containing species (46. 9, 22.5 and 18.8%, respectively). A significant amount of a 20:4-containing species (7.2%) was also detected in the acyl LPA fraction. We also confirmed that rat brain alkyl LPA consists of 16:0-, 18:0- and 18:1-containing species. Noticeably, either acyl or alkyl LPA is capable of stimulating neuroblastomaxglioma hybrid NG108-15 cells to elicit a Ca(2+) transient, the potencies being almost the same. Both acyl and alkyl LPAs also induce cell rounding upon addition to the cells. These results suggest that acyl and alkyl LPAs play important physiological roles as intercellular signaling molecules as well as the roles as metabolic intermediates in the nervous system.

2-Arachidonoyl-sn-glycero-3-phosphate, an arachidonic acid-containing lysophosphatidic acid : occurrence and rapid enzymatic conversion to 2-arachidonoyl-sn-glycerol, a cannabinoid receptor ligand, in rat brain

A substantial amount of lysophosphatidic acid (LPA) (15.66 nmol/g tissue) was found to occur in the brain isolated from rats killed in liquid nitrogen. We found that a significant portion of brain LPA was accounted for by the arachidonic acid-containing species (5.4%). We obtained evidence that both 2-arachidonoyl species and 1-arachidonoyl species of LPA are present. The occurrence of 2-arachidonoyl LPA in the brain (0.53 nmol/g tissue) is a notable observation, because of its structural resemblance to 2-arachidonoyl-sn-glycerol (2-AG), an endogenous cannabinoid receptor ligand. We then examined the biological activity of 2-arachidonoyl LPA and compared it with that of 2-AG using neuroblastoma x glioma hybrid NG108-15 cells which express both the LPA receptor and cannabinoid CB1 receptor. We found that 2-arachidonoyl LPA interacts with the LPA receptor(s) to elicit the elevation of intracellular free Ca(2+) concentrations, whereas 2-AG interacts exclusively with the cannabinoid CB1 receptor. Next, we examined the possible metabolic relationship between 2-arachidonoyl LPA and 2-AG and obtained clear evidence that rapid enzymatic conversion of 2-arachidonoyl LPA to 2-AG took place in the brain homogenate. It is noteworthy that two types of endogenous ligands, that interact with different types of receptors, are closely related metabolically and rapidly interconvert.

Hen egg yolk and white contain high amounts of lysophosphatidic acids, growth factor-like lipids: Distinct Molecular species compositions

Hen egg yolk and white were found to contain high amounts of lysophosphatidic acid (acyl LPA) in addition to small amounts of lysoplasmanic acid (alkyl LPA). The levels of acyl LPA in hen egg yolk (44.23 nmol/g tissue) and while (8.81 nmol/g tissue) were on the same order as or higher than the levels of acyl LPA known to be required to elicit biological responses in various animal tissues. Noticeably, there is a marked difference between the fatty acid composition of egg yolk acyl LPA and of egg white acyl LPA; egg yolk acyl LPA predominantly contains saturated fatty acids as the acyl moiety, whereas egg white acyl LPA primarily contains polyunsaturated fatty acids. We found that the level of acyl LPA, especially polyun-saturated fatty acid-containing acyl LPA, in egg white was augmented markedly during the incubation at 37°C, while there was no change in egg yolk. We confirmed that egg white contains both the substrate, i.e., polyunsaturated fatty acid-containing lysophosphatidylcholine (LPC), and the enzyme activity catalyzing the hydrolysis of polyunsaturated fatty acid-containing LPC to the corresponding acyl LPA. Egg yolk LPA and egg white LPA may play separate physiological roles in the development, differentiation, and growth of embryos.

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.

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.

Synthesis and biological activities of novel structural analogues of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand

Novel analogues of 2-arachidonoylglycerol (2-AG), an endogenous cannabinoid receptor ligand, were developed. Chemical synthesis of these analogues (2-AGA105 and 2-AGA109) was accomplished starting from 2-octyn-1-ol and diethyl malonate and employing Wittig coupling of triene phosphonate with an aldehyde intermediate in a convergent and stereoselective manner. These analogues should be useful lead compounds for the development of novel 2-AG mimetics.