Yoshio Ishima

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-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.

2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, induces rapid actin polymerization in HL-60 cells differentiated into macrophage-like cells.

Delta9-Tetrahydrocannabinol, a major psychoactive constituent of marijuana, interacts with specific receptors, i.e. the cannabinoid receptors, thereby eliciting a variety of pharmacological responses. To date, two types of cannabinoid receptors have been identified: the CB1 receptor, which is abundantly expressed in the nervous system, and the CB2 receptor, which is predominantly expressed in the immune system. Previously, we investigated in detail the structure-activity relationship of various cannabinoid receptor ligands and found that 2-AG (2-arachidonoylglycerol) is the most efficacious agonist. We have proposed that 2-AG is the true natural ligand for both the CB1 and CB2 receptors. Despite the potential physiological importance of 2-AG, not much information is available concerning its biological activities towards mammalian tissues and cells. In the present study, we examined the effect of 2-AG on morphology as well as the actin filament system in differentiated HL-60 cells, which express the CB2 receptor. We found that 2-AG induces rapid morphological changes such as the extension of pseudopods. We also found that it provokes a rapid actin polymerization in these cells. Actin polymerization induced by 2-AG was abolished when cells were treated with SR144528, a CB2 receptor antagonist, and pertussis toxin, suggesting that the response was mediated by the CB2 receptor and G(i/o). A phosphoinositide 3-kinase, Rho family small G-proteins and a tyrosine kinase were also suggested to be involved. Reorganization of the actin filament system is known to be indispensable for a variety of cellular events; it is possible that 2-AG plays physiologically essential roles in various inflammatory cells and immune-competent cells by inducing a rapid actin rearrangement.

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.

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.

Inositol 1,4,5-trisphosphate accumulation in brain of lithium-treated rats.

The mechanism of action of lithium as a drug for patients with affective disorders was investigated. Three-week-old male rats were orally administered 2.7 mEq Li2CO3/kg/d for 1 or 3 wk, and phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2), inositol phosphate (IP), inositol diphosphate (IP2) and inositol triphosphate (IP3) levels in brain were measured. The levels of IP were increased 1.7 and 2.4 times after 1 wk and 3 wk of lithium administration, respectively, while PI, PIP, PIP2, IP2 and IP3 levels were not altered. IP3 was further fractionated by high-performance liquid chromatography into I-1,3,4-P3 and I-1,4,5-P3. In the control rat brain, the relative percentages of I-1,3,4-P3 and I-1,4,5-P3 were 95.8 and 4.2, respectively. However, after 3 wk of lithium administration, the values were changed to 69.6 and 30.3%, respectively. This increase in the neurotransducer I-1,4,5-P3 in the brain may be relevant to the mechanism of action in the lithium treatment of patients with manic-depressive disorders.

Phospholipid analysis of the chick ventricles in the early stages of development when their excitability changes in tetrodotoxin or in sodium substitute media

Abstract 1. Phospholipid composition has first been analysed in the various stages of embryonic chick ventricles as early as day 4 of development. 2. Relatively high content of phosphatidylserine and low content of sphingomyelin (days 4–8 of development) were found in the ventricles which had tetrodotoxin-resistant action potentials. 3. This relation was inverted after day 10 of development when all of the ventricles lost the resistance to tetrodotoxin during the active state of the membrane.

Metabolism of Docosahexaenoic-Acid-Containing Species of Ether Phospholipid in Rat Brain

The metabolism of hexaene species of ethanolamine plasmalogen (1-alk-1’-enyl-2-acylglycero-3-phosphoethanol-amine, alkenylacyl-GPE) and 1-alkyl-2-acylglycero-3-phosphoethanolamine (alkylacyl-GPE) was studied by estimating the incorporation rates of two kinds of the precursor into brain phospholipids in vivo. In the first experiment, 1-3H-glycerol was intracerebrally injected into brain of 18-day-old rats. After selected periods, the lipids were extracted and 1-acyl-, 1-alkyl- and 1-alkenyl-2-acyl-3-acetylglycerols were prepared from the ethanolamine phosphoglycerides (EPG). Six molecular species were fractionated by AgNO3-impregnated TLC. Since it has been established that ethanolamine plasmalogen is biosynthesized from alkylacyl-GPE, the turnover time, synthetic rates and synthetic rate constant for each of the species of ethanolamine plasmalogen were calculated from the kinetic data of specific radioactivities for both ether-linked EPG. The synthetic rate constant of hexaene species was found to be approximately two times that of the other species. The high content of docosahexaenoic-acid- containing species in plasmalogen may be partially due to the rapid synthesis of this species from alkylacyl-GPE. In the second experiment, the incorporation rate of 14C- docosahexaenoic acid (22:6) was compared with that of 3h- arachidonic acid (20:4). Labelled 22:6 was distributed in the EPG fraction in much higher amount than 3H-201:4. Among the three types of EPG, the distribution of 14C-22:6 in both ether-linked EPG was much higher than in 1-3H-glycerol. The metabolic pathways which possibly contributed to the special retention of 22:6 are discussed.