Biosynthesis and degradation of anandamide and 2-arachidonoylglycerol and their possible physiological significance.

N -arachidonoylethanolamine (anandamide) was the first endogenous cannabinoid receptor ligand to be discovered. Dual synthetic pathways for anandamide have been proposed. One is the formation from free arachidonic acid and ethanolamine, and the other is the formation from N -arachidonoyl phosphatidylethanolamine (PE) through the action of a phosphodiesterase. These pathways, however, do not appear to be able to generate a large amount of anandamide, at least under physiological conditions. The generation of anandamide from free arachidonic acid and ethanolamine is catalyzed by a degrading enzyme anandamide amidohydrolase/fatty acid amide hydrolase operating in reverse and requires large amounts of substrates. As for the second pathway, arachidonic acids esterified at the 1-position of glycerophospholipids, which are mostly esterified at the 2-position, are utilized for the formation of N -arachidonoyl PE, a stored precursor form of anandamide. In fact, the actual levels of anandamide in various tissues are generally low except in a few cases. 2-Arachidonoylglycerol (2-AG) was the second endogenous cannabinoid receptor ligand to be discovered. 2-AG is a degradation product of arachidonic acid-containing glycerophospholipids such as inositol phospholipids. Several investigators have demonstrated that 2-AG is produced in a variety of tissues and cells upon stimulation. 2-AG acts as a full agonist at the cannabinoid receptors (CB1 and CB2). Evidence is gradually accumulating and indicates that 2-AG is the most efficacious endogenous natural ligand for the cannabinoid receptors. In this review, we summarize the tissue levels, biosynthesis, degradation and possible physiological significance of two endogenous cannabimimetic molecules, anandamide and 2-AG.

2-Arachidonoylglycerol and anandamide oppositely modulate norepinephrine release from the rat heart sympathetic nerves.

Anandamide (10(-7) and 10(-6) M) as well as a synthetic cannabinoid HU210 (10(-8) to 10(-6) M) suppressed the norepinephrine release evoked by perivascular nerve stimulation (PNS) of the rat heart Langendorff's preparation. The effects of HU210 and the lower dose of anandamide were completely blocked by the cannabinoid CB1-receptor antagonist AM251, while that of anandamide at 10(-6) M was partly mediated by arachidonate-derived metabolites. 2-Arachidonoylglycerol (2-AG), at 10(-6) M in the presence of DFP and indomethacin, increased PNS-evoked norepinephrine release, which was completely blocked by AM251. The present results suggest that the two endocannabinoids may oppositely participate in the CB1-receptor-mediated modulation of sympathetic norepinephrine release.

Endogenous cannabinoid, 2-arachidonoylglycerol, attenuates naloxone-precipitated withdrawal signs in morphine-dependent mice.

In the present study, we examined the effects of endogenous ligand 2-arachidonoylglycerol (2-AG) on naloxone-precipitated withdrawal in morphine-dependent mice, in comparison with that of two cannabinoid agonists, an ingredient of Cannabis sativa Delta(8)-tetrahydrocannabinol (Delta(8)-THC) and the synthetic cannabinoid CB1 receptor agonist HU-210. 2-AG at a dose of 10 microg per mouse (i.c.v.) significantly inhibited both jumping and forepaw tremor as signs of withdrawal following naloxone challenge in morphine-dependent mice. Furthermore, both Delta(8)-THC and HU-210 significantly attenuated these symptoms of withdrawal in morphine-dependent mice. Therefore, it is suggested that inactivation of the endogenous cannabinoid system is related to the induction of withdrawal syndrome in morphine-dependent mice. Moreover, hyperlocomotor activity in morphine-dependent mice was markedly increased by Delta(8)-THC 10 mg/kg, which had no effect in naive mice. This finding suggested that in morphine dependence, upregulation of cannabinoid CB1 receptors occurred. Non-psychoactive CB1 receptor agonists or accelerators of endocannabinoid synthesis may be potential as therapeutic drugs for opiate withdrawal symptoms.

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.

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 white (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 polyunsaturated fatty acid-containing acyl LPA, in egg white was augmented markedly during the incubation at 37 degrees 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.

Activation by 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, of p42/44 mitogen-activated protein kinase in HL-60 cells.

2-Arachidonoylglycerol (2-AG), an endogenous cannabinoid receptor ligand, was shown to induce rapid phosphorylation of p42/44 mitogen-activated protein kinase (MAP kinase) in HL-60 cells. We confirmed that the enzyme activity of p42/44 MAP kinase in HL-60 cells was augmented markedly when the cells were stimulated with 2-AG. The addition of SR144528, a cannabinoid CB2 receptor-specific antagonist, to the cells prior to the addition of 2-AG abolished the response induced by 2-AG, indicating that the CB2 receptor is involved in the response. G protein G(i) or G(o) is also assumed to be involved, because pertussis toxin treatment of the cells nullified the response induced by 2-AG. CP55940 and anandamide also induced the activation of p42/44 MAP kinase, although the activation by anandamide was less pronounced than that by 2-AG or CP55940. These results suggest that 2-AG may play an important physiological role in this type of cell through the activation of the p42/44 MAP kinase cascade.

ATP-independent fatty acyl-coenzyme A synthesis from phospholipid: coenzyme A-dependent transacylation activity toward lysophosphatidic acid catalyzed by acyl-coenzyme A:lysophosphatidic acid acyltransferase.

CoA-dependent transacylation activity in microsomes is known to catalyze the transfer of fatty acids between phospholipids and lysophospholipids in the presence of CoA without the generation of free fatty acids. We previously found a novel acyl-CoA synthetic pathway, ATP-independent acyl-CoA synthesis from phospholipids. We proposed that: 1) the ATP-independent acyl-CoA synthesis is due to the reverse reaction of acyl-CoA:lysophospholipid acyltransferases and 2) the reverse and forward reactions of acyltransferases can combine to form a CoA-dependent transacylation system. To test these proposals, we examined whether or not recombinant mouse acyl-CoA:1-acyl-sn-glycero-3-phosphate (lysophosphatidic acid, LPA) acyltransferase (LPAAT) could catalyze ATP-independent acyl-CoA synthetic activity and CoA-dependent transacylation activity. ATP-independent acyl-CoA synthesis was indeed found in the membrane fraction from Escherichia coli cells expressing mouse LPAAT, whereas negligible activity was observed in mock-transfected cells. Phosphatidic acid (PA), but not free fatty acids, served as an acyl donor for the reaction, and LPA was formed from PA in a CoA-dependent manner during acyl-CoA synthesis. These results indicate that the ATP-independent acyl-CoA synthesis was due to the reverse reaction of LPAAT. In addition, bacterial membranes containing LPAAT catalyzed CoA-dependent acylation of LPA; PA but not free fatty acid served as an acyl donor. These results indicate that the CoA-dependent transacylation of LPA consists of 1) acyl-CoA synthesis from PA through the reverse action of LPAAT and 2) the transfer of the fatty acyl moiety of the newly formed acyl-CoA to LPA through the forward reaction of LPAAT.

2-Arachidonoylglycerol, a candidate of endothelium-derived hyperpolarizing factor.

We investigated whether 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, is involved in acetylcholine- and calcium ionophore A23187-induced relaxations in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME) and indomethacin, which is considered to be mediated by endothelium-derived hyperpolarizing factor (EDHF). In rabbit mesenteric arterial rings pre-constricted with noradrenaline, 2-arachidonoylglycerol caused concentration-dependent relaxation. The 2-arachidonoylglycerol-induced relaxations were not affected by endothelium removal. N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-caroxamide (SR141716A) and 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morholinyl-1H-pyrazole-3-carboxamide (AM281), cannabinoid CB(1) receptor antagonists, significantly attenuated 2-arachidonoylglycerol-induced relaxation and the acetylcholine-induced relaxation only slightly, but not the calcium ionophore A23187-induced relaxation. On the other hand, charybdotoxin plus apamin, K(+) channel blockers, significantly attenuated acetylcholine and calcium ionohore A23187-induced relaxations but not 2-arachidonoylglycerol-induced relaxations. These results suggest that 2-arachidonoylglycerol can cause relaxations via cannabinoid CB(1) receptors, but is not involved in EDHF-mediated relaxations.

Rapid generation of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, in rat brain after decapitation.

Rat brain, frozen in liquid nitrogen immediately after decapitation, contains a substantial amount of 2-arachidonoylglycerol (0.34 nmol/g tissue), an endogenous cannabinoid receptor ligand. The level of 2-arachidonoylglycerol in the brain was rapidly augmented after decapitation, the peak being noted 30 s after decapitation (1.54 nmol/g tissue). Noticeably, there are two phases during the increase in the levels of 2-arachidonoylglycerol: a rapid transient increase and a subsequent gradual sustained increase, suggesting that at least two separate mechanisms are involved in the generation of 2-arachidonoylglycerol in the decapitated brain. Gradual sustained formation was also observed for other monoacylglycerols, (e.g. 2-palmitoylglycerol plus 2-oleoylglycerol and 2-cis-vaccenoylglycerol). Thus, it is important to minimize post-mortem changes to estimate the exact tissue levels of 2-arachidonoylglycerol as well as other monoacylglycerols in the brain.

2-Arachidonoylglycerol and the cannabinoid receptors.

2-Arachidonoylglycerol (2-AG) is a unique molecular species of monoacylglycerol isolated from rat brain and canine gut as an endogenous cannabinoid receptor ligand (Sugiura, T., Kondo, S., Sukagawa, A., Nakane, S., Shinoda, A., Itoh, K., Yamashita, A., Waku, K., 1995. 2-Arachidonoylglycerol: a possible endogenous cannabinoid receptor ligand in brain. Biochem. Biophys. Res. Commun. 215, 89-97; Mechoulam, R., Ben-Shabat, S., Hanus, L., Ligumsky, M., Kaminski, N. E., Schatz, A.R., Gopher, A., Almog, S., Martin, B.R., Compton, D.R., Pertwee, R.G., Giffin, G., Bayewitch, M., Brag, J., Vogel, Z., 1995. Identification of an endogenous 2-monoglyceride, present in canine gut, that binds to cannabinoid receptors. Biochem. Pharmacol. 50, 83-90). 2-AG binds to the cannabinoid receptors (CB1 and CB2) and exhibits a variety of cannabimimetic activities in vitro and in vivo. Recently, we found that 2-AG induces Ca(2+) transients in NG108-15 cells, which express the CB1 receptor, and in HL-60 cells, which express the CB2 receptor, through a cannabinoid receptor- and Gi/Go-dependent mechanism. Based on the results of structure-activity relationship experiments, we concluded that 2-AG but not anandamide is the natural ligand for both the CB1 and the CB2 receptors and both receptors are primarily 2-AG receptors. Evidences are gradually accumulating that 2-AG is a physiologically essential molecule, although further detailed studies appear to be necessary to determine relative importance of 2-AG and anandamide in various animal tissues. In this review, we described mainly our previous and current experimental results, as well as those of others, concerning the tissue levels, bioactions and metabolism of 2-AG.

Synthesis and biological activities of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, and its metabolically stable ether-linked analogues.

We synthesized 2-arachidonoylglycerol (1), an endogenous cannabinoid receptor ligand, and its metabolically stable ether-linked analogues. Compound 1 was synthesized from 1,3-benzylideneglycerol (6) and arachidonic acid in the presence of N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine followed by treatment with boric acid and trimethyl borate. An ether-linked analogue of 2-arachidonoylglycerol (2) was synthesized from 6 and 5,8,11,14-eicosatetraenyl iodide (9). The ether-linked analogues of 2-palmitoylglycerol (4) and 2-oleoyglycerol (5) were synthesized from 6 and hexadecyl iodide (12) and 9-octadecenyl iodide (14), respectively. We confirmed that 1 stimulates NG108-15 cells to induce rapid transient elevation of the intracellular free Ca2+ concentrations through a CB1 receptor-dependent mechanism. Noticeably, 2 exhibited appreciable agonistic activity, although its activity was significantly lower than that of 1. Compound 2 would be a useful tool in exploring the physiological significance of 1, because this compound is resistant to hydrolyzing enzymes in contrast to 1. On the other hand, the ether-linked analogues of either 4 or 5 failed to act as a CB1 receptor agonist. Compounds 4 and 5 would also be valuable as control molecules in experiments where 2 is employed.

Occurrence of a novel cannabimimetic molecule 2-sciadonoylglycerol (2-eicosa-5',11',14'-trienoylglycerol) in the umbrella pine Sciadopitys verticillata seeds.

The umbrella pine Sciadopitys verticillata seeds were found to contain a substantial amount (16.7 nmol/g) of sciadonic acid (all-cis-5,11,14-eicosatrienoic acid)-containing 2-monoacylglycerol, i.e., 2-sciadonoylglycerol (2-eicosa-5',11',14'-trienoylglycerol). Because the structure of 2-sciadonoylglycerol closely resembles that of 2-arachidonoylglycerol, the endogenous natural ligand for the cannabinoid receptor, we examined whether or not 2-sciadonoylglycerol exhibits cannabimimetic activity using NG108-15 neuroblastomaxglioma hybrid cells which express the cannabinoid CB1 receptor. We found that 2-sciadonoylglycerol induces rapid transient elevation of intracellular free Ca2+ concentration in NG108-15 cells through a cannabinoid CBI receptor-dependent mechanism similar to the case of 2-arachidonoylglycerol, yet the activity of 2-sciadonoylglycerol was apparently lower than that of 2-arachidonoylglycerol. The activity of 2-sciadonoylglycerol was detectable from 3-10 nM, reaching a maximum at around 10 microM. To our knowledge, this is the first report showing the occurrence of a cannabimimetic monoacylglycerol in higher plants.

Generation of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, in picrotoxinin-administered rat brain.

The levels of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, and other molecular species of monoacylglycerols in rat brain were examined. In this study, we sacrificed the animals in liquid nitrogen to minimize postmortem changes. We found that rat brain contains 0.23 nmol/g tissue of 2-arachidonoylglycerol, which accounts for 10.5% of the total monoacylglycerol present in this tissue. We next investigated the level of 2-arachidonoylglycerol after in vivo stimulation with picrotoxinin. We found that the level of 2-arachidonoylglycerol was elevated markedly in picrotoxinin-administered rat brain (4- to 6-fold over the control level). Changes in the levels of other molecular species were relatively small or negligible. Several cannabimimetic molecules as well as Delta(9)-tetrahydrocannabinol are known to depress neurotransmission and to exert anticonvulsant activities; endogenous 2-arachidonoylglycerol produced during neural excitation may play a regulatory role in calming the enhanced synaptic transmission.

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 Ca(2+) concentrations in HL-60 cells that express the cannabinoid CB2 receptor. We found that 2-arachidonoylglycerol induces a rapid transient increase in intracellular free Ca(2+) 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 Ca(2+) 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. G(i) or G(o) 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.

Giant hydronephrosis due to a ureteral stone, and elevated serum levels of CA 19-9.

CA 19-9 is a widely used tumor marker. However, an elevation in serum CA 19-9 can occur in some patients with benign disorders such as cholecystolithiasis in the absence of tumor. We treated a case of acquired ureteral stone-induced giant hydronephrosis with markedly elevated serum CA 19-9 values. After nephrectomy, the serum CA 19-9 level returned to normal. No malignant cells were found in the tissues of the resected kidney. Localization of CA 19-9 was confirmed by immunohistochemical staining of the renal pelvic mucosa. A detailed case report is presented with a review of the literature.

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.