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Evidence That the Cannabinoid CB1 Receptor Is a 2-Arachidonoylglycerol Receptor

STRUCTURE-ACTIVITY RELATIONSHIP OF 2-ARACHIDONOYLGLYCEROL, ETHER-LINKED ANALOGUES, AND RELATED COMPOUNDS*
Open AccessPublished:January 29, 1999DOI:https://doi.org/10.1074/jbc.274.5.2794
      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.
      It is well known that Δ9-tetrahydrocannabinol (Δ9-THC),
      The abbreviations used are: Δ9-THC, Δ9-tetrahydrocannabinol; GABA, γ-aminobutyric acid.
      1The abbreviations used are: Δ9-THC, Δ9-tetrahydrocannabinol; GABA, γ-aminobutyric acid.
      a psychoactive ingredient of marijuana, possesses a variety of pharmacological activities in vitro and in vivo(), although, until recently, the mechanism of the action of Δ9-THC had long been unelucidated. In 1988, Devaneet al. (
      • Devane W.A.
      • Dysarz III, F.A.
      • Johnson M.R.
      • Melvin L.S.
      • Howlett A.C.
      ) provided evidence that a specific binding site(s) for cannabinoids is present in the brain. Soon after, Matsuda et al. (
      • Matsuda L.A.
      • Lolait S.J.
      • Brownstein M.J.
      • Young A.C.
      • Bonner T.I.
      ) cloned a cDNA encoding a cannabinoid receptor (CB1) from a rat brain cDNA library. These findings raised the possibility that at least part of the action of Δ9-THC is mediated through such a specific receptor and prompted the search for endogenous cannabinoid receptor ligands in mammalian tissues.
      In 1992, Devane et al. (
      • Devane W.A.
      • Hanus L.
      • Breuer A.
      • Pertwee R.G.
      • Stevenson L.A.
      • Griffin
      • Gibson D.
      • Mandelbaum A.
      • Etinger A.
      • Mechoulam R.
      ) isolatedN-arachidonoylethanolamine (anandamide) from porcine brain as the first endogenous cannabinoid receptor ligand. They demonstrated that anandamide exhibits several cannabimimetic activitiesin vitro and in vivo (
      • Devane W.A.
      • Hanus L.
      • Breuer A.
      • Pertwee R.G.
      • Stevenson L.A.
      • Griffin
      • Gibson D.
      • Mandelbaum A.
      • Etinger A.
      • Mechoulam R.
      ,
      • Mechoulam R.
      • Fride E.
      ). So far, a number of studies have been carried out on anandamide, and it has been assumed that anandamide is one of the important lipid mediators in the nervous system as well as in other systems (
      • Mechoulam R.
      • Fride E.
      ). However, we (
      • Sugiura T.
      • Kondo S.
      • Sukagawa A.
      • Tonegawa T.
      • Nakane S.
      • Yamashita A.
      • Waku K.
      ,
      • Sugiura T.
      • Kondo S.
      • Sukagawa A.
      • Tonegawa T.
      • Nakane S.
      • Yamashita A.
      • Ishima Y.
      • Waku K.
      ) and others (
      • Schmid P.C.
      • Krebsbach R.J.
      • Perry S.R.
      • Dettmer T.M.
      • Maasson J.L.
      • Schmid H.H.O.
      ,
      • Kempe K.
      • Hsu F.-F.
      • Bohrer A.
      • Turk J.
      ,
      • Felder C.C.
      • Nielsen A.
      • Briley E.M.
      • Palkovits M.
      • Priller J.
      • Axelrod J.
      • Nguyen D.N.
      • Richardson J.M.
      • Riggin R.M.
      • Koppel G.A.
      • Paul S.M.
      • Becker G.W.
      ,
      • Cadas H.
      • di Tomaso E.
      • Piomelli D.
      ) have found that the levels of anandamide are very low in several mammalian tissues. In addition, the biosynthetic pathways for anandamide, either the N-acylphosphatidylethanolamine pathway (
      • Sugiura T.
      • Kondo S.
      • Sukagawa A.
      • Tonegawa T.
      • Nakane S.
      • Yamashita A.
      • Waku K.
      ,
      • Sugiura T.
      • Kondo S.
      • Sukagawa A.
      • Tonegawa T.
      • Nakane S.
      • Yamashita A.
      • Ishima Y.
      • Waku K.
      ,
      • Cadas H.
      • di Tomaso E.
      • Piomelli D.
      ,
      • Di Marzo V.
      • Fontana A.
      • Cadas H.
      • Schinelli S.
      • Cimino G.
      • Schwartz J.-C.
      • Piomelli D.
      ,
      • Schmid H.H.O.
      • Schmid P.C.
      • Natarajan V.
      ,
      • Hansen H.S.
      • Lauritzen L.
      • Moesgaard B.
      • Strand A.M.
      • Hansen H.H.
      ) or the condensation pathway (
      • Sugiura T.
      • Kondo S.
      • Sukagawa A.
      • Tonegawa T.
      • Nakane S.
      • Yamashita A.
      • Ishima Y.
      • Waku K.
      ,
      • Deutsch D.G.
      • Chin S.A.
      ,
      • Devane W.A.
      • Axelrod J.
      ,
      • Kruszka K.K.
      • Gross R.W.
      ,
      • Ueda N.
      • Kurahashi Y.
      • Yamamoto S.
      • Tokunaga T.
      ), do not appear able to provide large amounts of anandamide, at least under normal conditions in living tissues, because the availabilities of the substrates are very low. Furthermore, several investigators demonstrated that anandamide is produced mainly in the post-mortem period in the brain (
      • Schmid P.C.
      • Krebsbach R.J.
      • Perry S.R.
      • Dettmer T.M.
      • Maasson J.L.
      • Schmid H.H.O.
      ,
      • Felder C.C.
      • Nielsen A.
      • Briley E.M.
      • Palkovits M.
      • Priller J.
      • Axelrod J.
      • Nguyen D.N.
      • Richardson J.M.
      • Riggin R.M.
      • Koppel G.A.
      • Paul S.M.
      • Becker G.W.
      ). Thus, the physiological significance or meaning of anandamide, especially in the brain, has been questioned recently despite its high binding affinity toward the cannabinoid receptor(s).
      On the other hand, several years ago, we found that 2-arachidonoylglycerol, an arachidonic acid-containing monoacylglycerol, possesses binding affinity toward the cannabinoid receptor in rat brain synaptosomes and that a rat brain contains a significant amount of arachidonoylglycerol (
      • Sugiura T.
      • Itoh K.
      • Waku K.
      • Hanahan D.J.
      ,
      • Sugiura T.
      • Kondo S.
      • Sukagawa A.
      • Nakane S.
      • Shinoda A.
      • Itoh K.
      • Yamashita A.
      • Waku K.
      ). Indeed, the level of arachidonoylglycerol in the brain was found to be 800 times higher than that of anandamide present in the same tissue (
      • Sugiura T.
      • Kondo S.
      • Sukagawa A.
      • Tonegawa T.
      • Nakane S.
      • Yamashita A.
      • Ishima Y.
      • Waku K.
      ,
      • Sugiura T.
      • Kondo S.
      • Sukagawa A.
      • Nakane S.
      • Shinoda A.
      • Itoh K.
      • Yamashita A.
      • Waku K.
      ). We suggested the possibility that 2-arachidonoylglycerol is an endogenous ligand for the cannabinoid receptor in the brain (
      • Sugiura T.
      • Itoh K.
      • Waku K.
      • Hanahan D.J.
      ,
      • Sugiura T.
      • Kondo S.
      • Sukagawa A.
      • Nakane S.
      • Shinoda A.
      • Itoh K.
      • Yamashita A.
      • Waku K.
      ). Mechoulamet al. (
      • 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
      • Griffin G.
      • Bayewitch M.
      • Barg J.
      • Vogel Z.
      ) also isolated 2-arachidonoylglycerol from canine gut as another candidate for an endogenous cannabinoid receptor ligand; they demonstrated that 2-arachidonoylglycerol possesses binding activity toward cannabinoid receptors expressed on COS-7 cells transfected with cannabinoid receptor genes and induces the inhibition of adenylate cyclase in mouse spleen cells and twitch response in mouse vas deferens. Moreover, in recent studies, we found that 2-arachidonoylglycerol induces a rapid, transient elevation of intracellular free Ca2+ concentration ([Ca2+]i) through a cannabinoid CB1 receptor-dependent mechanism and proposed that the cannabinoid CB1 receptor is originally a 2-arachidonoylglycerol receptor (
      • Sugiura T.
      • Kodaka T.
      • Kondo S.
      • Tonegawa T.
      • Nakane S.
      • Kishimoto S.
      • Yamashita A.
      • Waku K.
      ,
      • Sugiura T.
      • Kodaka T.
      • Kondo S.
      • Nakane S.
      • Kondo H.
      • Waku K.
      • Ishima
      • Watanabe K.
      • Yamamoto I.
      ). Thus, the physiological significance of 2-arachidonoylglycerol came to receive increased attention (
      • Sugiura T.
      • Itoh K.
      • Waku K.
      • Hanahan D.J.
      ,
      • Sugiura T.
      • Kondo S.
      • Sukagawa A.
      • Nakane S.
      • Shinoda A.
      • Itoh K.
      • Yamashita A.
      • Waku K.
      ,
      • 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
      • Griffin G.
      • Bayewitch M.
      • Barg J.
      • Vogel Z.
      ,
      • Sugiura T.
      • Kodaka T.
      • Kondo S.
      • Tonegawa T.
      • Nakane S.
      • Kishimoto S.
      • Yamashita A.
      • Waku K.
      ,
      • Sugiura T.
      • Kodaka T.
      • Kondo S.
      • Nakane S.
      • Kondo H.
      • Waku K.
      • Ishima
      • Watanabe K.
      • Yamamoto I.
      ,
      • Lee M.
      • Yang K.H.
      • Kaminski N.E.
      ,
      • Bisogno T.
      • Sepe N.
      • Melck D.
      • Maurelli S.
      • De Petrocellis L.
      • Di Marzo
      ,
      • Di Marzo V.
      ,
      • Di Marzo V.
      ,
      • Stella N.
      • Schweitzer P.
      • Piomelli D.
      ,
      • Sugiura T.
      • Kodaka T.
      • Kondo S.
      • Tonegawa T.
      • Nakane S.
      • Kishimoto S.
      • Yamashita A.
      • Waku K.
      ,
      • Sugiura T.
      • Kodaka T.
      • Nakane S.
      • Kishimoto S.
      • Kondo S.
      • Waku K.
      ,
      • Kondo S.
      • Kondo H.
      • Nakane S.
      • Kodaka T.
      • Tokumura A.
      • Waku K.
      • Sugiura T.
      ,
      • Goparaju S.K.
      • Ueda N.
      • Yamaguchi H.
      • Yamamoto S.
      ,
      • Di Marzo V.
      • Bisogno T.
      • Sugiura T.
      • Melck D.
      • De Petrocellis L.
      ). Despite its possible physiological importance, however, the available information concerning this novel type of bioactive lipid is still limited. In addition, it is not clear whether or not 2-arachidonoylglycerol itself, but not its metabolites, is actually implicated in the observed biological effects, especially in cases of prolonged incubation of cells or in vivo experiments. In some cases, apparently, it is not easy to interpret the obtained experimental results because of the susceptibility of 2-arachidonoylglycerol to hydrolyzing enzymes, such as monoacylglycerol lipase.
      In this study, we examined the activities of 2-arachidonoylglycerol and its ether-linked metabolically stable analogues, as well as other structurally related compounds, to stimulate NG108–15 cells. We found that an ether-linked analogue of 2-arachidonoyl-glycerol, but not that of 1(3)-arachidonoylglycerol, exhibits appreciable cannabimimetic activity, although its activity was weak compared with that of 2-arachidonoylglycerol. The results obtained here provided strong evidence that the structure of 2-arachidonoylglycerol is strictly recognized by the cannabinoid CB1 receptor.

      RESULTS

      Fig. 1 illustrates the chemical structures of 2-arachidonoylglycerol, its analogues, and related compounds, the biological activities of which were examined using NG108–15 neuroblastoma × glioma hybrid cells. Here, we examined whether or not these compounds are capable of inducing rapid transient increases in [Ca2+]i. The dose response curves of the 24 compounds tested are depicted in Fig.2. First, we examined the activity of 2-arachidonoylglycerol and confirmed that low concentrations of 2-arachidonoylglycerol elicit rapid transient increases in [Ca2+]i (Fig. 2 A). The response was detectable from as low as 0.3 nm and was augmented with increased concentrations of 2-arachidonoylglycerol. We also found that 1-arachidonoylglycerol and 3-arachidonoylglycerol, isomers of 2-arachidonoylglycerol, exhibited substantial activities (Fig. 2,B and C), although their activities were apparently lower than that of 2-arachidonoylglycerol. We confirmed that free arachidonic acid failed to induce a similar response, even at high concentrations (Fig. 2 D).
      Figure thumbnail gr1
      Figure 1Chemical structures of 2-arachidonoylglycerol, its structural analogues, and several synthetic cannabinoids.
      Figure thumbnail gr2
      Figure 2The activities of 2-arachidonoylglycerol, its structural analogues, and several synthetic cannabinoids to induce rapid, transient increases in [Ca2+]i in NG108–15 cells. Cells, loaded with Fura-2/AM, were stimulated with 2-arachidonoylglycerol or other compounds in the presence of CaCl2 (1 mm), and changes in [Ca2+]i were analyzed in CAF-100. The mean and S.D. were calculated from the results of four separate experiments.
      The activities of metabolically stable ether-linked analogues of arachidonoylglycerols were next examined. We found that 2-eicosatetraenylglycerol, an ether-linked analogue of 2-arachidonoylglycerol, possesses some agonistic activity (Fig.2 E), although its activity was markedly lower than that of 2-arachidonoylglycerol. The response was detectable from 30 nm, and the response induced by 10 μm2-eicosatetraenylglycerol was about 50% of that induced by 10 μm 2-arachidonoylglycerol. On the other hand, the activity of 1(3)-eicosatetraenylglycerol, an ether-linked analogue of 1(3)-arachidonoylglycerol, was very weak, compared with that of 2-eicosatetraenylglycerol, even at high doses, such as 10 μm (Fig. 2 F). Such preference of the 2-isomer over the 1(3)-isomer coincides well with the results of ester-linked compounds previously described (Fig. 2, AC). We also found that another metabolically stable analogue of 2-arachidonoylglycerol, 2-hydroxymethyl-(allZ)-7,10,13,16-docosatetraen-1-ol (a methylene-linked analogue of 2-arachidonoylglycerol), lacked appreciable activity (Fig.2 G); this indicates that having an oxygen atom present in the linkage is important for agonistic activity.
      We then examined the activities of propanediol-type analogues of arachidonoylglycerols. We found that 2-hydroxypropyl arachidonate, one of two hydroxy groups lacking analogues of 1(3)-arachidonoylglycerol, possesses substantial agonistic activity (Fig. 2 H); its activity was almost comparable to that of 1(3)-arachidonoylglycerol. Interestingly, the activity of 3-hydroxypropyl arachidonate (Fig.2 I), another analogue of 1(3)-arachidonoylglycerol, was found to be much weaker than that of 2-hydroxypropyl arachidonate, suggesting that the presence of a free hydroxy group adjacent to the ester linkage is essential in exhibiting strong agonistic activity.
      Next, we examined the activities of anandamide and its analogues. As shown in Fig. 2 J, we confirmed that anandamide induces the elevation of [Ca2+]i. The response was detectable from as low as 3 nm, whereas the magnitude of the response induced by anandamide was not as pronounced as that induced by 2-arachidonoylglycerol even at high concentrations. We also found that (R)-1-methanandamide (Fig. 2 K) exhibits agonistic activity comparable to that of anandamide, although the activity of (S)-1-methanandamide (Fig. 2 L) appears to be somewhat lower than those of anandamide and (R)-1-methanandamide.
      The activities of 2-monoacylglycerols containing various species of saturated and unsaturated fatty acids were next examined. As shown in Fig. 2 M, 2-eicosatetraynoylglycerol, an eicosatetraynoic acid-containing analogue of 2-arachidonoylglycerol, failed to exhibit appreciable agonistic activity even at high concentrations, indicating that the presence of double bonds, but not triple bonds, is indispensable for agonistic activity. We also found that saturated or monoenoic 2-monoacylglycerols, such as 2-palmitoylglycerol (data not shown) and 2-oleoylglycerol (Fig. 2 N), were inactive. The activities of 2-linoleoylglycerol (Fig. 2 O) and 2-γ-linolenoylglycerol (Fig. 2 P) were also very low, suggesting that the acyl moiety of 2-arachidonoylglycerol is strictly recognized by the receptor.
      One striking observation shown here is the marked difference in the activities of three species of 2-eicosatrienoylglycerols. As shown in Fig. 2 Q, the activity of 2-(11,14,17-eicosatrienoyl(n-3))glycerol was very low. On the other hand, the activity of 2-(8,11,14-eicosatrienoyl(n-6))glycerol (Fig. 2 R) was greater than that of 2-(11,14,17-eicosatrienoyl(n-3))glycerol, although its activity was still weaker than that of 2-arachidonoylglycerol. Surprisingly, 2-(5,8,11-eicosatrienoyl(n-9))glycerol possessed strong agonistic activity, which was almost comparable to that of 2-arachidonoylglycerol (2-(5,8,11,14-eicosatetraenoyl(n-6))glycerol) (Fig.2 S), suggesting that the presence of the double bond at the Δ5 position of the C20 fatty chain is essential. In keeping with this, we found that 2-(5,8,11,14,17-eicosapentaenoyl(n-3))glycerol exhibits substantial agonistic activity (Fig. 2 T). In contrast to these C20 polyunsaturated fatty acid-containing 2-monoacylglycerols, C22 polyunsaturated fatty acid-containing 2-monoacylglycerols, such as 2-docosatetraenoyl(n-6)glycerol (Fig. 2 U) and 2-docosahexaenoyl(n-3)glycerol (Fig. 2 V), failed to exhibit strong agonistic activities.
      We then examined the activities of several synthetic cannabinoids and compared their activities to that of 2-arachidonoylglycerol. We found that HU-210 and CP55940 exhibited appreciable agonistic activities (Fig. 2, W and X). The response was detectable from as low as 0.3 nm, with a plateau at around 100 nm in each case. However, the maximal responses induced by these cannabinoids are apparently smaller than that induced by 2-arachidonoylglycerol.
      To confirm that the response induced by 2-arachidonoylglycerol is mediated through the cannabinoid CB1 receptor, we examined the effects of cell pretreatment with SR141716A, a cannabinoid CB1 receptor-specific antagonist (or an inverse agonist). As shown in Fig.3, the response induced by 1 μm 2-arachidonoylglycerol or 10 μm2-eicosatetraenylglycerol, an ether analogue of 2-arachidonoylglycerol, was inhibited by cell pretreatment with 1 μm SR141716A. We also confirmed that SR141716A (1 μm) blocked the response induced by either HU-210 (10 μm) or CP55940 (10 μm) (data not shown).
      Figure thumbnail gr3
      Figure 3Effects of SR141716A on 2-arachidonoylglycerol and an ether-linked analogue of 2-arachidonoylglycerol-induced rapid, transient increases in [Ca2+]i in NG108–15 cells. Cells, loaded with Fura-2/AM, were first treated with Me2SO (Aand B) or SR141716A (1 μm) (C andD). The cells were then challenged with 2-arachidonoylglycerol (2-AG) (1 μm) (A and C) or an ether-linked analogue of 2-arachidonoylglycerol (10 μm) (B andD). Changes in [Ca2+]i were analyzed in CAF-100.
      Finally, we examined the effects of cell pretreatment with various kinds of neurotransmitters or neuromodulators and related compounds (final concentration, 10 μm) known to interact with specific receptors on the response induced by 10 μm2-arachidonoylglycerol. As summarized in TableI, the cell pretreatment with various cannabinoid receptor agonists, such as Δ9-THC, HU-210, WIN55212–2, and CP55940, completely blocked the response induced by 2-arachidonoylglycerol, probably through the desensitization of the receptor molecule. On the other hand, cell pretreatment with various compounds other than cannabinoid receptor agonists did not affect the response induced by 2-arachidonoylglycerol.
      Table IEffects of cell pretreatment with various neurotransmitters, neuromodulators, and related compounds on 2-arachidonoylglycerol-induced rapid increases in [Ca2+]i
      PretreatmentInhibition
      Δ9-THC+
      HU-210+
      WIN55212–2+
      CP55940+
      [d-Ala2,d-Leu5]-enkephalin
      Dynorphin A
      Glutamic acid
      GABA
      Glycine
      Adenosine
      ATP
      Norepinephrine
      Epinephrine
      Acetylcholine
      Serotonin
      Histamine
      Dopamine
      Taurine
      Neurotensin
      Bradykinin
      Vasopressin
      Angiotensin II
      Substance P
      Substance K
      Thyrotropin-releasing hormone
      Lysophosphatidic acid
      Platelet-activating factor
      Prostaglandin E2
      Cells were pretreated with various compounds (10 μm) or the vehicle alone for 1 min. Then, cells were washed and resuspended in Hepes-Tyrode's solution. After adding Ca2+, cells were challenged with 2-arachidonoylglycerol (10 μm), and changes in [Ca2+]i were analyzed. Experiments were repeated at least twice using separate cell preparations to confirm the results.

      DISCUSSION

      In preceding studies, we investigated the mechanism and physiological meaning of 2-arachidonoylglycerol-induced transient increases in [Ca2+]i in NG108–15 cells (
      • Sugiura T.
      • Kodaka T.
      • Kondo S.
      • Tonegawa T.
      • Nakane S.
      • Kishimoto S.
      • Yamashita A.
      • Waku K.
      ,
      • Sugiura T.
      • Kodaka T.
      • Kondo S.
      • Nakane S.
      • Kondo H.
      • Waku K.
      • Ishima
      • Watanabe K.
      • Yamamoto I.
      ). We established that the response induced by adding 2-arachidonoylglycerol to the cells is mediated through the cannabinoid CB1 receptor and Gi or Go (
      • Sugiura T.
      • Kodaka T.
      • Kondo S.
      • Tonegawa T.
      • Nakane S.
      • Kishimoto S.
      • Yamashita A.
      • Waku K.
      ,
      • Sugiura T.
      • Kodaka T.
      • Kondo S.
      • Nakane S.
      • Kondo H.
      • Waku K.
      • Ishima
      • Watanabe K.
      • Yamamoto I.
      ), yet the detailed mechanism is not yet fully understood. One important issue to be verified is whether or not 2-arachidonoylglycerol itself, but not its metabolites, is actually involved in this rapid cellular response. Here, we provided clear evidence to show that this is the case. First, a metabolically stable analogue of 2-arachidonoylglycerol, 2-eicosatetraenylglycerol, was found to exhibit appreciable agonistic activity (Fig. 2 E). Second, free arachidonic acid is not capable of inducing the response (Fig. 2 D). In addition, we already confirmed that cell pretreatment with either indomethacin or nordihydroguaiaretic acid does not affect the cellular response (
      • Sugiura T.
      • Kodaka T.
      • Kondo S.
      • Tonegawa T.
      • Nakane S.
      • Kishimoto S.
      • Yamashita A.
      • Waku K.
      ). Thus, it is evident that the structure of 2-arachidonoylglycerol itself is recognized by the cannabinoid CB1 receptor as a receptor agonist.
      Importantly, among various cannabimimetic molecules, 2-arachidonoylglycerol exhibited the highest agonistic activity. We detected its activity from as low as 0.3 nm, and the magnitude of the response induced by 2-arachidonoylglycerol was the most prominent, compared with the responses of the other compounds (Fig. 2). HU-210 and CP55940 also exhibited appreciable agonistic activities from as low as 0.3 nm (Fig. 2, W andX), whereas these compounds were shown to act as partial agonists similar to Δ9-THC (
      • Sugiura T.
      • Kodaka T.
      • Kondo S.
      • Nakane S.
      • Kondo H.
      • Waku K.
      • Ishima
      • Watanabe K.
      • Yamamoto I.
      ) and anandamide (Fig.2 J). WIN55212–2 has also been shown to exhibit strong agonistic activity, whereas its activity was detectable only above 10 nm (
      • Sugiura T.
      • Kodaka T.
      • Kondo S.
      • Tonegawa T.
      • Nakane S.
      • Kishimoto S.
      • Yamashita A.
      • Waku K.
      ). These findings that the various cannabimimetic molecules are less active, compared with 2-arachidonoylglycerol, strongly support that the cannabinoid CB1 receptor is originally a 2-arachidonoylglycerol receptor and that 2-arachidonoylglycerol is the intrinsic physiological ligand.
      Table II summarizes the preferred structures of monoacylglycerols and their structural analogues as cannabinoid receptor agonists. Glycerol is the most suitable head group, and the 2-isomer is preferable over the 1(3)-isomer. As for the fatty acyl moiety, arachidonic acid is the most preferred fatty acid constituent of monoacylglycerols, among those examined, although the activity of eicosatrienoic acid (n-9)-containing species was almost comparable to that of arachidonic acid-containing species. Because the activities of 2-eicosatrienoyl(n-6)glycerol, 2-eicosatrienoyl(n-3)glycerol and 2-docosatetraenoyl(n-6)glycerol are apparently lower than those of 2-eicosatrienoyl(n-9)glycerol and 2-eicosapentaenoyl(n-3)glycerol (Fig. 2), we assume that the structure near the ester linkage, such as the presence of the double bond at the Δ5 position rather than the structure near the methyl end, is crucially important, probably for some characteristic conformation of the agonistic molecules.
      Table IIPreferred structures of monoacylglycerols and their structural analogues as cannabinoid receptor agonists
      BackboneGlycerol > ethyleneglycol
      From the data in Ref. 23.
      , propanediol
      Position2-Isomer > 1-isomer, 3-isomer
      BondEster > ether > methylene, amide
      From the data in Ref. 23.
      Acyl moiety20:4(n-6) > 20:3(n-9) > 20:5(n-3) > 20:3(n-6) > 20:3(n-3), 22:4(n-6),
      22:6(n-3), 18:3(n-6), 18:2(n-6), 18:1(n-9), 16:0
      2-a From the data in Ref.
      • Sugiura T.
      • Kodaka T.
      • Kondo S.
      • Nakane S.
      • Kondo H.
      • Waku K.
      • Ishima
      • Watanabe K.
      • Yamamoto I.
      .
      Another important feature of the structural requirement is that the presence of an ester linkage, especially one that is adjacent to a free hydroxy group, is essential for strong agonistic activity. A methylene-linked analogue of 2-arachidonoylglycerol possesses only weak agonistic activities (Fig. 2 G). Furthermore, the activity of an ether-linked analogue of 2-arachidonoylglycerol was also considerably lower than that of 2-arachidonoylglycerol (Fig.2 E). The activity of 3-hydroxypropyl arachidonate, which lacks an adjacent free hydroxy group, was considerably lower than that of an adjacent free hydroxy group-containing analogue, 2-hydroxypropyl arachidonate (Fig. 2, H and I). It is possible, therefore, that either the oxygen atoms in the ester bond or the adjacent free hydroxy group may have close interaction with the receptor molecule. Another possibility may be that the carbonyl group of the ester bond and an adjacent free hydroxy group of the glycerol moiety are linked by a hydrogen bond under some conditions; this possibility is predicted by computer analysis evaluating the minimum energy conformation of a single molecule of 2-arachidonoylglycerol in a vacuum: one of the most stable conformations of 2000 conformations has an intramolecular hydrogen bond (O−H = 2.1 Å). Such a linkage, possible in the ester-linked compound but not in ether-linked analogues and methylene-linked analogues, is effective in fixing the glycerol-head group and yields a ring structure consisting of three carbon atoms, three oxygen atoms, and a hydrogen atom. It will be of value to examine whether or not such a ring structure can be formed in 2-arachidonoylglycerol molecules under physiological conditions, especially at the receptor site, and whether or not such a possible ring structure is implicated in the induction of strong agonistic activities.
      Since the discovery of the cannabinoid receptor gene and an endogenous cannabinoid receptor ligand, anandamide, numerous studies have been conducted on cannabinoid receptors and their endogenous ligands. It is becoming evident that the cannabinoid receptor-endogenous ligand system plays important physiological roles in the nervous system. Herkenham (
      • Herkenham M.
      ) reported that whole brain cannabinoid receptor density is similar to the whole brain densities of receptors for amino acid transmitters, such as glutamic acid and GABA; this observation leads us to postulate that the endogenous cannabinoid receptor ligand(s) is also a common molecule and abundantly present in the brain, like glutamic acid and GABA. 2-Arachidonoylglycerol, but not anandamide, meets this requirement. In previous studies, we (
      • Sugiura T.
      • Itoh K.
      • Waku K.
      • Hanahan D.J.
      ,
      • Sugiura T.
      • Kondo S.
      • Sukagawa A.
      • Nakane S.
      • Shinoda A.
      • Itoh K.
      • Yamashita A.
      • Waku K.
      ) and others (
      • Stella N.
      • Schweitzer P.
      • Piomelli D.
      ) found that the level of 2-arachidonoylglycerol in the brain is on the order of nmol/g of tissue, which is several hundred times higher than that of anandamide in the same tissue. Indeed, 2-arachidonoylglycerol is one of the major species of monoacylglycerols in the brain and other tissues (
      • Kondo S.
      • Kondo H.
      • Nakane S.
      • Kodaka T.
      • Tokumura A.
      • Waku K.
      • Sugiura T.
      ). Furthermore, we recently found that a significant amount of 2-arachidonoylglycerol was generated in a rat brain homogenate during the incubation, especially in the presence of Ca2+ (
      • Kondo S.
      • Kondo H.
      • Nakane S.
      • Kodaka T.
      • Tokumura A.
      • Waku K.
      • Sugiura T.
      ). Stimulus-induced rapid formation of 2-arachidonoylglycerol occurred in neuroblastoma cells (
      • Bisogno T.
      • Sepe N.
      • Melck D.
      • Maurelli S.
      • De Petrocellis L.
      • Di Marzo
      ), hippocampal slices (
      • Stella N.
      • Schweitzer P.
      • Piomelli D.
      ), and dorsal root ganglion neurons (
      • Gammon C.M.
      • Allen A.C.
      • Morell P.
      ) besides vascular endothelial cells (
      • Sugiura T.
      • Kodaka T.
      • Nakane S.
      • Kishimoto S.
      • Kondo S.
      • Waku K.
      ), fibroblasts (
      • Hasegawa-Sasaki H.
      ), and platelets (
      • Prescott S.M.
      • Majerus P.W.
      ). In contrast to 2-arachidonoylglycerol, the level of anandamide in the brain was very low (
      • Sugiura T.
      • Kondo S.
      • Sukagawa A.
      • Tonegawa T.
      • Nakane S.
      • Yamashita A.
      • Ishima Y.
      • Waku K.
      ,
      • Schmid P.C.
      • Krebsbach R.J.
      • Perry S.R.
      • Dettmer T.M.
      • Maasson J.L.
      • Schmid H.H.O.
      ,
      • Kempe K.
      • Hsu F.-F.
      • Bohrer A.
      • Turk J.
      ,
      • Felder C.C.
      • Nielsen A.
      • Briley E.M.
      • Palkovits M.
      • Priller J.
      • Axelrod J.
      • Nguyen D.N.
      • Richardson J.M.
      • Riggin R.M.
      • Koppel G.A.
      • Paul S.M.
      • Becker G.W.
      ,
      • Cadas H.
      • di Tomaso E.
      • Piomelli D.
      ), and only small amounts of anandamide, if any, were produced in stimulated tissues and cells (
      • Di Marzo V.
      • Fontana A.
      • Cadas H.
      • Schinelli S.
      • Cimino G.
      • Schwartz J.-C.
      • Piomelli D.
      ,
      • Hansen H.S.
      • Lauritzen L.
      • Moesgaard B.
      • Strand A.M.
      • Hansen H.H.
      ,
      • Hansen H.S.
      • Lauritzen L.
      • Strand A.M.
      • Moesgaard B.
      • Frandsen A.
      ,
      • Di Marzo V.
      • De Petrocellis L.
      • Sepe N.
      • Buono A.
      ). All these observations validate our hypothesis: the cannabinoid CB1 receptor is originally a 2-arachidonoylglycerol receptor.
      The most striking and noticeable issue concerning 2-arachidonoylglycerol is that this unique lipid molecule links enhanced inositol phospholipid metabolism in stimulated neuronal cells with the function of cannabinoid receptors expressed mainly on presynaptic membranes (
      • Herkenham M.
      ,
      • Howlett A.C.
      ,
      • Deadwyler S.A.
      • Hampson R.E.
      • Childers S.R.
      ). As described previously, 2-arachidonoylglycerol is formed in neuronal cells upon stimulation (
      • Bisogno T.
      • Sepe N.
      • Melck D.
      • Maurelli S.
      • De Petrocellis L.
      • Di Marzo
      ,
      • Stella N.
      • Schweitzer P.
      • Piomelli D.
      ) and in a brain homogenate upon the addition of Ca2+ (
      • Kondo S.
      • Kondo H.
      • Nakane S.
      • Kodaka T.
      • Tokumura A.
      • Waku K.
      • Sugiura T.
      ), and the cannabinoid CB1 receptor is known to participate in the attenuation of neurotransmission (
      • Howlett A.C.
      ,
      • Deadwyler S.A.
      • Hampson R.E.
      • Childers S.R.
      ). Such a linkage should be effective in calming some neurons after excitation, which leads to negative feedback control of neurotransmission in some synapses in which the cannabinoid CB1/2-arachidonoylglycerol receptor is present. Similar negative feedback control mechanism may operate in the case of adenosine, which is assumed to be derived principally from ATP. In any case, the cannabinoid CB1/2-arachidonoylglycerol receptor-dependent negative feedback control system appears to be of great physiological significance, because sustained activation of neuronal cells is known to cause cell exhaustion and may lead to neuronal cell death. In relation to this, we recently found that 2-arachidonoylglycerol suppresses the activation of differentiated NG108–15 cells upon depolarization (
      • Sugiura T.
      • Kodaka T.
      • Kondo S.
      • Tonegawa T.
      • Nakane S.
      • Kishimoto S.
      • Yamashita A.
      • Waku K.
      ). Furthermore, Stella et al. (
      • Stella N.
      • Schweitzer P.
      • Piomelli D.
      ) reported that 2-arachidonoylglycerol reduces long term potentiation in hippocampal slices. In vivo administration of 2-arachidonoylglycerol has been shown to induce analgesia, immobility, and reduction of spontaneous activity in mice (
      • 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
      • Griffin G.
      • Bayewitch M.
      • Barg J.
      • Vogel Z.
      ). Thus, there is increasing evidence that 2-arachidonoylglycerol plays important physiological roles in the attenuation of neurotransmission and the protection of neuronal cells presumably with the cooperation of other inhibitory neurotransmitters or neuromodulators, such as GABA and adenosine.
      In conclusion, we obtained clear evidence that the cannabinoid CB1 receptor is originally a 2-arachidonoylglycerol receptor and that 2-arachidonoylglycerol is the intrinsic physiological ligand for the cannabinoid CB1 receptor. 2-Arachidonoylglycerol is assumed to play important modulatory roles in the attenuation of neurotransmission in the nervous system; thus, the details of the mechanism of synthesis, metabolic fate, and physiological implications of 2-arachidonoylglycerol in the nervous system are important issues to be clarified in the near future.

      Acknowledgments

      We thank Prof. H. Higashida (University of Kanazawa School of Medicine, Kanazawa, Japan) for providing NG108–15 cells and Dr. S. Miyamoto (Sankyo Co. Ltd., Tokyo, Japan) for conformational modeling of the molecule.

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