Tissue Levels of 2AG Continued

Rat skin Rat plasma Human milk a2-AG + 1(3)-AG

51.1 pmol/mg of lipid extract 0.012 nmol/ml 0.83 nmol/ml

Beaulieu et al. (2000)

Concerning the quantitative analysis of 2-AG, there is an important issue to be addressed as described in the following text. Previously, we found that the amount of 2-AG in the brains obtained from rats sacrificed by immersion in liquid nitrogen was 0.23 nmol/g tissue (Sugiura, Yoshinaga, et al., 2000), this value being about one fifteenth of the amount of 2-AG in the brains obtained from rats by decapitation without freezing (Kondo, S., Kondo, H., et al., 1998). This strongly suggests that a substantial amount of 2-AG was rapidly produced in the brain during the postmortem period. We confirmed that the rapid generation of 2-AG takes place in the rat brain immediately after decapitation (Sugiura et al., 2001). To estimate the exact tissue level of 2-AG under physiological conditions, it is therefore essential to minimize the postmortem changes in the levels of 2-AG. Even in the cases of frozen samples, however, there is a possibility of the generation of a large amount of 2-AG when frozen samples thaw in organic solvents. We found that a large amount of 2-AG was produced in frozen brains following the addition of chloroform:methanol unless lipid extraction was carried out very quickly (Sugiura et al., 2001). Thus, special care has to be exercised to minimize the possible artificial formation of 2-AG in the analysis of 2-AG in mammalian tissues, especially the brain.

Finally, it is necessary to mention the hydrolyzing-enzyme-resistant analog of 2-AG. Previously, we (Sugiura, Kodaka, et al., 1999) and Mechoulam, Fride, Ben-Shabat, et al., 1998) developed an ether-linked analog of 2-AG (2-AG ether or HU310). This compound is a useful tool in exploring the possible biological activities of 2-AG, especially in vivo, because this compound is quite stable against hydrolyzing enzymes. Recently, Hanus et al. (2001) reported that 2-AG ether is present in the pig brain (0.6 nmol/g tissue). They renamed it noladin ether. Fezza et al. (2002) also reported that a small amount of 2-AG ether is present in the rat brain (25.4 pmol/g tissue). However, we did not detect 2-AG ether in the brains of various mammalian species such as the rat, mouse, hamster, and pig (Oka et al., 2003). As for the ether-linked glycerolipids, it is well known that the ether bond is exclusively located at the 1-position of the glycerol backbone in mammalian tissues (for reviews, see Horrocks, 1970; Horrocks and Sharma, 1982; Sugiura and Waku, 1987). Accordingly, it is questionable that 2-AG ether is present in appreciable amounts in mammalian brains and acts as an endogenous ligand for the cannabinoid receptors.

Biosynthesis of 2-AG

About two decades ago, Prescott and Majerus (1983) reported the generation of AG in thrombin-stimulated platelets. The generation of AG in platelet-derived growth-factor-stimulated Swiss 3T3 cells (Hasegawa-Sasaki, 1985) and in bradykinin-stimulated rat dorsal ganglion neurons (Gammon et al., 1989) has also been reported. However, at that time, it was not known that 2-AG has an essential role acting as an endogenous ligand for the cannabinoid receptors. The generation of 2-AG as an endogenous cannabinoid receptor ligand was first described in ionomycin-stimulated N18TG2 cells (Bisogno, Sepe, et al., 1997), electrically-stimulated rat hippocampal slices, and ionomycin-stimulated neurons (Stella et al., 1997). We also investigated the generation of 2-AG and found that the rapid generation of 2-AG occurs in the rat brain homogenate during incubation in the presence of Ca2+ (Kondo, S., Kondo, H., et al., 1998), in thrombin- or A23187-stimulated human umbilical vein endothelial cells (Sugiura, Kodaka, Nakane, et al., 1998) and in the picrotoxinin-stimulated rat

Degradation of 2-AG

It has been shown that 2-AG is rapidly metabolized by a variety of cells to yield arachidonic acid and glycerol (e.g., Jarai et al., 2000; Bisogno, Sepe, et al., 1997; Di Marzo, Bisogno, et al., 1998, Di Marzo, Bisogno, et al., 1999; Beltramo and Piomelli, 2000; Maccarrone, Bari, et al., 2001). The most ubiquitous mechanism of the degradation of 2-AG is that by a monoacylglycerol lipase. Konrad et al. (1994) demonstrated that 2-AG can be hydrolyzed in a porcine islets homogenate. Goparaju et al. (1999) also demonstrated that 2-AG was hydrolyzed by a monoacylglycerol-lipase-like activity present in the porcine brain cytosol and particulate fractions. An enzyme activity catalyzing the hydrolysis of 2-AG was also found and partially characterized in macrophages (Di Marzo, Bisogno, et al., 1999). The properties of the monoacylglycerol lipase activity in the brain were also investigated by Horrocks and co-workers (Farooqui et al., 1990, 1993). Recently, Piomelli and co-workers (Dinh, Freund, et al., 2002; Dinh, Carpenter, et al., 2002) cloned a monoacylglycerol lipase from a rat brain cDNA library. This monoacylglycerol lipase contained 303 amino acids, and the molecular weight was calculated to be 33,367. Northern blot and in situ hybridization analyses showed that monoacylglycerol lipase mRNA is expressed in various regions of the rat brain, the highest levels being expressed in regions in which the CBj receptor is abundant, such as the hippocampus, cortex, anterior thalamus, and cerebellum. Importantly, the monoacylglycerol lipase is presynaptically expressed. They demonstrated that the increased expression of monoacylglycerol lipase in cortical neurons attenuated the level of 2-AG in these cells upon stimulation. These results strongly suggested a primary role of this enzyme in the inactivation of 2-AG in neurons.

In addition to being hydrolyzed by monoacylglycerol lipase, Di Marzo, Bisogno, et al. (1998) and Goparaju et al. (1998) provided evidence that 2-AG is metabolized by FAAH as well. 2-AG may be degraded by FAAH in addition to monoacylglycerol lipase under some circumstances.

2-AG can also be metabolized by several anabolic enzymes. For example, 2-AG can be metabolized to 2-arachidonoyl LPA through the action of a kinases (Figure 7.5). The enzyme activity involved in the formation of 2-acyl LPA from the corresponding 2-monoacylglycerol has already been studied by several investigators (e.g., Kanoh et al., 1986; Shim et al., 1989). This pathway is probably important in recycling 2-AG to form glycerophospholipids such as PI. Simpson et al. (1991) presented evidence that [3H]glycerol-labeled 2-AG, but not free [3H]glycerol, was gradually incorporated into PI when added to Swiss 3T3 cells. They demonstrated that these cells contain a monoacylglycerol kinase activity and the resultant 2-arachidonoyl LPA can be metabolized to 1-stearoyl-2-arachidonoyl PA. 1-Stearoyl-2-arachidonoyl PA then enters the "PI cycle" or the de novo synthesis of PC and PE. Another possible metabolic pathway for 2-AG is the enzymatic acylation of 2-AG, which is well known to take place in the intestine. Di Marzo, Bisogno, et al. (1998) and Di Marzo, Bisogno, et al. (1999) demonstrated that [3H]arachidonic-acid-containing 2-AG was gradually converted to phospholipids prior to its hydrolysis to arachidonic acid in N18TG2 cells, in RBL-2H3 cells, and in murine macrophages. In this case, it seems likely that a part of 2-AG was first metabolized into the lipid intermediates mentioned in the preceding text (2-arachidonoyl LPA or diacylglycerol) and then converted to phospholipids.

Oxygenation of 2-AG

An additional pathway for the metabolism of 2-AG is the enzymatic oxygenation of the molecule. Recently, Marnett and co-workers (Kozak et al., 2000, Kozak, Prusakiewicz, et al., 2001) demonstrated that 2-AG can be oxygenated by COX-2 to yield PGH2 glycerol ester (PGH2-G), and to a lesser extent, hydroxyeicosatetraenoic acid glycerol esters (HETE-G). The resultant PGH2-G isomerizes to provide PGE2-G and PGD2-G. In contrast to COX-2, COX-1 failed to metabolize 2-AG. 2-AG was also shown to be metabolized to PGH2-G and then to PGD2-G when added to RAW264.7 cells. In addition, the conversion of 2-AG to PGI2-G or TXA2-G was found to occur when COX-2 was added together with prostacyclin synthase or TX synthase (Kozak,

Crews, et al., 2002). Notably, human COX-2 and murine COX-2 metabolize 2-AG as efficiently as arachidonic acid. Among the various arachidonoyl esters, 2-AG is the most preferred substrate for COX-2. These results suggest that 2-AG acts as a natural COX-2 substrate in mammalian tissues, although concrete evidence has not yet been obtained as to whether such an enzymatic reaction actually takes place in vivo.

2-AG has also been shown to be metabolized by lipoxygenases in vitro. The incubation of 2-AG with soybean 15-lipoxygenase produced 15-HETE-G (Kozak, Gupta, et al., 2002). The leukocyte-type 12-lipoxygenase metabolized 2-AG 40% as efficiently as arachidonic acid to yield 12(S)-hydroperoxyeicosatetraenoic acid glycerol esters (12-HpETE-G) (Moody et al., 2001). On the other hand, platelet-type 12-lipoxygenase did not metabolize 2-AG effectively as in the case with anandamide (Moody et al., 2001).

It is not known whether the oxygenated derivatives of 2-AG have specific physiological or pathophysiological functions distinct from those of 2-AG and eicosanoids such as PGs. Further studies are necessary to answer this question. One possibility is that these oxygenated derivatives of 2-AG may act as a kind of prodrug in tissues. These oxygenated derivatives of 2-AG are metabolically more stable compared with PGs derived from free arachidonic acid and may have long half-lives (Kozak, Crews, et al., 2001). These oxygenated derivatives of 2-AG are transferred from the site of generation to remote target tissues in which hydrolases are expressed and then release bioactive eicosanoids such as PGs (See Kozak and Marnett, 2002, for review). The elucidation of whether this hypothesis is the case awaits further investigation.

Physiological Significance of 2-AG

As mentioned earlier, 2-AG possesses several notable features as a signaling molecule: (1) 2-AG exhibits potent cannabimimetic activities in vitro and in vivo; (2) importantly, unlike anandamide, 2-AG acts as a full agonist at both the CBj receptor and the CB2 receptor; (3) 2-AG is rapidly and selectively produced in a variety of cells upon stimulation and is easily released into the extracellular milieu; and (4) different from anandamide, 2-AG does not bind to receptors other than the cannabinoid receptors, such as the vanilloid receptor. Based on these observations, we have previously proposed that 2-AG is the true natural ligand of the cannabinoid receptors (Sugiura, Kodaka, Kondo, Nakane, et al., 1997; Sugiura, Kodaka, et al., 1999; Sugiura, Kondo, et al., 2000; for review, see Sugiura and Waku 2000).

It seems unlikely that 2-AG induces various psychedelic reactions in normal living animals. The CBj receptor is mainly present in the presynapses and is assumed to be involved in the attenuation of neurotransmission. We have previously proposed that the physiological role of 2-AG, the natural ligand of the CBj receptor, in the synapse is as follows: 2-AG generated through an increased phospholipid metabolism, especially inositol phospholipid breakdown, in neurons (presynapses and/or postsynapses) during accelerated synaptic transmission plays an important role in calming the excitation of neuronal cells by acting at the CB j receptor, thereby diminishing any subsequent neurotransmitter release (Sugiura, Kondo, et al., 1998; Sugiura and Waku, 2000). We have already demonstrated that 2-AG inhibited the depolarization-induced rapid elevation of the intracellular free Ca2+ concentration in NG108-15 cells (Sugiura, Kodaka, Kondo, Tonegawa, et al., 1997). Such a negativefeedback-regulation mechanism should be effective in calming stimulated neurons after excitation. In this context, it is noteworthy that several investigators have recently provided evidence indicating that endogenous cannabinoid receptor ligands derived from the postsynapse as retrograde messenger molecules play important roles in the attenuation of neurotransmission (Wilson and Nicoll, 2001; Kreitzer and Regehr, 2001; Ohno-Shosaku et al., 2001). Stella et al. (1997) also previously reported that 2-AG suppresses long-term potentiation in rat hippocampal slices, and Ameri and Simmet (2000) demonstrated that 2-AG reduces neuronal excitability in rat hippocampal slices in a cannabinoid CBj-receptor-dependent manner. In addition, Sinor et al. (2000) and Panikashvili et al. (2001)

reported that 2-AG plays an important protective role in the brain. Thus, the elucidation of the exact physiological functions of 2-AG in the synapses is essential to better understand the details of the regulatory mechanisms of neurotransmission in the mammalian nervous system.

In addition to the roles in the central nervous system, 2-AG is also suggested to play important physiological roles in other biological systems such as the immune system (for reviews, see Sugiura and Waku, 2000, 2002; Sugiura et al., 2002; Sugiura et al., 2003). Kaminski and co-workers (Lee et al., 1995; Ouyang et al., 1998) have conducted pioneering studies concerning the effects of 2-AG on the functions of murine lymphocytes. Gallily et al. (2000) reported that 2-AG suppressed the production of TNF- in LPS-stimulated mouse macrophages in vitro and in LPS-administered mice in vivo. However, the precise physiological roles of 2-AG as a CB2 receptor agonist in acute and chronic inflammation and/or immune responses have not yet been fully elucidated. Recently, we found that 2-AG induces the activation of MAP kinases (Kobayashi et al., 2001) and the acceleration of the generation of chemokines such as IL-8 and MCP-1 in HL60 cells (Kishimoto et al., 2004). We also found that 2-AG induces the migration of HL60 cells differentiated into macrophage-like cells (Kishimoto et al., 2003). 2-AG-induced migration has also been observed with myeloid leukemia cells and mouse splenocytes (Jorda et al., 2002) as well as with mouse microglia cells (Walter et al., 2003). Notably, Iwamura et al. (2001) demonstrated that JTE-907, a CB2 receptor antagonist/ inverse agonist, suppresses inflammatory reactions in vivo. Taking these results together, it is tempting to assume that 2-AG stimulates inflammatory reactions through acting at the CB2 receptor, at least under some circumstances. Whether 2-AG actually plays important stimulative roles during the course of inflammation and immune responses in vivo should be clarified in the near future.

There is also growing evidence showing that endogenous cannabinoid receptor ligands play some essential roles in the cardiovascular system (for reviews, see Kunos et al., 2000; Hogestatt and Zygmunt, 2002; Randall et al., 2002; Kunos et al., 2002). Human umbilical vein endothelial cells generate 2-AG when stimulated with thrombin, and the CB1 receptor mRNA is present in human aorta smooth muscle cells (Sugiura, Kodaka, Nakane, et al., 1998). The administration of 2-AG or 2-AG ether elicited hypotension in experimental animals (Mechoulam, Fride, Ben-Shabat, et al., 1998; Jarai et al., 2000). 2-AG also induced the relaxation of blood vessels in vitro (Kagota et al., 2001) and affected the norepinephrine release from rat heart sympathetic nerves (Kurihara et al., 2001). 2-AG has also been shown to counteract with endothelin-1-induced cerebral microvascular endothelial responses (Chen et al., 2000). Moreover, Stefano et al. (2000) reported that 2-AG induced the generation of nitric oxide from human vascular tissues. Despite these previous data, however, details of the physiological significance of 2-AG in the cardiovascular system still remain to be determined. Further studies are necessary for a full understanding of the physiological roles of 2-AG in the cardiovascular system as well as in other biological systems, such as the reproductive system and the endocrine system, under various physiological and pathophysiological conditions.

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