The earlier hypothesis concerning the mechanism of action of !9-THC, the major psychoactive component in marijuana, by general membrane perturbation may not be completely abandoned with the discovery of specic cannabinoid receptors on cell membranes that are activated by endogenous ligands. This is because these endogenous ligands and the cannabinoid receptors they activate may be derived from lipid precursors that are associated with lipid raft domains in the plasma membrane. If some components of the EPCS are localized in lipid raft microdomains, this may be important for the complex nature and multiple signaling mechanisms associated with the regulation of cannabinoid receptors following the activation of these receptors or after smoking marijuana and release of endocannabinoids. The characterization of lipid raft microdomains in the action of cannabinoids in vivo is poorly understood, but the identication of mouse transient-receptor homologs, including VR1 and lipid rafts from spermatogenic cells and sperm has been reported (Trevino et al., 2001). In vitro and in some cells, lipid rafts contain, besides sphingolipids and cholesterol, all classes of glycerophospholipids (Rouquette-Jazdanian et al., 2002). Thus, lipid rafts that contain glycerophospholipids from which some components of the EPCS are derived may be involved in the signaling pathways associated with mediating the effects of endocannabinoids, cannabinoids, or marijuana use. Many proteins are also partitioned to lipid rafts that can affect their function (Brown, 2000), and the possible involvement of lipid rafts in the apoptosis induced by anandamide in a variety of cells has been demonstrated (Sarker and Maruyama, 2003), thus supporting a role for specialized membrane microdomains in anandamide signaling. A previous study had shown that smoking hashish alters various blood cell and human platelet phospholipids (Kalofoutis et al., 1980). It also showed that phosphatidylethanolamine was increased after hashish intake, and we now know that these may be endogenous marijuana-like substances called endocannabinoids, such as anandamide and 2-AG. Therefore, membrane lipid rafts may be involved in mediating some of the biological actions of endocannabinoids after smoking marijuana. Anandamide is an endogenous ligand at both VR1 and cannabinoid receptors, and a component of the EPCS that may be associated with lipid rafts. In addition to the noncannabinoid-receptor-mediated effects, the unraveling of novel signaling pathways linked to the activation of different components of the EPCS along with the cross talk of endocannabinoids with a number of ion channels may warrant a fresh look at cannabinoid-induced membrane perturbation. Since the rst plant-derived cannabinoid, !9-THC, was structurally dened and synthesized over 30 yr ago, analogs have been synthesized exogenously which include synthetic cannabinoids CP55940, HU210, and WIN55212 (an aminoalkylindole), all of which bind to receptors (CB1, CB2…CBn), inducing cannabimimetic activity (see Table 3.1). There was considerable speculation as to whether or not endogenous ligands existed for the cloned cannab-inoid receptors. Endogenous ligands for mammalian cannabcannab-inoid receptors and their entourage ligands have been discovered and characterized. These endocannabinoids include anandamide, 2-AG, noladin
ether, and virodhamine (Table 3.1). The occurrence of a novel cannabimimetic molecule 2-scia-donoylglycerol (2-SG) in the plant seeds of umbrella pine (Sciadopitys verticillata) has also been reported (Nakane et al., 2000). 2-SG was found to have effects on the CB1R similar to, but with lower activity than, 2-AG, demonstrating the occurrence of these interesting molecules, not only in plants and animals but also in disparate organisms such as ticks. This widespread occurrence of endocannabinoids and related fatty acid amides and their receptors appears to be highly conserved in nature, indicating a fundamental role in biological systems. For example, the salivary glands of ticks, which are ectoparasitic and obligate blood-feeding arthropods, can make endocannabinoids and their congeners with analgesic and anti-inammatory activity, which possibly participate in the inhibition of the host defense reactions (Fezza et al., 2003). Apparently, the EPCS plays a critical role in the survival and mechanisms of cell death. Previously, the existence of anandamide analogs in chocolate had been demonstrated (di Tomaso et al., 1996). It is thought that chocolate and cocoa contain N-acylethanolamines, which are chemically and pharmacologically related to anandamide. These lipids could mimic cannabinoid ligands either directly by activating CBRs or indirectly by increasing anandamide levels (Bruinsma and Taren, 1999). These observations dem-onstrate that endocannabinoid analogs exist in plants and animals and further illustrate the evolu-tionary conservation of the cannabinoid system in nature. In this section, we will briey review the properties and functions of these endocannabinoids. Thus, the EPCS represented by CBrs, endocannabinoids, and enzymes for the biosynthesis and degradation of these ligands is conserved throughout evolution. Endocannabinoids are present in peripheral as well as in brain tissues and have recently been demonstrated to be present in breast milk. In addition, the recent demonstration of the expression of functional CB1R in the preimplantation embryo and synthesis of anandamide in the pregnant uterus of mice suggested that cannabinoid ligand-receptor signaling is operative in the regulation of preimplantation embryo development and implantation (Paria and Dey, 2000).
2-AG has been characterized as a unique molecular species of the monoacylglycerol isolated from rat brain and canine gut as an endogenous CBR ligand (Sugiura and Waku, 2000). 2-AG also exhibits a variety of cannabimimetic activities in vitro and in vivo, and clearly further studies are necessary to determine the relative importance of 2-AG and anandamide in the human body and brain. This is because the levels of anandamide (800 times lower than the levels of 2-AG) found by some investigators in several mammalian tissues, and its production mainly in the postmortem period in the brain, have led to questions about the physiological signicance of anandamide, especially in the brain, despite its high-afnity binding to CBRs (Sugiura and Waku, 2000). These research ndings undoubtedly have advanced cannabis research and have allowed us to hypothesize that the EPCS consists of a previously unrecognized but elaborate network of endocannabinoid neuromodulators complete with their accompanying biosynthetic, uptake, and degradation pathways just like the monoaminergic or opioidergic systems.
Because of its rapid progress and the transformation of marijuana research into mainstream science, a number of reviews of the effects of endocannabinoids in various in vivo and in vitro systems have appeared (Kozak et al., 2004; Walter and Stella, 2004; Park et al., 2003; Castellano et al., 2004;
Piomelli, 2003). Briey, a role for endocannabinoids has been suggested in brain development through the activation of second-messenger-coupled cannabinoid receptors (Fernandez-Ruiz et al., 2000). The evidence for a role of endocannabinoids in neural development was derived from studies in which the presence of CBRs and endocannabinoids during brain development and in neuronal or fetal glial cell cultures was shown to produce cellular responses. These responses, along with the neurotoxicological changes produced in pups from pregnant rats treated with phytocannabinoids, indicate the existence of a endocannabinoid system early in the development of the CNS (Fernandez-Ruiz et al., 2000). The stimuli for the production and release and the general physiological roles and signicance of endocannabinoids are incompletely understood. Their interaction with CBRs pro-duces a wide range of effects. The administration of endocannabinoids to experimental animals produces several of the pharmacological and behavioral actions associated with cannabinoids
(Onaivi et al., 1996; Martin et al., 1999; Salzet et al., 2000). Nevertheless, there appears to be cannabinoid-dependent and cannabinoid-independent actions of the endocannabinoids. Evidence for non-CB1, non-CB2, CNR-mediated actions of anandamide in CB1 knockout mouse brain was demonstrated by Di Marzo et al. (2000a). They reported that anandamide levels in the hippocampus and the striatum were lower in the CB1 knockout homozygotes (CB1#/#) than in the wild-type (CB1+/+), whereas there was no change in the 2-AG levels in both the homozygotes and wild-type controls. In addition, the effects of anandamide, unlike !9-THC, were not decreased in CB1#/#
mice. Furthermore, anandamide, but not !9-THC, stimulated GTP)S binding in brain membranes from CB1#/# mice, and this stimulation was insensitive to CB1 and CB2 antagonist. It was, therefore, suggested that non-CB1, non-CB2, G-protein-coupled receptors might mediate some of the behav-ioral actions of anandamide in mice. Thus, exo- and endogenous cannabinoids exert pleiotropic actions in the human brain and body.
Other supporting evidence that some of these effects involve non-CB1 and non-CB2 actions of anandamide is its activation of the capsaicin, the VR1 vanilloid receptor. It is also conceivable that there are other CBRs (e.g., CB3, CB4, CB5…CBn), which are yet to be identied and characterized, as discussed in this chapter. In the absence of such multiplicity of cannabinoid receptor subtypes, perhaps posttranscriptional modication of cannabinoid receptor gene expression yields splice variants that are involved in the myriad behavioral, physiological, and biochemical effects of smoking mari-juana or follow the administration of cannabinoids. Interestingly, there are some pharmacological differences between the plant-derived THC and endocannabinoids that could be due to the pharma-codynamic or pharmacokinetics proles. Some of the similarities between the classical cannabinoids and the endocannabinoids, which are structurally dissimilar, have been reviewed (Salzet et al., 2000).
These include neuromodulatory effects through which they inuence motor behavior, memory and learning, and sensory, autonomic, and neuroendocrine responses. A pathological consequence of marijuana use is the modulation of immune responses, and endocannabinoids are physiological immune regulators (Samson et al., 2003). Endocannabinoids also induce hypotension and bradycardia, inhibit cell growth, affect energy metabolism, and modulate immune and inammatory responses (Onaivi et al., 1996; Martin et al., 1999; Salzet et al., 2000; Sugiura and Waku, 2000).
The pathways for the synthesis and degradation of endocannabinoids have also been studied intensively. The endocannabinoids are unique from other neurotransmitters in that they are not known to be stored in vesicles but, rather, are synthesized on demand. In the nervous and immune systems the endocannabinoids, 2-AG, and anandamide, appear to be derived from the hydrolysis of phospho-lipid precursors from membrane phosphoglycerides. Anandamide can be generated by hydrolysis of N-arachidonoyl phoshatidylethanolamine (N-arachidonoyl PE) catalyzed by phospholipase D (PLD).
An N-acyl transferase activity (NAT) mediates the synthesis of new N-arachidonoyl PE by detaching an arachidonate moiety from the sn-1 position of other phospholipids — such as phosphatidylcholine (PC) — and by transferring it to the primary amino group of PE (Giuffrida and Piomelli, 2000).
The NAT/PLD action is known to give rise to a family of saturated and monounsaturated acyleth-anolamides, such as palmitoylethanolamide and oleylethanolamide (Schmid et al., 1996). The anandamide formed is released into extracellular space where it can activate the CBR. As described below, functional studies show that anandamide is inactivated by a carrier-mediated transport (AT), which can be inhibited by the transport inhibitor AM404. In the cells, anandamide is hydrolyzed into arachidonic acid and ethanolamine by a membrane-bound anandamide amidohydrolase (AAH).
Although anandamide has been extensively studied as a ligand for CBRs, Sugiura and Waku (2000) and co-workers believe that 2-AG, but not anandamide, may be the natural ligand for both the CB1 and the CB2 CBRs. Based on structure activity relationships and the ability of endocan-nabinoids to induce Ca2+ transients in cells expressing CBRs, Sugiura and Waku (2000) surmised that 2-AG appears to be the intrinsic optimum ligand among the compounds tested at the CB1 and CB2 CBRs. The generation of 2-AG from different types of cells under a number of conditions had been known for several years before it was recently established as endocannabinoid ligand
(Sugiura and Waku 2000). Therefore studies of the formation and inactivation of 2-AG indicate that the biosynthetic pathways for 2-AG appear to differ, depending on the types of tissues and cells and the types of stimuli (Sugiura and Waku, 2000; Giuffrida and Piomelli, 2000). 2-AG may be degraded like other monoacylglycerols or like anandamide, perhaps under different conditions.
Unlike the classical neurotransmitters, 2-AG and anandamide may be produced upon demand by receptor-stimulated cleavage of membrane lipid precursors and released from cells after their production, using similar but distinct receptor-dependent pathways, (Piomelli et al., 2000). It can, therefore be suggested that the nonsynaptic release of endocannabinoids and their short lifespans may be one determinant of their functional roles in vivo.
Nuclear lipid signaling is an established, widespread mechanism that operates in multiple cellular processes in response to a variety of stimuli (Gilmore and Mitchell, 2001). Knowledge of the role of endocannabinoids and their precursors in nuclear lipid signaling is scanty. A better understanding of their involvement will shed light on the signicance of the EPCS. The possible involvement of lipid rafts in the action of endocannabinoids, along with the localization of ion channels to lipid raft domains, implies a role for lipid rafts in the biological and physiological effects of endolipids and endocannabinoids that is poorly understood. If receptors and ion channels activated by endocannab-inoids, marijuana use, and other cannabinoids are in these lipid rafts, then Simons and Toomre (2000) suggest that localization and protection of cell signaling will allow these rafts to effectively act as platforms for receptors activated by lipid-signaling molecules like endocannabinoids, endovanilloids, and other endolipids. Although it is attractive to speculate on how cannabinoid receptors will behave if partitioned into lipid rafts, anandamide-induced apoptosis may be independent of cannabinoid or vanilloid receptors, but has been shown to involve lipid rafts (Sarker and Maruyama, 2003). Others have suggested a caveolae-related endocytic mechanism for the cellular uptake of anandamide because the depletion of cholesterol that disrupts caveolae and lipid rafts reduces the uptake of anandamide (McFarland and Barker, 2004). It has also been suggested that methanandamide induces cyclooxygenase-2 expression in human neuroglioma cells via a pathway linked to lipid raft micro-domains (Hinz et al., 2004). The physiological signicance is that this may help to explain the diverse biological processes that have been associated with the effects of marijuana use and possible therapeutic benets. There is also increasing awareness that lipid-signaling molecules and endoge-nous lipid modulators (endolipids) may be involved in a variety of processes associated with substance abuse and dependency. These endogenous “lipid-based” molecules, such as endocannab-inoids, endovanilloids, lysophospholipids, and the acyldopamine and arachidonoylglycine family of lipids, act on ligand-gated channels or GPCRs to regulate a number of peripheral and CNS functions.
Furthermore, these endolipid mediators are involved in a variety of functions such as neuronal migration, neurogenesis, and neuroinammatory effects. We have initiated studies to determine the nature of the interaction between cannabinoids and these endolipid molecules in drug and alcohol addiction.
Endocannabinoids have now been localized in the developing rat brain from early gesta-tional (fetal) stages, suggesting a specic role for these ligands as part of the EPCS in brain development. It was quantitated in peripheral tissues such as spleen and skin, in cells such as peritoneal macrophages and lymphocytes, and in very small amounts in such body uids as serum and cerebrospinal uid, suggesting that it is metabolized in tissues where it is synthesized and the mechanism of action is not hormonal in nature. Anandamide levels in the brain may be equivalent to those of other neurotransmitters such as dopamine and serotonin, whereas less than that of GABA and glutamate. As anandamide has been shown to compete for binding to CB1 in the brain in RIA assays, and as it was found in human and rat spleen, which express high levels of CB2R, it is considered an agonist at both CB1 and CB2 CBrs. There are, however, some reports of vast afnity differences that govern the binding of anandamide to CB1 and CB2, with a much greater Ki for ligand binding to CB1. With binding to the CB1R, anandamide has been shown to inhibit adenylate cyclase in vitro in cell lines where the receptor occurs naturally and in transfected cell lines. In addition, binding of this ligand is capable of inhibiting
N-type calcium channels in cell lines, although to a much lesser extent than synthetic cannab-inoids, suggesting that anandamide displays the properties of a partial cannabinoid CB1 receptor agonist. Reports of the binding of anandamide to CB2R are controversial, and inhibition of adenylate cyclase in CB2R expression in transfected CHO cells has not been observed.
A review of the plausible functions of anandamide will include functional aspects that range from involvement in the immune responses of the body to acting as a potent neuromodulator. It has a role as a developmental regulator during the formation of the brain, as an analgesic, and in neurobehavioral modication. The question immediately arising after the discovery of anandamide was to what extent this fatty acid amide shares common pharmacological properties with the plant-derived !9-THC. In summary, the many reports addressing this question concluded that anandamide shares many of the biological activities exhibited by THC, and the biological effects of anandamide include decreasing spontaneous motor activity, immobility, and production of hypothermia and analgesia. There are pharmacokinetic differences between the two substances. For example, when examining its effects on motor activity, anandamide has a faster onset and a shorter duration of action, most likely due to the rapid reuptake into neurons and astrocytes, and subsequent enzymatic breakdown. Anandamide was shown to be up to 20-fold less potent in producing hypomobility or decreased mobility than !9-THC. It was demonstrated that THC potentiates analgesic effects produced by anandamide but not vice versa (Welch and Eads, 1999). It, therefore, has been suggested that anandamide and THC bind to the same receptor but activate it in distinctively different ways. A future direction for endocannabinoid research will likely include the elucidation of anan-damide transduction mechanisms that may involve activation of Gs and Gi proteins, similar to that reported for the opioid receptors. The analgesic properties of cannabinoids have commanded considerable attention, because this class of analgesics has been shown to interact with other analgesics like opioids. In acute pain models in rodents, anandamide produces analgesia after systemic or local (paw) administration. This data proved that anandamide attenuates pain by interacting with CB1-like receptors located outside the CNS as the CB1 and not CB2 CBR antagonist inhibited the analgesic effects. In addition, it was also shown that after coadministration of both anandamide and palmitylethanolamide, the antinociceptive potency of each compound was increased 100-fold. Palmitylethanolamide is an analgesic agent and a CB1 agonist, which was found to be released together with anandamide from its putative precursor in the neurons of the PNS.
Together, these data indicate that the simultaneous activation of peripheral CB1 and CB2 CBRs results in a synergistic inhibition of peripheral pain transmission, and it has been shown by mass spectrometry that the skin contains 5 to 10 times more anandamide and palmitylethanolamide than in the brain, high enough to cause activation of local cannabinoid receptors.
A number of biological activities by 2-AG have been reported, in immune function, in cell proliferation, embryo development, long-term potentiation (LTP) in the hippocampus, neuropro-tection and neuromodulation, cardiovascular function, and inammatory responses (for a review, see Sugiura and Waku, 2000), most of which have been demonstrated for anandamide. For example, anandamide and 2-AG are known to protect cerebral cortical neurons subjected to 8 h hypoxia and glucose deprivation, through a mechanism independent of CBrs. In an animal model of Parkinson’s disease, the enhanced levels and presence of endocannabinoids in the basal ganglia were associated with movement disorders (Di Marzo et al., 2000b). This is not surprising because there is substantial evidence supporting a role for the cannabinoid system as a modulator of dopaminergic activity in the basal ganglia (for a review, see Giuffrida and Piomelli, 2000). Thus, available data point to a key role of the endogenous cannabinoid system in the regulation of psychomotor activity and suggest that this system may offer a therapeutic target in pathologies involving a dysregulation
A number of biological activities by 2-AG have been reported, in immune function, in cell proliferation, embryo development, long-term potentiation (LTP) in the hippocampus, neuropro-tection and neuromodulation, cardiovascular function, and inammatory responses (for a review, see Sugiura and Waku, 2000), most of which have been demonstrated for anandamide. For example, anandamide and 2-AG are known to protect cerebral cortical neurons subjected to 8 h hypoxia and glucose deprivation, through a mechanism independent of CBrs. In an animal model of Parkinson’s disease, the enhanced levels and presence of endocannabinoids in the basal ganglia were associated with movement disorders (Di Marzo et al., 2000b). This is not surprising because there is substantial evidence supporting a role for the cannabinoid system as a modulator of dopaminergic activity in the basal ganglia (for a review, see Giuffrida and Piomelli, 2000). Thus, available data point to a key role of the endogenous cannabinoid system in the regulation of psychomotor activity and suggest that this system may offer a therapeutic target in pathologies involving a dysregulation