Neurochemical Mechanisms in Disease P49 pps

Endogenous Antinociceptive Ligands 465 and experimental practice divides immunological cytokines into those that enhance cytokine responses, type 1 (IFN-γ,TGF-β etc.), and type 2 (IL-4, IL-10, IL-13, etc.), which favour antibody responses. Cytokines are critical to the development and functioning of both the innate and adaptive immune response, although not limited to just the immune system. There is increasing evidence that a number of cytokines and their receptors are involved in the processes that lead to the development and maintenance of pain states. A diverse range of cytokines and other inflammatory mediators are known to be secreted by activated glia, many of which have been shown to modulate nociception/allodynia ( Scholz and Woolf, 2007). These include the pronociceptive cytokines: IL-1β, IL-12, IL-18, IFNγ,TNF-α and the antinociceptive cytokines: IL-2, IL-4, and IL-10 (Vale et al., 2003; Yao et al., 2002a). IL-2 produces analgesic effect in both CNS and PNS (Jiang et al., 2000b;Yao et al., 2002a). Thus, microinjection of IL-2 in ICV, hippocampus, or LC increases the pain threshold, and the antinociceptive effect was related to the increase of Leu- ENK or SP (Guo and Zhao, 2000; Jiang et al., 2000a; Wu et al., 1999b). IT delivery of IL-2 or IL-2 gene also inhibits nociceptive responses by the activation of opioid receptors or the decrease of SP release in the spinal cord and the reduction of Fos protein in superficial SDH (Guo and Zhao, 2000; Song and Zhao, 2000; Wang et al., 1996; Wu et al., 1999b; Yao et al., 2002b; Yao et al., 2002a). The class of anti-inflammatory cytokines includes IL-4, IL-10, and IL-13 and transforming growth factor-β (Callard et al., 1996; Fiorentino et al., 1991;Hart et al., 1989). These cytokines are produced by several cell types, including T-helper2 lymphocytes, monocytes, macrophages, and mast cells. They are believed to play a role in inhibiting hypersensitivity reactions of macrophage functions, the synthesis of proinflammatory cytokines and the expression of cyclooxygenase-2 (COX2) and inducible nitric oxide synthase (iNOS) (Fernandes et al., 2002). Neither of these cytokines affected the acute pain sensitivity, but they inhibit the inflammatory mechanical hyperalgesia (after systemic administration) (Poole et al., 1995;Vale et al., 2003). This analgesic effect could be related to a peripheral mechanism, prob- ably via the inhibition of the release of the proinflammatory cytokine by resident peritoneal macrophages. Many studies examined the hyperalgesic action of chemokines, but recent evidence has also pointed towards their antinociceptive effects (Rittner et al., 2008). In early inflammation, granulocytes are activated by chemokines, and their activation induces opioid release leading to antinociception. Thus, chemokines may play an important role in the trafficking of opioid-containing cells to injured tissues and in the release of opioid peptides in inflamed tissue. Levels of CX3CR1 (the receptor for the chemokine fractalkine) mRNA, but not in the levels of fractalkine mRNA, in lumbar DRG significantly increase in neuropathic pain models (Holmes et al., 2008). IT or intra-PAG administration of fractalkine to rats produces pain facilitation (Chen et al., 2007; Johnston et al., 2004). The number of CX3CR1- positive macrophages and the expression of CX3CR1 in macrophages are markedly increased in the nerve proximal to the site of the injury, and intraneural injection of fractalkine significantly delays the development of allodynia, whereas CX3CR1 466 G. Horvath knock-out mice display an increase in allodynia (Holmes et al., 2008). Thus, fractalkine may play opposing and site-dependent nociceptive roles, although the summation of the two seems to be inhibitory, at least in mice, on the basis of the increased allodynia in the CX3CR1 knock-out animals. 6 Lipids Lipids are a diverse group of compounds, and they may be divided into eight categories: fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, saccha- rolipids, polyketides, sterol lipids, and prenol lipids. Once viewed primarily as structural constituents of cell membranes and energy storage sources, lipids are now recognized as also serving as valuable signalling functions. As do other chemical transmitters, lipids bind and activate specific protein receptors to produce their bio- logical effects. In the early 1990s, Mechoulam’s group opened the door to a new class of fatty acid derivatives that serve naturally to modulate pain (Devane et al., 1992; Martin et al., 1999). 6.1 Endocannabinoid System and Related Fatty Acid Derivatives Cannabinoids (CBs, e.g.,  9 -tetrahydrocannabinol) are a distinct class of psychoac- tive compounds, which produce a wide array of effects on specific receptors (CB1 . and CB2) (Calignano et al., 1998; Hohmann, 2002; Martin et al., 1999). The endogenous cannabinoids are lipid derivaties (except hemopressin; see Section 5.3.2), and much has ben written about their signalling mechanisms and their role in physiological regulation. A feature that distinguishes endocannabinoids from many other neuromodulators is that they are not synthesized in advance and stored in vesicles. Rather, their precursors exist in cell membranes (lipids) and are cleaved by specific enzymes on demand, and endocannabinoids release generally postsynaptically, and they act presynaptically (Walker and Hohmann, 2005). The endocannabinoid system consists of endogenous cannabinoids, cannabinoid receptors and the enzymes responsible for synthesis and degradation of endocannabinoids. The first endocannabinoid identified was arachidonoyl-ethanolamine (anandamide: AEA), and the second one was 2-arachidonoyl-glycerol (2-AG). The concentration of 2-AG in the brain is 50–500-fold higher than the concentration of AEA (Sugiura et al., 2006). Other putative endogenous ligands of cannabinoid receptors are palmityl- ethanolamide (PEA) and virodhamine (O-arachidonoyl-ethanolamine), a derivative of anandamide (Di Marzo et al., 1998; Porter et al., 2002; Walker et al., 2005). The derivative of 2-AG, 2-arachidonyl-glycerylether or noladin ether was also suggested to be an endocannabinoid (Hanus et al., 2001). A new subgroup contains molecules that consist of a lipid moiety conjugated to an amino acid and have been termed lipoamino acids; they are likely to serve a variety of regulatory functions in the brain and other tissues. (Huang et al., 2001). Several endogenous lipoamino acids were detected in a variety of tissues in the rat, that is, N-arachidonoyl-glycine (NAGly), Endogenous Antinociceptive Ligands 467 N-arachidonoyl-alanine (NAAla), N-arachidonoyl-serine (NASer), N-arachidonoyl- taurine (NaTau), and N-arachidonoyl-GABA (NAGABA) (De Petrocellis et al., 2004; Devane et al., 1992; Huang et al., 2001; Huang et al., 2002; Walker et al., 2005). NAGly has been intensively studied (see Section 6.1.3), but only one report has investigated the antinociceptive potency of NAGABA and NAAla. It has been found that their IT administration does not produce an increase in mechanical and thermal pain threshold in the inflammatory pain model (Succar et al., 2007). N-arachidonoyl-dopamine (NADA), oleamide, N-oleyl-dopamine (OLDA), and N-palmitoyl-glycine (PalGly) are also fatty acid derivates, and they have also been identified as endogenous lipids (Huang et al., 2002). All of these ligands constitute a family of ubiquitous endogenous lipids present in varying levels throughout the body, and several of them produce their effects through modula- tion of CB receptors, whereas other receptor activation/inhibition has also been suggested. Cannabinoid receptors (CB1 and CB2) are among the most abundant GPCRs (Pan et al., 2008). The CB1 receptor is widely distributed in the CNS and PNS; its density is especially high in the brain, and it preferentially presents on axons and their terminals. CB2 receptors are expressed predominantly peripherally, where they are localized extensively to cells of the immune system, but they can be found on the peripheral nerve terminals as well (Guindon and Hohmann, 2007; Szabo, 2008). As regards its expression in the CNS, neural CB2 receptor expression is very low under normal conditions, but it can be induced in nonneuronal cells under pathological conditions (Guindon and Hohmann, 2007; Pan et al., 2008; Szabo, 2008;Van Sickle et al., 2005; Zhang et al., 2003a). Both CB1 and CB2 receptors primarily sig- nal through the inhibitory GPCR proteins (Gi/o), however, under certain conditions and with certain agonists, coupling via Gs and Gq/11 has also been demonstrated (Mackie, 2008; Pertwee, 2001). Stimulation of CB1 receptors leads to the inhibition of AC, the inhibition of certain voltage-gated calcium channels, and the activation of G protein-linked inwardly rectifying potassium channels and these effects are associated with depression of neuronal excitability and transmitter release. The complexity of the actions of CB2 agonists on neuronal and nonneuronal cells and their signalling properties are only beginning to be explored. Activation of CB2 receptors inhibits AC and in contrast to CB1 receptors, CB2 receptors do not cou- ple to ion channels, but both receptors can activate the MAPK signaling cascade (Howlett et al., 2004). Considerable progress has been made in understanding the physiological functions of the endocannabinoids, and their corresponding potential pathological implications. Most of the above-mentioned ligands are now recognized as potent modulators of pain and inflammation (Hohmann, 2002; Hohmann et al., 2005; Pertwee, 2001; Quartilho et al., 2003). Cannabinoids induce antinociceptive effects at several levels, and they can mediate the opioid-independent SIA as well (Hohmann et al., 2005). Recent studies have demonstrated the antinociceptive effi- cacy of cannabinoids in several pain models acting primarily at the central CB receptors (Guindon and Hohmann, 2007; Walker et al., 2005; Walker and Hohmann, 2005). However, several data suggest the antinociceptive potential of peripherally 468 G. Horvath acting cannabinoid agonists (Agarwal et al., 2007; Dogrul et al., 2003; Yesilyurt et al., 2003). Cannabinoids can reduce the production and release of proinflammatory signalling molecules and enhance the release of anti-inflammatory cytokines; moreover, CB2 receptor activation may stimulate the local release of endorphins from cells such as keratinocytes (Ibrahim et al., 2005; Walter and Stella, 2004). Cannabinoids inhibit the release of calcitonin gene-related peptide (CGRP) in iso- lated skin preparations, suggesting that one mechanism by which these drugs may modulate pain is the inhibition of neuropeptide release from peripheral sensory terminals (Ellington et al., 2002). Thus, nonneuronal substrates as well as neuronal substrates may be responsible for the ability of CB2-selective agonists to influence pain sensitivity. The peripheral action may possibly be extremely important, because low doses of these endogenous ligands may reduce pain without disphoric side effects, and without the abused potential typical of centrally acting cannabimimetic drugs. As mentioned above, several of the fatty acid derivates can also interact with other GPCRs and ion channels. Thus, they modulate several types of potassium channels, α7-nAChRs, 5-HT3 receptors, and some orphan receptors (GPR55, GPR92, and GPR18) (Demuth and Molleman, 2006; Kohno et al., 2006; Oh et al., 2008; Pertwee, 2007). The orphan GPR55 is an especially serious candidate to become an additional cannabinoid receptor (Pertwee, 2007). As these ligands are lipophilic, they may partition into the cell membrane, where they may reach high local concentrations and thereby influence the actions of membrane proteins via so-called “receptor-independent” mechanisms (Oz, 2006). The best known and characterized ion channel interaction is the activation of TRPV1 channels. TRPV1 is a ligand-gated nonselective cation channel that is considered to be an important integrator of various pain stimuli such as capsaicin, heat, and low pH. Several endogenous lipids represent “chimeric” ligands (AEA, OLDA, and NADA) acting on both cannabinoid and TRPV1 receptors (Starowicz et al., 2008). Because CBs and TRPV1 receptors show coexpression in brain neurons, their coactivations can lead to a cross-talk between them. The role of peripheral TRPV1 receptor in pain has been the subject of several detailed studies (Jancso and Jancso-Gabor, 1980; Nagy et al., 2004; Starowicz et al., 2008; Szolcsanyi, 2000; Szolcsanyi, 2004), and its crucial role in nociception and hyperalgesia has been confirmed in the TRPV1 knock-out mice as well, in which impaired nociception and reduced sensitivity to painful heat in behavioral tests have been reported (Barton et al., 2006; Bolcskei et al., 2005; Caterina et al., 2000;Davis et al., 2000). The TRPV1 receptor activation at spinal level by capsaicin or AEA causes temporary painful behavior and a prolonged antinociception (Di Marzo et al., 2000a; Horvath et al., 2008b; Yaksh et al., 1979). However, TRPV1 antagonists effectively reduce thermal hyperalgesia and mechanical allodynia through both spinal and peripheral mechanisms (Cui et al., 2006). The expression of TRPV1 in supraspinal structures such as PAG, RVM, the LC, and thalamus suggests its involvement in descending and ascending supraspinal pain processing (Cristino et al., 2006; Maione et al., 2006; Mezey et al., 2000;Starowicz et al., 2008). Microinjection of capsaicin into the PAG increases the latency to Endogenous Antinociceptive Ligands 469 thermal nociceptive responses, an effect blocked by NMDA and metabotropic glutamate receptor antagonists (Palazzo et al., 2002; Starowicz et al., 2008). These data suggest that TRPV1 activation in the PAG increases glutamate release, and this leads to activation of postsynaptic glutamate receptors. However, the response to intra- PAG injected capsaicin depends on the location of the injection. Its injection into the dorsolateral-PAG decreases the pain threshold, whereas the capsaicin administration into the ventrolateral-PAG produces antinociception by the increased glutamate release in t he RVM, which leads to enhanced activity of antinociceptive OFF cells, and decreased firing of pronociceptive ON cells. Furthermore, capsaicin-induced excitation of LC neurons might also be involved, in part, in its analgesic properties (Hajos et al., 1987). TRPV1 is located presynaptically on afferents to the LC, and its activation may serve to potentiate the release of glutamate and norepinephrine in this brain region (Marinelli et al., 2002). Nociceptive neurons of the medial thalamus also respond to capsaicin, in agreement with the high density of TRPV1 in this area (Cristino et al., 2006). TRPV1 activation evokes glutamate release from the hypothalamus and cerebral cortex as well (Sasamura and Kuraishi, 1999). Both regions send their projections to PAG (Millan, 2002), and these data also suggest a mechanism by which TRPV1 activation may modulate neuronal activity in these central areas. 6.1.1 N-Arachidonoyl-Ethanolamine (Anandamide; AEA) Anandamide, the first identified and best-studied endocannabinoid, can be found both centrally and peripherally (Calignano et al., 1998; Devane et al., 1992;Walker et al., 2005). It is principally formed from glycerophospholipid by two succes- sive enzymatic reactions: N-acylation of phosphatidyl-ethanolamine to generate N-acylphosphatidyl-ethanolamine (NAPE) by Ca 2+ -dependent N-acyltransferase, and release of AEA from NAPE by a phosphodiesterase of the PLD type (NAPE- PLD) (Okamoto et al., 2007). It has been hypothesized that AEA could be recycled by the cell to form new endocannabinoid molecules and released into the extra- cellular space (Placzek et al., 2008). AEA is extremely short-lived, being rapidly inactivated by the enzymes fatty acid amide hydrolase (FAAH) (Cravatt et al., 1996). Termination of AEA signalling appears to involve a two-step process that begins with transport across the plasma membrane, followed by enzymatic hydrolysis into arachidonic acid and ethanolamine. AEA binds to both CB1 and CB2 receptors, behaving as a partial agonist, but it also activates TRPV1 receptors, and it has CB receptor-independent G protein-coupled antinociceptive potency through the activation of the GPR55 (Di Marzo et al., 2000b; Pertwee, 2007; Ryberg et al., 2007). Furthermore, AEA may directly affect the GlyRs and functionally antagonizes the transient receptor potential melastatin 8 (TRPM8) receptor-mediated responses (De Petrocellis et al., 2007; Hejazi et al., 2006; Lozovaya et al., 2005). Furthermore, AEA targets potassium channels, T-type calcium channels, and gap junctions. It is a substrate for COX2 giving rise to amino acid conjugates of the prostaglandins, and induces the expression of COX2 enzyme as well (Chemin et al., 2001; Chen et al., 2005a; Maingret et al., 2001). 470 G. Horvath Only a few studies have investigated the antinociceptive potency of AEA in vivo, and it seems that the CB1 receptor is the predominant target mediating anandamide’s antinociceptive effect (Wise et al., 2007). Systemic administration of AEA is able to attenuate the visceral hyperreflexia induced by inflammation of the urinary bladder and to reduce the second phase response to formalin (Jaggar et al., 1998). FAAH inhibitor significantly increases the potency of anandamide in a mild thermal injury model (Palmer et al., 2008). A recent study has shown that AEA (IV) produces a cannabinoid receptor-independent antinociception, and its effects were inhibited by 5-HT3 receptor antagonist suggesting that the activation of these receptors contributes to the anandamide-induced analgesia (Racz et al., 2008). The ICV administration of AEA also induces dose-related antinociception in the TF-test, and this effect is reduced by pertussin toxin, but not cholera toxin (Calignano et al., 1998; Raffa et al., 1999). Furthermore, the inhibition of FAAH at PAG level also decreases pain sensitivity which was reversed by CB1 and TRPV1 antagonist (De Novellis et al., 2008; Maione et al., 2006; Suplita et al., 2005). AEA is a potent short- lasting antinociceptive ligand at the spinal level in acute and inflammatory pain models (Horvath et al., 2008b; Smith et al., 1994; Welch et al., 1998). The effect of AEA is inhbited by CB1 antagonist, but also by TRPV1 antagonist (Horvath et al., 2008b; Welch et al., 1998). Local administration of anandamide significantly decreases the formalin-induced pain behavior but not the paw edema (Calignano et al., 1998; Guindon et al., 2006a,b). Furthermore, it also inhibits the TRPV1 receptor activation-induced drop in HP latency by activation CB1 receptors (Almasi et al., 2008). Thus, anandamide may activate cannabinoid CB1 receptors located on capsaicin-sensitive primary afferents, resulting in the decreased responsiveness of these afferents to noxious stimuli. However, others have shown that locally administered anandamide activates nociceptors in normal and arthritic rat by stimulating TRPV1 receptors on primary sensory neurons, suggesting a pain-inducing potential of anandamide at this level (Gauldie et al., 2001). 6.1.2 2-Arachidonoyl-Glycerol (2-AG) 2-AG is a 2-acyl-glycerol ester, and its concentration in the brain is 50–500-fold higher than the concentration of anandamide (Mackie, 2008; Sugiura et al., 2006). 2-AG is also short-lived, being rapidly inactivated mainly by the enzyme monoglyc- eride lipase (MAGL), but it might also be metabolized by FAAH (Bisogno, 2008; Cravatt et al., 1996; Dinh et al., 2002; Saario and Laitinen, 2007; Sugiura et al., 2006). It is a full agonist for CB1 and CB2 receptors with no direct binding to the TRPV1 receptor (Mechoulam et al., 1995; Pertwee, 2001; Sugiura et al., 2006). It is also a substrate for COX2, and 2-AG is capable of suppressing elevation of COX2 expression by activating the CB1 receptors (Bleakman et al., 2006; Zhang and Chen, 2008). As regards its antinociceptive potency, only a few data are available in this respect; systemic administration of 2-AG has produced antinociception in TF test, and its in vivo potency is similar to anandamide (Mechoulam et al., 1995). 2-AG (IP) does not decrease hyperalgesia after mild thermal injury, but it is effective if it Endogenous Antinociceptive Ligands 471 is administered together with a FAAH inhibitor (Palmer et al., 2008). Furthermore, the coadministration of 2-AG (in an ineffective dose IP) with two other endogenous lipids (2-linoleoyl-glycerol and 2-palmitoyl-glycerol) increases the potency of 2-AG, although these lipids are ineffective by themselves (Ben Shabat et al., 1998). This entourage effect might be due to the decreased inactivation of 2-AG. MAGL inhibitor has induced a CB1-mediated enhancement in endocannabinoid-mediated SIA following local administration into either the PAG or SDH (Hohmann et al., 2005; Hohmann, 2007; Suplita II et al., 2006; Suplita et al., 2005). This effect is associated with a profound increase in levels of 2-AG, but not anandamide, suggesting a physiological role for 2-AG in the suppression of pain sensitivity. Topical (IPL) administration of 2-AG and MAGL inhibitor decreased the pain behavior in the late phase of the formalin test and produced antihyperalgesic and antiallodynic effects in a neuropathy pain model (Desroches et al., 2008; Guindon et al., 2007; Hohmann, 2007). Moreover, the antinociceptive effects of 2-AG are prevented by a selective CB2 receptor antagonist, but not by a CB1 receptor antagonist in the formalin test, whereas both antagonists inhibit the antiallodynic and antihyperalgesic effects of 2- AG (Desroches et al., 2008; Guindon et al., 2007). However, local administration of CB1 and CB2 antagonists by themselves failed to induce hyperalgesia, suggesting that the endocannabinoids do not act tonically in the periphery to dampen senstivitiy to pain (Guindon et al., 2007). 6.1.3 N-Arachidonoyl-Glycine (NAGly) N-Arachidonoyl-glycine (NAGly) was first synthesized as a structural analogue of the AEA. It is expressed within the CNS, with particularly high levels within the spinal cord, but it can be detected in the skin as well (Burstein, 1999; Huang et al., 2001; Rimmerman et al., 2008). NAGly is formed via oxidation of AEA and by conjugation of glycine with arachidonic acid by arachidonyl-CoA, and being rapidly inactivated by FAAH (Burstein, 1999; Huang et al., 2001). The pharmacology of NAGly is still poorly understood, however, several targets for NAGly are emerging. NAGly has no affinity for the CB1 and TRPV1 receptors, although it can activate CB2 binding sites (Devane et al., 1992; Huang et al., 2001; Sipe et al., 2005). It is also a substrate for COX2 giving rise to amino acid conjugates of the prostaglandins, and it inhibits activation of COX2 and 5-lipoxygenase enzymes (Burstein, 1999; Prusakiewicz et al., 2002). Thus, it has a complex effect on prostaglandin synthesis, and a role for COX2 cannot be excluded in its antinociceptive effect (Burstein et al., 2007). Furthermore, NAGly inhibits FAAH, and it is a substrate for this enzyme, therefore, NAGly can regulate the levels of AEA in tissues (Grazia Cascio et al., 2004; Huang et al., 2001). NAGly is a ligand for the orphan receptors GPR18, and it activates this receptor in a pertussis t oxin-sensitive manner (Kohno et al., 2006). NAGly has also been shown to stimulate another orphan receptor GPR92, which is highly expressed in DRG and colocalized with TRPV1 receptors and has been postulated to play a role in sensory perception ( Oh et al., 2008). Alternatively, the coexpression of GPR92 and TRPV1 in the DRG raises the possibility that NAGly can exert its pain suppressive effects through GPR92 in the sensory nervous system. 472 G. Horvath In addition, NAGly inhibits the glycine transporter GLYT2, but it can also influence the GlyRs, thus GlyRs could also mediate some of the analgesic effects of NAGly (Wiles et al., 2006; Yang et al., 2009). As regards its effects after systemic (SC, IP, or oral) administration, the results are inconsistent. Thus, NAGly produces analgesia administered in acute pain models including the HP and formalin tests and has anti-inflammatory activity (Burstein et al., 2007; Huang et al., 2001). However, systemic administration of NAGly, at a dose similar to that used IT, is without effect, and NAGly does not produce antinociception in mild thermal injury model (Palmer et al., 2008; Vuong et al., 2008). IT administration of NAGly reduces the mechanical allodynia and thermal hyperalgesia, and its effect is not influenced by CB1 and CB2 antagonists (Succar et al., 2007; Vuong et al., 2008). In addition, NAGly does not produce the motor side effects associated with exogenous cannabinoid receptor agonists. Consistent with its high levels in skin, NAGly produces analgesia administered peripherally in HP and formalin tests, and it also has anti-inflammatory activity (Burstein et al., 2007; Huang et al., 2001). 6.1.4 2-Arachidonyl-Glycerylether (Noladin Ether) Mechoulam’s laboratory in 2001 has identified noladin ether, the derivative of 2-AG, from porcine brain (Hanus et al., 2001). This compound is more stable compared to 2-AG and anandamide. It has higher affinity to CB1 than CB2 receptors (Hanus et al., 2001), but another study has shown that it has high affinity for CB2 receptors as well, and it is a full agonist at this receptor (Shoemaker et al., 2005). Newer studies have investigated the activity of noladin ether on TRPV1 receptors, and it has been shown that its effects are not connected with this receptor (Duncan et al., 2004). Noladin ether inhibits the CGRP-induced vasorelaxation, and this effect is unaffected by both CB1 and CB2 antagonists, but is inhibited by pertussis toxin (Duncan et al., 2004). These results suggest that noladin ether produces its effects through non-CB1/CB2 GPCR activation, but noladin ether can decrease the MOR expression by acting on CB2 receptors (Paldyova et al., 2008). Only one study has investigated its antinociceptive potency after systemic (IP) administration and it has been shown that noladin ether produced antinociception on HP test in mice (Hanus et al., 2001). 6.1.5 N-Arachidonoyl-Dopamine (NADA) NADA was first synthesized, and then identified in the brain at the beginning of this century (Bisogno et al., 2000; Huang et al., 2002; Yang et al., 2007a). It is synthesized through a condensation reaction between arachidonic acid and dopamine or between arachidonic acid and tyrosine, and then converted to NADA (Huang et al., 2002). NADA can be inactivated by conversion into the less active 3-O-methyl-NADA by catechol O-methyltransferase, and it is slowly hydrolysed by FAAH as well (Huang et al., 2002). As regards its action mechanism, NADA can activate either TRPV1 or CB1 receptors depending on the location and cir- cumstance (Bisogno et al., 2000; Bisogno, 2008; De Petrocellis et al., 2000; Huang et al., 2002; Mackie, 2008). Thus, NADA activates DRG neurons, and increases the Endogenous Antinociceptive Ligands 473 intracellular calcium concentration and the CGRP release by activation of TRPV1 receptors, but it has lower potency than capsaicin (Huang et al., 2002; McDonald et al., 2008; Medvedeva et al., 2008). NADA also functionally antagonizes the TRPM8-mediated responses (De Petrocellis et al., 2007). It elicits analgesia following systemic administration in acute heat pain test (Bisogno et al., 2000). NADA produced slight allodynia after IT administration, and a dose-dependent antihyperalgesic effect was also observed; this effect was inhibited both by CB1 and TRPV1 antagonists (Horvath et al., 2008a; Pitcher et al., 2007). In addition, it causes nocifensive behavior and hyperalgesia when administered peripherally (Huang et al., 2002; Price et al., 2004). 6.1.6 N-Palmitoyl-Ethanolamide (PEA) and N-Palmitoyl-Glycine (PalGly) Although the subfamily of arachidonoyl amides has received considerable attention, much less is known about the presence and activity of their saturated counterparts (Rimmerman et al., 2008). The most studied member of the saturated acyl amides is N-palmitoyl-ethanolamide (PEA) (Di Marzo et al., 1998; Walker et al., 2005). PEA, found in neural and nonneural tissues, inhibits mast-cell activation and reduces inflammatory responses by a mechanism that may involve binding to CB2 receptors (Calignano et al., 1998; Martin et al., 1999). However, because PEA does not produce an effective activation of cannabinoid receptors, it is generally classified as a cannabimemic compound. Furthermore, PEA is an agonist at the peroxisome proliferator-activated receptor α (PPARα), and at the orphan receptor GPR55 (Lo Verme et al., 2005; Ryberg et al., 2007). An “entourage” effect on anandamide- mediated action may be due to the PEA-induced inhibition of FAAH that leads to an increase of tissue levels of AEA (Costa et al., 2008). Thus, a recent study demonstrated that CB1, PPARα, and TRPV1 receptors mediate the antinociception induced by systemic PEA in the neuropathic pain model, and repeated PEA treatment significantly decreased the enhanced NGF, GDNF, and NT-3 levels in the spinal cord (Costa et al., 2008). Orally administered PEA also reduced inflammatory hyperalgesia and edema by inhibiting mast cell degranulation (Mazzari et al., 1996). It can also attenuate the visceral pain sensitivity and the second phase response to formalin (Jaggar et al., 1998; Lo Verme et al., 2006). Its ICV administration was ineffective in acute heat pain test, whereas it showed marked antinociceptive properties at the peripheral level (Calignano et al., 1998; Calignano et al., 2001;Lo Verme et al., 2006). Local administration of PEA produced antinociception in the formalin test that was blocked by a CB2 receptor-selective antagonist (Calignano et al., 1998). Another study has proved the role of PPARα receptor activation in this respect (Lo Verme et al., 2006). It is noteworthy that local coadministration of PEA with exogenous anandamide produced a synergistic analgesic effect in both phases of the formalin test through a mechanism that involves both CB1 and CB2 receptor subtypes (Calignano et al., 1998; Calignano et al., 2001). PalGly also has been identified in rat brain, skin, and spinal cord (Rimmerman et al., 2008). It induces transient calcium influx in native adult DRG cells, and 474 G. Horvath stimulates the NO production. PalGly potently inhibits heat-evoked firing of nociceptive neurons in rat SDH, and its effects are not inhibited by CB1 and CB2 antagonists, but are blocked by TRP channel antagonists. No in vivo studies have been performed in this respect yet. 6.1.7 N-Oleoyl-Ethanolamide (OEA), N-Oleoyl-Dopamine (OLDA), and Oleamide All these substances are derivatives of oleic acid (monounsaturated omega-9 fatty acid). N-oleoyl-ethanolamide (OEA) is an endogenous regulator of food intake, and may have some potential as an antiobesity drug, however, some studies investigated its effects on sensory neurons as well (Fu et al., 2003; Hansen and Artmann, 2008). It does not bind to CB1 and CB2 receptors, but it is an endogenous agonist of TRPV1 and PPARα, however, there are some contradictions in this respect (Ahern, 2003; Almasi et al., 2008; Fu et al., 2003; Lo Verme et al., 2006; Wang et al., 2005a). Only a few inconsistent results suggest its role in the pain. Thus, IP administration of OEA has decreased the pain behavior in formalin and visceral models, and this effect was independent from PPARα activation, although high dose causes writhing behavior (Suardiaz et al., 2007; Wang et al., 2005a). Its IPL administration does not change the acute heat-pain latency, but reverses the thermal hyperalgesia (Almasi et al., 2008). Another study has found nocifensive behavior after its local injection, which could not be observed in TRPV1 knock-out animals (Lo Verme et al., 2006). The endogenous presence of OLDA has recently been confirmed i n the mam- malian brain (Chu et al., 2003; Huang et al., 2002). The in vivo pathways of OLDA synthesis are unsettled. The most probable pathway seems to be N-acylation of tyrosine by a fatty acid, with tyrosine entering then the normal pathway of dopamine synthesis to form N-acyl-dopamine, and it is inactivated by FAAH (Chu et al., 2003). However, it can weakly activate CB1 receptors (Bisogno et al., 2000; Chu et al., 2003). OLDA possesses activity at TRPV1 receptors with potency similar to that of capsaicin, and produces long-lasting nocifensive behavior and thermal hyperalgesia, which is blocked by TRPV1 antagonists (Chu et al., 2003; Szolcsanyi et al., 2004; Walker et al., 2005). Therefore, OLDA may function as either a peripheral or central mediator of TRPV1 activation. Oleamide (cis-9,10-octadecenoamide or oleic acid amide) originally was found in the cerebrospinal fluid (CSF) of sleep-deprived cats, and has received much attention due to its sleep-inducing properties in mammals (Cravatt et al., 1995;Farrell and Merkler, 2008). The primary site of action of oleamide in the central nervous system remains unclear. It does not interact directly with CB1 receptors, but it interacts with other neurotransmitter–receptor systems (GABAergic, dopamin- ergic, and serotonergic transmission) (Boring et al., 1996; Walker et al., 2005). Many of oleamide’s behavioral effects are consistent with its being an indirect cannabimimetic, increasing either the levels or activity of endogenous cannabinoids (e.g., AEA) (Fedorova et al., 2001). The mechanism by which this occurs remains unclear and may include the suppression of AEA uptake, although it also shares with AEA the same degradatory enzyme, FAAH (Mechoulam et al., 1997). Hence . the chemokine fractalkine) mRNA, but not in the levels of fractalkine mRNA, in lumbar DRG significantly increase in neuropathic pain models (Holmes et al., 2008). IT or intra-PAG administration. administration, the results are inconsistent. Thus, NAGly produces analgesia administered in acute pain models including the HP and formalin tests and has anti -in ammatory activity (Burstein. supraspinal pain processing (Cristino et al., 2006; Maione et al., 2006; Mezey et al., 2000;Starowicz et al., 2008). Microinjection of capsaicin into the PAG increases the latency to Endogenous Antinociceptive