Endogenous Antinociceptive Ligands 445 NRM, or amygdala generates antinociception in the acute pain test (Reny-Palasse et al., 1989; Webster et al., 1983). ICV administration of TRH has induced analgesic effect with similar or higher potency than morphine in mechanical but not thermal pain tests (Boschi et al., 1983). Its antinociceptive effects are mediated via activa- tion of both descending monoaminergic and serotoninergic pathways (Tanabe et al., 2007), and others have found that TRH activity was resistant to modifications of NE, dopamine, and 5-HT systems (Boschi et al., 1983). The TRH effect was not antag- onized by naloxone, but TRH at a nonanalgesic dose prevented the hyperalgesia induced by naloxone (Boschi et al., 1983). 5.1.9 Somatostatin (SST) Somatostatin (in 14 and 28 amino-acid-containing forms) was originally described as a hypothalamic polypeptide that inhibits the secretion of pituitary growth hor- mone. It exerts a wide range of effects such as modulation of hormone and neurotransmitter release, cognitive and behavioral processes, the gastrointestinal tract, the cardiovascular system and tumour cell proliferation, but it also has an important neuromodulator function (Gamse et al., 1981; Pan et al., 2008; Pinter et al., 2006). SST is synthesised and stored in capsaicin-sensitive transient recep- tor potential vanilloid 1 (TRPV1) receptor expressing nociceptive afferents, but it also has been identified in SDH neurons (Pan et al., 2008; Willis, 1988). The effects of SST are mediated via five different GPCR subtypes which can be divided into two main groups on the basis of their sequence similarities and their binding pro- file towards synthetic somatostatin analogues. SST2, 3, and 5 mediate the endocrine and antiproliferative effects of SST, whereas SST1 and 4 may be responsible for the anti-inflammatory and antinociceptive actions (Helyes et al., 2000; Pinter et al., 2006; Sandor et al., 2006). Exogenously administered SST has been shown to inhibit neurogenic inflammation and nociception in several experimental assessments, and it is effective in the treatment of patients with certain pain conditions, including dif- ferent types of headaches (Pan et al., 2008; Yu et al., 2004). Central administration of SST (ICV or into the caudate nucleus) increases the pain threshold, suggesting an antinociceptive role of SST at the supraspinal level (Tashev et al., 2001; Zheng and Li, 1995). IT injection of SST has failed to influence TF latency at low doses, how- ever, higher doses have caused motor impairments (Cridland and Henry, 1988a). Much progress has been made in the past ten years in the understanding of the important roles of SST in the regulation of pain transmission at the peripheral level (Helyes et al., 2000; Sandor et al., 2006). Somatostatin released from the activated capsaicin-sensitive sensory nerve terminals reaches the circulation, and it is able to elicit systemic anti-inflammatory and antinociceptive actions. This endogenous counterregulatory mechanism of neurally derived somatostatin has been termed as its “sensocrine function” (Szolcsanyi, 2004; Than et al., 2000). 5.1.10 Prolactin Prolactin is a polypeptide hormone (199 amino acids) whose major biological actions are related to normal lactation and reproduction. After hormone binding, signal transduction occurs via the cytokine receptor superfamily. It is well known 446 G. Horvath that prolactin level increases during painful stimuli, but only a few data suggest its antinociceptive role. Thus it has been shown that systemic administration of prolactin induced antinociception in visceral pain model, and it can contribute to postictal antinociception as well (Portugal-Santana et al., 2004; Ramaswamy et al., 1985). No other data are available in this context. 5.1.11 Ghrelin Ghrelin (28 amino acids), a gastric-derived hormone, was discovered as a ligand for the growth hormone secretagogue receptor (GHSR). It has gained increasing attention as a brain–gut hormone with GH-releasing and appetite-inducing func- tions (Arora and Anubhuti, 2006). GHSR is a GPCR and two isoforms (type 1a and 1b) have been detected (Kojima and Kangawa, 2005). Recent studies have reported that, in addition to the stomach, ghrelin and its receptors are expressed in vari- ous peripheral tissues and in the brain, including the pituitary, hypothalamus, pons medulla, oblongata, and SDH, the regions implicated in the control of pain trans- mission (Kojima and Kangawa, 2005; Vergnano et al., 2008). It is very important in this context that ghrelin specifically inhibits the expression of the proinflam- matory cytokines; therefore it may attenuate proinflammatory cytokine-mediated neuropathic pain (Dixit et al., 2004; Guneli et al., 2007;Lietal.,2004). IP and ICV administration of ghrelin reduced the inflammatory hyperalgesia and edema in a naloxone-reversible manner (Sibilia et al., 2006). Ghrelin is able to stimulate the neural activity in the hypothalamic ARC, where it increases the endogenous opi- oid synthesis and/or activity (Sibilia et al., 2006). Ghrelin increased the inhibitory postsynaptic currents and prevented the capsaicin-induced increase of Fos-LI in the deep SDH (Vergnano et al., 2008). These data suggest that the effect of the ghrelin is mainly due to an action potential-dependent presynaptic release of inhibitory neu- rotransmitters, and it may be tonically active in the spinal cord. Ghrelin promotes neuronal release of neuropeptide Y, which is another antinociceptive ligand (see below Section 5.2.8; Cowley et al., 2003). IPL administration of ghrelin increased the inflammatory pain threshold, suggesting a peripheral role of this ligand, too (Sibilia et al., 2006). 5.1.12 Orexins Orexins, also called hypocretins, are the common names given to a pair of highly excitatory neuropeptide hormones that were discovered in rat brain, and they are implicated in body mass regulation (Arora and Anubhuti, 2006; Trivedi et al., 1998). The two related peptides (orexin-A: 33 and orexin-B: 28 amino acids), with approx- imately 50% sequence identity, are produced by cleavage of a single precursor protein. Orexin-A has two intrachain disulfide bonds and has greater biological importance, and orexin-B is a linear peptide (Smart, 1999). Although these pep- tides are produced by a very small population of cells in the lateral and posterior hypothalamus, they send projections throughout the brain and to the spinal cord (Marcus et al., 2001; Van Den Pol, 1999; Yamamoto et al., 2002a). The orexin peptides bind to the GPCR orexin receptors (OX-1 and OX-2), which are widely Endogenous Antinociceptive Ligands 447 distributed in the CNS (Kukkonen et al., 2002; Trivedi et al., 1998). The deficiency in orexin induced an increased hyperalgesia and less SIA (Watanabe et al., 2004), and ICV administration of orexin was effective on acute and neuropathic pain tests (Mobarakeh et al., 2005; Yamamoto et al., 2003). At the spinal level orexin was also effective in different pain models (visceral, formalin, postoperative, and neuro- pathic), and the inhibition of the NMDA receptors might play a significant role in this process (Cheng et al., 2003; Kajiyama et al., 2005; Peng et al., 2008; Yamamoto et al., 2002a; Yamamoto et al., 2003). Its peripheral administration inhibits the neu- ronal vasodilation and this effect can also contribute to a decreased pain sensitivity (Holland et al., 2005). 5.1.13 Bombesin-Related Peptides Bombesin (BN; amidated tetradecapeptide) was isolated from frog skin. Subsequently, in mammals two BN-like peptides were identified: gastrin-releasing peptide (GRP; 27 amino acids) and neuromedin B (NMB; 10 amino acids) (Jensen et al., 2008). On the basis of the preceding molecular studies, three classes of mammalian bombesin receptors (BB1-3, GPCRs acting primarily through PLC system) were proposed. The BB1 is an NMB-preferring receptor, the BB2 is a GRP-preferring receptor, and the BB3 has low affinity for these peptides. Studies of GRP and NMB immunoreactivity as well as mRNA studies have demonstrated that these peptides and their receptors are widely distributed in mammals in both the nervous system and peripheral tissues, especially in the gastrointestinal tract (Jensen et al., 2008). Only a few studies suggest the role of these peptides in the nociception. GRP or BB1 receptor-deficient mice did not show any impairment in pain threshold (Sun and Chen, 2007; Yamada et al., 2003), but intra-PAG injection of bombesin produced antinociception in the HP and TF tests (Pert et al., 1980;Yu et al., 2004). Furthermore, the IT administration of bombesin and neuromedin B produced nocifensive behavior (Cridland and Henry, 1992). 5.2 Neuropeptides 5.2.1 Opioid-Related Peptides Morphine, the main alkaloid of opium, is utilized for the treatment of severe pain, and is the gold standard to which all analgesics are compared. Early efforts to under- stand the endogenous targets of opiate drugs led to the identification of receptor sites. Binding studies suggested four main classes of opioid receptors, named μ- (MOR), δ- (DOR), κ- (KOR), and opioid receptor-like ( ORL1) receptors. Opioid receptors comprise a subfamily of structurally homologous GPCRs. Activation of these receptors inhibits the formation of cAMP, close voltage-gated Ca 2+ -channels and opens inwardly rectifying potassium channels (Dhawan et al., 1996; Jordan et al., 2000; Lambert, 2008). The net effect of these cellular actions is to reduce neuronal excitability and neurotransmitter release. Opioid receptors and their endogenous ligands are widely distributed in the organism, thus both central and peripheral activation of this system might lead to 448 G. Horvath effective antinociception (Akil et al., 1984; Basbaum and Fields, 1984; Bodnar, 2008; Menetrey and Basbaum, 1987; Palkovits, 2000; Pan et al., 2008). A high dose of naloxone (opioid antagonist) produces hyperalgesia, suggesting a significant role of endogenously released opioids in the development of normal pain sensitivity (Boschi et al., 1983). For example, the distribution of the endomorphins (EMs) along the nociceptive pathway implicates them as particularly important for the modu- lation of pain (Horvath, 2000). Thus, the EMs have been found unequally in the brain; they are stored in neurons and axon terminals with heterogenous distribution and they are released from synaptosomes by depolarization (Horvath, 2000; Zadina et al., 1997). Nociceptin is also widely distributed in central structures involved in sensory, emotional, and cognitive processing, and in the periphery including the immune cells (Lambert, 2008; Reinscheid et al., 2000). Furthermore, nocistatin (the other opioid-related peptide) is also present in the brain and the spinal cord (Boom et al., 1999; Lee et al., 1999; Okuda-Ashitaka et al., 1998), and its distribu- tion appears to be almost identical to that of nociceptin (Okuda-Ashitaka and Ito, 2000). As regards the actions of opioids at the supraspinal level, several centers are involved in this process. Some of the analgesic actions of opioids may be due to modulation of the descending pathways to reduce nociceptive transmission in the spinal dorsal horn (Anderson et al., 1977; Basbaum and Fields, 1984). Thus, spinally projecting RVM neurons expressing opioid receptors can mediate the opioid analgesia triggered from the PAG (Anderson et al., 1977; Basbaum and Fields, 1984; Fields and Basbaum, 1999; Millan, 2002), and microinjection of MOR agonists into the RVM elicits analgesia because opioids can reduce synap- tic GABA release to spinally projecting neurons (Connor et al., 1999; Fields et al., 1991; Fields and Basbaum, 1999; Hurley et al., 2003). In addition, through presynaptic inhibition of GABA release, activation of opioid receptors may disin- hibit spinally projecting noradrenergic neurons in the LC (Pan et al., 2002). It is well known that opioids reduce the sensory discriminative and affective compo- nent of pain as well. Thus, microinjection of morphine into the ACC decreases the affective component of pain processing, and activation of presynaptic MOR attenuates GABAergic synaptic input in the amygdala (Finnegan et al., 2005; Finnegan et al., 2006; LaGraize et al., 2006). Furthermore, both the prefrontal cortex and thalamic nuclei are involved in the actions of opioids (Zhao et al., 2007). It is well known that opioids produce very effective antinociception at spinal levels as well. Opioid receptors in the DRG of sensory neurons undergo axonal transport to reach peripheral nerve terminals, and inflammation induces increases in MOR binding within DRG leading to an improved antinociceptive potency in these circumstances (Endres-Becker et al., 2007; Mousa et al., 2007; Zollner et al., 2003). The endogenous opioid ligands can induce antinociception at peripheral levels as well. During inflammation of the peripheral tissues leukocytes are the important source of the endogenous opioid peptides, and β-endorphin, Met-ENK, dynorphins, and endomorphins are produced and released by these cells (Labuz et al., 2006; Mousa et al., 2002; Rittner et al., 2008). Endogenous Antinociceptive Ligands 449 β-Endorphin Since the discovery and characterization of β-endorphin (31 amino acids) as an opi- oid peptide in 1976, the opinion has been widely held that this peptide has a role in the control of pain (Akil et al., 1984; Basbaum and Fields, 1984; Loh et al., 1976; Rossier et al., 1977). POMC-derived β-endorphin is considered to be a key component of the endogenous antinociceptive system attenuating the stress- and inflammation-induced hyperalgesia (Rossier et al., 1977; Stein et al., 1990; Sun et al., 2003). It binds with high affinity to both MOR and DOR (Akil et al., 1984). Pain stimulation induces PAG release of β-endorphin and the ICV administration of β-endorphin produces analgesia (Akil et al., 1984). Similarly, both spinal and peripheral administration of β-endorphin evokes antinociceptive effects in different pain models (Chung et al., 1994; Stein et al., 1990; Suh et al., 1994; Suh et al., 1996). Leu-enkephalin and Met-enkephalin (Leu-ENK, Met-ENK) Methionine-enkephalin (Tyr-Gly-Gly-Phe-Met) and leucine-enkephalin (Tyr-Gly- Gly-Phe-Leu) were isolated and characterized as the first endogenous peptidic ligands for DOR receptors (Hughes et al., 1975). ENKs, synthesizing from pre- proenkephalin, possess antinociceptive activity at both spinal and supraspinal levels (Lee et al., 1980; Maldonado et al., 1994; Takemori and Portoghese, 1993; Yu et al., 2004). For example, pain stimulation induces PAG release of Leu-ENK and Met- ENK, and their antinociceptive effects are mediated in the brain through interactions mainly at DOR1 (Yang et al., 2006). Leu-ENK inhibited the nociceptin-induced allodynia in a dose-dependent manner at the spinal level (Honda et al., 2001). In the spinal cord, the ENKs interact with DOR2 receptors, and the release of ENKs in the SDH inhibit the projecting neurons (Mizoguchi et al., 1997; Willis, 1988). Furthermore, ENKergic neurons in the rat SDH are innervated by serotonin t ermi- nals, and 5-HT3A receptors colocalized with ENK, thus it seems that the activation of these neurons might be involved in 5-HT-induced antinociception (Huang et al., 2008). As regards their peripheral action, the results showed that clonidine (an α 2 - adrenoceptor agonist) can induce peripheral antinociception by the local release of ENKs (Nakamura and Ferreira, 1988). Dynorphins Dynorphin A 1–17 , a heptadecapeptide, and dynorphin A 1–13 , the N-terminal tride- capeptide of dynorphin A, were isolated from porcine pituitary tissue, and they are produced from preprodynorphin. Both dynorphins possess high affinity for the KOR (Szeto, 2003). The high density of KORs in the spinal cord, medulla, amygdala, hypothalamus, and periphery suggests a possible involvement of dynorphins in the regulation of pain mechanisms (Lai et al., 2008; Menetrey and Basbaum, 1987; Palkovits, 2000; Szeto, 2003). Several reports indicate hyperalgesic or allodynic effects of dynorphins (Fujimoto et al., 1990; Lai et al., 2008; Qu and Isaac, 1993; Rady and Fujimoto, 2002; Wang et al., 2001b; Wen et al., 1985), however, KOR 450 G. Horvath deletion significantly exacerbated mechanical and thermal inflammatory hypersen- sitivity but there was no change in the formalin-induced pain behavior (Schepers et al., 2008; Xu et al., 2004b). ICV administration of dynorphins can produce antinociception, which is reversed by the KOR antagonist (Fujimoto et al., 1990; Shukla et al., 1992). As regards their effects at the spinal level, the results are inconsistent. Dynorphins can induce nocifensive behavior or hyperalgesia, and arthritic rats displayed a pro- nounced rise in immunoreactive dynorphins in the lumbosacral spinal cord, which correlated both with the intensity and time-course of hyperalgesia (Arcaya et al., 1999; Gardell et al., 2004; Millan et al., 1985; Vanderah et al., 1996; Wang et al., 2001b). However, other data suggest that spinal KOR activation is involved in the antinociceptive effects of some opioids, and the selective blockade of KORs increases the formalin-induced nocifensive behavior (Ossipov et al., 1996; Tseng and Collins, 1993). As regards the explanation for these opposing data, increas- ing evidence suggests that the dynorphin-induced antinociception is KOR-mediated, whereas its pronociceptive effects are elicited by binding of its enzymatic degrada- tion peptide fragments to nonopioid receptors. Therefore, the pronociceptive effecs of dynorphins is mediated by activation of NMDA and/or bradykinin receptors lead- ing to the release of SP and CGRP from primary sensory neurons (Arcaya et al., 1999; Lai et al., 2008; Wang et al., 2001b). However, peripheral application of dynorphins A 1–17 produced antiallodynia, and this effect was reversed by KOR antagonists (Ko et al., 2000). Endomorphin-1 and Endomorphin-2 (EM1, EM2) More than ten years ago, a new group of MOR agonists was discovered and named endomorphins (EMs) by Zadina et al. (1997). Endomorphin-1 (EM-1: Tyr- Pro-Trp-Phe-NH 2 ) and endomorphin-2 (EM-2: Tyr-Pro-Phe-Phe-NH 2 ) differ from conventional endogenous opioid receptor ligands in their N-terminal sequence, pep- tide length, and C-terminal amidation. The pathway for their synthesis is unknown, but they are converted enzymatically by endopeptidases (Horvath, 2000; Zadina et al., 1997). They interact specifically and with high affinity with MOR (Horvath, 2000; Zadina et al., 1997), and they possess partial rather than full agonist properties at MOR (Sim et al., 1998). EM-1 and EM-2 produce their effects through different subtypes of MOR, EM-1 affecting predominantly the MOR2 receptor and EM-2 the MOR1 (Sakurada et al., 2000). The administration of EMs elicits short-lasting antinociception, and tolerance was also observed (Csullog et al., 2001;Horvath et al., 1999; Tseng et al., 2000; Yu et al., 2004; Zadina et al., 1997). The antinociceptive effects are produced by peripheral, spinal and supraspinal levels as well (Przewlocki et al., 1999). ICV or intrathalamic administration of EMs produced antinociception in both acute and chronic pain models (Zadina et al., 1997; Zhao et al., 2007; Zubrzycka et al., 2005; Zubrzycka and Janecka, 2008). The EMs displayed lower potencies in the mechanical (paw pressure) test than in the heat-pain (TF) test in rats after IT administration (Horvath et al., 1999; Przewlocka et al., 1999), but they exerted high analgesic potency in different inflammatory pain Endogenous Antinociceptive Ligands 451 models as well (Csullog et al., 2001; Hao et al., 1999; Przewlocka et al., 1999; Przewlocki et al., 1999; Wang et al., 1999). Neuropathic pain has been assumed to be resistant to treatment with opioids, therefore it is of particular interest that the EMs have high potency in decreasing neuropathic pain (Przewlocka et al., 1999). EM-1, but not EM-2, dose-relatedly reduced the Aβ-fibre evoked responses, there- fore, spinal EM-2 exerts selective effects on noxious responses, whereas EM-1 is nonselective (Chapman et al., 1997). IPL administration of EM1 dose-dependently decreased the mechanical allodynia and the thermal hypersensitivity in neuropathic and inflammatory pain models (Labuz et al., 2006; Obara et al., 2004; Mecs et al., 2009). Tyr-MIF Peptides The Tyr-MIF (melanocyte-inhibiting factor) family of neuropeptides includes MIF- 1 (Pro-Leu-Gly-NH2), Tyr-MIF-1 (Tyr-Pro-Leu-Gyl-NH2), Tyr-W-MIF-1 (Tyr-Pro- Trp-Gly-NH2), and Tyr-K-MIF-1 (Tyr-Pro-Lys-Gly-NH2). All have been isolated from bovine hypothalamus and the cortex of human brain (Zadina et al., 1992; Zadina et al., 1994). They bind to MORs, but Tyr-K-MIF-1 primarily interacts with specific Tyr-MIF-1 binding sites (Zadina et al., 1992; Zadina et al., 1994; Zamfirova et al., 2007). Both ICV and IT administrations of Tyr-W- MIF-1 and/or Tyr-MIF-1 induce prolonged, naloxone-reversible analgesia (Bell et al., 1999; Gergen et al., 1996; Zadina et al., 1993; Zamfirova et al., 2007; Yu et al., 2004). However, the spinal effect is about 75 times stronger than the supraspinal one (Zadina et al., 1996). IP administration of Tyr-K-MIF-1 also produced antinociception by the activation of MORs and stimulated the histaminergic system, too (Zamfirova et al., 2007). However, others have shown that the peripherally and systemically applied Tyr- MIF-1 acts as an opioid antagonist in the TF test (Kastin et al., 1984; Kavaliers, 1987). Hemorphins Hemorphins are endogenous peptides belonging to the family of “nonclassical” or “atypical” opioid peptides, derived from hemoglobin (Nyberg et al., 1997). The hemorphin family member peptides vary in size from 4 to 10 amino acids, and they have been identified in the brain, plasma, and cerebrospinal fluid (Nyberg et al., 1997). These peptides include: hemorphin-4 (Tyr-Pro-Trp-Thr), hemorphin- 5 (Tyr-Pro-Trp-Thr-Gln), hemorphin-6 (Tyr-Pro-Trp-Thr-Gln-Arg), hemorphin-7 (Tyr-Pro-Trp-Thr-Gln-Arg-Phe), LVV-hemorphin-4 (Leu-Val-Val-Tyr-Pro-Trp-Thr; spinorphin), LVV-hemorphin-6 (Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg), and LVV- hemorphin-7 (Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe). These peptides display affinities for MOR, DOR, and KOR (Davis et al., 1989; Liebmann et al., 1989; Zadina et al., 1992), except for spinorphin, which has an ENK-degrading activ- ity (Nishimura and Hazato, 1993). Only a few studies investigated their role in pain mechanisms. Thus, the ICV administration of hemorphin-4 and hemorphin-5 showed potent antinociceptive effects in the acute pain model in a naloxone- reversible manner, but they did not influence formalin-induced pain behavior (Davis 452 G. Horvath et al., 1989). Peripheral administration of hemorphin-7 decreased the acute inflam- mation. which may also contribute to its antinociceptive effect (Sanderson et al., 1998). Spinorphin has antinociceptive potency, and its effect may be due to the inhibi- tion of the degradation of endogenous opioids (Honda et al., 2001; Maldonado et al., 1994; Schmidt et al., 1991; Yamamoto et al., 2002b; Yu et al., 2004). Spinorphin administered ICV did not influence acute pain sensitivity, but potentiated the effects of Leu-ENK. It inhibited the nociceptin-induced allodynia in a dose-dependent man- ner after IT administration, which was reversed by naloxone (Honda et al., 2001). Thus, spinorphin is not a real endogenous opioid neurotransmitter, but it might enhance the effect of Leu-ENK through inhibition of the degradation of ENK. Nociceptin Shortly after the cloning of the three known opioid receptors, a fourth member of this family was identified, the opioid-like receptor (ORL-1), which was found not to bind any of the known natural or synthetic opioid ligands (Reinscheid et al., 2000). In 1995, the natural ligand for this receptor was isolated and named orphanin FQ or nociceptin (Reinscheid et al., 1995). It is a 17-amino acid peptide, the amino terminus of which displays a striking s imilarity to the known mammalian opioid peptides. It is derived from pronociceptin together with another peptide, nocistatin. Nociceptin has been reported to be an active ligand at multiple sites of nocicep- tive transmission, ranging from peripheral nociceptors to nociceptive centers in the brain. Pharmacologically, the actions of nociceptin are complex and contradictory. ICV administration of this peptide exerts a pronociceptive action. The neuroanatom- ical site underlying the pronociceptive actions of nociceptin might be the RVM, where it inhibits the actions of OFF cells (Lambert, 2008). However, its effect at the spinal level depends on the dose applied; that is, a low dose produces nociception, whereas higher doses result in antinociception in acute and neuropathic pain mod- els (Calo et al., 2000; Ma et al., 2003; Mogil and Pasternak, 2001; Yu et al., 2004). It is suggested that low doses may increase the release of SP, whereas high doses inhibit the glutamate release (Lambert, 2008). ORL-1 receptors and nociceptin can be found peripherally as well, and their activation can lead to peripheral antinocicep- tion (Lambert, 2008; Obara et al., 2005), whereas other data suggest that nociceptin has pain-inducing effects (McDougall and Larson, 2006). Nocistatin A further endogenous peptide that has been implicated in the modulation of pain transmission is the heptadecapeptide nocistatin, produced by the proteolytic cleav- age of prepronociceptin (Lee et al., 1999; Okuda-Ashitaka et al., 1998). It has been detected in different parts of the body and exerts its effect through the activation of an unknown GPCR (Joseph et al., 2007; Zeilhofer et al., 2000). Some data have shown that its ICV administration increased the acute and inflammatory pain thresh- old (Nakagawa et al., 1999; Zhao et al., 1999), whereas other data suggest that it does not influence the normal pain latency, but prevented the nociceptin-induced Endogenous Antinociceptive Ligands 453 hyperalgesia (Liu et al., 2006; Nakagawa et al., 1999; Scoto et al., 2005). It presum- ably acts as a neuromodulator in pain processing at the spinal level as well, because it blocks the hyperalgesia and allodynia induced by nociceptin (its name originates from this observation) or prostaglandin-E2 (Ito et al., 2001; Okuda-Ashitaka et al., 1998; Okuda-Ashitaka and Ito, 2000). It does not influence the TF latency (Zeilhofer et al., 2000) and can decrease the neuropathic pain (in low doses) (Muth-Selbach et al., 2004), but higher doses could increase hyperalgesia or block nociceptin- induced analgesia in neuropathic rats (Ma et al., 2003; Muth-Selbach et al., 2004). However, it increases the flexor reflex response (Xu et al., 1999b), and inconsistent data are available on its effect on the formalin test (Nakano et al., 2000; Yamamoto and Sakashita, 1999; Zeilhofer et al., 2000). Cumulative administration of its C- terminal octapeptide, BPNP-3-8P, also significantly decreased heat hyperalgesia but it did not change the paw withdrawal (PWD) latency on the normal side (Csullog et al., 2001). As regards its peripheral effects, its pronociceptive potency has been reported (Inoue et al., 2003). 5.2.2 Kyotorphin Kyotorphin (Tyr-Arg), was isolated from bovine brain by Takagi et al. (1979). It is formed by kyotorphin synthase in the presence of ATP and Mg 2+ in the brain (Kawabata et al., 1995). High concentrations of the dipeptide were found in the brainstem and SDH, and several studies have demonstrated its analgesic properties (Ueda et al., 1980). Kyotorphin binds to its specific receptors (kyotorphin receptor; GPCR) and activates PLC (Lopes et al., 2006; Ueda et al., 1989). ICV administra- tion of kyotorphin produced antinociception in the acute pain test (Kawabata et al., 1994b). Kyotorphin excites cortical neurons directly, and it also exerts indirect opi- oid action to produce analgesia via the release of Met-ENK (Shiomi et al., 1981). Systemic administration of a kyotorphin receptor agonist leads to antinociception in the acute pain test, and this effect has been antagonized by IT injection of kyotorphin receptor antagonist, but not by ICV application (Ochi et al., 2000). Furthermore, IT kyotorphin also produced antinociception in an acute mechanical pain model (Ochi et al., 2002). Kyotorphin has a nonopioid analgesic effect at peripheral level, which makes it quite appealing for chronic pain treatment (Inoue et al., 1997). 5.2.3 Tachykinins The mammalian tachykinins are a family of evolutionary conserved peptides that share the common C-terminal motif. Until recently, the family consisted of three peptides: substance P (SP; undecapeptide), neurokinin A (10 amino acids), and neurokinin B (10 amino acids). Since the discovery of a third preprotachykinin gene (TAC4), the number of tachykinins has more than doubled to reveal several species-divergent peptides. This group includes hemokinin-1 (HK-1) in mouse and rat, endokinin-1 (EK-1) in rabbit, and EKA, EKB in humans (Page, 2004). Their peripheral expression has led to the proposal that they are the endogenous periph- eral SP-like endocrine/paracrine agonists where SP is not expressed. Additionally, 454 G. Horvath three orphan tachykinin gene-related peptides are identified, in rabbit, endokinin- 2 (EK-2), and in humans, EKC and EKD (Page, 2004). The biological actions of tachykinins are mediated by at least three different transmembrane GPCRs, namely NK1, NK2, and NK3. SP is preferential, but not exclusive, for NK1, NKA for NK2, and NKB for NK3, therefore, each ligand can interact with all receptors (Maggi et al., 1993; Morteau et al., 2001; Patacchini and Maggi, 2001). The NK1 recep- tor is widely expressed in both CNS and PNS, and the effects of NK1 receptor involvement in nociceptive transmission are very complex (Quartara and Maggi, 1998). NK1 knock-out mice have substantial impairments of endogenous pain con- trol mechanisms (Bester et al., 2001). At the supraspinal level, the dorsal raphe nucleus and PAG have numerous NK1 and GABA double-labelled neurons (Ma and Bleasdale, 2002). SP and HK1, acting upon NK1 receptors, might be relevant in descending pain control, because their administration in different brain areas increased the pain threshold, and this effect was reversed by opioid and GABAA receptor antag- onists (Altier and Stewart, 1993; Altier and Stewart, 1998; Altier and Stewart, 1999; Fu et al., 2008; Holden and Pizzi, 2008; Rosen et al., 2004; Yu et al., 2004). Furthermore, the injection of SP into the ventrolateral PAG has induced analgesia, and morphine has increased the SP release in this area (Rosen et al., 2004). The activation of SP-containing neurons in the lateral hypothalamus also increases the pain threshold by activating NK-1 receptors in the RVM (Holden and Pizzi, 2008). Therefore, SP may activate the descending antinociceptive path- ways through activation of NK1 receptors. Several data suggest that SP, HK1, and EKA/B decrease the pain threshold both spinally and peripherally (Abbadie et al., 1996; Afrah et al., 2001; Beyer et al., 1991; Cridland and Henry, 1988b; Dirig and Yaksh, 1999; Donnerer et al., 1992). However, NK1 receptor activation may also increase the inhibitory neurotransmission by activating inhibitory interneurons in the SDH (Vergnano et al., 2004). Furthermore, both induction of scratching behav- ior and thermal hyperalgesia by IT administration of SP and EKA/B as well as enhancement of c-Fos-LI following noxious thermal stimulation are suppressed by pretreatment with EKC/D, suggesting that the EKC/D peptide is an antagonist of the NK1 receptor (Naono et al., 2007). 5.2.4 Calcitonin Gene-Related Peptide (CGRP) Calcitonin gene-related peptide (CGRP; 37 amino acids) expresses predominantly in the nervous system and it influences multiple physiological activities. As regards its action mechanism, molecular correlates for discrete CGRP receptor types are still lacking. Functional CGRP receptors represent a multiprotein entity composed of at least three discrete proteins, that is, the seven-transmembrane receptor calcitonin- receptor-like receptor (CRLR), receptor-activity-modifying protein 1 (RAMP1), and the cytoplasmic receptor component protein (RCP) (Wu et al., 2002). CGRP is involved in many stages of the transmission of nociceptive information, because CGRP-LI has been found to colocalize with that of SP-LI in capsaicin-sensitive nerve terminals in the periphery and SDH, but it has been identified in the DRG and . administration of hemorphin-4 and hemorphin-5 showed potent antinociceptive effects in the acute pain model in a naloxone- reversible manner, but they did not in uence formalin-induced pain behavior (Davis 452. (SP; undecapeptide), neurokinin A (10 amino acids), and neurokinin B (10 amino acids). Since the discovery of a third preprotachykinin gene (TAC4), the number of tachykinins has more than doubled. bovine hypothalamus and the cortex of human brain (Zadina et al., 1992; Zadina et al., 1994). They bind to MORs, but Tyr-K-MIF-1 primarily interacts with specific Tyr-MIF-1 binding sites (Zadina