Endogenous Antinociceptive Ligands 455 several brain areas as well (Ballet et al., 1998; Brain and Cox, 2006; Olgiati et al., 1983; Skofitsch and Jacobowitz, 1985a). Thus, CGRP-Li fibers and CGRP recep- tors distribute densely in the amygdala, and these fibers originate from parabrachial nucleus and the thalamic nuclei (Oliver and Keyvan-Fouladi, 2000; Shimada et al., 1989). ICV, intra-PAG and intra-amygdala injection of CGRP induce antinocicep- tion (Candeletti and Ferri, 1990; Xu et al., 2003; Yu et al., 2003). It is probable that in the amygdala CGRP-containing terminals activate ENKergic neurons, which project to the PAG releasing ENK (Palkovits, 2000). However, other data have shown that ICV administration of CGRP did not modify pain sensitivity in the TF test and did not affect the antinociceptive action of morphine (Azarov et al., 1995). As regards its effects at spinal and peripheral levels, it is well known that it has a facilitation role in the nociceptive information, and this effect may be mediated via SP mech- anism (Ballet et al., 1998;Lietal.,2008; Morton et al., 1991; Nahin and Byers, 1994; Santicioli et al., 1993). 5.2.5 Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Pituitary adenylate cyclase-activating polypeptide-38 (PACAP-38) is a member of the vasoactive intestinal peptide family (VIP) that was originally isolated from ovine hypothalamus (Miyata et al., 1989). Two forms of PACAP have been iden- tified, containing 27 and C-terminally extended 38 amino acids. PACAP acts via GPCRs (activation of AC and PLC): the PAC1 receptors, which specifically bind PACAP and the VPAC1/VPAC2 receptors, which have a similar binding affinity for PACAP and VIP (see Section 5.2.6). Both PACAP and its receptors have been widely described on central and peripheral neurons, smooth muscle cells, and sev- eral inflammatory cells (Bartho et al., 2000; Delgado et al., 2003; Dickinson and Fleetwood-Walker, 1999; Narita et al., 1996). On the basis of the morphological and molecular biological results, PACAP has been suggested to be involved in pain transmission, but very few functional data are available to support this theory. Decreased response to different pain stimuli has been observed in PACAP or PAC1 receptor deficiencies (Mabuchi et al., 2004), and PAC1 receptor activation can lead to stimulation of NMDA receptors and synthesis of brain-derived neurotrophic fac- tor (BDNF), and these processes can lead to enhanced nociception (Jongsma et al., 2001a; Mabuchi et al., 2004; Martin et al., 2003). ICV administration of PACAP did not influence the acute heat pain thresh- old (TF) in mice, but significantly decreased morphine-induced analgesia (Macsai et al., 2002). As regards its effects at the spinal level, it can also facilitate spinal nociceptive flexor reflexes, and pronociceptive effects were observed, whereas PAC1-antagonists potently reduced mechanical allodynia in a neuropathic nerve model and were also effective in reducing thermal hyperalgesia in the carrageenan model (Davis-Taber et al., 2008; Narita et al., 1996; Ohsawa et al., 2002; Shimizu et al., 2004). However, other studies have shown that IT PACAP 27 or 38 were anal- gesic in inflammatory and neuropathic pain models (Narita et al., 1996; Yamamoto and Tatsuno, 1995; Zhang et al., 1996). PACAP inhibits the release of proinflamma- tory/pronociceptive sensory neuropeptides: SP and CGRP from peripheral terminals 456 G. Horvath of capsaicin-sensitive nerves, and it also inhibited acute neurogenic and nonneuro- genic inflammatory processes in both mice and rats (Helyes et al., 2007; Nemeth et al., 2006). IPL PACAP-38 did not alter basal mechanical or heat thresholds, but it inhibited the carrageenan- or heat injury-induced hypersensitivities, as well as nocif- ensive behaviors in the early and late phase of the formalin test (Sandor et al., 2009). In mice, it significantly diminished acetic acid-induced abdominal contractions but exerted no effect on neuropathic mechanical hyperalgesia. In contrast, it markedly increased rotation-induced firing of afferent fibres in the inflamed rat knee joint, clearly demonstrating a peripheral sensitization in this organ (Sandor et al., 2009). 5.2.6 Vasoactive Intestinal Peptide (VIP) VIP contains 28 amino acids, and it was first isolated from the porcine small intes- tine, but it has also been detected in the PNS and CNS (Fuji et al., 1983; Jancso et al., 1981). Both VAPC1 and VPAC2 receptors bind VIP with high affinity, there- fore, similarly to PACAP, VIP can also regulate different aspects of pain. Thus, its ICV administration elicits analgesia in acute heat pain tests, but impairs the antinoci- ceptive effect of morphine (Macsai et al., 1998). Bilateral application of VIP into the amygdala persistently suppressed the heat-evoked reflexes by exiting amygdala- originating neurons that innervate the PAG antinociceptive cells (Shin, 2005). VIP can produce both analgesia and hyperalgesia at the spinal level, depending on its molecular conformation (Yeomans et al., 2003). However, most of the data have shown that IT VIP induces nocifensive behavior, potentiates the effects of SP and decreased the pain threshold in acute pain models (Beyer et al., 1991; Cridland and Henry, 1988a; Wiesenfeld-Hallin, 1987; Xu and Wiesenfeldhallin, 1991;Yu et al., 2004). As regards its peripheral effect, intra-articular injection of VIP caused increased allodynia in rats with osteoarthritis probably by the activation of PKA (McDougall et al., 2006). 5.2.7 Galanin (GAL) Galanin is a neuropeptide consisting of 29 or 30 (in humans) amino acids and was originally isolated from porcine intestine (Bartfai et al., 1992). GAL exerts its biological effects by interacting with three high-affinity cell surface receptors GALR1-3, which all belong to the family of GPCRs (Branchek et al., 2000). All three receptors couple to Gi/o and inhibit AC, although GalR2 also signals via Gq/11 to activate PLC and PKC (Wittau et al., 2000). GAL and its receptors are widely distributed in the nervous system and have been implicated in a number of important body functions, including feeding, cognition, endocrine modulation, and nociception (Holmberg et al., 2005; Wiesenfeld-Hallin et al., 1992). Most neurons of the hypothalamic PVN and SO also contain GAL, colocalized with AVP, OT, and opioids (Zubrzycka and Janecka, 2008). In the rat, cell bodies and fibres containing GAL-LI and GAL receptors have been identified in DRG and in laminae I, II, VII, and X of SDH (Rokaeus et al., 1984; Skofitsch and Jacobowitz, 1985b; Wiesenfeld- Hallin et al., 2005). As GAL-LI and receptor numbers in the SDH are decreased following dorsal rhizotomy or capsaicin treatment, it has been suggested that one Endogenous Antinociceptive Ligands 457 source of GAL is derived primarily from unmyelinated primary afferent fibres (Xu et al., 1996b). In most systems the effect of GAL on nociception appears to be pre- dominantly inhibitory, mediated at least partially by GALR1 s (Bacon et al., 2002; Blakeman et al., 2003; Grass et al., 2003b; Ji et al., 1994a; Xu et al., 1998). Mice overexpressing GAL or deficient in GAL have become available, and have provided a genetic approach to analyze the role of GAL in nociception. GAL knock-out mice have a lower nociceptive t hreshold to heat stimulation, and have changed neuro- pathic pain behaviors (Blakeman et al., 2003; Grass et al., 2003a; Kerr et al., 2000; Malkmus et al., 2005). Furthermore, mice deficient in GALR1 or GALR2 have impaired pain-like behavior (Blakeman et al., 2003; Grass et al., 2003b; Hobson et al., 2006). Mice overexpressing GAL exhibit significant elevation of the nociceptive thresh- old to thermal stimulation, but no change in response to mechanical or cold stimulation was seen (Blakeman et al., 2001). ICV perfusion of GAL concentration dependently inhibited pain-induced responses, and its effect was blocked by GAL receptor and by MOR antagonists, whereas it was potentiated by EM2, AVP, and OT (Zubrzycka and Janecka, 2008). Similarly, the analgesic effect of GAL admin- istered i n the PAG or arcuate nucleus was also attenuated by naloxone (Sun and Yu, 2005; Wang et al., 2000). These data suggest that GAL can induce antinociception at the supraspinal level by release of the endogenous opioid ligands. The antinocicep- tive role of GAL at the spinal level has been extensively studied (Wiesenfeld-Hallin et al., 2005). IT administration of GAL produced a dose-dependent increase in the TF latency, but surprisingly, it lowered the threshold to von Frey stimulus (Cridland and Henry, 1988a). The effect of exogenous GAL is predominantly inhibitory under normal conditions, and due to blocking the excitatory effect of SP and CGRP (Hua et al., 2004; Xu et al., 1998; Yu et al., 2001), but GAL can also modulate the release of endogenous opioid ligands (Zhang et al., 2000a). However, other reports pro- posed that GAL produces a biphasic, dose-dependent effect on nociception through activation of antinociceptive (inhibitory) GALR1 or pronociceptive (excitatory) GALR2 receptors, thus endogenous GAL can potentiate nociceptive processing dur- ing inflammation (Kerr et al., 2001; Liu and Hokfelt, 2002). Some results suggest that peripheral GAL has an excitatory role in inflammatory pain, likely mediated by peripheral GALR2 and that GAL can modulate TRPV1 function, whereas activa- tion of peripheral GalR1 results in antinociception (Jimenez-Andrade et al., 2004; Jimenez-Andrade et al., 2006). 5.2.8 Neuropeptide Y (NPY) NPY is an abundant neuroactive peptide (containing 36 amino acids) that exerts numerous physiological actions, including pain modulation. NPY is expressed in the CNS and PNS, and can be released from sensory, enteric, and sympathetic neu- rons but also from glial cells (Arora and Anubhuti, 2006; Chronwall and Zukowska, 2004; Dumont et al., 1992). There are six receptors (Y1-6) of NPY which are GPCRs. They exhibit dynamic alterations in signalling pathways, leading to neu- ronal excitatory or inhibitory effects after receptor activation (Lin et al., 2004). NPY 458 G. Horvath and Y1 and Y2 (most prevalent) receptors are located at key pain signalling centers throughout the nervous systems, particularly the SDH, and their effects on noci- ceptive modulation has been extensively studied (Allen et al., 1984; Bannon et al., 2000; Gibbs and Hargreaves, 2008; Ji et al., 1994b; Shi et al., 1998; Smith et al., 2007). In contrast to the cellular localization of Y1 receptors, spinal Y2 receptors are located on primary afferent terminals (Brumovsky et al., 2005). Y1 receptor deletion increased acute heat, inflammatory, and neuropathic pain sensitivities (Kuphal et al., 2008). Furthermore, lack of Y1 receptor or antagonism at this receptor inhibited the antinociceptive potency of NPY at the spinal level. These data suggest that the Y1-receptor system exerts tonic inhibitory control and it mediates the antiallodynic effects of NPY during inflammatory and neuropathic pain syndromes. Several studies have proved the antinociceptive potential of supraspinal NPY (Illes et al., 1993;Lietal.,2005a). After the microinjection of NPY into the RVM, the PAG, the ARC, or in the nucleus accumbens, withdrawal reflexes to noxious heat or tactile stimuli are decreased (Li et al., 2005a;Lietal.,2002b; Wang et al., 2001a; Zhang et al., 2000b). However, intracranial administration of anti-NPY antiserum or Y1 receptor antagonist into the nucleus gracilis reversed nerve injury-mediated mechanical allodynia (Ossipov et al., 2002). IT administration of NPY inhibits tran- sient, inflammatory, and neuropathic pain (Hua et al., 1991; Intondi et al., 2008; Mahinda and Taylor, 2004; Smith et al., 2007; Taiwo and Taylor, 2002). Evidence from pharmacological studies suggests that both Y1 and Y2 agonists can attenuate the flexor reflex in axotomized animals (Xu et al., 1999a). The neurophysiological mechanism of this antinociception involves inhibition of pronociceptive, excita- tory neurotransmitter release from primary afferent neurons through activation of Y2 receptors, whereas Y1 receptor activation inhibits GABAergic inhibition on the substantia gelatinosa (Martire et al., 2000; Smith et al., 2007). NPY is colocalized with TRPV1 receptors, and it can inhibit the excitatory transmitter release from the capsaicin-sensitive primary sensory neurons leading to peripheral antinociception (Gibbs et al., 2006a; Gibbs and Hargreaves, 2008). 5.2.9 RFamide Neuropeptides There is a new family of mammalian neuropeptides, that is, RF (Arg-Phe) amide neuropeptides including neuropeptide FF (NPFF, octapeptide), prolactin-releasing peptide (PrRP, 31 amino acids), RF-amide related peptides (RFRP); RFRP1: Leu- Pro-Leu-Arg-Phe-amide; RFRP2: gonadotropin-inhibitory hormone:dodecapeptide and RFRP3: Leu-Pro-Gln-Arg-Phe-amide, kisspeptins (10, 13 and 14 amino acids), and the 26Rfa (RF[Arg-Phe]amide family 26-amino acid peptide, also known as P518) (Fukusumi et al., 2006). RF-amides represent a group of peptides sharing a C-terminal RF-terminus, and they are involved in many regulatory functions in the body by action on different GPCRs. Both NPFF and RF-amide related peptides pro- duce their effects by activation of NPFF receptors (1 and 2). PrRP is a ligand for an orphan receptor, the GPR10-like receptor, but it can also activate NPFF2 receptors (Engstrom et al., 2003). Kisspeptins are the products of the gene Kiss1 and they are ligands for GPR54, whereas 26Rfa produces its effects by activation of GPR103, a Endogenous Antinociceptive Ligands 459 receptor that is widely distributed in the spinal cord (Bruzzone et al., 2007). These peptides and their binding sites are expressed in the CNS (Engstrom et al., 2003; Pertovaara et al., 2005; Sullivan et al., 1991; Yang and Iadarola, 2006). NPFF has both potent pro-opioid antinociceptive and antiopioid-like effects, depending on the sites of administration and the dose (Frances et al., 2001; Panula et al., 1996; Roumy and Zajac, 1998; Wei et al., 1998; Yang et al., 1985). It produces antinociception after ICV or intra-PAG administration in neuropathic and inflammatory models in a naloxone reversible manner (Altier et al., 2000; Kalliomaki et al., 2004; Wei et al., 1998; Wei et al., 2001). On the other hand, NPFF either decreases or does not influ- ence the acute mechanical and thermal pain sensitivities and can antagonize the effect of morphine (Altier et al., 2000; Wei et al., 2001; Yang et al., 1985). As regards its role at the spinal level, several reports have proved its effec- tivities in neuropathic and inflammatory pain models; it can potentiate the effect of morphine, but the results about the effects on acute pain tests are inconsistent (Altier et al., 2000; Gouarderes et al., 2000; Kontinen and Kalso, 1995; Wei et al., 2001; Yamamoto et al., 2008; Yang and Iadarola, 2006). Peripheral administration of an NPFF analogue did not produce antinociceptive effect (Wei et al., 2001). The potential role of PrRP in pain was addressed by its ICV and IT injections in both neuropathic and normal rats (Kalliomaki et al., 2004). It was ineffective at the spinal level, but with administration in the dorsal medulla, PrRP produced sig- nificant antinociception in normal rats and an antiallodynic effect in neuropathic rats. The PrRP-induced antinociception is not mediated by MOR because it is not reversible by naloxone. However, other data have shown that ICV administration of PrRP promoted hyperalgesia, and it reversed the morphine-induced antinociception (Laurent et al., 2005). Furthermore, PrRP knock-out animals showed increased pain threshold. As regards the RF-amide-related peptides, their injection into the brain (nucleus of solitary tract) inhibited mechanical hyperalgesia, whereas IT administra- tion significantly decreased the acute heat pain sensitivity and the tactile allodynia in a neuropathic pain model (Pertovaara et al., 2005). Spinally applied 26Rfa also sig- nificantly decreased the nocifensive behavior in the formalin test, and attenuated the level of mechanical allodynia in a carrageenan-induced inflammatory pain model, but it did not influence the normal heat and mechanical pain sensitivity (Yamamoto et al., 2008). The relation of kisspeptin to the pain has been suggested as well. Thus, a marked elevation in the levels of kisspeptin and GPR54 mRNA as well as protein was observed in the SDH and DRG during inflammation, indicating a possible involvement of the kisspeptin/GPR54 system in chronic inflammatory pain (Mi et al., 2009). 5.2.10 Neurotensin (NeT) Another endogenous peptide which has been implicated in pain transmission and the central integration of pain responses is neurotensin (NeT) (Dobner, 2006;Gui et al., 2004; Pettibone et al., 2002). NeT is a brain–gut tridecapeptide that fulfils a dual function: as a neurotransmitter/neuromodulator in the nervous system, and as a paracrine and circulating hormone at the periphery. Three NeT receptors, NTR1, 460 G. Horvath NTR2, and NTR3, have been cloned to date (Dubuc et al., 1999). NTR1 and NTR2 belong to the GPCR family, whereas NTR3 is a single transmembrane domain pro- tein that belongs to a recently identified family of sorting receptors (Mazella, 2001; Mazella and Vincent, 2006). Most of the known peripheral and central effects of NeT are mediated through NTR1. NTR2 may possibly take part in the analgesic response elicited by the central administration of NeT; the biological roles of NTR3 are yet to be discovered in detail (Dubuc et al., 1999; Kitabgi, 2002; Mazella and Vincent, 2006). Various in vivo data support its modulatory role i n pain transmis- sion (Dobner, 2006;Yuetal.,2004). NeT normally facilitates visceral nociception, whereas an increased NeT expression can be observed under high stress condi- tions, and this ligand is required for SIA (Gui et al., 2004). The results indicate that the supraspinal antinociceptive effect of NeT is largely MOR independent (Osbahr et al., 1981). ICV, intra-PAG or intra-RVM administrations of NeT produce anal- gesic effects in different pain models (HP, TF, writhing, colorectal distension tests) by NTR1 and/or NTR2 activations (Behbehani and Pert, 1984; Dobner, 2006; Dubuc et al., 1999; Nemeroff et al., 1979; Osbahr et al., 1981; Pettibone et al., 2002; Urban et al., 1999). It seems that NeT can excite PAG neurons, which leads to activation of the descending pain inhibitory system (Behbehani and Pert, 1984; Dobner, 2006). NeT also induces analgesia through stimulation of NTR1 and NTR2 at spinal level, as was shown in acute and inflammatory pain models (Roussy et al., 2008; Sarret et al., 2005). 5.2.11 Neurotrophic Factors Neurotrophic factors are a unique family of polypeptide growth factors that influence the proliferation, differentiation, survival, and death of neuronal and nonneuronal cells. Neurotrophic factors are synthesized as high-molecular-weight precursors and their release from cells is constitutive as well as activity depen- dent. The nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), glial cell line-derived neurothrophic factor (GDNF), and insulin-like growth factor-1 (IGF-1) are essential for the health and well being of the nervous system, and also mediate additional higher-order activities, such as learning, memory, and behaviour. Alterations in their levels have been implicated in neurodegenerative disorders, such as Alzheimer’s disease and Huntington’s disease, as well as psychiatric disorders, including depression and substance abuse. Most of the neurotrophic factors interact with two types of cell-surface receptors, the low-affinity p75 receptor and TrK family of high- affinity tyrosine kinase receptors (TrKA, B, and C). Whereas all neurotrophins (NGF, BDNF, NT-3, and NT-4) bind the p75 receptor with similar affinity, NGF bind TrKA receptors, BDNF and NT-4 bind TrKB receptors, and NT-3 preferen- tially binds TrKC receptors, and to a lesser extent, TrKA receptors (Huang and Reichardt, 2003). There is now strong evidence that two neurotrophins, NGF and BDNF act as important mediators and modulators of pain in a variety of circumstances. Particularly, NGF can promote the sensitization and activation of nociceptors (Pezet Endogenous Antinociceptive Ligands 461 et al., 2002; Zhao et al., 2006). NGF is produced in the periphery and taken up by SP and BDNF containing sensory nerve terminals, it binds to the TrKA receptors and it is retrogradely transported to their cell bodies within DRG (Delcroix et al., 2003). The altered retrograde supply of NGF contributes t o the neuronal response to injury and inflammation by the modulation of SP and CGRP release from the primary sensory neurons (Donnerer et al., 1992; Woolf and Costigan, 1999). Furthermore, NGF acts on mast cells to induce release of 5-HT, which s ensitizes nociceptors as well (Theodosiou et al., 1999). Thus, endoneural injection of NGF is sufficient to produce transient histological and behavioral effects like those seen in neuropathic pain models, whereas sequestration of NGF prevents hyperalgesia, which normally accompanies inflammation (Dmitrieva et al., 1997; Lewin et al., 1994; Ruiz et al., 2004). NGF can also increase opioid binding sites, and it enhances the opioid sus- ceptibility of sensory neurons towards better pain control by an upregulation in the number and efficacy of sensory neuron MOR (Chen et al., 1997; Inoue and Hatanaka, 1982; Mousa et al., 2007). Furthermore, NGF may increase the amount of PACAP in sensory neurons, and this effect can also decrease its pain-inducing effects (Jongsma et al., 2001b). This suggests its therapeutic potential for patholog- ical conditions with a reduced susceptibility to opioids such as certain neuropathic pain states. Among the neurotrophins, BDNF is the most abundant and widely distributed in the CNS. In the sensory s ystem BDNF is constitutively produced by nociceptive- sensitive primary sensory neurons in the DRG, t hen it is transported anterogradely to the central terminals of sensory neurons in the SDH, where it can be released (together with SP) (Malcangio and Lessmann, 2003; Michael et al., 1997). In sen- sory neurons the concentration of BDNF and SP depends on the availability of NGF, therefore, the increase in NGF concentration in peripheral tissues that fol- lows an inflammatory insult enhances the expression of these ligands (Malcangio and Lessmann, 2003). BDNF may act as a regulator of neuronal excitability and modulator of synaptic plasticity, playing an important role in pain pathways (Kerr et al., 1999; Malcangio and Lessmann, 2003; Thompson et al., 1999). Conditional BDNF knock-out (in the primary sensory neurons) mice showed several alterations, including a reduced baseline thermal threshold and a decreased inflammatory hyper- algesia, whereas neuropathic pain behavior developed normally (Zhao et al., 2006). IT grafts of BDNF-secreting neurons or overexpression of BDNF in the spinal cord have been shown to alleviate chronic neuropathic pain, and other data suggest an ineffectivity after IT administration of BDNF (Boucher et al., 2000; Cejas et al., 2000; Eaton et al., 1999; Eaton et al., 2002). BDNF can depress sensory neu- rone transmission in the SDH by an indirect mechanism that requires the release of GABA from interneurons and the activation of GABAB receptors located in the terminals of sensory neurons. Therefore, BDNF decreased neuropathic pain by increasing the GABA release in SDH (Lever et al., 2003). However, other reports have shown that the expression of BDNF is dramatically upregulated in models of inflammatory pain, it enhances the NMDA-receptor-mediated responses and TrKB antagonism significantly reduced the inflammatory pain (Garraway et al., 2005; Kerr et al., 1999; Pezet et al., 2002). Thus, BDNF released from nociceptors along 462 G. Horvath with SP and glutamate appears necessary for the full activation of s econd-order DH neurons (Pezet et al., 2002). The role of NT-3 in pain transmission has not been fully worked out. NT-3 significantly attenuates neuronal expression of voltage-gated s odium channels and elevated levels of galanin and PACAP in DRG neurons, and all these effects can influence the pain sensitivity (Wilson-Gerwing and Verge, 2006). It is upregulated in models of neuropathic pain, and can contribute to the mechanical hyperalgesia (Zhou et al., 2000). However, IT administration NT-3 suppressed thermal hyperal- gesia associated with neuropathic pain, although others did not find any effects in a similar model (Boucher et al., 2000; Wilson-Gerwing et al., 2005). Glial cell line-derived neurothrophic factor (GDNF) binds to its high-affinity receptor, glial cell line-derived neurotropic factor receptor α-1 (GFRα1), and a sub- population of DRG neurons expresses GFRα1 (Lindfors et al., 2006). The effect of GDNF on nociception is still a matter of debate, but it seems that GDNF expression decreases in neuropathic pain states (Nagano et al., 2003). IT GDNF exerts potent analgesic effects on hyperalgesia as well as protective effects against the development of neuropathic pain (Boucher et al., 2000; Sakai et al., 2008; Wang et al., 2003a). In association with the analgesic effects of GDNF, several molecules, including sodium channels, purinergic receptors, and neuropeptides, have been reported to exhibit changes in expression (Boucher et al., 2000; Issa et al., 2001; Wang et al., 2003a). Recently, GDNF was shown to bind to neu- ral cell adhesion molecule (NCAM) via GFRα1, and NCAM signalling plays a role in mediating the analgesic effect of GDNF in rats with nerve injury (Sakai et al., 2008). IPL injection of GDNF induces thermal hyperalgesia, and inflamma- tory hyperalgesia is attenuated by treatment with an antibody against GDNF (Malin et al., 2006). Insulin-like growth factor-1 (IGF-1) is a 70-amino acid polypeptide that exerts effects on peripheral growth, differentiation, and survival in a variety of cells and tissues (Daftary and Gore, 2005). IGF-1 is an important regulator of synap- tic plasticity and neuronal survival in response to injury. The secretion of growth hormone (GH) stimulates the production of peripheral IGF-1 from its primary target, the liver, as well as from secondary targets such as lungs, thymus, heart, neurons and glia. Immunoreactivity and mRNA transcripts for IGF-1 and its recep- tor have been reported to exist in the brain and spinal cord, and IGF-1-LI is also transported retrogradely and anterogradely in axons of the peripheral nerve (Bitar et al., 1996; Daftary and Gore, 2005; Hansson et al., 1987). IGF-1 produces its effects through activation of its tyrosine kinase receptor, TrK IGF-1R that signals through the phosphoinositol-3 kinase and mitogen-activated protein kinase (MAPK) signalling cascade (Daftary and Gore, 2005). Only few studies suggest its role in antinociception. Thus, IT administration of IGF-1 produces a dose-dependent antinociception, and the increased IGF-1 level may play a significant role in the serotoninergicic antinociception at the spinal level (Bitar et al., 1996; Bonnefont et al., 2007) Endogenous Antinociceptive Ligands 463 5.3 Other Peptides 5.3.1 Endothelins (ET) It is now firmly established that endothelins, a family of 21-amino-acid residue pep- tides produced by many cell types, can exert multiple and important actions in many tissues and systems, including those implicated in nociceptive signaling functions (Kedzierski and Yanagisawa, 2001). The potent and widespread actions of ET-1 and other isopeptides of the family (ET- 2 and ET-3) are mediated by specific GPCR receptors (ETA and ETB), and the signal transduction pathways involve increases in intracellular calcium levels (Rubanyi and Polokoff, 1994). ET-1-LI and specific binding sites have been found in the SDH and in different brain areas (Gulati and Srimal, 1992; Rubanyi and Polokoff, 1994). Some data imply that endothelin might have a role in neurotransmission that is important for an animal’s pro- and antinoci- ception. Injection of ET-1 into the PAG reduces pain response in mice subjected to the HP paradigm (D’Amico et al., 1996). Furthermore, astrocytes produce endoge- nous cannabinoids (CBs) in response to treatment with ET1 through ETA receptor activation, and these effects can contribute to its antinociceptive potency (Walter et al., 2002; Walter and Stella, 2003). Intrathecal administration of ET1 also induced antinociception in the acute heat pain model (Kamei et al., 1993b; Kamei et al., 1993a). It is suggested that ET-1 induces SP release, which provokes endogenous opioid release in the SDH (Kamei et al., 1993b). The peripheral administration of ET produces pronociceptive effects primarily through ETA receptor activation, but ETB receptor also can contribute to the effect (Da Cunha et al., 2004; Daher et al., 2004; Piovezan et al., 1997; Raffa et al., 1996). One study has shown that ETB receptors normally can display antihyperalgesic and antinociceptive functions in the rat knee-joint incapacitation test (Daher et al., 2004) 5.3.2 Hemopressin Hemopressin is a nonapeptide derived from the α1-chain of hemoglobin, which was originally isolated from rat brain homogenates (Rioli et al., 2003). It is the first endogenous peptide ligand for cannabinoid-1 (CB1) receptors, and it behaves as an inverse agonist (Dale et al., 2005; Heimann et al., 2007; Lippton et al., 2006; Rioli et al., 2003). Hemopressin causes hypotension in anaesthetised rats and is metabolised in vivo and in vitro by endopeptidase 24.15 (EP24.15), neurolysin (EP24.16), and ACE (Lippton et al., 2006; Rioli et al., 2003). There are no data available on its distribution in the organism including the brain. This peptide selec- tively binds to CB1 receptors, but did not affect on CB2, MOR, DOR, α2- and β2-adrenergic, AT1 and AT2 and bradykinin B2 receptors (Heimann et al., 2007). The only in vivo experiment showed that IPL, IT and oral hemopressin reduced inflammatory pain sensitivity (Heimann et al., 2007). IP hemopressin also decreased the visceral nociception. Because a large body of evidence has clearly demonstrated 464 G. Horvath the antinociceptive action of cannabinoid CB1 receptor agonists, a possible expla- nation for these paradoxical effects could be that after CB1 receptor blockade by the antagonist, the released endocannabinoids could induce antinociception by affecting another pain transmission mechanism (see below Section 6.1). 5.3.3 Annexin-A1 Annexin-A1 (37 kDa glucocorticoid-regulated protein), formerly known as lipocortin-1, is a member of the annexin family of calcium and phospholipids- binding proteins (Buckingham and Flower, 1997). It is widely distributed in different tissues including the CNS, and annexin-A1 mediates the anti-inflammatory actions of glucocorticoids (Yang et al., 2004). Few studies have addressed the question of whether annexin-A1 is involved in the activation/modulation of pain pathways. Systemic administration of annexin-A1 peptidomimetics produced anal- gesia in inflamed rat paws and neutralizing antisera t o annexin-A1 prevented the antihyperalgesic activity of glucocorticoids (Ferreira et al., 1997). Annexin-A1 knock-out mice were more susceptible to visceral pain stimulus compared with wild-type, and increased levels of prostaglandin E2 (PGE2) in the spinal cord of knock-out compared with normal mice suggest that annexin-A1 modulates noci- ceptive processing at the spinal level by downregulating PGE2 spinal nociceptive facilitation (Ayoub et al., 2008). Inhibition of the formalin-induced nociceptive behavior by annexin-A1, administered centrally (ICV) or locally (IPL), is dependent on activation of the receptors of the formylated peptide family, which is a GPCR family (Pieretti et al., 2004). IPL administration of annexin-A1 also significantly decreases the intensity of hyperalgesia by inhibition of neutrophil accumulation (Ferreira et al., 1997; Pieretti et al., 2004). Furthermore, using a rat model of C-fibre modulated bradykinin-induced plasma extravasation, Green et al. (1998) suggest that the inhibitory action of a glucocorticoid on C-fibre activation is mediated by the release of annexin-A1. These results may provide a possible mechanism for the analgesic action of the glucocorticoids, which are routinely given to patients with postoperative pain. 5.4 Cytokines The term cytokine encompasses a large and diverse family of polypeptide regula- tors that are produced widely throughout the body by cells of diverse embryological origin. Historically, the term “cytokine” has been used to refer to the immunomod- ulating agents (interleukins, interferons, etc.). Virtually all nucleated cells, but especially endo/epithelial cells and resident macrophages are potent producers of different cytokines (e.g., IL-1, IL-6, and TNF-α) (Cannon, 2000). The action of cytokines, similarly to hormones, may be autocrine, paracrine, and endocrine. Cytokines have been classified as lymphokines, interleukins, and chemokines, based on their presumed function, cell of secretion, or target of action. Because cytokines are characterized by considerable redundancy and pleiotropism, such distinctions, allowing for exceptions, are obsolete. A classification that proves useful in clinical . in the levels of kisspeptin and GPR54 mRNA as well as protein was observed in the SDH and DRG during in ammation, indicating a possible involvement of the kisspeptin/GPR54 system in chronic in ammatory. PKA (McDougall et al., 2006). 5.2.7 Galanin (GAL) Galanin is a neuropeptide consisting of 29 or 30 (in humans) amino acids and was originally isolated from porcine intestine (Bartfai et al., 1992). GAL. antinociceptive functions in the rat knee-joint incapacitation test (Daher et al., 2004) 5.3.2 Hemopressin Hemopressin is a nonapeptide derived from the α1-chain of hemoglobin, which was originally