Neurochemical Mechanisms in Disease P50 pot

Endogenous Antinociceptive Ligands 475 the cannabimimetic effects have largely been attributed to the indirect entourage effect on the endocannabinoid system (Mechoulam et al., 1998). Only one study has reported its effects on pain sensation. Systemic administration of oleamide induces cannabimimetic effects, and it produces relatively long-lasting antinociceptive effects (HP and TF tests), but repeated administration of oleamide causes tolerance as well (Fedorova et al., 2001). 6.2 Eicosanoids The derivatives of arachidonic acid are eicosanoids, which have four families: the prostaglandins, the prostacyclins, the thromboxanes, and the leukotrienes. Prostanoid is the term used to describe a subclass of eicosanoids consisting of the prostaglandins, the thromboxanes, and the prostacyclins. They are important lipid mediators involved in the transmission of nociceptive pain. Their synthesis is initiated by the generation of arachidonic acid by phospholipase A2 (PLA2), which is metabolized by COX1 enzymes (1–3) to generate short-lived mediators that act as precursors for the synthesis of the biologically active prostanoids: prostaglandin-E2 (PGE2), PGD2, PGI2, PGF2α, or thromboxane A2. In the spinal cord both COX1 and COX2 are expressed. The major prostaglandins produced in the spinal cord are PGE2 and PGD2. The role of PGE2 is to enhance synaptic transmission and increase spinal responses to peripheral stimulation, and it thus plays a major role in the induction of hyperexcitability during peripheral inflammation (Ahmadi et al., 2002; Vanegas and Schaible, 2001; Vasquez et al., 2001). By contrast, much less is known about the role of PGD2. In general, PGD2 is the most produced postanoid in the CNS of mammals. A profound basal release of PGD2 in the spinal cord has been reported, and peripheral nociceptive stimulation and systemic inflammation increases spinal PGD2 biosynthesis (Grill et al., 2008; Willingale et al., 1997). As regards its action mechanism, PGD2 activates two GPCRs, the DP1 and DP2 receptors. Activation of DP1 stimulates AC and increases cAMP concentration, whereas DP2 receptors couple to inhibitor G-proteins and decrease cAMP concentration (Hata et al., 2003; Kostenis and Ulven, 2006). Both DP1 and DP2 receptors are localized in neurons of all laminae within the ventral and dorsal horn (Grill et al., 2008). As regards its action on the pain threshold, IT application of PGD2 evokes hyperalgesia and allodynia (Minami et al., 1994), and allodynia cannot be elicited in mice lacking the PGD synthase (Eguchi et al., 1999), suggesting a pronociceptive role of PGD2. A recent study has shown that spinally administered PGD2 does not change the responses to mechanical stimulation in normal animals, and neither DP1 nor DP2 receptor agonists influences this reflex (Telleria-Diaz et al., 2008). However, IT PGD2 can also inhibit the PGE2-induced allodynia supporting an antinociceptive effect of PGD2 as well (Minami et al., 1996). Furthermore, either PGD2 or a DP1 receptor agonist decreases responses to mechanical stimulation in rats with inflamed joints and the facilitatory effects of PGE2, and the inhibitory effect of PGD2 could have been resulted from the activation of GABAergic inhibitory interneurons (Eguchi et al., 1999; Minami 476 G. Horvath et al., 1997; Telleria-Diaz et al., 2008). Another prostaglandin, which can produce antinociception is the prostaglandin J, which is dominant during t he resolution of an inflammatory condition (Burstein et al., 2007). Thus, compounds that pro- mote the synthesis of this PG without significantly raising the level of the other prostaglandins could be considered as good candidates for the treatment of inflammation. Thus, NAGly produces a favorable prostaglandin ratio and is effective in reducing in vivo responses to proinflammatory agensts (Burstein et al., 2007). Cytochrome P450 genes catalyze formation of epoxyeicosatrienoic acids (EETs) from arachidonic acid. The effects of 5,6EET, 8,9EET, 11,12EET, and 14,15EET microinjected into the ventrolateral PAG on the thermally produced TF-response have been studied in rats (Terashvili et al., 2008). 14,15EET dose-dependently increased the TF-latency, whereas other EETs were inactive. The effect of 14,15EET has been blocked by antiserum against β-endorphin or Met-ENK, suggesting that this ligand evokes β-endorphin and Met-ENK releases. 6.3 Gangliosides Gangliosides are glycosphingolipids that occur in nearly all cellular membranes and are particularly concentrated in the nervous tissue (Zeller and Marchase, 1992). Gangliosides include all sialic acid-containing glycosphingolipids possessing a spe- cific sequence of neutral sugars (in different numbers). Members of the ganglioside family are designated by the capital letter G and are defined by the characteristic neutral sugar chain sequence. The sialic acid content of a ganglioside is designated by a capital letter: A (asialo), M (monosialo), or D (disialo). These agents have been shown to be effective in treating features of a variety of diabetic and toxic peripheral neuropathies. The mechanisms of ganglioside action in peripheral nerves include the enhancement of mean sprouting length, and increase of the number of regenerating axons (Zeller and Marchase, 1992). The protection afforded by gangliosides may be attributable to their ability to attenuate the neural injury induced by glutamate and/or to block the translocation of PKC (Vaccarino et al., 1987; Vorwerk et al., 1999; Zeller and Marchase, 1992). Furthermore, GM 1 ganglioside displays a broad spectrum of neurotrophic effects in vivo and in vitro. Some articles have reported the antinociceptive potency of GM 1 and the pronociceptive effect of anti-GD anti- body (Fromm et al., 1993; Goettl et al., 2000; Mao et al., 1992; Sorkin et al., 2002), but opposite data have also been published (Crain and Shen, 1992; Crain and Shen, 2000). The systemic or IT administrations of gangliosides to control animals have no effect on sensory thresholds, but they suppress thermal and mechanical hyperalgesia and also spontaneous pain behavior in neuropathic pain models (Fromm et al., 1993; Mao et al., 1992). The chronic administration of GM 1 to aged rats partially restored the pain responses, but it had no effect on any sensory modality tested in young rats, suggesting a normalgesic effect (Goettl et al., 2000). The coadminis- tration of a highly purified bovine brain ganglioside mixture (without GM 1 ) with pure GM 1 produced a potentiated antinociceptive effect in a model of peripheral mononeuropathy (Hayes et al., 1992). Endogenous Antinociceptive Ligands 477 6.4 Steroids Steroids are a large group of compounds, any of which have important biologi- cal actions. These effects are typically brought about by steroid binding to nuclear receptors and subsequent changes in gene expression (Beato and Sanchez-Pacheco, 1996). However, steroids can also exert faster effects by activating membrane surface receptors (Losel and Wehling, 2003). Some of the best-characterised membrane surface steroid receptors in mammals are ion channels. For example, GlyRs and GABA receptors display different sensitivities to many neuroactive steroids (Webb and Lynch, 2007). However, most neurosteroids are active at a wide range of receptors so their potential as therapeutic agents seems limited. 6.4.1 Neurosteroids Neurosteroids are steroid hormones synthesized in the brain that can modulate neuronal function through both gene expression and by direct modulation of neuronal excitability (Rupprecht, 2003). Nongenomic rapid effects of neurosteroids are particularly efficient. Two metabolites of progesterone, allopregnanolone (3α5αTHP: 3α5α-tetrahydro-progesterone) and pregnanolone (3α5βTHP: 3α5β- tetrahydroprogesterone), and sulfated steroids as well, act on GABAA receptors and potentiate their inhibitory function in the CNS (Covey et al., 2000; Keller et al., 2004; Majewska, 1992; Schlichter et al., 2006). Apart from GABAA, pregnanolone significantly reduced GlyR function as well (Jiang et al., 2006). Pregnenolone sul- phate (PES) has been shown to modulate the activity of NMDA receptors and a variety of other ionotropic receptors, therefore PES increases neurotransmitter release from a variety of preparations and it affects the strength of synaptic transmission; the chronic IP administration of PES prevents the development of morphine tolerance (Gibbs et al., 2006b; Mameli et al., 2005; Reddy and Kulkarni, 1997). Both PES and the androgen dehydroepiandrosterone sulfate (DHEAS; see below in the section “Androgens”) bind to sigma receptors (sigma1 and 2), which are nonopioid, nonphencyclidine receptors (Monnet et al., 1995). The sigma1 receptor has been cloned and its sequence does not resemble that of any mammalian protein, whereas sigma2 receptors have not been cloned. Sigma1 agonists, although having no effects by themselves, caused the amplification of signal transductions incurred upon the stimulation of the glutamatergic, dopaminergic, IP3-related metabotropic, or nerve growth factor-related systems. Inasmuch as this receptor has a significant role in the pain mechanisms, we may not exclude the role of its activation by these neurosteroids in their antinociceptive effects (Guitart et al., 2004), but the activation of this receptor at spinal level induces mechanical allodynia by the activation of spinal NMDA receptors (Roh et al., 2008). ICV administration of allopregnanolone significantly and dose-dependently increases the pain thresholds to heat stimulus, an effect which was mediated by GABAARs (Kavaliers and Wiebe, 1987). Endogenous neurosteroids are produced in the SDH and the elevated concentration of neurosteroids seen in inflammatory pain states significantly reduces thermal heat hyperalgesia (Poisbeau et al., 2005). It has been proposed that neurosteroids could be part of an 478 G. Horvath endogenous modulatory/compensatory mechanism in response to a strong and/or sustained activation of the spinal nociceptive system (Vergnano et al., 2007). The IT administration of allopregnanolone effectively decreased both the mechanical and thermal hyperalgesia, whereas pregnanolone was only efficient on mechanical allodynia and had no effect on thermal heat hyperalgesia (Charlet et al., 2009). 6.4.2 Sexual Hormones Estrogen and Progesterone It is well known that women are more sensitive to several types of pain than men and functional bowel disorders are 2–3 times more prevalent in women (Berkley, 1997). The severity of pain symptoms fluctuates with the menstrual cycle suggesting female gonadal hormones modulate pain processing (Bartok and Craft, 1997; Kayser et al., 1996; Houghton et al., 2002; Hucho et al., 2006). Furthermore, pregnancy and parturition are associated with an opioid-mediated maternal analgesia (Dawson-Basoa and Gintzler, 1997). Estrogen has a permissive rather than a modulating function in this respect, and progesterone seems to specifically inhibit GlyRs (Mogil et al., 2003; Webb and Lynch, 2007). The change in estrogen status alone is sufficient to modify the processing of noxious sensory input to the CNS. The classical estrogen receptor exists as two subtypes, ERα and ERβ, and is expressed in the primary sensory neurons, sensory ganglia, dorsal horn, and supraspinal brain regions associated with pain modulation (Bereiter et al., 2005; Merchenthaler et al., 2004; Okamoto et al., 2008). Estrogen receptor signalling dramatically affects uterine cervical structure, and may also enhance pain responses at this level (Ji et al., 2005; Yan et al., 2007). Compared to intact rats, ovariectomy reduces the magnitude of the visceromotor responses and the response of SDH neurons, which is reversed by estradiol replacement (Ren et al., 2000; Tang et al., 2008). The data suggest that estrogens play an important role in modulating visceral nociceptive processing by increasing the spinal NMDA receptor expression and activation (Tang et al., 2008). Furthermore, chronic estrogen treatment increases spontaneous activity of afferents that innervate the uterine cervix, and enhances afferent firing in response to cervical distension, and TRPV1 receptor function is important for estrogen-induced sensitization (Yan et al., 2007). The effects of progesterone are mediated by two distinct nuclear receptor proteins, PRA and PRB. Some studies reported antinociceptive effects of progesterone. Lactating rats with a high level of progesterone demonstrated significantly less hyperalgesia and progesterone replacement in ovariectomised rats significantly attenuated inflammation-induced hyperalgesia (Ji et al., 2005; Ren et al., 2000). It is thought that progesterone’s antihyperalgesic effects include suppression of NMDA receptor activation at the level of the spinal cord (Ren et al., 2000). Because the increase in estradiol and that in progesterone coincide during the estrous cycle, the pronociceptive effect of estradiol and the antinociceptive effect of progesterone may obscure each other, reducing fluctuations during the course of the estrous cycle. Dawson-Basoa and Gintzler (1996, 1997, 1998) have performed studies of the interaction of β-estradiol and progesterone and their potential mechanism of action in Endogenous Antinociceptive Ligands 479 respect of pain sensitivity. Simulation of the pregnancy blood profile of β-estradiol and progesterone in nonpregnant, ovariectomised rats have resulted in a statistically significant elevation of the pain threshold in the electric foot shock test, suggesting that the entire pregnancy profile of steroid hormones is responsible for the mani- festation of analgesia. As regards the mechanism, it is proposed that the analgesia during pregnancy may result from direct effects of estrogen and progesterone on the CNS. The activation of the receptors caused a 52% increase in the opioid receptor binding density and in the concentration of β-endorphin in the preoptic area (Bridges and Ronsheim, 1987). Additionally, estrogen has been shown to positively regulate pro-ENK mRNA levels in the ventrolateral aspect of the ventromedial hypothala- mic nucleus (Romano et al., 1989). These observations may suggest t hat the opioid systems mediating the analgesic effects of estrogen and progesterone are modulated in a synergistic fashion; accordingly, pregnancy provides a special case of antinociceptive interaction between these endogenous ligands. Androgens Men are typically reported to have higher pain thresholds than women, and gonadal hormones, particularly testosterone for males, contribute to this effect. Gonadectomy in adult male rats enhanced inflammation-induced sensitivity to mechanical stimulation and the effects was reversed by testosterone, and the antihyperalgesic potential of morphine decreased in neonatally gonadectomised male animals (Cicero et al., 2002). According to another study, castration reduces both opioid and nonopioid SIA in rats, which was reversed by testosterone replacement (Romero et al., 1988). However, other reports suggest pronociceptive potential of testosterone, because castration induces analgesia in the late phase of the formalin test, which correlates with increased 5-HT level in the SDH (Nayebi and Ahmadiani, 1999; Nayebi and Rezazadeh, 2004). IT administration of testosterone caused analgesia in neuropathic rats (Kibaly et al., 2008). As regards the weak androgens originated primarily from the adrenal cortex (i.e. dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS)), they are abundant in the brain even after adrenalectomy and gonadectomy (Corpechot et al., 1985). Both DHEA and DHEAS can inhibit the GABA- and Gly-induced current, and the chronic systemic (IP) administration of DHEA or DHEAS elevates the basal nociceptive threshold, and prevents the development of morphine tolerance (Majewska, 1992; Ren et al., 2004; Webb and Lynch, 2007). As regards the role of DHEA at the spinal level, its level drops in neuropathy in the SDH (Kibaly et al., 2008). Behavioral anal- ysis shows a rapid pronociceptive and a delayed antinociceptive action of acute DHEA treatment, and the inhibition of its synthesis evokes analgesia. In contrast, the peripheral administration of DHEAS has significant hyperalgesic and vasodilatory actions through histamine release (Uchida et al., 2003). 6.4.3 Glucocorticoids Glucocorticoids (mainly cortisol) are considered to be essential stress hormones and their levels increase immediately after injury, pain, and the like. The basal 480 G. Horvath level of glucocorticoids is critical for the expression of analgesia, playing a permissive role in this process and they have an important role in the SIA (Panocka et al., 1987; Sutton et al., 1994). The glucocorticoid receptors are widely distributed both centrally and peripherally, and similarly to other steroids, the glucocorticoids also possess genomic and nongenomic actions (DeLeon et al., 1994; Joels, 1997). At the genomic level, the glucocorticoids repress transcription of a number of proinflammatory gene products that include the proinflammatory cytokines and enzymes such as PLA2, COX2, and inducible NOS (Niederberger et al., 2007; Sorrells and Sapolsky, 2007). Glucocorticoids are also involved in the induction of anti-inflammatory products such as interleukin-10 and annexin-A1 (Ayoub et al., 2008; Sorrells and Sapolsky, 2007). The nongenomic actions of the glucocorticoids are very rapid, occurring within minutes, and are dependent on the cytoplasmic glucocorticoid receptor (Buckingham et al., 2006). Glucocorticoids reduce PGE2 biosynthesis by either inhibition of PLA2 activity or inhibition of COX2 protein induction, the former effect considered to be a nongenomic, and the latter a genomic, effect. All of these actions decrease the release of pronociceptive ligands. Therefore, systemic, local, and IT administrations of glucocorticoids decrease the hyperalgesia, and cortisols are routinely given to patients with different pain syndroms (Ferreira et al., 1997; Taguchi et al., 2007). However, other studies have shown that chronic stress induces a long-lasting hyperalgesia, which is inhibited by glucocorticoid receptor antagonists (Khasar et al., 2008). Furthermore, the ICV administration of glucocorticoids decreases both the opioid- and clonidine-induced antinociception (Capasso et al., 1992; Capasso and Loizzo, 2001). Thus, the glucocorticoids influence both the antinociceptive and pronociceptive processes, and the net effects may depend on the qualities of activating factors and their duration. 7 Gases 7.1 Nitric Oxide (NO) It has been recognized that NO serves as an important intracellular and intercellular messenger molecule in the PNS and CNS, and functions in a variety of physiological and pathophysiological processes (Mizutani and Layon, 1996; Wu and Morris, 1998). NO, a free radical gas, is not stored in synaptic vesicles and released by exocytosis. Three different NOSs are responsible for NO synthesis: neuronal (nNOS or NOS1), endothelial (eNOS or NOS2), and inducible NOS (iNOS or NOS-3) (Boehning and Snyder, 2003). Modulation of nNOS activity by multiple signalling cascades permits the regulated production of NO in response to neuronal stimulation. Unreacted NO has been assumed to simply diffuse away from target areas, but recent studies have suggested an enzymatic inactivation system, thus, myeloperoxidase, an enzyme highly enriched in leukocytes, also regulates NO bioavailability. Once NO is synthesized from L-arginine, it quickly diffuses from one neuron to another neuron and acts on the soluble guanylyl cyclase (sGC) to Endogenous Antinociceptive Ligands 481 stimulate the formation of cGMP. This appears to be the principal mode for mediating the effects of NO in the neurons. However, NO also acts by modifying the transition metal centers of a wide variety of proteins. It can function by selectively and reversibly S-nitrosylating cysteine residues on a wide variety of proteins with precise spatial and temporal resolution. These proteins may be ion channels, pumps, or metabolic enzymes; for example, S-nitrosylation activates L-type Ca 2+ channels, Ca 2+ -activated K + -channels, and GABAA receptors, but it inhibits NMDA receptors and several classes of Na + -channels. The role of NO in the nociceptive processes is very controversial. Several lines of evidence have shown that inhibition of NO production reduces pain hypersensitivity; however, other studies suggest the opposite role of NO. Systemic administration of NOS inhibitors have reversed pain hypersensitivity (Chu et al., 2005; Crosby et al., 1995; Mabuchi et al., 2003). Disruption of nNOS partially reduced inflammation- induced mechanical pain hypersensitivity (but not thermal hyperalgesia) ( Chu et al., 2005), whereas others have not found this effect in other pain models (Crosby et al., 1995; Tao et al., 2004). Findings support the view that a condition of chronic stress can enhance hyperalgesia induced by systemic nitroglycerin administration (Costa et al., 2005). These observations may be relevant to pain disorder, and particularly to migraine, because nitroglycerin is able to induce spontaneous-like pain attacks in humans. NO appears to play a promoting role in supraspinal pain transmission, inasmuch as a number of NOS inhibitors exhibit potent antinociceptive activities on systemic or ICV administration (Pelligrino et al., 1996). In contrast, an antinociceptive property of brain NO has also been reported in acute mechanical and heat pain tests (Kawabata et al., 1992). Considerable evidence has demonstrated that NO and its enzymes are involved in the central mechanisms of pain at the spinal cord level (Chu et al., 2005; Malmberg and Yaksh, 1993; Meller et al., 1992a;Tao et al., 2003). IT NOS inhibitors have decreased the neuropathic and inflammatory pain sensitivity, and significantly decreased the C-fiber-evoked activity (Chu et al., 2005; Meller et al., 1992b; Meller et al., 1994b; Meller et al., 1994a; Zhang et al., 2005). Moreover, many of the effects of NMDA receptors in inflammatory hyperalgesia appear to be mediated through the production of NO (Chu et al., 2005;Li et al., 1994b; Mabuchi et al., 2003; Meller et al., 1992a; Tao et al., 2004). Given the link between NMDA receptor activation and production of nitric oxide, it is not surprising that activation of NOS and subsequent production of NO is one of the signal transduction systems shown to be involved in the behavioral response to pain stimuli (Meller et al., 1994c). All of these data indicate that the spinal NOS is essential for pain hypersensitivity. However, the effects of NO on neuronal firing differ among cell types, and NO production can lead to inhibition of neuronal firing and transmission at spinal level (Ma and Eisenach, 2007; Song et al., 1998; Xu et al., 1996a). Thus, spinal NO is directly involved in the analgesic effects of morphine and clonidine and acetyl- choline (Lauretti et al., 2000; Ma and Eisenach, 2007; Pan et al., 1998; Song et al., 1998; Xu et al., 1996a; Xu et al., 1997; Xu et al., 2000). As regards the role of NO at the peripheral level, locally released NO also plays a dual role in nociceptive modulation, inducing either nociceptive or antinociceptive responses depending on 482 G. Horvath its amount and tissue level (Kawabata et al., 1994a). Noxious heat induces NO generation causing SP release from the peripheral endings of small-diameter primary afferent neurons (Yonehara and Yoshimura, 1999). Inflammation also enhances peripheral release of NO, which may contribute to edema and hyperalgesia (Lawand et al., 1997; Omote et al., 2001). NO may play an important role in neurogenic inflammation through enhancement of the release of neuropeptides by activating small-diameter primary afferent neurons (Yonehara and Yoshimura, 1999). In contrast, the local increase in NO level can decrease the mechanical hyperalgesia through an increase in cGMP (Steiner et al., 2001). Furthermore, the peripheral MT-induced antinociception is inhibited by NOS inhibitor, because NO production can lead to opening K + -channels (Hernandez-Pacheco et al., 2008). Several other observations also indicate that NO donors inhibit the ongoing mechanical nociceptor supersensitivity, and NOS inhibitors enhance the hypersensitivity (Ferreira et al., 1991; Ferreira et al., 1992; Lorenzetti and Ferreira, 1996). The simplest explanation for these conflicting observations may be that the role and importance of the pathway varies among the groups of primary sensory neurons mobilized by different types of nociceptive stimuli. 7.2 Carbon Monoxide (CO) It has been long recognized that the gaseous compound CO is noxious and harmful. CO is generated by haeme oxygenase (HO) that degrades haeme in aging red blood cells giving rise to biliverdin, iron, and CO (Boehning and Snyder, 2003). HO activity can be induced in almost all cell types by cellular stressors. Molecular cloning has revealed three types of HOs, the highly inducible isoform termed HO1, and two constitutive expressed isoforms termed HO2 and HO3. HO2 is selectively concentrated in the brain and testes, and it is colocalized with sGC throughout multiple brain regions. CO activates sGC to generate cGMP, but it can also directly activate different K + -channels (Boehning and Snyder, 2003). Since the beginning of the 1990s, a growing body of evidence has given support to the physiological actions of CO as a vasoactive substance and a neurotransmitter/modulator. Thus, as with NO, CO is also a labile gaseous messenger in the nervous system (Boehning and Snyder, 2003; Verma et al., 1993). Because the nociceptor activity and excitability may be modulated by intracellular cGMP, CO can influence the pain sensitivity in a cGMP-dependent manner (Duarte et al., 1992; Sousa and Prado, 2001). Various studies have suggested that CO regulates nociception and a part of them indicate pronociceptive effects. Thus, the lack of HO2 enzyme has not changed the normal heat and mechanical threshold, but it reduced the hyperalgesia in inflammatory or nerve injury models; it has not modified the potency of morphine, but it has prevented morphine tolerance (Li and Clark, 2002; Li and Clark, 2003; Liang et al., 2003; Liang et al., 2004). Furthermore, the IT administration of HO inhibitors has not influenced acute heat pain latency, but inhibited pain-related behaviors and increased the potency of morphine (Li and Clark, 2001; Li and Clark, 2002). Thus, the production of CO at the spinal level is important in the behavioral expression of Endogenous Antinociceptive Ligands 483 acute mechanical hyperalgesia, but is not involved in thermal hyperalgesia (Meller et al., 1994c). However, another study has found opposite results, that is, IT HO inhibitor increased, whereas CO substrate decreased the formalin-induced behavior (Nascimento and Branco, 2008). IPL administration of an HO inhibitor potentiated mechanical hyperalgesia and the formalin-induced behavior, and the increased CO level decreased the hypersensitivity by increasing the intracellular level of sGC (Nascimento and Branco, 2007; Rosa et al., 2008; Steiner et al., 2001). The effect of CO has been prevented by the NOS blocker, suggesting that the effect of CO depends on the integrity of the NO pathway. In conclusion, our knowledge of the role of CO in the nociceptive processes is incomplete, therefore further studies are required to reveal its role at different levels. 7.3 Hydrogen Sulfide (H 2 S) H 2 S is now considered a novel gasotransmitter in peripheral tissues and the CNS (Boehning and Snyder, 2003; Qu et al., 2008). Similarly to CO and NO, H 2 Salso exists in the brain in relatively high concentrations (50–160 μM), and there is a cross-talk between H 2 S and NO. H 2 S is formed from cysteine by cystathione β-synthase (CBS) and cystathione γ-lyase (CSE) (Szabo, 2007). CBS transcript levels are high in the brain, with little or no CSE. CBS is activated by stimulation of ionotropic glutamate receptors in the presence of Ca 2+ . Much progress has been made in the past decade in elucidating the roles of H 2 S in physiological and pathological conditions at the cellular level (Bhatia et al., 2005; Qu et al., 2008; Szabo, 2007). It increases cAMP level, which stimulates PKA to phosphorylate and activate postsynaptic NMDA receptors. H 2 S also upregulates GABAB receptors, therefore, H 2 S can act presynaptically to inhibit neurotransmission (Boehning and Snyder, 2003). However, H 2 S directly activates T-type Ca 2+ channels as well, and H 2 S may play a part in maintaining the excitation/inhibition balance (Kawabata et al., 2007). Systemic administration of H 2 S donors inhibits acute and inflammatory visceral nociception by opening ATP-sensitive potassium channels (Distrutti et al., 2006b; Distrutti et al., 2006a). However, the H 2 S may play a dual role in inflammatory hypernociception (Cunha et al., 2008). Production of endogenous H 2 S during inflammation mediates the induction of mechanical hypernociception. The pronociceptive role of H 2 S seems to be closely associated with upregulation of neu- trophil migration to the inflammatory site, and the activation of T-type Ca 2+ channel activity. On the other hand, the direct action of H 2 S on peripheral nociceptive neurons can produce antinociception by the activation of peripheral K + ATP channels. Only one study demonstrated the effect of H 2 S at the spinal level (Maeda et al., 2009). In isolated DRG neurons, H 2 S donor (sodium hydrosulfide: NaHS) facili- tated T-type calcium channel-dependent currents, and caused hyperalgesia, and this effect was blocked by a T-type channel inhibitor. More results are available about the peripheral effect of H 2 S. H 2 S biosynthesis is increased following IPL injection of carrageenan, local administration of NaHS induces a mechanical hypernociception, and the hyperalgesia was decreased by H 2 S synthesis inhibitor or by T-type 484 G. Horvath calcium channel blockers (Bhatia et al., 2005; Cunha et al., 2008; Kawabata et al., 2007; Maeda et al., 2009). NaHS also excites capsaicin-sensitive primary afferents and evokes a peripheral release of neurokinins (Patacchini et al., 2004). Intracolonic administration of NaHS caused visceral pain-like nociceptive behavior and referred abdominal hyperalgesia (Matsunami et al., 2009). Retrograde injection of NaHS into the pancreatic duct induced expression of Fos-protein in the superficial layers of the SDH, and the pancreatitis-induced referred pain was decreased by inhibition of the H 2 S enzyme (Nishimura et al., 2009). All of these experiments suggest aroleofH 2 S as a nociceptive messenger in the periphery. The mechanisms of H 2 S action in these processes are dependent on the direct modulation of T-type Ca 2+ channel activity in nociceptors and independent of K + ATP channels (Maeda et al., 2009). However, another study provides evidence suggesting a nociceptive- intensity-dependent role for peripheral H 2 S in nociception. Topical administration of H 2 S donor increases the nociceptive behavior of formalin, whereas the H 2 S level decreases in the s pinal cord with hind paw injection of formalin (Lee et al., 2008). Because H 2 S inhibits microglia production of proinflammatory cytokines and nitric oxide (Hu et al., 2007), a decrease in spinal H 2 S is pronociceptive i n the formalin test by virtue of disinhibition of microglial function. All these preliminary reports suggest a complex role of H 2 S in the pain mechanism, and further studies are required to reveal the central role of H 2 S in these process. 8 Conclusions Knowledge of the pathophysiology of pain has evolved substantially inasmuch as the more current hypotheses incorporate gene–environment interactions, endocrine, immunological, and metabolic mediators, and cellular, molecular, and epigenetic factors of plasticity. However, enormous gaps in the knowledge of pain and its treatment persist. The data reveal that the actions of the endogenous substances are very different depending on the type of ligands, the pain tests, and the route of administrations. Activation of the different receptors may produce anti- or pronociception depending on the types and localization of the binding sites. In an ideal case the ligand induces analgesia by the presynaptic inhibition of excitatory neurotransmitter release, and the postsynaptic inhibition of the effects of excitatory neurotransmitters or increase of the release of endogenous inhibitory transmitters from the neurons. Accordingly, the simultaneous engagement of pre- and postsynaptic mechanisms by a combination of drugs may magnify the effects produced by either drug acting at one site independently. Furthermore, both the coordination and plasticity of cellular responses to different receptor activation could be influenced by variables such as the types and numbers of receptors present in each cell type, physical or functional compartmentalisation of the signalling components, and differential and/or overlapping sensitivities to various ligands and/or costimulation with other receptor types. Another crucial factor that underlies the efficacy of a drug is the “robustness” of the network that the compound targets, because the ligands could express their effects at several levels of the pathways. Moreover, all of these variables need to . relevant to pain disorder, and particularly to migraine, because nitroglycerin is able to induce spontaneous-like pain attacks in humans. NO appears to play a promoting role in supraspinal pain transmission, inasmuch. 2004). Furthermore, the IT administration of HO inhibitors has not in uenced acute heat pain latency, but inhibited pain-related behaviors and increased the potency of morphine (Li and Clark, 2001;. increase in NO level can decrease the mechanical hyperalgesia through an increase in cGMP (Steiner et al., 2001). Furthermore, the peripheral MT-induced antinociception is inhibited by NOS inhibitor,