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Genes Dev 18:1241–1250 Zhang XH, Kangsamaksin T, Chao MS, Banerjee JK, Chasin LA (2005) Exon inclusion is dependent on predictable exonic splicing enhancers. Mol Cell Biol 25:7323–7332 Zorio DA, Blumenthal T (1999) Both subunits of U2AF recognize the 3  splice site in Caenorhabditis elegans. Nature 402:835–838 Neurochemistry of Endogenous Antinociception Gyongyi Horvath Abstract It is well known that a multitude of ligands and receptors are involved in the nociceptive system, and some of them increae, whereas others inhibit the pain sensation both peripherally and centrally. These substances, including neuro- transmitters, neuromodulators, hormones, cytokines, and the like, may modify the activity of nerves involved in the pain pathways. The organism itself can express very effective antinociception under different circumstances (e.g., stress), and during such situations the levels of various endogenous ligands change. Accordingly, a very exciting field of pain research relates to the roles of endogenous ligands. This chapter provides a comprehensive overview of the endogenous ligands that can produce antinociception, discusses their effects on different receptors and focuses on their action in different parts of the pain pathways. The results show that the net effect of a ligand is determined by the activation/inhibition of the different types of receptors and the location of these receptors, however, only a part of the endogenous substances has been characterized extensively in this respect. Keywords Pain · Receptor · Endogenous · Antinociception Contents 1 Introduction 418 2 Small-Molecules 420 2.1 Class I. Acetylcholine (ACh) 420 2.2 Class II. Amines 424 2.3 Class III. Amino Acids and Derivatives 431 3Purines 436 3.1 Adenosine 436 3.2 Nucleotides 437 G. Horvath (B) Department of Physiology, Faculty of Medicine, University of Szeged, H-6701 Szeged, Hungary e-mail: horvath@phys.szote.u-szeged.hu 417 J.P. Blass (ed.), Neurochemical Mechanisms in Disease, Advances in Neurobiology 1, DOI 10.1007/978-1-4419-7104-3_15, C  Springer Science+Business Media, LLC 2011 418 G. Horvath 4 Other Nonpeptide Molecules 438 4.1 Ouabain 438 5 Peptides 439 5.1 Peptide Hormones 439 5.2 Neuropeptides 447 5.3 Other Peptides 463 5.4 Cytokines 464 6Lipids 466 6.1 Endocannabinoid System and Related Fatty Acid Derivatives 466 6.2 Eicosanoids 475 6.3 Gangliosides 476 6.4 Steroids 477 7 Gases 480 7.1 Nitric Oxide (NO) 480 7.2 Carbon Monoxide (CO) 482 7.3 Hydrogen Sulfide (H 2 S) 483 8 Conclusions 484 References 485 1 Introduction Suffering from pain is a major medical, social, and economic burden worldwide, however, the ideal solution for effective pain-relief remains elusive. Understanding the neurochemistry of antinociception has advanced considerably in recent years. Pain is a dynamic phenomenon resulting from the activity of both excitatory and inhibitory endogenous modulation systems. It is well known that a multitude of substances and receptors are involved in the nociceptive system; some of them increase, and others inhibit the pain sensation both peripherally and centrally (Furst, 1999; Sandkuhler, 1996). Virtually no ligands/receptors are to be found that have not been investigated in this respect. These substances, which include neurotransmit- ters, neuromodulators, hormones, cytokines, and the like, can modify the activity of nerves involved in the pain pathways. One of the physiological functions of the endogenous system is to tonically regulate nociceptive transmission; therefore the ratio of the pronociceptive and antinociceptive ligands determines the pain sensitivity. The balance between these actions ensures effective modulation of acute pain, whereas during chronic pain the pronociceptive effects appear to prevail. It is also well known that the organism can express very effective antinociception in different circumstances, and during such situations the levels of various endogenous ligands change. Thus, endogenous antinociceptive mechanisms play an important role in the regulation of behavior under stressful circumstances. One of the first explicit nota- tions of stress-induced analgesia (SIA) came from observations of soldiers’ behavior in World War II (Beecher, 1957). Endogenous opioid peptides have been associated with SIA as its chemical mediators but other, nonopioid, mediators of SIA are known to exist as well (Ortiz et al., 2008). Not only stress may influence pain Endogenous Antinociceptive Ligands 419 sensitivity, but several psychiatric diseases can also change it. Thus, the pain threshold is increased in schizophrenic patients and animal models, and depression can result in increased pain sensitivity or increased analgesic requirement (Becker et al., 2006; Blumensohn et al., 2002;Dworkin,1994; Jackson and St Onge, 2003; Tuboly et al., 2009). Furthermore, migraine disease and other chronic pain syndromes are also based on the imbalance between pro- and antinociceptive endogenous ligands (Gagnier, 2001). The endogenous ligands can produce their effects at both peripheral and central (spinal and supraspinal) levels. The first relay in pain pathways activated by Aδ- and C-nociceptors is the spinal dorsal horn (SDH) and, as such, this represents an important site for the modulation of the pain signal. The activation of several pathways is involved in the production of analgesia including pathways that project from the amygdala, hypothalamus (arcuate nucleus: ARC, and lateral area of anterior hypothalamus: LAAH), the somatosensory cortex and the anterior cingulated cortex (ACC) to the midbrain periaqueductal grey matter (PAG) (Millan, 2002; Pilcher et al., 1988). ACC and amygdala are particularly related to the affective component of pain and ACC is also implicated in the cognitive processing of pain (Fields, 2004; Ji and Neugebauer, 2008; Neugebauer et al., 2004; Rainville et al., 1997). The hypothalamus is known to be one of the key structures involved in pain modulation and transmission (Dafny et al., 1996), and the hypothalamic fibers containing opioid neurons terminate i n PAG (Pilcher et al., 1988). The LAAH has the capacity to differentially modulate components of the pain signal (i.e., activation of this nucleus inhibits the responses to unmyelinated C-fiber activation) and not change the activity of Aδ fibers (Simpson et al., 2008). The overall effect of this would be to safeguard sensory-discriminative information that could direct motivational behaviors and, at the same time, filter out those components of the pain signal that are less relevant to emergency situations. The thalamus contributes to the emotional component of pain and in particular, the intralaminar parafascicular nucleus receives nociceptive information from the spinal cord by both the spinothalamic and spinopontothalamic tracts and its output is to the ACC. PAG represents the mechanisms whereby cortical and other inputs act to control the nociceptive “gate” in the dorsal horn of the spinal cord. PAG projects rostrally to the medial thalamus and orbital frontal cortex, and also interacts with several brainstem structures to modulate nociception including the rostroventral medulla (RVM) (Jensen and Yaksh, 1989; Sandkuhler, 1996; Smith et al., 1988; Zhao et al., 2007). RVM is considered an important source of descending control of spinal nociceptive neurons (Fields and Basbaum, 1999). RVM is the principal relay in the integration of ascending nociceptive inputs with descending outputs from rostral sites (Fields and Basbaum, 1999), as well as the major source of bulbospinal projections that terminate in laminas I, II, and V of the SDH, mostly via OFF (antinociceptive) and ON (pronociceptive) cells. Descending control of spinal nociception, which originates from the locus ceruleus (LC), is another major determinant of pain sensitivity in different behavioral and emotional states (Willis and Westlund, 1997). These descending modulations are exerted by three main neurochemical systems – noradrenergic, serotonergic and opioidergic – which interact in an intricate manner (Millan, 2002). 420 G. Horvath A very exciting and rapidly developing field of pain research relates to the roles of different endogenous ligands. The endogenous antinociceptive ligands may have potentially advantageous features: their synthesizing and breakdown enzymes (or the mechanism of their excretion) are available in the body; thus, in general they have short half-lives and they may have lower toxicity. On the other hand, most of the endogenous ligands exhibit lower specificity and affinity for their receptors as compared with exogenous drugs, and/or they exert their effects at several types of receptors at different parts of the body. Therefore, the net effect depends on the localization of the ligands/receptors, and on which receptors and where they will be influenced by a ligand. Accordingly, their effectiveness might be lower than that of synthetic drugs, suggesting that these ligands alone would not be ideal drugs for pain therapy. This chapter provides a comprehensive overview of endogenous ligands with antinociceptive potential, discussing their effects on different receptors and focusing on their action at distinct levels of neural axis. 2 Small-Molecules 2.1 Class I. Acetylcholine (ACh) Acetylcholine, the first neurotransmitter to be identified, is an ester of acetic acid and choline with the name 2-acetoxy-N,N,N-trimethylethanaminium. It plays piv- otal roles in a diverse array of physiological processes, and its activity is controlled through enzymatic degradation by acetylcholinesterase. The effects of ACh receptor (AChR) agonists and enzyme inhibitors, collectively termed cholinomimetics, in antinociception/analgesia are widely investigated. These compounds successfully inhibit pain signaling in both humans and animals, and are efficacious in a number of different preclinical and clinical pain models, suggesting a broad therapeutic potential (Jones and Dunlop, 2007; Wess et al., 2007). Both peripheral and central cholinergic components may be involved in the antinociception. For example, cholinergic stimulation of lateral hypothalamus increases the pain threshold by activating the descending inhibitory pathways (Holden et al., 2002). However, a major site of action of ACh is the spinal cord (Xu et al., 2000; Zhuo and Gebhart, 1991). Intrathecal cholinergic agents cause antinociception by mimicking the release of ACh from the spinal cholinergic nerves, whereas the inhibition it effects decreases the pain threshold suggesting a tonic activity of these neurons (Hood et al., 1997; Krukowski et al., 1997; Pan et al., 2008). Dorsal root ganglion (DRG) neurons express several markers for cholinergic neurons, and it seems that ACh is synthesized both in unmyelinated and myelinated DRG neurons (Khan et al., 2003; Matsumoto et al., 2007; Sann et al., 1995; Takeda et al., 2003; Tata et al., 2004; Vincler and Eisenach, 2004). Other data have shown that painful stimuli increase ACh level in the spinal cord releasing from the cholinergic interneurons in the SDH, and these neurons are activated by the inhibitory descending noradrenergic Endogenous Antinociceptive Ligands 421 and serotoninergicic pain modulatory pathways (Detweiler et al., 1993; Eisenach et al., 1996b; Jones and Dunlop, 2007; Zhuo and Gebhart, 1990). ACh exerts its physiological actions by binding to and activating two structurally and functionally distinct families of cell-surface receptors, the nicotinic ACh receptors (nAChRs) and the muscarinic ACh receptors (mAChRs). The nAChRs function as ACh-gated cation channels, whereas the mAChRs are members of the superfam- ily of G-protein-coupled receptors (GPCRs). The fast actions of ACh are mediated by its interaction with nAChRs, a family of pentameric ligand-gated ion channels composed of 1 or more of 17 different subunits, and these receptors are distributed widely throughout the central nervous system (CNS) and the periphery (Kalamida et al., 2007). These subunits are divided into muscle-type and neuronal-type. In the CNS the predominant nAChRs are the homomeric α7 and the heteromeric α4/β2 receptor. These receptors have been widely r eferred to as the neuronal nAChRs, and they mediate synaptic transmission of ACh by gating inward flux of Na + and Ca 2+ at diverse synapses. Molecular-cloning studies have revealed the existence of five molecularly distinct mammalian mAChR subtypes, M1–M5 (Wess et al., 2007). The M1–M5 receptors can be subdivided into two major functional classes according to their G-protein coupling preference. The M1, M3, and M5 receptors selectively couple to G-proteins of the G q /G 11 family, whereas the M2 and M4 receptors prefer- entially activate G i /G o -type G-proteins (Wess et al., 2007). Thus, M1, M3, and M5 are linked to phospholipase-C (PLC), and their stimulation leads to formation of inositol phosphates (inositol-triphosphate: IP3 and diacylglycerol: DAG) and a con- sequent increase in intracellular calcium, whereas M2 and M4 receptor activation inhibits formation of cyclic adenosine monophosphate (cAMP) through inhibition of adenylate cyclase (AC) (Jones and Dunlop, 2007). Agonist-induced activation of mAChRs leads to a wide range of biochemical and electrophysiological responses, and the precise nature of these responses and the resulting physiological effects primarily depend on the location and the molecular identity of the activated mAChR subtypes (Wess, 1996). Each of the five mAChR subtypes exhibits a distinct pattern of distribution; they are expressed in many regions of the CNS (in both neurons and glial cells) and in various peripheral tissues (Wess et al., 2007). The M1, M4, and M5 receptors are predominantly expressed in the CNS, whereas the M2 and M3 receptor subtypes are widely distributed both in the CNS and in peripheral tissues. Initial observations that nicotine might have an analgesic activity dates back to 1932 {9808}. The concept of nicotinic analgesia being superior to opioids led the songwriter, Paul Simon to memorialize the event in a song (Arneric et al., 2007). It has been found that nAchRs play a role in modulating pain transmission both centrally and peripherally, however, the results are controversial. Multiple populations of nACh receptors at both spinal and supraspinal level can modulate the transmission of nociceptive stimuli (Damaj et al., 2000; Guimaraes et al., 2000; Jones and Dunlop, 2007; Matsumoto et al., 2007). As regards the activation of nAChRs supraspinally, the ACh administered in the dorsal PAG increased the spinally orga- nized pain threshold, and this effect was inhibited by nAChR antagonists, suggesting an nAChR mediated descending pain control (Guimaraes et al., 2000). Furthermore, 422 G. Horvath the activation of neuronal nAchR in the nucleus raphe magnus (NRM) produces an antinociceptive effect as well (Bannon et al., 1998; Jones and Dunlop, 2007). Stimulation of spinal nAChRs may produce both pronociceptive and antinociceptive behaviors via stimulation of separable populations of nAChRs (Khan et al., 1998; Li and Eisenach, 2002). However, most studies found that intrathecal (IT) administration of nicotine produced antinociception, and the inhibition of nAChRs produced hyperalgesia (Li and Eisenach, 2002; Matsumoto et al., 2007; Rashid et al., 2006; Rashid and Ueda, 2002; Vincler and Eisenach, 2004; Young et al., 2008). Activation of nAChRs may enhance the inhibitory GABAergic (γ-amino- butyric acid) and glycinergic activities in the SDH (Genzen and McGehee, 2005; Kiyosawa et al., 2001; Takeda et al., 2003). It has been proposed that the increased expression of α3 and α4 subunits may contribute to the neuropathic pain, whereas inhibition of α3/β2 subunits produces a pronociceptive effect (Vincler and Eisenach, 2004; Young et al., 2008). The peripheral stimulation of nAChR excites or sensitizes peripheral sensory nerve fibres, but can also mediate cholinergic antinociception (Bernardini et al., 2001; Gilbert et al., 2001). NAChR stimulates nitric oxide syn- thase (NOS) in DRG neurons, and the synthesized nitric oxide (NO) is able to block ion channels in DRG (Haberberger et al., 2004; Renganathan et al., 2002), however, other studies suggest the activation of calcium channels by NO (Bernardini et al., 2001; Haberberger et al., 2004). There is controversy about the role of α7 nAChR at the periphery, inasmuch as Haberberger et al. (2004) found these receptors on all nociceptive neurons and activation of α7-nAChR elicited antinociceptive effects in an inflammatory pain model by peripheral mechanism (Wang et al., 2005b). However, Lang et al. (2003) could not detect these receptors peripherally, and the deficiency in this receptor did not influence pain sensitivity (Rashid et al., 2006). Centrally active muscarinic agonists are known to induce robust analgesic effects via activation of spinal and supraspinal mAChRs (Gomeza et al., 1999b;Iwamoto and Marion, 1993; Wess et al., 2007). Perhaps the clearest indication of the role of the individual mAChR subtype in antinociception has been provided by receptor knock-out (KO) mice, but plastic changes can mask the real role of the receptors/ligands (Wess et al., 2003). These data suggest that almost all the mAChRs play a significant role in the decrease of pain sensitivity (Jones and Dunlop, 2007; Wess et al., 2007). Independent of the route of administration, the analgesic effi- cacy of mAChR agonists was greatly reduced, but not abolished, in M2R –/– mice (Duttaroy et al., 2002; Gomeza et al., 1999a). On the other hand, in M2R –/– /M4R –/– double-knock-out mice, the agonist was virtually devoid of analgesic activity. These findings suggest that the M2 receptor is the predominant mAChR mediating muscarinic antinociception at the spinal and the supraspinal level, but M4 receptors also contribute to the analgesic activity (Chen et al., 2005b). Some data suggest that muscarinic antinociception is mediated by M1/M2 receptors or M1/M3 receptors in rats (Naguib and Yaksh, 1997), whereas others have failed to demonstrate the role of these receptors in antinociception (Velligan et al., 2002). It seems that M5 receptors do not play a significant role in acute pain sensitivity (Wang et al., 2004). Endogenous Antinociceptive Ligands 423 As regards the activation of mAChRs supraspinally, painful stimuli have been reported to increase the neuronal activity in the thalamus and ACh through the activation of M1 AChRs can inhibit this effect (Harte et al., 2004; Jones and Dunlop, 2007). Furthermore, M1 receptors located in the nuclei of RVM are involved in the opioid-induced antinociception (Abe et al., 2003). Data suggest that about 90% of all mAChRs in the spinal cord represent M2Rs, which provides a molecular basis for the predominant functional role of M2Rs at the spinal level (Duttaroy et al., 2002). Presynaptic M2R activation inhibits the glutamate release in the spinal cord and ACh induces a presynaptic stimulatory effect on the release of GABA by activating M1R/M2R, whereas the release of glycine (Gly) from spinal cord interneurons was increased through the activation of M3Rs (Jones and Dunlop, 2007;Lietal.,2002a; Wang et al., 2006c). The spinal cholinergic system also plays a role in the actions of opiates because spinally administered atropine can reduce the analgesic effects of systemically administered morphine in rats, and inhibition of ACh-esterase by neostigmine enhances the ability of morphine to reduce pain sensitivity (Chiang and Zhuo, 1989; Eisenach and Gebhart, 1995; Pan et al., 2008). Thus, there is strong evidence that activation of mAChR in the spinal cord results in an increased release of inhibitory transmitters along with a decrease in the release of excitatory transmitters, and this may mediate their antinociceptive effects. Evidence is also accumulated for a peripheral site of action for mAChRs in antinociception (Bernardini et al., 2001; Wess et al., 2007; Wess et al., 2003). Electrophysiological and neurochemical studies using skin and skin- saphenous nerve preparations demonstrated that muscarine-induced peripheral antinociception was abolished in M2R –/– mice, indicating that this activity is mediated by the M2R subtype (Bernardini et al., 2002; Pan et al., 2008). Stimulation of peripheral mAChRs reduces the heat-stimulated release of calcitonin gene-related peptide (CGRP) from mouse skin, this effect being absent in tissue taken from M2R knock-out mice (Bernardini et al., 2002; Wess et al., 2003). In summary, both the muscarinic and nicotinic receptor activations are very important in the antinociceptive effects of ACh, and the cholinergic system may offer a number of tractable targets for the development of pain therapeutics. 2.1.1 Choline Choline, a quaternary saturated amine (and a precursor of ACh), is generally thought of as a relatively inactive molecule, although several studies have shown that it can have direct effects on various biological systems and signal transduction pathways. Choline interacts with both nAChRs and mAChRs as a full and selective agonist at α7-containing nAChRs, and at the M1Rs as well (Alkondon et al., 1997; Carriere and El-Fakahani, 2000). The systemic administration of choline did not change the acute heat pain latency (HP), but decreased the inflammatory pain (Wang et al., 2005b). Both intracerebroventricular (ICV) and intrathecal (IT) administrations of choline produces antinociception in acute heat pain tests, and its effects are blocked by the α7-receptor antagonist, but not by atropine or naloxone (Damaj et al., 2000; Wang et al., 2005b). 424 G. Horvath 2.2 Class II. Amines 2.2.1 Epinephrine (E)/Norepinephrine (NE) Norepinephrine (4-(2-amino-1-hydroxyethyl) benzene-1,2-diol) and epinephrine ((R)-4-(1-hydroxy-2-(methylamino) ethyl) benzene-1,2-diol) are monoamines orig- inating from the adrenal medulla, sympathetic nerve terminals and the CNS. In the CNS, adrenergic cells can be found primarily in the brainstem (RVM, LC) (Milner et al., 2002; Stone et al., 2003). NE is synthesized from tyrosine as a precursor, and E is s ynthesized via methylation of NE. E/NE perform their actions on the target cell by binding to and activating adrenergic receptors (α 1 , α 2 , β 1 , β 2 , and β 3 ). They are widely distributed both centrally and peripherally, including most motor, sensory, autonomic, and neuroendocrine-related areas (Delfs et al., 2000; Egan et al., 1983; Nicholson et al., 2005; Stone et al., 2003). All of these receptors are GPCRs; α 1 -adrenoceptors couple to G q , which results in increased intracellular Ca 2+ . α 2 -Receptors, couple to G i and decrease the level of cAMP, whereas β-receptors couple to G s , and increase intracellular cAMP activity. Several data suggest that the α 2 -receptors play the most important role in pain mechanisms, primarily through the inhibition of transmitter release presynaptically, but they also inhibit the project- ing neurons (Willis, Jr., 1988). In vitro studies have suggested that α 2 -adrenoceptor agonists decrease glutamate release, and inhibit glutamate-mediated neuronal activation, and this action can also contribute to their antinociceptive potency (Faber et al., 1998; Li and Eisenach, 2001). There is conflicting evidence concerning the role of α 2 -receptors located supraspinally (Mansikka et al., 1996; Mansikka and Pertovaara, 1995; Ossipov and Gebhart, 1983). The noradrenergic innervation of the spinal cord arises from noradrenergic nuclei in the brainstem, including the A6 (locus ceruleus), the A5 (lateral reticu- lar nucleus: LRN), and A7 (in the dorsolateral pontine tegmentum) nuclei (Guo et al., 1996; Kwiat and Basbaum, 1992; Proudfit and Clark, 1991). The activity of these neurons can be modulated by ligands acting at α 2 -adrenoceptors, chang- ing the descending noradrenergic effects (Aghajanian and Vandermaelen, 1982; Andrade and Aghajanian, 1982; Mansikka and Pertovaara, 1995). Stimulation of α 2 - adrenoceptors in the LRN did not influence the mechanical hyperalgesia, whereas α 2 -adrenoceptor antagonist reversed the central hyperalgesia induced by mustard oil, without having any effects on nocifensive withdrawal thresholds of an intact limb (Mansikka et al., 1996). The RVM does not contain noradrenergic cells, but receives a dense noradrenergic projection from the A5 and A7 neurons, and these inputs affect pain modulation by RVM neurons (Fields and Basbaum, 1999). It has been shown that excitatory α 1 -adrenoceptor is present on both On and Off cells, but the inhibitory α 2 -adrenoceptor is present only on the Off cells, and the activation of On cells can be involved in the increased pain sensitivity during opioid withdrawal (Bie et al., 2003). Thus, activation of α 2 -adrenoceptors in NRM may induce antinociceptive effects (Haws et al., 1990; Proudfit, 1988). The activation of LC neurons by α 2 -adrenoceptor agonists also produces antinociception, and α 2 - adrenoceptor activation might contribute to the antinociceptive effects of amygdala . Horvath 2.2 Class II. Amines 2.2.1 Epinephrine (E)/Norepinephrine (NE) Norepinephrine (4-(2-amino-1-hydroxyethyl) benzene-1,2-diol) and epinephrine ((R)-4-(1-hydroxy-2-(methylamino) ethyl) benzene-1,2-diol). shown that painful stimuli increase ACh level in the spinal cord releasing from the cholinergic interneurons in the SDH, and these neurons are activated by the inhibitory descending noradrenergic Endogenous. central cholinergic components may be involved in the antinociception. For example, cholinergic stimulation of lateral hypothalamus increases the pain threshold by activating the descending inhibitory