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The neuropeptide galanin is found in the central and peripheral nervous systems. It may have excitatory or inhibitory actions via three subtypes of G-protein-coupled receptor, and it modulates the mechanosensitivity of somatic sensory fibres. We aimed to determine if galanin also modulates vagal afferent mechanosensitivity, and to localize endogenous sources. The responses of ferret and mouse gastro-oesophageal vagal afferents to graded mechanical stimuli were investigated in vitro. The effects of galanin and/or the galanin receptor antagonist galantide on these responses were quantified. Immunohistochemistry for galanin was performed in ferret and mouse proximal stomach and nodose ganglion. In ferrets, retrograde labelling of gastric afferents to the nodose ganglion was combined with immunohistochemistry. When exposed to galanin (1–10 nm), 18/31 ferret and 12/15 mouse gastro-oesophageal afferents (tension, mucosal and tension/mucosal receptors) showed inhibition of mechanosensitivity. Four of 31 ferret afferents showed potentiation of mechanosensitivity, and 9/31 were unaffected (2/15 and 1/15 in mouse, respectively). Galanin effects were reversed after washout or by galantide (10–30 nm). Galantide given alone increased mechanosensitivity. Galanin immunoreactivity was found in nodose neurones, including those innervating the stomach in ferret. Enteric neurones were also galanin immunoreactive, as were endings associated with myenteric ganglia and smooth muscle. We conclude that galanin potently modulates mechanosensitivity of gastro-oesophageal vagal afferents with either facilitatory or inhibitory actions on individual afferent fibres. Both intrinsic and extrinsic (vagal) neurones contain galanin and are therefore potential sources of endogenous galanin.
Therapies that reduce afferent signalling of mechanical stimuli from the upper gut to the central nervous system may prove to be effective in a number of gastrointestinal (GI) diseases. These include functional dyspepsia and gastro-oesophageal reflux disease (GORD), the rationale being that functional dyspepsia is characterized by increased perception of nonpainful stimuli, including gastric distension and contraction (Tack et al. 2004), and in GORD that gastric distension leads to triggering of transient lower oesophageal sphincter (LOS) relaxations, and thence GOR (Mittal et al. 1995; Blackshaw, 2001). We have developed methods to evaluate the modulatory effects of endogenous and exogenous compounds on the mechanosensitivity of extrinsic GI primary afferents. So far we have shown that metabotropic receptors to the amino acids γ-amino butyric acid (GABA) and glutamate are coupled to potent inhibition of vagal afferent mechanosensitivity (Page & Blackshaw, 1999; Page et al. 2005), and that these effects may be accompanied by inhibition of transient LOS relaxations and gastro-oesophageal reflux (Blackshaw, 2001; Zhang et al. 2002). Another group has demonstrated that κ-opioid receptors may also inhibit vagal afferent fibres (Ozaki et al. 2000), suggesting that peptidergic receptors may also represent targets on these neurones.
Galanin is a 29–30 amino acid peptide found throughout the central and enteric nervous systems. It may have either excitatory or inhibitory effects on motor function and neural excitability, depending on which of the three galanin receptors it binds to (Branchek et al. 2000). For example, it may have both pro- and antinociceptive actions in the spinal cord (Wynick et al. 2001), and may contract or relax gastrointestinal smooth muscle (see Liu et al. 2003). It has recently been shown to affect signalling in central gastric vagal pathways, with both inhibitory and excitatory effects being demonstrated (Yuan et al. 2002; Tan et al. 2004). Its direct effects on intrinsic neurones of the enteric nervous system are specific to sensory (AH) neurones (Liu et al. 2003). We considered the possibility that galanin may also have peripheral actions on extrinsic sensory neurones which may be of importance in modulation of visceral sensation and reflex control of GI function. The aims of this study were therefore to determine the effects of galanin on mechanosensitivity of vagal afferents in two species (rodent and nonrodent), and the distribution of the peptide peripherally and in vagal sensory ganglia.
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Our data indicate that galanin potently influences responses to mechanical stimuli in several types of gastro-oesophageal vagal afferents in ferret and mouse. It may have either facilitatory or inhibitory actions, with facilitation seen mostly in TM receptors in the population studied. Actions were restricted to changes in mechanical responsiveness, as changes in spontaneous discharge were not seen. However, whether galanin affects mechanotransduction mechanisms separately from general excitability could not be determined. The effects of galanin are reversible upon washout or with a competitive galanin receptor antagonist. The effect of the antagonist alone is an indication that endogenous release of galanin may be able to modulate mechanosensitivity. This is not surprising as there are several peripheral sources of galanin. We have shown using immunohistochemistry in mouse and ferret that both intrinsic (myenteric) and extrinsic (vagal) neurones contain galanin, and importantly that vagal afferent galanin-containing neurones project to the stomach.
The presence of galanin in enteric nerves has been established in a number of mammalian species (Melander et al. 1985; Furness et al. 1987) including humans (Singaram et al. 1991, 1994), and its presence in vagal afferents has been shown in the rat (Calingasan & Ritter, 1992). Our data extend these observations, and confirm them in both locations in the ferret and mouse. Moreover, we have shown vagal afferents that project to the stomach contain galanin as do other vagal afferent cell bodies in the nodose ganglia. Both vagal and enteric sources are candidates for providing ongoing modulation of mechanosensitivity by endogenous release of galanin. Galanin is released from the isolated perfused gut by distension at a rate of 2.4 pmol min −1. This would most probably result in local concentrations equivalent to or higher than those we administered in this study (Harling et al. 1991). Circulating levels of galanin are approximately 18 pmol l−1 in humans, but would be several orders of magnitude higher at the site of release. These considerations implicate galanin as a strong candidate for an ongoing modulator of extrinsic mechanosensitive reflexes. Galanin has also been suggested to play a predominantly inhibitory role in intrinsic reflexes based on anatomical and functional evidence (Pham et al. 2002; Liu et al. 2003). Because galanin may have facilitatory or inhibitory effects on vagal afferents, the resulting change in the signal received by the central nervous system from the periphery would depend on the net effect on the whole population, or on the specific central targets of each population of facilitated and inhibited fibres.
The combined observations that galanin modulates mechanosensitivity in vagal afferents, and that it is contained within their cell bodies, indicate the possibility of an autoregulatory role of galanin, in addition to the role of galanin from other sources. Although we have shown evidence of galanin in the cell bodies of gastric afferents, an autoregulatory role would require galanin to be present at peripheral vagal endings from which it could be released. Endings of vagal tension receptors in the stomach and oesophagus have been identified as intraganglionic laminar endings, or IGLEs (Zagorodnyuk & Brookes, 2000; Zagorodnyuk et al. 2001), which have been shown to exist in mouse (Fox et al. 2000). These vagal endings have collaterals that form intramuscular arrays, or IMAs, in the longitudinal and circular muscle layers. It is possible that the abundant galanin positive endings we found in the mouse stomach may be IGLEs and IMAs; however, we did not see evidence for a laminar structure, and the endings were not identified by anterograde tracing.
In order to mediate both positive and negative effects on neuronal function, galanin acts on three different receptor subtypes: GALR1 and GALR3 are coupled positively to K+ channels and may also reduce cyclic AMP, giving rise to hyperpolarization (Branchek et al. 2000); GALR2, on the other hand, is coupled positively to phospholipase C, resulting in cellular excitation (Branchek et al. 2000). Therefore, it is highly likely that the inhibitory effects we observed are mediated via GALR1 and/or GALR3, and the potentiating effects are via GALR2. The divergent effects of galanin we observed on vagal afferents (see Fig. 2) could be accounted for by widely different expression of different GALR subtypes on different afferent fibres, although this remains to be demonstrated. So far there is only evidence for localization of GALR1 in the GI tract (Pham et al. 2002). Unfortunately, at the time of this study, there are no selective antagonists for any of these receptors, so more precise identification of the receptors mediating increases and decreases in mechanosensitivity is not possible. The potency of galanin was high in both positive and negative effects we observed, being within the range of affinity found for both human and rat GALR1 and GALR2. GALR3 has a slightly lower affinity for galanin and analogues (Branchek et al. 2000), but none of these analogues can as yet be used to distinguish the predominant receptor active in a system. Galantide is a nonselective GALR agonist, and the reasons for its use in this study were firstly to gain insight into the role of endogenous galanin, and secondly to determine if effects of galanin are reversible by an antagonist and therefore mediated via conventional galanin receptors. It was effective in achieving both of these aims. However, clearly there will have to be major advances in drug development before better tools are available, and the possibility remains that galantide may have as yet undiscovered actions other than as a GALR antagonist.
Although ours is the first report of the actions of galanin on vagal afferents, there are existing accounts of its actions on somatic afferents that are interesting to compare. Studies on rat knee joint afferents (Heppelmann et al. 2000) and on rat skin nociceptors (Flatters et al. 2003) both found subpopulations of afferents whose mechanical or thermal sensitivity was increased or decreased by galanin. Effects were observed both in vitro and in vivo, and proportions of fibres affected in each direction are comparable with our data. The antagonist galantide was similarly able to increase mechanosensitivity of knee joint afferents (Heppelmann et al. 2000). Interestingly, in a model of neuropathic pain, the proportion of afferents inhibited by galanin increased compared to controls (Flatters et al. 2003), suggesting that GALR1 and/or GALR3 may be upregulated in this condition. This has led to speculation that galanin receptors may be therapeutic targets in conditions involving somatic pain (Branchek et al. 2000; Wynick et al. 2001). We propose that they may also be targets in diseases that involve disordered afferent signalling from the GI tract. The relatively higher frequency at which we encountered inhibitory effects compared to excitatory effects suggests that agonists aimed at GALR1 or GALR3 may have more therapeutic potential for reducing mechanosensory function than antagonists for GALR2.
In conclusion, we have discovered a novel role for a peptide that was discovered in the gut some two decades ago. This new role is in the modulation of extrinsic afferent function of the upper GI tract, and fits alongside the roles already demonstrated for galanin in intrinsic control of gut secretion and motility. It shows parallels with the function of galanin in the somatic sensory innervation, and further exemplifies the sophisticated control of the extrinsic sensory innervation of the gut.