Molecular basis of mechanosensitivity

Autonomic Neuroscience: Basic and Clinical 153 (2010) 58–68

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Autonomic Neuroscience: Basic and Clinical j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a u t n e u

Molecular basis of mechanosensitivity Stuart M. Brierley ⁎ Nerve–Gut Research Laboratory, Department of Gastroenterology & Hepatology, Hanson Institute, Royal Adelaide Hospital, Adelaide, South Australia, 5000 Australia Discipline of Physiology, School of Molecular and Biomedical Sciences, The University of Adelaide, Adelaide, South Australia, 5000 Australia

a r t i c l e

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Article history: Received 1 April 2009 Received in revised form 16 July 2009 Accepted 20 July 2009 Keywords: Visceral afferents Mechanotransduction ASIC channels TRP channels Mechanosensation

a b s t r a c t An organism's ability to perceive mechanical stimuli is vital in determining how it responds to environmental challenges. External mechanosensation is responsible for the senses of touch, hearing, proprioception and aspects of somatic pain. Internally, mechanosensation underlies the initiation of autonomic reflex control and all manner of visceral sensations including chronic pain. Despite our increased knowledge of the molecular identity of invertebrate proteins that convert mechanical stimuli into electrical signals, understanding the complete molecular basis of mammalian mechanotransduction is currently a major challenge. Although the number of candidate molecules that serve as mechanotransducers is ever increasing, debate currently rages as to whether or not they contribute directly or indirectly to mammalian mechanotransduction. Despite these controversies novel molecules have been identified and their contribution to mechanosensation, be it direct or indirect, have improved our understanding of the mechanisms underlying visceral mechanosensation. Moreover, they have provided potential new pharmacological strategies for the control of visceral pain. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved.

1. Introduction Mechanotransduction is the conversion of mechanical forces into electrical signals. The speed required for this conversion suggests ion channels are gated in direct response to mechanical force. Therefore to be considered a molecular component of mechanotransduction a given protein must be located within the mechanoreceptor, particularly at the site where mechanical stimuli is detected (Gillespie and Walker, 2001). Genetic and localization studies in Caenorhabditis elegans (C-elegans) and Drosophila have provided much of the groundwork for determining the families of ion channels implicated in all other forms of mechanotransduction (Fig. 1). These studies suggest the Degenerin/Epithelial Na+ Channels (DEG/ENaC) and Transient Receptor Potential (TRP) families of ion channels form molecular components of the mechanotransduction complex which detects mechanical stimuli. These channels have been located within

a wide variety of mechanoreceptors in a multitude of species and disruption of these channels results in alterations in the detection of the mechanical environment (Gillespie and Walker, 2001; Welsh et al., 2002; Goodman et al., 2004; Lumpkin and Bautista, 2005; Corey, 2006; Lumpkin and Caterina, 2007). However, a currently overlooked area for review is the field of visceral mechanotransduction. As such, this review will focus on recent published work on the molecular mechanisms with which visceral neurons transduce mechanical signals. In particular this review will focus on the mechanisms underlying intestinal mechanosensation as bladder sensation will be discussed elsewhere in this issue. Other models and systems of mechanosensation will be discussed initially for a historical perspective to understand the current leading candidates in mechanotransduction. However, it is becoming clear that visceral afferents might detect mechanical stimuli using different mechanisms than somatic afferents, inner ear hair cells or neurons in lower species (Figs. 1 and 3).

2. Mechanisms underlying the transduction of mechanical stimuli Abbreviations: ASIC, Acid Sensing Ion Channel; C-elegans, Caenorhabditis elegans; CRD, colorectal distension; DEG/ENaC, Degenerin/Epithelial Na+ Channels; DRG, dorsal root ganglia; IGLES, intraganglionic laminar endings; PAR2, Protease Activated Receptor2; QRT-PCR, Quantitative Reverse Transcription Polymerase Chain Reaction; RA, rapidly adapting; SA, slowly adapting; TRP, Transient Receptor Potential; TRPA, Transient Receptor Potential Ankyrin; TRPC, Transient Receptor Potential Canonical; TRPV, Transient Receptor Potential Vanilloid; VMR, visceromotor response; +/+, wild-type mice; −/−, null mutant mice. ⁎ Nerve–Gut Research Laboratory, Level 1 Hanson Institute, Frome Road, Adelaide, South Australia, 5000 Australia. Tel.: +61 8 8222 2077; fax: +61 8 8222 5934. E-mail addresses: [email protected], [email protected].

The key components in the process of mechanotransduction are speed and sensitivity. First, mechanotransduction needs to be fast and therefore mechanical forces need be focused directly to transduction channels (Gillespie and Walker, 2001). Secondly, mechanotransduction requires exquisite sensitivity so that varying grades of mechanical forces can be immediately directed to a mechanotransduction complex in the membrane. This rapidly opens the channel and amplifies the signal by allowing entry of large numbers of ions

1566-0702/$ – see front matter. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2009.07.017

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Fig. 1. Schematic diagrams comparing the molecular basis of mechanotransduction in a variety of cell types. A) Mechanotransduction machinery required for touch sensation in C-elegans. To date this is the most completely understood system of mechanotransduction with the identification of several known ion channels with intra and extracellular proteins. i) For body touch, MEC-4 and MEC-10 are pore-forming subunits belonging to the DEG/ENaC family. MEC-2 and MEC-6 are auxiliary subunits required for channel function in vivo. This channel complex is bound intracellularly to MEC-2 (homologous to stomatin) which in turn binds to microtubule proteins MEC-7 and MEC-12. MEC-9 is an extracellular protein connecting the channel to unique collagen proteins MEC-1 and MEC-5. In contrast ii) nose touch requires the TRP channels osm-9 and ocr-2 (Syntichaki and Tavernarakis, 2004; Drew and Wood, 2005; O'Hagan et al., 2005; Christensen and Corey, 2007; Brown et al., 2008). B) Mechanotransduction machinery of the inner ear hair cell. The mechanosensitive organelle, the hair bundle, is highly defined with an elaborated structure of stereocilia. The key proteins of the transduction apparatus are the transduction channel, the tip link, and the adaptation motor. Deflection of these sterocilia opens a non-selective cation ion channel (located in the distal tips of the stereocilia) with a high Ca2+ permeability and specific pharmacology. The proteins associated with these channels appear well defined with the Tip link (comprised of Cadherin-23 and protocadherin 15) having a clear role in mechanosensation, whilst adaptation of the channel is mediated by Myosin-1c. However, the identities of the ion channels remain controversial and enigmatic. Recent candidates have included TRPV4, TRPA1, TRPN1 (structurally similar to TRPA1 but not found in mammals) and TRPML3 (Corey, 2006, 2007; Christensen and Corey, 2007; Vollrath et al., 2007).

(Gillespie and Walker, 2001), resulting in a graded receptor potential and action potential generation (Hu et al., 2006). For the purists there are two preferred hypothesis of how mechanotransduction occurs. The first is via direct activation of a channel due to mechanical force causing tension in the lipid bilayer (Fig. 2A). The second is via linking or tethering of a channel to the extracellular matrix and/or the intracellular cytoskeleton (Fig. 2A) (Gillespie and Walker, 2001; Welsh et al., 2002; Barritt and Rychkov, 2005). Movement of this structure changes tension in all elements of the system, and the transduction channel responds by changing its open probability, therefore altering the mechanosensory signal (Gillespie and Walker, 2001; Barritt and Rychkov, 2005; Christensen and Corey, 2007). 3. Controversies: direct or indirect channel activation? In principle these mechanisms appear relatively straight forward. However, in many instances it is difficult to demonstrate how a particular channel contributes to mechanosensation. The channel may be

directly activated by mechanical force, as above, or alternatively it may act indirectly as part of a downstream signalling pathway from the mechanotransduction complex (Fig. 2B). Contrasting data from different systems has lead to controversies surrounding the mechanosensitive nature of various DEG/ENaC and TRP channels. In the case of the Acid Sensing Ion Channels (ASIC), ASIC2 and 3 negative results in one system, a heterogeneous population of isolated dorsal root ganglion (DRG) neurons, (Drew et al., 2004) lead to questions over previous findings implicating the direct involvement of these channels in mechanical signalling in specific populations of somatic afferents (Price et al., 2000, 2001). More recently a member of the Transient Receptor Potential Canonical family, TRPC1 was claimed to be a vertebrate stretch sensitive channel (Maroto et al., 2005), however, these findings could not be replicated with alternate mechanical stimuli (Gottlieb et al., 2008). It could also be argued that reduced responses to mechanical stimulation in −/− mice reflect reduced neuronal excitability rather than a specific deficit in mechanical gating. As such providing sufficient evidence that a

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Fig. 2. Proposed models by which a putative mechanotransduction channel is gated by mechanical stimuli. Putative mechanotransduction channels can be activated either directly or indirectly (Gillespie and Walker, 2001; Welsh et al., 2002; Lumpkin and Caterina, 2007) by mechanical force (black arrows). Each channel gating model can occur via two different processes. (A) Direct activation. In this model mechanical force causes (1) tension in the lipid bilayer which directly opens the channel allowing ionic influx. This would represent the model of a stretch activated channel. Alternatively direct activation may require a (2) tethered channel. This model suggests that accessory proteins, either intracellular, extracellular or both, are bound to the channel itself. Mechanical force is transferred through these proteins directly opening the channel allowing ionic influx. In both cases direct gating has a rapid time-course of channel activation. (B) Indirect activation. In this model the putative channel itself is not directly gated by mechanical force but relies on intermediate stages for activation. This potentially means a much slower time-course of activation compared with direct activation models. (1) Adjacent mechanosensitive protein, which is not directly connected to the putative channel. Mechanical force activates the adjacent mechanosensitive protein which then triggers diffusible second messenger molecules or kinase activation allowing channel gating and ionic influx. It has been suggested that this model makes such a channel mechanically sensitive but not mechanically gated (Christensen and Corey, 2007). (2) Ligand release. Mechanical force causes release of an extracellular ligand that activates the putative channel causing ionic influx.

candidate channel directly transduces mechanical stimuli is becoming increasingly difficult. In order to try and address these issues a number of criteria have recently been proposed for establishing direct mechanical activation of ion channels (Christensen and Corey, 2007). Briefly, the candidate channel should be located within the cell at the site where mechanical transduction takes place. It should be a pore-forming subunit and the response latency to mechanical stimuli should be quicker than second messenger systems (b5 ms). This is in itself a fundamental issue as many stimuli (e.g. osmotic or pressure) lack the rapid rise-time needed to determine such a fast latency. Deletion of the candidate channel should reduce or abolish the transduction current or action potential response evoked by mechanical force, whilst over-expressing the channel should increase the response. Selected mutation of the candidate channels pore-forming region should alter the pharmacology and kinetics of the mechanotransduction conductance. If the channel itself is directly activated by mechanical force, without the need for tethers, it should retain this property when transfected into other cell types (Christensen and Corey, 2007). These criteria are more easily addressed in lower species where there is greater access and understanding of the neuronal pathways underlying certain behaviours. As yet some of these criteria have not been systematically addressed for somatic or visceral mechanotransduction due to the physical and anatomical limitations which include prevention of recording mechanosensitive receptor potentials from afferent fibers (Hu et al., 2006). Determining the full contribution of a particular candidate protein to mechanotransduction may require a multitude of complimentary techniques (Christensen and Corey, 2007; Wetzel et al., 2007). To date visceral mechanosensation studies have utilized null mutant mice and combined localization studies (in neuronal cell bodies and in the peripheral nerve fibers) with electrophysiological, pharmacological and whole animal recordings to determine the contribution of a particular channel in the detection of mechanical stimuli in the viscera.

Side bar 1 Somatic Mechanosensation Somatic mechanoreceptors have cell bodies located in the dorsal root ganglia (DRG), whilst their axonal projections into the periphery can be grouped into 5 different subtypes. Briefly, afferents are classified based on their conduction velocities (Aβfibers N 10 m/s; Aδ-fibers 1–10 m/s; and C-fibers 1 m/s) and responses to mechanical stimuli. Aβ and Aδ fibers can be classified into four major mechanoreceptor subtypes, three of which are low-threshold mechanoreceptors termed slowly adapting (SA), rapidly adapting (RA) and D-hair mechanoreceptors. The fourth subtype are high-threshold mechanoreceptors, termed Aδ-fiber mechanonociceptors. The fifth class afferent are C-fiber mechanonociceptors which are unmyelinated and respond to noxious mechanical stimuli (Lewin and Moshourab, 2004).

3.1. Visceral mechanosensation Vagal and spinal afferents innervating the gut have cell bodies in either the nodose ganglia or DRG. These afferent neurons project centrally to the brainstem or spinal cord respectively, with their peripheral projections terminating at various levels within the gut wall including the mucosa, muscle layers, enteric ganglia, serosa and mesenteric attachments (Blackshaw et al., 2007). In mice at least, all vagal and spinal afferent classes tested have conduction velocities in the C-fiber range (Jones et al., 2005; Bielefeldt and Davis, 2008; Brierley et al., 2008, 2009; Malin et al., 2009). However, unlike somatosensation, visceral sensation does not have a standardised nomenclature for defining sensory afferents. This is primarily due to different investigators using varying in vitro or in vivo techniques in

S.M. Brierley / Autonomic Neuroscience: Basic and Clinical 153 (2010) 58–68

different species whilst utilizing an assortment of mechanical stimuli in different regions of the gut. This non-standardised nomenclature also stems from the heterogeneity of afferents recorded from throughout the gut. Although the structural and functional relationship of some of these afferents will be discussed elsewhere in this issue, for the basis of this review it is crucial to understand the functional classes of afferent innervating the murine gut. This is important as studies conducted on null mutant mice investigating visceral mechanotransduction mechanisms have used functional criteria to determine afferent classes.

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latency of visceral mechanotransduction is quicker than murine lowthreshold cutaneous D-hair receptors (8 ms) and markedly faster than murine cutaneous high-threshold mechanoreceptors (80 ms) (Shin et al., 2003). However, this doesn't necessarily mean that lowthreshold afferents use a direct mechanism whilst higher-threshold afferents use an indirect mechanism. The amount of mechanical force required to open a channel in a high-threshold afferent may be significantly greater which underlies the afferents ability to only respond to higher mechanical intensities. 6. Ion channels contributing to visceral mechanosensation

4. Functional classes of afferent in the murine gastrointestinal tract

6.1. Acid Sensing Ion Channel 1 (ASIC1)

Vagal gastro-oesophageal afferents can be classed into 2 subtypes, mucosal or tension (Page et al., 2002). Mucosal afferents respond to very fine mucosal stroking whilst tension sensitive afferents respond to stretch or distension (Page et al., 2002; Bielefeldt and Davis, 2008). These tension sensitive afferents are also known as, distension sensitive afferents, wide-dynamic range afferents or intraganglionic laminar endings (IGLES) in other species (Blackshaw et al., 2007). In the jejunum 3 different subtypes of vagal or spinal afferent can be classified on the basis of their response to distension and are termed: low-threshold, wide-dynamic range and high-threshold afferents (Rong et al., 2004). Lower down the gut the sensory information from the distal colon and rectum travels to the central nervous system through 2 distinct anatomic pathways: the lumbar splanchnic nerves, which terminate in the thoracolumbar spinal cord, and the sacral pelvic nerves, which terminate in the lumbosacral spinal cord. Mucosal afferents respond to very fine mucosal stroking and are prevalent in the pelvic pathway but very rare in the splanchnic pathway (Brierley et al., 2004). Muscular afferents respond to low distension pressures (e.g. b20 mm Hg; (Malin et al., 2009) or low intensity stretch stimuli (b3 g; (Hughes et al., 2009) and are prevalent in the pelvic pathway but rare in the splanchnic pathway (Brierley et al., 2004). In the pelvic pathway only a class of afferent termed muscular/mucosal responds to both mucosal stroking and low intensity stretch (Brierley et al., 2004). Serosal afferents are generally unresponsive to low-threshold stimuli and respond to much higher intensities of distension (N40 mm Hg; (Brierley et al., 2008) or stretch (N9 g;(Hughes et al., 2009). Serosal afferents are common to both the splanchnic and pelvic pathways and are most robustly characterised by compression of their receptive field with von Frey Hairs. Mesenteric afferents are found in the splanchnic innervation but unlike other classes discussed above; are not located in the intestine itself but on the mesenteric attachment (Brierley et al., 2004). Mesenteric afferents are generally unresponsive to lowthreshold stimuli and respond to much higher intensities of distension (N40 mm Hg; (Brierley et al., 2008) or stretch (N9 g; (Hughes et al., 2009) and are most robustly characterised with by compression of their receptive field with von Frey Hairs.

Interest in ASIC1 as a mechanosensory molecule stemmed from its close relationship to invertebrate DEG/ENaC channels and observations by the Welsh and Lewin laboratories of alterations in somatic mechanosensory function in ASIC2 and ASIC3 −/− mice (discussed below). However, loss of ASIC1 has no significant effect on any of the 5 populations of cutaneous mechanoreceptor (Page et al., 2004). In the viscera the Blackshaw laboratory demonstrated, with fluorescence in situ hybridisation in conjunction with retrograde tracing from the distal colon, ASIC1 expression in 30% of splanchnic afferents innervating the thoracolumbar DRG (Hughes et al., 2007). Retrograde tracing combined with laser capture microdissection and QRT-PCR determined a 1.8-fold greater expression of ASIC1 transcript in gastrooesophageal innervating nodose ganglion neurons compared to splanchnic colonic neurons innervating the thoracolumbar DRG (Page et al., 2007). Functionally, deletion of ASIC1 causes a modest increase in the sensitivity of vagal gastro-oesophageal mucosal and tension receptors (Table 1). Correspondingly gastric emptying, which is modulated by vagovagal reflexes, was decreased with ASIC1 −/− mice displaying significantly slower gastric emptying rate (Page et al., 2004, 2005). Similarly in the distal colon ASIC1 gene disruption increased the sensitivity of splanchnic colonic mesenteric and serosal afferents, as indicated by increased stimulus response functions (Table 1) (Page et al., 2004, 2005). Thresholds to mechanosensory stimuli were unaffected in these afferent subtypes whilst faecal output was unaffected in ASIC1 −/− mice. These data suggested a striking molecular difference in mechanoreceptor function in viscera compared with skin (Figs. 3 and 4) (Page et al., 2004). However, even at this early stage differences were beginning to emerge between upper gut and lower gut afferents, with differences in the sensitivity to the non-selective DEG/ENaC blocker benzamil. The mechanosensitivity of splanchnic colonic afferents was potently reduced and virtually abolished by benzamil, whereas gastrooesophageal afferents were only marginally inhibited. It was also clear that in the colon deletion of ASIC1 had no effect on the ability of benzamil to inhibit splanchnic serosal afferent mechanosensitivity (Page et al., 2007).

5. Is visceral mechanosensation quick enough for direct mechanical gating?

6.2. Acid Sensing Ion Channel 2 (ASIC2)

The speed of visceral mechanosensation in the upper and lower gut suggests that activation via mechanogated ion channels located on afferent endings is responsible for visceral mechanotransduction. In guinea-pig the mechanotransduction delay for low intensity stretch sensitive (muscular like) afferents was shown to be b2 ms in the rectum and 6 ms for vagal oesophageal mechanoreceptors. Mechanosensory responses are unaltered in 0 mM Ca2+, which prevents rapid exocytotic transmitter release (Zagorodnyuk et al., 2003, 2005). This is also the case in higher-threshold mouse colonic serosal afferents (unpublished observation). These factors combined provide strong evidence against the involvement of secondary chemical mediators in these mechanotransduction mechanisms. As such, the mechanical

ASIC2 plays a consistent role in somatic mechanosensation with disruption of ASIC2 reducing the mechanical responsiveness of slowly adapting (SA) and rapidly adapting (RA) mechanoreceptors (Fig. 3). These deficits were consistent with the somatic structures expressing ASIC2 protein in wild-type mice (Price et al., 2000). In the viscera 47% of splanchnic afferent DRG neurons expressed ASIC2 (Hughes et al., 2007), with a quantitatively similar expression in vagal gastrooesophageal neurons (Page et al., 2007). Deletion of ASIC2 caused an increase in gastro-oesophageal mucosal receptor sensitivity, similar to that observed in ASIC1 null mutants. In contrast, tension receptors had marked decreases in mechanosensory function with a 50% deficit in stimulus response functions. However, ASIC2 −/− mice displayed no change in gastric emptying (Table 1) (Page et al., 2005). In the

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Table 1 Insights into visceral mechanosensation gained from utilizing null mutant mice.

Gastro-oesophageal Mucosal afferents Tension afferents Gastric emptying

ASIC1

ASIC2

ASIC3

TRPV1

TRPV4

TRPA1

↑ ↑ ↓

↑ ↓↓ ↔

↔ ↓↓ ↔

↓

↔ ↔

↓ ↔

Jejunal Low-threshold afferents Wide dynamic range afferents High-threshold afferents Distal colon and rectum Splanchnic mesenteric afferents Splanchnic serosal afferents Splanchnic mucosal afferents Splanchnic muscular afferents Pelvic serosal afferents Pelvic mucosal afferents Pelvic muscular afferents Pelvic muscular/mucosal afferents Stool output Visceromotor response References

↔ ↓ ↔

↑

↔

↓↓

↓↓↓

↓↓↓

↑

↑↑

↓↓

↓↓↓

↓↓↓

↔ ↔ ↔ ↓↓

↓↓↓ ↔ ↔ ↔

↓↓↓ ↓ ↔ ↔

↔

↓

Page et al. (2004, 2005)

Page et al. (2004, 2005)

↔ ↔ ↔ ↓↓ ↔ ↓↓ Jones et al. (2005, 2007), Page et al. (2005), Bielefeldt and Davis (2008)

↓↓ Rong et al. (2004), Jones et al. (2005, 2007), Bielefeldt and Davis (2008)

↓↓↓ Brierley et al. (2008), Cenac et al. (2008), Sipe et al. (2008)

↓↓ Brierley et al. (2009)

This table summarizes the effects of 1) ion channel disruption on intestinal afferent mechanosensory responses and 2) the effects on whole animal functional outcomes. Arrows indicate the degree of change whilst blank cells indicate afferent classes or functional outcomes were not investigated in a particular null mutant.

colon splanchnic afferents were affected differently by ASIC2 disruption according to the subclass of fibers. Mesenteric afferents were completely unaffected by ASIC2 deletion, whereas serosal afferents became more sensitive, with almost double the response to mechanical stimulation (Table 1) (Page et al., 2005). Other parameters such as adaptation, mechanical activation thresholds, and spontaneous activity, were not affected for either afferent class. ASIC2 −/− mice also displayed a decreased number of faecal pellets produced per day. Other differences between −/− mice were also appearing with deletion of ASIC2 significantly reducing the ability of benzamil to attenuate the mechanical response of splanchnic serosal afferents (Page et al., 2007). There are limited data suggesting against a role for ASIC2 in visceral mechanosensory function (Roza et al., 2004), however, the methods utilized are somewhat indirect and are unlikely to detect the subtle differences observed between different afferent classes. 6.3. Acid Sensing Ion Channel 3 (ASIC3) ASIC3 plays contrasting roles in somatic mechanosensation as disruption of ASIC3 reduced the responsiveness of Aδ mechanociceptors, whereas RA mechanoreceptors showed increased mechanosensitivity (Fig. 3) (Price et al., 2001). In the viscera, 73% of splanchnic colonic DRG neurons express ASIC3 (Hughes et al., 2007), which equated to a 4.5-fold greater expression of ASIC3 in these neurons than gastro-oesophageal neurons in the nodose ganglia (Page et al., 2007). Correspondingly, gastro-oesophageal mucosal afferents showed no significant differences between genotypes, whereas tension receptors were consistently reduced in their sensitivity (Table 1) (Page et al., 2005). These effects were subsequently confirmed by Bielefeldt and Davis (2008), with a novel sharp electrode recording preparation of vagal neurons innervating the gastro-oesophageal region, demonstrating blunted responses to distension in ASIC3 −/− compared with wild-type controls (Table 1). In the colon ASIC3 deletion caused major and universal mechanosensory deficits (Table 1). In the splanchnic innervation loss of ASIC3 significantly reduced the responses of mesenteric and serosal splanchnic afferents (Page et al., 2005). Deletion of ASIC3 also reduced

the ability of benzamil to attenuate serosal afferent mechanosensitivity (Page et al., 2007). Subsequently using the same in vitro techniques the Gebhart laboratory showed that ASIC3 −/− pelvic muscular/ mucosal afferents had significantly reduced mechanosensory responses (Table 1). However, three other classes of afferent; serosal, mucosal and muscular remained unchanged (Jones et al., 2005). These reductions in mechanosensory function translated to a significantly reduced visceromotor response (VMR) to colorectal distension (CRD) in the whole animal (Jones et al., 2005), but no deficit in gastric emptying or stool output (Table 1) (Page et al., 2005). The Gebhart laboratory also established a key role for ASIC3 in visceral peripheral sensitization, demonstrating intra-colonic zymosan produced increased VMR in control mice, which was lost in ASIC3 −/− mice (Jones et al., 2007). The widespread and contrasting changes in mechanoreceptor sensitivity between respective ASIC −/− mice demonstrates clear differences in the mechanotransduction mechanisms between somatic, gastro-oesophageal and splanchnic/pelvic colonic afferents. It is clear from the literature that ASICs form heteromultimers (Welsh et al., 2002) and the current comparisons demonstrate different functionally active ASIC heteromultimeric complexes which is reflected in the predominant expression of ASIC3 in splanchnic colonic neurons and ASIC1 transcript in gastro-oesophageal afferents (Page et al., 2007). Generally speaking in the viscera ASIC1, 2, or 3 are all required for normal mechanotransduction (Figs. 3 and 4), whilst in somatosensation only ASIC2 or 3 are required (Fig. 3). ASIC3 is highly expressed in visceral sensory neurons; its deletion consistently decreases mechanosensory function across three different pathways (vagal, splanchnic and pelvic). ASIC3 therefore appears to make a positive contribution to mechanosensitivity, a conclusion that is emphasized by its key role in visceral peripheral sensitization. However, findings of both positive and negative effects of ASIC mutations on mechanosensitivity suggest a great complexity in the way ASICs contribute to mechanotransduction. As such ASICs are unlikely to function simply as individual mechanically gated cation channels, and in some cases they may in fact dampen the mechanotransduction process. This is the case for ASIC1 which appears to make little if any direct contribution to mechanotransduction, because

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Fig. 3. Schematic diagrams comparing the molecular basis of somatic and visceral mechanotransduction. A) Mechanotransduction channels and tethers of somatic mechanoreceptors. ASIC2 contributes to normal RA and SA mechanoreceptor function (Price et al., 2000), whilst ASIC3 contributes to normal Aδ mechanociceptor and RA mechanoreceptor function (Price et al., 2001). The stomatin like protein, SPL3, is critical for the function of a subset of cutaneous mechanoreceptors where it is thought to function as a tether for ASIC2 and ASIC3 (Wetzel et al., 2007). The T-type Ca2+ channel CaV3.2 plays a secondary role in normal D-hair receptor function probably via regulating mechanoreceptor excitability (Shin et al., 2003). TRPA1 contributes to a wide range of somatic afferents including C-fibers, Aδ-fiber mechanonociceptors, Aβ and D-hair mechanoreceptors (Kwan et al., 2009). Question marks remain over the involvement of TRPV4 in somatosensation. Although null mutant mice display behavioural reductions in their sensitivity to noxious mechanical stimuli (Liedtke and Friedman, 2003; Suzuki et al., 2003), it has yet to be shown if these changes are due to reduced sensitivity of primary afferent nociceptors innervating the skin. B) Mechanotransduction channels of visceral mechanoreceptors. A multitude of ASIC and TRP channels have been implicated in visceral mechanosensation. Interestingly ASIC1 contributes to mechanosensation in the upper and lower gut, but there is no role for this channel in somatic mechanosensation (Page et al., 2004). ASIC2 (Page et al., 2005), ASIC3 (Jones et al., 2005; Page et al., 2005; Bielefeldt and Davis, 2008) and TRPV1 (Rong et al., 2004; Jones et al., 2005; Bielefeldt and Davis, 2008) contribute to mechanosensation in the upper and lower gut, whilst TRPV4 only contributes in the lower gut (Brierley et al., 2008; Sipe et al., 2008). Recent evidence suggests TRPA1 has a major role in mechanosensation, both in the upper and lower gut respectively (Brierley et al., 2009). As yet no putative tether proteins have been identified which contribute to visceral mechanosensation.

without it mechanosensitivity is universally increased in visceral afferents, suggesting that the heteromultimeric mechanotransduction complex becomes more efficient. This complexity is highlighted by the effect of benzamil on splanchnic colonic afferent mechanosensitivity, which is markedly reduced in ASIC2 and ASIC3 null mutants, but unchanged in ASIC1 null mutants (Page et al., 2007). The complexity of issues associated with ASICs plus the sudden emergence of TRP channels, a family of evolutionarily conserved ligand-gated ion channels that contribute to detection of physical stimuli in lower species, lead to a change in focus in the search for

mammalian mechanotransduction molecules. TRP channels are also intriguing by their ability to interact with a multitude of inflammatory mediators, which (Levine and Alessandri-Haber, 2007) can alter their functionality. 6.4. Transient Receptor Potential Vanilloid 1 (TRPV1) TRPV1 is probably the best known TRP channel, through its activation by capsaicin, which when administered intra-colonically causes pronounced visceral pain (Laird et al., 2001). Deletion of TRPV1

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Fig. 4. Channels contributing to mechanosensation in the gut. A) Upper gut: the vagal pathway innervating the oesophagus and stomach possess two different types of mechanoreceptive endings termed mucosal and tension receptors. ASIC1 (Page et al., 2004), ASIC2 (Page et al., 2005) and TRPA1 (Brierley et al., 2009) contribute to mucosal receptor function. ASIC1 (Page et al., 2004), ASIC2 (Page et al., 2005) and ASIC3 (Page et al., 2005; Bielefeldt and Davis, 2008) contribute to tension receptor function. TRPV1 contributes to the mechanosensory function of distension sensitive (tension) mechanoreceptors innervating the oesophagus and stomach (Bielefeldt and Davis, 2008). Furthermore, TRPV1 also contributes to the mechanosensory function of wide-dynamic range (tension) mechanoreceptors innervating the jejunum (not shown), however these fibers may be of vagal or spinal origin (Rong et al., 2004). B) Lower gut: two different spinal pathways, termed splanchnic and pelvic, innervate the distal colon and rectum. In total five different classes of mechanosensitive afferents have been characterised, three of which (serosal, muscular, mucosal) are in both pathways. Mesenteric and muscular/mucosal afferents are specific to the splanchnic and pelvic pathways respectively (Brierley et al., 2004). 1) In the splanchnic pathway ASIC1 (Page et al., 2004, 2005), ASIC3 (Page et al., 2005), TRPV4 (Brierley et al., 2008) and TRPA1 (Brierley et al., 2009) contribute to mesenteric mechanoreceptor function, whilst ASIC1,2,3 (Page et al., 2004, 2005), TRPV4 (Brierley et al., 2008) and TRPA1 (Brierley et al., 2009) all contribute to serosal mechanoreceptor function. 2) In the pelvic pathway ASIC3 and TRPV1 (Jones et al., 2005) contribute to muscular/mucosal mechanoreceptor function, whilst TRPV4 (Brierley et al., 2008; Sipe et al., 2008) and TRPA1 (Brierley et al., 2009) contribute to serosal mechanoreceptor function. In addition TRPA1 contributes to mucosal mechanoreceptor function (Brierley et al., 2009).

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has no significant effect on somatic mechanosensory function; however intriguingly there are deficits in visceral mechanosensation particularly within bladder (Birder et al., 2002; Daly et al., 2007) and intestinal (Rong et al., 2004; Jones et al., 2005; Bielefeldt and Davis, 2008) afferents. Notably, TRPV1 expression is greater in colonic innervating neurons that those innervating the skin (Christianson et al., 2006), with 82% of thoracolumbar and 50–60% of lumbosacral colonic DRG neurons displaying TRPV1-LI (Robinson et al., 2004; Brierley et al., 2005a; Christianson et al., 2006). In the gastro-oesophageal region Bielefeldt and Davis (2008) showed deletion of TRPV1 significantly blunted responses to distension compared with wild-type controls, with deletion of TRPV1 having a more significant effect than ASIC3 deletion. In the jejunum, the Grundy laboratory demonstrated TRPV1 deletion significantly reduces the pressure response curve of wide-dynamic range fibers but has no affect low or high-threshold afferents (Rong et al., 2004). In the distal colon/rectum the Gebhart laboratory showed TRPV1 deletion significantly reduced the mechanosensory response of pelvic muscular/ mucosal afferents. However, three other classes of pelvic afferent, serosal, mucosal and muscular remained unchanged (Jones et al., 2005). Therefore, TRPV1 deletion consistently reduces the mechanosensitivity of distension sensitive gastro-oesophageal, jejunal and pelvic colonic afferents (Table 1, Figs. 3 and 4). TRPV1 −/− mice also displayed decreased VMR to CRD (Jones et al., 2005). TRPV1 plays a key role in visceral peripheral sensitization, evoked by intra-colonic zymosan administration (Jones et al., 2007) and initiates and maintains colonic hypersensitivity induced by colonic irritation in neonatal rats (Winston et al., 2007). TRPV1 is not considered to be mechanically gated therefore the effects on mechanosensation may be due to indirect effects on neuron excitability or via interactions with other receptors or TRP channels. This is highlighted by several observations; 1) 5-HT enhances TRPV1 sensitivity to capsaicin-, heat-, and proton-evoked currents in colonic DRG neurons (Sugiura et al., 2004), 2) the protease receptor PAR2 sensitizes TRPV1 through protein kinase C (PKC) activation (Amadesi et al., 2004), 3) capsaicin causes pronounced mechanical desensitization in splanchnic but not pelvic colonic serosal afferents (Brierley et al., 2005a). However, this desensitization does not occur in TRPA1 −/− mice (Brierley et al., 2009), indicating a key link between TRPV1 and TRPA1 (Levine and Alessandri-Haber, 2007) in the change in splanchnic serosal afferent function. 6.5. Transient Receptor Potential Vanilloid 4 (TRPV4) TRPV4 is the mammalian homologue of C-elegans gene Osm-9 which is critical in detecting nose touch (Liedtke, 2005). TRPV4 itself is crucial in the transduction of osmotic stimuli and is located at sites of mechanical stimulus detection including inner ear hair cells, sensory neurons, and somatic mechanosensory structures (Liedtke and Friedman, 2003). These observations combined with TRPV4 −/− mice displaying behavioural reductions in their sensitivity to noxious somatic mechanical stimuli (Liedtke and Friedman, 2003; Suzuki et al., 2003) lead to TRPV4 being considered as a mechanosensor. However, such behavioural observations should be treated with caution, as these reflexes rely on peripheral input which is strongly modulated by other systemic factors. It has not been shown if these behavioural deficits are due to reduced sensitivity of somatic primary afferent nociceptors. For each mechanotransduction candidate its role should be tested using direct recordings from primary afferents before making conclusions about involvement in mechanotransduction. However, this issue has been addressed in the viscera. In terms of expression TRPV4 appears to be enriched within certain populations of visceral DRG neurons. QRT-PCR analysis demonstrates greater TRPV4 mRNA expression in laser-captured colonic cells than in whole DRG. Within the thoracolumbar DRG TRPV4 was 20-fold more abundant in identified splanchnic colonic neurons than in whole

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thoracolumbar DRG. TRPV4 is localized in 38% of gastro-oesophageal vagal neurons (Brierley et al., 2008), 65–76% of splanchnic colonic DRG neurons (Brierley et al., 2008; Sipe et al., 2008) and 58% of pelvic colonic DRG neurons (Brierley et al., 2008). Differences between pathways are also evident; with colonic thoracolumbar and lumbosacral neurons expressing 8- and 3-fold the levels of TRPV4 in identified gastric sensory neurons in nodose ganglia respectively. In the periphery TRPV4 protein co-localized with CGRP in colonic nerve fibers in the outermost (serosal and mesenteric) layers but is scarce in intramuscular or mucosal endings (Brierley et al., 2008). Consistent with the lack of TRPV4 expression in gastro-oesophageal vagal neurons deletion of TRPV4 had absolutely no effect on vagal afferent function (Brierley et al., 2008). However, in colonic afferents, where TRPV4 is enriched, mechanosensory responses were dramatically reduced in TRPV4 −/− mice. This was the case for both splanchnic serosal and mesenteric afferents, which was consistent with TRPV4 expression in the periphery (Table 1, Fig. 4). Critically TRPV4 deletion also increased the mechanosensory thresholds of these afferents, changes which could not be attributed to altered afferent excitability as electrical thresholds and conduction velocities were identical in TRPV4 −/− and TRPV4 +/+ mice. Pelvic serosal afferents displayed similar deficits in mechanosensory responses and thresholds to those seen in the splanchnic pathway. However, as was the case in the vagal pathway, pelvic afferents sensitive to mucosal stroking and muscular tension showed no deficits in TRPV4 −/− mice. Thus, TRPV4 makes a specific and major contribution to colonic serosal and mesenteric afferent mechanosensory function (Brierley et al., 2008). If TRPV4 acts as a mechanosensor then its activation should result in an increased mechanical response to a given force. This was the case as the endogenous TRPV4 agonist 5,6-EET (arachidonic acid metabolite) caused significant potentiation of mechanosensory responses in wild-type mice, which was lost in TRPV4-null mutants. Moreover, the non-selective TRP channel blocker ruthenium red reduced mechanosensitivity in these afferents in wild-type mice, but it had no effect on those from TRPV4-null mutants (Brierley et al., 2008; Sipe et al., 2008). Consistent with TRPV4 activation in colonic afferent endings, the Vergnolle Laboratory showed that 4α-PDD, another TRPV4 agonist, caused significant TRPV4 mediated calcium influx in isolated colonic DRG neurons (Cenac et al., 2008). From these data it is clear that alterations in the pharmacology of splanchnic and pelvic colonic afferents parallel the changes in mechanosensitivity in TRPV4-null mutants, providing increased evidence for TRPV4 as a peripheral mechanosensor. Changes in colonic neuron function translated to decreased VMR to CRD in TRPV4 −/− mice or in mice with down-regulated TRPV4 expression (via intervertebral small interfering RNA delivery), the extent of which was particularly apparent at higher distension pressures (Table 1) (Brierley et al., 2008; Cenac et al., 2008). Intra-colonic administration of 4α-PDD in wild-type mice increased fos expression in the lumbosacral spinal cord and caused dose-dependent visceral hypersensitivity (Cenac et al., 2008). Therefore TRPV4's role is not only linked to visceral mechanosensation but also to visceral hypersensitivity (Brierley et al., 2008; Cenac et al., 2008; Sipe et al., 2008). The Bunnett, Vergnolle, Blackshaw and Vanner Laboratories concurrently showed evidence for a strong interaction of TRPV4 with the Protease Activated Receptor-2 (PAR2), which underlies visceral hypersensitivity (Cenac et al., 2008; Sipe et al., 2008). First, TRPV4 and PAR2 are anatomically and functionally co-expressed in large proportions of colonic innervating neurons (Cenac et al., 2008; Sipe et al., 2008). In isolated colonic DRG neurons the PAR2-activating peptide, SLIGRL, sensitizes TRPV4 mediated currents, whilst SLIGRL also activates splanchnic serosal colonic afferent fibers by a TRPV4dependent mechanism (Sipe et al., 2008). Intra-colonic administration of SLIGRL also caused enhanced VMR to CRD at higher distending pressures (N40 mm Hg) measured 6 h (Cenac et al., 2008; Sipe et al., 2008) and 24 h (Sipe et al., 2008) after administration. However, SLIGRL

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did not affect the VMR to CRD in TRPV4 −/− mice (Cenac et al., 2008; Sipe et al., 2008) at either time point, suggesting TRPV4 mediates both acute and delayed hyperalgesia induced by PAR2 activation (Sipe et al., 2008).

tivity observed in splanchnic serosal afferents (Brierley et al., 2005b) is lost in fibers from TRPA1 −/− mice (Brierley et al., 2009). However, unlike TRPV4 there appears to be little interaction between PAR2 and TRPA1 in splanchnic colonic serosal afferents (Brierley et al., 2009).

6.6. Transient Receptor Potential Ankyrin 1 (TRPA1)

7. P2X purinoceptors

TRPA1 is the mammalian orthologue of the Drosophila gene painless which is expressed in polymodal nociceptor endings and contributes to the detection of intense mechanical stimuli. For a mammalian TRP channel it has an extraordinarily large number of ankyrin repeats, thought to act as a spring, which makes it characteristically similar to TRPN1 a proposed mechanosensory transducer in Drosophila (Vollrath et al., 2007). TRPA1 was a good candidate to participate in inner ear hair cell transduction. However, negative results in TRPA1 −/− mice mean this now appears unlikely, unless another channel compensates for TRPA1 (Bautista et al., 2006; Kwan et al., 2006). In one line of TRPA1 deficient mice there is increased somatic mechanical nociceptive thresholds (Kwan et al., 2006), which is complementary to the deficits of painless Drosophila mutants. However, such changes were not evident in a separate line of TRPA1 deficient mice (Bautista et al., 2006), although these mice may still have membrane expression of a truncated non-functional TRPA1 channel (Story and Gereau, 2006). Whilst this review was under editorial consideration two publications emerged describing TRPA1 as a modulator of mechanotransduction in cutaneous sensory neurons, either through the use of TRPA1 gene deletion (Kwan et al., 2009) or via the use of a specific TRPA1 antagonist (Kerstein et al., 2009). Mustard oil, which we now know to be a TRPA1 agonist, has long been used as a visceral inflammatory model to sensitize nociceptors and provoke tissue damage (Laird et al., 2001). Increased knowledge of the function of TRPA1 gives us a more complete understanding as to how this compound causes these effects in the viscera. In other systems TRPA1 has been shown to be directly activated by irritants such as mustard oil, garlic, acrolein and the endogenous aldehyde, 4Hydroxynonenal, but it can also be indirectly activated by inflammatory mediators such as bradykinin (Levine and Alessandri-Haber, 2007; Trevisani et al., 2007). In the viscera, TRPA1 is localized in 55% of gastro-oesophageal, 54% of splanchnic colonic and 58% of pelvic colonic innervating DRG neurons (Brierley et al., 2009). TRPA1 −/− mice have markedly reduced mechanosensitivity of mucosal afferents in vagal and pelvic pathways. TRPA1 deletion also reduced the stimulus response functions of high-threshold colonic splanchnic and pelvic serosal and mesenteric afferents (Table 1, Figs. 3 and 4). Importantly, TRPA1 deletion increased the mechanosensory thresholds of these serosal and mesenteric afferents (Brierley et al., 2009), which was not be attributable to altered afferent excitability. TRPA1 −/− mice also display decreased VMR to high intensity CRD (Brierley et al., 2009). If TRPA1 acts as a mechanosensor activation of it should result in an increased mechanical response to a given force. Correspondingly, in wild-type, but not −/− mice the TRPA1 agonists mustard oil or cinnamaldehyde increased the mechanosensitivity of pelvic mucosal and serosal afferents and splanchnic serosal and mesenteric afferents (Brierley et al., 2009). In contrast, TRPA1 deletion does not alter the mechanosensory function of vagal tension receptors or pelvic muscular and muscular/mucosal afferents. Taken together these data suggest TRPA1 acts as a peripheral mechanosensor in select afferent subtypes. TRPA1 also has a role in visceral hyperalgesia in rats, with intrathecal administration of TRPA1 antisense oligodeoxynucleotide (to reduce TRPA1 expression in DRG) suppressing the colitis-induced hyperalgesia to nociceptive colonic distension and intra-colonic mustard oil (Yang et al., 2008). It is also clear that TRPA1 mediates bradykinin-induced mechanical hypersensitivity in the guinea-pig oesophagus (Yu and Ouyang, 2009), whilst in the colon bradykinin-induced mechanical hypersensi-

ATP is released from damaged cells and as such has a major role in signalling nociceptive events. P2X agonists evoke direct excitation of intestinal afferents. However, this can induce varying effects on intestinal mechanosensitivity ranging from either no change, to increased or decreased mechanosensitivity (Kirkup et al., 1999; Wynn et al., 2003, 2004; Zagorodnyuk et al., 2003, 2005; Brierley et al., 2005a; Rong et al., 2009). Of all the candidates discussed in this review the mechanism by which ATP has its effects on mechanosensitivity is probably the most indirect (Fig. 2, Ligand binding). It is likely that mechanical force results in ATP release, from certain cell types, which then act on afferents, setting their sensitivity (Cockayne et al., 2000; Cook and McCleskey, 2000). The relatively slow speed of this mechanism suggests against a direct involvement of these channels and against P2X receptors being a mechanically gated channel which is a core component of the intestinal mechanotransduction complex. 8. Concluding remarks It is becoming clear from the literature that a variety of mechanisms underlie mechanosensation. It is apparent that visceral afferents may detect mechanical stimuli using fundamentally different mechanisms than those required in other systems. There are clear similarities in the molecular mechanisms underlying mechanosensation between different regions of gut (ASIC3, TRPV1 and TRPA1). However, there is also clear disparity between different regions of gut and between different afferent subtypes innervating the same region of gut (ASIC2 and TRPV4). The clear, overriding theme is most of these channels (ASIC3, TRPV1, TRPV4, TRPA1 and P2X3) play a role in the mechanisms underlying visceral hypersensitivity. Although questions remain unanswered as to whether or not certain channels contribute directly or indirectly to mechanotransduction, the specialised function of various afferent subtypes with unique combinations of channel expression may provide potential new pharmacological strategies for the control of visceral pain and hyperalgesia. Acknowledgements I thank the National Health and Medical Research Council of Australia for an NHMRC Australian Biomedical Fellowship and project grant support at the Royal Adelaide Hospital and The University of Adelaide (LAB, SMB, GYR). I am especially grateful to Prof L. Ashley Blackshaw and Dr Grigori Y. Rychkov for ongoing discussions concerning the mechanistic idiosyncrasies and nuances of mechanotransduction. I thank Prof Michael J. Welsh, Dr Margaret P. Price, Dr John A. Wemmie (University of Iowa, USA), Prof Wolfgang Liedtke (Duke University, USA), Prof David P. Corey and Dr Kelvin Y. Kwan (Harvard Medical School, USA) for the generous use of their respective ASIC1/2/3, TRPV4 and TRPA1 null mutant mice. These mice were utilized for visceral mechanosensation studies, the results of which are discussed in this review. I also thank Dr Amanda J. Page and Dr Patrick A. Hughes who were co-investigators on many of these studies. References Amadesi, S., Nie, J., Vergnolle, N., Cottrell, G.S., Grady, E.F., Trevisani, M., Manni, C., Geppetti, P., McRoberts, J.A., Ennes, H., Davis, J.B., Mayer, E.A., Bunnett, N.W., 2004. Protease-activated receptor 2 sensitizes the capsaicin receptor transient receptor potential vanilloid receptor 1 to induce hyperalgesia. J. Neurosci. 24, 4300–4312.

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