Voltage‐gated sodium channels: (Na_V)igating the field to determine their contribution to visceral nociception

Abstract

Chronic visceral pain, altered motility and bladder dysfunction are common, yet poorly managed symptoms of functional and inflammatory disorders of the gastrointestinal and urinary tracts. Recently, numerous human channelopathies of the voltage‐gated sodium (Na_V) channel family have been identified, which induce either painful neuropathies, an insensitivity to pain, or alterations in smooth muscle function. The identification of these disorders, in addition to the recent utilisation of genetically modified Na_V mice and specific Na_V channel modulators, has shed new light on how Na_V channels contribute to the function of neuronal and non‐neuronal tissues within the gastrointestinal tract and bladder. Here we review the current pre‐clinical and clinical evidence to reveal how the nine Na_V channel family members (Na_V1.1–Na_V1.9) contribute to abdominal visceral function in normal and disease states.

Introduction

Chronic visceral pain, altered intestinal motility and bladder dysfunction remain poorly managed symptoms of functional and inflammatory disorders of the gastrointestinal and urinary tracts. A lack of suitable treatments for these disorders is a major contributing factor to their debilitating nature and the large socio‐economic cost accrued by patients, their families and society (NIH, 2009; Gaskin & Richard, 2012; Enck et al. 2016). Conventional analgesics, such as opioids and non‐steroidal anti‐inflammatory drugs (NSAIDs), are unsuitable for treating chronic pain originating in the gastrointestinal and lower urinary tract, as they are associated with severe side effects. This includes tolerance, a lack of efficacy and importantly for some inflammatory gastrointestinal disorders the potential to exacerbate the disease (Sikandar & Dickenson, 2012; Farrell et al. 2014). The colon, rectum and bladder are innervated by specialised sensory afferents travelling via the splanchnic and pelvic nerves that terminate within the dorsal horn of the thoracolumbar and lumbosacral spinal cord, respectively (Brierley et al. 2004; Harrington et al. 2012; Brierley & Linden, 2014). These neurons detect both non‐noxious physiological stimuli, including muscle stretch during organ distension, and noxious mechanical and chemical stimuli such as bloating, intense distension/contraction, or the presence of inflammatory mediators (Brierley & Linden, 2014; Brierley, 2016). To encode for such wide‐ranging stimuli, visceral organs rely on an array of stimuli‐activated primary ‘sentinel’ transducers, including transient receptor potential (TRP) channels, acid‐sensing ion channels (ASIC), mechanosensitive two‐pore domain K (K2P) channels and Piezo channels (Grundy, 2002; Brierley, 2010; Christianson & Davis, 2010; La & Gebhart, 2011; Brierley, 2016; Alcaino et al. 2017). Furthermore, primary transducers and ion channels involved in sensory signalling can be modulated and controlled by G‐protein coupled receptors (GPCRs) and regulators of GPCR signalling proteins, in response to endogenous mediators (Geppetti et al. 2015; Salaga et al. 2016).

Voltage‐gated sodium (Na_V) channels are secondary in the neuronal response to non‐noxious or noxious stimuli. They perform the crucial role of regulating neuronal excitability and the key function of amplifying cation influx generated by the primary transducers to generate and propagate action potentials (Catterall, 2012; King & Vetter, 2014). Voltage‐gated potassium (K_V) channels repolarise the membrane potential following Na⁺ influx and modulate firing frequency, and have been reported to contribute to visceral hypersensitivity in peripheral neurons in animal models (Hirano et al. 2007; Qian et al. 2009; Luo et al. 2011; Du & Gamper, 2013); however, this family of ion channels is not covered within the scope of this review.

The Na_V channel family contains nine isoforms (Na_V1.1–Na_V1.9), which are encoded by nine SCN genes (SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A, SCN10A, SCN11A). Functionally, these channels are historically categorised as either tetrodotoxin‐sensitive (TTX‐S: Na_V1.1‐Na_V1.4, Na_V1.6 and Na_V1.7), or tetrodotoxin‐resistant (TTX‐R: Na_V1.5, Na_V1.8 and Na_V1.9). Anatomically, these channels display wide and diverse expression patterns across neuronal and smooth muscle cells throughout the body (Table 1), as well as cells of the immune system (including macrophages and mast cells) where they are involved in migration and phagocytosis (Bradding et al. 2003; Roselli et al. 2006; Carrithers et al. 2011; Black & Waxman, 2013). Na_V1.1, Na_V1.2, Na_V1.3 and Na_V1.6 are traditionally considered to be the predominant isoforms expressed in the brain and spinal cord, whilst Na_V1.7, Na_V1.8 and Na_V1.9 are preferentially expressed in the peripheral nervous system (PNS). Na_V1.4 is found predominantly within skeletal muscle and Na_V1.5 is the major isoform in cardiac myocytes (Catterall et al. 2005). Furthermore, Na_V channels are regulated by a range of enzymes and structural proteins, including auxiliary β‐subunits (β1, β1_B, β2, β3, β4) (Qin et al. 2003; Tseng et al. 2007), kinases and ubiquitin‐protein ligases (Feng et al. 2012; Savio‐Galimberti et al. 2012; Laedermann et al. 2015), which collectively regulate Na_V channel biophysical properties and expression.

Table 1.

Expression of Na_V isoforms in neuronal and non‐neuronal cells in different species relevant for visceral sensation and processing

NaV isoform	Species	System or tissue	Found in:	Not found in:	Reference
NaV1.1	Human	CNS	Cerebral cortex, cerebellum, hypothalamus, caudate, hippocampus, amygdala, C1 level spinal cord		GTEx Consortium et al. 2017)
	Rat	CNS	Hippocampus, cerebellum, spinal cord (dorsal horn, ventral horn, primarily grey matter restricted)	Embryonic brain and spinal cord	(Beckh et al. 1989; Westenbroek et al. 1989)
	Mouse	CNS	Cerebral cortex, cerebellum, hippocampus, thalamus, central grey, pons, medulla	Fimbria, corpus callosum	(Duflocq et al. 2008)
	Human	PNS	L3–L5		(Chang et al. 2018)
	Rat	PNS	L4–L5; L5		(Black et al. 1996; Fukuoka et al. 2008; Wang et al. 2011)
	Mouse	PNS	Colonic neurons in T10–L1 and L5–S1; T10–L1	L3–L6 dorsal and ventral roots	(Duflocq et al. 2008; Osteen et al. 2016; Hockley et al. 2017)
	Human	ENS	Colonic myenteric plexus		(Hetz et al. 2014)
	Guinea pig	ENS		Duodenal myenteric plexus	(Sage et al. 2007)
NaV1.2	Human	CNS	Cerebral cortex, cerebellum, hypothalamus, caudate, hippocampus, amygdala, C1 level spinal cord		(GTEx Consortium et al. 2017)
	Rat, cat	CNS	Cortex, hippocampus, cerebellum, hypothalamus, spinal cord grey matter		(Jarnot & Corbett, 2006)
	Rat	CNS	Hippocampus and cerebellum; embryonic brain and spinal cord		(Beckh et al. 1989; Westenbroek et al. 1989)
	Human	PNS	L3–L5		(Chang et al. 2018)
	Rat	PNS	L4–L5; L5		(Black et al. 1996; Fukuoka et al. 2008)
	Mouse	PNS	Colonic neurons in T10–L1 and L5–S1		(Chang et al. 2018; Hockley et al. 2017)
	Human	ENS	Colonic myenteric plexus		(Hetz et al. 2014)
	Guinea pig	ENS		Duodenal myenteric plexus	(Sage et al. 2007)
NaV1.3	Human	CNS	Caudate, cerebellum, cerebral cortex, hippocampus, hypothalamus, amygdala, C1 level spinal cord		(GTEx Consortium et al. 2017)
	Rat	CNS	Embryonic brain and spinal cord	Adult brain and spinal cord	(Beckh et al. 1989)
	Human	PNS	L3–L5		(Chang et al. 2018)
	Rat	PNS	L4–L5	L5	(Black et al. 1996; Fukuoka et al. 2008)
	Mouse	PNS	Colonic neurons in T10–L1 and L5–S1; DRG		(Chang et al. 2018; Hockley et al. 2017)
	Human	ENS	Colonic myenteric plexus		(Hetz et al. 2014)
	Guinea pig	ENS	Duodenal myenteric plexus		(Sage et al. 2007)
	Human, mouse	Neuroendocrine	Jejunal and colonic enterochromaffin cells		(Bellono et al. 2017; Strege et al. 2017a,b)
NaV1.4	Human	CNS		Brain	(GTEx Consortium et al. 2017)
	Rat	PNS		L5	(Fukuoka et al. 2008)
	Mouse	PNS		Colonic neurons in T10–L1 and L5–S1	(Hockley et al. 2017)
	Human	ENS		Colonic myenteric plexus	(Hetz et al. 2014)
	Human	Muscle	Oesophageal smooth muscle		(Deshpande et al. 2002)
NaV1.5	Human	CNS		Brain	(GTEx Consortium et al. 2017)
	Mouse	PNS	Colonic neurons in T10–L1 and L5–S1		(Hockley et al. 2017)
	Human	ENS	Colonic myenteric plexus		(Hetz et al. 2014)
	Mouse	ENS	Duodenal myenteric plexus		(Osorio et al. 2014)
	Human	Interstitial cells	Jejunal interstitial cells of Cajal		(Strege et al. 2003)
	Human, dog, rat	Muscle	Jejunal circular smooth muscle		(Holm et al. 2002; Ou et al. 2002; Strege et al. 2007; Beyder et al. 2016)
	Human, rat	Muscle	Colonic circular smooth muscle		(Strege et al. 2003; Beyder et al. 2016)
	Human, mouse	Muscle		Jejunal longitudinal smooth muscle	(Ou et al. 2002; Strege et al. 2007)
	Pig, guinea pig	Muscle		Jejunal circular smooth muscle	(Strege et al. 2007)
	Human	Macrophages	Macrophages		(Carrithers et al. 2007, 2011; Black & Waxman, 2013)
NaV1.6	Human	CNS	Cerebral cortex, cerebellum, hypothalamus, caudate, hippocampus		(Whitaker et al. 1999; GTEx Consortium et al. 2017)
	Rat	CNS	Cerebellum, hippocampus, spinal cord (white and grey matter)		(Tzoumaka et al. 2000)
	Mouse	CNS	Spinal cord white and grey matter		(Duflocq et al. 2008)
	Human	PNS	L3–L5		(Chang et al. 2018)
	Rat	PNS	L4–L5; L5		(Tzoumaka et al. 2000; Fukuoka et al. 2008)
	Mouse	PNS	L3–L6 dorsal and ventral roots; DRG		(Duflocq et al. 2008; Chang et al. 2018)
	Mouse	PNS	Colonic neurons in T10–L1 and L5–S1; T10–L1; T9–T13; L6		(King et al. 2009; Feng et al. 2015; Hockley et al. 2017; Inserra et al. 2017)
	Human	ENS	Colonic myenteric plexus		(Hetz et al. 2014)
	Guinea pig	ENS		Duodenal myenteric plexus	(Sage et al. 2007)
	Human	Macrophages	Macrophages		(Carrithers et al. 2007, 2011; Black & Waxman, 2013)
NaV1.7	Human	CNS	Hypothalamus		(GTEx Consortium et al. 2017)
	Rat	CNS	Hypothalamus, subfornical organ, intermediolateral cell column	Cerebellum, cerebral cortex, hippocampus, striatum, septum, thalamic nuclei	(Morinville et al. 2007)
	Mouse	CNS	Hypothalamus		(Branco et al. 2016)
	Human	PNS	L3–L5; DRG		(Flegel et al. 2015; Chang et al. 2018)
	Rat	PNS	L5		(Fukuoka et al. 2008)
	Mouse	PNS	Colonic neurons in T10–L1 and L5–S1; T10–L1; L6		(Feng et al. 2015; Hockley et al. 2017; Inserra et al. 2017)
	Human	ENS	Colonic myenteric plexus		(Hetz et al. 2014)
	Guinea pig	ENS	Duodenal myenteric plexus		(Sage et al. 2007)
NaV1.8	Human	CNS		Brain	(Flegel et al. 2015; GTEx Consortium et al. 2017)
	Human	PNS	L3–L5; DRG		(Flegel et al. 2015; Chang et al. 2018)
	Rat	PNS	Colonic neurons T13–L2		(Hu et al. 2013a, 2016; Lin et al. 2017)
	Mouse	PNS	Colonic neurons in T10–L1 and L5–S1; T10–L1; T9–T13; T9–L1; L6		(Beyak et al. 2004; Hillsley et al. 2006; King et al. 2009; Feng et al. 2015; Hockley et al. 2017; Inserra et al. 2017)
	Human	ENS		Colonic myenteric plexus	(Hetz et al. 2014)
	Mouse	ENS		Duodenal myenteric plexus	(Osorio et al. 2014)
NaV1.9	Human	CNS		Brain	(GTEx Consortium et al. 2017)
	Human	PNS	L3–L5; DRG		(Flegel et al. 2015; Chang et al. 2018)
	Rat	PNS	L4–L5		(Dib‐Hajj et al. 1998)
	Mouse	PNS	Colonic neurons T9–T13; trigeminal ganglia		(Beyak et al. 2004; Padilla et al. 2007; King et al. 2009)
	Human	ENS	Colonic submucosal and myenteric plexus		(Hetz et al. 2014; O'Donnell et al. 2016)
	Rat	ENS	Duodenal myenteric plexus		(Rugiero et al. 2003)
	Mouse	ENS	Sensory, Dogiel type II, myenteric and submucosal neurons		(Padilla et al. 2007; Osorio et al. 2014)
	Guinea pig	ENS	Duodenal intrinsic primary afferent neurons; duodenal myenteric plexus		(Rugiero et al. 2003; Copel et al. 2009)
	Human	Muscle	Colonic smooth muscle		(O'Donnell et al. 2016)
	Human	Mast cells	lung, skin and cord blood‐derived mast cells		(Bradding et al. 2003)

Recently, numerous studies have reported Na_V isoform channelopathies, including for Na_V1.7 (SCN9A), Na_V1.8 (SCN10A) and Na_V1.9 (SCN11A) as the primary cause of increased pain or loss of pain phenotypes in humans (Yang et al. 2004; Cox et al. 2006; Fertleman et al. 2006; Klein et al. 2013; Leipold et al. 2013; Huang et al. 2014, 2017; Waxman et al. 2014; Dib‐Hajj et al. 2015; Han et al. 2015). Pharmacological modulation of Na_V channels supports these genetic observations, including the finding that activation of all Na_V channels by Pacific ciguatoxin 1 (P‐CTX‐1) or veratridine due to accidental consumption manifests as acute and severe gastrointestinal disturbances associated with abdominal pain in humans (Schep et al. 2006; Stewart et al. 2010). Intracolonic administration of purified P‐CTX‐1 also causes pain behaviour in mice (Inserra et al. 2017). On the other hand, TTX (which blocks Na_V1.1–Na_V1.4, Na_V1.6 and Na_V1.7) poisoning in humans is associated with paralysis rather than pain (Lago et al. 2015). Whilst potentially fatal upon consumption, administration of Na_V‐selective agents such as TTX and neosaxitoxin has been shown to decrease pain responses in a range of pain modalities including visceral pain in humans (Hagen et al. 2011, 2017; Manriquez et al. 2015) and rodents (Marcil et al. 2006; Gonzalez‐Cano et al. 2017). Similarly, intrarectal administration of lidocaine (lignocaine) in irritable bowel syndrome (IBS) patients reduces rectal sensitivity and abdominal pain, suggesting Na_V channels and activation of peripheral afferent endings in the colon play key roles in the pathogenesis of chronic visceral pain in IBS patients (Verne et al. 2005).

Human genetic studies have triggered widespread investigation into the therapeutic potential of Na_V channels in the treatment of acute and chronic pain and also prompted studies to identify the wider roles of these channels throughout the body. It is also clear from most studies utilising inflammatory, nematode or bacterial models that gut‐ and bladder‐innervating neurons become hyperexcitable after the initial insult, which involves changes in TTX‐R and TTX‐S Na_V currents, amongst others. This is apparent in neurons innervating the stomach (Gebhart et al. 2002; Bielefeldt et al. 2002a, b; Dang et al. 2004), small intestine (Moore et al. 2002; Stewart et al. 2003; Hillsley et al. 2006; Keating et al. 2008), the colon (Beyak et al. 2004; Ibeakanma et al. 2009; King et al. 2009) and the bladder (Yoshimura & deGroat, 1997). This review presents recent evidence on the specific roles of Na_V1.1–Na_V1.9 in transmitting sensation and nociception from the distal gut and bladder in healthy and pathological states.

Na_V1.1

Na_V1.1 is predominantly expressed in cell bodies, axon initial segments and at the nodes of Ranvier in the central nervous system (CNS) (Westenbroek et al. 1989; Duflocq et al. 2008; Carithers et al. 2015; Uhlen et al. 2015; GTEx Consortium et al. 2017). It is also expressed in human, rat and mouse PNS (Fukuoka et al. 2008; Wang et al. 2011; Osteen et al. 2016; Chang et al. 2018), and in human, but not guinea pig, myenteric plexus (Sage et al. 2007; Hetz et al. 2014) (Table 1). In thoracolumbar (T10–L1) and lumbar (L5) dorsal root ganglia (DRG) neurons, which contain the cell bodies of sensory neurons innervating the colon, rectum, bladder and skin, Na_V1.1 is expressed in 15–35% of all neurons. Expression is predominantly in Tropomyosin‐related kinase C (TrkC)‐ and Tropomyosin‐related kinase A (TrkA)‐expressing myelinated A‐fibres of medium to large diameter and nearly absent in C‐fibre small diameter neurons innervating the skin (Fukuoka et al. 2008; Wang et al. 2011; Osteen et al. 2016). However, Na_V1.1 mRNA transcript is detected in approximately half of thoracolumbar (T10–L1) and lumbosacral (L5–S1) mouse DRG neurons innervating the colon (Osteen et al. 2016; Hockley et al. 2017). As colonic afferents are predominantly peptidergic C‐fibres, there are clearly key differences in the populations of afferent neurons expressing Na_V1.1 when comparing between the colon and the skin. In colon‐innervating DRG neurons, Na_V1.1 is frequently co‐localised with Na_V1.2, Na_V1.3, Na_V1.6, Na_V1.7, Na_V1.8 and Na_V1.9 (Osteen et al. 2016; Hockley et al. 2017). Functional studies of colonic afferents reveal that Na_V1.1 plays a crucial role in the signalling of mechanical pain from the colon (Osteen et al. 2016). Application of the selective Na_V1.1 agonist, δ‐theraphotoxin‐Hm1a (Hm1a), enhances mechanically evoked firing in a subpopulation of high‐threshold colonic nociceptors. Notably, the mechanical hypersensitivity evoked by Hm1a was blocked by incubation with the Na_V1.1/Na_V1.3 antagonist ICA‐121431 (Table 2) (Osteen et al. 2016). Furthermore, Hm1a also induces hyperexcitability of isolated colon‐innervating DRG neurons from healthy control mice (Osteen et al. 2016). Notably, the percentage of colon‐innervating afferents/neurons affected by Hm1a is similar to the percentage of colon‐innervating DRG neurons expressing Na_V1.1, as determined by single cell PCR (Osteen et al. 2016; Hockley et al. 2017). Importantly, colon‐innervating DRG neurons isolated from mice with chronic visceral hypersensitivity (CVH) show significantly enhanced responsiveness to Hm1a compared to healthy control mice, suggesting that Na_V1.1 may be essential for the development and maintenance of chronic visceral pain conditions (Osteen et al. 2016). As such, antagonism of Na_V1.1 may be a future target for the treatment of disorders accompanied by chronic visceral pain originating from the colon. There are currently no reports on the expression profile or function of Na_V1.1 in the bladder or bladder‐innervating sensory neurons.

Table 2.

Predominant Na_V isoforms contributing functionally to visceral sensation

A. Healthy states
Species	Test	Response	Reference
Human	Appendix distension (ex vivo extracellular recordings of mesenteric afferents) before and after exposure to PF‐5198007 (Nav1.7 antagonist)	No difference in mesenteric afferent peak firing	(Hockley et al. 2017)
Mouse	Colonic mechanical stimulation (ex vivo extracellular recordings of colonic afferents in the splanchnic nerve) after Hm1a (highly selective Nav1.1 agonist, mucosal application)	Increase in colonic nociceptor response to mechanical stimuli in a sub‐population of afferents.	(Osteen et al. 2016)
	Colonic stretch (ex vivo extracellular recordings of colorectal afferents in the pelvic nerve) μ‐conotoxin GIIIa, and μ‐conotoxin PIIIa, serosal/mucosal application)	Reduced action potential firing of stretch‐sensitive afferent response	(Feng et al. 2015)
	Colonic stretch (ex vivo extracellular recordings of colorectal afferents in the pelvic nerve) ‐ ProTxII (Nav1.7 antagonist, serosal/mucosal application)	No difference in stretch‐sensitive afferent response	(Feng et al. 2015)
	Ciguatoxin (pan‐Nav agonist) (intracolonic)	Increased pain behavioural response	(Inserra et al. 2017)
	Colonic incubation with A‐803467 (Nav1.8 antagonist) (ex vivo extracellular recordings of colorectal nociceptors), followed by ciguatoxin	Inhibited afferent firing induced by ciguatoxin
	Incubation with supernatant from colitis patients	Increased excitability of colonic DRG neurons associated with enhanced NaV1.8 currents	(Ibeakanma & Vanner, 2010)
	Tumour necrosis factor‐α incubation

B. Knock‐out and knock‐down models
Model	Species	Test	Response	Reference
NaV1.7Nav1.8	Mouse	Formalin (intraplantar)	Reduction in pain behavioural response in phase I and phase II of formalin response	(Nassar et al. 2004)
		Complete Freund's adjuvant (intraplantar)	Reduction in thermal hyperalgesia and mechanical allodynia from day 1 to day 10
		Carrageenan (intraplantar)	Reduction in thermal hyperalgesia from 1 to 4 h
		Nerve growth factor (intraplantar)	Absence of phase I thermal hyperalgesia and reduction in phase II
		Colonic distension (ex vivo extracellular recordings of lumbar splanchnic nerve activity)	No difference in afferent firing in physiological range (0–80 mmHg)	(Hockley et al. 2017)
			Reduction in firing in supramaximal range (80–145 mmHg)
		Capsaicin (intracolonic)	Normal pain behavioural response
		Mustard oil (intracolonic)
		Cyclophosphamide‐induced cystitis	Normal level of referred mechanical hyperalgesia responses
NaV1.8−/−	Mouse	Whole‐cell patch clamp	Reduced action potential amplitude in retrogradely labelled neurons projecting to the peritoneal cavity (DRG, T9–T13)	(Hillsley et al. 2006)
		Nippostrongylus brasiliensis post‐infectious stage, whole‐cell patch clamp	Absence of neuronal hyperexcitability 19–25 days post‐infection in retrogradely labelled neurons projecting to the peritoneal cavity (DRG, T9–T13)
		Acetylcholine (intraperitoneal injection)	Normal pain behavioural response	(Laird et al. 2002)
		Capsaicin (intracolonic)	Reduced pain behavioural response
		Mustard oil (intracolonic)
		Cyclophosphamide‐induced cystitis	Normal pain and inflammatory responses
NaV1.8 knock‐down (L6–S1)	Rat	Cystometry (saline)	No change in intercontraction intervals	(Yoshimura et al. 2001)
		Acetic acid (intravesical)	Hyper‐reflexia attenuated
NaV1.9−/−	Mouse	Whole‐cell patch clamp	Normal excitability and action potential characteristics in colonic neurons (DRG T9–T13)	(Hillsley et al. 2006)
		Nippostrongylus brasiliensis post‐infectious stage, whole‐cell patch clamp	No change in neuronal hyperexcitability 19–25 days post‐infection in retrogradely labelled neurons projecting to the peritoneal cavity (DRG, T9–T13)
		Colorectal distension	Normal pain behavioural response	(Martinez & Melgar, 2008)
		R‐848 (toll‐like receptor 7 activator)‐induced colonic inflammation, colorectal distension	Reduced pain behavioural response
		Colonic distension (ex vivo extracellular recordings of splanchnic nerve activity)	Reduced afferent discharge	(Hockley et al. 2014)
		Ex vivo extracellular recordings of lumbar splanchnic nerve activity following inflammatory soup (bradykinin, ATP, histamine, PGE2 and 5‐HT), or inflammatory bowel disease patient colonic supernatant application	Reduced afferent fibre responses
		Ex vivo extracellular recordings of lumbar splanchnic nerve activity following UTP (P2Y2 and P2Y4 agonist) or ADP (P2Y1, P2Y12 and P2Y13 agonist), application	Reduced afferent fibre responses	(Hockley et al. 2016a)
		Cystometry (saline)	No change in basal urodynamics	(Ritter et al. 2009)
		Cyclophosphamide‐induced cystitis
		Bladder distension (ex vivo extracellular recordings of bladder nerve activity) following PGE2 bladder infusion application	Reduced afferent excitability

C. Inflammatory hypersensitivity models
Model/disease	Species	Test	Response	Reference
Neonatal induced colitis	Rat	Protein expression	Increase in NaV1.7 and Nav1.8 protein in colonic (DRG, T13–L2) neurons post‐inflammation	(Qu et al. 2013)
		Whole‐cell patch clamp	Increase in Na+ current in colonic neurons (DRG, T13–L2) 6 weeks post‐inflammation
			No change in Na+ current in colonic neurons (DRG, T13–L2) 10 weeks post‐inflammation
			No change in Na+ current in non‐colonic neurons (DRG, L4–L5) 6 or 10 weeks post inflammation
Acute TNBS‐induced colitis	Mouse	Whole‐cell patch clamp	Increased slow TTX‐R Na+ current in colonic neurons (DRG, T9–L1) 7–10 days post‐ induction	(Beyak et al. 2004)
			No change in persistent TTX‐R Na+ currents in colonic neurons (DRG, T9–L1) 7–10 days post‐induction
		Gene and protein expression	No change in NaV1.7 mRNA or protein in retrogradely labelled colonic neurons (DRG, T9–T13) 1 week post‐induction	(King et al. 2009)
		Gene expression	Tenfold reduction in NaV1.8 mRNA 2–4 days post‐induction, no change at day 7, in retrogradely labelled colonic neurons (DRG, T9–T13)
		Protein expression	No change in NaV1.8 protein 2–4 days post‐induction, up‐regulation at day 7, in retrogradely labelled colonic neurons (DRG, T9–T13) 1 week post‐induction
			No change in Nav1.9 protein in colonic neurons (DRG, T9–T13) day 7 post‐induction
Post‐TNBS‐induced colitis	Mouse	Whole‐cell patch clamp in the presence of Hm1a (Nav1.7 agonist)	Pronounced increase in excitability of colonic DRG neurons: significant lowering of rheobase and a dramatic increase in the number of action potentials fired at 2× rheobase	(Osteen et al. 2016)
		Gene expression	Up‐regulation of NaV1.7 mRNA in retrogradely labelled colonic neurons (DRG, L6–S1) 4 weeks post‐induction	(Campaniello et al. 2016)
Nippostrongylus brasiliensis post‐infectious stage	Mouse	Gene expression	No change in NaV1.8 or Nav1.9 mRNA 19–25 days post ‐infection in retrogradely labelled neurons projecting to the peritoneal cavity (DRG, T9–T13)	(Hillsley et al. 2006)
Interstitial cystitis/bladder pain syndrome	Human	Neosaxitoxin (blocker of TTX‐S Nav channels) (bladder infiltration)	Analgesia and reduced frequency lasting up to 90 days	(Manriquez et al. 2015)
Cyclophosphamide‐induced cystitis	Rat	A‐803467 administration (intraperitoneal)	No change in pain behavioural response	(Jarvis et al. 2007)

D. Non‐inflammatory hypersensitivity models
Model/disease	Species	Test	Response	Reference
Clinical rectal hypersensitivity	Human	Protein expression (full thickness rectal biopsies)	Increased NaV1.7‐immunoreactive nerve fibres in mucosal, submucosal and muscle layers	(Yiangou et al. 2007)
Maternal separation model (visceral hypersensitivity)	Rat	Gene expression	No change in NaV1.8 mRNA in colonic neurons (DRG, T13–L2)	(Hu et al. 2013a)
		Protein expression	Increase in NaV1.8 protein in colonic neurons (DRG, T13–L2)
		Whole‐cell patch clamp	Increased TTX‐R Na+ current in colonic neurons (DRG, T13–L2)
Streptozotocin‐induced diabetes (visceral hypersensitivity)		Protein expression	Increase in NaV1.7 and NaV1.8 protein in colonic neurons (DRG, T13–L2)	(Hu et al. 2016)
		Whole‐cell patch clamp	Increased TTX‐R Na+ current in colonic neurons (DRG, T13–L2)
Partial colonic obstruction (visceral hypersensitivity)		Gene expression	Increase in NaV1.8 mRNA in colonic neurons (DRG, T13–L2)	(Lin et al. 2017)
		Whole‐cell patch clamp	Increased TTX‐R Na+ current in colonic neurons (DRG, T13–L2)
T8 spinal transection		Whole‐cell patch clamp	Reduced TTX‐R Na+ current in bladder neurons	(Yoshimura & deGroat, 1997)

Na_V1.2

Na_V1.2 is extensively expressed in the CNS (Jarnot & Corbett, 2006) but has also been detected at low levels in small‐diameter DRG neurons (Black et al. 1996; Fukuoka et al. 2008; Chang et al. 2018). Conversely, in colon‐innervating DRG neurons of the mouse, Na_V1.2 mRNA transcript is present in 69% of thoracolumbar (T10–L1) neurons and at a similar level in lumbosacral (L5–S1) neurons (Hockley et al. 2017) (Table 1). Despite this mRNA expression, there is currently no functional data to support a role for Na_V1.2 in colonic sensory signalling or pain. Similarly, there are currently no reports on the expression profile or function of Na_V1.2 in the bladder or bladder‐innervating sensory neurons.

Na_V1.3

Na_V1.3 is highly expressed in sensory neurons during embryogenesis in rats, but its expression traditionally subsides in fully developed neurons (Beckh et al. 1989). The major body of Na_V1.3 research in nociception focuses on its role in neuropathic pain, as Na_V1.3 is re‐expressed following neuropathic injury in large diameter, myelinated A‐fibre neurons where it may contribute to ectopic discharge and painful neuropathy (Waxman et al. 1994; Zang et al. 2010). However, due to the limited expression of this channel in adult tissues and lack of channelopathy‐associated pain syndromes, studies investigating the role of Na_V1.3 in other pain pathways are few. In relation to the viscera, Na_V1.3 mRNA is detected in adult guinea‐pig enteric nervous system (ENS) neurons (Sage et al. 2007), but its functional role has yet to be determined. Initial experiments indicate that Na_V1.3 expression is low in rat lumbar (L5) DRG neurons (Fukuoka et al. 2008). However, Na_V1.3 mRNA transcripts are detected in approximately half of the colon‐innervating thoracolumbar (T10–L1) and lumbosacral (L5–S1) DRG neurons in the mouse (Hockley et al. 2017) (Table 1).

More recent studies show a key role for Na_V1.3 in non‐neuronal tissues, specifically within enterochromaffin cells located within the epithelium from the small and large intestine of humans and mice (Bellono et al. 2017; Strege et al. 2017a,b). Voltage‐gated sodium currents generated by Na_V1.3 likely allow enterochromaffin cells to respond to the detection of mechanical and chemical stimuli within the lumen of the intestine (Bellono et al. 2017; Strege et al. 2017b). In contrast, expression of the other eight Na_V isoforms is very low, or indeed lacking from both intestinal enterochromaffin cells and the wider population of intestinal epithelial cells (Bellono et al. 2017). There are currently no reports on the role of Na_V1.3 in the bladder or bladder‐innervating sensory neurons.

Na_V1.4

Na_V1.4 is the predominant Na_V isoform in skeletal muscle (Trimmer et al. 1990) but is also found in human oesophageal smooth muscle tissue (Deshpande et al. 2002). In peripheral neurons, Na_V1.4 transcripts are nearly absent in rat lumbar (L5) DRG (Fukuoka et al. 2008) and in colon‐innervating mouse DRG neurons (Hockley et al. 2017) (Table 1). In agreement with tissue distribution, Na_V1.4 channelopathies appear to exclusively involve deficits in skeletal muscle function, and to date no involvement in colon or bladder function has been shown.

Na_V1.5

Na_V1.5 channels have been identified in circular smooth muscle of the jejunum of human, dog, rat and mouse but are absent in pig and guinea pig. Na_V1.5 is also absent from human and mouse jejunal longitudinal smooth muscle (Holm et al. 2002; Ou et al. 2002; Strege et al. 2007; Beyder et al. 2016). Na_V1.5 has been found in colonic circular smooth muscle of human and rat (Strege et al. 2003), in jejunal interstitial cells of Cajal in human (Strege et al. 2003), and in myenteric plexuses of human and mouse (Hetz et al. 2014; Osorio et al. 2014).

Na_V1.5 in circular smooth muscle may contribute to normal intestinal motility through modulation of slow‐wave activity and muscle contractility (Ou et al. 2002; Strege et al. 2007). These findings are supported by data showing that ranolazine, a treatment for chronic angina, is able to inhibit Na_V1.5 currents in human colonic smooth muscle cells (Neshatian et al. 2015), which is likely to be responsible for the constipation seen during long‐term ranolazine treatment (Nash & Nash, 2008). These data strongly point towards a primary role for Na_V1.5 channels in mediating gastrointestinal motility and transit (Beyder & Farrugia, 2016). Similarly, several loss‐of‐function mutations in SCN5A, the gene encoding Na_V1.5 channels, are associated with IBS and abdominal pain (Saito et al. 2009; Beyder et al. 2014; Strege et al. 2017c). Whether this is purely a consequence of reduced gastrointestinal contractility or whether Na_V1.5 channels also play a direct role in visceral sensation remains unclear, as Na_V1.5 mRNA transcripts are expressed in 18% of thoracolumbar and 51% of lumbosacral colon‐innervating DRG neurons (Hockley et al. 2017) (Table 1). Whether this translates into channel expression and a functional role remains to be determined. There are currently no reports on the expression profile or function of Na_V1.5 in the bladder or bladder‐innervating sensory neurons.

Na_V1.6

Na_V1.6 is extensively expressed within the CNS and PNS (Whitaker et al. 1999; Tzoumaka et al. 2000; Catterall et al. 2005; Catterall, 2012; Chang et al. 2018), commonly located in clusters at the nodes of Ranvier (Duflocq et al. 2008), indicating that Na_V1.6 may have a primary role in transmitting rather than initiating action potentials. In rat lumbar (L5) DRG neurons, Na_V1.6 transcripts are detected in a third of all neurons and selectively expressed in TrkC‐ and TrkA‐expressing myelinated A‐fibre nociceptors (Fukuoka et al. 2008). In colon‐innervating mouse DRG neurons, Na_V1.6 mRNA transcript is present in 63–87% of thoracolumbar (T10–L1) neurons, and in 51% of lumbosacral (L5–S1) neurons (Hockley et al. 2017; Inserra et al. 2017). Immunohistochemical and western blot analysis show that Na_V1.6 protein is present in the cell bodies of sensory neurons and on sensory afferent nerve endings innervating the distal colon and rectum in mice (Feng et al. 2015) (Table 1). Antagonism of Na_V1.6 reduces action potential firing of stretch‐sensitive colorectal afferents in vitro (Feng et al. 2015) (Table 2). Whether these effects are altered in animal models of inflammatory or chronic visceral pain remains to be investigated. It has, however, been reported that there is no change in Na_V1.6 expression in colon‐innervating DRG neurons (T9–T13) during the acute inflammatory phase of the mouse model of trinitrobenzenesulphonic acid (TNBS)‐induced colitis (King et al. 2009). This corresponds with the phase when colorectal afferent hypersensitivity also occurs (Hughes et al. 2009). Activation of low‐threshold stretch‐sensitive afferents is essential for normal physiological function of the colon (Brierley et al. 2004; Kyloh et al. 2011) and Na_V1.6 appears to play a key integrative role in this process. Whether Na_V1.6 contributes to aberrant colonic afferent sensory signalling during chronic visceral hypersensitivity remains to be determined. There are currently no reports on the expression profile or function of Na_V1.6 in the bladder or bladder‐innervating sensory neurons.

Na_V1.7

Na_V1.7 has become a key target of interest as several human mutations in the SCN9A gene, which encodes Na_V1.7, lead to either a loss of pain or increased pain perception (Bennett & Woods, 2014). For example, a loss‐of‐function mutation of SCN9A results in a congenital insensitivity to pain (CIP) (Cox et al. 2006; Goldberg et al. 2007), whereas gain‐of‐function mutations produce distinct pain syndromes, such as erythromelalgia, small‐fibre neuropathy and paroxysmal extreme pain disorder (Fertleman et al. 2006). Na_V1.7 is extensively expressed in sensory and sympathetic neurons of the PNS, as well as ENS neurons, and is highly restricted in the CNS (Klugbauer et al. 1995; Catterall et al. 2005; Morinville et al. 2007; Sage et al. 2007; Branco et al. 2016; Chang et al. 2018). In rat lumbar (L5) DRG neurons, Na_V1.7 transcripts are preferentially expressed in TrkA‐expressing C‐fibre neurons, and in a subset of A‐fibre neurons (Fukuoka et al. 2008). Robust immunolabelling of Na_V1.7 is present within the peripheral endings of sensory nerves in the skin (Black et al. 2012).

From mouse knock‐out studies, it appears that Na_V1.7 in Na_V1.8‐expressing cells (Na_V1.7^Nav1.8) does not contribute in the development of neuropathic pain, nor noxious cold or heat detection (Nassar et al. 2004, 2005; Minett et al. 2012, 2014; Hockley et al. 2017). However, Na_V1.7^Nav1.8 mice have significantly reduced behavioural responses to inflammatory mediators (formalin, complete Freund's adjuvant, carrageenan and nerve growth factor) when injected into the sole of the hind paw (Nassar et al. 2004) and impaired somatic noxious mechanosensation (Minett et al. 2012, 2014). Thus far, only the deletion of Na_V1.7 in sympathetic and sensory (Wnt1‐expressing) neurons and the global Na_V1.7 knock‐out have been able to significantly reduce pain responses to a range of stimuli and recapitulate the human SCN9A‐associated CIP phenotype (Gingras et al. 2014; Minett et al. 2014). Recent studies also show that endogenous opioids contribute to pain insensitivity in both humans and mice lacking Na_V1.7, as the opioid antagonist naloxone reverses analgesia associated with the loss of Na_V1.7 expression (Minett et al. 2015). This suggests that Na_V1.7 channel blockers alone may not replicate the analgesic phenotypes of Na_V1.7 null mutants, but may be potentiated with exogenous opioids. Na_V1.7‐selective inhibitors are currently in clinical trial for different types of pain (Pennington et al. 2017; Yekkirala et al. 2017).

In relation to visceral sensation, Na_V1.7 is highly abundant in human lumbar DRG, and is expressed in 100% of mouse colon‐innervating thoracolumbar (T10–L1) DRG neurons, and in most colon‐innervating lumbosacral (L5–S1) DRG neurons (Chang et al. 2018; Hockley et al. 2017; Inserra et al. 2017) (Table 1). Accordingly, Na_V1.7 constitutes the most prevalent TTX‐S isoform within colon‐innervating DRG neurons. It is of interest to note that ‘paroxysmal extreme pain disorder’, caused by the human gain of function SCN9A mutation, was originally called ‘familial rectal pain syndrome’. As the name implies, this disorder is characterised by excruciating rectal and abdominal pain commonly associated with defecation (Fertleman et al. 2006), suggesting a key role for Na_V1.7 in visceral pain. Moreover, pain perception in a subset of patients with interstitial cystitis/bladder pain syndrome (IC/BPS) is shown to correlate with a polymorphism in SCN9A (Reeder et al. 2013). IC/BPS patients treated with a bladder infiltration of neosaxitoxin, a blocker of TTX‐S Na_V channels, resulted in significant analgesia and reduced bladder overactivity for 90 days after the treatment (Manriquez et al. 2015). Normal physiological function of the bladder, however, appears to be independent of Na_V1.7, as SCN9A‐associated CIP individuals have normal bladder control, and no increased incidence of urinary infections, incontinence, or retention (Cox et al. 2006).

Despite these studies, the initial promise of Na_V1.7's contribution to visceral pain is somewhat tempered by experimental studies showing that Na_V1.7^Nav1.8 mice exhibit normal nocifensive responses to intracolonic administration of capsaicin (TRPV1 agonist) and mustard oil (TRPA1 agonist), indicating that Na_V1.7 is not crucial for acute visceral pain signalling (Hockley et al. 2017). Low‐threshold stretch‐sensitive pelvic afferents are unaffected by the Na_V1.7 antagonist ProTX‐II (Feng et al. 2015) (Table 2). Similarly, ex vivo extracellular recordings of mesenteric afferents from resected human appendices show that peak firing before and after exposure to a novel Na_V1.7‐selective antagonist, PF‐5198007, is unchanged during repeat noxious ramp distensions (Hockley et al. 2017). Afferent responses in mouse ex vivo colorectal recordings are attenuated by application of TTX (Feng et al. 2015), indicating that TTX‐S channels other than Na_V1.7 may be important in responding to innocuous and noxious mechanical stimuli. Accordingly, intracolonic co‐administration of TTX and P‐CTX‐1 did not significantly alter the pain response induced by P‐CTX‐1 (Inserra et al. 2017).

Ex vivo extracellular recordings of splanchnic nerve activity from the distal colon of Na_V1.7^Nav1.8 mice show no difference in peak firing between Na_V1.7^Nav1.8 and littermate control afferents in the physiological and supraphysiological pressure range (0–80 mmHg) (Hockley et al. 2017). However, significantly less action potential firing in afferents from Na_V1.7^Nav1.8 mice at distension pressures in the supramaximal range (80–145 mmHg) is observed, suggesting that Na_V1.7 in Na_V1.8‐positive colonic afferent neurons may be involved in transducing non‐physiological extremes of pressure. This may be important and more relevant to chronic visceral pain states, when splanchnic afferents show mechanical hypersensitivity and decreased activation thresholds to mechanical stimuli (Hughes et al. 2009; Castro et al. 2013, 2017; de Araujo et al. 2014; Osteen et al. 2016). In the bladder, Na_V1.7^Nav1.8 mice have comparable levels of referred hyperalgesia in an acute cyclophosphamide‐induced cystitis model compared to littermates (Hockley et al. 2017). Overall, these findings suggest that Na_V1.7 has a role in mediating acute inflammatory pain in somatic but not visceral pathways. While studies on visceral nociception using Na_V1.7^Nav1.8 mice have provided valuable insight, replication of these studies in mice with sensory neuron‐specific deletion of Na_V1.7 (e.g. Na_V1.7^Advill) will be beneficial to strengthen conclusions concerning Na_V1.7 in visceral pain signalling.

Diseases that have a significant visceral pain component are commonly chronic and have unmet needs in terms of clinical treatment. Therefore, further investigations into the role of Na_V1.7 in long term and chronic visceral pain models, which are more clinically relevant to pathological chronic visceral pain states, are critical. For example, significant up‐regulation of Na_V1.7 mRNA occurs 4 weeks after induction of colitis in colon‐innervating DRG (L6–S1) neurons (Campaniello et al. 2016). Similarly, rats with streptozotocin‐induced diabetes show hypersensitivity to colonic distension, which corresponds with the up‐regulation of Na_V1.7 protein in thoracolumbar (T13–L2) DRG neurons 4 weeks post‐induction (Hu et al. 2016). In support of these findings, rat neonatal colitis‐induced visceral hypersensitivity induces up‐regulation of Na_V1.7 protein levels in DRG from higher spinal levels (T13–L2), but not lower spinal levels (L4–L5) at 6 weeks post‐colitis compared to control animals (Qu et al. 2013). Taken together, these findings suggest that Na_V1.7 may have an acquired role during chronic visceral pain states. It is well documented that inflammation, tissue damage and healing of visceral organs can induce structural, synaptic or intrinsic neuroplasticity, altering neuronal and gastrointestinal function in the long term (Brierley & Linden, 2014). For example, rectal samples from patients with physiologically characterised rectal hypersensitivity show significantly increased numbers of Na_V1.7‐immunoreactive nerve fibres in the mucosal, submucosal and muscle layers compared to control tissues (Yiangou et al. 2007). In addition to these findings, changes in the ratios of Na_V1.7 to a pan‐neuronal structural marker, PGP9.5, indicate that increased Na_V1.7 expression and nerve sprouting occurs in rectal mucosa, which may contribute to enhanced sensitivity in these patients (Yiangou et al. 2007).

Overall, the role of Na_V1.7 in pain sensation is complicated, and species differences in expression, assumed translatability of isoform‐compound interaction, and effects of Na_V knock‐out on other genes may confound overall conclusions. Furthermore, few studies using human tissue have been completed, and healthy tissue is often obtained from patients with colonic or rectal carcinoma (Yiangou et al. 2007; Hetz et al. 2014; Hockley et al. 2017). In addition to species‐dependent differences in tissue distributions (Table 1), there are also differences in relative isoform distributions, for example, Na_V1.7 is the most abundant isoform in human lumbar DRG, whereas Na_V1.8 is more abundant in mouse (Chang et al. 2018). Na_V1.7 isoforms from different species can also have different compound selectivity in heterologous expression systems that should be carefully considered during experimental design. For example, human, monkey, dog and mouse Na_V1.7 isoforms were found to be largely insensitive to a small molecule inhibitor of Na_V1.1/Na_V1.3 (ICA‐121431) and potently inhibited by a small molecule inhibitor of Na_V1.7 (PF‐04856264), whereas rat Na_V1.7 was potently inhibited by ICA‐121431, but largely insensitive to PF‐04856264 (McCormack et al. 2013). A ProTxII analogue, JNJ63955918, on the other hand was equipotent at human and rat Na_V1.7 (Flinspach et al. 2017). Single cell studies have shown that Na_V channel expression is heterologous across cells, and there is high co‐localisation of Na_V1.7 with Na_V1.6, Na_V1.8 and Na_V1.9 in colon‐innervating thoracolumbar and lumbosacral neurons in mice (Hockley et al. 2017). However, functional relationships of co‐expression and investigations of redundancy between Na_V channels are unclear. In knock‐out models, deletion of one Na_V gene can lead to a change in expression levels of over 190 genes (Minett et al. 2015). Studies investigating Na_V channel contribution to pain signalling using knock‐out models or pharmacological modification may benefit from collecting data on regulation of other Na_V family genes and auxiliary β‐subunits in parallel, and other key genes where possible. Furthermore, inducible knock‐out models offer the advantage of normal development and being able to compare Na_V channel contribution pre‐ and post‐induction of visceral hypersensitivity in the adult, thereby increasing therapeutic potential of these findings.

Na_V1.8

Na_V1.8 mediates slowly inactivating TTX‐R Na⁺ currents and carries the majority of the current underlying the upstroke of the action potential in nociceptive neurons. Hence, they are considered to play an important role in action potential electrogenesis (Renganathan et al. 2001). Na_V1.8‐null mice display reduced sensitivity to noxious mechanical stimuli (tail pressure) and noxious thermal stimuli (radiant heat), but normal sensitivity to acute noxious colonic distension by isotonic saline and intraperitoneal acetylcholine (Akopian et al. 1999; Laird et al. 2002). Na_V1.8 is the most abundant isoform expressed in mouse lumbar DRG (Chang et al. 2018), and is prevalently expressed in thoracolumbar (96%) and lumbosacral (91%) colonic sensory DRG neurons, with almost complete co‐expression with Na_V1.7 (Hockley et al. 2017). Consistent with this, knock‐down of Na_V1.8 in DRG neurons results in action potentials with reduced peak amplitude and slower rise times, but similar baseline excitability (Renganathan et al. 2001; Hillsley et al. 2006). Similarly, A‐803467, a selective Na_V1.8 antagonist, does not significantly affect the frequency of action potential firing from low‐threshold mechanosensitive colonic afferent nerve endings (Feng et al. 2015). Together, these data seem to suggest that Na_V1.8 channels do play a major role in mediating visceral sensations and pain under physiological conditions.

Na_V1.8 channels also have a major role in visceral signalling under pathophysiological conditions. Several studies support increased expression of Na_V1.8 protein in colon‐innervating sensory DRG neurons in murine models of visceral hypersensitivity (Beyak et al. 2004; Hillsley et al. 2006; King et al. 2009; Qu et al. 2013; Hu et al. 2013a,b; Inserra et al. 2017; Lin et al. 2017) (Table 2). In most studies, increased channel expression correlates with enhanced TTX‐R Na⁺ current density in colon‐innervating DRG neurons in vitro, and with visceral hypersensitivity in vivo, as the visceromotor response to noxious colonic distension in rats is significantly reduced following intraperitoneal administration of the Na_V1.8‐specific antagonist A‐803467 (Jarvis et al. 2007). Similarly, colonic co‐administration of A‐803467 with P‐CTX‐1 significantly reduces P‐CTX‐1‐induced nocifensive behaviours in mice (Inserra et al. 2017). These findings are consistent with studies in Na_V1.8‐null mice, which do not develop visceral hypersensitivity after intracolonic administration of sensitising agents such as capsaicin (TRPV1 agonist) and mustard oil (TRPA1 agonist). Furthermore, unlike their wild‐type littermates, DRG neurons from Na_V1.8‐null mice do not display enhanced neuronal hyperexcitability following intestinal infection with Nippostrongylus brasiliensis (Laird et al. 2002; Hillsley et al. 2006).

Inflammatory mediators acting via GPCRs are powerful modulators of Na_V1.8 currents, and are believed to underlie increased excitability of nociceptive DRG neurons and associated hyperalgesia (Beyak et al. 2004). In this regard, colon‐innervating DRG neurons incubated with supernatant from colonic biopsies from patients with active ulcerative colitis (a chronic inflammatory bowel disease) show increased action potential discharge and enhanced Na_V1.8 currents (Ibeakanma & Vanner, 2010). These effects were replicated by incubation with tumour necrosis factor α (TNFα), whose levels are enhanced in the ulcerative colitis supernatant. Similar sensitising effects have been reported for prostaglandin E2 (PGE2), adenosine, serotonin (5‐HT), ATP, as well as nerve growth factor (NGF), which may persist during and possibly after the inflammation as a result of increased expression of Na_V1.8 channels (Gold, 1999; Gold et al. 2002; Beyak et al. 2004). Recent data, however, indicate that these effects are not limited to inflammatory conditions, but may extend to non‐inflammatory chronic pain states. Partial colonic obstruction is associated with an increase in Na_V1.8 mRNA expression, as well as enhanced TTX‐R Na⁺ currents and referred in vivo hyperalgesia, effects that were abolished by anti‐NGF treatment (King et al. 2009; Ibeakanma & Vanner, 2010).

Decreased TTX‐R currents occur in bladder‐innervating DRG neurons from T8 spinal transected rats (Yoshimura & deGroat, 1997), which has since been attributed to a down‐regulation of Na_V1.8 (Black et al. 2003). This change is only seen in bladder‐innervating DRG neurons, and is accompanied by an up‐regulation of TTX‐S current, which may also enhance the excitability of these afferent neurons. Knock‐down of Na_V1.8 in rats at spinal levels L6–S1, known to contain the majority of bladder sensory terminals, does not have an effect on intercontraction intervals following cystometry with saline; however, intravesical acetic acid‐induced hyper‐reflexia is attenuated in knock‐down rats (Yoshimura et al. 2001). Na_V1.8‐null mice develop normal pain and inflammatory responses during cyclophosphamide‐induced cystitis compared to littermates (Laird et al. 2002), and pain behaviours are sustained in rats with cyclophosphamide‐induced cystitis following intraperitoneal administration of A‐803467 (Jarvis et al. 2007).

Cross‐organ sensitisation of the gastrointestinal and lower urinary tract is evident clinically and in animal models (Malykhina et al. 2004, 2012; Lei & Malykhina, 2012), highlighting the importance of understanding the mechanisms of viscero‐visceral crosstalk. Several studies report increases in TTX‐resistant Na⁺ current in bladder‐innervating DRG neurons following colitis, implicating some involvement of TTX‐R channels in bladder pain as a consequence of gastrointestinal tract inflammation. C‐fibre bladder‐innervating DRG neurons, involved in the transduction of noxious stimuli signalling (Fowler et al. 2008), in the majority express TTX‐R currents (Yoshimura & deGroat, 1997). Collectively, experimental findings to date indicate that Na_V1.8 is not crucial for visceral pain signalling from the bladder in response to several noxious stimuli, but it may have an important role during referred hyperalgesia and in response to certain irritants.

Na_V1.9

Several human Na_V1.9 channelopathies are associated with congenital episodic pain syndromes, painful neuropathy, and an insensitivity to pain (Huang et al. 2014, 2017). Na_V1.9 channels are preferentially expressed in small‐diameter nociceptors (Dib‐Hajj et al. 1998; Tate et al. 1998), and mediate ultraslow or persistent TTX‐R Na⁺ currents. Due to their kinetic properties, Na_V1.9 channels are unlikely to contribute to action potential generation, but instead regulate neuronal excitability by setting the resting membrane potential closer to threshold (Dib‐Hajj et al. 1998, 2002; Tate et al. 1998). In colonic afferents, action potential firing in response to colonic ramp distension is reduced in Na_V1.9^−/− mice, and accompanied by a run‐down of responses to repeated phasic distension (Hockley et al. 2014). Similar to Na_V1.8 channels, several studies indicate that Na_V1.9 currents can be enhanced via GPCRs (Maingret et al. 2008; Ostman et al. 2008; Vanoye et al. 2013; Hockley et al. 2016b). Colonic afferent excitatory responses to the application of multiple inflammatory mediators (applied at once, either in the form of supernatants from chronically inflamed human bowel or as an experimental inflammatory soup containing ATP, PGE2, bradykinin, histamine and 5‐HT) are significantly reduced in visceral afferents from Na_V1.9^−/− mice (Hockley et al. 2014, 2016a) (Table 2).

Na_V1.9^−/− mice have similar baseline visceromotor responses to colonic distension to wild‐type littermates, but reduced visceral hypersensitivity in vivo after colonic inflammation induced by activation of toll‐like receptor 7 (Martinez & Melgar, 2008). Neuronal hyperexcitability following Nippostrongylus bransiliensis infection is unchanged in Na_V1.9^−/− mice compared to wild‐type littermates, reporting similar action potential characteristics and excitability of colon‐innervating DRG neurons (Hillsley et al. 2006). Likewise, others do not see changes in Na_V1.9 protein expression in colon‐innervating DRG neurons, nor differences in either the numbers of neurons expressing persistent TTX‐R (Na_V1.9) currents or the magnitude of these currents in acute TNBS‐induced colitis (Beyak et al. 2004; King et al. 2009). It is unclear whether these discrepancies in the contribution of Na_V1.9 to neuronal (hyper)excitability relates to differences in knock‐out constructs and mice strains, or to differences in the inflammatory insult studied. The latter may be of considerable importance as inflammatory mediators such as bradykinin, ATP, histamine, PGE2 and noradrenaline (norepinephrine), potentiate Na_V1.9 channel activity when applied conjointly, but fail to modulate Na_V1.9 currents when applied separately (Maingret et al. 2008).

Na_V1.9 channels are also present in myenteric plexus neurons in human, mouse, rat and guinea‐pig (Rugiero et al. 2003; Padilla et al. 2007; Copel et al. 2009; Osorio et al. 2014) pointing towards an additional role in intestinal motor function. In line with this, colonic migrating motor complex patterns are altered in Na_V1.9^−/− mice (Copel et al. 2013). Moreover, expression of Na_V1.9 channels is decreased in submucosal and myenteric plexus neurons (most likely intrinsic primary afferent neurons) in Hirschsprung's disease (O'Donnell et al. 2016). Interestingly, these findings apply not only to aganglionic bowel sections, but in some patients extend to those sections containing normal ganglia numbers, which could explain some of the post‐surgery bowel dysmotility issues frequently encountered by these patients (O'Donnell et al. 2016). Conversely, a gain‐of‐function mutation (L811P) in the Na_V1.9 gene, SCN11A, identified in three unrelated individuals with congenital insensitivity to pain, is associated with severe gastrointestinal dysmotility, including alternating episodes of diarrhoea and constipation (Leipold et al. 2013; Woods et al. 2015). In contrast, other gain‐of‐function mutations are predominantly linked to chronic pain syndromes such as autosomal‐dominant episodic pain and small fibre neuropathy (Zhang et al. 2013; Huang et al. 2014; Han et al. 2015).

No difference is observed in basal urodynamics between wild‐type and Na_V1.9^−/− mice; however, the change of urodynamic parameters associated with cyclophosphamide‐induced cystitis is absent in Na_V1.9^−/− mice, as well as attenuation of PGE2‐induced afferent excitability during bladder distension (Ritter et al. 2009). It remains to be investigated whether this involvement of Na_V1.9 in bladder nociception is due to functional up‐regulation of Na_V1.9 in bladder afferents, or whether Na_V1.9 has a role in central processing of bladder nociceptive pathways.

Conclusion

Recent findings highlight the diversity in expression patterns of Na_V isoforms in abdominal visceral organs. This diversity extends across neurons (enteric, extrinsic sensory DRG innervating the intestine or bladder) and non‐neuronal cells (intestinal enterochromaffin cells, intestinal smooth muscle cells, and interstitial cells of Cajal). Na_V channels have a range of functions in health and disease and we are only now, with the development of novel pharmacological and genetic tools, beginning to unpick their complex physiological and pathophysiological interactions. Na_V1.1, Na_V1.6, Na_V1.8 and Na_V1.9, contribute to visceral hypersensitivity, particularly within colonic pathways, and respond to inflammatory mediators in pathophysiological models (Fig. 1).

Figure 1. Current understanding of how specific voltage‐gated sodium channels (Na_V) contribute to the functioning of neurons and non‐neuronal cells within visceral organs.

 

Whilst Na_V1.3 contributes to enterochromaffin cell function and Na_V1.5 contributes to intestinal smooth muscle cells and interstitial cells of Cajal function, there is currently no determined function in visceral afferents for Na_V1.2, Na_V1.3, Na_V1.4 or Na_V1.5, despite significant mRNA expression of Na_V1.2 and Na_V1.5 in visceral afferent pathways. Na_V1.7 is one of the most extensively expressed and studied Na_V channels, but a role in visceral pain, like that attributed to Na_V1.7 in somatic pain studies is currently unclear. Although many of these Na_V channels have been investigated under physiological conditions or in models of acute pain, chronic visceral pain models are necessary for the determination of a precise role in long term pathological visceral pain. Future studies would benefit from the further development of novel, specific agonists and antagonists, as we have seen with recent advances in the role of Na_V1.1 in mechanical pain. Likewise, selective Na_V modulators with low systemic uptake for in vivo studies will advance our understanding of Na_V channels in visceral pain signalling and the suitability of targeting Na_V channels in the treatment of pain originating in the distal gut and bladder.

Additional information

Competing interests

In relation to the content covered within this review, the authors have nothing to declare.

Author contributions

All authors contributed to searching the published literature and to writing the review. All authors approved the final version and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

Funding

S.M.B. is a National Health and Medical Research Council of Australia (NHMRC) R.D. Wright Biomedical Research Fellow (APP1126378) and is funded by NHMRC Australia Project Grants 1083480, 1139366 and 1140297. A.M.H. receives funding via the Australian Research Council (ARC) Discovery Early Career Research Award. A.M.H. and S.M.B. receive funding via the ARC Discovery Project DP180101395.

Biographies

Joel Castro, Andrea Harrington, Luke Grundy, Annemie Deiteren and Sonia Garcia‐Caraballo are postdoctoral research fellows within the Visceral Pain Research Group, whilst Andelain Erickson and Ashlee Caldwell are PhD students enrolled via the University of Adelaide. Our research comprises pre‐clinical and translational science investigating the causes and cures of chronic abdominal and pelvic pain associated with highly prevalent gastrointestinal disorders such as irritable bowel syndrome and inflammatory bowel disease, and bladder disorders such as interstitial cystitis/painful bladder syndrome.

Stuart M. Brierley is an NHMRC R.D. Wright Fellow and Matthew Flinders Research Fellow in Gastrointestinal Neuroscience. He is Head of the Visceral Pain Research Group located at Flinders University and the South Australian Health and Medical Research Institute (SAHMRI) in Adelaide, Australia.