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Endometriosis, an estrogen-dependent chronic inflammatory disease, is the most common cause of chronic pelvic pain. Here, we investigated the effects of linaclotide, a Food and Drug Administration-approved treatment for IBS-C, in a rat model of endometriosis. Eight weeks after endometrium transplantation into the intestinal mesentery, rats developed endometrial lesions as well as vaginal hyperalgesia to distension and decreased mechanical hind paw withdrawal thresholds. Daily oral administration of linaclotide, a peripherally restricted guanylate cyclase-C (GC-C) agonist peptide acting locally within the gastrointestinal tract, increased pain thresholds to vaginal distension and mechanical hind paw withdrawal thresholds relative to vehicle treatment. Furthermore, using a cross-over design, administering linaclotide to rats previously administered vehicle resulted in increased hind paw withdrawal thresholds, whereas replacing linaclotide with vehicle treatment decreased hind paw withdrawal thresholds. Retrograde tracing of sensory afferent nerves from the ileum, colon, and vagina revealed that central terminals of these afferents lie in close apposition to one another within the dorsal horn of the spinal cord. We also identified dichotomizing dual-labelled ileal/colon innervating afferents as well as colon/vaginal dual-labelled neurons and a rare population of triple traced ileal/colon/vaginal neurons within thoracolumbar DRG. These observations provide potential sources of cross-organ interaction at the level of the DRG and spinal cord. GC-C expression is absent in the vagina and endometrial cysts suggesting that the actions of linaclotide are shared through nerve pathways between these organs. In summary, linaclotide may offer a novel therapeutic option not only for treatment of chronic endometriosis-associated pain, but also for concurrent treatment of comorbid chronic pelvic pain syndromes.

α-Conotoxins are disulfide-bonded peptides from cone snail venoms and are characterized by their affinity for nicotinic acetylcholine receptors (nAChR). Several α-conotoxins with distinct selectivity for nAChR subtypes have been identified as potent analgesics in animal models of chronic pain. However, a number of α-conotoxins have been shown to inhibit N-type calcium channel currents in rodent dissociated dorsal root ganglion (DRG) neurons via activation of G protein-coupled GABA B receptors (GABA B R). Therefore, it is unclear whether activation of GABA B R or inhibition of α9α10 nAChRs is the analgesic mechanism. To investigate the mechanisms by which α-conotoxins provide analgesia, we synthesized a suite of Vc1.1 analogues where all residues, except the conserved cysteines, in Vc1.1 were individually replaced by alanine (A), lysine (K), and aspartic acid (D). Our results show that the amino acids in the first loop play an important role in binding of the peptide to the receptor, whereas those in the second loop play an important role for the selectivity of the peptide for the GABA B R over α9α10 nAChRs. We designed a cVc1.1 analogue that is >8000-fold selective for GABA B R-mediated inhibition of high voltage-activated (HVA) calcium channels over α9α10 nAChRs and show that it is analgesic in a mouse model of chronic visceral hypersensitivity (CVH). cVc1.1[D11A,E14A] caused dose-dependent inhibition of colonic nociceptors with greater efficacy in ex vivo CVH colonic nociceptors relative to healthy colonic nociceptors. These findings suggest that selectively targeting GABA B R-mediated HVA calcium channel inhibition by α-conotoxins could be effective for the treatment of chronic visceral pain.

Functional bowel disorder patients can suffer from chronic abdominal pain, likely due to visceral hypersensitivity to mechanical stimuli. As there is only a limited understanding of the basis of chronic visceral hypersensitivity (CVH), drug-based management strategies are ill defined, vary considerably, and include NSAIDs, opioids, and even anticonvulsants. We previously reported that the 1.1 subtype of the voltage-gated sodium (NaV; NaV1.1) channel family regulates the excitability of sensory nerve fibers that transmit a mechanical pain message to the spinal cord. Herein, we investigated whether this channel subtype also underlies the abdominal pain that occurs with CVH. We demonstrate that NaV1.1 is functionally upregulated under CVH conditions and that inhibiting channel function reduces mechanical pain in 3 mechanistically distinct mouse models of chronic pain. In particular, we use a small molecule to show that selective NaV1.1 inhibition (a) decreases sodium currents in colon-innervating dorsal root ganglion neurons, (b) reduces colonic nociceptor mechanical responses, and (c) normalizes the enhanced visceromotor response to distension observed in 2 mouse models of irritable bowel syndrome. These results provide support for a relationship between NaV1.1 and chronic abdominal pain associated with functional bowel disorders.

Understanding the sensory mechanisms innervating the bladder is paramount to developing efficacious treatments for chronic bladder hypersensitivity conditions. The contribution of Mas-gene-related G protein-coupled receptors (Mrgpr) to bladder signaling is currently unknown. Using male and female mice, we show with single-cell RT-PCR that subpopulations of DRG neurons innervating the mouse bladder express MrgprA3 (14%) and MrgprC11 (38%), either individually or in combination, with high levels of coexpression with Trpv1 (81%-89%). Calcium imaging studies demonstrated MrgprA3 and MrgprC11 agonists (chloroquine, BAM8-22, and neuropeptide FF) activated subpopulations of bladder-innervating DRG neurons, showing functional evidence of coexpression between MrgprA3, MrgprC11, and TRPV1. In ex vivo bladder-nerve preparations, chloroquine, BAM8-22, and neuropeptide FF all evoked mechanical hypersensitivity in subpopulations (20%-41%) of bladder afferents. These effects were absent in recordings from Mrgpr-clusterÎ"-/- mice. In vitro whole-cell patch-clamp recordings showed that application of an MrgprA3/C11 agonist mixture induced neuronal hyperexcitability in 44% of bladder-innervating DRG neurons. Finally, in vivo instillation of an MrgprA3/C11 agonist mixture into the bladder of WT mice induced a significant activation of dorsal horn neurons within the lumbosacral spinal cord, as quantified by pERK immunoreactivity. This MrgprA3/C11 agonist-induced activation was particularly apparent within the superficial dorsal horn and the sacral parasympathetic nuclei of WT, but not Mrgpr-clusterÎ"-/- mice. This study demonstrates, for the first time, functional expression of MrgprA3 and MrgprC11 in bladder afferents. Activation of these receptors triggers hypersensitivity to distension, a critically valuable factor for therapeutic target development. [ABSTRACT FROM AUTHOR]

Key points: Voltage‐gated sodium channels are critical for peripheral sensory neuron transduction and have been implicated in a number of painful and painless disorders.The β‐scorpion toxin, Cn2, is selective for NaV1.6 in dorsal root ganglion neurons.NaV1.6 plays an essential role in peripheral sensory neurons, specifically at the distal terminals of mechanosensing fibres innervating the skin and colon.NaV1.6 activation also leads to enhanced response to mechanical stimulus in vivo.This works highlights the use of toxins in elucidating pain pathways moreover the importance of non‐peripherally restricted NaV isoforms in pain generation. Peripheral sensory neurons express multiple voltage‐gated sodium channels (NaV) critical for the initiation and propagation of action potentials and transmission of sensory input. Three pore‐forming sodium channel isoforms are primarily expressed in the peripheral nervous system (PNS): NaV1.7, NaV1.8 and NaV1.9. These sodium channels have been implicated in painful and painless channelopathies and there has been intense interest in them as potential therapeutic targets in human pain. Emerging evidence suggests NaV1.6 channels are an important isoform in pain sensing. This study aimed to assess, using pharmacological approaches, the function of NaV1.6 channels in peripheral sensory neurons. The potent and NaV1.6 selective β‐scorpion toxin Cn2 was used to assess the effect of NaV1.6 channel activation in the PNS. The multidisciplinary approach included Ca2+ imaging, whole‐cell patch‐clamp recordings, skin–nerve and gut–nerve preparations and in vivo behavioural assessment of pain. Cn2 facilitates NaV1.6 early channel opening, and increased persistent and resurgent currents in large‐diameter dorsal root ganglion (DRG) neurons. This promotes enhanced excitatory drive and tonic action potential firing in these neurons. In addition, NaV1.6 channel activation in the skin and gut leads to increased response to mechanical stimuli. Finally, intra‐plantar injection of Cn2 causes mechanical but not thermal allodynia. This study confirms selectivity of Cn2 on NaV1.6 channels in sensory neurons. Activation of NaV1.6 channels, in terminals of the skin and viscera, leads to profound changes in neuronal responses to mechanical stimuli. In conclusion, sensory neurons expressing NaV1.6 are important for the transduction of mechanical information in sensory afferents innervating the skin and viscera. Key points: Voltage‐gated sodium channels are critical for peripheral sensory neuron transduction and have been implicated in a number of painful and painless disorders.The β‐scorpion toxin, Cn2, is selective for NaV1.6 in dorsal root ganglion neurons.NaV1.6 plays an essential role in peripheral sensory neurons, specifically at the distal terminals of mechanosensing fibres innervating the skin and colon.NaV1.6 activation also leads to enhanced response to mechanical stimulus in vivo.This works highlights the use of toxins in elucidating pain pathways moreover the importance of non‐peripherally restricted NaV isoforms in pain generation. [ABSTRACT FROM AUTHOR]

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 (NaV) 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 NaV mice and specific NaV channel modulators, has shed new light on how NaV 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 NaV channel family members (NaV1.1–NaV1.9) contribute to abdominal visceral function in normal and disease states. [ABSTRACT FROM AUTHOR]

Irritable bowel syndrome (IBS) is a chronic gastrointestinal disorder characterized by abdominal pain and altered bowel habit that affects ~11% of the global population. Over the past decade, preclinical and clinical studies have revealed a variety of novel mechanisms relating to the visceral analgesic effects of guanylate cyclase-C (GC-C) agonists. Here we discuss the mechanisms by which GC-C agonists target the GC-C/cyclic guanosine-3′,5′-monophosphate (cGMP) pathway, resulting in visceral analgesia as well as clinically relevant relief of abdominal pain and other sensations in IBS patients. Due to the preponderance of evidence we focus on linaclotide, a 14-amino acid GC-C agonist with very low oral bioavailability that acts within the gut. Collectively, the weight of experimental and clinical evidence supports the concept that GC-C agonists act as peripherally acting visceral analgesics. Linaclotide and plecanatide are synthetic peptide agonists of guanylate cyclase-C (GC-C), a transmembrane receptor that is predominantly located on intestinal epithelial cells. These medications have very low oral bioavailability, which, when combined with the expression profile of GC-C, provides a mechanism of action 'targeted to the gut'. Linaclotide has been shown to inhibit colonic nociceptors and reduce peripheral drive from the colon, resulting in reduced numbers of activated dorsal horn neurons within the spinal cord and reduced pain responses to noxious colorectal distension. These effects are greatest in animal models of chronic visceral hypersensitivity that drive the 'bottom-up' and/or 'top-down' sensitization relevant to irritable bowel syndrome (IBS). Activation of GC-C results in the generation and release of cyclic guanosine-3′,5′-monophosphate (cGMP) from intestinal epithelial cells. When released into the submucosal space through the basolateral membrane, extracellular cGMP acts as a neuromodulator to inhibit pain-sensing nerve fibers innervating the colon. cGMP has also been demonstrated to inhibit pain-sensing nerves from human donors. In all human Phase III clinical studies conducted to date in patients with irritable bowel syndrome with constipation (IBS-C) or chronic idiopathic constipation (CIC), linaclotide significantly improved abdominal pain, discomfort, and bloating. Recent preclinical studies also provide the prospect that linaclotide may offer a novel therapeutic option, not only for the treatment of chronic abdominal pain in IBS-C, but also comorbid bladder dysfunction and endometriosis-associated pain. [ABSTRACT FROM AUTHOR]

Irritable bowel syndrome and inflammatory bowel disease are major forms of chronic visceral pain, which affect over 15% of the global population. In order to identify new therapies, it is important to understand the underlying causes of chronic visceral pain. This review provides recent evidence demonstrating that inflammation or infection of the gastrointestinal tract triggers specific changes in the neuronal excitability of sensory pathways responsible for the transmission of nociceptive information from the periphery to the central nervous system. Specific changes in the expression and function of a variety of ion channels and receptors have been documented in inflammatory and chronic visceral pain conditions relevant to irritable bowel syndrome and inflammatory bowel disease. An increase in pro-nociceptive mechanisms enhances peripheral drive from the viscera and provides an underlying basis for enhanced nociceptive signalling during chronic visceral pain states. Recent evidence also highlights increases in anti-nociceptive mechanisms in models of chronic visceral pain, which present novel targets for pharmacological treatment of this condition. [ABSTRACT FROM AUTHOR]

Supplemental Digital Content is Available in the Text. Bladder sensory neurons express T-type calcium channel Cav3.2, but not Cav3.1 or Cav3.3. Blocking Cav3.2 inhibits bladder afferent sensitivity and bladder pain responses to distension. The bladder wall is innervated by a complex network of afferent nerves that detect bladder stretch during filling. Sensory signals, generated in response to distension, are relayed to the spinal cord and brain to evoke physiological and painful sensations and regulate urine storage and voiding. Hyperexcitability of these sensory pathways is a key component in the development of chronic bladder hypersensitivity disorders including interstitial cystitis/bladder pain syndrome and overactive bladder syndrome. Despite this, the full array of ion channels that regulate bladder afferent responses to mechanical stimuli have yet to be determined. Here, we investigated the role of low-voltage-activated T-type calcium (CaV3) channels in regulating bladder afferent responses to distension. Using single-cell reverse-transcription polymerase chain reaction and immunofluorescence, we revealed ubiquitous expression of CaV3.2, but not CaV3.1 or CaV3.3, in individual bladder-innervating dorsal root ganglia neurons. Pharmacological inhibition of CaV3.2 with TTA-A2 and ABT-639, selective blockers of T-type calcium channels, dose-dependently attenuated ex-vivo bladder afferent responses to distension in the absence of changes to muscle compliance. Further evaluation revealed that CaV3.2 blockers significantly inhibited both low- and high-threshold afferents, decreasing peak responses to distension, and delayed activation thresholds, thereby attenuating bladder afferent responses to both physiological and noxious distension. Nocifensive visceromotor responses to noxious bladder distension in vivo were also significantly reduced by inhibition of CaV3 with TTA-A2. Together, these data provide evidence of a major role for CaV3.2 in regulating bladder afferent responses to bladder distension and nociceptive signalling to the spinal cord. [ABSTRACT FROM AUTHOR]