Dorsal root ganglion là gì

α-Conotoxin Vc1.1 inhibits human dorsal root ganglion neuroexcitability and mouse colonic nociception via GABAB receptors

1. Joel Castro1,
2. Andrea M Harrington1,
3. Sonia Garcia-Caraballo1,
4. Jessica Maddern1,
5. Luke Grundy1,
6. Jingming Zhang2,
7. Guy Page2,
8. Paul E Miller2,
9. David J Craik3,
10. David J Adams4,
11. Stuart M Brierley1

1. 1Visceral Pain Group, Centre for Nutrition and Gastrointestinal Diseases, Discipline of Medicine, Faculty of Health Sciences, The University of Adelaide, South Australian Health and Medical Research Institute [SAHMRI], Adelaide, South Australia, Australia
2. 2Anabios, San Diego, California, USA
3. 3Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia
4. 4Illawarra Health & Medical Research Institute [IHMRI], University of Wollongong, Wollongong, NSW, Australia

1. Correspondence to Professor Stuart M Brierley, Visceral Pain Group, Centre for Nutrition and Gastrointestinal Diseases, The University of Adelaide, Level 7, South Australian Health and Medical Research Institute [SAHMRI], North Terrace, Adelaide, SA 5000, Australia; stuart.brierley{at}adelaide.edu.au

Abstract

Objective α-Conotoxin Vc1.1 is a small disulfide-bonded peptide from the venom of the marine cone snail Conus victoriae. Vc1.1 has antinociceptive actions in animal models of neuropathic pain, but its applicability to inhibiting human dorsal root ganglion [DRG] neuroexcitability and reducing chronic visceral pain [CVP] is unknown.

Design We determined the inhibitory actions of Vc1.1 on human DRG neurons and on mouse colonic sensory afferents in healthy and chronic visceral hypersensitivity [CVH] states. In mice, visceral nociception was assessed by neuronal activation within the spinal cord in response to noxious colorectal distension [CRD]. Quantitative-reverse-transcription-PCR, single-cell-reverse-transcription-PCR and immunohistochemistry determined γ-aminobutyric acid receptor B [GABABR] and voltage-gated calcium channel [CaV2.2, CaV2.3] expression in human and mouse DRG neurons.

Results Vc1.1 reduced the excitability of human DRG neurons, whereas a synthetic Vc1.1 analogue that is inactive at GABABR did not. Human DRG neurons expressed GABABR and its downstream effector channels CaV2.2 and CaV2.3. Mouse colonic DRG neurons exhibited high GABABR, CaV2.2 and CaV2.3 expression, with upregulation of the CaV2.2 exon-37a variant during CVH. Vc1.1 inhibited mouse colonic afferents ex vivo and nociceptive signalling of noxious CRD into the spinal cord in vivo, with greatest efficacy observed during CVH. A selective GABABR antagonist prevented Vc1.1-induced inhibition, whereas blocking both CaV2.2 and CaV2.3 caused inhibition comparable with Vc1.1 alone.

Conclusions Vc1.1-mediated activation of GABABR is a novel mechanism for reducing the excitability of human DRG neurons. Vc1.1-induced activation of GABABR on the peripheral endings of colonic afferents reduces nociceptive signalling. The enhanced antinociceptive actions of Vc1.1 during CVH suggest it is a novel candidate for the treatment for CVP.

• ABDOMINAL PAIN
• VISCERAL NOCICEPTION
• IRRITABLE BOWEL SYNDROME
• ION CHANNELS
• REAL TIME PCR

This is an Open Access article distributed in accordance with the Creative Commons Attribution Non Commercial [CC BY-NC 4.0] license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: //creativecommons.org/licenses/by-nc/4.0/

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• ABDOMINAL PAIN
• VISCERAL NOCICEPTION
• IRRITABLE BOWEL SYNDROME
• ION CHANNELS
• REAL TIME PCR

Significance of this study

What is already known on this subject?

• Patients with IBS suffer from chronic visceral pain [CVP]; however, there are limited analgesic therapeutic options currently available for treatment.

• A rich source of novel agents to treat chronic pain is the α-conotoxin family of peptides from the venom of marine cone snails.

• α-Conotoxin Vc1.1 has antinociceptive and antihyperalgesic actions in neuropathic pain models; however, its ability to inhibit human sensory dorsal root ganglion [DRG] neurons remains unknown.

• Vc1.1's applicability in reducing CVP is also unknown.

What are the new findings?

• Vc1.1 reduces human sensory DRG neuroexcitability, via a γ-aminobutyric acid receptor B [GABABR]-mediated mechanism.

• We show that human DRG neurons express GABABR and the voltage-gated calcium channels CaV2.2, and CaV2.3, which are the direct and downstream targets of Vc1.1, respectively.

• Vc1.1 inhibits mouse colonic nociceptors and also low-threshold distension-sensitive colonic afferents with greatest effect during chronic visceral hypersensitivity [CVH].

• Peripheral in vivo Vc1.1 administration inhibits the signalling of noxious information from the colon to the spinal cord. This antinociceptive effect is also greater in mice with CVH.

• During CVH, mouse colonic DRG neurons show upregulation of the CaV2.2 exon-37a variant, which may explain the increased inhibitory effect of Vc1.1 in CVH states.

How might it impact on clinical practice in the foreseeable future?

• Vc1.1 has been tested in human clinical trials for the treatment of neuropathic pain, where it has been demonstrated to have a clean safety and side-effect profile.

• Our current findings show Vc1.1 inhibits human DRG neurons, via activation of the GABABR, which is a key finding for clinical translatability.

• This inhibitory effect in human neurons combined with the enhanced antinociceptive action of Vc1.1 in colonic pathways in a mouse model of CVH suggests it is a novel candidate for the treatment for CVP associated with IBS.

• We show that by reducing nociceptive signalling from the periphery, Vc1.1 has potential therapeutic value in treating CVP.

• These findings put GABABR agonists in the spotlight as potential peripheral agents for the treatment of CVP.

Introduction

IBS is a prevalent, chronic GI disorder that negatively impacts the quality of life in up to 14% of the population.1 ,2 It is characterised by abdominal pain and discomfort associated with altered bowel habits.3–5 Although the pathophysiology of IBS is not completely understood, it is becoming clear that changes to peripheral cellular and sensory mechanisms play key roles in the associated pain.6 ,7 In particular, chronic visceral hypersensitivity [CVH] of colonic afferents is implicated in the development and maintenance of chronic visceral pain [CVP] in IBS.4 ,5 Characteristic features of CVH include nociceptor hypersensitivity8 and increased signalling of noxious colorectal distension [CRD] within the spinal cord.9–11 Recent evidence suggests sensory afferents display upregulation of numerous ion channels and receptors in animal models of CVH,7 ,10 ,12 making them targets for analgesic treatment.

A recently introduced treatment for patients with IBS and constipation involves a small disulfide-rich peptide that is restricted to the GI tract, where it inhibits peripheral nociceptive pathways and produces clinically relevant pain relief.9 Given the limited treatments available for patients with other subtypes of IBS, additional analgesic therapeutic options are needed. A rich source of novel small disulfide-rich agents comes from the α-conotoxin family of peptides from the venom of marine cone snails.13 These peptides target a wide variety of membrane receptors and ion channels.14 In particular α-conotoxin Vc1.1, a 16-amino acid synthetic version of a peptide derived from the marine cone snail Conus victoriae, has antinociceptive actions in vitro and antihyperalgesic actions in numerous in vivo neuropathic pain models.15–17 Interestingly, in a chronic constriction injury model of neuropathic pain, Vc1.1 relieves tactile allodynia.17 These inhibitory effects were similar to those obtained with gabapentin, a ligand recently proposed as a potential IBS therapeutic,18 but were achieved at far lower doses.17 Notably, Vc1.1 [also called ACV1] has been used in phase I and phase IIA clinical trials for the treatment of neuropathic pain.19–21 These studies showed Vc1.1 was safe and well tolerated with a clean safety and side-effect profile. Despite this promise, therapeutic trials were discontinued as Vc1.1 was shown to be less potent at the human α9α10 nicotinic acetylcholine receptor [nAChR], which was thought to mediate the inhibitory action of Vc1.1. However, more recent recombinant cell line studies have clearly demonstrated that the human γ-aminobutyric acid receptor B [GABABR] is the primary and high affinity target for Vc1.1.17 ,22 ,25–27 These studies also demonstrated GABABR activation by Vc1.1 causes downstream inhibition of the voltage-gated calcium channels CaV2.2 and CaV2.3, which underlies Vc1.1's inhibitory actions.14 ,28 These recent findings are intriguing; as both oral and intravenous administration of baclofen, the archetypal GABABR agonist has been shown to reduce the pseudo-affective responses to CRD in animal models.29 ,30 Although it is unclear if this baclofen-induced inhibition is centrally or peripherally mediated, we wondered if Vc1.1 represents a novel peripheral gut analgesic for the treatment of CVP. Therefore, we determined if Vc1.1 inhibits human sensory dorsal root ganglion [DRG] neurons, the primary transducers at the start of the pain-processing pathway. Second, we determined if Vc1.1 inhibits sensory pathways within the splanchnic and pelvic innervation of the colon and whether these actions are enhanced in an animal model of CVH. Third, we determined if the inhibitory actions of Vc1.1 are mediated via activation of GABABR on the peripheral endings of colonic afferents.

Materials and methods

For comprehensive descriptions of the methodologies used, see the online supplementary information.

Human DRG

Thoracolumbar [TL] DRG [T9–L1] were acquired from five [three female, two male] human adult organ donors [22.2±2.08 years of age] during the removal of the vital organs for transplantation. The harvested DRG were immediately processed for downstream patch clamp or RNA studies. Intact DRG were kept for quantitative-reverse-transcription-PCR [qRT-PCR] mRNA expression studies from each spinal level [T9, T10, T11, T12, L1] while additional DRG were dissociated to allow individual DRG neurons to be studied with single-cell-reverse-transcription-PCR [RT-PCR] studies, or to allow patch clamp recordings to be performed.

Human DRG patch clamp recordings

Whole-cell patch clamp recordings of cultured human DRG neurons were performed in current clamp mode in response to depolarising current pulses [20 or 50 pA current steps, 500 ms duration]. This allowed the rheobase [amount of current needed to initiate action potential generation] to be assessed in the presence and absence of Vc1.1 [1000 nM] and a synthetic analogue of Vc1.1 [[P6O]Vc1.1;1000 nM], which is inactive at GABABR. An increased rheobase indicates more current is required to fire an action potential and therefore the neuron displays reduced excitability.

Mouse model of CVH

Intracolonic trinitrobenzene-sulfonic acid [TNBS] was administered as described previously.8–10 TNBS-treated mice were allowed to recover for 28 days, at which stage inflammation had resolved and chronic colonic afferent mechanical hypersensitivity was evident.8–10 ,12

Ex vivo electrophysiology

Recordings of splanchnic and pelvic afferents were made from healthy control and CVH mice as described previously.8–10 Briefly, colonic nociceptors were recorded from the splanchnic pathway. They respond to noxious distension [40 mm Hg], stretch [≥7 g] or von Frey hair filaments [2 g]8 ,31 and become mechanically hypersensitive in models of CVP.8–10 ,12 Muscular–mucosal afferents were recorded from the pelvic pathway and respond to both low-intensity circular stretch [