In a recent article in Gut, Hughes et al1 identified distinct patterns of immune dysfunction in IBS patients compared with healthy subjects. In particular, they showed that peripheral blood mononuclear cell (PBMC) supernatants from healthy subjects inhibited colonic afferents in a μ-opioid receptor (MOR)-mediated manner. These findings correlated with β-endorphin from T lymphocytes providing an important MOR-mediated antinociceptive influence in the healthy gut.2 Intriguingly, these inhibitory effects were lost with PBMC supernatants from constipation-predominant IBS patients suggesting a loss of MOR-mediated inhibition, coupled with increased excitatory mediators (TNF-α, IL-1β, IL-6), contributes to abdominal pain.1

We evaluated if this alteration was MOR specific or whether it extended to other members of the opioid receptor family. As clinical studies have shown varying outcomes on visceral pain perception with κ-opioid receptor (KOR) agonists,3^–5 we postulated KOR expression and function are altered during visceral hypersensitivity. Therefore, we determined if the peripherally restricted selective KOR agonist, asimadoline, was able to modify colonic nociceptor function in health and during inflammatory and postinflammatory chronic visceral mechanical hypersensitivity (CVH).6

We performed in vitro afferent recordings from mouse splanchnic high-threshold nociceptors, which respond to focal compression and noxious stretch/distension.1 ,6 They also display pronounced mechanical hypersensitivity and lowered activation thresholds in models of acute and chronic visceral pain.6 We assessed nociceptor mechanosensitivity before and after increasing doses of asimadoline, which has a 500-fold selectivity for KOR versus MOR and δ-opioid receptor subtypes and is 500–1000-fold less potent at sodium channels.5

We found healthy nociceptor mechanosensitivity was not affected by asimadoline at any of the doses tested (figure 1A), which were in the range of KOR affinity and not high enough to block sodium channels.5 In contrast, when we performed recordings from mice with colonic inflammation we found that asimadoline dose-dependently inhibited colonic nociceptors (figure 1B). Furthermore, asimadoline also dose-dependently inhibited colonic nociceptors from CVH mice (figure 1C), an effect that was prevented by the prior application of a KOR antagonist (figure 1D). Overall, these data indicate KOR expression is functionally upregulated during inflammation and CVH. Correspondingly, retrogradely-traced colonic dorsal root ganglia (DRG) neurones from CVH mice displayed KOR immunoreactivity (figure 1E) and significant upregulation of KOR mRNA (figure 1F).

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Figure 1

(A) Asimadoline had no effect on healthy splanchnic colonic serosal nociceptor mechanosensitivity (p>0.05, n=6), but (B) caused dose-dependent inhibition of colonic nociceptors from mice with inflammatory hypersensitivity (7 days post-trinitrobenzene-sulphonic acid (TNBS) administration; ***p<0.001, n=10) and (C) chronic visceral hypersensitivity (CVH; 28 days post-TNBS administration; *p<0.05; ***p<0.001, n=10). (D) The κ-opioid receptor (KOR) antagonist Nor-BNI (100 nM) did not alter CVH nociceptor mechanosensitivity (NS, p>0.05, n=8), but did block asimadoline-induced inhibition (p>0.05, n=8). (E) (i) Retrogradely-traced colonic neurones within the thoracolumbar (T10-L1) dorsal root ganglia (DRG). (ii) KOR immunoreactivity (KOR-IR) using immunohistochemistry. Yellow arrows: CVH colonic neurones expressing KOR-IR. White arrows: CVH colonic neurones lacking KOR-IR. Scale bar: 50 μm. (F) Quantitative-RT-PCR showing significant upregulation of KOR mRNA in retrogradely labelled colonic thoracolumbar DRG neurones from CVH mice (***p<0.001).

Our findings are consistent with recent somatosensory studies showing KOR mRNA is maintained in a dormant state, but is subjected to transcriptional and post-transcriptional influences. Induction of functional KOR occurs via sensitising agents, such as bradykinin, activating the phospholipase-C pathway and membrane integrins or via neuronal depolarisation stimulating KOR mRNA, axonal transport and protein translation.7 ,8

Although previous studies of acute trinitrobenzene-sulphonic acid-induced inflammation show greater potency of KOR agonists in vivo,9 our findings are the first to demonstrate increased KOR agonist efficacy during CVH. Notably, we used doses in the range of KOR affinity and were able to block the inhibitory effects of asimadoline with a KOR antagonist. As bouts of gastroenteritis can contribute to IBS onset and prolonged low-grade intestinal inflammation is apparent in IBS patients,10 our findings provide further information on the potential therapeutic benefit of KOR agonists for abdominal pain relief. In healthy volunteer barostat studies, asimadoline did not significantly reduce pain perception scores, even at noxious levels of distension.3 This suggests KOR activation does not significantly alter noxious sensation in health, which is consistent with our lack of effect of asimadoline on healthy colonic nociceptors. However, in IBS patients a single dose of asimadoline significantly decreased pain perception across a range of colonic distension pressures,4 while in a phase IIb study of 596 patients asimadoline's efficacy against IBS symptoms was greatest in D-IBS patients, with at least moderate pain at baseline.5 This suggests a preferential efficacy of asimadoline in the relief of abdominal pain in patients with visceral hypersensitivity, which is consistent with our findings of increased KOR expression and function during CVH. However, in a confirmatory clinical trial in D-IBS patients, efficacy was not observed with asimadoline.

These findings add another layer of complexity to KOR signalling, whereby KOR expression is upregulated during inflammation and CVH. Correspondingly, asimadoline inhibits nociceptors during inflammation and CVH, but not in health, via peripheral KOR on colonic afferent endings. Overall, KOR appears to be a silent receptor system that is activated ‘on demand’ in response to neuronal sensitisation and remains active during CVH.