Tu1707 Endogenous Opioids From Human IBD Supernatants Modulate TRPV1 on DRG Neurons During Chronic Colitis

Abstract

Our previous mouse studies have shown that endogenous opioid signaling is enhanced during the evolution of colonic inflammation, altering pain signaling. The mechanisms underlying this action are unknown but given that TRPV1 channels are important in mechano and chemosensitive nociceptive signaling, and that TRPV1 is co-expressed with opioid receptors on dorsal root ganglia (DRG) neurons, we postulated that TRPV1 signaling may be involved. Therefore, in the present work we sought to investigate if the endogenous opioids released during the chronic inflammation model regulate TRPV1 activity. The 2% chronic DSS (cDSS) colitis model was employed in C57/BL6 mice. Human colonic biopsies from patients with chronic ulcerative colitis, crohn's desease or healthy controls and colonic tissue from cDSS colitis were used to generate supernatants (sup). DRG neurons were incubated overnight with mouse or human supernatants or μ and δ opioid agonists DAMGO (10 nM) and DADLE (1 μM) respectively and with or without the opioid receptor antagonist naloxone (10 μM). For Ca2+ imaging 1 μM Fura-2 AM and capsaicin (250 nM) was used as a TRPV1 agonist. T-test or one way ANOVA were used to analyze the data obtained, n= number of neurons. The capsaicin-evoked Ca2+ signal was inhibited 66% by cDSS sup compared to control sup (F340/380: Ctrl=2.08±0.33 n=19, cDSS=0.70±0.14 n=16, P= <0.01). This effect was blocked by naloxone 10 μM (F340/380: cDSS+NLX= 2.32±0.65, n=6, P= <0.05), demonstrating that endogenous opioids were involved. Moreover, the supernatant effect was mimicked by the use of low concentration DAMGO (10 nM) or DADLE (1 μM) alone. (F340/380: Ctrl=5.66±0.52 n=26, DAMGO=2.42±0.55 n=19, DADLE=3.45±50, P= <0.001), DRG neurons incubated with the human UC sup showed a similar effect with a 58% reduction of the Ca2+ signal (F340/380: Ctrl=5.51±0.51 n=18, UC sup=2.30±0.67 n= 9, P= 0.0011). In contrast, in a symptomatic IBD patient where inflammation had resolved (post-inflammatory IBS) there was no difference in the Ca2+ signal compared to control, thus suggesting the endogenous opioid effect was lost (F340/380: Ctrl=4.59±0.58 n=15, Crohn's sup=3.95±0.79 n=11). These results suggest that endogenous opioids released during chronic inflammation have an important role in inhibiting TRPV1 channels in nociceptive neurons. Following resolution of the inflammation in some patients however, the pain persists but the endogenous opioid system appears to be inactive.