Genomic Determinants of a Relaxation Response Resiliency Program in Inflammatory Bowel Disease and Irritable Bowel Syndrome

G A A b st ra ct s exogenous application of proteases on neuronal excitability (decrease in rheobase and/or increased number of action potentials discharged at twice the rheobase) were studied. Incubation of neurons with supernatants from diarrhea predominant IBS patients induced hyperexcitability compared to controls (n = 18 cells). Mean action potential discharge increased by > 100% compared to controls (mean = 1.6 ± 0.4 control vs. 4.3 ± 0.9 PI-IBS, n = 11 and 9 cells respectively, p = 0.014). In contrast, the effect of supernatants from constipation predominant IBS patients did not differ from controls. To determine if cysteine proteases may be important mediators in the supernatant, the effects of exogenous cysteine proteases (cathepsin-s 500 nM) were studied and the effect of the cysteine protease inhibitor E-64 (100 μM) was determined. Incubation of neurons in cysteine proteases for 2 hours increased excitability in patch clamp recordings. Action potential discharge increased by more than 100% (mean = 1.5 ± 0.2 control vs. 3.8 ± 0.6 cathepsin-s, n = 12 and 11 respectively, p = 0.002). When enemas containing cathepsins (500 nM) were applied rectally for 2 hours, visceromotor reflexes evoked by rectal distentions (0 60 mmHg) were significantly increased compared to vehicle enemas (p = 0.039). In patch clamp studies, the increased action potential discharge mediated by the incubation with cysteine proteases was blocked by E-64 (mean = 3.8 ± 0.6 cathepsin-s vs. 1.9 ± 0.4 cathepsin-s plus E-64, n = 11 and 14 cells respectively, p = 0.017). To test the selectivity of this inhibitor, the effects of the serine protease trypsin was studied. Incubation in trypsin (50 nM) for 2 hours also increased excitability but this effect was not blocked by E-64, suggesting E-64 was selective for cysteine proteases. IBS supernatant from diarrhea predominant IBS patients was then incubated with E-64 and compared to supernatant alone. Action potential discharge was decreased ~ 100% by E-64 (p< 0.05) and the rheobase increased 20% (did not reach significance) compared to supernatant alone. These data demonstrate that supernatants from diarrhea-predominant IBS patients increase the intrinsic excitability of colonic DRG neurons. Cysteine proteases appear to be important mediators underlying this effect and could play a role in generating visceral hyperalgesia.