Modulation of Visceral Hypersensitivity in IBS by Lipid Mediators

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Visceral hyperalgesia (VH) is frequently observed in irritable bowel syndrome (IBS) patients. The responsible mechanism is unclear. Our preliminary data show colonic biopsies from diarrhea-prone IBS pts contain pro- inflammatory molecule(s) which can modify the electrophysiological properties of the DRG neurons and induce visceral hypersensitivity (VH). In vivo and in vitro studies show the pro-nociceptive effects can be inhibited by either blocking mast cell (MC) dependent PGE2 synthesis pathway or by injecting EP2 receptor antagonists intrathecally. We also show resolvins, a novel class of endogenous anti-inflammatory lipid mediators, are synthesized in the colonic mucosa and may be therapeutically useful to reverse the actions of PGE2 on DRG excitability. We hypothesize PGE2 is a key pro-inflammatory molecule released by colonic mucosa MC of IBS pts, can activate G protein coupled EP2 receptors and stimulates adenylyl cyclase (AC) resulting in an increase in cAMP, which modulates IH, IA and IC currents via different signal transduction pathways. On the other hand, resolvins activating via FPR2/Giα reduce cAMP formation and decrease excitability of sensory neurons and consequently reduce VH. Aim 1. Demonstrate IBS colonic biopsies contain elevated levels of PGE2 generated by increased proteolytic activities in colonic mucosa of IBS-D pts. Proteases, PGE2 and other polyunsaturated fatty acids will be quantified in colonic biopsies and biopsy supernatants from IBS pts and healthy controls. In vivo pain behavior studies will examine if pretreatment with an EP2 antagonist or silencing EP2 receptor can prevent VH in rats exposed to colonic instillation of IBS biopsy supernatant. Whole-cell patch-clamp studies will characterize the changes in basic membrane properties and excitability of colon projecting DRG neurons exposed to IBS biopsy supernatant and show these changes are mediated by PGE2. Aim 2. Western blot, RT- PCR and whole-cell patch-clamp studies will examine the intracellular signaling pathways and membrane channels that mediate the actions of PGE2 in IBS biopsy supernatant. Use siRNA technologies and pharmacological inhibitors to show PGE2 in IBS colonic supernatants acts via the EP2-AC-cAMP-PKA pathway to modulate IH, IA and IC currents. Demonstrate HCN2, Kv4.2 and TRPV1 participation by silencing expression of these molecules using fluorescein labeled siRNAs. Aim 3. To demonstrate resolvins is an effective antidote for the excitatory actions of PGE2 in the IBS-D supernatant, whole-cell patch-clamp recordings will examine the effects of resolvins on DRG neurons treated with IBS-D supernatant or PGE2. Signal transduction pathways will be studied by silencing the expressions of formyl peptide receptor (FPR2) and treatment with pertussis toxin to inactivate Giα. In vivo pain behavior studies following intrathecal injections of resolvins or chronic docosahexaenoic acid (DHA) (precursor of resolvin) feeding will demonstrate therapeutic significance of this novel class of compounds. Our proposal will identify a new mechanism responsible for VH observed in IBS pts and explore the use of resolvins as a class of novel therapeutic agent to treat VH.