Introduction: Hydrogen sulphide (H2S) is recognised as an endogenous neuromodulator in the peripheral and central nervous system.It has been shown to directly influence sensory signalling via a variety of ion channels including KATP, T-type Ca2+ channels, and several TRP family members including TRPV1 and TRPA1.ATP is considered to be a key signalling molecule in the gastrointestinal tract and is important for sensory signal transduction.We hypothesised that H2S could influence gastrointestinal afferent signalling via release of ATP from the mucosal epithelium triggered by TRP channel activation.Methods: Three experimental approaches were used.First, we examined the sensitivity of jejunal mesenteric afferents in a mouse in vitro preparation to exogenous NaHS, a donor of H2S.Secondly, ATP release from whole thickness jejunal segments was quantified using a luciferin-luciferase assay.Thirdly, the effect of H2S on intracellular Ca2+ was determined in HEK-293 cells expressing P2X receptors and various TRP channels using Fluo-4 imaging.Results: H2S (NaHS, 0.03-1 mM, for 2 min) evoked a concentration-dependent increase in mesenteric afferent nerve discharge.The threshold response occurred around 0.03 mM NaHs and maximal activation occurred at 0.3 mM.The P2 receptor antagonist, PPADS (0.03 mM), markedly attenuated the nerve response profile to 0.3 mM NaHS.Similarly, the P2X3 and P2X2/3 selective antagonist-A317491 at 0.01 mM inhibited NaHS-induced afferent discharge to a similar extent.NaHS-induced peak afferent firing was also significantly reduced by RR and 2-APB.Afferent response to luminal distension at low but not high distension pressures was also attenuated by 2-APB.HC-030031 had no effect on afferent response to distension.However, it significantly attenuated afferent responses to NaHS.In the ATP release assay NaHS caused a concentration dependent release of ATP with a peak response to 0.3 mM NaHS compared to the baseline.ATP release in response to 0.3 mM NaHS was attenuated by pre-treatment with RR.Using recombinant expression, NaHS (0.3-100 μM) failed to evoke calcium transients in cells stably expressing P2X2, or P2X2/3 receptors despite robust responses to 30 μM ATP and 30 μM α,β-methylene-ATP respectively.Negative responses to NaHiS were also observed in cells transfected with TRPV1.In contrast calcium transients were observed from varying proportions of cells transfected with TRPC4, TRPC5, TRPM2 or TRPM4b receptors.H2S-induced calcium transients were blocked by lanthanum, gadolinium, and removal of extracellular calcium but not by the PLC inhibitor U73122.Conclusions: These data suggest that H2S activates a subset of TRPC/M and possibly TRPA1, but not TRPV, channels to induce the release of ATP, which excites mechanosensitive afferent terminals via P2X receptors.This mechanism may play an important role in gut physiology and pathophysiology.Supported by the BBSRC.