Role of hydrogen sulphide in visceral afferent transmission

Hydrogen sulphide (H2S), the smell of rotten egg, is a serious environmental hazard in many industries, e.g. at paper mills, on farms and in oil production. Much research concerning environmental H2S has been carried out over the past 25 years, but it is very recent data pointing to roles of endogenous H2S in mammals both normal and pathological functions of the peripheral nervous system. The gastrointestinal tract (bowel) contains the highest concentration of H2S generated either by gut bacteria or by enzyme systems in the bowel wall. It was this observation that prompted us to perform some preliminary experiments to determine what H2S would do to sensory signals generated from the gut. The sensory nerves that project from the gut to the brain respond to a diverse range of chemicals that are released during normal digestion or inflammation. These nerves help coordinate gut function, influence feeding behaviour and generate abdominal sensations such as discomfort and pain. We made the surprising finding that physiological concentrations of H2S excite intestinal sensory nerves and that it may act by activating specific cell membrane proteins that were only discovered in about the past decade. These are the TRP ion channels now thought to be responsible for many forms of thermal, mechanical, chemical, and/or painful sensations. The overall aims of this proposal are to systemically investigate the physiological role of H2S signalling in abdominal sensory communication by using a multi-disciplinary approach to find out where and how H2S acts on these TRP channels to excite the intestinal sensory neurones, what types of sensory neurones are activated, and whether H2S is released to activate sensory neurones during normal gut function and/or when the bowel is inflamed. The proposed work involves the integration of molecular and cellular physiology with systems neuroscience by an internationally recognised group of experts at the University of Sheffield. A number of different experimental approaches will be used that allow us to detect the electrical signals generated by sensory nerves in the mouse intestine in response to the application of H2S. Using drugs that interact with TRP channels and other receptors in the gut we will be able to determine the site of action of H2S and in particular whether H2S acts directly on the intestinal sensory nerve endings or indirectly by causing release of other chemical mediators from cells in the gut wall. Other drugs block the enzymes that are needed by the body to generate H2S. These can therefore be used to determine how locally produced H2S contributes to the sensitivity of gut sensory afferents and particularly its role in inflammation when the sensory endings become hypersensitive leading to pain and discomfort. Research into sensory signalling from the gut is at the cutting edge of enteric neurobiology. The systematic characterisation of the mechanisms underlying sensory signalling will enhance the competitiveness of the UK pharmaceutical industry and may in the long term lead to improvement in new drugs to treat bowel disorders.

Our preliminary data has shown that physiological concentrations of H2S excite sensory neurons in the gut by causing release of neurotransmitters and has identified a subset of TRP ion channels that may be the direct target of H2S. The main objectives are to determine the site of action of H2S; to investigate the mechanism of its action and to determine the roles of H2S signalling in visceral sensory transmission in normal mice and mice with post-inflammatory visceral hypersensitivity. Extracellular afferent nerve recordings combined with several molecular and cellular biological techniques will be employed to achieve these specific objectives. The detailed procedures include: 1. afferent recording from an isolated segment of intestinal mesenteric nerve bundles in (a) normal, (b) capsaicin pre-treated, (c) chronic sub-diaphragmatic vagotomized mice, and (d) post-inflammatory mice infected with Trichinella spiralis. 2. constructing quantitative afferent discharge rate dose-response curves to H2S under these various conditions 3. examining the dose related effects of non-selective TRP receptor antagonists ruthenium red, lanthanum, 2APB and gadolinium 4. examining the distribution of cystathionine-?-synthetase (CBS) and cystathionine-?-lyase (CSE) expression, the specific enzymes for H2S production, in the gut wall using immunohistochemistry 5. determining the effect of endogenous H2S using PAG and hydroxylamine (specific inhibitors of CBS and CSE) on afferent sensitivity under the different conditions 6. identifying gut-specific sensory neurones by retrograde labelling for patch clamp electrophysiology, calcium imaging, immunocyctochemistry and quantitative RT-PCR to determine role of specific TRP channels in afferent (hyper-)sensitivity. 7. assessing the generation of H2S under the various experimental conditions using polarographic sensors to detect H2S.