Development of Analytical Tools for Concentration and Real-Time Control of Dissolved Gases and Their Regulation of Tissue Function

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Project Summary The major goal of the proposed research is to develop tools to enable the study of dissolved gases on biological samples. Both blood gases (O2, and CO2) as well as trace signaling gases (NO, CO and H2S) play critical roles in regulation of wide array of tissue functions as diverse as regulation of heart rate, blood flow, immune responses, hormone and neurotransmitter secretion, as well as cytoprotective and anti-inflammatory properties. Indeed, most if not all tissues produce and are regulated by NO, CO and H2S on top of the central role of O2 in regulating bioenergetics and metabolic pathways. Research by academic institutions as well as pharmaceutical companies are endeavoring to harness the beneficial effects of gas signals in order to treat a range of conditions including diabetes, transplant rejection, sepsis, atherosclerosis and cancer. Despite the scientific and clinical importance of dissolved gases, quantitative methods to measure real time effects of dissolved gases on tissue/cells are not available. Investigators who have studied the beneficial signaling initiated by trace gases and/or who are developing drugs to trigger the same benefits almost exclusively use water soluble surrogates/donors of each signaling gas. A critical point for this proposal is that such chemical donors of gases may not allow for accurate control of gas levels within tissue, and due to their low aqueous solubility, rapidly deplete from culture media. Preliminary data we have generated and published revealed opposite effects of dissolved H2S vs. that obtained with an aqueous provider of H2S, indicating a need to re- evaluation the effects of what is established regarding NO, H2S and CO from studies using donor molecules. As most life science researchers do not have the ability to study the direct effects of trace gases at physiologically relevant concentrations, we will develop a turnkey, automated instrumentation to control the concentration and exposure time of tissue to media containing user-specified levels of 6 gases including NO, CO, H2S, O2, CO2 and N2. This automated and user-friendly system will be constructed to supply gas mixtures to a variety of widely used tissue assessment modalities including fluidics systems, static culture in plates, and cuvette systems for studying gas binding interactions to purified proteins. We have assembled a team consisting of Bio- and Mechanical/Combustion Engineers with many years of experience designing and constructing gas fluidics system applied to biological analysis, as well as an Applied Mathematician, an Analytical Chemists and Cellular Physiologists who will be involved with the validation of the instrumentation. This technology will have broad impact on fundamental research by facilitating the study of kinetic and concentration-dependent effects of signaling gases on tissue function, signaling and bioenergetics, and evaluation of therapeutics being developed to mimic the cytoprotective properties of the gases.