NSF Award
0641516
Nitric oxide (NO) is synthesized in a variety of biological systems, fulfilling a wide range of signaling roles. Although the biological consequences of its synthesis are widely documented, its chemical modes of action and function, in vivo, remain controversial and largely unclear. One difficulty in pinpointing the biochemistry of NO stems from the lack of instrumentation capable of (1) tracing NO from its sources, through chemical intermediates, and to its protein targets, while (2) affording sufficient sensitivity to detect these products at levels found in most biological samples, which typically lie in the low picomole range. <br/>The first goal of this project is to perfect an experimental technique currently under development in the Rodriguez laboratory for tracking the fate of NO in biological systems, using forms of NO labeled with stable isotopes. The technique is based on existing analytical assays for the quantification of NO-related products, including NO-modified proteins, which were originally developed for NO-ozone chemiluminescence detection. Steps are incorporated in the project for use of mass spectrometry in these assays, in a way that will enable detection of low picomole levels of NO-related products with isotope specificity.<br/>The second goal of the project is to validate the use of this newly-developed technique in biological fluids and matrices. To this end, the technique will be applied to a well-characterized biological model capable of producing NO at high fluxes, namely the macrophage immune cell. As part of this process, the mechanism that allows these cells to cope with potentially toxic levels of the NO related product, nitrite, and how they may use this substance to fulfill their immunological role will be investigated. Preliminary results suggest these cells may possess a mechanism that prevents intracellular levels of nitrite from reaching excessive levels when their immune machinery is activated. With the aid of 15N-labelled nitrite and NO precursor L-arginine, the temporal changes in intra- and extra-cellular concentrations of nitrite, as well as its rate of production, metabolism, and transport in and out of these cells will be quantitated. Knowledge of these quantities, combined with mathematical modeling, will permit elucidation of which processes are actively involved in the regulation of nitrite. The same modeling will allow determination of whether these processes allow toxic levels of nitrite to accumulate within phagosomes, the intracellular spaces that engulf invading pathogens.<br/>Broader impact. Education: This project will allow undergraduate students with diverse mathematical and scientific backgrounds to collaborate in biological research that interfaces mathematics, chemistry, physics, and engineering. In the process they will discover, and hopefully pass on to others, the realization that training in these disciplines helps to advance the understanding of modern biology. Improving research infrastructure: The hardware design, and techniques developed in this project will be disseminated through meetings and publications. In addition, the instrumentation will be made available to other investigators, particularly those in Northwest Louisiana, many of whom have an interest in understanding the role of NO in mammalian and plant systems. The technique will also provide plant scientists with improved capability to study nitrogen uptake and metabolism in plants.
