Inhibition and Inactivation of NO Synthase by Tobacco

描述(申请人提供)：拟议研究的目的是表征烟草对一氧化氮合酶(NOS)的抑制和失活，一氧化氮合酶是一种细胞色素P450样酶。在这些研究过程中，我们的目标是分离和识别烟草中导致这些影响的化学物质。这在一定程度上将通过不同的代谢谱方法以及高通量生物分析和最先进的LC-MS/MS技术来解决。众所周知，接触烟草中的化学物质会导致一氧化氮的产生和供应功能障碍。吸烟与多种疾病有关，由于一氧化氮在多种生理功能中发挥核心作用，这种相互作用很可能在临床上很重要。通过使用烟草和烟草烟雾的粗提物，我们已经确定，存在对一氧化氮合酶的即时、可逆的抑制，以及基于新陈代谢的、时间依赖的、不可逆转的一氧化氮合酶的失活。对提取物的进一步研究表明，小的有机水溶性化合物是导致一氧化氮合酶功能障碍的原因。此外，当L-精氨酸存在时，这些提取物特异性地灭活内皮型一氧化氮合酶，而当四氢生物蝶呤过量时，则特异性地灭活神经元型一氧化氮合酶。具体目标如下：1.研究烟草成分对神经元型一氧化氮合酶的失活作用。这包括识别导致失活的化学物质。2.研究烟草成分对eNOS失活的影响。四氢生物蝶呤氧化的机制，以及负责灭活的化学物质的身份将被讨论。3.研究烟草烟雾抑制eNOS和nNOS的作用机制，并确定其作用成分。这些目标的成功完成将确定潜在的新型一氧化氮合酶抑制剂和灭活剂作为药理学工具，并允许对这些化学物质对吸烟者的影响进行更详细的研究。由于每天有4000多万人接触到这些化学品，有关这些化学品的性质及其与重要生物系统相互作用的信息将是有用的。这些研究还应有助于确定涉及一氧化氮合酶的烟草相关疾病的生物标记物，并促进一氧化氮合酶功能障碍的治疗方法的开发。

DESCRIPTION (provided by applicant): The objective of the proposed research is to characterize the tobacco-mediated inhibition and inactivation of NO-synthase (NOS), a cytochrome P450-like enzyme. In the course of these studies, we aim to isolate and identify the chemicals in tobacco that are responsible for these effects. This will be addressed, in part, by a differential metabolic profiling approach, as well as the use of high-throughput bioassays and state of the art LC-MS/MS techniques. Exposure to chemicals in tobacco is known to cause a dysfunction in NO production and availability. Cigarette smoking is associated with a variety of diseases and, since NO plays a central role in a myriad of physiological functions, it is likely that such interactions may be clinically important. We have established, with the use of crude extracts derived from tobacco and tobacco smoke, that there is an immediate, reversible inhibition of NOS, as well as a metabolism-based, time-dependent, irreversible inactivation of NOS. Further studies on the extracts indicate that small, organic, water-soluble compounds are responsible for causing NOS dysfunction. Moreover, these extracts specifically inactivated endothelial NOS (eNOS) when L-arginine was present, whereas specific inactivation of neuronal NOS (nNOS) was observed when tetrahydrobiopterin was in excess. The following specific aims are proposed: 1. To characterize the inactivation of nNOS caused by tobacco components. This includes identification of the chemicals responsible for inactivation. 2. To characterize the inactivation of eNOS caused by tobacco components. The mechanism of tetrahydrobiopterin oxidation, as well as the identity of the chemicals responsible for inactivation will be addressed. 3. To characterize the mechanism of inhibition of eNOS and nNOS caused by tobacco smoke, as well as determine the identity of the components responsible. The successful completion of these aims will identify potentially novel inhibitors and inactivators of NOS as pharmacological tools, as well as allow for a more detailed study of the effects of these chemicals in smokers. Since over 40 million people are exposed to these chemicals on a daily basis, information on the nature of these chemicals and their interaction with important biological systems would be useful. These studies should also lead to identification of biomarkers for tobacco-related diseases involving NOS and facilitate development of therapies for NOS dysfunction.