Inhibition and Inactivation of NO Synthase by Tobacco

描述（由申请人提供）：拟议研究的目的是表征烟草介导的no合成酶（NOS）的抑制和失活，NOS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。这将部分通过差异代谢分析方法，以及高通量生物测定和最先进的LC-MS/MS技术的使用来解决。已知接触烟草中的化学物质会导致一氧化氮产生和可得性障碍。吸烟与多种疾病有关，由于一氧化氮在许多生理功能中起着核心作用，因此这种相互作用可能在临床上很重要。我们已经确定，使用从烟草和烟草烟雾中提取的粗提取物，对NOS有直接的，可逆的抑制作用，以及基于代谢的，时间依赖性的，不可逆的NOS失活。对提取物的进一步研究表明，小的，有机的，水溶性化合物是导致NOS功能障碍的原因。此外，当l -精氨酸存在时，这些提取物特异性灭活内皮细胞NOS (eNOS)，而当四氢生物蝶呤过量时，观察到神经元NOS （nNOS）特异性失活。提出了以下具体目标：表征烟草成分引起的nNOS失活。这包括鉴定导致失活的化学物质。2. 表征烟草成分引起的eNOS失活。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解决。3. 研究烟草烟雾对eNOS和nNOS的抑制机制，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NOS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4 000多万人接触这些化学品，因此关于这些化学品的性质及其与重要生物系统的相互作用的信息将是有用的。这些研究还将有助于鉴定涉及NOS的烟草相关疾病的生物标志物，并促进NOS功能障碍治疗的发展。

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.