Mechanisms of Biological Nitrosation from Nitric Oxide

说明(申请人提供)：一氧化氮(一氧化氮，NO)在体内容易氧化，导致产生活性物种，形成含氮氧化物的加合物、氧化的细胞成分或相对不活性的氧阴离子。在NO的生物作用中，亚硝化(单电子氧化物种亚硝化亚硝胺(NO+)的化学作用)历来(无疑)在化学病理学方面受到最大的关注。亲核细胞靶标的亚硝化既会造成损害(例如亚硝胺的形成和DNA碱基的脱氨基)，也会产生可能的调节效应(亚硝硫醇的形成)。然而，亚硝化在细胞中发生的机制(S)尚不清楚。我们的总体目标是从NO中确定亚硝化的生化机制(S)。为了实现这一目标，我们将在这两个领域以往工作的基础上，沿着两条主线(包括我们的四个具体目标)前进。在AIMS I-II中，我们将描述在疏水环境(如膜)中NO与O2反应的动力学和机理特征。我们以前曾提出，从生物学上讲，这是这个反应的主要部位，该反应产生了强有力的亚硝化物种。我们将确定这种效应的动力学特征，我们估计这种效应在生物条件下可以加速NO/O2反应约300倍。此外，我们将研究这种分配的机理效应，特别是膜双层的疏水内部在影响小的未带电自由基物种之间的反应(与水溶液中的反应相反)时可能具有的溶剂化和碰撞效应。在AIMS Ill-IV中，我们将描述非红系细胞O2依赖的NO消耗的特征，这是发生在血管管腔外的NO的主要转化，我们已经展示了在亚硝酸盐(亚硝化化学的标志)的产生中的结果。这一现象有别于AIMS I-II中的膜效应，我们将确定该反应的生化机制(S)，包括参与该反应的细胞物种和负责NO氧化的氧物种(二氧化氧或超氧化物)的性质以及过渡金属离子的参与。我们还将从外源性和内源性NO两个方面描述亚硝化细胞化学。这些研究将为NO及其衍生物的基本生物化学提供新的见解，也可能为各种病理条件下的治疗开发提供新的可能途径，包括癌症发生、炎症刺激和氧化应激。

DESCRIPTION (provided by applicant): Nitric oxide (nitrogen monoxide, NO) is subject to oxidation in vivo, which results in the production of reactive species that either form nitrogen oxide-containing adducts, oxidized cellular components, or relatively unreactive oxyanions. Among the biological actions of NO, nitrosation (the chemistry of the one-electron oxidized species nitrosonium (NO+)) has historically (and undoubtedly) received the most attention in terms of chemical pathology. Nitrosation of nucleophilic cellular targets results in both damaging (e.g., nitrosamine formation and DNA base deamination) and also possible regulatory effects (formation of nitrosothiols). However, the mechanism(s) whereby nitrosation takes place in cells is unknown. Our overall objective is to define the biochemical mechanism(s) of nitrosation from NO. To accomplish this objective, we will proceed along two lines (encompassing our four Specific Aims), based on our previous work in these two areas. In Aims I-II, we will delineate the kinetic and mechanistic characteristics of the reaction of NO with O2 within hydrophobic environments such as membranes. We have previously proposed that this is the major site biologically for this reaction that produces potent nitrosating species. We will determine the kinetic characteristics of this effect, which we have estimated accelerates the NO/O2 reaction by a factor of approximately 300-fold under biological conditions. In addition, we will examine the mechanistic effects of this partitioning, specifically, the solvation and collisional effects which the hydrophobic interior of membrane bilayers may have in influencing these reactions between small uncharged radical species (as opposed to reactions in aqueous solution). In Aims Ill-IV, we will delineate the characteristics of non-erythroid cellular O2-dependent consumption of NO, which is quantitatively the major transformation of NO that occurs outside the vascular lumen, and which we have shown results in the production of nitrite (a marker for nitrosative chemistry). This phenomenon is distinct from the membrane effect in Aims I-II, and we will identify the biochemical mechanism(s) of this reaction, including the cellular species involved and nature of the oxygen species responsible for NO oxidation (dioxygen or superoxide) as well as the involvement of transition metal ions. We will also delineate the nitrosative cellular chemistry from both exogenous and endogenous NO. These studies will provide both new insight into the basic biochemistry of NO and its derivatives, and also potentially provide new possible avenues for therapeutic development in a variety of pathological conditions, including carcinogenesis, inflammatory stimulation, and oxidative stress.