Mechanisms of Biological Nitrosation from Nitric Oxide

描述（由申请人提供）：一氧化氮（一氧化氮，NO）在体内会被氧化，从而产生活性物质，形成含氮氧化物的加合物、氧化的细胞成分或相对不活泼的氧离子。在NO的生物作用中，亚硝化作用（单电子氧化物质亚硝基铵（NO+）的化学反应）历来（毫无疑问）在化学病理学方面受到了最多的关注。亲核细胞靶点的亚硝化会导致损伤（例如亚硝胺的形成和DNA碱基脱胺）和可能的调节作用（亚硝基硫醇的形成）。然而，亚硝化在细胞内发生的机制尚不清楚。我们的总体目标是确定一氧化氮亚硝化的生化机制。为了实现这一目标，我们将根据我们以前在这两个领域的工作，沿着两条路线（包括我们的四个具体目标）进行。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，这是该反应的主要生物学位点，产生强大的亚硝化物种。我们将确定这种效应的动力学特性，我们估计在生物条件下，它将NO/O2反应加速了大约300倍。此外，我们将研究这种分配的机制效应，特别是，膜双层疏水内部在影响小的不带电自由基之间的反应时可能产生的溶剂化和碰撞效应（与水溶液中的反应相反）。在Aims ii - iv中，我们将描述非红系细胞对一氧化氮的依赖消耗的特征，这是在血管腔外发生的一氧化氮的主要转化，我们已经证明了亚硝酸盐（亚硝化化学的标志）的产生结果。这一现象与Aims I-II中的膜效应不同，我们将确定该反应的生化机制，包括参与NO氧化（双氧或超氧）的细胞种类和氧气种类的性质，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（包括致癌、炎症刺激和氧化应激）的治疗开发提供新的可能途径。

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.