Central and Peripheral Actions of Nitric Oxide

一氧化氮（NO）是一种在植物和动物中都能产生的高活性小分子。它被用来保护组织免受感染，并在脊椎动物的脉管系统和大多数物种的中枢神经系统（CNS）中充当信号分子。因为一氧化氮可以溶解在细胞膜和细胞周围的液体中，所以它被认为是从它产生的地方广泛传播的。然而，一氧化氮信号的范围还没有严格的定义，可能在不同的组织和物种之间有所不同。在中枢神经系统中，200微米的有效范围将使NO能够控制数百个神经元，但在肌肉等外周组织中，这个范围可能将NO限制在非常局部甚至细胞内的作用。因此，NO靶组织在大小、代谢功能和细胞结构上的巨大差异，对NO在不同部位的信号传导机制提出了重要的问题。本实验利用昆虫模型系统（Manduca sexta幼虫）中具有良好特征的NO信号系统来确定NO在中枢和外周部位的功能作用。对这种昆虫进行研究的一个关键优势是，信号可以在完整的、自由移动的幼虫中被操纵，也可以在从昆虫解剖出来的组织中被操纵，并在规定的条件下保持存活数小时。药物工具可用于阻断或补充NO的产生，并阻断或增加其有效性。现在还可以通过注射双链RNA在分子水平上改变完整动物体内关键酶的合成。这种处理会干扰完全发育的动物或中枢神经系统选定部分特定基因的表达。初步的生理实验结果，以及产生或响应NO的酶（一氧化氮合酶NOS和可溶性鸟苷环化酶sGC）的解剖分布表明，NO参与控制摄食（咀嚼运动和前肠活动）、调节正常的体壁张力和直接的细胞免疫反应。将电极和压力传感器植入正常幼虫和实验中缺乏NOS或sGC的幼虫，记录其神经和肌肉活动。一氧化氮对运动模式和神经肌肉生理的这些作用也将在分离组织中进行药理学检查。通过将荧光微球植入体腔，并评估正常和NO缺乏幼虫的包封程度，来检测NO在昆虫免疫应答中的作用。预计饲料（和生长）、肠道运动和体壁感觉神经元的活动都将受到NO的强烈调节。控制体肿胀、运动协调和非生物包封也可能是非常重要的作用部位。拟议活动的长期目标是更好地了解NO如何执行其各种功能。特别是，结果将有助于定义在非常不同的组织环境中的NO信号的专门化和局限性。由于NO参与了这种草食性昆虫的运动和摄食行为，该结果也可能影响作物保护害虫特异性抗食性的开发。

Nitric oxide (NO) is a small, highly reactive molecule produced in both plants and animals. It is used to protect tissues from infections and acts as a signaling molecule in the vasculature of vertebrates and the central nervous system (CNS) of most species. Because NO can dissolve in both cell membranes and the fluid around cells it is thought to spread widely from where it is produced. However, the range of NO signaling has not been rigorously defined and may vary between tissues and species. In the CNS an effective range of 200 microns would enable NO to control hundreds of neurons, but in peripheral tissues such as muscles this range could limit NO to a very local or even intracellular role. Hence, the immense differences in size, metabolic function and cellular architecture of NO target tissues raise important questions about the mechanisms of NO signaling at different locations. The proposed experiments take advantage of a well-characterized NO signaling system in an insect model system (larval Manduca sexta) to establish the functional roles of NO at central and peripheral sites. A key advantage of studies in this insect is that signaling can be manipulated in the intact, freely moving larva, or in tissues dissected from the insect and maintained alive for several hours under defined conditions. Pharmacological tools are available to block or supplement NO production and to block or increase its effectiveness. It is now also possible to alter the synthesis of key enzymes at the molecular level in intact animals using injections of double-stranded RNA. This treatment interferes with the expression of specific genes in the fully developed animal or in selected parts of the CNS. The results of preliminary physiological experiments, together with the anatomical distribution of enzymes that make or respond to NO (nitric oxide synthase, NOS; and soluble guanylyl cyclase, sGC; respectively), suggest that NO is involved in the control of feeding (chewing movements and foregut activity), the regulation of normal body wall tension and the direct cellular immune response. The neural and muscular activity will be recorded using electrodes and pressure sensors implanted in normal larvae and in larvae that are experimentally deficient in NOS or sGC. These actions of NO on motor patterns and neuromuscular physiology will also be examined pharmacologically in isolated tissues. The role of NO in the insect immune response will be tested by implanting fluorescent micro spheres into the body cavity and assessing the degree of encapsulation in normal and NO-deficient larvae. It is expected that feeding (and growth), gut movements and activity of body wall sensory neurons will all be strongly modulated by NO. The control of body turgor, motor coordination, and abiotic encapsulation are also potentially very important sites of action.The long-term goal of the proposed activity is to better understand how NO carries out its diverse functions. In particular, the results will help to define the specializations and limitations of NO signaling in very different tissue environments. Because NO is involved in the locomotion and feeding behavior of this herbivorous insect, the results could also impact the development of pest-specific antifeedants for crop protection.