Neuromuscular Transmission in Arteries and Veins

描述（由申请人提供）：本实验室的长期目标是促进我们对与多化学神经传递特别相关的血管神经效应器机制的理解。有大量证据表明，内脏循环中的电阻性和电容性血管对交感神经刺激的机械、电和神经化学反应不同。然而，动脉和静脉中神经效应器过程不同的机制仍然未知。在目的1中，我们提出了一个假设，即神经释放的去甲肾上腺素（NE）和腺苷5 '-三磷酸（ATP）的量和/或比例在动脉和静脉中不同，由于不同的表达水平或功能活性的神经元钙通道蛋白。目的2是基于我们最近观察到的肠系膜动、静脉节后神经末梢的激活引起腺苷5 '-二磷酸核糖（ADPR）的释放，以及ATP、ADP、AMP、腺苷和NE的沿着。这代表了一个新的发现，可能导致识别神经血管控制的新机制。动脉和静脉在ADPR释放方面表现出质和量的差异。关于这一点，我们建议检查的假设，即形成（主要是CD 38介导的）和释放ADPR或其前体，NAD+和环ADPR，差异调制的激活蛋白激酶A（PKA）依赖的途径在动脉和静脉。在目的3中，我们假设对神经刺激和NE的电反应是由肾上腺素受体/PI 3-激酶/PKC调节Ca 2+激活的Cl-通道介导的，这可能在动脉和静脉中具有不同的患病率。这些研究将涉及犬肠系膜动脉和静脉中的实验，以确定神经诱发的核苷酸、核苷和NE溢出（HPLC-FLD和ECD技术）;神经元Ca 2+通道表达和功能（Western印迹、神经递质释放）; PKA/CD 38相互作用（免疫印迹、共沉淀、蛋白磷酸化、RT-PCR）以及NE激活ICaCI的机制（膜片钳技术）。这种前所未有的方法组合应该为动脉和静脉神经控制的复杂机制提供重要的见解。这些结果可能为中断或预防与交感神经功能亢进和血管功能障碍相关的病理状况，如高血压、心源性休克、充血性心力衰竭、出血提供策略。

DESCRIPTION (provided by applicant): The long-term goal of this laboratory is to forward our understanding of vascular neuroeffector mechanisms specifically related to plurichemical neurotransmission. The evidence is substantial that the resistive and capacitative vessels in the splanchnic circulation differ in their mechanical, electrical, and neurochemical responses to sympathetic nerve stimulation. However, the mechanisms by which neuroeffector processes differ in the arteries and veins remain unknown. In Aim 1 we propose to examine the hypothesis that the amount and/or ratio of neurally released norepinephrine (NE) and adenosine 5'-triphosphate (ATP) differ in artery and vein due to different expression levels or functional activity of neuronal Ca2+ channel proteins. Aim 2 is based on our recent observation that activation of postganglionic nerve terminals in mesenteric arteries and veins evokes release of adenosine 5'-diphosphoribose (ADPR), along with ATP, ADP, AMP, adenosine and NE. This represents a novel finding that may lead to identifying new mechanisms of neurovascular control. Arteries and veins show quantitative and qualitative differences in ADPR release. With regard to this, we propose to examine the hypothesis that the formation (primarily CD38-mediated) and release of ADPR or its precursors, NAD+ and cyclic ADPR, are differentially modulated by the activation of protein kinase A (PKA)-dependent pathways in artery and vein. In Aim 3 we hypothesize that electrical responses to nerve stimulation and NE are mediated by adrenoceptor/PI3-kinase/PKC regulation of Ca2+-activated CI- channels, which may have differential prevalence in artery and vein. These studies will involve experiments in canine mesenteric artery and vein to determine the nerve-evoked overflow of nucleotides, nucleosides and NE (HPLC-FLD and ECD techniques); neuronal Ca2+ channel expression and function (Western blotting, neurotransmitter release); PKA/CD38 interactions (immunoblotting, coprecipitation, protein phosphorylation, RT-PCR), as well as mechanisms of NE activation of ICaCI (patch-clamp techniques). This unprecedented combination of approaches should provide important insight into the complex mechanisms governing neural control in arteries and veins. The results may suggest strategies for interrupting or preventing pathological conditions associated with sympathetic hyperactivity and vascular dysfunctions such as hypertension, cardiogenic shock, congestive heart failure, hemorrhage.