Neuromuscular Transmission in Arteries and Veins

描述(申请人提供)：这个实验室的长期目标是促进我们对血管神经效应机制的理解，特别是与多种化学神经传递相关的机制。大量证据表明，内脏循环中的阻性血管和容量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，神经效应器过程在动脉和静脉中的不同机制仍不清楚。在目标1中，我们提出了一种假设，即由于神经元钙通道蛋白的表达水平或功能活性不同，动脉和静脉中神经释放的去甲肾上腺素(NE)和5‘-三磷酸腺苷(ATP)的数量和/或比例不同。目的2是基于我们最近观察到的肠系膜动脉和静脉节后神经末梢的激活引起腺苷5‘-二磷酸核糖(ADPR)以及ATP、ADP、AMP、腺苷和NE的释放。这代表了一项新的发现，可能会导致识别神经血管控制的新机制。动脉和静脉在ADPR释放方面表现出数量和质量上的差异。对此，我们建议检验这样一种假设，即ADPR或其前体NAD和环状ADPR的形成和释放受到动脉和静脉中依赖于蛋白激酶A(PKA)的通路的激活的不同调控。在目标3中，我们假设对神经刺激和去甲肾上腺素的电反应是由肾上腺素受体/PI3-激酶/PKC对钙激活的CI通道的调节所介导的，这种调节在动脉和静脉中可能有不同的流行。这些研究将涉及犬肠系膜动脉和静脉的实验，以确定神经诱发的核苷酸、核苷和NE的溢出(高效液相色谱-FLD和电子捕获技术)；神经元钙通道的表达和功能(Western blotting，神经递质释放)；PKA/CD38的相互作用(免疫印迹、共沉淀、蛋白质磷酸化、RT-PCR)，以及NE激活ICaCI的机制(膜片钳技术)。这一史无前例的方法组合应该为控制动脉和静脉中的神经控制的复杂机制提供重要的洞察。这一结果可能为干预或预防与交感神经过度活动和血管功能障碍相关的病理情况提供策略，如高血压、心源性休克、充血性心力衰竭、出血。

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.