Purinergic neurotransmission in the gut

描述(申请人提供)：肠道神经系统(ENS)是位于肠壁内的自主神经系统的分支。ENS控制胃肠(GI)的运动、分泌和局部血流。ENS可以执行这些复杂的功能，因为它包含了GI反射和整合所需的所有神经元元素(感觉神经元、中间神经元和运动神经元)。ENS包含14种不同类型的神经元，它们释放不同的神经递质。每个神经递质也有多个受体。此外，ENS中的突触可能由突触前神经末梢释放的神经递质和突触后细胞表达的受体编码。拟议的研究将使用细胞内电生理、免疫组织化学和分子生物学方法来研究肠道神经肌肉传递。这项建议有三个具体目标。具体目标1将检验这样的假设，即有两个独立的抑制神经群体供应肌层。其中一组使用一氧化氮(NO)作为主要的神经递质，而第二组是嘌呤能的(ATP和/或b-烟酰胺腺嘌呤二核苷酸是神经递质)。这些研究表明，神经末梢的ATP/b-NAD和NO的释放受不同的钙通道类型的控制。针对泡核苷酸(VNUT)抗体的抗体将用于对嘌呤能神经进行定位。这些研究还将利用P/Q型和R型钙通道突变小鼠。具体目标2将集中在肌间神经丛中的中间神经元表达的钙通道。在口腔-肛门方向投射的中间神经元释放乙酰胆碱(ACh)和ATP作为快速突触递质，而在肛门-口腔方向投射的神经元则释放ACh。这些研究将验证这一假设，即R-、N-和P/Q型钙通道由口腔投射通路中的神经元表达，而N-和P/Q型钙通道仅由肛门投射通路中的神经末梢表达。这些研究还将使用野生型、P/Q型和R型钙通道突变小鼠。具体目标3将重点放在K+通道作为肠道平滑肌张力和结肠神经肌肉传递的调节器。这些研究将利用大电导钙激活K+(BK)通道基因敲除小鼠的b1亚单位。意义：肠道突触机制的紊乱导致胃肠动力障碍。肠道神经元及其突触功能的改变也可能导致内脏疼痛。因此，对肠道神经回路和突触传递的更全面的了解将为胃肠动力障碍的病理生理学提供更深入的认识。这些信息将有助于开发治疗常见运动障碍的新药。

DESCRIPTION (provided by applicant): The enteric nervous system (ENS) is the division of the autonomic nervous system that resides within the gut wall. The ENS controls gastrointestinal (GI) motility, secretion and local blood flow. The ENS can perform these complex functions because it contains all the neuronal elements (sensory neurons, interneurons and motorneurons) required for GI reflexes and integration. The ENS contains 14 different types of neurons that release different neurotransmitters. There are also multiple receptors for each neurotransmitter. In addition, synapses in the ENS may be coded by the neurotransmitters released from presynaptic nerve terminals and by receptors expressed by postsynaptic cells. The proposed studies will use intracellular electrophysiological, immunohistochemical and molecular biological methods to study enteric neuromuscular transmission. There are 3 specific aims in this proposal. Specific aim 1 will test the hypothesis that there are two separate populations of inhibitory nerves supplying the muscle layers. One subset uses nitric oxide (NO) as the primary neurotransmitter while the second population is purinergic (ATP and/or b-nicotinamide adenine dinucleotide are the neurotransmitters). These studies will show that release of ATP/b-NAD and NO from nerve terminals is controlled by different Ca2+ channel types. An antibody against the vesicular nucleotide (VNUT) antibody will be used to localize purinergic nerves. These studies will also make use of P/Q type and R-type Ca2+ channel mutant mice. Specific aim 2 will focus on Ca2+ channels expressed by interneurons in the myenteric plexus. Interneurons which project in an oral-anal direction release acetylcholine (ACh) and ATP as fast synaptic transmitters, while neurons that project in an anal-oral direction release ACh. These studies will test the hypothesis that R-, N- and P/Q type Ca2+ channels are expressed by neurons in the orally-projecting pathway while only N- and P/Q type Ca2+ channels are expressed by nerve terminals in the anally-projecting pathway. These studies will also use wild type and P/Q-type and R-type Ca2+ channel mutant mice. Specific aim 3 will focus on K+ channels as regulators of gut smooth muscle tone and neuromuscular transmission in the colon. These studies will make use of a b1 subunit of the large conductance Ca2+-activated K+ (BK) channel knockout mouse. Significance: Disturbances in enteric synaptic mechanisms contribute to GI motility disorders. Changes in the function of enteric neurons and their synapses might also contribute to visceral pain. Therefore, a more complete understanding of enteric neural circuits and synaptic transmission would provide insights into the pathophysiology of GI motility disorders. This information would help to develop new drug treatments for common motility disorders.