Transmitter Repletion: Key to Phrenic-Diaphragm Function

描述(申请人提供)：膈神经-隔膜神经肌肉传递失败导致高碳酸血症呼吸衰竭。这不仅发生在明显病变的神经肌肉接头(例如，重症肌无力，肉毒杆菌中毒)，但也在正常连接受到高强度激活在机械负荷期间由肺部疾病(例如。慢性阻塞性肺病)或暴露于全身因素，如缺氧和体温过低。神经传递需要充分的节前释放乙酰胆碱(ACh)以确保肌肉收缩。在重复激活的过程中，ACh的释放减少，严重时会导致传输失败。可供释放的ACh的恢复依赖于两个独立但相互关联的过程：从突触间隙回收递质，以及从一个或多个储备池中重新填充立即可释放的囊泡池。呼吸肌持续活跃，因此递质补充需要足够强大，以确保ACh的持续供应可供释放。这项建议的总体目标是进一步研究递质补充的作用，以及调节补充的因素，以确定呼吸神经肌肉连接中传递的完整性。需要检验的具体假设如下。1)ACh补充的速度和时间是膈-隔神经肌肉接头传递的关键决定因素，尤其是在病变的神经肌肉接头。2)高频刺激加速了ACh的补充，以适应高频激活对释放和耗尽的不利影响。此外，突触前[Ca]升高介导了补充的加速。3)低氧和低温在很大程度上是通过减缓递质的补充来损害神经传递，而不是主要通过直接抑制递质的释放。4)突触前钾通道不仅调节Ach的释放，而且还调节递质的补充，提供了两种通过药物调控K通道电导来改善神经递质传递的机制。神经肌肉传递的评估将使用力测量的组合来量化疲劳的神经肌肉成分，电生理记录来确定ACh的释放和从递质衰竭中恢复，以及使用荧光类型1染料(FM1-43，FM2-10)的光学方法来评估囊泡池的动力学。这些研究可能导致呼吸肌损伤的新治疗方法，以及导致神经肌肉连接功能障碍的高碳酸血症呼吸衰竭。

DESCRIPTION (provided by applicant): Failure of phrenic nerve-diaphragm neuromuscular transmission leads to hypercapnic respiratory failure. This occurs not only in overtly diseased neuromuscular junctions (eg. myasthenia gravis, botulism), but also in normal junctions subjected to high intensity activation during mechanical loading by lung disease (eg. COPD) or during exposure to systemic factors such as hypoxia and hypothermia. Neurotransmission requires sufficient prejunctional release of acetylcholine (ACh) to ensure muscle contraction. During repetitive activation, ACh release diminishes, which when severe leads to transmission failure. Restoration of ACh available for release depends on two separate but interrelated processes: recycling of transmitter from the synaptic cleft, and repletion of the immediately releaseable vesicle pool from one or more reserve pools. Respiratory muscles are active continuously, so that transmitter replenishment needs to be sufficiently robust to ensure that a constant supply of ACh is available for release. The overall objective of this proposal is to further examine the role of transmitter replenishment, and the factors which regulate replenishment, in determining the integrity of transmission in respiratory neuromuscular junctions. The specific hypotheses to be tested are as follows. 1) The rapidity of, and time available for, ACh replenishment are critical determinants of transmission at the phrenic-diaphragm neuromuscular junction, especially in diseased neuromuscular junctions. 2) ACh replenishment is hastened by high frequency stimulation, an accommodation to the adverse effects of high frequency activation on release and depletion. Furthermore, the acceleration of replenishment is mediated by elevated presynaptic [Ca+ +]. 3) Hypoxia and hypothermia impair neurotransmission to a large extent by slowing transmitter replenishment, rather than primarily by a direct inhibition of transmitter release. 4) Presynaptic K+ channels regulate not only Ach release but also transmitter replenishment, providing two mechanisms of improving neurotransmission by pharmacologic manipulation of K- channel conductances. Neuromuscular transmission will be assessed using a combination of force measurements to quantify the neuromuscular component of fatigue, electrophysiological recording to determine ACh release and recovery from transmitter rundown, and optical approaches using fluorescent styry1 dyes ( FM1-43, FM2-10) to assess vesicle pool dynamics. These studies may lead to novel therapeutic approaches to respiratory muscle impairment and resulting hypercapnic respiratory failure for conditions which produce neuromuscular junction dysfunction.