Transmitter Repletion: Key to Phrenic-Diaphragm Function

描述（由申请方提供）：膈神经-膈肌神经肌肉传递失败导致高碳酸血症性呼吸衰竭。这不仅发生在明显病变的神经肌肉接头（如。重症肌无力，肉毒杆菌中毒），而且在肺部疾病的机械负荷期间受到高强度激活的正常连接中（例如，COPD）或暴露于全身因素（如缺氧和体温过低）期间。神经传递需要足够的连接前乙酰胆碱（ACh）释放，以确保肌肉收缩。在重复激活过程中，ACh释放减少，严重时会导致传输故障。可供释放的ACh的恢复取决于两个独立但相互关联的过程：突触间隙中递质的再循环，以及一个或多个储备池中可立即释放的囊泡池的补充。呼吸肌持续活动，因此递质补充需要足够强大，以确保ACh的持续供应可用于释放。本建议的总体目标是进一步研究的作用，递质补给，和调节补给的因素，在确定呼吸神经肌肉接头传输的完整性。待检验的具体假设如下。1)ACh补充的速度和时间是膈-膈肌神经肌肉接头（尤其是患病的神经肌肉接头）传递的关键决定因素。2)高频率刺激加速ACh的补充，这是对高频率激活对释放和消耗的不利影响的调节。此外，突触前[Ca+ +]升高介导了补充的加速。3)缺氧和体温过低在很大程度上通过减缓递质补充而损害神经传递，而不是主要通过直接抑制递质释放。4)突触前K+通道不仅调节Ach的释放，还调节递质的补充，提供了通过药物操纵K-通道电导来改善神经传递的两种机制。神经肌肉传递将使用力测量的组合进行评估，以量化疲劳的神经肌肉成分，电生理记录，以确定ACh释放和从发射器耗尽恢复，并使用荧光苯乙烯染料（FM 1 -43，FM 2 -10）的光学方法，以评估囊泡池动力学。这些研究可能会导致新的治疗方法，呼吸肌损伤和导致高碳酸呼吸衰竭的条件下，产生神经肌肉接头功能障碍。

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.