Center for Swallowing & Motility Disorders, VA Boston HealthCare System and Harvard Medical School, Boston, Massachusetts, USA.
Am J Physiol Gastrointest Liver Physiol. 2011 Sep;301(3):G498-507. doi: 10.1152/ajpgi.00164.2011. Epub 2011 Jun 16.
Nitrergic neurotransmission at the smooth muscle neuromuscular junctions requires nitric oxide (NO) release that is dependent on the transport and docking of neuronal NO synthase (nNOS) α to the membrane of nerve terminals. However, the mechanism of translocation of nNOSα in actin-rich varicosities is unknown. We report here that the processive motor protein myosin Va is necessary for nitrergic neurotransmission. In wild-type mice, nNOSα-stained enteric varicosities colocalized with myosin Va and its tail constituent light chain 8 (LC8). In situ proximity ligation assay showed close association among nNOSα, myosin Va, and LC8. nNOSα was associated with varicosity membrane. Varicosities showed nitric oxide production upon stimulation with KCl. Intracellular microelectrode studies showed nitrergic IJP and smooth muscle hyperpolarizing responses to NO donor diethylenetriamine-NO (DNO). In contrast, enteric varicosities from myosin Va-deficient DBA (for dilute, brown, non-agouti) mice showed near absence of myosin Va but normal nNOSα and LC8. Membrane-bound nNOSα was not detectable, and the varicosities showed reduced NO production. Intracellular recordings in DBA mice showed reduced nitrergic IJPs but normal hyperpolarizing response to DNO. The nitrergic slow IJP was 9.1 ± 0.7 mV in the wild-type controls and 3.4 ± 0.3 mV in the DBA mice (P < 0.0001). Deficiency of myosin Va resulted in loss of nitrergic neuromuscular neurotransmission despite normal presence of nNOSα in the varicosities. These studies reveal the critical importance of myosin Va in nitrergic neurotransmission by facilitating transport of nNOSα to the varicosity membrane.
在平滑肌神经肌肉接头处的氮能神经传递需要依赖于神经元型一氧化氮合酶（nNOS）α向神经末梢膜的转运和对接的一氧化氮（NO）释放。然而，nNOSα 在富含肌动蛋白的轴突中的易位机制尚不清楚。我们在此报告，动力蛋白肌球蛋白 Va 是氮能神经传递所必需的。在野生型小鼠中，nNOSα 染色的肠神经末梢与肌球蛋白 Va 及其尾部成分轻链 8（LC8）共定位。原位接近连接测定显示 nNOSα、肌球蛋白 Va 和 LC8 之间密切相关。nNOSα 与轴突末梢膜相关。用 KCl 刺激后，轴突末梢显示一氧化氮产生。细胞内微电极研究表明，氮能 IJPs 和平滑肌对一氧化氮供体二乙三胺五乙酸-NO（DNO）的超极化反应。相比之下，肌球蛋白 Va 缺陷的 DBA（用于稀释、棕色、非阿古伊）小鼠的肠神经末梢显示出几乎不存在肌球蛋白 Va，但正常的 nNOSα 和 LC8。膜结合的 nNOSα 不可检测，轴突末梢显示 NO 产生减少。在 DBA 小鼠中进行细胞内记录显示，氮能 IJPs 减少，但对 DNO 的超极化反应正常。在野生型对照中，氮能缓慢 IJPs 为 9.1±0.7 mV，而在 DBA 小鼠中为 3.4±0.3 mV（P<0.0001）。尽管 nNOSα 在轴突末梢正常存在，但肌球蛋白 Va 的缺乏导致氮能神经肌肉传递丧失。这些研究揭示了肌球蛋白 Va 通过促进 nNOSα 向轴突末梢膜的转运在氮能神经传递中的关键重要性。
