Myosin Phosphorylation and Neurotransmitter Secretion

递质释放和其他分泌过程已经被 与肌肉收缩相比， 受Ca ~（2+）调节，依赖于代谢能。 虽然 分泌最初被认为类似于收缩， 骨骼肌，最近的实验表明，它可能更多 非常类似于平滑肌收缩。 已知Ca 2 + 调节平滑肌收缩通过激活的 Ca 2 +/钙调蛋白依赖性酶肌球蛋白轻链激酶。 的 活性酶磷酸化平滑肌肌球蛋白， 在肌动蛋白对ATP水解速率的刺激中， 肌球蛋白 在这项研究中，平滑肌收缩的知识 将被用作研究肌球蛋白的作用的基础 在儿茶酚胺分泌中的磷酸化。 这些研究将 在PC 12大鼠嗜铬细胞瘤细胞和牛上进行 嗜铬细胞 肌球蛋白和肌球蛋白轻链激酶将被 从嗜铬细胞中纯化的，和肌球蛋白上的位点， 被肌球蛋白轻链激酶和蛋白激酶磷酸化 C将被识别。 多克隆抗体 嗜铬细胞肌球蛋白和肌球蛋白轻链激酶将被 在以下实验中产生并使用：（1）抗体 将用于定量关联肌球蛋白磷酸化 在完整的嗜铬细胞中，细胞内Ca 2+和 儿茶酚胺分泌反应的各种 （2）它们将用于研究 肌球蛋白磷酸化，以确定激酶 在完整的嗜铬细胞中磷酸化肌球蛋白;（3）抗- 肌球蛋白和抗肌球蛋白轻链激酶抗体将被 电注入嗜铬细胞，研究 这些抗体对儿茶酚胺分泌的影响。 这些实验 旨在阐明位点特异性肌球蛋白的作用 磷酸化在调节神经递质分泌中的作用。 此外，通过同时研究肌球蛋白磷酸化， 钙和分泌，这些研究将提供重要 生理学、药理学和生物化学信息 发射器释放

Transmitter release and other secretory processes have been compared to muscle contraction in that both processes are regulated by Ca2+ and depend upon metabolic energy. Although secretion was thought initially to resemble the contraction of skeletal muscle, recent experiments suggest that it may more closely resemble smooth muscle contraction. Ca2+ is known to regulate smooth muscle contraction through the activation of the Ca2+/calmodulin-dependent enzyme myosin light chain kinase. The active enzyme phosphorylates smooth muscle myosin, which results in a stimulation of the rate of ATP hydrolysis by actin and myosin. In this study, knowledge of smooth muscle contraction will be used as a basis to investigate the role of myosin phosphorylation in catecholamine secretion. The studies will be performed on PC12 rat pheochromocytoma cells and on bovine chromaffin cells. Myosin and myosin light chain kinase will be purified from chromaffin cells, and the sites on myosin that are phosphorylated by myosin light chain kinase and by protein kinase C will be identified. Polyclonal antibodies against purified chromaffin cell myosin and myosin light chain kinase will be raised and used in the following experiments: (1) Antibodies will be used to correlate quantitatively myosin phosphorylation in intact chromaffin cells with intracellular Ca2+ and with catecholamine secretion in response to a variety of secretagogues; (2) They will be used to study the sites on myosin that are phosphorylated in order to identify the kinases that phosphorylate myosin in intact chromaffin cells; (3) Anti- myosin and anti-myosin light chain kinase antibodies will be electroinjected into chromaffin cells to study the effects of these antibodies on catecholamine secretion. These experiments are designed to elucidate the role of site-specific myosin phosphorylation in the regulation of neurotransmitter secretion. Moreover, by simultaneously studying myosin phosphorylation, cell calcium and secretion, these studies will provide important information on the physiology, pharmacology and biochemistry of transmitter release.