Myosin Phosphorylation and Neurotransmitter Secretion

递质释放和其他分泌过程已经与肌肉收缩进行了比较，因为这两个过程都是由Ca2+调节的，并依赖于代谢能量。虽然分泌最初被认为类似于骨骼肌的收缩，但最近的实验表明，它可能更接近于平滑肌的收缩。已知Ca2+通过激活Ca2+/钙调素依赖性酶肌球蛋白轻链激酶来调节平滑肌收缩。这种活性酶使平滑肌肌球蛋白磷酸化，从而刺激肌动蛋白和肌球蛋白对ATP的水解速率。在本研究中，平滑肌收缩的知识将作为研究肌球蛋白磷酸化在儿茶酚胺分泌中的作用的基础。研究将在PC12大鼠嗜铬细胞瘤细胞和牛嗜铬细胞上进行。将从染色质细胞中纯化肌球蛋白和肌球蛋白轻链激酶，并鉴定肌球蛋白上被肌球蛋白轻链激酶和蛋白激酶C磷酸化的位点。针对纯化的染色质细胞肌球蛋白和肌球蛋白轻链激酶的多克隆抗体将被提出并用于以下实验：(1)抗体将用于定量地将完整的染色质细胞中肌球蛋白磷酸化与细胞内Ca2+和儿茶酚胺分泌相关联，以响应各种分泌物；(2)它们将用于研究肌球蛋白磷酸化的位点，以确定完整的染色质细胞中磷酸化肌球蛋白的激酶；(3)将抗肌球蛋白和抗肌球蛋白轻链激酶抗体电注入嗜铬细胞，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、细胞钙和分泌，这些研究将为递质释放的生理、药理学和生物化学提供重要信息。

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.