Myosin Phosphorylation and Neurotransmitter Secretion

递质释放和其他分泌过程被比作肌肉收缩，因为这两个过程都受钙调节，依赖于代谢能量。虽然最初认为分泌物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通过激活钙/钙调蛋白依赖的酶肌球蛋白轻链激酶来调节平滑肌收缩。该活性酶使肌球蛋白磷酸化，从而刺激肌动蛋白和肌球蛋白对ATP的水解率。在这项研究中，肌球蛋白磷酸化对儿茶酚胺分泌儿茶酚胺的作用将以平滑肌收缩知识为基础。这些研究将在PC12大鼠嗜铬细胞瘤细胞和牛嗜铬细胞上进行。肌球蛋白和肌球蛋白轻链激酶将从嗜铬细胞中纯化出来，肌球蛋白上被肌球蛋白轻链激酶和蛋白激酶C磷酸化的位点将被鉴定。纯化的嗜铬细胞肌球蛋白和肌球蛋白轻链激酶的多克隆抗体将被用于下列实验：(1)抗体将被用来定量地关联完整嗜铬细胞的肌球蛋白磷酸化与细胞内钙以及多种促分泌剂对儿茶酚胺的分泌；(2)它们将用于研究肌球蛋白上的磷酸化位点，以确定完整嗜铬细胞中肌球蛋白磷酸化的激酶；(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神经递质分泌调节中的作用。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学、药理学和生化提供重要信息。

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.