Allen C M, Ely C M, Juaneza M A, Parsons S J
Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
J Neurosci Res. 1996 Jun 1;44(5):421-9. doi: 10.1002/(SICI)1097-4547(19960601)44:5<421::AID-JNR2>3.0.CO;2-H.
Adrenal medullary chromaffin cells derive from the neural crest during embryogenesis and differentiate into dedicated secretory cells that release catecholamines in response to acetylcholine in vivo or nicotinic agonists in vitro. Previous studies have indicated that tyrosine kinases participate in early secretagogue-induced events in these cells and are required for exocytosis. Abundant levels of the cytoplasmic tyrosine kinases, c-Src and c-Yes, have been detected in chromaffin cells, thereby implicating them as kinases relevant to these events. However, c-Src has been found to undergo a decrease in activity following secretagogue-stimulation, and c-Yes appears to exist in a constituitively low activity state, suggesting that other tyrosine kinases are involved. Furthermore, other members of the Src family of tyrosine kinases have been implicated as playing roles in secretion in a variety of cell types. Therefore, we sought to determine if other Src family members were present in chromaffin cells, and if so, to examine them for subcellular localization and changes in activity following treatment with nicotinic agonists. To this end, antibodies for Fyn, Lck, Lyn, and Fgr were assembled and used in immunoprecipitation, in vitro autokinase, and Western immunoblotting assays. Of these four kinases, only Fyn was found to be expressed at detectable levels. Differential centrifugation studies revealed that Fyn resides predominantly (> 95%) in the crude plasma membrane fraction and undergoes nicotinic-and carbachol-induced activation. This activation is reduced by the nicotinic antagonist, mecamylamine, is not elicited by muscarine, and is dependent upon the presence of extracellular Ca2+. These results suggest that Fyn is involved in signalling through the nicotinic receptor and may be one of the relevant kinases responsible for at least some of the tyrosine phosphorylations detected after stimulation.
肾上腺髓质嗜铬细胞在胚胎发育过程中起源于神经嵴，并分化为专门的分泌细胞，这些细胞在体内对乙酰胆碱或体外对烟碱激动剂作出反应时释放儿茶酚胺。先前的研究表明，酪氨酸激酶参与了这些细胞中早期促分泌剂诱导的事件，并且是胞吐作用所必需的。在嗜铬细胞中已检测到大量的细胞质酪氨酸激酶c-Src和c-Yes，因此暗示它们是与这些事件相关的激酶。然而，已发现c-Src在促分泌剂刺激后活性降低，而c-Yes似乎以组成性低活性状态存在，这表明其他酪氨酸激酶也参与其中。此外，酪氨酸激酶Src家族的其他成员已被认为在多种细胞类型的分泌中发挥作用。因此，我们试图确定嗜铬细胞中是否存在其他Src家族成员，如果存在，则检查它们的亚细胞定位以及用烟碱激动剂处理后的活性变化。为此，制备了针对Fyn、Lck、Lyn和Fgr的抗体，并用于免疫沉淀、体外自激酶和Western免疫印迹分析。在这四种激酶中，仅发现Fyn以可检测水平表达。差速离心研究表明，Fyn主要（>95%）存在于粗质膜部分，并经历烟碱和卡巴胆碱诱导的激活。这种激活被烟碱拮抗剂美加明降低，不被毒蕈碱引发，并且依赖于细胞外Ca2+的存在。这些结果表明，Fyn参与通过烟碱受体的信号传导，并且可能是负责至少一些刺激后检测到的酪氨酸磷酸化的相关激酶之一。
