Hartness M E, Wade J A, Walker J H, Vaughan P F
Institute for Cardiovascular Research, Worsley Medical and Dental Building, University of Leeds, Leeds LS2 9JT, UK.
Eur J Neurosci. 2001 Mar;13(5):925-34. doi: 10.1046/j.0953-816x.2001.01466.x.
The aim of this study was to investigate a possible role of the myristoylated alanine-rich C kinase substrate (MARCKS) in the mechanism of noradrenaline uptake and release in the human neuroblastoma cell line SH-SY5Y. A stable cell line showing a twofold overexpression of MARCKS was prepared by transfecting SH-SY5Y with pCEP4 containing MARCKS cDNA in the sense orientation. This cell line showed no changes in the expression of neurofilaments or markers of noradrenergic large dense-cored vesicles compared with both untransfected SH-SY5Y and SH-SY5Y transfected with pCEP4 only (mock transfected). Similarly, no differences in the rate of cell growth could be detected between these three cell lines. In contrast, specific uptake and depolarization-evoked (100 mM K(+)) release of noradrenaline from the cell line overexpressing MARCKS was inhibited by approximately 50% compared with mock-transfected SH-SY5Y. K(+)-evoked noradrenaline release enhanced by pretreatment with 12-O-tetradecanoylphorbol 13-acetate (100 nM) was also inhibited by 50%. In contrast, carbachol-evoked noradrenaline release was unaffected. Thus, in SH-SY5Y cells, overexpression of MARCKS leads to a decrease in the K(+)-evoked noradrenaline release possibly by increased actin cross-linking preventing the movement of noradrenaline containing large dense-cored vesicles to the plasma membrane in response to depolarization.
本研究的目的是探讨富含肉豆蔻酰化丙氨酸的蛋白激酶C底物（MARCKS）在人神经母细胞瘤细胞系SH-SY5Y去甲肾上腺素摄取和释放机制中可能发挥的作用。通过用含有正义方向MARCKS cDNA的pCEP4转染SH-SY5Y，制备了MARCKS表达量两倍于正常水平的稳定细胞系。与未转染的SH-SY5Y和仅用pCEP4转染的SH-SY5Y（空载体转染）相比，该细胞系在神经丝或去甲肾上腺素能大致密核心囊泡标志物的表达上没有变化。同样，这三种细胞系在细胞生长速率上也没有差异。相比之下，与空载体转染的SH-SY5Y相比，过表达MARCKS的细胞系对去甲肾上腺素的特异性摄取和去极化诱发（100 mM K(+)）释放被抑制了约50%。用12-O-十四烷酰佛波醇-13-乙酸酯（100 nM）预处理增强的K(+)诱发的去甲肾上腺素释放也被抑制了50%。相比之下，卡巴胆碱诱发的去甲肾上腺素释放不受影响。因此，在SH-SY5Y细胞中，MARCKS的过表达可能通过增加肌动蛋白交联，阻止去甲肾上腺素包被的大致密核心囊泡在去极化时向质膜移动，从而导致K(+)诱发的去甲肾上腺素释放减少。
