Stable RNA interference of synaptotagmin I in PC12 cells results in differential regulation of transmitter release

被引:10
作者
Roden, William H. [1 ]
Papke, Jason B. [1 ]
Moore, Johnnie M. [1 ]
Cahill, Anne L. [2 ]
Macarthur, Heather [1 ]
Harkins, Amy B. [1 ]
机构
[1] St Louis Univ, Sch Med, Dept Pharmacol & Physiol Sci, St Louis, MO 63104 USA
[2] Univ Chicago, Dept Neurobiol Pharmacol & Physiol, Chicago, IL 60637 USA
来源
AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY | 2007年 / 293卷 / 06期
关键词
neuropeptide Y; catecholamines; adenosine 5'-triphosphate; dopamine; norepinephrine;
D O I
10.1152/ajpcell.00482.2006
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
Stable RNA interference of synaptotagmin I in PC12 cells results in differential regulation of transmitter release. Am J Physiol Cell Physiol 293: C1742 - C1752, 2007. First published October 3, 2007; doi: 10.1152/ ajpcell. 00482.2006. -In sympathetic neurons, it is well-established that the neurotransmitters, norepinephrine (NE), neuropeptide Y (NPY), and ATP are differentially coreleased from the same neurons. In this study, we determined whether synaptotagmin (syt) I, the primary Ca2+ sensor for regulated release, could function as the protein that differentially regulates release of these neurotransmitters. Plasmid-based RNA interference was used to specifically and stably silence expression of syt I in a model secretory cell line. Whereas stimulated release of NPY and purines was abolished, stimulated catecholamine (CA) release was only reduced by similar to 50%. Although expression levels of tyrosine hydroxylase, the rate- limiting enzyme in the dopamine synthesis pathway, was unaffected, expression of the vesicular monoamine transporter 1 was reduced by 50%. To evaluate whether NPY and CAs are found within the same vesicles and whether syt I is found localized to each of these NPY- and CA-containing vesicles, we used immunocytochemistry to determine that syt I colocalized with large dense core vesicles, with NPY, and with CAs. Furthermore, both CAs and NPY colocalized with one another and with large dense core vesicles. Electron micrographs show that large dense core vesicles are synthesized and available for release in cells that lack syt I. These results are consistent with syt I regulating differential release of transmitters.
引用
收藏
页码:C1742 / C1752
页数:11
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