Submitted on September 8, 2006
Accepted on October 1, 2007
Stable RNA Interference of Synaptotagmin I in PC12 Cells Results in Differential Regulation of Transmitter Release
William Henry Roden1, Jason Brian Papke1, Johnnie Marie Moore1, Anne Lynn Cahill2, Heather Macarthur1, and Amy Beth Harkins1*
1 Pharmacological and Physiological Science, Saint Louis University, St. Louis, Missouri, United States
2 Neurobiology, Pharmacology and Physiology, University of Chicago, Chicago, Illinois, United States
* To whom correspondence should be addressed. E-mail: harkinsa{at}slu.edu.
In sympathetic neurons, it is well-established that the neurotransmitters, norepinephrine (NE), neuropeptide Y (NPY) and ATP are differentially co-released 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 RNAi was used to specifically and stably silence expression of syt I in a model secretory cell line. Whereas stimulated release of neuropeptide Y (NPY) and purines was abolished, stimulated catecholamine (CA) release was only reduced by ~50%. Although expression levels of tyrosine hydroxylase (TH), the rate-limiting enzyme in the dopamine synthesis pathway, was unaffected, expression of the vesicular monoamine transporter 1 (VMAT1) was reduced by 50%. In order 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 co-localized with large dense core vesicles, with NPY and with CAs. Furthermore, both CAs and NPY co-localized 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.
