Dr. M. E. Graham and Dr. A. W. Sudlow contributed equally to this work and should both be considered as first author.
Evidence Against an Acute Inhibitory Role of nSec-1 (Munc-18) in Late Steps of Regulated Exocytosis in Chromaffin and PC12 Cells
Version of Record online: 18 NOV 2002
Journal of Neurochemistry
Volume 69, Issue 6, pages 2369–2377, December 1997
How to Cite
Graham, M. E., Sudlow, A. W. and Burgoyne, R. D. (1997), Evidence Against an Acute Inhibitory Role of nSec-1 (Munc-18) in Late Steps of Regulated Exocytosis in Chromaffin and PC12 Cells. Journal of Neurochemistry, 69: 2369–2377. doi: 10.1046/j.1471-4159.1997.69062369.x
- Issue online: 18 NOV 2002
- Version of Record online: 18 NOV 2002
- Received May 2, 1997; revised manuscript received July 16, 1997; accepted July 16, 1997.
- nSec-1 (munc-18) protein;
- Chromaffin cells;
- PC12 cells;
- α-Soluble N-ethylmaleimide-sensitive fusion protein attachment protein;
- Growth hormone secretion
Abstract: nSec-1 (munc-18) is a mammalian homologue of proteins implicated in constitutive exocytosis in yeast and neurotransmission in Caenorhabditis elegans and Drosophila. Mutant phenotypes in these species suggest that nSec-1 is likely to be required for neurotransmission. Various other data have been interpreted as suggesting that nSec-1 could also be a negative regulator of Ca2+-dependent exocytosis. We have tested this possibility by introducing exogenous nSec-1 into permeabilised chromaffin or PC12 cells and examining its effects on Ca2+-induced and α-soluble N-ethylmaleimide-sensitive fusion protein attachment protein-stimulated exocytosis. No effects of exogenous nSec-1 were observed in these assays. In addition, the effect of nSec-1 overexpression in transiently transfected PC12 cells on reporter growth hormone (GH) secretion was examined. Overexpression of nSec-1 resulted in a marked increase in GH production, reflected in an increase in both cell-associated and medium GH levels. The relative amounts retained in the cells were unaffected by nSec-1 overexpression, indicating that GH storage was unaffected and that the major effect was on its synthesis. In contrast, nSec-1 overexpression did not affect the proportion of GH that was released following stimulation in intact or permeabilised cells. These results suggest either that nSec-1 is already expressed at sufficient levels and remains so following permeabilisation or that nSec-1 may not be an acute inhibitory regulator of Ca2+-dependent exocytosis in chromaffin or PC12 cells.
Abbreviations used: CMV, cytomegalovirus; GH, growth hormone; NSF, N-ethylmaleimide-sensitive fusion protein; PCR, polymerase chain reaction; SDS, sodium dodecyl sulphate; α-SNAP, α-soluble N-ethylmaleimide-sensitive fusion protein attachment protein; SNAP-25, synaptosomal protein of 25 kDa; VAMP, vesicle-associated membrane protein.