Evidence against an acute inhibitory role of nSec-1 (munc-18) in late steps of regulated exocytosis in chromaffin and PC12 cells.
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 alpha-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.
Graham
M E
ME
The Physiological Laboratory, University of Liverpool, England.
Sudlow
A W
AW
Burgoyne
R D
RD
D000313:Adrenal Medulla / Q000166:cytology; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002352:Carrier Proteins / Q000494:pharmacology; D002417:Cattle; D002463:Cell Membrane Permeability / Q000502:physiology; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D003470:Culture Media / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects*; D013006:Growth Hormone / Q000378:metabolism; D008565:Membrane Proteins / Q000494:pharmacology; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins / Q000494:pharmacology*; D016716:PC12 Cells / Q000502:physiology*; D051381:Rats; D011994:Recombinant Proteins / Q000494:pharmacology; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D003470:Culture Media; D008565:Membrane Proteins; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins; D013006:Growth Hormone; D005033:Ethylmaleimide; D002118:Calcium