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Keywords:

  • nSec-1 (munc-18) protein;
  • Exocytosis;
  • Chromaffin cells;
  • PC12 cells;
  • α-Soluble N-ethylmaleimide-sensitive fusion protein attachment protein;
  • Growth hormone secretion

Abstract

  1. Top of page
  2. Abstract

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.