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Keywords:

  • exocytosis;
  • protein kinase C substrate;
  • stable transfected cell line;
  • sympathetic neuron

Abstract

The aim of this study was to investigate a possible role of the myristoylated alanine-rich C kinase substrate (MARCKS) in the mechanism of noradrenaline uptake and release in the human neuroblastoma cell line SH-SY5Y. A stable cell line showing a twofold overexpression of MARCKS was prepared by transfecting SH-SY5Y with pCEP4 containing MARCKS cDNA in the sense orientation. This cell line showed no changes in the expression of neurofilaments or markers of noradrenergic large dense-cored vesicles compared with both untransfected SH-SY5Y and SH-SY5Y transfected with pCEP4 only (mock transfected). Similarly, no differences in the rate of cell growth could be detected between these three cell lines. In contrast, specific uptake and depolarization-evoked (100 mm K+) release of noradrenaline from the cell line overexpressing MARCKS was inhibited by approximately 50% compared with mock-transfected SH-SY5Y. K+-evoked noradrenaline release enhanced by pretreatment with 12-O-tetradecanoylphorbol 13-acetate (100 nm) was also inhibited by 50%. In contrast, carbachol-evoked noradrenaline release was unaffected. Thus, in SH-SY5Y cells, overexpression of MARCKS leads to a decrease in the K+-evoked noradrenaline release possibly by increased actin cross-linking preventing the movement of noradrenaline containing large dense-cored vesicles to the plasma membrane in response to depolarization.