Overexpression of the myristoylated alanine-rich C kinase substrate decreases uptake and K(+)-evoked release of noradrenaline in the human neuroblastoma SH-SY5Y.
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.
Institute for Cardiovascular Research, Worsley Medical and Dental Building, University of Leeds, Leeds LS2 9JT, UK.
M E
ME
Hartness
J A
JA
Wade
J H
JH
Walker
P F
PF
Vaughan
D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D003598:Cytoskeletal Proteins; D005089:Exocytosis / Q000502:physiology; D020555:GTP-Binding Protein Regulators; D005822:Genetic Vectors / Q000502:physiology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D009447:Neuroblastoma; D009638:Norepinephrine / Q000378:metabolism* / Q000493:pharmacokinetics; D011188:Potassium / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D016601:RNA-Binding Proteins; D014162:Transfection / Q000379:methods; D014316:Tritium / Q000493:pharmacokinetics; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism*; D020764:cdc42 GTP-Binding Protein; D020741:rho GTP-Binding Proteins
C422985:CDC42EP4 protein, human; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D020555:GTP-Binding Protein Regulators; D047908:Intracellular Signaling Peptides and Proteins; C000619710:MARCKS protein, human; D008565:Membrane Proteins; D011506:Proteins; D016601:RNA-Binding Proteins; D014316:Tritium; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C; D020764:cdc42 GTP-Binding Protein; D020741:rho GTP-Binding Proteins; D011188:Potassium; D009638:Norepinephrine