Overexpression of the myristoylated alanine‐rich C kinase substrate decreases uptake and K⁺‐evoked release of noradrenaline in the human neuroblastoma SH‐SY5Y

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.

Myristoylated alanine rich C kinase substrate (MARCKS) heterozygous mutant mice exhibit deficits in hippocampal mossy fiber-CA3 long-term potentiation

“…MARCKS binds and crosslinks F-actin in a PKC phosphorylation-dependent manner (Hartwig et al, 1992;Bubb et al, 1999;Yarmola et al, 2001), and PKC-mediated phosphorylation of MARCKS results in the movement of synaptic vesicles from reserve pools to active release sites in cultured cells and synaptosomes (Vitale et al, 1995;Stevens and Sullivan, 1998;Walaas and Sefland, 2000;Rose et al, 2001;Yang et al, 2002). Furthermore, anti-MARCKS antibodies, or F-actin destabilizing agents, increase spontaneous neurotransmitter release in PC12 cells (Shoji-Kasai et al, 2002), whereas MARCKS overexpression (two-fold) impaired PKCinduced enhancement of neurotransmitter release in human neuroblastoma SH-SY5Y cells (Hartness et al, 2001). Therefore, a 50% reduction in MARCKS in mossy fiber axon terminals may compromise the regulation of F-actin cytoskeletal rigidity, resulting in less restriction in the movement of synaptic vesicles from reserve pools to active release sites.…”

Notch activation induces neurite remodeling and functional modifications in SH‐SY5Y neuronal cells

“…Noradrenaline (NA) release from SH-SY5Y differentiated cells was measured according to Hartness et al (2001 …”

“…After differentiation, these cells are able to release NA after secretagogue treatment or depolarization, suggesting the acquisition of the neurosecretory competence (Encinas et al, 2000;Hartness et al, 2001). Therefore, the possible functional effects of Jagged1 were analyzed by measuring NA release in basal condition and after K + -evoked depolarization.…”

New Aspects of Neurotransmitter Release and Exocytosis: Rho-Kinase-Dependent Myristoylated Alanine-Rich C-Kinase Substrate Phosphorylation and Regulation of Neurofilament Structure in Neuronal Cells

“…These reactions including MARCKS phosphorylation, disassembly of cortical F-actin structure, and the potentiation of Ca 2+ -evoked secretion are reported to be inhibited by a synthetic peptide of the PSD of MARCKS. In SH-SY5Y cells, overexpression of MARCKS leads to a decrease in the K + -evoked noradrenaline release (8). Although it is well-known that MARCKS phosphorylation responds to several kinds of stimuli, it is not plausible that all these stimuli activate PKC to phosphorylate PSD.…”

“…On the other hand, it is demonstrated that PKC-dependent phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) occurs coincidently with F-actin disassembly and potentiation of catecholamine release in a permeabilized chromaffin cells (7). Overexpression of MARCKS is reported to decrease catecholamine release from K + -stimulated human neuroblastoma (8). These results seem to be attributed to MARCKS-dependent reorganization of F-actin structure, which may allow the vesicles to access the presynaptic membrane and its adjacent area, the so-called "active zone".…”