Mechanistic and functional changes in Ca 2+ entry after retinoic acid-induced differentiation of neuroblastoma cells

Institute for Cell and Molecular Biosciences, The Medical School, University of Newcastle, Framlington Place, Newcastle-upon-Tyne NE2 4HH, UK.
Biochemical Journal (Impact Factor: 4.4). 07/2005; 388(Pt 3):941-8. DOI: 10.1042/BJ20042127
Source: PubMed


We have investigated effects of neuronal differentiation on hormone-induced Ca2+ entry. Fura-2 fluorescence measurements of undifferentiated SH-SY5Y neuroblastoma cells, stimulated with methacholine, revealed the presence of voltage-operated Ca2+-permeable, Mn2+-impermeable entry pathways, and at least two voltage-independent Ca2+- and Mn2+-permeable entry pathways, all of which apparently contribute to both peak and plateau phases of the Ca2+ signal. Similar experiments using 9-cis retinoic acid-differentiated cells, however, revealed voltage-operated Ca2+-permeable, Mn2+-impermeable channels, and, more significantly, the absence or down-regulation of the most predominant of the voltage-independent entry pathways. This down-regulated pathway is probably due to CCE (capacitative Ca2+ entry), since thapsigargin also stimulated Ca2+ and Mn2+ entry in undifferentiated but not differentiated cells. The Ca2+ entry components remaining in methacholine-stimulated differentiated cells contributed to only the plateau phase of the Ca2+ signal. We conclude that differentiation of SH-SY5Y cells results in a mechanistic and functional change in hormone-stimulated Ca2+ entry. In undifferentiated cells, voltage-operated Ca2+ channels, CCE and NCCE (non-CCE) pathways are present. Of the voltage-independent pathways, the predominant one appears to be CCE. These pathways contribute to both peak and plateau phases of the Ca2+ signal. In differentiated cells, CCE is either absent or down-regulated, whereas voltage-operated entry and NCCE remain active and contribute to only the plateau phase of the Ca2+ signal.

Download full-text


Available from: Chris Redfern, Feb 02, 2016
  • Source
    • "In HEK293T cells down-regulation of STIM1 allowed TRPC1 to function as a ROC, insensitive to store depletion [30]. If TRPC1 functions as a ROC in differentiated N-type cells this could explain our previous observation that in 9cRA-differentiated SH-SY5Y cells a non-SOCE pathway becomes up-regulated [19]. TRPC1 may be associated with differentiation itself; TRPC1 was required for neurite outgrowth in differentiating PC12 cells but was independent of SOCE [51] . "
    [Show abstract] [Hide abstract]
    ABSTRACT: Neuroblastoma cell lines are heterogeneous, comprised of at least three distinct cell phenotypes; neuroblastic N-type cells, non-neuronal substrate-adherent S-type cells and intermediate I-type cells. N- and S-type cell populations were enriched from the parental SH-SY5Y neuroblastoma cell line and induced to differentiate by the addition of retinoic acid (RA), a drug used in the treatment of neuroblastoma. N- and S-type cells were identified based on their differential expression of β-tubulin III, vimentin and Bcl-2. Store-operated Ca(2+) entry (SOCE) was then measured in proliferating and differentiated N- and S-type cell populations and the expression of STIM1, Orai1 and TRPC1, three proteins reported to play a key role in SOCE, was determined. In N-type cells the RA-induced switch from proliferation to differentiation was accompanied by a down-regulation in SOCE. STIM1 and Orai1 expression became down-regulated in differentiated cells, consistent with their respective roles as ER Ca(2+) sensor and store-operated Ca(2+) channel (SOC). TRPC1 became up-regulated suggesting that TRPC1 is not involved in SOCE, at least in differentiated N-type cells. In S-type cells SOCE remained active following the RA-induced switch from proliferation to differentiation and the expression of STIM1 and Orai1 remained unchanged. TRPC1 was not expressed in S-type cells. Our results indicate that differentiation of neuronal cells is associated with a remodelling of SOCE. Therapeutic targeting of SOCE proteins could potentially be a means of promoting neuronal differentiation in the treatment of neuroblastoma.
    Full-text · Article · Dec 2012 · Biochimica et Biophysica Acta
  • Source
    • "Roughly half of the neurons initially generated die by apoptosis in a well-defined interval, coincident with the establishment of synaptic connections. This cell death is known as naturally occurring programmed cell death and it is believed to match the number of innervating neurons to the size of the target cell population, as well as to eliminate aberrant synaptic contacts (Encinas et al., 2000; C. Rasetti-Escargueil et al. Brown et al., 2005). The human neuroblastoma SH-SY5Y cell line was originally derived from a sympathetic ganglion and contains tyrosine hydroxylase and dopamine β-hydroxylase: the requisite enzymes for synthesis of norepinephrine (Ross and Biedler, 1985; Ciccarone et al., 1989). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Research on more sensitive cell based assays is essential to assess neutralising activities of Botulinum neurotoxins antibodies. A reproducible and robust differentiation protocol was applied on human neuroblastoma SH-SY5Y cells generating neuronal cultures stable for 2–3 weeks. The presence of specific neuronal markers was assessed with labelled antibodies. Accurate and reproducible noradrenaline release levels were obtained which were significantly higher in differentiated cells and correlated with vesicle turnover assessments. The suitability of differentiated SH-SY5Y cells for detecting low levels of Botulinum type A and for toxin neutralisation assay was evidenced using functional end points relevant to Botulinum neurotoxicity.
    Full-text · Article · Aug 2011 · The Botulinum J

  • No preview · Article ·
Show more