Localization of voltage-sensitive Ca2+ fluxes and neuropeptide Y immunoreactivity to varicosities in SH-SY5Y human neuroblastoma cells differentiated by treatment with the protein kinase inhibitor staurosporine.
The distribution of voltage-sensitive elevations of the level of Ca2+ in untreated SH-SY5Y cells and cells that had been induced to differentiate with staurosporine was investigated by monitoring fura-2 fluorescence in cell suspensions, and by using microfluorometry and quantitative fluorescence imaging on cell bodies and on cellular processes. Cell bodies of both types of cells displayed small Ca2+ elevations, which were composed of transient and sustained components. Elevations were partially sensitive to the L- and N-channel blockers nifedipine (1 microM) and omega-conotoxin GVIA (100 nM) respectively. Up to ten times Ca2+ elevations were observed in varicosities of treated cells than in cell bodies of treated and cells. These elevations were insensitive to compounds known to release Ca2+ from intracellular stores. Elevations of Ca2+ were sustained, and they were insensitive to 5 microM nifedipine, 100 nM omega-agatoxin IVA and 100 nM omega-conotoxin GVIA, and partially sensitive to 2 microM omega-conotoxin GVIA, indicating predominance of non-L-type, non-N-type, non-P-type channel activity. The intracellular localization of neuropeptide Y, a marker of differentiation in these cells, was also investigated by fluorescence immunocytochemistry. Varicosities of treated cells displayed marked fluorescence when viewed in a confocal microscope. These findings show that the varicosities of staurosporine-treated cells exhibit some of the functional properties of nerve terminals. The varicosities resemble boutons en passant nerve endings and they seem to express Ca2+ channels different from those in the cell body.
Kukkonen
J P
JP
Department of Biochemistry and Pharmacy, Abo Akademi University, Turku, Finland.
Shariatmadari
R
R
Courtney
M J
MJ
Akerman
K E
KE
D001932:Brain Neoplasms / Q000378:metabolism* / Q000648:ultrastructure*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D003470:Culture Media; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005456:Fluorescent Dyes; D016257:Fura-2; D006801:Humans; D007150:Immunohistochemistry; D015640:Ion Channel Gating / Q000187:drug effects; D009447:Neuroblastoma / Q000378:metabolism* / Q000648:ultrastructure*; D009478:Neuropeptide Y / Q000378:metabolism*; D047428:Protein Kinase Inhibitors; D019311:Staurosporine / Q000494:pharmacology*
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D003470:Culture Media; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D009478:Neuropeptide Y; D047428:Protein Kinase Inhibitors; D019311:Staurosporine; D016257:Fura-2