Depolarization-induced ERK phosphorylation depends on the cytosolic Ca2+ level rather than on the Ca2+ channel subtype of chromaffin cells.

The contribution of Ca2+ entry through different voltage-activated Ca2+ channel (VACC) subtypes to the phosphorylation of extracellular signal regulated kinase (ERK) was examined in bovine adrenal-medullary chromaffin cells. High K+ depolarization (40 mM, 3 min) induced ERK phosphorylation, an effect that was inhibited by specific mitogen-activated protein kinase kinase inhibitors. By using selective inhibitors, we observed that depolarization-induced ERK phosphorylation completely depended on protein kinase C-alpha (PKC-alpha), but not on Ca2+/calmodulin-dependent protein kinase nor cyclic AMP-dependent protein kinase. Blockade of L-type Ca2+ channels by 3 microm furnidipine, or blockade of N channels by 1 micromomega-conotoxin GVIA reduced ERK phosphorylation by 70%, while the inhibition of P/Q channels by 1 micromomega-agatoxin IVA only caused a 40% reduction. The simultaneous blockade of L and N, or P/Q and N channels completely abolished this response, yet 23% ERK phosphorylation remained when L and P/Q channels were simultaneously blocked. Confocal imaging of cytosolic Ca2+ elevations elicited by 40 mm K+, showed that Ca2+ levels increased throughout the entire cytosol, both in the presence and the absence of Ca2+ channel blockers. Fifty-eight percent of the fluorescence rise depended on Ca2+ entering through N channels. Thus, ERK phosphorylation seems to depend on a critical level of Ca2+ in the cytosol rather than on activation of a given Ca2+ channel subtype.

Establishment and characterization of immortalized neuronal cell lines derived from the spinal cord of normal and trisomy 16 fetal mice, an animal model of Down syndrome.

We report the establishment of continuously growing cell lines from spinal cords of normal and trisomy 16 fetal mice. We show that both cell lines, named M4b (derived from a normal animal) and MTh (trisomic) possess neurological markers by immunohistochemistry (neuron specific enolase, synaptophysin, microtubule associated protein-2 [MAP-2], and choline acetyltransferase) and lack glial traits (glial fibrillary acidic protein and S100). MTh cells were shown to overexpress mRNA of Cu/Zn superoxide dismutase, whose gene is present in autosome 16. We also studied intracellular Ca2+ signals ([Ca2+]i) induced by different agonists in Indo-1 loaded cells. Basal [Ca2+]i was significantly higher in MTh cells compared to M4b cells. Glutamate (200 microM) and (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACDP) (100 microM) induced rapid, transient increases in [Ca2+]i in M4b and MTh cells, indicating the presence of glutamatergic metabotropic receptors. N-methyl-D-aspartate (NMDA) and kainate, but not alpha-amino-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), produced [Ca2+)]i rises in both cell types. MTh cells exhibited faster time-dependent decay phase kinetics in glutamate-induced responses compared to M4b cells. Nicotine induced a transient increase in [Ca2+]i in M4b and MTh cells, with significantly greater amplitudes in the latter compared to the former. Further, both cell types responded to noradrenaline. Finally, we examined cholinergic function in both cell lines and found no significant differences in the [3H]-choline uptake, but fractional acetylcholine release induced by either K+, glutamate or nicotine was significantly higher in MTh cells. These results show that M4b and MTh cells have neuronal characteristics and the MTh line shows differences which could be related to neuronal pathophysiology in Down's syndrome.