VANADATE STIMULATES DIFFERENTIATION AND NEURITE OUTGROWTH IN RAT PHEOCHROMOCYTOMA PC12 CELLS AND NEURITE EXTENSION IN HUMAN NEUROBLASTOMA SH-SY5Y CELLS

We show here that a protein tyrosine phosphatase inhibitor, sodium ort hovanadate, induces rat pheochromocytoma cells to express neurites, a prominent morphological marker of neuronal phenotype. Vanadate-induced differentiation and neurite outgrowth in pheochromocytoma cells was n ot as extensive as that induced by the positive control employed, nerv e growth factor. However, neurite outgrowth responses were comparable between nerve growth factor-treated pheochromocytoma cells and cells p rimed and then restimulated with vanadate. In the human neuroblastoma cell line, SH-SY5Y, a single exposure to vanadate induced neurite exte nsion in this cell line equal to that initiated by nerve growth factor . In both cell lines vanadate treatment resulted in tyrosine phosphory lation of several high-molecular-weight proteins and using anti-phosph otyrosine antibodies, intense fluorescence was observed in the cell bo dy and neurites of pheochromocytoma cells exposed to vanadate. Vanadat e mediated differentiation and neurite outgrowth in pheochromocytoma c ells could be ablated by the tyrosine kinase inhibitor erbstatin, wher eas nerve growth factor-induced neurite outgrowth was only partially i nhibited. In SH-SY5Y cells, erbstatin mediated partial inhibition of b oth vanadate and nerve growth factor-induced neurite elongation with s imilar kinetics. In contrast, K252b, a trk tyrosine kinase inhibitor, exhibited only a 30% reduction of neurite outgrowth in vanadate treate d pheochromocytoma cells but an 80% reduction in nerve growth factor-t reated cells. In SH-SY5Y cells, K252a did not have a statistically sig nificant effect on neurite elongation induced by vanadate in contrast to a 60% reduction in nerve growth factor-treated cells. The membrane impermeable analogue K252b, had no effect on neurite elongation induce d with either vanadate or nerve growth factor in these cells. The effe cts of vanadate were not mimicked by ouabain (0.1-50 mu M) indicating that vanadate does not induce differentiation and/or neurite extension by inhibiting ion channel Na,K-ATPase, which is one of its other well -characterised inhibitory activities. Evidence for the selective actio n of vanadate on some but not all neuronal cell lines comes from the f act that it did not induce neurite extension in the human neuroblastom a cell line SK-N-MC. These data imply that vanadate-induced neurite ou tgrowth responses in pheochromocytoma and SH-SY5Y cells can be induced by the inhibition of tyrosine phosphatases and appears not to simply mimic nerve growth factor signals. The target(s) of vanadate action in the two cell lines are currently being sought.