EFFECTS OF THE NEUROPROTECTANT LUBELUZOLE ON THE CYTOTOXIC ACTIONS OFVERATRIDINE, BARIUM, OUABAIN AND 6-HYDROXYDOPAMINE IN CHROMAFFIN CELLS

1 Incubation of bovine adrenal chromaffin cells with veratridine (10-1 00 mu M) during 24 h, caused a concentration-dependent release of the cytosolic lactate dehydrogenase (LDH) into the bathing medium, an indi cator of cell death. Lubeluzole or its R(-) enantiomer, R91154, did no t enhance LDH release. Both lubeluzole and R91154 (0.3-10 mu M) decrea sed the veratridine-induced LDH release.2 Penfluridol did not increase LDH release at concentrations 0.003-1 mu M; 3-10 mu M increased LDH r elease to 50-60%, after 24 h exposure. Penfluridol (0.03-0.3 mu M) did not protect against the cytotoxic effects of veratridine; at 1 mu M, 15% protection was produced. Higher concentrations (3-10 mu M) enhance d the cytotoxic effects of veratridine. 3 Ba2+ ions caused a concentra tion-dependent increase of LDH release. This cytotoxic effect was part ially prevented by 3 mu M lubeluzole and fully counteracted by 1 mu M penfluridol. R91154 was less potent than lubeluzole and only protected against the lesion induced by 0.5 mM Ba2+. 4 Ouabain (10 mu M during 24 h) increased LDH release to about 30%. Both lubeluzole (0.3-10 mu M ) and the lower concentrations of penfluridol (0.003-0.3 mu M) prevent ed the ouabain cytotoxic effects. At higher concentrations (3 mu M), p enfluridol increased drastically the ouabain cytotoxic effects. 5 6-Hy droxydopamine (6-OHDA) caused significant cytotoxic effects at 30 and 100 mu M. Lubeluzole (3-10 mu M) or penfluridol (0.03-0.3 mu M) had no cytoprotective effects against 6-OHDA. 6 Lubeluzole (3 mu M), R91154 (3 mu M) and penfluridol (1 mu M) blocked the current through Na+ chan nels in voltage-clamped chromaffin cells (I-Na) by around 20-30%. Ca2 current through Ca2+ channels (I-Ca) was inhibited 57% by lubeluzole and R91154 and 50% by penfluridol. The effects of penfluridol were not washed out, but those of lubeluzole and R91154 were readily reversibl e. 7 Lubeluzole (3 mu M) induced reversible blockade of the oscillatio ns of the cytosolic Ca2+, [Ca2+](i), in fura-2-1oaded cells exposed to 30 or 100 mu M veratridine. Penfluridol (1 mu M) inhibited those osci llations in an irreversible manner. 8 The results suggest that lubeluz ole,and its R-isomer caused cytoprotection against veratridine cell da mage, by blocking the veratridine stimulated Na+ and Ca2+ entry, as we ll as the [Ca2+](i) oscillations. The Ba2+ and ouabain cytotoxic effec ts were prevented more efficiently by penfluridol, likely by blocking the plasmalemmal Na+/Ca2+ exchanger. It remains dubious whether these findings are relevant to the reported neuroprotective action of lubelu zole in stroke; the doubt rests in the stereoselective protecting effe cts of lubeluzole in in vivo stroke models, as opposed to its lack of stereoselectivity in the in vitro model reported here.