Effects of the neuroprotectant lubeluzole on the cytotoxic actions of veratridine barium ouabain and 6-hydroxydopamine in chromaffin cells

摘要

class="enumerated" style="list-style-type:decimal">Incubation of bovine adrenal chromaffin cells with veratridine (10–100 μM) during 24 h, caused a concentration-dependent release of the cytosolic lactate dehydrogenase (LDH) into the bathing medium, an indicator of cell death. Lubeluzole or its R(−) enantiomer, , did not enhance LDH release. Both lubeluzole and (0.3–10 μM) decreased the veratridine-induced LDH release.Penfluridol did not increase LDH release at concentrations 0.003–1 μM; 3–10 μM increased LDH release to 50–60%, after 24 h exposure. Penfluridol (0.03–0.3 μM) did not protect against the cytotoxic effects of veratridine; at 1 μM, 15% protection was produced. Higher concentrations (3–10 μM) enhanced the cytotoxic effects of veratridine.Ba²⁺ ions caused a concentration-dependent increase of LDH release. This cytotoxic effect was partially prevented by 3 μM lubeluzole and fully counteracted by 1 μM penfluridol. was less potent than lubeluzole and only protected against the lesion induced by 0.5 mM Ba²⁺.Ouabain (10 μM during 24 h) increased LDH release to about 30%. Both lubeluzole (0.3–10 μM) and the lower concentrations of penfluridol (0.003–0.3 μM) prevented the ouabain cytotoxic effects. At higher concentrations (3 μM), penfluridol increased drastically the ouabain cytotoxic effects.6-Hydroxydopamine (6-OHDA) caused significant cytotoxic effects at 30 and 100 μM. Lubeluzole (3–10 μM) or penfluridol (0.03–0.3 μ class="small-caps">M) had no cytoprotective effects against 6-OHDA.Lubeluzole (3 μ class="small-caps">M), (3 μ class="small-caps">M) and penfluridol (1 μ class="small-caps">M) blocked the current through Na⁺ channels in voltage-clamped chromaffin cells (INa) by around 20–30%. Ca²⁺ current through Ca²⁺ channels (ICa) was inhibited 57% by lubeluzole and and 50% by penfluridol. The effects of penfluridol were not washed out, but those of lubeluzole and were readily reversible.Lubeluzole (3 μ class="small-caps">M) induced reversible blockade of the oscillations of the cytosolic Ca²⁺, [Ca²⁺]i, in fura-2-loaded cells exposed to 30 or 100 μ class="small-caps">M veratridine. Penfluridol (1 μ class="small-caps">M) inhibited those oscillations in an irreversible manner.The results suggest that lubeluzole and its R-isomer caused cytoprotection against veratridine cell damage, by blocking the veratridine stimulated Na⁺ and Ca²⁺ entry, as well as the [Ca²⁺]i oscillations. The Ba²⁺ and ouabain cytotoxic effects 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 lubeluzole in stroke; the doubt rests in the stereoselective protecting effects of lubeluzole in in vivo stroke models, as opposed to its lack of stereoselectivity in the in vitro model reported here.