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

Abstract

1. 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, R91154, did not enhance LDH release. Both lubeluzole and R91154 (0.3–10 μM) decreased the veratridine-induced LDH release.

2. 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.

3. 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. R91154 was less potent than lubeluzole and only protected against the lesion induced by 0.5 mM Ba²⁺.

4. 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.

5. 6-Hydroxydopamine (6-OHDA) caused significant cytotoxic effects at 30 and 100 μM. Lubeluzole (3–10 μM) or penfluridol (0.03–0.3 μM) had no cytoprotective effects against 6-OHDA.

6. Lubeluzole (3 μM), R91154 (3 μM) and penfluridol (1 μM) blocked the current through Na⁺ channels 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 reversible.

7. Lubeluzole (3 μM) induced reversible blockade of the oscillations of the cytosolic Ca²⁺, [Ca²⁺]_i, in fura-2-loaded cells exposed to 30 or 100 μM veratridine. Penfluridol (1 μM) inhibited those oscillations in an irreversible manner.

8. 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.

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