Differential effects of the neuroprotectant lubeluzole on bovine and mouse chromaffin cell calcium channel subtypes.

Abstract

1. The effects of lubeluzole (a neuroprotective benzothiazole derivative) and its (-) enantiomer R91154 on whole-cell currents through Ca2+ channels, with 10 mM Ba2+ as charge carrier (IBa), have been studied in bovine and mouse voltage-clamped adrenal chromaffin cells. Currents generated by applying 50 ms depolarizing test pulses to 0 mV, from a holding potential of -80 mV, at 10 s intervals had an average magnitude of 1 nA. 2. Lubeluzole and R91154 blocked the peak IBa of bovine chromaffin cells in a time and concentration-dependent manner; their IC50s were 1.94 microM for lubeluzole and 2.54 microM for R91154. In a current-voltage protocol, lubeluzole (3 microM) inhibited peak IBa at all test potentials. However, no obvious shifts of the I-V curve were detected. 3. After 10 min exposure to 3 microM lubeluzole, the late current (measured at the end of the pulse) was inhibited more than the peak current. Upon wash out of the drug, the inactivation reversed first and then the peak current recovered. 4. Blockade of peak current was greater at more depolarizing holding potentials (i.e. 35% at -110 mV and 87% at -50 mV, after 10 min superfusion with lubeluzole). Inactivation of the current was pronounced at -110 mV, decreased at -80 mV and did not occur at -50 mV. 5. Intracellular dialysis of bovine voltage-clamped chromaffin cells with 3 microM lubeluzole caused neither blockade nor inactivation of IBa. The external application of 3 microM lubeluzole to those dialysed cells produced inhibition as well as inactivation of IBa. 6. The effects of lubeluzole (3 microM) on IBa in mouse chromaffin cells were similar to those in bovine chromaffin cells. At -80 mV holding potential, a pronounced inactivation of the current led to greater blockade of the late IBa (66%) as compared with peak IBa (46% after 10 min superfusion with lubeluzole). 7. In mouse chromaffin cells approximately half of the whole-cell IBa was sensitive to 3 microM nifedipine (L-type Ca2+ channels) and the other half to 3 microM omega-conotoxin MVIIC (non-L-type Ca2+ channels). In omega-conotoxin MVIIC-treated cells, 3 microM lubeluzole caused little blockade and inactivation of IBa. However in nifedipine-treated cells, lubeluzole caused a pronounced blockade and inactivation of IBa that reversed upon wash out of the compound. 8. The results are compatible with the idea that lubeluzole preferentially blocks non-L-types of voltage-dependent Ca2+ channels expressed by bovine and mouse chromaffin cells. The higher concentrations of the compound also block L-type Ca2+ channels. The mechanism of inhibition involves the access of lubeluzole to the open channel from the outside of the cell and promotion of its inactivation. The differential blockade of Ca2+ channel subtypes might contribute to the neuroprotective actions of lubeluzole (which exhibit stereoselectivity). However, in view of the lack of stereoselectivity in blocking Ca2+ channels, this effect cannot be the only explanation for the protective activity of lubeluzole in stroke.