Cyanocobalamin, vitamin B12, depresses glutamate release through inhibition of voltage-dependent Ca2+ influx in rat cerebrocortical nerve terminals (synaptosomes)

The effect of cyanocobalamin, vitamin B12, on glutamate release in isolated nerve terminals (synaptosomes) prepared from rat prefrontal cortex was examined. Cyanocobalamin inhibited the release of glutamate evoked by 4-aminopyridine in a concentration-dependent manner. The inhibitory action of cyanocobalamin was blocked by the vesicular transporter inhibitor bafilomycin A1, not by the glutamate transporter inhibitor L-transpyrrolidine-2,4-dicarboxylic acid or the nontransportable glutamate inhibitor DL-threo-beta-benzyloxyaspartate, indicating that this release inhibition results from a reduction of vesicular exocytosis and not from an inhibition of Ca2+-independent efflux via glutamate transporter. Examination of the effect of cyanocobalamin on cytosolic free Ca2+ concentration revealed that the inhibition of glutamate release could be attributed to a reduction in voltage-dependent Ca2+ influx. Consistent with this, the N- and P/Q-type Ca2+ channel blocker ω-conotoxin MVIIC, largely attenuated the inhibitory effect of cyanocobalamin on 4-aminopyridine-evoked glutamate release, but the Ca2+ release inhibitor dantrolene had no effect. Cyanocobalamin did not alter the resting synaptosomal membrane potential or 4-aminopyridine-mediated depolarization; thus, the inhibition of 4-aminopyridine-evoked Ca2+ influx and glutamate release produced by cyanocobalamin was not due to its decreasing synaptosomal excitability. In addition, cyanocobalamin-mediated inhibition of 4-aminopyridine-evoked Ca2+ influx and glutamate release was significantly attenuated by protein kinase C inhibitors GF109203X and Ro318220. Furthermore, 4-aminopyridine-induced phosphorylation of protein kinase C was significantly reduced by cyanocobalamin. These results suggest that cyanocobalamin effects a decrease in protein kinase C activation, which subsequently reduces the Ca2+ entry through voltage-dependent N- and P/Q-type Ca2+ channels to cause a decrease in evoked glutamate release.