Involvement of pentylenetetrazole in synapsin I phosphorylation associated with calcium influx in synaptosomes from rat cerebral cortex

Abstract

To determine precisely how pentylenetetrazole (PTZ) is involved in the biochemical processes at the presynaptic nerve terminal, the effect of PTZ, under various conditions, on the phosphorylation of synapsin I (previously called protein I) was investigated, using 32P i, in synaptosomes from rat cerebral cortex. PTZ markedly stimulated the incorporation of 32P into this protein as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and autoradiography, but it failed to stimulate protein phosphorylation in Ca 2+-free medium containing ethylene glycol bis-(β-aminoethylether)- N', N'-tetraacetic acid (EGTA). Moreover, the PTZ-stimulated synapsin I phosphorylation was reversed by addition of EGTA sufficient to chelate all external free Ca 2+ PTZ also stimulated synaptosomal accumulation of Ca 2+. The PTZ-stimulatory effects of both synapsin I phosphorylation and synaptosomal accumulation of Ca 2+ were inhibited markedly by tetrodotoxin as well as by cobalt chloride and lanthanum chloride. The calmodulin antagonists N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide (W-7, strongly) and N-(6-aminohexyl)-l-naphthalenesulfonamide (W-5, weakly) reduced the PTZ-stimulatory effect on synapsin I phosphorylation by about 75 and 15%, respectively, whereas these antagonists had essentially no effect on PTZ-stimulated synaptosomal accumulation of Ca 2+. These results suggest that PTZ causes the influx of Ca 2+ into the presynaptic nerve terminal secondary to the elevated Na + and is consequently involved in the synapsin I phosphorylation step, facilitating the Ca 2+/ calmodulin-mediated presynaptic event leading to seizure discharge.