Release of dopamine from human neocortex nerve terminals evoked by different stimuli involving extra- and intraterminal calcium

1 The release of [H-3]-dopamine ([H-3]-DA) from human neocortex nerve terminals was studied in synaptosomes prepared from brain specimens removed in neurosurgery and exposed during superfusion to different releasing stimuli. 2 Treatment with 15 mM KCl, 100 mu M 4-aminopyridine, 1 mu M ionomycin or 30 mM caffeine elicited almost identical overflows of tritium. Removal of external Ca2+ ions abolished the overflow evoked by K+ or ionomycin and largely prevented that caused by 4-aminopyridine; the overflow evoked by caffeine was completely independent of external Ca2+. 3 Exposure of synaptosomes to 25 mu M of the broad spectrum calcium channel blocker CdCl2 strongly inhibited the 4-aminopyridine-induced tritium overflow while that evoked by ionomycin remained unaffected. 4 The Ca2+ chelator, 1,2-bis-(2-aminophenoxy)ethane-N,N, N',N' tetrancetic acid (BAPTA), reduced significantly the K+- and the caffeine-induced tritium overflow. The effect of caffeine was attenuated by exposure to the ryanodine receptor blocker dantrolene or when the membrane-impermeant inositol trisphosphate receptor antagonist, heparin, was entrapped into synaptosomes; the combined treatment with dantrolene and heparin abolished the release elicited by caffeine. 5 Tetanus toxin, entrapped into human neocortex synaptosomes to avoid prolonged incubation, inhibited in a concentration-dependent manner the K+- or the 4-aminopyridine-evoked tritium overflow; in contrast, the release stimulated by ionomycin and by caffeine were both totally insensitive to the same concentrations of tetanus toxin. Western blot analysis showed about 50% reduction of the content of the vesicular protein, synaptobrevin, in synaptosomes poisoned with tetanus toxin. 6 In conclusion, the release of dopamine from human neocortex nerve terminals can be triggered by Ca2+ ions originating from various sources. It seems that stimuli not leading to activation of voltage-sensitive Ca2+ channels elicit Ca2+-dependent, probably exocytotic, release that is insensitive to tetanus toxin.