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Abstract: The mechanisms by which an elevated KCl level and the K + ‐channel inhibitor 4‐aminopyridine induce release of transmitter glutamate from guinea‐pig cerebral cortical synaptosomes are contrasted. KC1 at 30 m M caused an initial spike in the cytosolic free Ca 2+ concentration (Ca 2+ c ), followed by a partial recovery to a plateau 112 ± 13 n M above the polarized control. The Ca 2+ ‐dependent release of endogenous glutamate, determined by continuous fluorimetry, was largely complete by 3 min, by which time 1.70 ± 0.35 nmol/ mg was released. Ca 2+ c elevation and glutamate release were both insensitive to tetrodotoxin. KCl‐induced elevation in Ca 2+ c could be observed in both low‐Na + medium and in the presence of low concentrations of veratridine. 4‐Aminopyridine at 1 m M increased Ca 2+ c by 143 ± 18 n M to a plateau similar to that following 30 m M KCl. The initial rate of increase in Ca 2+ c following 4‐aminopyridine administration was slower than that following 30 m M KCl. and a transient spike was less apparent. Consistent with this, the 4‐aminopyridine‐induced net uptake of 45 Ca 2+ is much lower than that following an elevated KCl level. 4‐Aminopyridine induced the Ca 2+ ‐dependent release of glutamate, although with somewhat slower kinetics than that for KCl. The measured release was 0.81 nmol of glutamate/mg in the first 3 min of 4‐aminopyridine action. In contrast to KCl, glutamate release and the increase in Ca 2+ c with 4‐aminopyridine were almost entirely blocked by tetrodotoxin, a result indicating repetitive firing of Na + channels. Basal Ca 2+ c and glutamate release from polarized synaptosomes were also significantly lowered by tetrodotoxin. Addition of 30 m M KCl to 4‐ami‐nopyridine‐pretreated synaptosomes caused a large transient spike in Ca 2+ c and further release of glutamate. 4‐Aminopyridine failed to increase Ca 2+ c in low‐Na + media or after addition of low concentrations of veratridine. It is proposed that an unstable membrane potential in the presence of 4‐aminopyridine is amplified by repetitive firing of Na + channels and that this leads to random opening of transient Ca 2+ channels in the synaptosomal population. In contrast, KCl would induce a synchronous activation of transient Ca 2+ channels followed by a plateau of low residual channel conductance. By mimicking repetitive stimulation in vivo, 4‐aminopyridine may more closely model physiological excitation than does an elevated KCl level. Our results are difficult to reconcile with a major role for Na + /Ca 2+ exchange in the elevation of Ca 2+ c and contradict proposals that 4‐aminopyridine induces release of transmitter from isolated nerve terminals by a mechanism not linked to Na + ‐channel firing orCa 2+ entry
Tibbs et al. (Fri,) studied this question.