Repetitive Action Potentials in Isolated Nerve Terminals in the Presence of 4‐Aminopyridine: Effects on Cytosolic Free Ca²⁺ and Glutamate Release

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. KCl at 30 mM caused an initial spike in the cytosolic free Ca2+ concentration ([Ca2+]c), followed by a partial recovery to a plateau 112 +/- 13 nM above the polarized control. The Ca2+-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. [Ca2+]c elevation and glutamate release were both insensitive to tetrodotoxin. KCl-induced elevation in [Ca2+]c could be observed in both low-Na+ medium and in the presence of low concentrations of veratridine. 4-Aminopyridine at 1 mM increased [Ca2+]c by 143 +/- 18 nM to a plateau similar to that following 30 mM KCl. The initial rate of increase in [Ca2+]c following 4-aminopyridine administration was slower than that following 30 mM KCl, and a transient spike was less apparent. Consistent with this, the 4-aminopyridine-induced net uptake of 45Ca2+ is much lower than that following an elevated KCl level. 4-Aminopyridine induced the Ca2+-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 [Ca2+]c with 4-aminopyridine were almost entirely blocked by tetrodotoxin, a result indicating repetitive firing of Na+ channels. Basal [Ca2+]c and glutamate release from polarized synaptosomes were also significantly lowered by tetrodotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Nicotinic receptor-evoked hippocampal norepinephrine release is highly sensitive to inhibition by isoflurane

“…1B). In contrast, depolarization by 4AP, a K þ channel blocker that mimics repetitive action-potentialmediated depolarization, 16 evoked significant release of NE, GABA, and glutamate (Fig. 1B).…”

Dendrotoxin and charybdotoxin increase the cytosolic concentration of free Ca2+ in cerebrocortical synaptosomes: An effect not shared by apamin

“…Dendrotoxin (DTX) and 4-aminopyridine (4-AP) increase the spontaneous excitability of synaptosomes [1][2][3] as a consequence of a reduction in the K ÷ permeability [3][4][5]. Rapidlyactivating, voltage-dependent, Ca2+-insensitive K ÷ channels inhibited by both ligands have been characterized [6,7].…”

“…Thus, it was of interest to ascertain if either of these toxins could modulate the excitability of isolated nerve terminals either independently or synergistically with that of DTX. [Ca2+]c was determined using fura-2 acetoxymethyl ester (fura-2/AM) as described previously [2]. Briefly, pellets were resuspended in incubation medium (122 mM NaCl, 3.1 mM KC1, 0.4 mM KH2PO4, 5 mM NaHCO3, 20 mM Tes-Na, 1.2 mM MgSO4, 5 mM glucose and 1 mg/ml bovine serum albumin, pH 7.4) to a concentration of 1.34 mg synaptosomal protein/ml and incubated at 37°C for 35 min in the presence of 5 #M fura-2/AM.…”

The glutamatergic nerve terminal

“…Na' channels occasionally 'flicker' in polarized synaptosomes, since addition of the Na' channel inhibitor tetrodotoxin can slightly inhibit respiration (Kauppinen et al, 1986) and lower [Ca2+Ic (Tibbs et al, 1989a). Inactivation of these 'flickering' channels can be prevented by veratridine and, in a few seconds, Na' channels accumulate in the open state, causing a long-lasting increase in Na" conductance and associated depolarization.…”

“…However, in addition to the channel which is active under polarizing conditions and which gives the dominant K' conductance to the membrane, there is speculative evidence that at least three other K' channels are present: a Ca2+-activated K' conductance (Bartschat and Elauskein, 1985b;Benishin et al, 1988a), a 'delayed rectifier' which controls the intensity and duration of presynaptic action potentials (Colby and Blaustein, 1988;Blaustein et al, 1991) and a K: channel, closed at normal resting potentials but producing transient, rapidly inactivating, currents on depolarization (Bartschat and Blaustein, 1985a;Tibbs et al, 1989a). K; channels may control the excitability of the nerve terminal and limit statistical fluctuations of the plasma membrane potential by opening rapidly and repolarizing the membrane.…”