Effects of a 4‐Aminopyridine in Calcium Movements and Changes of Membrane Potential in Pinched‐Off Nerve Terminals from Rat Cerebral Cortex

Abstract

A study was conducted on the influence of 4‐aminopyridine (4‐AP) on the radiocalcium uptake and membrane potential of rat cortical synaptosomes previously depolarized by biochemical procedures. The initial calcium entry into isolated nerve terminals was substantally enhanced in the presence of 10‐−4M 4‐AP in potassium‐rich media (60 mM). The fast initial phase of potassium‐stimulated calcium entry involves different kinetic characteristics in the presence and the absence of 4‐AP. In the presence of 4‐AP, the fast component of calcium entry reached a peak during the first 15 s immediately following depolarization. The potassium‐stimulated synaptosomal calcium influx at 15 s was 3.5 ± 0.17 times higher in the presence of 4‐AP (10‐−4M) and 2.6 ± 0.11 times higher in the absence of 4‐AP as compared with the respective unstimulated uptake values. In the absence of 4‐AP, however, the calcium entry did not show a peak until 45 s after depolarization. The total amount of calcium accumulated in the synaptosome treated and untreated by 4‐AP is equal at the end of this initial uptake period. The effect of 4‐AP on membrane potential during depolarization of synaptosomes evoked by potassium‐rich medium was also determined by means of a potential sensitive fluorescent dye. It was found that 4‐AP had no effect on the final level of membrane potential, induced by high [K+]o; however, the kinetics showed significant differences. The initial phase of synaptosomal membrane depolarization was slower in the presence of 4‐AP: τ‐4‐AP: 42 ± 4.0 s, τ‐control: 18 ± 2.5 s. The initial calcium entry into the nerve terminals was increased during the 4‐AP treatment in potassium‐rich media—as resulted from our calcium uptake studies—and thus, during this higher calcium influx, the degree of membrane depolarization was decreased. This faster initial calcium influx constitutes a feasible explanation for the unique effect of 4‐AP termed “chemical potentiation” on transmitter release at chemical synapses.

Mobilization of Calcium from Intracellular Stores, Potentiation of Neurotransmitter-Induced Calcium Transients, and Capacitative Calcium Entry by 4-Aminopyridine

“…All of the therapeutic activities of 4-AP are currently explained by blockade of voltageactivated K ϩ channels (Vislobokoe et al, 1983;Davies et al, 1991;Choquet and Korn, 1992;Kirsch and Drewe, 1993;Castle et al, 1994). However, effects of 4-AP on calcium homeostasis have also been reported (Agoston et al, 1983;Pant et al, 1983;Tapia et al, 1985;Gibson and Manger, 1988;Campbell et al, 1993). Here, we show that 4-AP causes a complex change of calcium homeostasis, which includes mobilization of calcium from ICS, likely caused by InsP 3 elevation, modulation of InsP 3 -linked calcium transients, and potentiation of CCE activated by ICS depletion.…”

“…There are, however, a number of reports addressing a possible direct effect of 4-AP on calcium homeostatic mechanisms (Glover, 1981;Agoston et al, 1983;Furukawa et al, 1985;Fu et al, 1987;Gibson and Manger, 1988;Gobet et al, 1995;Fryer and Glover, 1997). In this study, we analyzed how 4-AP may affect calcium homeostasis.…”

The glutamatergic nerve terminal

“…respiration can be stimulated S -10-fold by the addition of mitochondria1 uncouplers Kauppinen and Nicholls, 1986). They maintain a plasma membrane potential of -60 mV to -80mV in low K+ medium (Blaustein and Goldring, 1975;Scott and Nicholls, 1980;Agoston et al, 1983) together with a volumeaverage cytoplasmic free Ca2+ concentration, [Ca2+Ic, of 0.1 -0.2 pM (Ashley et al, 1984;Nachshen, 198Sa;Verhage et al, 1988;Kauppinen et al, 1988). It may thus be concluded that the preparation is energetically 'intact'.…”

“…Thus inhibition of the channel by the addition to synaptosomes of either nanomolar dendrotoxin or millimolar 4-aminopyridine (4AP) abolishes this negative feedback and causes synaptosomes to undergo spontaneous 'epileptic' action potentials (Fig. 3), involving the repetitive firing of Na' channels, as though the terminal was still receiving action potentials down its severed axon (Agoston et al 1983;Tibbs et al, 1989a,b,c;Muniz et al, 1990). 4AP thus more closely mimics the physiological mechanism of terminal depolarization than does elevated KCl.…”

Ion channels and the regulation of neurotransmitter glutamate release

“…In addition to adenosine, a muscarinic inhibition of endogenous glutamate release from hippocampal synaptosomes has been reported [4 I], while 5-hydroxytryptamine (SHT,) receptors inhibit glutamate release from cerebellar synaptosomes [42] and D, dopaminergic receptors regulate the release of glutamate from striatal synaptosomes Although the small size of synaptosomes precludes electrical stimulation, it is possible to induce the firing of spontaneous repetitive action potentials in synaptosomes by inhibiting the K+-A-channel [44,451. This channel normally operates to stabilize the plasma membrane potential against spontaneous fluctuations by opening rapidly in response to slight depolarizations, aiding the restoration of the resting membrane potential.…”