María Sitges

Mechanism of the calcium-dependent stimulation of transmitter release by 4-aminopyridine in synaptosomes

The mechanism of the Ca2+-dependent stimulation of neurotransmitter release by 4-aminopyridine in synaptosomes was studied. The stimulation of gamma-[3H]aminobutyric acid and [3H]acetylcholine release by 4-aminopyridine was not significantly affected either by tetrodotoxin or by the absence of Na+ in the medium, whereas the toxin notably inhibited the release of both transmitters induced by veratridine. On the other hand, the release of labeled gamma-aminobutyric acid induced by 4-aminopyridine was inhibited by both La3+ and ruthenium red, two blockers of Ca2+ transport in synaptosomes. In other experiments, 4-aminopyridine had only a slight stimulatory effect, if any, on the influx of 45Ca2+ into synaptosomes, under both resting and K+-depolarizing conditions. Ruthenium red inhibited the stimulation by K+ of the 45Ca2+ uptake, and 4-aminopyridine did not antagonize this inhibition. We conclude that the transmitter-releasing action of 4-aminopyridine in synaptosomes does not involve an excitatory effect on the membrane which may result in the opening of voltage-sensitive Na+ channels. 4-Aminopyridine does not seem to act either by enhancing Ca2+ entry into the synaptosomes. It is proposed that 4-aminopyridine facilitates the coupling between Ca2+ binding and transmitter secretion at the presynaptic membrane.

Effects of carbamazepine, phenytoin, lamotrigine, oxcarbazepine, topiramate and vinpocetine on Na+ channel-mediated release of [3H]glutamate in hippocampal nerve endings.

Several of the most effective antiepileptic drugs are believed to stop the paroxysmal neuronal activity acting as Na(+) channel blockers. However, no single study comparing in parallel the potency and efficacy of the most commonly used antiepileptic drugs on brain Na(+) channel-mediated responses is available. In the present study the effects of increasing concentrations of carbamazepine, phenytoin, lamotrigine, oxcarbazepine and topiramate, which are among the most frequently used antiepileptic drugs, and of the new putative antiepileptic drug, vinpocetine, on the release of glutamate (Glu) elicited by the Na(+) channel opener, veratridine were investigated in hippocampal isolated nerve endings preloaded with the labeled excitatory amino acid neurotransmitter. The present results show that carbamazepine, phenytoin, lamotrigine and oxcarbazepine, in the range from 150 to 1500 microM, progressively inhibit [(3)H]Glu release induced by veratridine. Also vinpocetine progressively inhibits the veratridine-induced response, but in a much lower range of concentrations (from 1.5 to 15 microM), whereas topiramate only exerts a modest inhibition (20%) of Glu release to veratridine at the highest dose tested (1500 microM). These results indicate that the mechanism of action of several of the most widely used antiepileptic drugs involves reduction in cerebral presynaptic voltage sensitive Na(+) channels permeability. Considering that the high doses of antiepileptic drugs required to control seizures are frequently accompanied by adverse secondary effects, the higher potency of vinpocetine to reduce Na(+) channels permeability might be advantageous.

Effects of carbamazepine, phenytoin, valproic acid, oxcarbazepine, lamotrigine, topiramate and vinpocetine on the presynaptic Ca2+ channel-mediated release of [3H]glutamate: Comparison with the Na+ channel-mediated release

The effect of carbamazepine, phenytoin, valproate, oxcarbazepine, lamotrigine and topiramate, that are among the most widely used antiepileptic drugs (AEDs), and of the new putative AED vinpocetine on the Ca(2+) channel-mediated release of [(3)H]Glu evoked by high K(+) in hippocampal isolated nerve endings was investigated. Results show that carbamazepine, oxcarbazepine and phenytoin reduced [(3)H]Glu release to high K(+) to about 30% and 55% at concentrations of 500 microM and 1500 microM, respectively; lamotrigine and topiramate to about 27% at 1500 microM; while valproate failed to modify it. Vinpocetine was the most potent and effective; 50 microM vinpocetine practically abolished the high K(+) evoked release of [(3)H]Glu. Comparison of the inhibition exerted by the AEDs on [(3)H]Glu release evoked by high K(+) with the inhibition exerted by the AEDs on [(3)H]Glu release evoked by the Na(+) channel opener, veratridine, shows that all the AEDs are in general more effective blockers of the presynaptic Na(+) than of the presynaptic Ca(2+) channel-mediated response. The high doses of AEDs required to control seizures are frequently accompanied by adverse secondary effects. Therefore, the higher potency and efficacy of vinpocetine to reduce the permeability of presynaptic ionic channels controlling the release of the most important excitatory neurotransmitter in the brain must be advantageous in the treatment of epilepsy.

Stimulation of [3H]γ-Aminobutyric Acid Release by Calcium Chelators in Synaptosomes

Abstract: The effect of EGTA on the release of labeled γ‐aminobutyric acid (GABA), glutamate, acetylcholine, and dopamine was studied in superfused synaptosomes from mouse brain. In the absence of both Ca2+ and Mg2+, EGTA and also EDTA at 50 μM or higher concentrations induced a 2.5‐5‐fold stimulation of [3H]GABA release, similar to that produced by potassium depolarization, whereas only a slight effect, or no effect at all, was observed on the release of the other transmitters studied. The GABA‐releasing action of EGTA was practically abolished in the presence of Mg2+. In contrast, the effect of EDTA was also observed when the medium contained Mg2+. Studies on the ionic dependence showed that the stimulation of GABA release by EGTA was abolished in a Na+‐free medium. Li+ did not substitute Na+ for the EGTA effect, which was also independent of chloride. This Na+ dependence does not seem to involve voltage‐sensitive channels, since tetrodotoxin did not affect the GABA‐releasing action of EGTA, whereas in parallel su‐perfusion chambers it blocked over 80% the stimulation of GABA release by veratridine. In contrast, two calcium channel blockers in synaptosomes, La3+ and the cationic dye ruthenium red, greatly inhibited the GABA‐releasing effect of EGTA. L‐2,4‐Diaminobutyric acid, an inhibitor of the Na+‐dependent GABA carrier, did not affect the releasing action of EGTA, whereas in a parallel experiment this drug inhibited by more than 90% the exchange of labeled GABA with unlabeled GABA. It is concluded that the Na+‐dependent releasing action of EGTA and EDTA on GABA is probably due to a destabilization of the synaptosomal membrane by chelation of endogenous membranal Ca2+, which can be prevented by Mg2+. Such destabilization results in Na+ influx through Ca2+ channels, and the consequent increase in the intrater‐minal Na+ concentration induces the release of GABA by a mechanism probably not involving the amino acid carrier. The possible participation of mitochondrial Na+ ‐Ca2+ exchange is considered improbable in view of the lack of effect of EGTA on the release of other neurotrans‐mitters.

Vinpocetine blockade of sodium channels inhibits the rise in sodium and calcium induced by 4-aminopyridine in synaptosomes

The objective of this study was to get a more understandable picture of the mechanism underlying the anticonvulsant action of vinpocetine. The question of how the cerebral excitability is affected was investigated by determining the effect of vinpocetine on the changes on the internal concentrations of Na(+) (Na(i)) and Ca(2+) (Ca(i)) induced by different concentrations of the convulsing agent 4-aminopyridine (4-AP) in striatal isolated nerve endings. The cytosolic concentrations of Na(i) and Ca(i) were detected fluorimetrically with sodium-binding benzofuran isophthalate (SBFI) and fura-2, respectively. Vinpocetine, like the Na(+) channel blocker, tetrodotoxin, abolished the increase in Na(i) induced by 0.1 mM 4-AP and only inhibited in 30% the rise in Na(i) induced by 1mM 4-AP. In contrast with the different sensitivity of the rise in Na(i) induced by 0.1 and 1mM 4-AP to vinpocetine and tetrodotoxin, the rise in Ca(i) induced by the two concentrations of 4-AP was markedly inhibited by vinpocetine (and tetrodotoxin), indicating that only the voltage-sensitive sodium channels (VSSC)-mediated fraction of the rise in Na(i) induced by 4-AP is linked with the activation of pre-synaptic Ca(2+) channels. The elevation of Ca(2+) induced by high K(+) (30 mM) does not require a Na(+) gradient and is vinpocetine and tetrodotoxin insensitive. In contrast, the elevation of Ca(i) induced by 4-AP, requires a physiological (out/in) Na(+) gradient and is vinpocetine and tetrodotoxin-sensitive. It is concluded that by blocking the tetrodotoxin-sensitive fraction of the rise in Na(i) induced by 4-AP, vinpocetine inhibits the concomitant rise in Ca(i) induced by 4-AP. The inhibitory effect of vinpocetine on pre-synaptic voltage-sensitive sodium channels may underlie the in vivo anticonvulsant action of vinpocetine.

Effect of Organic and Inorganic Calcium Channel Blockers on γ-Amino-n-Butyiic Acid Release Induced by Monensin and Veratrine in the Absence of External Calcium

Abstract: The effects of two organic Ca2+ antagonists (verapamil and nitrendipine) and of two inorganic Ca2+ channel blockers (Co2+ and ruthenium red) on the Na+‐dependent release of γ‐amino‐n‐butyric acid (GABA) triggered by veratrine and monensin in the absence of external Ca2+ were studied in mouse brain synaptosomes. Ca2+‐independent release of GABA stimulated by the Na+ channel activator veratrine was inhibited with micromolar concentrations of verapamil and nitrendipine. In contrast, GABA release induced by the Na+ ionophore monensin was insensitive to the organic Ca2+ antagonists. Verapamil also failed to modify A23187‐stimulated release of GABA in the presence of Ca2+ but inhibited high K+‐induced release of the transmitter. Co2+ partially diminished veratrine‐induced release but did not change monensin‐induced release. Releasing responses to monensin and veratrine were insensitive to ruthenium red, which inhibited the Ca2+‐dependent component of GABA release evoked by high K+ depolarization. These data demonstrate that the mechanism of inducing GABA release is different for veratrine and monensin, as evidenced by their differing sensitivities to inhibition by Ca2+ channel antagonists and organic Ca2+ blockers. It is concluded that voltage‐sensitive Ca2+ channels of the presynaptic membrane are not involved in the inhibitory action of Ca2+ antagonists on the Na+‐de‐pendent, Ca2+‐independent mechanism of GABA release.

Vinpocetine selectively inhibits neurotransmitter release triggered by sodium channel activation.

The effects of vinpocetine on internal Na+ (Nai), cAMP accumulation, internal Ca2+ (Cai) and excitatory amino acid neurotransmitters release, under resting and under depolarized conditions, was investigated in rat striatum synaptosomes. Veratridine (20 μM) or high K+ (30 mM) were used as depolarizing agents. Results show that vinpocetine in the low μM range inhibits the elevation of Nai, the elevation of Cai and the release of glutamate and aspartate induced by veratridine depolarization. In contrast, vinpocetine fails to inhibit the rise of Cai and the neurotransmitter release induced by high K+, which are both TTX insensitive responses. Results also show that the inhibition exerted by vinpocetine on all the above veratridine-induced responses is not reflected in PDE activity. Our interpretation of these results is that vinpocetine inhibits neurotransmitter release triggered by veratridine activation of voltage sensitive Na+ channels, but not that triggered by a direct activation of VSCC. Thus, the main mechanism involved in the neuroprotective action of vinpocetine in the CNS is unlikely to be due to a direct inhibition of Ca2+ channels or PDE enzymes, but rather the inhibition of presynaptic Na+ channel-activation unchained responses.

The anti‐seizure drugs vinpocetine and carbamazepine, but not valproic acid, reduce inflammatory IL‐1β and TNF‐α expression in rat hippocampus

In the present study, the effects of the two classical anti‐epileptic drugs, carbamazepine and valproic acid, and the non‐classical anti‐seizure drug vinpocetine were investigated on the expression of the pro‐inflammatory cytokines IL‐1β and TNF‐α in the hippocampus of rats by PCR or western blot after the administration of one or seven doses. Next, the effects of the anti‐seizure drugs were investigated on the rise in cytokine expression induced by lipopolysaccharides (LPS) inoculation in vivo. To validate our methods, the changes induced by the pro‐convulsive agents 4‐aminopyridine, pentylenetetrazole and pilocarpine were also tested. Finally, the effect of the anti‐seizure drugs on seizures and on the concomitant rise in pro‐inflammatory cytokine expression induced by 4‐aminopyridine was explored. Results show that vinpocetine and carbamazepine reduced the expression of IL‐1β and TNF‐α from basal conditions, and the increase in both pro‐inflammatory cytokines induced by LPS. In contrast, valproic acid failed to reduce both the expression of the cytokines from basal conditions and the rise in IL‐1β and TNF‐α expression induced by LPS. Tonic‐clonic seizures induced either by 4‐aminopyridine, pentylenetetrazole or pilocarpine increased the expression of IL‐1β and TNF‐α markedly. 4‐aminopyridine‐induced changes were reduced by all the tested anti‐seizure drugs, although valproic acid was less effective. We conclude that the anti‐seizure drugs, vinpocetine and carbamazepine, whose mechanisms of action involve a decrease in ion channels permeability, also reduce cerebral inflammation.

Simultaneous action of MK-801 (dizclopine) on dopamine, glutamate, aspartate and GABA release from striatum isolated nerve endings.

The simultaneous effect of MK-801 on the baseline- and depolarization (20 microM veratridine or 30 mM high K+)-evoked release of endogenous dopamine, glutamate (Glu), aspartate (Asp), and GABA is investigated in the same preparation of rat striatum isolated nerve endings. MK-801, in the microM range, selectively increases the baseline and high K+ depolarization-evoked release of dopamine, without causing any effect on the baseline or on the high K+-evoked release of Glu, Asp and GABA. In addition to this selective action on dopamine release, MK-801 inhibits the veratridine depolarization-evoked release of all the neurotransmitters tested, including dopamine. In SBFI and fura-2 preloaded striatal synaptosomes, MK-801 inhibits the elevation of internal Na+ (Na(i)) and the elevation of internal Ca2+ (Ca(i)) induced by veratridine depolarization. The elevation of Ca(i) induced by high K+ depolarization is unchanged by MK-801. This study reveals two separate MK-801 actions. (1) The voltage-independent action, which increases dopamine release selectively, and might contribute to the effects of MK-801 on motor coordination. (2) The voltage-dependent action, which inhibits all the veratridine-evoked responses including the evoked release of the excitatory amino acids (which are particularly concentrated in striatum nerve endings), and might contribute to the anticonvulsant and neuroprotective effects of MK-801.

Synthetic peptides corresponding to the sequence of noxiustoxin indicate that the active site of this K+ channel blocker is located on its amino-terminal portion

A nonapeptide Thr-Ile-Ile-Asn-Val-Lys-Cys-Thr-Ser (NTX1–9) and a decapeptide Met-Asn-Gly-Lys-Cys-Lys-Cys-Tyr-Asn-Asn (NTX30–39) corresponding to the N-terminal and C-terminal sequences respectively of Noxiustoxin (NTX) were synthesized by the solid phase method of Merrifield (1963). The first synthetic peptide (NTX1–9) was shown to be toxic to mice independently of the route of administration: intraperitoneally, subcutaneously or intraventricularly (100–200 μg/20 g mouse weight). The second (NTX30–39) was not toxic even at higher dose (400 μg/20 g mouse). When the effects of the peptide NTX1–9 and of the authentic toxin (Noxiustoxin) were studied on the liberation of [3H] 4-aminobutyric acid (3H-GABA) from mouse synaptosomes, both gave essentially the same results, except that peptide NTX1–9 was needed at higher concentration. Synthetic peptide NTX30–39 had no effect in the same preparation at even higher doses. The GABA release produced by toxic peptide NTX1–9 was not affected by tetrodotoxin but was completely abolished by the presence of the K+ ionophore valinomycin, mimicking the effect of native NTX in the same system (Sitges et al., 1986). These results indicate that the toxic active site of Noxiustoxin is possibly located in or near the N-terminal amino acid portion of the molecule.