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

https://doi.org/10.1016/j.neuint.2005.02.001Get rights and content

Abstract

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+ (Nai) and Ca2+ (Cai) induced by different concentrations of the convulsing agent 4-aminopyridine (4-AP) in striatal isolated nerve endings. The cytosolic concentrations of Nai and Cai were detected fluorimetrically with sodium-binding benzofuran isophthalate (SBFI) and fura-2, respectively. Vinpocetine, like the Na+ channel blocker, tetrodotoxin, abolished the increase in Nai induced by 0.1 mM 4-AP and only inhibited in 30% the rise in Nai induced by 1 mM 4-AP. In contrast with the different sensitivity of the rise in Nai induced by 0.1 and 1 mM 4-AP to vinpocetine and tetrodotoxin, the rise in Cai 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 Nai induced by 4-AP is linked with the activation of pre-synaptic Ca2+ channels. The elevation of Ca2+ induced by high K+ (30 mM) does not require a Na+ gradient and is vinpocetine and tetrodotoxin insensitive. In contrast, the elevation of Cai 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 Nai induced by 4-AP, vinpocetine inhibits the concomitant rise in Cai 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.

Introduction

Voltage-sensitive sodium channels (VSSC) play a fundamental role in the normal function of the CNS because they are responsible for the initiation and conduction of neuronal action potentials. Most of the energy demand coupled to brain functional activity is used for ion transport and to restore the ionic gradients, degraded by excitation, back to resting membrane potentials. Therefore, the pharmacological down-modulation of VSSC represents a reasonable therapeutic strategy against brain tissue damage in situations such as epilepsy, in which the energy demand surpasses the energy supply (Urenjak and Obrenovitch, 1996).

Many of the most widely used antiepileptic drugs suppress the abnormal neuronal excitability associated with seizures by means of complex voltage- and frequency-dependent inhibition of ionic currents through Na+ channels (Ragsdale and Avoli, 1998). Consistently, several of the most commonly used antiepileptic drugs displace radioligand (3H-batracotoxin) binding to Na+ channels in cerebral membranes and synaptosomes (Lingamaneni and Hemmings, 2003).

In previous studies in vitro, we have shown that the nootropic drug with neuroprotective capabilities, vinpocetine (ethyl apovincamine-22-oate), selectively inhibits the increase in the internal concentration of Na+ (Nai) and the release of glutamate, aspartate and dopamine induced by activation of pre-synaptic VSSC with veratridine, and failed to inhibit the rise in Cai and the release of the above neurotransmitters induced by high K+ (Sitges and Nekrassov, 1998, Sitges and Nekrassov, 1999, Trejo et al., 2001). In a recent study in vivo, we have shown that vinpocetine prevents the epileptic cortical activity induced by the convulsing agent, 4-aminopyridine (4-AP) (Sitges and Nekrassov, 2004). The epileptiform activity induced by 4-AP is amply documented in both brain slices in vitro and animal models of epilepsy in vivo (Ives and Jefferys, 1990, Psarropoulou and Avoli, 1996, Armand et al., 1999, Morales-Villagrán et al., 1999, Nekrassov and Sitges, 2003). With the aid of the Na+ channel blocker, tetrodotoxin, we have recently characterized the participation of VSSC in the rise in Nai and in the rise in the internal concentration of Ca2+ (Cai) induced by 4-AP in striatum synaptosomes (Galván and Sitges, 2004). In an attempt to explore the mechanism underlying the prevention exerted by vinpocetine on the epileptic cortical activity induced by 4-AP in vivo, the effect of vinpocetine on the rise in Nai and Cai induced by 4-AP in striatal synaptosomes was investigated here.

Section snippets

Materials

Sodium-binding benzofuran isophthalate (SBFI), fura-2 (1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2′-amino-5′-methylphenoxy)-ethane-N,N,N′,N′-tetraacetic acid) in their cell-permeant acetoxymethyl ester (AM) forms and pluronic acid F-127 were from molecular probes. Vinpocetine (eburnamenine-14-carboxylic acid ethyl ester), 4-aminopyridine, veratridine, tetrodotoxin, gramicidin D, digitonin and probenecid were obtained from Sigma Chemical Co. (St. Louis, MO). All other reagents were

Effect of vinpocetine on the elevation of Nai induced by various 4-AP concentrations in striatal isolated nerve endings

The effect of vinpocetine at a concentration of 15 μM on the rise in Nai induced by increasing concentrations of 4-AP (0.1, 0.2 and 1 mM) is shown in Fig. 1a. We choose 15 μM vinpocetine to test its effect on the ionic changes induced by 4-AP because we have previously shown that at this concentration vinpocetine exerts a marked inhibition of the rise in Nai induced by veratridine in striatal synaptosomes (Sitges and Nekrassov, 1999). Fig. 1a shows that 15 μM vinpocetine abolishes the rise in Nai

Discussion

In the present study, the effect of vinpocetine on the rise in Nai and Cai induced by 4-AP was investigated in striatal synaptosomes pre-loaded with SBFI and fura-2, respectively.

The parallelism between the degree of inhibition exerted by vinpocetine and the Na+ channel blocker, tetrodotoxin, on the rise in Nai induced by 4-AP at increasing concentrations shown in Fig. 1 confirms the involvement of VSSC in the vinpocetine mechanism of action (Erdö et al., 1996). The inhibition exerted by

Acknowledgments

The authors thank Luz María Chiu and Araceli Guarneros for their excellent technical assistance. This work was financially supported by project P42046352Q from SEP-CONACYT and by Psicofarma S.A. de C.V.

References (29)

Cited by (0)

View full text