Neurochemical Research, Vol. 24, No. 12, 1999, pp. 1585-1591
Vinpocetine Selectively Inhibits Neurotransmitter Release Triggered by Sodium Channel Activation Maria Sitges1,3 and Vladimir Nekrassov2 (Accepted June 30, 1999)
The effects of vinpocetine on internal Na+ (Nai), cAMP accumulation, internal Ca2+ (Cai) and excitatory ammo acid neurotransmitters release, under resting and under depolarized conditions, was investigated in rat striatum synaptosomes. Veratridine (20 UM) or high K+ (30 mM) were used as depolarizing agents. Results show that vinpocetine in the low uM 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.
KEY WORDS: Neuroprotection; presynaptic sodium; excitatory amino acids-release; striatum synaptosomes; veratridine; cyclic nucleotides; PDE.
INTRODUCTION Several evidences in a variety of experimental models suggest that vinpocetine (ethyl apovincamine22-oate; or cavinton), is a neuroprotective drug that exerts beneficial effects on neurological symptoms and cerebrovascular disease accompanyed by hypoxia and ischemia (1-4). Among the proposed mechanisms of neuronal damage are those involving glutamate and free radicals (5,6), but also a role of voltage sensitive Na+ channels Depto. de Biologia Celular, Instituto de Investigaciones Biomedicas, UNAM 2 Instituto Nacional de la Comunicacion Humana, SSA. 3 Address reprint requests to Dr. Maria Sitges, Instituto de Investigaciones Biom6dicas, Apartado Postal 70228, Ciudad Universitaria 04510, Mexico, D.F. Phone 622-38-66 FAX (525) 622 38 97, e-mail:
[email protected] 1
in neuronal damage has been proposed (7). In a recent study we found that the elevation in the internal concentration of Na+ (Nai) induced by activation of presynaptic Na+ channels is inhibited by anticalmodulin drugs such as W7 and trifluoperazine (8). Interestingly those drugs are classified, along with vinpocetine, as Ca2+ and calmodulin dependent phosphodiesterase (PDE) inhibitors. The formers acting on the regulatory domain of the enzyme, while vinpocetine selectively inhibiting PDE catalytic site (9-12). Various studies indicating the inhibitory action of vinpocetine on Na+, Ca2+ and K+ channels have been carried out in different preparations (13-17). The possible relationship between the effects of vinpocetine on channels with its action as PDE inhibitor or with the effect of vinpocetine on excitatory amino acids physiology have not however been explored.
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1586 In the present study the effect of vinpocetine on internal Na+ (Nai), cAMP accumulation (as indicator of PDE activity), internal Ca2+ (Cai) and excitatory amino acid neurotransmitters release was investigated in the striatal synaptosomal preparation. We chose this preparation because of the extremely high concentration of PDE1B1 in the striatum compared to other brain regions (18,19), that facilitates detection of cyclic nucleotides as a measure of PDE activity, and because in striatal synaptosomes glutamatergic nerve endings originating from the cortex are present, and this allows to test the effect of vinpocetine on excitatory amino acid neurotransmitters release in the same preparation. Part of this work has been published previously in an Abstract form (20).
EXPERIMENTAL PROCEDURE Source of Materials. SBFI-AM, fura-2AM, and pluronic acid F-127 were from Molecular Probes. Veratridine, gramicidin D, digitonin and probenecid were obtained from Sigma Chemical Co. (St. Louis, MO). The TRK 432 cAMP assay kit was from Amersham International. The o-phthalaldehyde reagent solution (OPA) was from Pierce (Rockford, ILL). Forskolin from Research Biochemicals International and vinpocetine (cavinton) was a kind gift of Armstrong Laboratories of Mexico. All other reagents were of analytical grade. Preparation of Striatum Synaptosomes. Dissected striata of 46 male Wistar rats (250-300 g) were immediately placed in cold isotonic sucrose (1:10, wt/vol) and homogenized (6 strokes at 2000 rpm, 0.15mm pestle-vessel clearence). The resulting suspensions were centrifuged at 1500x g for 10 min, and supernatants obtained from this centrifugation were centrifuged for another 20 min at 9000x g. The resulting pellets containing striatal synaptosomes were resuspended in standard Kreb's Ringer HEPES (KRH, in mM: 127 NaCl, 1.2 KH2PO4, 3.37 KCl, 1 CaCl2, 1.18 MgSO4, 20 HEPES and 5.6 mM dextrose, pH 7,4, bubbled with a O2/CO2 mixture). Estimation of Nai and Cai Concentrations in Striatal Synaptosomes. The methods used to load synaptosomes with fura-2 or with SBFI and to monitor their fluorescence were previously reported (8,21-23). Briefly, striatal synaptosomes (3 mg/ml) were incubated with SBFI-AM (10 uM) or with fura-2 (5 uM) for 45 min at 37°C. Incubation was stop by dilution and centrifugation. After washing out the unincorporated fluorescent dye, the final synaptosomal pellets were resuspended in 1 ml of low Na+ KRH (for the case of SBFI) or KRH (for the case of fura-2-loaded synaptosomes). The synaptosomal suspensions were kept at 4°C in the dark and used within 2 h. Aliquots (200 ul) of these suspensions were transferred to acrylic cuvettes, diluted 10 fold to a final volume of 2 ml with KRH and stirred continuously. Nai and Cai were estimated from fluorescence monitored on-line, in a Perkin-Elmer LS-50 spectrofluorometer interfaced with an IBM-compatible computer. Excitation wavelengths were set at 340 and 380 nm, emission wavelength at 505 nm and slit widths at 10 nm. Experiments were performed at room temperature (22-25°C). Data points were collected at 1.8 s intervals. After monitoring the 340/380 nm baseline ratio for 2 min, the drug to be tested was added to the cuvette and data points were collected for another 2 or 3 min. For determinations under depolar-
ized conditions, an aliquot of a veratridine concentrated solution was directly added to the cuvette for obtaining the desired final concentration (20 uM). The method used for estimation of Nai in mM from SBFI fluorescence was previously reported (22). Cai was estimated following the ratio method (24). Estimation of cAMP Accumulation. Striatum synaptosomes from 4 male Wistar rats (4.8 mg) were resuspended in 4 ml of ice cold KRH. Aliquots of 500 ul (containing 600ug synaptosomal protein aproximately) were distributed in eppendorf tubes and preincubated for 10 min at 37°C. After the preincubation period 10 ul of a concentrated solution of the drug to be tested (vinpocetine, veratridine, forskolin) was rapidly added to the respective tubes in order to obtain the desired final concentration and the preparation was incubated for another 10 min. The incubation was stopped by centrifugation. The supernatant from this centrifugation was eliminated, and the pellet (synaptosomes containing cAMP) thoroughly suspended in 100 ul perchloric acid (0.1 M) for cAMP extraction. These suspensions were centrifuged, the pellets used for protein determination and 80 ul of the resulting supernatants transferred to tubes containing 109 ul of a Trisma base 100 mM, EGTA 8 mM, pH 9.3 solution. With the last manipulation an appropriate pH (7.4) and reasonable concentrations of Tris and EDTA (57 and 4.6 mM respectively) are achieved to determine the content of cAMP by radioimmunoassay with the Amersham TRK 432 cAMP assay kit. The cAMP concentration is expressed in pmoles cAMP/mg protein. Endogenous Excitatory Amino Acid Neurotransmitters Release Experiments. Rat striatum synaptosomes suspended (400 ug/500 ul) in: KRH or in veratridine (20 uM) or high K (30 mM) depolarizing media without or with vinpocetine (15 uM) or TTX (1 uM) were incubated at 37°C for 10 min. In the high K+ buffer 30 mM KCl replaced an equimolar concentration of NaCl. After the incubation period, the preparation was centrifuged. An aliquot of perchloric acid was added to the resulting supernatants to obtain 0.1 M final. The resulting pellets were used for protein determination following the method of Lowry. A 10 ul volume of sample (perchloric acid treated supernatants) was mixed with 20 ul of OPA reagent. After 120s (optimun derivatization time for the reaction) a 10 ul aliquot was injected to the HPLC system. Amino acids were eluted from the analytical column (Nova-pak C-18, 75 x 3.9 mm internal diameter, particle size 10 um set at 25°C) using a lineal gradient elution program performed in 30 min: eluent A (30 mM sodium acetate buffer, pH 6.8) from 100% to 50%, and eluent B (methanol) from 0% to 50% at a flow rate 1 ml/min. A fluorescence detector set at 360 nm (excitation wavelength) and at 450 nm (emission wavelength) was used. The amino acid concentrations in the experimental samples were calculated with calibration curves obtained from the injection of increasing concentrations of standard amino acid mixtures after OPA derivatization into the HPLC system. Statistics. Student's t test was used for statistical evaluations. From p < 0.05, differences between data were considered statistically significant.
RESULTS Effect of Vinpocetine on the Elevation of Nai Induced by Veratridine in Rat Striatum Synaptosomes. Veratridine (20 uM) progressively elevates the concentration of Nai from the basal level (23 mM in average)
Vinpocetine Effects in Striatum Synaptosomes to a sustained average plateau value of 120 mM in less than 60 s (Fig. 1a). This elevation of Nai induced by veratridine is markedly diminished in synaptosomes pretreated with 5 uM vinpocetine (Fig. 1b), and completely inhibited with a higher (15 uM) vinpocetine concentration (Fig. 1c). Single and Combined Effects of Vinpocetine and Veratridine on cAMP Accumulation. The concentration of cAMP in rat striatum synaptosomes incubated in KRH for 10 min is 21.9 ± 1.3 pmol/mg synaptosomal protein. This concentration in control synaptosomes is not significantly modified by the single or combined treatment with vinpocetine and veratridine. The concentration of cAMP in synaptosomes exposed to 15 uM vinpocetine is 23.6 ± 3.2 pmol/mg, and the
Fig. 1. Effect of vinpocetine on the elevation of Nai induced by veratridine depolarization. Striatal synaptosomes loaded with SBFI were suspended in KRH and Nai estimated as described in "Methods". Data points were taken at 1.8 s intervals. After measuring the basal Nai level, the preparation was exposed to: (a) vehicle (VEH) and then to 20 uM veratridine (VTRD); (b) to 5 uM vinpocetine (VIN 5 uM) and then to 20 uM veratridine (VTRD); (c) to 15 uM vinpocetine (VIN 15 uM) and then to 20 uM veratridine (VTRD), and Nai measured for 2-3 min after each addition. Data shown are representative of four independent experiments. In each independent experiment the three conditions shown were run at least once. The highest variability among responses under one condition between experiments was 12%.
1587 concentration of cAMP in synaptosomes exposed to 20 uM veratridine is 24.7 ± 1.8 pmol/mg. In synaptosomes simultaneously exposed to 15 uM vinpocetine and 20 uM veratridine the concentration of cAMP is 25.6 ± 2.3. These data are the mean ± SEM values of six independent preparations. Single and Combined Effect of Vinpocetine and Forskolin on cAMP Levels. The concentration of cAMP in rat striatum synaptosomes incubated in KRH for 10 min in the presence of 10 uM forskolin is increased from respective control values (21.2 + 1.18 pmol/mg) to 29.2 + 1.5 pmol/mg. Exposure to 15 uM vinpocetine simultaneously with 10 uM forskolin further increases the response to 10 uM forskolin alone. When synaptosomes are incubated in the presence of 100 uM forskolin cAMP accumulation is even higher (43.4 ± 1.4 pmol/mg) (Fig. 2). Effect of Forskolin on the Elevation of Nai Induced by Veratridine. The elevation of Nai induced by veratridine in control and 30 uM forskolin treated striatal synaptosomes is similar (Fig. 3). Effect of Vinpocetine on the Elevation of Cai Induced by High K+ and Veratridine. When striatum synaptosomes are exposed to high K+ depolarization Cai rapidly increases and then declines to a plateau value. The subsequent addition of veratridine (20uM) further increases Cai, but in a progressive manner that also reaches a plateau value (Fig. 4a). The elevation of Cai induced by high K+ is insensitive to either TTX or vinpocetine, whereas the subsequent elevation of Cai induced by veratridine is completely abolished by both treatments (Fig. 4b and 4c).
Fig. 2. cAMP accumulation in striatal synaptosomes exposed to vinpocetine or/and forskolin. Synaptosomes (600 ug/500 ul) incubated (37°C) for 10 min in: KRH (control, CTR), KRH containing 15 uM vinpocetine (VIN 15), KRH containing 10 uM forskolin (FK 10), KRH containing 15 uM vinpocetine plus 10 uM forskolin (VIN 15 + FK 10), or KRH containing 100 uM forskolin (FK 100) were treated for cAMP determination, as indicated under "Methods". Data are the mean ± SEM of seven independent preparations, except for forskolin 100 uM, in which data are the mean ± SEM of three independent preparations.
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Fig. 3. Effect of forskolin on Nai under resting and depolarized conditions. After measuring the basal Nai level, SBFI loaded synaptosomes suspended in KRH were exposed to: (D) 20 uM veratridine (VTRD) and Nai measured for 180 s, (o) to 30 uM forskolin (FK) and Nai measured for 100 s and then to 20 uM veratridine (VTRD) and Nai measured for 180 s. Data shown are representative of three independent experiments. In each independent experiment both conditions were tested at least once.
Effect of Vinpocetine on the Veratridine Evoked Release of Excitatory Amino Acid Neurotransmitters. The baseline release of glutamate and aspartate from striatum synaptosomes is increased about fourfold by veratridine depolarization: from 13.1 ± 2 to 40.4 ± 3.1 nmol/mg and from 4.4 ± 0.3 to 16.8 ± 0.5 nmol/ mg, respectively. The presence of vinpocetine (15 uM) does not change the baseline release of the endogenous amino acid neurotransmitters, but markedly inhibits the veratridine depolarization evoked release of glutamate and aspartate (Fig 5a and b). Effect of Vinpocetine on high K+ Evoked Release of Excitatory Amino Acid Neurotransmitters. In rat striatum synaptosomes, the baseline release of endogenous glutamate and aspartate is increased about twice by the elevation of external K+ to 30 mM; from 12.1 + 0.9 and 5.2 ± 0.3 nmol/mg to 22.1 ± 1.3 and to 7.5 + 0.3 nmol/mg, respectively. This high K+ evoked release of glutamate and aspartate is neither inhibited by vinpocetine nor by the Na+ channel blocker, TTX (Fig. 6).
DISCUSSION The present study shows that vinpocetine inhibits with the same potency the elevations of Nai and Cai induced by veratridine depolarization, and that these effects are not reflected on cAMP accumulation. Vinpocetine also inhibits the Na+ dependent, veratridine depolarization evoked release of glutamate and aspartate. In contrast, vinpocetine was uncapable to inhibit the elevation of Cai induced by high K+, as well as the high K+ evoked release of the above excitatory amino acid neurotransmitters.
Fig. 4. Vinpocetine inhibits the elevation of Cai induced by veratridine, but not by high K+ depolarization. Striatal synaptosomes loaded with Fura-2 were suspended in KRH and Cai estimated as described under "Methods". Data points were taken at 1.8 s intervals. After measuring the baseline Cai level, synaptosomes were subsequently exposed to (a): vehicle (VEH), 30 mM K+ (High K+) and 20 uM veratridine (VTRD), (b): 1 uM TTX, 30 mM K+ (High K+) and 20 uM veratridine (VTRD), (c): 15 uM vinpocetine (VIN), 30 mM K+ (High K+) and 20 uM veratridine (VTRD), and Cai measured for 2-3 min after each addition. Data shown are representative of four independent experiments. In each independent experiment the three conditions were run at least once. The highest variability among responses under one condition between experiments was 10%.
The inhibition exerted by vinpocetine on the elevation of Nai induced by veratridine in rat striatal synaptosomes found here is in agreement with previous findings (14,16,25). Rat brain Na+ channels phosphorylation (26,27), and Na+ currents modulation mediated by cAMP dependent protein kinase A (PKA), by Ca2+ and phospholipid dependent protein kinase C (PKC), or by both kinases concurrently are documented (28-31). Although, the incipient effect of vinpocetine as PDE inhibitor, unmasked when cAMP levels are already elevated by forskolin, does not suggest an involvement of PDE on the marked inhibition exerted by vinpocetine on the veratridine-induced elevation of
Vinpocetine Effects in Striatum Synaptosomes
Fig. 5. Vinpocetine inhibits veratridine depolarization evoked release of glutamate and aspartate. Striatal synaptosomes were incubated (37°C) in: KRH without (CTR) or containing 20 uM veratridine (CTR, VTRD), and in KRH with 15 uM Vinpocetine without (VIN) or containing 20 uM veratridine (VIN, VTRD) for 10 min. Incubation was stopped by centrifugation. The supernatants containing the glutamate (a) or the aspartate (b) released were prepared for HPLC analysis and the pellets used for protein determination. Results are the mean ± SEM values of three independent determinations. (*) The difference between veratridine evoked neurotransmitter release in the absence and presence of vinpocetine is statistically significant.
Nai. In addition, forskolin, that clearly increased cAMP levels in striatal synaptosomes, hence PKA activity, was unable to significantly modify Nai, neither under resting nor under depolarized conditions, in which also PKC is expected to be activated by the increase in Ca2+ induced by veratridine. The inhibition exerted by vinpocetine on the elevation of Nai induced by veratridine in rat striatum synaptosomes found here, that confirms recent results in guinea pig brain cortex synaptosomes (16), suggested us that vinpocetine was inhibiting glutamate and aspartate release evoked by veratridine by decreasing presynaptic Na+ channels permeability. Because, using the same experimental methods we have previously found that the elevation of Nai and the release of the amino acid neurotransmitters induced by veratridine were both, sensitive TTX, but independent on external Ca2+ (8). In the absence of external Ca2+, the elevation of Nai elicited by veratridine evokes neurotransmitter release at expense of the cytoplasmic neurotransmitter pools by reversal of the Na+ dependent transporters (21,32,33). Nevertheless, in the presence of external Ca2+ veratridine depolarization also increases Cai in striatal synaptosomes. This elevation of Cai induced by veratridine depolarization might also be triggered by the activation of voltage sensitive Na+ channels, because in a previous study from our laboratory in mouse whole brain synaptosomes we have demonstrated that TTX also blocks the elevation of Cai to veratridine (21). Thus, by blocking the depolarization caused by the elevation of Nai, vinpocetine indirectly inhibits the acti-
1589 vation of VSCC and the concomitant elevation of Cai induced by veratridine. On the other hand, high K+ evoked responses are strictly dependent on the activation of VSCC, but do not involve voltage sensitive Na+ channels. The rise of Ca2+ and the neurotransmitter released to high K+ are both, insensitive to the absence of external Na+ or to the presence of TTX, but blocked by w-AGA IVA or by the absence of external Ca2+ (21,34,35). Thus, our findings that, like TTX, vinpocetine abolishes the Na+ dependent, veratridine depolarization evoked release of glutamate and aspartate, but not the elevation of Cai and the release of the above transmitters evoked by high K+, further demonstrates that vinpocetine inhibits
Fig. 6. Vinpocetine fails to inhibit high K+ depolarization evoked release of glutamate and aspartate. Striatal synaptosomes were incubated (37°C) for 10 min in: KRH (CTR), high (30 mM) K+ depolarizing buffer without (K 30) or with 15 uM vinpocetine (K30, VIN) or 1 uM TTX (K30, TTX). Incubation was stopped by centrifugation. The supernatants containing the glutamate (a) and the aspartate (b) released were prepared for HPLC analysis and the pellets used for protein determination. Results are the mean ± SEM values of 6 independent determinations. The differences between high K+ evoked neurotransmitter release in the absence and presence of vinpocetine or TTX are not statistically significant.
1590 the release of glutamate and aspartate evoked by a direct activation of voltage sensitive Na+ channels, but not that evoked by a direct activation of VSCC. In summary, by blocking Na+ permeability vinpocetine prevents the reversal of the Na+ dependent neurotransmitter transporters and the concomitant release of the cytoplasmic pools of the excitatory amino acid neurotransmitters. This effect might contribute importantly to the neuroprotective action of vinpocetine in the CNS. Finally, this study supports the hypothesis that inhibitors of voltage gated Na+ channels are potential neuroprotective agents (7,36,37). ACKNOWLEDGMENTS The authors thank Luz Mania Chiu and Araceli Guarneros for their excellent technical assistance. This work was financially supported by project IN212398 from PAPIIT, UNAM.
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