Introduction

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Neurotransmitter release from varicosities of nerve axon terminals in the central nervous system is triggered by Ca²⁺ influx through voltage-dependent Ca²⁺ channels (VDCCs; L-, N-, P/Q-type) in response to action potentials or other depolarizing stimuli (Nachshen, 1985; Suszkiw et al., 1989; Tareilus et al., 1993). Depolarization-induced increase in cytosolic concentration of calcium [Ca²⁺]_i in cerebral synaptosomes reflects presynaptic Ca²⁺ influx, which is predominantly mediated by VDCCs. A synaptosomal preparation is composed of a mixed population of varicosities characteristic of the respective brain region; i.e., predominantly glutamatergic and -aminobutyric acid varicosities in the cerebral cortex (Nieuwenhuys, 1994). Blocking presynaptic VDCCs is of potential interest for treatment of seizures and ischemic brain injury. Because Ca²⁺ influx in synaptosomes cannot be induced by electrical impulses, chemical depolarization methods must be applied, such as high K⁺ concentration (Blaustein and Goldring, 1975) or veratridine, which opens Na⁺ channels and prevents their inactivation (Blaustein, 1975; Adam-Vizi and Ligeti, 1986).

Presynaptic VDCCs have been extensively studied in rat brain (Hillyard et al., 1992; Mintz et al., 1992; Luebke et al., 1993; Tareilus et al., 1993; Meder et al., 1997) but not in human brain. Therefore, it was the aim of the present study to investigate which kind of VDCCs are involved in K⁺- and veratridine-induced increase in [Ca²⁺]_i in human cerebral cortical synaptosomes. For this purpose, toxins that exhibit different selectivity patterns of VDCC blockade (for a recent review see Mori et al., 1996) were applied; in particular, the N-type channel blocker -conotoxin GVIA (-CTx GVIA; Kerr and Yoshikami, 1984), the P- and Q-type channel blocker -agatoxin IVA (-AgaTx IVA; Mintz et al., 1992) and the N-, P-, and Q-type channel blocker -conotoxin MVIIC (-CTx MVIIC; Hillyard et al., 1992). In addition, our study included Bay K 8644 and nifedipine, which selectively activate and block the L-type Ca²⁺ channel, respectively (Franckowiak et al., 1985; Nowycky et al., 1985; Takasu et al., 1987), mibefradil, which blocks all types of VDCCs mentioned so far plus R- and T-type VDCCs (Mishra and Hermsmeyer, 1994; Bezprozvanny and Tsien, 1995; Meder et al., 1997) and ifenprodil, which resembles mibefradil in its ability to block the L-, N-, P-, Q- and, potentially, the R-type VDCC (Biton et al., 1994; Church et al., 1994; Meder et al., 1997).

Using these pharmacological tools, it can be evaluated whether different VDCC types are activated in response to different depolarization methods such as potassium elevation or veratridine.

	Materials and Methods

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Synaptosomes were prepared as described earlier (Meder et al., 1997) from human cerebral cortical tissue (temporo-basolateral region) obtained from 24 patients (8 male and 16 female; age 11-55 years, 31.2 ± 2.8 years) who underwent neurosurgery for otherwise intractable epilepsy; antiepileptic medication was not interrupted for surgery. After resection the tissue was instantly immersed in cold (4°C) Krebs-Henseleit with the following composition: 118 mM NaCl, 4.8 mM KCl, 25 mM NaHCO₃, 1.2 mM KH₂PO₄, 1.3 mM CaCl₂, 1.2 mM MgSO₄, 11.1 mM D-glucose, 0.06 mM ascorbic acid, and 0.03 mM disodium EDTA (equilibrated with 95% O₂ and 5% CO₂). Synaptosomes were prepared within 1 h after resection. Patients were under antiepileptic medication (carbamazepine and valproinate) and were premedicated with flunitrazepam and atropine, and anesthetized with thiopental (for introduction), fentanyl, and enflurane or isoflurane (for maintenance). Pancuronium was used to achieve muscle relaxation. The cerebral cortical specimens had to be removed to yield access for amygdalahippocampectomy. The study was approved by the local ethics committee.

Briefly, cerebral cortex specimens were homogenized with a Potter-Elvehjem glass homogenizer (800 rpm, 6 strokes/2 min) in 40 volumes (w/v) of 0.32 M sucrose and the homogenate was centrifuged (10 min, 1000g at 4°C) to remove nuclei and debris. The supernatant was then centrifuged at 1200g for 10 min. The buffy layer of pelleted synaptosomes was resuspended by gentle agitation in Ca²⁺-free, physiological salt solution (PSS). The solution contained: 133 mM NaCl, 4.8 mM KCl, 10 mM HEPES, 1.2 mM Na₂HPO₄, 1.2 mM MgSO₄, and 10 mM D-glucose; the pH was adjusted to 7.4 with NaOH.

The synaptosomal suspension (2 ml; about 3 mg of protein/ml) was incubated with fura-2 acetoxymethyl ester (fura-2/AM; 5 µM) for 40 min at 37°C while gently shaking. Fura-2-loaded synaptosomes were centrifuged at 1300g and the pellet was washed once with Ca²⁺-free PSS and centrifuged again. The pellet was resuspended and stored on ice until use. Aliquots (200 µl) of the washed synaptosomal suspension containing 450 to 600 µg of protein were diluted with 1.8 ml PSS containing 1.3 mM CaCl₂, placed in a quartz cuvette at 37°C and preincubated for 6 min. The synaptosomes were kept in suspension by means of a magnetic stirrer. K⁺ and veratridine were added from the 360th second of incubation onward. Substances under investigation were present in the buffer from the beginning of the incubation onward until the end of the experiments. The measurements were made with a spectrofluorometer (Perkin-Elmer LS 50B; Perkin-Elmer Cetus Instruments, Eden Prairie, MN). The intrasynaptosomal free calcium concentration [Ca²⁺]_i was determined by calculating the ratios of the fluorescence at 510 nm induced by excitation at 340 and 380 nm; 10 values of fluorescence per second for each wavelength were recorded. Calibration was accomplished by lysis of the synaptosomes with Triton X-100 to obtain the maximum fluorescence ratio, followed by the addition of 7.5 mM ethyleneglycol-bis (-aminoethyl ether)-N,N,N',N'-tetraacetic acid (pH 8) to obtain the minimum ratio. For every experiment the autofluorescence of the synaptosomal suspension without fura-2 was measured and subtracted automatically from the total fluorescence of the loaded synaptosomes. For determination of extrasynaptosomal fura-2, Mn²⁺ (40 µM) was added to quench the extracellular fluorescence; this fluorescence of extracellular fura-2 amounted to about 10% of the total fluorescence and was stable throughout the experiments. After correction for extracellular dye, [Ca²⁺]_i was calculated according to Grynkiewicz et al. (1985). When the K⁺ concentration was not elevated and no veratridine was added to the incubation solution, spontaneous time-dependent increases in [Ca²⁺]_i occurred from the 360th until the 370th and 480th second of incubation, respectively; this spontaneous increase in [Ca²⁺]_i was routinely subtracted from the corresponding depolarization-evoked increase in [Ca²⁺]_i.

From each patient's tissue specimen one synaptosomal preparation was made. Results are given as means ± S.E.M. of n experiments. For comparison of mean values, Student's t test was used. In case of multiple comparisons, one-way or two-way ANOVA was applied, followed by Dunnett's post hoc tests. P < .05 was considered significant.

Fura-2/AM(1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2'-amino-5'-methylphenoxy) ethane-N,N,N',N'-tetraacetic acid pentaacetoxy methyl ester), veratridine free base, tetrodotoxin (TTX), nifedipine, and -CTx GVIA were purchased from Sigma (Deisenhofen, Germany), -AgaTx IVA from RBI (Natick, MA), and -CTx MVIIC from Alomone Labs (Jerusalem, Israel). Ifenprodil tartrate (Synthélabo, Paris, France), mibefradil dihydrochloride (Hoffmann-La Roche, Grenzach-Wyhlen, Germany), KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate; Kanebo, Osaka, Japan), and Bay K 8644 (1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)-phenyl] pyridine-3-carboxylic acid methyl ester; Bayer, Leverkusen, Germany) were kind gifts of the respective companies.

For stock solutions, fura-2/AM and veratridine were dissolved in dimethyl sulfoxide. The final dimethyl sulfoxide concentration did not exceed 0.1% (v/v) which had no effect in our experiments. All other compounds were dissolved in deionized water.

	Results

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Basal [Ca²⁺]_i, Depolarization-Evoked Increase in [Ca²⁺]_i, and Effects of TTX and Omission of Ca²⁺. Under control conditions, the basal [Ca²⁺]_i in cerebral cortical synaptosomes, measured after 360 s of incubation in PSS, was 207 ± 12 nM (n = 70). Omission of Ca²⁺ from the incubation medium decreased [Ca²⁺]_i by 85% (P < .001) after K⁺ depolarization and by 98% (P < .001) after veratridine depolarization (Fig. 1). It was not affected by the presence of the drugs at the concentrations investigated in this study (results not shown; for drugs and concentrations, see Figs. 1-3 and Table 1).

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Effects of TTX (1 µM), mibefradil (10 µM), ifenprodil (320 µM), or omission of Ca2+ ions on K+ (A; elevation of concentration by 30 mM)- or veratridine (B; 10 µM)-induced increase in [Ca2+]i in synaptosomes preloaded with fura-2/AM (5 µM). The effects on the evoked increase in [Ca2+]i are shown as percentages of the controls. Means + S.E.M. of 6 to 10 experiments; *P < .05, compared with the corresponding controls. Insets: K+ (A)- or veratridine (Ver; B)-induced increase in [Ca2+]i in synaptosomes preloaded with fura-2/AM (5 µM) and effects of TTX (1 µM) or omission of Ca2+ ions. Abscissa: time after onset of incubation of the synaptosomes in PSS containing 1.3 mM Ca2+ or Ca2+-free PSS. Excitation wavelengths were 340 and 380 nm and fluorescence emission was determined at 510 nm. Ca2+ was omitted from, or TTX was added to, the medium from the onset of incubation of the synaptosomes onward until the end of the experiment. K+ (elevation of concentration in the PSS by 30 mM) or veratridine (10 µM) was present from the 360th min of incubation until its end. The stimulated increases in [Ca2+]i shown in Figs. 1 to 3 and Table 1, were determined after 10 s of exposure to high K+ and 100 s of exposure to veratridine. Representative experiments shown.

Effects of Ca²⁺ Channel Blockers on K⁺-Induced Increase in [Ca²⁺]_i. The nonselective Ca²⁺ channel blockers mibefradil and ifenprodil and the more selectively acting drugs nifedipine, -CTx GVIA, and -CTx MVIIC were applied at concentrations which, according to the literature on rat and human brain or vascular tissue, should completely block the VDCCs at which the respective compounds act. Mibefradil (10 µM) and ifenprodil (320 µM) virtually abolished the K⁺-induced increase in [Ca²⁺]_i (Fig. 1A). Nifedipine (1 µM) failed to alter the K⁺-induced increase in [Ca²⁺]_i, whereas -CTx GVIA (0.1 µM) reduced it by about 20% (Fig. 2B). Because -AgaTx IVA was not completely characterized in human cortical synaptosomes, we provide a concentration response curve for the inhibition of K⁺-induced [Ca²⁺]_i increase. -AgaTx IVA inhibited K⁺-induced [Ca²⁺]_i increase concentration-dependently at concentrations higher than 1 nM (IC₅₀ 113 nM; Fig. 2A). Maximum inhibition was achieved at 0.2 µM, but the effects of 0.1 and 1 µM did not differ significantly. Hence, 0.2 µM -AgaTx IVA was used in our experiments to block P/Q-type VDCCs. Combined application of -CTx GVIA (0.1 µM) and -AgaTx IVA (0.2 µM) inhibited the K⁺-induced increase in [Ca²⁺]_i by about 85%; a similar degree of inhibition was induced by -CTx MVIIC (0.2 µM; by about 80%; Fig. 2B).

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Effects of Ca2+ channel blockers on K+ (A and B; elevation of concentration by 30 mM)- or veratridine (C; 10 µM)-induced increase in [Ca2+]i in synaptosomes preloaded with fura-2/AM (5 µM). The effects on the evoked increase in [Ca2+]i are shown as percentages of the controls. Means + S.E.M. of 4 to 11 experiments; *P < .05, compared with the corresponding controls. For further details concerning the time schedule of application of the stimulus and addition of the drugs, see legend to Fig. 1.

Effects of Ca²⁺ Channel Blockers on Veratridine-Induced Increase in [Ca²⁺]_i. Both mibefradil (10 µM) and ifenprodil (320 µM) inhibited the veratridine-induced increase in [Ca²⁺]_i by about 90% (Fig. 1B). In contrast to the lack of an effect of nifedipine (1 µM) on K⁺-induced increase in [Ca²⁺]_i (Fig. 2B), this drug caused an inhibition of the veratridine-evoked response by about 30% (Fig. 2C). A reduction of the response to veratridine also occurred after application of -CTx GVIA and -AgaTx IVA (by about 25 and 45%, respectively; effects of both toxins were partially additive (inhibition by 50%; Fig. 2C). When nifedipine was applied in addition to -CTx GVIA and -AgaTx IVA an inhibition by 78% was observed (Fig. 2C).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Effects of nifedipine on veratridine (10 µM)-induced increase in [Ca2+]i in synaptosomes preloaded with fura-2/AM (5 µM) and interaction with Bay K 8644 (3 µM). The effects on the evoked increase in [Ca2+]i are shown as percentages of the corresponding controls (i.e., in the absence and presence of Bay K 8644, respectively). Means ± S.E.M. of 6 to 19 experiments. For further details concerning the time schedule of application of the stimulus and addition of the drugs, see legend to Fig. 1. *P < .05 compared with corresponding controls without nifedipine. +P < .05 compared with corresponding experiments in the absence of Bay K 8644.

Effects of KB-R7943 on K⁺- and Veratridine-Induced Increase in [Ca²⁺]_i. In view of the possibility that the veratridine-induced increase in synaptosomal [Ca²⁺]_i might be partly due to the operation of a Na⁺/Ca²⁺ exchanger (Bouron and Reuter, 1996), we examined the influence of KB-R7943, an inhibitor of Na⁺/Ca²⁺ exchanger (NCX1; Iwamoto et al., 1996; Watano et al., 1996) on the depolarization-evoked increase in [Ca²⁺]_i. Whereas KB-R7943 up to 3 µM did not affect the K⁺-evoked increase in [Ca²⁺]_i, it inhibited the veratridine-evoked increase in [Ca²⁺]_i at a concentration of 3 µM by 20% (Table 1). Higher concentrations of KB-R7943 were not investigated because the compound itself would interfere with the fura fluorescence.

	Discussion

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In this study we attempted to identify VDCCs involved in depolarization-induced [Ca²⁺]_i increase in human cortical synaptosomes, which represent predominantly glutamatergic and -aminobutyric acid axon terminals. Human tissue from patients undergoing neurosurgery was histologically normal. It cannot be excluded, however, that the patients' disease, antiepileptic drugs, surgical manipulations, or anesthetics might have led to changes in synaptosomal function compared with that in tissue from healthy individuals, which is unavailable.

Basal [Ca²⁺]_i in human cerebral cortical synaptosomes was in the same range as we (Meder et al., 1997) and others (Tibbs et al., 1989; Duarte et al., 1991; Fontana and Blaustein, 1993) have reported on rat cerebral synaptosomes. We demonstrate here that: 1) the plateau of the K⁺-evoked increase in [Ca²⁺]_i was reached within 10 s, whereas it took 100 s to reach the plateau when veratridine was used for depolarization; and 2) TTX practically abolished the veratridine-induced increase in [Ca²⁺]_i, leaving that in response to high K⁺ unaffected. These findings indicate that in human cerebral cortical synaptosomes, high K⁺ in the extracellular fluid directly and rapidly depolarizes the synaptosomal membrane, whereas veratridine causes an indirect depolarization by opening voltage-dependent Na⁺ channels.

The increase in [Ca²⁺]_i in human synaptosomes induced by high K⁺ or by veratridine was due to Ca²⁺ ion influx, because it was not observed in the absence of Ca²⁺ in the incubation buffer. The evoked accumulation of Ca²⁺ in human synaptosomes occurred, at least predominantly, via VDCCs because mibefradil, which blocks all types of VDCCs (Bezprozvanny and Tsien, 1995), and ifenprodil, which blocks at least L-, N-, and P/Q-type channels (Church et al., 1994; Bath et al., 1996; Meder et al., 1997), virtually abolished the increase in [Ca²⁺]_i after K⁺ depolarization and reduced it by about 90% after veratridine depolarization. In rat cerebral synaptosomes, mibefradil and ifenprodil abolished not only the K⁺- but also the veratridine-induced increase in [Ca²⁺]_i. The small Ca²⁺ channel blocker-resistant fraction of the veratridine-induced increase of [Ca²⁺]_i in human synaptosomes may be caused by an intrasynaptosomal Ca²⁺ accumulation via the Na⁺/Ca²⁺ exchanger. The results discussed so far are compatible with the previous suggestion that intracellular Ca²⁺ stores do not substantially contribute to the generation of presynaptic Ca²⁺ signals (Okada et al., 1989; Mulkey and Zucker, 1991; Tareilus and Breer, 1992).

K⁺-induced increase in [Ca²⁺]_i in human cerebral synaptosomes was not changed by nifedipine, but was inhibited by about 20 and 65% by -CTx GVIA and -AgaTx IVA, respectively, whereas effects of both toxins were additive. These findings suggest that N- and P/Q- but not L-type VDCCs mediate Ca²⁺ influx after K⁺ (30 mM) depolarization. In agreement with this conclusion, Ca²⁺ accumulation was inhibited by about 80% when N- and P/Q-type channels were simultaneously blocked by -CTx MVIIC. This finding represents a substantial difference from rat cortical synaptosomes in which -CTx MVIIC at 0.2 µM, i.e., at the same concentration as in the present study, produced an inhibition of no more than 50%, whereas the inhibition amounted to 80% (as in the present study) when N- and P/Q-type channels were blocked by a combination of -CTx GVIA and -AgaTx IVA; it was suggested that in rat synaptosomes -CTx MVIIC at this concentration may not yet substantially block N- and P/Q-type but predominantly Q-type VDCCs (Meder et al., 1997), which apparently cannot be postulated for human cortical synaptosomes.

Veratridine-induced increase of [Ca²⁺]_i in human cerebral synaptosomes was also sensitive to blockade by -CTx GVIA and, to a greater extent, by -AgaTx IVA, suggesting that Ca²⁺ influx in response to this kind of depolarization also occurs via N- and P/Q-type VDCCs, respectively. Similar to K⁺ depolarization, the contribution of the P/Q-type VDCCs appears to be most important. However, involvement of L-type VDCCs suggested by the inhibition of Ca²⁺ influx by nifedipine was observed only after veratridine depolarization.

For closer evaluation of the role of L-type VDCCs in the veratridine-induced increase of [Ca²⁺]_i in human synaptosomes, the concentration dependence of the effect of nifedipine and its interaction with Bay K 8644 was determined. The IC₅₀ of nifedipine (4.9 nM) was in the same range as in human vascular tissue in which L-type VDCCs are involved in electromechanical coupling (4.7 nM; Godfraind et al., 1987). Bay K 8644 itself increased the veratridine-evoked response, suggesting that L-type VDCCs are not maximally activated after veratridine depolarization. In addition, Bay K 8644 potently counteracted the inhibitory effect of nifedipine, because in the presence of 3 µM Bay K 8644, the concentration-response curve for nifedipine was significantly shifted to the right (by a factor of 57). It is difficult to explain this differential contribution of the influx of Ca²⁺ ions to the overall increase in [Ca²⁺]_i evoked by high K⁺ on the one hand and veratridine on the other. It is conceivable that L-type VDCCs are activated only after veratridine (10 µM) depolarization because their threshold potential of 20 mV is not reached after K⁺ (30 mM) depolarization. N- and P/Q-type VDCCs may get involved after either depolarization method because they have lower threshold potentials (30 mV for N- and 40 mV for P/Q-type; Tareilus and Breer, 1992). However, the difference could also be related to different time intervals at which [Ca²⁺]_i was measured after the onset of depolarization (10 and 100 s in the case of high K⁺ and veratridine, respectively).

It is not clear why, after veratridine-induced depolarization, the effects of -CTx GVIA and -AgaTx IVA were only partially additive. In agreement with this, application of the N- plus P/Q-type channel blocker, -CTx MVIIC, resulted in an about 55% inhibition, which is also not significantly different from that of -AgaTx IVA alone. Coadministration of nifedipine with -AgaTx IVA and -CTx GVIA induced 78% inhibition, which seems to reflect the addition of 30% inhibition by nifedipine and 50% by -AgaTx IVA plus -CTx GVIA. Irrespective of whether depolarization was induced by high K⁺ or by veratridine, the inhibitory effect of mibefradil, which blocks all types of VDCCs identified so far (Bezprozvanny and Tsien, 1995), was more pronounced than that yielded by drug combination for blockade of N-, P/Q-, and in case of veratridine depolarization, L-type channels. Therefore, it is conceivable that R-type or other so far unknown VDCCs may play a role, albeit minor, in Ca²⁺ influx into human cortical synaptosomes.

KB-R7943, an inhibitor of the Na⁺/Ca²⁺ exchanger NCX1 (Iwamoto et al., 1996) inhibited veratridine- but not K⁺-induced [Ca²⁺]_i increase, suggesting that the small fraction of the veratridine-evoked increase in [Ca²⁺]_i which was resistant to VDCC blockade, might represent intrasynaptosomal Ca²⁺ accumulation via the Na⁺/Ca²⁺ exchanger NSX1. NSX1 is expressed at high density in synaptosomal membranes (Reuter and Porzig, 1995; Blaustein et al., 1996; Juhaszova et al., 1996).

Taken together, there are considerable differences in presynaptic VDCCs between human and rat brain cortex. It may be concluded that the K⁺- and veratridine-evoked increase of free cytosolic [Ca²⁺] in human cerebral cortical synaptosomes is caused mainly by Ca²⁺ influx via P/Q-type VDCCs and to a lesser degree via N-type channels. In case of veratridine-induced depolarization, L-type channels also substantially contribute to Ca²⁺ influx. A minor part of the veratridine-induced increase in [Ca²⁺]_i may be due to Ca²⁺ accumulation via Na⁺/Ca²⁺ exchanger.