The conditions of Ca2+ entry via L-type channels for induction of serotonin release from rabbit hippocampus

European Journal of Pharmacology, 198 (1991) 37-42 © 1991 Elsevier Science Publishers B.V. 0014-2999/91/$03.50 ADONIS 0014299991003728

37

EJP 51875

The conditions of Ca2+ entry via L-type channels for induction of serotonin release from rabbit hippocampus T h o m a s J. Feuerstein

1,2, Eric

N e u s c h w a n d e r 1, Willi S a u e r m a n n 2 and Amelie L u p p 1

t Neuropharmakologisches Labor der Neurologischen Unioersitiitsklinik, and 2 GiJdecke AG, Kiinische Forschung und Entwicklung, Mooswaldallee 1-9, D- 7800 Freiburg, F.R.G. Received 8 November 1990, revised MS received 5 February 1991, accepted 5 March 1991

rhe L-type voltage-sensitive calcium channel (VSCC) agonists of the dihydrcpyridine (DHP) type, Bay K 8644 and (+)-202-791, concentration dependently enhanced the K ÷ (26.2 mM)-induced 5-HT release from slices of rabbit hippocampus prelabelled with [3H]5-HT when the slices were treated with the monoamine oxidase (MAO) inhibitor, pargyline. The DHP agonists were ineffective on K + (26.2 mM)-induced release in the absence of pargyline. However, when ~-conotoxin GVIA pretreatment of the slices irreversibly blocked N-type VSCCs, (+)-202-791 markedly enhanced the release of 5-HT evoked by 26.2 mM K ÷. Thus, at this rather strong stimulus intensity either an increase in the (preferentially cytoplasmic) transmitter pool or blockade of N-type VSCCs was necessary in order to unmask agonist-activated L-type VSCCs. Reduction of the depolarization intensity from 26.2 to 17.2 mM K ÷, given for 8 min, strongly intensified the stimulatory effects of L-type VSCC agonists irrespective of the use of pargyline under these conditions. The concentration-response curve of (+)-202-791 was "competitively" shifted to the fight by the enantiomer, ( - )-202-791, with a pA 2 value of 8.6. In conclusion, N- and L-type VSCCs seem to differ in their relation to the cellular machinery for 5-HT release, the latter getting markedly operative when a weak and sustained depolarization is applied or when N-type VSCCs are blocked or when the cytoplasmic transmitter pool is expanded by inhibition of MAO. 5-HT release; Hippocampus; Voltage-sensitive Ca 2+ channels; Dihydropyridine ligands; t~-Conotoxin GVIA

1. Introduction

Despite the presence of a high density of binding sites for L-type voltage-sensitive calcium channel (VSCC) ligands in brain tissue (e.g. Dooley et al., 1987b; Feuerstein et al., 1990) the role of these channels on nerve terminals is still unclear. Middlemiss and Spedding (1985) postulated different subtypes of L-VSCCs to explain the facilitatory effects of the dihydropyridine (DHP) agonist, Bay K 8644, on serotonin (5-HT) release from potassium-stimulated rat cortex slices. The potassium-induced release of endogenous dopamine from rat striatal slices was modulated to a relative small extent by D H P drugs which were totally ineffective when electrical field stimulation was applied to evoke release (Herdon and Nahorski, 1989). This lack of effect of L-VSCC ligands, in contrast to N-type VSCC antagonists, on electrically evoked release of neurotransmitters has been confirmed by several authors (e.g.

Correspondence to: T.J. Feuerstein, GiSdecke AG, Mooswaldallee 1-9, D-7800 Freiburg, F.R.G.

Dooley et al., 1988; Feuerstein et al., 1990). In the present study, we investigated whether the extent of depolarization and other conditions, such as inhibition of the enzyme, monoamine oxidase (MAO), or blockade of N-type VSCCs, were critical to reveal strong effects of L-type VSCC ligands on 5-HT release, which may be not only statistically, but (patho)physiologically significant. Some of the present results were reported at the 19th annual meeting of the Society for Neuroscience (Phoenix, 1989).

2. Materials and methods

Rabbit hippocampal slices with approximate dimensions 0.3 × 2.5 × 3.5 mm, wet weight 4-6 mg, were prepared and incubated in 4 ml medium containing 0.1/tM [ 3H]5-HT (hydroxytryptamine, 5-[1,2- 3H(N)]creatinine sulfate, 28.3 Ci/mmol, NEN, Dreieich, FRG) at 37°C for 50 min in the absence or presence of t~-conotoxin GVIA 1 /tM (Peninsula Laboratories, St. Helens, UK) or the MAO inhibitor, pargyline HCI 10 #M (Deutsche

3S

Abbt~tt, lngelheim, FRG), as indicated. When pargyline x~as added, it was also present during the next superfusion experiment. After incubation the slices were rinsed, transferred to glass superfusion chambers, and superfused with prevearmed medium saturated with 5% CO295% O,, pH 7.4, at a rate of 1 ml/min. After 75 min of continuous washing the superfusate was collected as 5-min samples for tritium determination by liquid scintillation counting. The composition of the incubation and superfusion medium was (mM): NaCI 118, KCI 4.8, CaCI, 1.3 or 2.6, as indicated, MgSO4 1.2, NaHCO 3 25, KH_,PO~ 1.2, glucose 11, ascorbic acid 0.57, disodium EDTA 0.03, During superfusion the slices were stimulated twice after 90 and 145 rain (S~, S,) by raising the K + concentration to substitute isosmotically for Na +. K + depolarization was applied either for 4 rain with 26.2 mM K + (instead of 6 mM K +) or for 8 rain with 17.2 mmol K ÷ (instead of 6 mM K +). Bay K 8644 (methyl-l,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoro-methylphenyl)-p)~dine-5-carboxylate; Bayer, Leverkusen, FRG), ( + )- and ( - )-202-791 (isopropyl 4-(2,1,3-benzoxadiazol-4-yl)-I A-dihydro-2,6-dimethyl-5-nitro-3-pyridinecarboxylate, Sandoz, Basel, Switzerland). Fluvoxamine maleate (Duphar, Weesp, The Netherlands), and metitepin maleate (Hoffmann-La Roche, Basel, Switzerland) were added to the medium from 15 rain before $2 onwards or were present in the medium throughout the superfusion, as indicated. None of these substances, at the concentrations used, affected the basal tritium outflow, quantified as ratio of the fractional 3H-efflux (see below) during the 5-rain collection period before S2 and S~. Controls without drugs added before S2 were always run in parallel to drug experiments. At the end, the slices were dissolved in Soluene-350 for tritium determination. The 3H-efflux of a 5-min collection period was expressed as percent of tissue tritium at the beginning of this pcriod (fractional rate of 3 H-effluxj.\ Stimulationevoked overflow (S~ and S2 value) was calculated by subtraction of the basal from the total fractional rate of 3H-efflux during each 4-min stimulation and the subsequent 21 min (at 26.2 mM K ÷) or during each 8-rain stimulation and the following 17 min (at 17.2 mM K+), if not stated otherwise. Basal efflux was assumed to decline linearly from the 5-rain period before to that 25 min after the start of stimulation. The K+-induced 3H-overflow at the first stimulation was expressed as S 1 value per min of depolarization, if not stated otherwise in order to allow for the different duration (i.e. 4 or 8 rnin) of K ÷ depolarization. Thus, the whole S~ values were divided by either 4 or 8. The effects of drugs given before the second stimulation were evaluated by calculating the ratio $2/S~ of the 3H-overflow evoked by the two stimulation periods. The evoked tritium overflow is assumed to represent the 'release of 5-HT'; therefore

this expression is used below for stimulation-evoked tritium efflux. [3H]5-HT was separated from its metabolite, [ 3 H ] 5 hydroxyindole acetic acid, in some experiments as described recently (Feuerstein et al., 1986). The slices were depolarized by 17.2 mM K + twice for 8 min: the first stimulation was carried out in the absence of L-type VSCC agonists, whereas Bay K 8644 (1 #M) or ( + ) 202-791 (1 #M) was added to the superfusion fluid from 45 min (instead of 15 min) before the second stimulation until the end of superfusion, since a possible process of diffusion to the intraterminal enzyme, MAO, should not be limited by a too short time interval. The 5-min samples of the superfusate before and during the first 5 rain of each stimulation and the two following samples were analyzed for unmetabolized [3H]5-HT and the deaminated metabolite. The recovery from standard samples of [3H]5-HT was 88.5 + 2.2%, n = 6. All results are expressed as means + S.E.M., if not indicated otherwise. The law of error propagation was considered when mean values are given as percentages of corresponding control means. The existence of differences between the means of treatment or corresponding control groups was tested with a one-way analysis of variance, the preconditions of which were considered. Subsequently, Scheffr's test was used to determine the significance of differences between the groups. The shift to the right due to (-)-202-791 of the concentration-response curve for (+)-202-791 was estimated by analysis of covariance, with concentration as the covariate based on the assumption of equal slopes of the concentrationresponse curves as verified in a pretest.

3. Results

3.1. Interactions of DHP agonists with pargyline In the presence, but not in the absence, of pargyline (10 #M) throughout the experiment the DHP agonist, Bay K 8644, at 1 # M enhanced the K + (26.2 mM)-induced 5-HT release from [3H]5-HT-prelabelled slices (fig. 1), when the Ca a+ concentration of the superfusion medium was 1.3 mM. The increase in 5-HT release in the presence of pargyline amounted to 139.49 + 10.05% when expressed as percentage of the corresponding controls, as compared to the insignificant change of a release of 115.81 + 7.42% following Bay K 8644 in the absence of pargyline. The enhancement by Bay K 8644 in the presence of pargyline was slightly increased to 156.90 + 13.53% of the corresponding control mean by doubling the concentration of Ca 2÷ in the medium from 1.3 to 2.6 mM. The effects of Bay K 8644 became concentration-dependent at 0.1 #M (fig. 1). Therefore, all subsequent experiments were performed at 2.6 mM external Ca 2÷. As seen with Bay K 8644 (1 #M) at 1.3

39

mM Ca 2+ throughout superfusion, another D H P agonist, (+)-202-791, was ineffective with a stimulus intensity of 26.2 mM K ÷ at 2.6 m M Ca 2÷ throughout superfusion in the absence of an inhibitor of MAO, but significantly increased the K ÷ (26.2 mM)-evoked 5-HT release in the presence of pargyline (table 1).

3.2. Effects of reduction of the stimulus intensity to 17.2 mMK + The time course and the facilitation of release by Bay K 8644 (1 /~M) at stimulation intensities of 26.2 and 17.2/tM K ÷ are shown in fig. 2. The comparison of S: and $2 values as $2/S~ ratios does not take into account the different 'absolute' amounts of release (see S~ values in the legends of figures). Additional experiments were performed with a stimulation intensity of 26.2 mM K ÷, applied for 1.8 min only, instead of 4 min to demonstrate that the different size of the D H P agonist-in-

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TABLE 1 Effects of ( + )-202-791 on K ÷ (26.2 mM)-induced 5-HT release from hippocampal slices of the rabbit. Experiments were performed at 2.6 mM Ca 2+ throughout, without or with ~-conotoxin GVIA pretreatment or with pargyline present during the experiment. Significant differences from the corresponding mean $2/S 1 ratio of controls (no drug given before $2): "~P < 0.05, b p < 0.01, c p < 0.001; the number of values per mean was >/7. S 1 values: see Results.

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duced enhancement at 26.2 and 17.2 mM K ÷ of 5-HT release, expressed as the 'relative' $2/$1 ratio (e.g. figs. 1 and 3), was not due to the decreased release as such at the lower K ÷ stimulation. Thus, S] values were obtained which were even lower than those with a stimulation intensity of 17.2 mM K ÷ (see legend of fig. 2). The Bay K 8644 (1/tM)-induced increase was smaller at 26.2 m M K ÷ applied for 4 or for 1.8 min only than at 17.2 mM K ÷ applied for 8 min (compare the areas representing the S] and $2 values in fig. 2). Therefore, stimulus intensity but not the absolute amount of release was critical for the amount of facilitation of release by the D H P agonist. Bay K 8644 and (+)-202-791 enhanced the $2/S] ratio much more at 17.2 mM K ÷ (figs. 1, 3), irrespective of the presence or the absence of pargyline (not shown). As evident from figs. 1 and 3, the S.E.M.s of the $2/S] ratios increased markedly with increasing mean values.

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3. 3. A gonist / antagonist interaction experiments Fig. 1. Effects of the D H P agonist, Bay K 8644, on K J-induced 5-HT release from hippoeampal slices of the rabbit. Stimulations (S], $2) were performed by raising the K + concentration to 26.2 mM (obliquely hatched, dotted aad horizontally hatched columns) or to 17.2 mM (open columns. Bay K 86,14 enhanced the K + (26.2 mM)-induced release only in the presence (dotted and horizontal hatched columns), but not in the absence (oblique hatched columns) of pargyline (10 /~M). The stimulatory effect of Bay K 8644 was slightly enhanced by elevating the Ca 2+ concentration from 1.3 (dotted columns) to 2.6 mM (horizontal hatched columns). The relative effects of Bay K 8644 were markedly enhanced when only 17.2 mM K + was used instead of 26.2 mM to induce release at 2.6 mM Ca 2 + and in presence of pargyline (open columns). The mean S: values per rain induced by 26.2 mM K + were 0.863:t:0.038%, n = 6 0 (absence of pargyline, 1.3 mM Ca 2+) and 0.647+0.027%, n = 8 4 (presence of pargyline, 1.3 mM Ca2+; similar S i values per rain at 2.6 mM Ca2÷; not shown). K +, 17.2 mM, induced a mean S I value per min of 0.104+0.003%, n =177 (presence of pargyline). Asterisks indicate significant differences from the corresponding mean $2/S] ratio of controls (no drug given before $2): * P < 0.05, * * P < 0.01. The number of values per mean was >I 6.

The concentration-response curve of (+)-202-791, obtained in the absence of pargyline with the low stimulus of 17.2 m M K ÷, was shifted to the right in a parallel manner in the presence of the enantiomer ( - ) 202-791 (fig. 3). Since the experimental points of the concentration-response relationships presumably reflected the linear segment of sigmoid curves, a linear regression analysis was used to fit the points of the two concentration-response curves. As the equivalence of the slopes of the two regression lines (legend of fig. 3) was evident, an analysis of covariance with equal slopes could be applied, which yielded a shift of 1.607 log units with a 95% confidence interval of (0.646, 3.110), estimated according to the method of Fieller (1940). Thus, the potency of the antagonist (-)-202-791 in the present model was roughly assessed by a pA 2 value of 8.6

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Fig. 2. Outflow and stimulation-induced overflow of tritium (5-HT release) from rabbit hippocampal slices preincubated with [3H]5-HT. After labelling, the slices were superfused in the presence of pargyline 10/~M at 2.6 mM Ca 2 " and stimulated ~wice (S l, $2) by elevating the K ÷ concentration to 26.2 mM for 4 min (upper curve), or to 17.2 mM for 8 min (middle curve), or to 26.2 mM for 1.8 mia (lower curve). Bay K 8644 (1 # M ) was added 15 min before Sz onwards. The means+ S.E.M. of fractional rates are given. The S 1 and S2 values are visualized as areas under the stimulation-evoked peaks of tritium overflow: the areas representing the effects of Bay K 8644 are more densely dotted, as compared to the S2 peaks of controls. The corresponding $ 2 / S l means had the following values: S z / S I = 1 . 1 9 4 + 0.083, n = 7; $ 2 / S i of controls = 0.761 +0.039, n = 1 0 (P < 0.01: upper curve; compare fig. 1); $ 2 / S ] = 1 . 9 3 1 ± 0 . 2 4 5 , n = 7 : $ 2 / S ] of controls = 0.984±0.040, n = 6 (P < 0.01, middle curve, compare fig. 1 }, and S=/S ! = 1.252 +0.088, n = 9: S2/St of controls = 0.799 ± 0.078, n = 6 (P < 0.01: lower curve).

Fig. 3. Effects of the D H P agonist, ( + )-202-791, on K +-induced 5-HT release from hippocampal slices of the rabbit, Stimulations (S l, $2) were performed by raising the K + concentration to 17.2 mM. Open symbols represent experiments without and filled symbols represent experiments xvith (-)-202-791 (0.1 # M ) throughout superfusion. K +, 17.2 mM, ind~:ced a mean S 1 value per min of 1.61 +0.004%, n = 209 (no drug given throughout), and of 0.158+0.008%, n = 30 (presence of ( - )-202-791 0.1 ~tM). The slopes of the two regression lines were 0.382 +0.128 (absence of the antagonist) and 0.357 +0.127 (presence of the antagonist). All values greater than 1.4 were significantly different from the corresponding control mean (P at least < 0.05). The number of values per mean was >t 6.

which were incubated in to-conotoxin GVIA (1 #M) in addition to [3H]5-HT. to-Conotoxin GVIA was used to block N-type VSCCs. Under these conditions, (+)-202791 strongly enhanced the release of 5-HT as compared to the absence of to-conotoxin GVIA during incubation (table 1). ~0-Conotoxin GVIA (0.1 #M), added 15 min before the second stimulation onward, was ineffective to modulate 5-HT release when the slices had been preincubated for 50 min with u-conotoxin GVIA (1 ~M; not shown). Without such a pretreatment, t0-conotoxin GVIA (0.1 #M) diminished the evoked release by about 40% when given 15 min before $2 at the stimulation intensity of 26.2 mM K + (the mean S2/St ratio of controls, 0.877 _+ 0.045, n = 11, was significantly (P < 0.01) decreased to 0.510 + 0.059, n = 6). An even greater reduction (about 50%) of the K + (26.2 mM)-evoked release by to-conotoxin GVIA at the higher concentration of 1 /tM was seen when the St values per min of depolarization without pretreatment (0.863 _+ 0.038% of tissue tritium, n = 60) were compared with those following pretreatment with to-conotoxin GVIA (0.415 + 0.019% of tissue tritium, n = 46; P < 0.01).

3.5. No interactions between DHP agonists and blockade of the 5-HT autoreceptor or of the 5-HT uptake pump The stimulatory effects of the L-type VSCC agonists were affected by neither the 5-HT autoreceptor antagonist, metitepin (0.1 #M), nor the 5-HT uptake inhibitor, fluvoxamine (1 #M), present throughout su-

perfusion (not shown). These 5-HT-related drugs did not enhance the S 1 values at the weak stimulus intensity of 17.2 mM K + that was used. The respective S l values per min of 0,154 _+ 0.009% n = 26 (metitepin) and of 0.140 +_0.009%, n = 8 (fluvoxamine) were similar to the S~ values per rain of the controls, i.e. 0.161_ 0.004%, n -- 209.

3.6. Separation of [3H]5-HT from [3H]5-hydroxyindole acetic acid In order to exclude direct interactions of L-type VSCC agonists with the enzyme, MAO, which deaminates 5-HT, additional separation experiments were performed. (+)-202-791 (1 #M) did not change the fraction of [3H]5-HT in the basal tritium outflow or in the K+-evoked 3H-overflow during and after stimulation. The percentage of [3H]5-HT in the basal outflow in the absence of ( + )-202-791 was 19.6 _+0.9%, n = 20, as compared to 18.9 _+ 1.2%, n = 8, in the presence of (+)-202-791. The percentages of [3H]5-HT in the superfusate samples were also similar during and after stimulation (27.2 _+ 1.2%, n = 15, absence of (+)-202-791 vs. 27.1 _+ 1.4%, n = 9, presence of (+)-202-791, samples obtained during the first 5 min of stimulation). The samples obtained in the intervals 5-10 and 10-15 rain after the onset of stimulation also yielded very similar fractions of [3HI5-HT (28.7 +_ 0.9%, n = 15, absence of (+)-202-791, paralleled 29.3 _+ 2.0%, n = 9, presence of ( + )-202-791 (first 5-rain interval), and 23.0 +_ 0.5%, n = 15, absence of (+)-202-791 paralleled 24.5 _+ 1.8%, n = 9, presence of (+)-202-791 (second 5 rain interval)). The respective fractions of [3H]5-HT were also not affected by Bay K 8644 (1 #M; not shown). The presence of fluvoxamine (1 /_tM) throughout superfusion raised the percentage of [3H]5-HT in both tritium outflow and the evoked 3H-overflow. Again, (+)-202-791 remained ineffective to change the percentage of [3H]5HT also in the presence of fluvoxamine (not shown).

4. Discussion

Our first intention was to test if inhibition of MAO was critical for the effects of L-type VSCC agonists. Using the stimulus intensity of 26.2 mM K + and pargyline throughout tbe experiment, the results of Middlemiss and Speddin3 (1985) with Bay K 8644 were reproduced and confirmed with another L-type VSCC agonist, (+)-202-791. However, under these conditions of stimulation, the L-type VSCC agonists, which did not interact directl~ with MAO acti¢lty, were ineffective if this enzyme was not inhibited. Thus, at the rather strong stimulus intensity of 26.2 mM K ÷ an increase in the (preferentially cytoplasmic) transmitter pool of 5-HT seems necessary in order to make agonist-activated L-

type VSCCs visible. It is well known that a rather strong stimulus intensity of 26.2 mM or more K ÷ mainly involves N-type VSCCs for Ca 2+ entry, as indicated by the predominant effects of the N-type VSCC antagonist, o~-conotoxin GVIA, under such conditions (Hirning et al., 1988; Herdon and Nahorski, 1989; present results with a 50% reduction of release by t~-conotoxin GVIA). Thus, it seems that an additional significant contribution of L-type VSCCs on Ca 2+ entry and resulting transmitter release requires an abundant cytoplasmic transmitter pool at higher stimulus intensities if N-type VSCCs are also operative, at least in the case of 5-HT. In order to test if the reduction of the intenisty of depolarization unmasked the role of activated L-type VSCCs by diminishing the predominance of N-type VSCCs, only 17.2 mM K +, applied for 8 min, was used to evoke 5-HT release. Indeed, the relative effects of the L-type VSCC agonists were much stronger at this low stimulus intensity. The durafon of the weak K ÷ depolarization also seems to be critical for the effects presented, since depolarization conditions of 15 mM K +, applied for 3 s only in order to investigate the fast-phase entry of C a 2+ only, revealed a rather small Bay K 8644-induced increase in the release of endogenous dopamine (about 50%; Woodward and Leslie, 1986) as compared to our approach with an increase in release up to 150% (fig. 3). However, the (single) concentration of Bay K 8644 of I nM used by these auhtors was much lower than the concentrations of L-type VSCC agonists used in the present experiments, a condition which may reflect differences between the neurotransmitters investigated and between the experimental conditions. N-type VSCCs still markedly contributed to the mediation of 5-HT release induced by 17.2 mM K +, as was evident from the 40% inhibition of release by o~-conotoxin GVIA (0.1 #M). The involvement of typical L-type VSCCs with discrete binding sites for different classes of agents was confirmed by the use of an antagonist. The effect of the DHP agonist, ( + )-202-791, was blocked competitively by the DHP antagonist, ( - ) 202-791. The potency of (-)-202-791 in our model was comparable to that on smooth muscle as reported in the literature for this antagonist (Hof et al., 1985). The enhanced stimulatory effect of (+)-202-791 in t0-conotoxin GVIA-pretreated slices indirectly reflects the extent of the contribution of L-type VSCC~, as compared to N-type VSCCs, on 5-HT release at a stimulus intensity of 26.2 mM K ÷. In addition, it supports the idea of a somewhat specific action of o~-conotoxin GVIA on N-type channels despite the rather high concentration of 1 #M, since the effects of the L-type agonist (+)-202-791 were preserved. These findings seem to contradict some electrophysiological studies in which o~-conotoxin GVIA was reported to block equipotently both N- and L-type VSCCs (e.g. Mc-

42 Cleskey et al., 1987), In line with our evidence a recent electrophysiological report stated that neuronal L-type channels are insensitive to ~-conotoxin G V l A ( P l u m m e r et al., 1989), The completeness of the blockade, i.e. the quasi-irreversible action of ~-conotoxin G V I A (Dooley et a L 1987a) on N-type VSCCs, by p r e t r e a t m e n t of the slices vAth this toxin, was shown by the lack of effect of ~-conotoxin G V I A (0.1 # M ) when a d d e d additionally before, the second stimulation. In contrast to the results of H c r d o n a n d N a h o r s k i (1989) we could not find any evidence for an endogenous ligand activating the L-type VSCCs in our model, since the antagonist (--)-202-791 did not depress the release of 5-HT. This finding corresponds to reports of Starke et al. (1984) and of other authors who showed a relative resistance of the release of neurotransmitters on L-type VSCC antagonists. At first glance, this result makes a (patho)physiological significance of L-type VSCCs subserving transmitter release rather unlikely. Beneficial effects of L-type VSCC antagonists, however, have been shown in not only experimental but clinical ischemic brain lesions (Gelmers et al., 1988). With the weak and long-lasting depolarization applied we tried to reflect appropriately such a pathophysiological, i.e., ischemic condition. In the p e n u m b r a region of an infarction a weak and sustained rather t h a n a sudden a n d phasic depolarization of neurones a n d of nerve endings may take place, since the lack of energy no longer maintains the resting potential of the neurones. Despite the lack of effect of L-type V S C C antagonists when given alone, our approach, with a weak and long-lasting depolarization which very clearly u n m a s k s L-type VSCCs involved in the process of transmitter release, could be a promising way for further studies to explore Ca :+ entry into nerve terminals, which, in addition to entry into neuronal cell bodies, m a y play a harmful role in ischemic neuronal damage.

Acknowledgements The helpful discussions with Dr. D. Dooley are gratefully acknowledged. We thank the various pharmaceutical firms for donating drugs.

References Dooley, D.J., A. Lupp and G. Hertting, 1987a, Inhibition of central neurotransmitter release by to-conotoxin GVIA, a peptide modulator of the N-type voltage-sensitive calcium channel, NaunynSchmiedeb. Arch. Pharmacol. 336, 467. Dooley, D.J., A. Lupp, G. Hertting and H. Osswald, 1988, t0-Conotoxin GVIA and pharmacological modulation of hippocampal noradrenaline release, European J. Pharmacol. 148, 261. Dooley, D.J., H. M~ihlmann, O. Brenner and H. Osswald, 1987b, Characterization of the dihydropyridine binding sites of rat neocortical synaptosomes and microvessels, J. Neurochem. 49, 900. Feuerstein, T.J., D.J. Dooley and W. Seeger, 1990, Inhibition of norepinephrine and acetylcholine release from human neocortex by w-conotoxin GVIA, J. Pharmacol. Exp. Ther. 252, 778. Feuerstein, T.J., G. Hertting, A. Lupp and B. Neuf~ng, 1986, False labelling of dopaminergic terminals in the rabbit caudate nucleus: uptake and release of [3H]-5-hydroxytryptamine, Br. J. Pharmacol. 88, 677. Fieller, E.C., 1940, The biological standardization of insulin, J.R. Statist. Soc. (Suppl.) 7, 1. Gelmers, H.J., K. Gorter, C.J. De Weert and H.J.A. Wiezer, 1988, A controlled trial of nimodipine in acute ischemic stroke, N. Engi. J. Med. 318, 203. Herdon, H. and S,R. Nahorski, 1989, Investigations of the roles of dihydropyridine and w-conotoxin-sensitive calcium channels in mediating depolarization-evoked endogenous dopamine release from striatal slices, Naunyn-Schmiedeb. Arch. Pharmacol. 340, 36. Hirning, L.D., A.P. Fox, E.W. McCleskey, B.M. Olivera, S.A. Thayer, R.J. Miller and R.W. Tsien, 1988, Dominant role of N-type Ca 2+ channels in evoked release of norepinephrine from sympathetic neurons, Science 239, 57. Hof, R.P., U.T. Rtiegg, A. Hof and A. Vogel, 1985, Stereeselectivity at the calcium channel: opposite action of the enantiomers of a 1,4-dihydropyridine, J. Cardiovasc. Pharmacol. 7, 689. McCleskey, E.W., A.P. Fox, D.H. Feldman, L.J. Cruz, B.M. Olivera, R.W. Tsien and D. Yoshikami, 1987, w-Conotoxin: direct and persistent blockade of specific types of calcium channels in neurons but not muscle, Proc. Natl. Acad. Sci. U.S.A. 64, 4327. Middlemiss, D.N. and M. Spedding, 1985, A functional correlate for the dihydropyridine binding site in rat brain, Nature 314, 94. Plummer, M.R., D.E. Logothetis and P. Hess, 1989, Elementary properties and pharmacological sensitivies of calcium channels in mammalian peripheral neurons, Neuron 2, 1453. Starke, K., L. Spiith and T. Wichmann, 1984, Effects of verapamil, diltiazem and ryosidine on the release of dopamine and acetylcholine in rabbit caudate nucleus slices, Naunyn-Schmiedeb. Arch. Pharmacol. 325, 124. Woodward, J.J. and S.W. Leslie, 1986, Bay K 8644 stimulation of calcium entry and endogenous dopamine release in rat striatal synaptosomes antagonized by nimodipine, Brain Res. 370, 397.