Effect
of Ca 2+ Antagonists
on High-K+
Evoked
Increase
in [Ca2±1iin Rat Cerebral Synaptosomes and Tsutomu Biological
Research
Hippocampal
Kobayashi,
Laboratory, Received
Tanabe November
Rikako Seiyaku
Neurons
Yamauchi Co., Ltd.,
22, 1991
and Sakae Murata
2-2-50
Accepted
Kawagishi,
December
Toda,
Saitama
335, Japan
27, 1991
ABSTRACT-By fura-2 fluorometry, we investigated the direct effects of Ca2+ antago nists including a new benzothiazepine, clentiazem, on the high-K+-evoked increase in the concentration of cytosolic free Ca2+ ([Ca2+]i) in rat cerebral synaptosomes and cultured hippocampal neurons. In both preparations, metal ions inhibited the high-K+ induced increase in [Ca2+]i, in the following order: La 3+ > Cd2+ >> Ni2 Although flunarizine and nicardipine inhibited the K+-induced increase in [Ca2+]i in synapto somes, other Ca2+ antagonists, including clentiazem and nitrendipine, had little effect at 10,uM. In hippocampal neurons, clentiazem inhibited the K+-induced increase in [Ca2+]; at 10,uM, as did flunarizine and nicardipine. However, nifedipine and nitren dipine had little effect in either cultured neurons or in synaptosomes.
Calcium antagonists may be useful in the treatment of many central nervous system dis orders, including brain ischemia and epilepsy (1). These agents are generally regarded as vasodilators, because they antagonize the ac tion of Ca 2+ in vascular smooth muscle cells, but it is unknown whether their effects on the central nervous system are due to increased cerebral circulation or to direct inhibition of neuronal Ca 2+ influx. The direct action of Ca2+ antagonists on neurons is not clear, because neurons have at least three types of voltage sensitive Ca2+ channels (VSCC), and there is evidence that they differ from those in the cardiovascular system (2). The effects of Ca2+ antagonists on neuronal VSCC have not been studied in de tail. We studied the effects of various metal ions and Ca 2+ antagonists, including a new ben zothiazepine, clentiazem, and its metabolite
MB-1 (a major metabolite found in the rat brain), on the high-K+-induced increase in the concentration of cytosolic free Ca2+ ([Ca2+1i) in rat synaptosomes and cultured hippocampal neurons. The effects of some Ca2+ antagonists on Ca 2+ influx into synaptosomes, which is re lated to transmitter release, have been studied with 45Ca2+ (3-5). However, in the present study we used the Ca 2+ indicator fura-2, which enabled us to monitor real-time changes in [Ca2+]i. The [Ca2+]i in hippocampal neurons is of interest because of the relationship between the vulnerability of neurons to ischemia and excess uptake of Ca2+ (6). However, little is known about the effects of Ca2+ antagonists on the high-K+ induced increase in [Ca2+1i in hippocampal neurons.
MATERIALS AND METHODS Materials Clentiazem (TA-3090, (+)-(2S,3S)-3-acetoxy 8-chloro-5-(2-(dimethylamino)ethyl)-2,3-dihydro 2-(4-methoxyphenyl)-1,5-benzothiazepine-4-(5 H)-one maleate), MB-1 (the desacetylated form of clentiazem), and other Ca2+ antago nists were synthesized at the Organic Chemis try Research Laboratory of our company (Tanabe Seiyaku Co., Ltd.). Fura-2 acetoxy methyl ester (fura-2 AM) was purchased from Dojin Chemicals (Tokyo, Japan). Other re agents were obtained from commercial sources. Measurement of intrasynaptosomal free Ca-2+ Synaptosomes were prepared from cerebral cortices of male Sprague-Dawley rats (200 300 g) by a modified version of the method of Nachshen and Sanchez-Armass (7). Brain samples were homogenized in ice-cold 0.32 M sucrose, with 5 mM HEPES (pH 7.4) and 0.1 mM EDTA. The homogenate was centrifuged at 1000 X g for 10 min. The supernatant was then centrifuged at 12000 X g for 20 min, and the resulting supernatant was decanted. The pellet was resuspended in the medium de scribed above and then layered onto a gra dient consisting of 7.5% and 17% Ficoll-70 (Pharmacia) in the sucrose medium described above. The gradient was centrifuged at 63000 X g for 60 min. The synaptosomes, which banded at the 7.5%/17% interface, were re moved; diluted with 10 volumes of ice-cold sodium solution (composition: 145 mM NaCI, 5 mM KCI, 3 mM MgC12, 10 mM glucose, 10 mM HEPES, pH adjusted to 7.4 with Tris); and then centrifuged. The pellet was resus pended in sodium solution as descrived above, containing 0.1 mM CaC12, and incubated with fura-2 AM (3 u M) at 30°C for 30 min. The synaptosomes were washed twice with fresh sodium solution, without calcium, by centri fugation. The synaptosomes suspended in sodium solution, containing 1 mM CaC12, were incu bated in the presence or absence (control) of
Ca 2+ antagonists or metal ions for 5 min at 30°C and put in the cuvette of a fluorometer which was designed to measure fura-2-derived fluorescence (CAF-100 Japan Spectroscopic, Tokyo, Japan). The magnetically stirred sus pension was illuminated alternately (at 48 Hz) with two excitation wavelengths (340 nm and 360 nm). Light at 500 nm (F340, F360)emitted from the synaptosomes and the ratio (R340/360) were recorded. Depolarization was evoked by the addition of KCI solution to the sample sus pensions. An increase in R340/360after de polarization indicated that [Ca2+]i increased. In the sample suspension, some fluorescence came from high doses of some drugs or from extravesicular fura-2 which had leaked out of the synaptosomes. These contaminants were constant during depolarization. Therefore, if we use the increase in R340/360(which is equal to F340/constant), instead of the increase in R340/380as an index of the relative increase in [Ca2+]i, the effect of these contaminants will cancel out. The effects of the various drugs were esti mated by comparing the increase in R340/360 ratios with that measured in the controls. At first, we tried to calculate [Ca21]i. In practice, however, the calculation was impossible, be cause of the significant scatter (8) and the con tribution of autofluorescence which could not be estimated. All experiments were performed at 30°C. Microfluorometry of fura-2-loaded neurons Hippocampal neurons were isolated from cerebra of 18 19 day-old embryonic Sprague Dawley rats (9), and they were cultured for 4 7 days on monolayers of hippocampal glial cells precultured in specially designed dishes composed of a glass coverslip and a silicon rubber wall (10). The culture medium was re moved, and the neurons were incubated for 1 hr at 37°C in normal external solution (NES, composition, 145 mM NaCI, 5 mM KCI, 2 mM CaC12, 1 MM MgC12, 10 mM glucose, 10 mM HEPES, pH adjusted to 7.4 with Tris) con taining 2 ,uM fura-2 AM. After the loading-in cubation, the cells were washed with NES and
the dishes were mounted on an inverted microscope (TMD, Nikon, Japan) equipped with a specially designed fluorometer (CAM 200, Japan Spectroscopic, Japan). Neurons were illuminated alternately (at 10011z) by 340 nm and 380 nm light, and the light emitted at 500 nm (F340and F380) from an area con taining about 10 neurons was collected into a photomultiplier through an objective lens (Nikon Fluor X20). In this fluorometer, the excitation wavelengths were set automatically so that one was 40 nm longer than the other. Therefore, R340/360could not be monitored. Fortunately there was no contamination by light from the drugs or extracellular fura-2. High-K+ depolarization was evoked by total replacement of the extracellular solution with a high-K+ solution prepared by substituting KCl for NaCI in NES. Drugs were adminis tererd 2 min before and during high-K+ sti mulation. All experiments were performed at room temperature (22-24'C). The absolute [Ca2+]i was calculated after calibration. To calibrate, CaC12was added to a solution (100 mM KC1, 10 mM PIPES, 10 mM EGTA, pH 6.8) containing 10 ,uM fura-2. Free calcium concentration was calculated by assuming an apparent association constant of 8.42 X 105M-1 for CaEGTA (11). A plot of log[Ca2+] vs R340/380gave a straight line from about 130 nM to 900 nM of [Ca 2+1. The equa tion of the line was: R340/380=0.429 log[Ca2+] -0.664 However, to estimate drug effects, the in crease in R340/380was used as an index of rela tive increase in [Ca2+]i increase (12).
added, while it did not increase when the same amount of NaCI was added (data not shown). Therefore, the increase in [Ca 2+]i caused by KCI can be attributed to the K+ ion. The increase in R3401360 was related to the external K+ concentration ([K+]o) (Fig. 1B). The R340/36oresponse evoked by 50 mM KCl
RESULTS
[Ca2+]i of cerebral synaptosomes and the effects of Ca2+ antagonists and metal ions When synaptosomes were depolarized with 50 mM KCI, F340 rapidly increased, which in dicates that synaptosomal [Ca 2+]i increased (Fig. 1A). [Ca2+1i was maximal at 5 sec and gradually decreased. However, F360decreased by dilution of the incubation medium with KC1 solution. R340/360increased when KCl was
Fig. 1. High-K+ evoked increase in [Ca 211i in rat cerebral synaptosomes. (A) Typical fluorescence re cordings of a fura-2-loaded synaptosomal suspension stimulated by 55 mM K+ (KC1). Changes in F34o and F36o are shown by relative values, with the resting level as 100%. (B) Relation between the K+ concen tration used for stimulation and the increase in the R3401360.Each point and bars show a mean ± S.E. of 4 rats.
was used to estimate the inhibitory effects of Ca2+ antagonists and metal ions. The effects of metal ions on the K+-induced increase in [Ca2+]i are shown in Fig. 2. La 3+ and Cd2+ inhibited the increase in [Ca 2+]i in the synaptosomes. The increase in R340/360 were 15.6% and 59.7% of the control at 10 ,uM, respectively. Ni2+ had little effect on the increase in R340/360(the response was 90.0% of control at 10 ,uM) . The concentrations of La 3+ and Cd2+ that attenuated the increase in R340/360(not the increase in [Ca 2+]i) to half that of the control (ID50) were 3 ,uM and 20 ,uM, respectively. The increase in R340/360was 81.2% of the control at 1 mM of Ni2+ Ni2+ concentrations over 1 mM and Cd2+ concen
Fig. 2. Effect of La3+, Cd2; and Ni2+ on the R340/36o response in rat synaptosomes. The increases in R340/36o evoked by 50 mM KCI in the presence of metal ions were compared with that of the control. Each point and bars show a mean ± S.E. of 4 rats.
Fig.
3.
Typical
of Ca 2+ antagonists:
recordings
of R340/360
clentiazem,
MB-1,
of synaptosomes flunarizine
tration over 100,uM affected the fluorescence measurement (F34o was increased and de creased by high doses of Cd2+ and Nit , re spectively). Figure 3 shows recordings of R340/360 in the presence of 10 ,uM of various Ca2+ antago nists. Clentiazem,its metabolite MB1 and nit rendipine had little effect on the high K+ evoked-increasein [Ca2+]i, whereas 10,uM of flunarizine almost completely inhibited the in crease in [Ca2+]i. Figure4 shows the concentration-inhibition curvesfor various Ca2+ antagonists. The ID50s of flunarizine and nicardipine were 3.0,uM and 4.0,uM, respectively. However, other Ca2+ antagonists, including nifedipine, dil tiazem, verapamil, nitrendipine, clentiazem and MB-1, had much weaker effects on the high-K+-evokedincrease in [Ca2+]i at 10,uM. Thus the effects of nicardipine differ from those of its analogues nifedipine and nitrendi pine. [Ca2+J, in hippocampal neurons and the effects of Ca2+ antagonists and metal ions Figure 5A shows records of F340, F38o and their ratio. In an isotonic high-K+ solution (80 mM K+), F340 increased, F380 decreased and R340/380increased rapidly, which indicates that neuronal [Ca 2+]i increased. In an area without neurons, but with non-neuronal flat cells, high-K+ stimulation did not increase [Ca 2+]i (data not shown). Cultured astroglial cells have VSCCs (13). Therefore, they may have responded, but if the magnitude of the re sponse was much smaller than that of the
stimulated and
nitrendipine.
by 50 mM
KCl
in the
presence
of 10,uM
Fig. 4. Effects of Ca 2+ antagonists on the R340/360 response in rat synaptosomes. The increases in R340/360 evoked by 50 mM KCI in the presence of drugs were compared with that of the control. Each point and bars show a mean ± S.E. of 5 rats.
neurons, our system would not have detected it. The rapid increase in R340/380was followed by a gradual decrease during the sustained de polarization. The depolarization-induced high level of [Ca2+]i decreased rapidly to the rest ing level after the neurons were washed with NES. The increase in R340/38obecame greater as [K+]o increased (Fig. 5B). The R340/38ore sponse evoked by 80 mM K+ was used to esti mate the inhibitory effects of metal ions and Ca2+ antagonists. The effects
of metal
ions on the
increase
in
Fig. 5. High-K+ evoked increase in [Ca2+]; in rat cultured hippocampal neurons. (A) Typical fluores cence recordings from fura-2-loaded neurons stimu lated by 80 mM K+ (80 K). The fluorescence was the total of all those from an area with about 10neurons. (B) Relation between the K+ concentration used for stimulation and the increase in R340/380.Each point and bars show a mean ± S.E. of 4 wells.
[Ca2+]i are shown in Figs. 6 and 7. La 3+ and Cd2+ inhibited the high-K+-evoked response of hippocampal neurons (R340/38oresponses were 20% and 50% of control at 10 ,u M, re spectively), but Ni2+ did not inhibit the in crease at 10 MM (the response was 95% of control). Cd2+-induced inhibition was re versed by a wash with fresh NES, but the La 3+-induced blockade was not easily re versed. The concentration-inhibition curves are shown in Fig. 7. The ID50s (for the inhibi tion of the increase in R340/380)of La 3+ and
Fig. 6. Typical recordings of the R340/380 of rat cultured hippocampal neurons in the presence of 10,uM of La 3+ Cd2; and Nit Metal ions were added 2 min before stimulation with 80 mM K+ (80 K).
Cd2+
were
2,uM
and 10,uM,
respectively.
In
the case of Nit,, the response was 55% of the
Fig.
7.
Effects
of
80 mM-K+-induced campal mean
cultured ± S.E.
La 3+
Cd2+,
and
Ni2+
increase
in
R340/380
in
neurons. of 4 wells.
Each
point
and
on rat
bars
the
hippo show
a
control at 1 mM. Metal ions in these concen tration ranges did not affect the fluorescence measurements. Figure 8 shows records of R340/380in the presence of 10 ,uM of Ca2+ antagonists. Vera pamil, clentiazem and flunarizine inhibited the high-K+-evoked increase in [Ca2+],. Diltiazem and nifedipine had slight inhibitory effects (data not shown). These effects, except for the inhibition caused by a high-dose of flunarizine, were reversed by 2-3 washes with NES. Figure 9 shows the concentration-inhibition curves for various Ca2+ antagonists. The ID50s of flunarizine, nicardipine, clentiazem and verapamil were 8 ,uM, 10 ,uM, 15 ,uM and 30 ,uM, respectively.
Fig. 8. Typical recordings of the R340/380of rat hippocampal cultured neurons in the presence of 10,uM Ca 2+ antagonists. Drugs were added 2 min before stimulation with 80 mM K+ (80 K).
through VSCCs, and not to activationof Ca2+ efflux or to sequestration. Synaptosomes are isolated presynaptic ter minals which retain most of the functions of intact neurons. They have VSCCs which are responsiblefor the voltage and Ca2+-depend ent release of neurotransmitters, which are thought to be N-type channels (2, 14). In the present study, the high-K+-induced increase in synaptosomal[Ca2+1iwas inhibited by La3+ and Cd2, and not by Nit These effects are identical to the results obtained from 45Ca2+ uptake experiments (15). Since the Cd2+-in duced inhibition was stronger than that in
Fig.
9.
sponse creases ence
Effect
of Ca 2+ antagonists
in rat hippocampal in R340/3so evoked of drugs
were
compared
evoked by 80 mM K mean ± S.E. of 5 wells.
on
the
R340/38o
re
cultured neurons. The in by 80 mM K+ in the pres
Each
with point
that and
of
the bars
control show
a
DISCUSSION K+-depolarization induces Ca2+ influx through VSCCs, and this increases the [Ca 2+]i. The [Ca2+]i can increase not only by Ca2+ influx but also by Ca 2+ release from in tracellular stores, inhibition of Ca2+ efflux, or sequestration. However, the Ca2+ antagonists induced inhibition of the increase in [Ca2+]i that occurs immediately after K depolarization will be mainly due to inhibition of Ca2+ influx
duced by Nit;; these were not T-type chan nels. Dihydropyridine Ca2+ antagonists such as nifedipine and nitrendipine did not have an inhibitory effect, which indicates that the channels were not the L-type (16). These re sults suggest that the channels we observed were similar to N-channels. However, a recent detailed investigationre vealed that synaptosomalVSCCs are pharma cologicallysimilar to N-channels,but their in activationkinetics differ from those of N-chan nels (17). Ca2+ antagonists that affect the Ca2+ influx into synaptosomes may modulate neurotrans mitter release. In this preparation, flunarizine and nicardipine inhibited the depolarization evoked increase in [Ca2+]i, whereas nifedi pine, nitrendipine, verapamil and diltiazem had smaller effects. These observations are in agreement with those from 45Ca2+uptake ex periments (3-5). The new Ca antagonists clentiazem and MB-1 had little effect at 10 MM.These results suggestthat flunarizine and nicardipine suppress neurotransmitter release from nerve endings,while other Ca2+ antago nists have a lesser effect on transmitter re lease. In cultured hippocampal neurons, which contain many types of VSCCs, the high-K-in duced increase in [Ca2+1i was affected by La3+ and Cd2; but not by Nit This also occurred in synaptosomes. It has been said that there are L-type VSCCs in neuronal soma (2). However, it is not clear whether or not
the VSCCs involved in the phenomena observed in the present experiments were L type channels, because the increase in [Ca.2+]i was not sensitive to nifedipine or nitrendipine. The classification by Nowycky et al. (T, N and L types) (14) may be too simple to de scribe neuronal VSCCs (18). Whether or not that is true, VSCCs such as the L-type, with a large conductance and a slow inactivation, will cause more net Ca2+ influx and make a major contribution to the increase in [Ca2+]i. Considering the effects of metal ions and Ca.2+ antagonists, we see that this pharmaco logical property of VSCC in hippocampal neurons is similar to a property of synapto somes, although there were differences in the experimental conditions (for example, temper ature). Ca2+ current through low threshold VSCCs (T-type) has been measured in hippo campal and hypothalamic neurons (19, 20). Their pharmacological properties are similar to those observed in the present study. Both currents are inhibited by flunarizine and nicar dipine more than by diltiazem or D600. Therefore the pharmacological behaviors of neuronal VSCCs are similar to each other and are quite different from those of VSCCs in the cardiovascular systems. The high-K+-evoked increase in [Ca2-1Ji had two phases, the rapid increase and the high [Ca2+]i during the depolarization. Ca2+ antago nists inhibited the increase in [Ca2+]i during both phases. The pattern of inhibition seemed to be different between drugs. In the case of flunarizine, the latter, sustained phase was in hibited more strongly than the early rapid in crease. Therefore, the channels responsible for the sustained phase may be different from those responsible for the early phase. It is also possible that some drugs (especially flunar izine) may have more affinity for the depola rized channels (in the sustained phase) than for the resting channels (responsible for the initial [Ca.2+]i increase). The direct effects of Ca.2+ antagonists on the increase in [Ca2+]i in hippocampal neurons have only been studied for nitrendi pine and diltiazem (21, 22). In this study, we
showed that the high-K+-induced Ca.2+ influx into cultured hippocampal neurons can be in hibited by flunarizine, clentiazem and nicardi pine at more than 5 ,uM. In studies of whole animals, knowledge of the actual concentration of Ca2+ antagonists in the brain is important for the prediction of pharmacological effects. The concentration of clentiazem and its metabolite MB-1 in the brain are about 1 u M and 5 ,uM, respectively, 2 6 hr after oral administration (30 mg/kg) in the rat (S. Nakamura, unpublished data). Therefore, clentiazem will probably inhibit the Ca 2+ influx into hippocampal neurons in vivo, but only slightly. The function of Ca.2+ ions that enter neurons through VSCCs is still unknown. In hippocampal neurons, it is of interest because of the relationship between their vulnerability to ischemia and the excess uptake of Ca2+ (6). Nicardipine (23), nimodipine (24), flunar izine (25) and clentiazem (26) have been re ported to be effective against cerebral ische mia or stroke in whole animals. These com pounds may suppress neuronal Ca.2+ influx to some extent. However, it is unknown whether this contributes to the beneficial effect on cerebral ischemia, because the concentrations that inhibit neuronal Ca 2+ influx (10-6-10-5 M) are higher than those at which the drugs are vasodilators (10-8-10-6 M). Acknowledgements We
thank
Naito,
and
Prof. H.
T. Nagao
Narita
for
and the
Drs.
I. Yamaguchi,
encouragement
and
K. help
ful discussions.
REFERENCES 1 Raeburn, D. and Gonzales, R.E.: CNS disorders and calcium antagonists. TIPS 9, 117-118 (1988) 2 Miller, R.J.: Multiple calcium channels and neuronal function. Science 235, 46-52 (1987) 3 Nachshen, D.A. and Blaustein, M.P.: The effects of some organic calcium antagonists on calcium in flux in presynaptic nerve terminals. Mol. Pharma col. 16, 579-586 (1979) 4 Daniell, L.C., Barr, E.M. and Leslie, S. W.: 45Ca uptake into rat brain synaptosomes unaltered by dihydropyridine Ca antagonists. J. Neurochem. 41,
5
6
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12 13 14
15
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17
1455 -1459 (1983) Wibo, M. and Godfraind, T.: Calcium channels of synaptosomes of rat cerebral cortex. J. Pharmacol. (Fr.) 14, 651 (1983) Siesjo, B.K.: Cell damage in the brain: a specula tive synthesis. J. Cereb. Blood Flow Metab. 1, 155-185 (1981) Nachshen, D.A. and Sanchez-Armass, S.: Co-op erative action of calcium ions in dopamine release from rat brain synaptosomes. J. Physiol. (Lond.) 387, 415 423 (1987) Ashley, R.H., Brammer, M.J. and Marchbanks, R.: Measurement of intrasynaptosomal free cal cium by using the fluorescent indicator quin-2. Biochem. J. 219, 149-158 (1984) Banker, G.A. and Cowan, W.M.: Rat hippocam pal neurons in dispersed cell culture. Brain Res. 126, 397-425 (1977) Kudo, Y. and Ogura, A.: Glutamate-induced in crease in intracellular CA concentration in isolated hippocampal neurons. Br. J. Pharmacol. 89, 191 198 (1986) Harafuji, H. and Ogawa, Y.: Re-examination of the apparent binding constant of ethylene glycol bis(b-aminoethyl ether)-N, N, N',N'-tetraacetic acid with calcium around neutral pH. J. Biochem. 87, 1305 -1312 (1980) Karaki, H.: Ca localization and sensitivity in vascular smooth muscle. TIPS 10, 320-325 (1989) MacVicar, B.A.: Voltage-dependent calcium chan nels in glial cells. Science 226, 1345 1347 (1984) Nowycky, M.C., Fox, A.P. and Tsien, R.W.: Three types of neuronal calcium channel with different calcium agonist sensitivity. Nature 316, 440 443 (1985) Nachshen, D.A.: Selectivity of the Ca binding site in synaptosome Ca channel. J. Gen. Physiol. 83, 941 967 (1984) Fox, A.P., Nowycky, M.C. and Tsien, R.W.: Kinetic and pharmacological properties disting uishing three types of Ca currents in chick sensory neurons. J. Physiol. (Lond.) 394, 149-172 (1987) Suszkiw, J.B., Murawsky, M.M. and Shi, M.: Further characterization of phasic Ca influx in rat cerebrocortical synaptosomes: inferences regarding
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19
20
21
22
23
24
25
26
Ca channel types in nerve endings. J. Neurochem. 52, 1260-1269 (1989) Aosaki, T. and Kasai, H.: Characterization of two kinds of high-voltage-activatedCa channel currents in chick sensory neurons. Pflugers Arch. 414, 150 156 (1989) Takahashi, K. and Akaike, N.: Calcium antagonist effects on low threshold (T-type) calcium current in rat isolated hippocampal CA1 pyramidal neurons. J. Pharmacol. Exp. Ther. 256, 169-175 (1991) Akaike, N., Kostyuk, P.G. and Osipchuk, Y.V.: Dihydropyridine-sensitive low threshold calcium channels in rat isolated hypothalamic neurons. J. Physiol. (Lond.) 412, 181-195 (1989) Thayer, S.A., Murphy, S.N. and Miller, R.J.: Widespread distribution of dihydropyridine-sensi tive Ca channels in the central nervous system. Mol. Pharmacol. 30, 505-509 (1986) Kudo, Y., Ishibashi, A., Ogura, A. and Akita, K.: Mode of action of nitrendipine and diltiazem on the intracellular Ca elevations induced by KCI and L-glutamatein cultured hippocampal neurones of rats. Folia Pharmacol. Japon. 92, 47P (1988) (in Japanese) Alps, B.J., Calder, C., Hass, W.K. and Wilson, A.D.: The delayed post-ischemictreatment effects of nicardipine in a rat model of four vessel occlu sion. Br. J. Pharmacol. 91, 312P (1987) Steen, P.A., Newberg, L.A., Milde, J.H. and Michenfelder, J.D.: Nimodipine improves cerebral blood flow and neurologic recovery after complete cerebral ischemia in the dog. J. Cereb. Blood Flow Metab. 3, 38-43 (1983) Deshpande, J.K. and Wieloch, T.: Flunarizine, a calcium entry blocker, ameliorates ischemic brain damage in the rat. Anesthesiology 64, 215-224 (1986) Murata, S., Kikkawa, K., Iwasaki, H.O., Toriumi, W. and Nagao, T.: Effects of TA-3090, a novel 1,5-benzothiazepine calcium antagonist on neuro logical symptoms and cerebral lesions after stroke in SHRSP. Japan. J. Pharmacol. 52, Supp. I, 78P (1990)
of Ca 2+ Antagonists
on High-K+
Evoked
Increase
in [Ca2±1iin Rat Cerebral Synaptosomes and Tsutomu Biological
Research
Hippocampal
Kobayashi,
Laboratory, Received
Tanabe November
Rikako Seiyaku
Neurons
Yamauchi Co., Ltd.,
22, 1991
and Sakae Murata
2-2-50
Accepted
Kawagishi,
December
Toda,
Saitama
335, Japan
27, 1991
ABSTRACT-By fura-2 fluorometry, we investigated the direct effects of Ca2+ antago nists including a new benzothiazepine, clentiazem, on the high-K+-evoked increase in the concentration of cytosolic free Ca2+ ([Ca2+]i) in rat cerebral synaptosomes and cultured hippocampal neurons. In both preparations, metal ions inhibited the high-K+ induced increase in [Ca2+]i, in the following order: La 3+ > Cd2+ >> Ni2 Although flunarizine and nicardipine inhibited the K+-induced increase in [Ca2+]i in synapto somes, other Ca2+ antagonists, including clentiazem and nitrendipine, had little effect at 10,uM. In hippocampal neurons, clentiazem inhibited the K+-induced increase in [Ca2+]; at 10,uM, as did flunarizine and nicardipine. However, nifedipine and nitren dipine had little effect in either cultured neurons or in synaptosomes.
Calcium antagonists may be useful in the treatment of many central nervous system dis orders, including brain ischemia and epilepsy (1). These agents are generally regarded as vasodilators, because they antagonize the ac tion of Ca 2+ in vascular smooth muscle cells, but it is unknown whether their effects on the central nervous system are due to increased cerebral circulation or to direct inhibition of neuronal Ca 2+ influx. The direct action of Ca2+ antagonists on neurons is not clear, because neurons have at least three types of voltage sensitive Ca2+ channels (VSCC), and there is evidence that they differ from those in the cardiovascular system (2). The effects of Ca2+ antagonists on neuronal VSCC have not been studied in de tail. We studied the effects of various metal ions and Ca 2+ antagonists, including a new ben zothiazepine, clentiazem, and its metabolite
MB-1 (a major metabolite found in the rat brain), on the high-K+-induced increase in the concentration of cytosolic free Ca2+ ([Ca2+1i) in rat synaptosomes and cultured hippocampal neurons. The effects of some Ca2+ antagonists on Ca 2+ influx into synaptosomes, which is re lated to transmitter release, have been studied with 45Ca2+ (3-5). However, in the present study we used the Ca 2+ indicator fura-2, which enabled us to monitor real-time changes in [Ca2+]i. The [Ca2+]i in hippocampal neurons is of interest because of the relationship between the vulnerability of neurons to ischemia and excess uptake of Ca2+ (6). However, little is known about the effects of Ca2+ antagonists on the high-K+ induced increase in [Ca2+1i in hippocampal neurons.
MATERIALS AND METHODS Materials Clentiazem (TA-3090, (+)-(2S,3S)-3-acetoxy 8-chloro-5-(2-(dimethylamino)ethyl)-2,3-dihydro 2-(4-methoxyphenyl)-1,5-benzothiazepine-4-(5 H)-one maleate), MB-1 (the desacetylated form of clentiazem), and other Ca2+ antago nists were synthesized at the Organic Chemis try Research Laboratory of our company (Tanabe Seiyaku Co., Ltd.). Fura-2 acetoxy methyl ester (fura-2 AM) was purchased from Dojin Chemicals (Tokyo, Japan). Other re agents were obtained from commercial sources. Measurement of intrasynaptosomal free Ca-2+ Synaptosomes were prepared from cerebral cortices of male Sprague-Dawley rats (200 300 g) by a modified version of the method of Nachshen and Sanchez-Armass (7). Brain samples were homogenized in ice-cold 0.32 M sucrose, with 5 mM HEPES (pH 7.4) and 0.1 mM EDTA. The homogenate was centrifuged at 1000 X g for 10 min. The supernatant was then centrifuged at 12000 X g for 20 min, and the resulting supernatant was decanted. The pellet was resuspended in the medium de scribed above and then layered onto a gra dient consisting of 7.5% and 17% Ficoll-70 (Pharmacia) in the sucrose medium described above. The gradient was centrifuged at 63000 X g for 60 min. The synaptosomes, which banded at the 7.5%/17% interface, were re moved; diluted with 10 volumes of ice-cold sodium solution (composition: 145 mM NaCI, 5 mM KCI, 3 mM MgC12, 10 mM glucose, 10 mM HEPES, pH adjusted to 7.4 with Tris); and then centrifuged. The pellet was resus pended in sodium solution as descrived above, containing 0.1 mM CaC12, and incubated with fura-2 AM (3 u M) at 30°C for 30 min. The synaptosomes were washed twice with fresh sodium solution, without calcium, by centri fugation. The synaptosomes suspended in sodium solution, containing 1 mM CaC12, were incu bated in the presence or absence (control) of
Ca 2+ antagonists or metal ions for 5 min at 30°C and put in the cuvette of a fluorometer which was designed to measure fura-2-derived fluorescence (CAF-100 Japan Spectroscopic, Tokyo, Japan). The magnetically stirred sus pension was illuminated alternately (at 48 Hz) with two excitation wavelengths (340 nm and 360 nm). Light at 500 nm (F340, F360)emitted from the synaptosomes and the ratio (R340/360) were recorded. Depolarization was evoked by the addition of KCI solution to the sample sus pensions. An increase in R340/360after de polarization indicated that [Ca2+]i increased. In the sample suspension, some fluorescence came from high doses of some drugs or from extravesicular fura-2 which had leaked out of the synaptosomes. These contaminants were constant during depolarization. Therefore, if we use the increase in R340/360(which is equal to F340/constant), instead of the increase in R340/380as an index of the relative increase in [Ca2+]i, the effect of these contaminants will cancel out. The effects of the various drugs were esti mated by comparing the increase in R340/360 ratios with that measured in the controls. At first, we tried to calculate [Ca21]i. In practice, however, the calculation was impossible, be cause of the significant scatter (8) and the con tribution of autofluorescence which could not be estimated. All experiments were performed at 30°C. Microfluorometry of fura-2-loaded neurons Hippocampal neurons were isolated from cerebra of 18 19 day-old embryonic Sprague Dawley rats (9), and they were cultured for 4 7 days on monolayers of hippocampal glial cells precultured in specially designed dishes composed of a glass coverslip and a silicon rubber wall (10). The culture medium was re moved, and the neurons were incubated for 1 hr at 37°C in normal external solution (NES, composition, 145 mM NaCI, 5 mM KCI, 2 mM CaC12, 1 MM MgC12, 10 mM glucose, 10 mM HEPES, pH adjusted to 7.4 with Tris) con taining 2 ,uM fura-2 AM. After the loading-in cubation, the cells were washed with NES and
the dishes were mounted on an inverted microscope (TMD, Nikon, Japan) equipped with a specially designed fluorometer (CAM 200, Japan Spectroscopic, Japan). Neurons were illuminated alternately (at 10011z) by 340 nm and 380 nm light, and the light emitted at 500 nm (F340and F380) from an area con taining about 10 neurons was collected into a photomultiplier through an objective lens (Nikon Fluor X20). In this fluorometer, the excitation wavelengths were set automatically so that one was 40 nm longer than the other. Therefore, R340/360could not be monitored. Fortunately there was no contamination by light from the drugs or extracellular fura-2. High-K+ depolarization was evoked by total replacement of the extracellular solution with a high-K+ solution prepared by substituting KCl for NaCI in NES. Drugs were adminis tererd 2 min before and during high-K+ sti mulation. All experiments were performed at room temperature (22-24'C). The absolute [Ca2+]i was calculated after calibration. To calibrate, CaC12was added to a solution (100 mM KC1, 10 mM PIPES, 10 mM EGTA, pH 6.8) containing 10 ,uM fura-2. Free calcium concentration was calculated by assuming an apparent association constant of 8.42 X 105M-1 for CaEGTA (11). A plot of log[Ca2+] vs R340/380gave a straight line from about 130 nM to 900 nM of [Ca 2+1. The equa tion of the line was: R340/380=0.429 log[Ca2+] -0.664 However, to estimate drug effects, the in crease in R340/380was used as an index of rela tive increase in [Ca2+]i increase (12).
added, while it did not increase when the same amount of NaCI was added (data not shown). Therefore, the increase in [Ca 2+]i caused by KCI can be attributed to the K+ ion. The increase in R3401360 was related to the external K+ concentration ([K+]o) (Fig. 1B). The R340/36oresponse evoked by 50 mM KCl
RESULTS
[Ca2+]i of cerebral synaptosomes and the effects of Ca2+ antagonists and metal ions When synaptosomes were depolarized with 50 mM KCI, F340 rapidly increased, which in dicates that synaptosomal [Ca 2+]i increased (Fig. 1A). [Ca2+1i was maximal at 5 sec and gradually decreased. However, F360decreased by dilution of the incubation medium with KC1 solution. R340/360increased when KCl was
Fig. 1. High-K+ evoked increase in [Ca 211i in rat cerebral synaptosomes. (A) Typical fluorescence re cordings of a fura-2-loaded synaptosomal suspension stimulated by 55 mM K+ (KC1). Changes in F34o and F36o are shown by relative values, with the resting level as 100%. (B) Relation between the K+ concen tration used for stimulation and the increase in the R3401360.Each point and bars show a mean ± S.E. of 4 rats.
was used to estimate the inhibitory effects of Ca2+ antagonists and metal ions. The effects of metal ions on the K+-induced increase in [Ca2+]i are shown in Fig. 2. La 3+ and Cd2+ inhibited the increase in [Ca 2+]i in the synaptosomes. The increase in R340/360 were 15.6% and 59.7% of the control at 10 ,uM, respectively. Ni2+ had little effect on the increase in R340/360(the response was 90.0% of control at 10 ,uM) . The concentrations of La 3+ and Cd2+ that attenuated the increase in R340/360(not the increase in [Ca 2+]i) to half that of the control (ID50) were 3 ,uM and 20 ,uM, respectively. The increase in R340/360was 81.2% of the control at 1 mM of Ni2+ Ni2+ concentrations over 1 mM and Cd2+ concen
Fig. 2. Effect of La3+, Cd2; and Ni2+ on the R340/36o response in rat synaptosomes. The increases in R340/36o evoked by 50 mM KCI in the presence of metal ions were compared with that of the control. Each point and bars show a mean ± S.E. of 4 rats.
Fig.
3.
Typical
of Ca 2+ antagonists:
recordings
of R340/360
clentiazem,
MB-1,
of synaptosomes flunarizine
tration over 100,uM affected the fluorescence measurement (F34o was increased and de creased by high doses of Cd2+ and Nit , re spectively). Figure 3 shows recordings of R340/360 in the presence of 10 ,uM of various Ca2+ antago nists. Clentiazem,its metabolite MB1 and nit rendipine had little effect on the high K+ evoked-increasein [Ca2+]i, whereas 10,uM of flunarizine almost completely inhibited the in crease in [Ca2+]i. Figure4 shows the concentration-inhibition curvesfor various Ca2+ antagonists. The ID50s of flunarizine and nicardipine were 3.0,uM and 4.0,uM, respectively. However, other Ca2+ antagonists, including nifedipine, dil tiazem, verapamil, nitrendipine, clentiazem and MB-1, had much weaker effects on the high-K+-evokedincrease in [Ca2+]i at 10,uM. Thus the effects of nicardipine differ from those of its analogues nifedipine and nitrendi pine. [Ca2+J, in hippocampal neurons and the effects of Ca2+ antagonists and metal ions Figure 5A shows records of F340, F38o and their ratio. In an isotonic high-K+ solution (80 mM K+), F340 increased, F380 decreased and R340/380increased rapidly, which indicates that neuronal [Ca 2+]i increased. In an area without neurons, but with non-neuronal flat cells, high-K+ stimulation did not increase [Ca 2+]i (data not shown). Cultured astroglial cells have VSCCs (13). Therefore, they may have responded, but if the magnitude of the re sponse was much smaller than that of the
stimulated and
nitrendipine.
by 50 mM
KCl
in the
presence
of 10,uM
Fig. 4. Effects of Ca 2+ antagonists on the R340/360 response in rat synaptosomes. The increases in R340/360 evoked by 50 mM KCI in the presence of drugs were compared with that of the control. Each point and bars show a mean ± S.E. of 5 rats.
neurons, our system would not have detected it. The rapid increase in R340/380was followed by a gradual decrease during the sustained de polarization. The depolarization-induced high level of [Ca2+]i decreased rapidly to the rest ing level after the neurons were washed with NES. The increase in R340/38obecame greater as [K+]o increased (Fig. 5B). The R340/38ore sponse evoked by 80 mM K+ was used to esti mate the inhibitory effects of metal ions and Ca2+ antagonists. The effects
of metal
ions on the
increase
in
Fig. 5. High-K+ evoked increase in [Ca2+]; in rat cultured hippocampal neurons. (A) Typical fluores cence recordings from fura-2-loaded neurons stimu lated by 80 mM K+ (80 K). The fluorescence was the total of all those from an area with about 10neurons. (B) Relation between the K+ concentration used for stimulation and the increase in R340/380.Each point and bars show a mean ± S.E. of 4 wells.
[Ca2+]i are shown in Figs. 6 and 7. La 3+ and Cd2+ inhibited the high-K+-evoked response of hippocampal neurons (R340/38oresponses were 20% and 50% of control at 10 ,u M, re spectively), but Ni2+ did not inhibit the in crease at 10 MM (the response was 95% of control). Cd2+-induced inhibition was re versed by a wash with fresh NES, but the La 3+-induced blockade was not easily re versed. The concentration-inhibition curves are shown in Fig. 7. The ID50s (for the inhibi tion of the increase in R340/380)of La 3+ and
Fig. 6. Typical recordings of the R340/380 of rat cultured hippocampal neurons in the presence of 10,uM of La 3+ Cd2; and Nit Metal ions were added 2 min before stimulation with 80 mM K+ (80 K).
Cd2+
were
2,uM
and 10,uM,
respectively.
In
the case of Nit,, the response was 55% of the
Fig.
7.
Effects
of
80 mM-K+-induced campal mean
cultured ± S.E.
La 3+
Cd2+,
and
Ni2+
increase
in
R340/380
in
neurons. of 4 wells.
Each
point
and
on rat
bars
the
hippo show
a
control at 1 mM. Metal ions in these concen tration ranges did not affect the fluorescence measurements. Figure 8 shows records of R340/380in the presence of 10 ,uM of Ca2+ antagonists. Vera pamil, clentiazem and flunarizine inhibited the high-K+-evoked increase in [Ca2+],. Diltiazem and nifedipine had slight inhibitory effects (data not shown). These effects, except for the inhibition caused by a high-dose of flunarizine, were reversed by 2-3 washes with NES. Figure 9 shows the concentration-inhibition curves for various Ca2+ antagonists. The ID50s of flunarizine, nicardipine, clentiazem and verapamil were 8 ,uM, 10 ,uM, 15 ,uM and 30 ,uM, respectively.
Fig. 8. Typical recordings of the R340/380of rat hippocampal cultured neurons in the presence of 10,uM Ca 2+ antagonists. Drugs were added 2 min before stimulation with 80 mM K+ (80 K).
through VSCCs, and not to activationof Ca2+ efflux or to sequestration. Synaptosomes are isolated presynaptic ter minals which retain most of the functions of intact neurons. They have VSCCs which are responsiblefor the voltage and Ca2+-depend ent release of neurotransmitters, which are thought to be N-type channels (2, 14). In the present study, the high-K+-induced increase in synaptosomal[Ca2+1iwas inhibited by La3+ and Cd2, and not by Nit These effects are identical to the results obtained from 45Ca2+ uptake experiments (15). Since the Cd2+-in duced inhibition was stronger than that in
Fig.
9.
sponse creases ence
Effect
of Ca 2+ antagonists
in rat hippocampal in R340/3so evoked of drugs
were
compared
evoked by 80 mM K mean ± S.E. of 5 wells.
on
the
R340/38o
re
cultured neurons. The in by 80 mM K+ in the pres
Each
with point
that and
of
the bars
control show
a
DISCUSSION K+-depolarization induces Ca2+ influx through VSCCs, and this increases the [Ca 2+]i. The [Ca2+]i can increase not only by Ca2+ influx but also by Ca 2+ release from in tracellular stores, inhibition of Ca2+ efflux, or sequestration. However, the Ca2+ antagonists induced inhibition of the increase in [Ca2+]i that occurs immediately after K depolarization will be mainly due to inhibition of Ca2+ influx
duced by Nit;; these were not T-type chan nels. Dihydropyridine Ca2+ antagonists such as nifedipine and nitrendipine did not have an inhibitory effect, which indicates that the channels were not the L-type (16). These re sults suggest that the channels we observed were similar to N-channels. However, a recent detailed investigationre vealed that synaptosomalVSCCs are pharma cologicallysimilar to N-channels,but their in activationkinetics differ from those of N-chan nels (17). Ca2+ antagonists that affect the Ca2+ influx into synaptosomes may modulate neurotrans mitter release. In this preparation, flunarizine and nicardipine inhibited the depolarization evoked increase in [Ca2+]i, whereas nifedi pine, nitrendipine, verapamil and diltiazem had smaller effects. These observations are in agreement with those from 45Ca2+uptake ex periments (3-5). The new Ca antagonists clentiazem and MB-1 had little effect at 10 MM.These results suggestthat flunarizine and nicardipine suppress neurotransmitter release from nerve endings,while other Ca2+ antago nists have a lesser effect on transmitter re lease. In cultured hippocampal neurons, which contain many types of VSCCs, the high-K-in duced increase in [Ca2+1i was affected by La3+ and Cd2; but not by Nit This also occurred in synaptosomes. It has been said that there are L-type VSCCs in neuronal soma (2). However, it is not clear whether or not
the VSCCs involved in the phenomena observed in the present experiments were L type channels, because the increase in [Ca.2+]i was not sensitive to nifedipine or nitrendipine. The classification by Nowycky et al. (T, N and L types) (14) may be too simple to de scribe neuronal VSCCs (18). Whether or not that is true, VSCCs such as the L-type, with a large conductance and a slow inactivation, will cause more net Ca2+ influx and make a major contribution to the increase in [Ca2+]i. Considering the effects of metal ions and Ca.2+ antagonists, we see that this pharmaco logical property of VSCC in hippocampal neurons is similar to a property of synapto somes, although there were differences in the experimental conditions (for example, temper ature). Ca2+ current through low threshold VSCCs (T-type) has been measured in hippo campal and hypothalamic neurons (19, 20). Their pharmacological properties are similar to those observed in the present study. Both currents are inhibited by flunarizine and nicar dipine more than by diltiazem or D600. Therefore the pharmacological behaviors of neuronal VSCCs are similar to each other and are quite different from those of VSCCs in the cardiovascular systems. The high-K+-evoked increase in [Ca2-1Ji had two phases, the rapid increase and the high [Ca2+]i during the depolarization. Ca2+ antago nists inhibited the increase in [Ca2+]i during both phases. The pattern of inhibition seemed to be different between drugs. In the case of flunarizine, the latter, sustained phase was in hibited more strongly than the early rapid in crease. Therefore, the channels responsible for the sustained phase may be different from those responsible for the early phase. It is also possible that some drugs (especially flunar izine) may have more affinity for the depola rized channels (in the sustained phase) than for the resting channels (responsible for the initial [Ca.2+]i increase). The direct effects of Ca.2+ antagonists on the increase in [Ca2+]i in hippocampal neurons have only been studied for nitrendi pine and diltiazem (21, 22). In this study, we
showed that the high-K+-induced Ca.2+ influx into cultured hippocampal neurons can be in hibited by flunarizine, clentiazem and nicardi pine at more than 5 ,uM. In studies of whole animals, knowledge of the actual concentration of Ca2+ antagonists in the brain is important for the prediction of pharmacological effects. The concentration of clentiazem and its metabolite MB-1 in the brain are about 1 u M and 5 ,uM, respectively, 2 6 hr after oral administration (30 mg/kg) in the rat (S. Nakamura, unpublished data). Therefore, clentiazem will probably inhibit the Ca 2+ influx into hippocampal neurons in vivo, but only slightly. The function of Ca.2+ ions that enter neurons through VSCCs is still unknown. In hippocampal neurons, it is of interest because of the relationship between their vulnerability to ischemia and the excess uptake of Ca2+ (6). Nicardipine (23), nimodipine (24), flunar izine (25) and clentiazem (26) have been re ported to be effective against cerebral ische mia or stroke in whole animals. These com pounds may suppress neuronal Ca.2+ influx to some extent. However, it is unknown whether this contributes to the beneficial effect on cerebral ischemia, because the concentrations that inhibit neuronal Ca 2+ influx (10-6-10-5 M) are higher than those at which the drugs are vasodilators (10-8-10-6 M). Acknowledgements We
thank
Naito,
and
Prof. H.
T. Nagao
Narita
for
and the
Drs.
I. Yamaguchi,
encouragement
and
K. help
ful discussions.
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