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Differential effect of a dihydropyridine derivative to Ca2+ entry pathways in neuronal preparations
Brain Research, 301 (1984) 323-330 Elsevier
323
BRE 10029
Differential Effect of a Dihydropyridine Derivative to Ca 2+ Entry Pathways in Neuronal Preparations AKIHIKO OGURA and MASAMI TAKAHASHI
Laboratory of Neurochemistry, Mitsubishi-Kasei Institute of Life Sciences, Machida, Tokyo 194 (Japan) (Accepted October llth, 1983)
Key words: nicarpidine - - Ca2+ channel blocker - - Ca 2+ influx - - transmitter release - pheochromocytoma cell - - brain synaptosome
Nicardipine is one of the 1,4-dihydropyridine derivatives known as blockers for the voltage-dependent Ca2÷ channels in muscle cells. The effects of nicardipine on the neuronal functions were studied in several neuronal preparations including clonal rat pheochromocytoma (PC12) cells, rat brain synaptosomes and slices. Nicardipine failed to block the Ca2+-dependent action potentials and the after-spike hyperpolarizations evoked by intracellularly injected current pulses in rat pheoehromocytoma cells, while the high K +stimulated Ca 2+ influx and ATP release were dose-dependently inhibited in the same cells. In rat cerebral synaptosomes and cortical slices, nicardipine showed no blockade on the high K+-stimulated Ca2÷ influx and transmitter releases. It was then suggested that the voltage-dependent Ca2÷ channels are polymorphic among tissues or even in a single cell from the viewpoint of dihydropyridine susceptibility. INTRODUCTION Calcium ions play critical roles in various neuronal functions, including m e m b r a n e excitation ll, neurotransmitter release 14 and p r o t e i n p h o s p h o r y l a t i o n 15. The v o l t a g e - d e p e n d e n t Ca 2÷ channel is one of the key factors in determining the cellular Ca 2÷ level. The molecular nature of the neuronal Ca 2÷ channel, however, still remains u n r e v e a l e d , partly because ofunavailability of ligands which bind to the neuronal Ca 2÷ channels with high affinity and specificity. Recently, 1,4-dihydropyridine derivatives, known as Ca2+ channel blockers in cardiac, smooth and skeletal muscle cells 28, were r e p o r t e d to bind with high affinity to the m e m b r a n e fractions not only of muscle cells but of brain homogenatesl,6-9,18. This led to an expectation that the d i h y d r o p y r i d i n e derivatives might serve as m o l e c u l a r p r o b e s for the supposed molecules of Ca2+ channels in n e u r o n a l cells. But for using as such m o l e c u l a r probes, it is necessary to show a parallelism b e t w e e n the binding affinities and the potencies to interfere the Ca 2+ channels as in the case of muscle cells. Toll 27 and Takahashi and Ogura 25 r e p o r t e d that the d i h y d r o p y r i d i n e derivatives bound specifically to clonal rat p h e o c h r o m o c y t o m a 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.
cells (PC12) and that high K+-stimulated Ca 2+ influx into and catecholamine release from the same cells were blocked by the derivatives. To apply this conclusion to the neuronal Ca 2+ channels in general, we investigated here the effects of the d i h y d r o p y r i d i n e derivative to the Ca 2÷ channel functions in several neuronal preparations. C o n t r a r y to our expectations, electrophysiological examinations revealed that the Ca2+-dependent action potential in PC12 cells was not suppressed at all by the derivative, even at high concentrations where the high K+-stimulated Ca 2÷ influx was considerably inhibited. W e also recognized that the derivative suppressed neither by the high K+-stimulated Ca 2÷ influx into, nor the transmitter release from synaptosomes and slices of rat cerebral cortices. These results suggest a p o l y m o r p h i s m of the voltage-dependent Ca 2÷ channels among their sources as judged from dihydropyridine-sensitivity. MATERIALS AND METHODS
Chemicals The chemicals and drugs used in this study were
324 obtained from the suppliers indicated below. 45CaCi2 (spec. act. 32 Ci/mg Ca) and [3H]y-aminobutyric acid (spec. act. 57 Ci/mmol), Amersham; nicardipine hydrochloride, Yamanouchi Pharmaceuticals, Tokyo; verapamil, Eisai Pharmaceuticals, Tokyo; luciferin/ luciferase, Lumac, Schaesberg, The Netherlands.
PC12 cells Rat pheochromocytoma clone PC12 cells 10 (subclone h 13was used throughout this study) were plated on a poly-D-lysine-coated plastic dish (Falcon) and maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco) containing 5% precolostrum newborn calf serum (Mitsubishi Chemical Industries, Tokyo) and 5% heat-inactivated horse serum (Gibco). For the assay of a high K+-stimulated ATP release, the culture medium was replaced with a balanced salt solution-1 (BSS-1, composed of 130 mM NaC1, 5.4 mM KCI, 1.8 mM CaC12, 0.8 mM MgSO4, 5.5 mM glucose and 50 mM HEPES-NaOH, pH 7.3). After rinsing 7 times by replacements with BSS-1, a stimulation medium (=BSS-1 plus additional 46 mM KC1) was introduced in the dish. All replacements were done at 1 min intervals and each replaced medium was separately collected. Amounts of ATP released in the media were measured on a Lumac Biocounter M2010 photometer using luciferin/luciferase reaction. Nicardipine of the desired concentration was included in the BSS-1 for 2 min before the high K ÷stimulation. After the stimulation, the cells were lysed by 0.5 N NaOH and measured for protein by biuret reaction. For electrophysiological examinations, the cells were treated with a 1:1 mixture of polyethylene glycol (mean molecular weight 1000), and DMEM following the method of Davidson and Gerald4 5 days prior to the experiment. The obtained multinuclear cells with diameters of approximately 30 pm were used. A conventional set-up was employed, including a bridge circuit for a simultaneous intracellular recording and stimulation through a 3 M potassium acetate-filled microelectrode. Before electrode insertion, the culture medium was replaced with a recording medium composed of 120 mM Tris-HCl, pH 7.3; 5.4 mM KCI; 5.0 mM CaC12; 0.8 mM MgCI:; 5.5 mM glucose; and 20 mM tetraethylammonium chloride (TEA-C0. Action potentials were triggered by injecting brief depolarizing current pulses. The first
time-derivative of membrane potential was obtained electronically. After electrode insertion, the microscope illumination was turned off and the recording medium supplemented with nicardipine or CoC12 was introduced into the dish. Data were taken after at least 2 min of incubation. A high K+-stimulated Ca2+ influx was assayed using 45CAC12 as described in ref. 25, with a modification of the media. Briefly, the culture medium was replaced with the recording medium for the electrophysiological assay and, 2 min after an addition of nicardipine, another replacement with a high K + medium (the recording medium plus 46 mM KC1, 1 #Ci/ml 45CAC12 and 1 pM nicardipine) was carried out. After 5-40 s, the medium was removed and the cells were washed and then alkali-lysed. The radioactivity of the lysate was measured as the amount of Ca 2+ taken up.
Synaptosome Synaptosomal fraction was prepared from rat cerebral cortices following the method of Hajos12. Synaptosomes were suspended in a basal salt solution-2 (BSS-2, composed of 145 mM NaC1, 5 mM KC1, 1 mM MgSO n, 0.02 mM CaC12, 10 mM glucose and 10 mM Hepes-Tris, pH 7.5). A high K+-stimulated Ca 2+ influx was assayed by the method of Nachsen and Blaustein 19 with modifications. Briefly, 2 min after an addition of nicardipine to the synaptosomal suspension, an equal amount of a high K + medium (BSS-2 plus 140 mM KC1, 2 mM CaCI 2 and 2 ~Ci/ml 45CAC12) was rapidly mixed. After 5-20 s, the suspension was diluted with ice-cold BSS-2 and filtered through a Whatman GF/A filter. After several rinses with BSS-2, the filter was counted for radioactivity which was taken as an uptaken Ca 2+ amount. A high K+-stimulated y-aminobutyric acid (GABA) release was assayed as follows. The synaptosomal suspension was incubated for 40 rain in the presence of [3H]GABA (1 /~Ci/ml) with a 95% 02/5% CO2 perfusion and then trapped on a Millipore disposable filter unit (Milex 0.45/~m pore size). A balanced salt solution-3 (BSS-3, composed of 130 mM NaC1, 5.4 mM KC1, 1.0 mM CaC12, 0.8 mM MgSO4, 5.5 mM glucose, 50 mM Hepes-NaOH pH 7.3 and 10 pM aminooxyacetic acid (AOAA)) was perfused through the filter unit by a peristaltic pump
325 (Gilson Minipuls 2) at a constant flow rate of 1 ml/min. After 15 min perfusion, the perfusing medium was switched to a stimulating medium (46 mM KCI and 100/~M diaminobutyric acid ( D A B A ) were added to BSS-3 with omission of A O A A ) . After 2 min, perfusion was done with BSS-3 (100/tM D A B A was added instead of 10/~M A O A A ) . The filtrate was collected at 30 s intervals and was counted for radioactivity. Finally, distilled water was perfused to disrupt the synaptosomes and the radioactivity of the perfusate was taken as an amount of [3H]GABA retained in the synaptosomes. Nicardipine, verapamil and CoC12 was added in the perfusion media since 2 min before the high K+-stimulation. The assay for a high K+-stimulated ATP release from synaptosomes was made on the principle of White 29 as follows. Fifty microliters of a high K ÷ medium (=BSS-2 supplemented with 420 raM KCI) was flushed into 400/~1 of synaptosomal suspension set in a Jobin Yvon PicoATP photometer. The drugs in test, CaCI 2 (2 mM) and luciferin/luciferase were added to the suspension 2 min before the flush. The
emitted luminescence was recorded and the peak in 5-8 recordings were averaged.
Brain slice A 300 btm thin slice of adult rat cerebral cortex was divided into 6 pieces, which were enveloped in nylonmesh bags and soaked in BSS-3 containing [3H]GABA (1/~Ci/ml) for 25 min. After rinsing 6 times in BSS-3, the bags carrying the brain slices were transferred sequentially in a series of 1.5 ml wells at 2 min intervals (3 wells contained BSS-3 supplemented with the drugs in test and 3 wells contained BSS-3 added with 46 mM KC1 plus drug). The radioactivity released in each well was counted. Each slice was lysed by 0.5 N N a O H and the radioactivity in the lysate was taken as an amount of [3H]GABA retained in the tissue. All wells were continuously perfused with 95% 02/5% C O 2. All experiments using nicardipine were made under darkness or dimmed sodium lamp. All experiments were done at 37 °C except for the preparation of synaptosomes. RESULTS
~ ~...51.4
Inhibitory effect of nicardipine on high K+-stimulated A TP release from PC12 cells
mM K÷
It is known that ATP is stored in adrenal gland and various neuronal cells. Upon stimulation in the presence of external Ca 2÷, ATP is released from these sources together with catecholamine and acetylcho1ine16,22,24 (reviewed by Brunstock2). We found that PC12 cells also released ATP by high K÷-stimula tion. Fig. 1 shows the dose-inhibition effect of nicardipine on the high K+-stimulated ATP release from PC12 cells. Nicardipine at over 1 nM suppressed the ATP release and a full suppression was seen at 100 nM. A 50% inhibition dose was about 15 nM, which was close to that previously reported on a high Kstimulated norepinephrine release from PC12 cells (7 nM) 2s.
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Fig. 1. Dose-dependent inhibitory effect of nicardipine on a high K+-stimulated ATP release from PC12 cells. Closed circles indicate the amounts of ATP released over 1 min upon exposures to a 51.4 mM K+-environment. Open circles indicate those released during 1 min immediately before high K+-stimulations. Lines were fitted by eye.
Effects of nicardipine on electrophysiological properties of PC12 cells Since the small size of PC12 cells limited the stable membrane potential recording, we fused the cells by an exposure to 50% polyethylene glycol. The membrane excitability of the cell was reported to be unal-
326
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b
c
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40mYI 200msl Fig. 2. Membrane potential responses to applied current pulses in PC12 cells. Responses were recorded in the absence of drug (A,a), in the presence of 1/~M nicardipine (B,b) and in the presence of 10 mM CoOl 2 (C,c), A-C: action potentials were triggered by current pulses superimposed on constant currents that shifted the membrane to --100 mV. Upper traces show membrane potentials (Vm) and lower traces show their time-derivatives ('v'm). a-c: after-spike hyperpolarizations following action potentials triggered from resting levels. In a Co2+-treated cell, an electrotonic depolarization with amplitude and duration comparable with the action potential was induced. Dashed lines demarcate 0 mV levels. tered by the treatment21. For isolation and enhancement of the Ca2+-dependent membrane electrogenesis, Na ÷ was removed from the conventional medium and replaced with isotonic Tris-HCl. For the same purpose TEA-C1 was added up to 20 mM, and Ca 2+ was raised to 5 mM. Fig. 2A shows the Ca2+-dependent action potential in a PC12 cell evoked by a depolarizing current pulse injection in the Na+-free Ca2+-raised medium. The peak level of the action potential and the rate of potential rise fully developed due to a preceding shift of the membrane potential to - - 1 0 0 mV to remove the inactivation of the voltage-dependent Ca 2+ channels. A slow recording of a spike evoked from the resting potential (Fig. 2a) shows a profound afterspike hyperpolarization which is attributable to an activation of a Ca2+-dependent K + conductance. Cobaltous ion, a widely used inorganic Ca 2÷ channel blocker, totally inhibited the generation of action potential and negative after-potential (Fig. 2C and c) at
the concentration of 10 mM. On the other hand, as shown in Fig. 2B and b, an addition of 1/~M nicardipine had no effect on the peak level and shape of the action potential and negative after-potential. Even higher concentration of nicardipine had no effect (now shown here). This consequence was quantified in Table I. Neither the passive membrane properties such as resting potential level, cell's input resistance and capacitance nor the active properties such as peak level and rate of rise of the action potential and negative after-potential were influenced by the presence of 1 p M of nicardipine. The concentration of nicardipine as 1 p M was 50 times higher than a half inhibition dose on a high K+-stimn lated Ca 2+ influx in PC12 cells (20 nM) 25. Claims might be raised that the increased external Ca 2+ concentration from conventional 1.8 mM to 5 mM could have antagonized the effect of nicardipine. We therefore repeated the high K+-stimulated Ca 2+ influx measurements in the condition parallel to that
327 TABLE I Comparison of membrane properties of PC12 cells before and after a nicardipine treatment All values represent mean __ 1 S,D. of 9 randomly selected cells. Resting input resistance determined by membrane potential shifts by 0.1 nA hyperpolarizing current injections. Resting input capacitance determined by rates of potential fall in response to 0.1 nA hyperpolarizing current injections. Action potential peak level and maximum rate of rise: action potentials were evoked from membrane potential shifted to --100 mV. After-spike hyperpolarization level: action potentials were evoked from resting potential levels. Condition
Nicardipine
Resting membrane potential (mV) Resting input resistance (MS) Resting input capacitance (pF) Action potential peak level (mV) Maximum rate of rise (V/s) After-spike hyperpolarization level (mV)
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Time(sec) Fig. 3. Time courses of high K+-stimulated Ca2+ influxes into rat cerebral cortical synaptosomes. Circles and triangles represent the influxes in the absence and the presence of 1/~M nicardipine, respectively. Open symbols indicate basal Ca2+ influxes measured without high K+-stimulations. Closed symbols indicate the high K÷-stimulated Ca2+ influxes. Typical result out of 3 similarly repeated experiments is shown.
in the electrophysiological assay. The result was that nicardipine at 1 p M still remarkably suppressed the high K+-stimulated Ca 2+ influx into PC12 cells (inhibited 55-76% for high K+-stimulation periods of
5--40 s). Effects o f nicardipine on high K +-stimulated Ca 2+ influx and neurotransmitter release in rat cerebral neurons Next we examined the effect of nicardipine on the Ca 2+ channel function of rat cerebral cortical neurons using synaptosomes and slices. Fig. 3 shows time courses of the Ca 2+ influxes into the rat cerebral cortical synaptosomes triggered by high K ÷ stimulations. As reported by Nachsen and Blaustein 20, the extrapolating lines of the time-courses did not intersect the origin, implying an initial rapid Ca 2+ influx within the first 5 seconds. Nachsen and Blaustein 19 also showed that under low Ca 2+ concentrations (0.06 mM) nifedipine, another dihydropyridine derivative, failed to suppress the Ca 2÷ influx during the first 5 s. We observed that the Ca 2+ influx into synaptosomes under 2 m M external Ca 2÷ was also unaltered through 30 s by 1/~M nicardipine, which completely suppressed the high K+-stimulated Ca 2+ influx into PC12 cells 25. The lack of inhibition of nicardipine in rat brain neuronal Ca 2+ influx systems was confirmed by the experiments on transmitter releases. Tritiated G A B A preloaded in the synaptosomes or the brain slices was released in response to an exposure to a high K+ environment (Fig. 4A and B). Known Ca 2+ channel blockers, verapamil (30 p M ) and Co2+ (5 raM) significantly suppressed the high K+-stimulated [ 3 H ] G A B A releases from both synaptosomes and brain slices, whereas nicardipine even at 1/~M little affected the amounts and time-courses of the [3H]GABA releases. Unaffected G A B A release from brain slices might exclude the possibility that a nicardipine-sensitivity would have been lost during the preparation of synaptosomes. The assay for high K+-stimulated A T P release from the synaptosomes gave similar results; inhibited significantly (down to 51 + 10% of control) by the presence of 30/~M verapamil but suppressed only slightly (88 + 11% of control) by 1/~M nicardipine.
328
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Time (rain) Fig. 4. Time courses of high K+-stimulated [3H]GABA releases from rat cerebral cortical synaptosomes (A) and thin slices (B). Circles, triangles, squares and reversed triangles represent the releases under the absence of drug, the presence of 1 ~M nicardipine, the presence of 30/~M verapamil and the presence of 10 mM CoCI 2, respectively. Stimulation schedules are shown in upper bars. Typical result out of 4 similarly repeated experiments (A) or averaged values from 2 repeated experiments (B) are presented.
DISCUSSION In this study we showed that nicardipine, at the concentration which inhibited the high K+-stimulated Ca 2+ influx into PC12 cells (ref. 25 and this report) failed to affect the Ca2+-dependent membrane electrogenesis of the same cells. Some explanations are possible for this apparent discrepancy. First, high K+-stimulation activated the CaZ+ entry system other than the voltage-sensitive Ca2+ channel, such as Na + channel and Na+/Ca 2+ exchange. Participation of the Na + channel is not plausible, since high K+-stimulated Ca 2+ influx into PC12 cells was known to be blocked by Mn 2+, Co 2+ and veraparail, but not by tetrodotoxin 23,26. If nicardipine would enhance the Na+/Ca z+ exchange, the observed decrease in Ca2+ influx would result. But then we might expect a decrease of basal (K+-unstimulated) trans-
mitter release by nicardipine, which was not the case. The second explanation is that nicardipine would affect the open Ca2+ channels only, or that nicardipine's action would be facilitated under depolarized condition of high K +. But repetitive stimulation in a nicardipine-containing medium did not produce an inhibition of Ca2+-dependent action potential in PC12 cell (our unpublished observation, see also ref. 17). And Ca 2+ influx into synaptosomes was not inhibited, even by high K+ stimulation. The third and most plausible explanation might be that PC12 cells have two independent pathways for voltage-dependent Ca2+ incorporation; one is a dihydropyridine-insensitive spike-generating channel, the other is a dihydropyridine-sensitive channel with sustained activation state over 30 s. Coexistence of two populations of voltage-sensitive Ca 2+ pathways is also suggested by previous authors.
329 From the analyses on the high. K÷-stimulated Ca 2÷
Are there entirely different molecules? Alternative-
influx in brain synaptosomes, Nachsen and Blau-
ly, a molecule of Ca 2÷ channel consists of several
stein 19 showed two successive phases of the Ca 2÷ influx, fast-inactivating and slowly-inactivating phases.
functional sites (or subunits) including voltage sen-
From differences in La3+-sensitivity and p e r m e a n t cation selectivity, they suggested the presence of two types of voltage-dependent Ca 2+ channels in synap-
sor/gate, ionophore and drug receptor. A n d a pharmacological variety might owe to a difference in the drug receptor site. Recently a single channel current analysis3 showed a striking similarity in gating kinet-
tosomes. It is known that the Ca 2÷ channels in n e u r o n s are
ics of the voltage-dependent Ca 2÷ channels from var-
less sensitive than those in smooth and cardiac muscle cells to verapamil, another class of Ca 2÷ channel
ron, PC12), suggesting that the model of entirely dif-
blocker II. Thus dihydropyridine derivatives might be another tool for pharmacological classification and
ious sources (snail n e u r o n , vertebrate sensory neuferent molecules is less plausible. ACKNOWLEDGEMENTS
characterization of Ca 2÷ channels from various sources. We are still far from explaining what determines the difference in drug sensitivity of the Ca 2+ channel.
The authors are grateful to Dr. H. H a t a n a k a of this institute for providing PC12h ceils and to Dr. T. m m a n o for encouragement.
REFERENCES
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323
BRE 10029
Differential Effect of a Dihydropyridine Derivative to Ca 2+ Entry Pathways in Neuronal Preparations AKIHIKO OGURA and MASAMI TAKAHASHI
Laboratory of Neurochemistry, Mitsubishi-Kasei Institute of Life Sciences, Machida, Tokyo 194 (Japan) (Accepted October llth, 1983)
Key words: nicarpidine - - Ca2+ channel blocker - - Ca 2+ influx - - transmitter release - pheochromocytoma cell - - brain synaptosome
Nicardipine is one of the 1,4-dihydropyridine derivatives known as blockers for the voltage-dependent Ca2÷ channels in muscle cells. The effects of nicardipine on the neuronal functions were studied in several neuronal preparations including clonal rat pheochromocytoma (PC12) cells, rat brain synaptosomes and slices. Nicardipine failed to block the Ca2+-dependent action potentials and the after-spike hyperpolarizations evoked by intracellularly injected current pulses in rat pheoehromocytoma cells, while the high K +stimulated Ca 2+ influx and ATP release were dose-dependently inhibited in the same cells. In rat cerebral synaptosomes and cortical slices, nicardipine showed no blockade on the high K+-stimulated Ca2÷ influx and transmitter releases. It was then suggested that the voltage-dependent Ca2÷ channels are polymorphic among tissues or even in a single cell from the viewpoint of dihydropyridine susceptibility. INTRODUCTION Calcium ions play critical roles in various neuronal functions, including m e m b r a n e excitation ll, neurotransmitter release 14 and p r o t e i n p h o s p h o r y l a t i o n 15. The v o l t a g e - d e p e n d e n t Ca 2÷ channel is one of the key factors in determining the cellular Ca 2÷ level. The molecular nature of the neuronal Ca 2÷ channel, however, still remains u n r e v e a l e d , partly because ofunavailability of ligands which bind to the neuronal Ca 2÷ channels with high affinity and specificity. Recently, 1,4-dihydropyridine derivatives, known as Ca2+ channel blockers in cardiac, smooth and skeletal muscle cells 28, were r e p o r t e d to bind with high affinity to the m e m b r a n e fractions not only of muscle cells but of brain homogenatesl,6-9,18. This led to an expectation that the d i h y d r o p y r i d i n e derivatives might serve as m o l e c u l a r p r o b e s for the supposed molecules of Ca2+ channels in n e u r o n a l cells. But for using as such m o l e c u l a r probes, it is necessary to show a parallelism b e t w e e n the binding affinities and the potencies to interfere the Ca 2+ channels as in the case of muscle cells. Toll 27 and Takahashi and Ogura 25 r e p o r t e d that the d i h y d r o p y r i d i n e derivatives bound specifically to clonal rat p h e o c h r o m o c y t o m a 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.
cells (PC12) and that high K+-stimulated Ca 2+ influx into and catecholamine release from the same cells were blocked by the derivatives. To apply this conclusion to the neuronal Ca 2+ channels in general, we investigated here the effects of the d i h y d r o p y r i d i n e derivative to the Ca 2÷ channel functions in several neuronal preparations. C o n t r a r y to our expectations, electrophysiological examinations revealed that the Ca2+-dependent action potential in PC12 cells was not suppressed at all by the derivative, even at high concentrations where the high K+-stimulated Ca 2÷ influx was considerably inhibited. W e also recognized that the derivative suppressed neither by the high K+-stimulated Ca 2÷ influx into, nor the transmitter release from synaptosomes and slices of rat cerebral cortices. These results suggest a p o l y m o r p h i s m of the voltage-dependent Ca 2÷ channels among their sources as judged from dihydropyridine-sensitivity. MATERIALS AND METHODS
Chemicals The chemicals and drugs used in this study were
324 obtained from the suppliers indicated below. 45CaCi2 (spec. act. 32 Ci/mg Ca) and [3H]y-aminobutyric acid (spec. act. 57 Ci/mmol), Amersham; nicardipine hydrochloride, Yamanouchi Pharmaceuticals, Tokyo; verapamil, Eisai Pharmaceuticals, Tokyo; luciferin/ luciferase, Lumac, Schaesberg, The Netherlands.
PC12 cells Rat pheochromocytoma clone PC12 cells 10 (subclone h 13was used throughout this study) were plated on a poly-D-lysine-coated plastic dish (Falcon) and maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco) containing 5% precolostrum newborn calf serum (Mitsubishi Chemical Industries, Tokyo) and 5% heat-inactivated horse serum (Gibco). For the assay of a high K+-stimulated ATP release, the culture medium was replaced with a balanced salt solution-1 (BSS-1, composed of 130 mM NaC1, 5.4 mM KCI, 1.8 mM CaC12, 0.8 mM MgSO4, 5.5 mM glucose and 50 mM HEPES-NaOH, pH 7.3). After rinsing 7 times by replacements with BSS-1, a stimulation medium (=BSS-1 plus additional 46 mM KC1) was introduced in the dish. All replacements were done at 1 min intervals and each replaced medium was separately collected. Amounts of ATP released in the media were measured on a Lumac Biocounter M2010 photometer using luciferin/luciferase reaction. Nicardipine of the desired concentration was included in the BSS-1 for 2 min before the high K ÷stimulation. After the stimulation, the cells were lysed by 0.5 N NaOH and measured for protein by biuret reaction. For electrophysiological examinations, the cells were treated with a 1:1 mixture of polyethylene glycol (mean molecular weight 1000), and DMEM following the method of Davidson and Gerald4 5 days prior to the experiment. The obtained multinuclear cells with diameters of approximately 30 pm were used. A conventional set-up was employed, including a bridge circuit for a simultaneous intracellular recording and stimulation through a 3 M potassium acetate-filled microelectrode. Before electrode insertion, the culture medium was replaced with a recording medium composed of 120 mM Tris-HCl, pH 7.3; 5.4 mM KCI; 5.0 mM CaC12; 0.8 mM MgCI:; 5.5 mM glucose; and 20 mM tetraethylammonium chloride (TEA-C0. Action potentials were triggered by injecting brief depolarizing current pulses. The first
time-derivative of membrane potential was obtained electronically. After electrode insertion, the microscope illumination was turned off and the recording medium supplemented with nicardipine or CoC12 was introduced into the dish. Data were taken after at least 2 min of incubation. A high K+-stimulated Ca2+ influx was assayed using 45CAC12 as described in ref. 25, with a modification of the media. Briefly, the culture medium was replaced with the recording medium for the electrophysiological assay and, 2 min after an addition of nicardipine, another replacement with a high K + medium (the recording medium plus 46 mM KC1, 1 #Ci/ml 45CAC12 and 1 pM nicardipine) was carried out. After 5-40 s, the medium was removed and the cells were washed and then alkali-lysed. The radioactivity of the lysate was measured as the amount of Ca 2+ taken up.
Synaptosome Synaptosomal fraction was prepared from rat cerebral cortices following the method of Hajos12. Synaptosomes were suspended in a basal salt solution-2 (BSS-2, composed of 145 mM NaC1, 5 mM KC1, 1 mM MgSO n, 0.02 mM CaC12, 10 mM glucose and 10 mM Hepes-Tris, pH 7.5). A high K+-stimulated Ca 2+ influx was assayed by the method of Nachsen and Blaustein 19 with modifications. Briefly, 2 min after an addition of nicardipine to the synaptosomal suspension, an equal amount of a high K + medium (BSS-2 plus 140 mM KC1, 2 mM CaCI 2 and 2 ~Ci/ml 45CAC12) was rapidly mixed. After 5-20 s, the suspension was diluted with ice-cold BSS-2 and filtered through a Whatman GF/A filter. After several rinses with BSS-2, the filter was counted for radioactivity which was taken as an uptaken Ca 2+ amount. A high K+-stimulated y-aminobutyric acid (GABA) release was assayed as follows. The synaptosomal suspension was incubated for 40 rain in the presence of [3H]GABA (1 /~Ci/ml) with a 95% 02/5% CO2 perfusion and then trapped on a Millipore disposable filter unit (Milex 0.45/~m pore size). A balanced salt solution-3 (BSS-3, composed of 130 mM NaC1, 5.4 mM KC1, 1.0 mM CaC12, 0.8 mM MgSO4, 5.5 mM glucose, 50 mM Hepes-NaOH pH 7.3 and 10 pM aminooxyacetic acid (AOAA)) was perfused through the filter unit by a peristaltic pump
325 (Gilson Minipuls 2) at a constant flow rate of 1 ml/min. After 15 min perfusion, the perfusing medium was switched to a stimulating medium (46 mM KCI and 100/~M diaminobutyric acid ( D A B A ) were added to BSS-3 with omission of A O A A ) . After 2 min, perfusion was done with BSS-3 (100/tM D A B A was added instead of 10/~M A O A A ) . The filtrate was collected at 30 s intervals and was counted for radioactivity. Finally, distilled water was perfused to disrupt the synaptosomes and the radioactivity of the perfusate was taken as an amount of [3H]GABA retained in the synaptosomes. Nicardipine, verapamil and CoC12 was added in the perfusion media since 2 min before the high K+-stimulation. The assay for a high K+-stimulated ATP release from synaptosomes was made on the principle of White 29 as follows. Fifty microliters of a high K ÷ medium (=BSS-2 supplemented with 420 raM KCI) was flushed into 400/~1 of synaptosomal suspension set in a Jobin Yvon PicoATP photometer. The drugs in test, CaCI 2 (2 mM) and luciferin/luciferase were added to the suspension 2 min before the flush. The
emitted luminescence was recorded and the peak in 5-8 recordings were averaged.
Brain slice A 300 btm thin slice of adult rat cerebral cortex was divided into 6 pieces, which were enveloped in nylonmesh bags and soaked in BSS-3 containing [3H]GABA (1/~Ci/ml) for 25 min. After rinsing 6 times in BSS-3, the bags carrying the brain slices were transferred sequentially in a series of 1.5 ml wells at 2 min intervals (3 wells contained BSS-3 supplemented with the drugs in test and 3 wells contained BSS-3 added with 46 mM KC1 plus drug). The radioactivity released in each well was counted. Each slice was lysed by 0.5 N N a O H and the radioactivity in the lysate was taken as an amount of [3H]GABA retained in the tissue. All wells were continuously perfused with 95% 02/5% C O 2. All experiments using nicardipine were made under darkness or dimmed sodium lamp. All experiments were done at 37 °C except for the preparation of synaptosomes. RESULTS
~ ~...51.4
Inhibitory effect of nicardipine on high K+-stimulated A TP release from PC12 cells
mM K÷
It is known that ATP is stored in adrenal gland and various neuronal cells. Upon stimulation in the presence of external Ca 2÷, ATP is released from these sources together with catecholamine and acetylcho1ine16,22,24 (reviewed by Brunstock2). We found that PC12 cells also released ATP by high K÷-stimula tion. Fig. 1 shows the dose-inhibition effect of nicardipine on the high K+-stimulated ATP release from PC12 cells. Nicardipine at over 1 nM suppressed the ATP release and a full suppression was seen at 100 nM. A 50% inhibition dose was about 15 nM, which was close to that previously reported on a high Kstimulated norepinephrine release from PC12 cells (7 nM) 2s.
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Fig. 1. Dose-dependent inhibitory effect of nicardipine on a high K+-stimulated ATP release from PC12 cells. Closed circles indicate the amounts of ATP released over 1 min upon exposures to a 51.4 mM K+-environment. Open circles indicate those released during 1 min immediately before high K+-stimulations. Lines were fitted by eye.
Effects of nicardipine on electrophysiological properties of PC12 cells Since the small size of PC12 cells limited the stable membrane potential recording, we fused the cells by an exposure to 50% polyethylene glycol. The membrane excitability of the cell was reported to be unal-
326
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40mYI 200msl Fig. 2. Membrane potential responses to applied current pulses in PC12 cells. Responses were recorded in the absence of drug (A,a), in the presence of 1/~M nicardipine (B,b) and in the presence of 10 mM CoOl 2 (C,c), A-C: action potentials were triggered by current pulses superimposed on constant currents that shifted the membrane to --100 mV. Upper traces show membrane potentials (Vm) and lower traces show their time-derivatives ('v'm). a-c: after-spike hyperpolarizations following action potentials triggered from resting levels. In a Co2+-treated cell, an electrotonic depolarization with amplitude and duration comparable with the action potential was induced. Dashed lines demarcate 0 mV levels. tered by the treatment21. For isolation and enhancement of the Ca2+-dependent membrane electrogenesis, Na ÷ was removed from the conventional medium and replaced with isotonic Tris-HCl. For the same purpose TEA-C1 was added up to 20 mM, and Ca 2+ was raised to 5 mM. Fig. 2A shows the Ca2+-dependent action potential in a PC12 cell evoked by a depolarizing current pulse injection in the Na+-free Ca2+-raised medium. The peak level of the action potential and the rate of potential rise fully developed due to a preceding shift of the membrane potential to - - 1 0 0 mV to remove the inactivation of the voltage-dependent Ca 2+ channels. A slow recording of a spike evoked from the resting potential (Fig. 2a) shows a profound afterspike hyperpolarization which is attributable to an activation of a Ca2+-dependent K + conductance. Cobaltous ion, a widely used inorganic Ca 2÷ channel blocker, totally inhibited the generation of action potential and negative after-potential (Fig. 2C and c) at
the concentration of 10 mM. On the other hand, as shown in Fig. 2B and b, an addition of 1/~M nicardipine had no effect on the peak level and shape of the action potential and negative after-potential. Even higher concentration of nicardipine had no effect (now shown here). This consequence was quantified in Table I. Neither the passive membrane properties such as resting potential level, cell's input resistance and capacitance nor the active properties such as peak level and rate of rise of the action potential and negative after-potential were influenced by the presence of 1 p M of nicardipine. The concentration of nicardipine as 1 p M was 50 times higher than a half inhibition dose on a high K+-stimn lated Ca 2+ influx in PC12 cells (20 nM) 25. Claims might be raised that the increased external Ca 2+ concentration from conventional 1.8 mM to 5 mM could have antagonized the effect of nicardipine. We therefore repeated the high K+-stimulated Ca 2+ influx measurements in the condition parallel to that
327 TABLE I Comparison of membrane properties of PC12 cells before and after a nicardipine treatment All values represent mean __ 1 S,D. of 9 randomly selected cells. Resting input resistance determined by membrane potential shifts by 0.1 nA hyperpolarizing current injections. Resting input capacitance determined by rates of potential fall in response to 0.1 nA hyperpolarizing current injections. Action potential peak level and maximum rate of rise: action potentials were evoked from membrane potential shifted to --100 mV. After-spike hyperpolarization level: action potentials were evoked from resting potential levels. Condition
Nicardipine
Resting membrane potential (mV) Resting input resistance (MS) Resting input capacitance (pF) Action potential peak level (mV) Maximum rate of rise (V/s) After-spike hyperpolarization level (mV)
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Time(sec) Fig. 3. Time courses of high K+-stimulated Ca2+ influxes into rat cerebral cortical synaptosomes. Circles and triangles represent the influxes in the absence and the presence of 1/~M nicardipine, respectively. Open symbols indicate basal Ca2+ influxes measured without high K+-stimulations. Closed symbols indicate the high K÷-stimulated Ca2+ influxes. Typical result out of 3 similarly repeated experiments is shown.
in the electrophysiological assay. The result was that nicardipine at 1 p M still remarkably suppressed the high K+-stimulated Ca 2+ influx into PC12 cells (inhibited 55-76% for high K+-stimulation periods of
5--40 s). Effects o f nicardipine on high K +-stimulated Ca 2+ influx and neurotransmitter release in rat cerebral neurons Next we examined the effect of nicardipine on the Ca 2+ channel function of rat cerebral cortical neurons using synaptosomes and slices. Fig. 3 shows time courses of the Ca 2+ influxes into the rat cerebral cortical synaptosomes triggered by high K ÷ stimulations. As reported by Nachsen and Blaustein 20, the extrapolating lines of the time-courses did not intersect the origin, implying an initial rapid Ca 2+ influx within the first 5 seconds. Nachsen and Blaustein 19 also showed that under low Ca 2+ concentrations (0.06 mM) nifedipine, another dihydropyridine derivative, failed to suppress the Ca 2÷ influx during the first 5 s. We observed that the Ca 2+ influx into synaptosomes under 2 m M external Ca 2÷ was also unaltered through 30 s by 1/~M nicardipine, which completely suppressed the high K+-stimulated Ca 2+ influx into PC12 cells 25. The lack of inhibition of nicardipine in rat brain neuronal Ca 2+ influx systems was confirmed by the experiments on transmitter releases. Tritiated G A B A preloaded in the synaptosomes or the brain slices was released in response to an exposure to a high K+ environment (Fig. 4A and B). Known Ca 2+ channel blockers, verapamil (30 p M ) and Co2+ (5 raM) significantly suppressed the high K+-stimulated [ 3 H ] G A B A releases from both synaptosomes and brain slices, whereas nicardipine even at 1/~M little affected the amounts and time-courses of the [3H]GABA releases. Unaffected G A B A release from brain slices might exclude the possibility that a nicardipine-sensitivity would have been lost during the preparation of synaptosomes. The assay for high K+-stimulated A T P release from the synaptosomes gave similar results; inhibited significantly (down to 51 + 10% of control) by the presence of 30/~M verapamil but suppressed only slightly (88 + 11% of control) by 1/~M nicardipine.
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Time (rain) Fig. 4. Time courses of high K+-stimulated [3H]GABA releases from rat cerebral cortical synaptosomes (A) and thin slices (B). Circles, triangles, squares and reversed triangles represent the releases under the absence of drug, the presence of 1 ~M nicardipine, the presence of 30/~M verapamil and the presence of 10 mM CoCI 2, respectively. Stimulation schedules are shown in upper bars. Typical result out of 4 similarly repeated experiments (A) or averaged values from 2 repeated experiments (B) are presented.
DISCUSSION In this study we showed that nicardipine, at the concentration which inhibited the high K+-stimulated Ca 2+ influx into PC12 cells (ref. 25 and this report) failed to affect the Ca2+-dependent membrane electrogenesis of the same cells. Some explanations are possible for this apparent discrepancy. First, high K+-stimulation activated the CaZ+ entry system other than the voltage-sensitive Ca2+ channel, such as Na + channel and Na+/Ca 2+ exchange. Participation of the Na + channel is not plausible, since high K+-stimulated Ca 2+ influx into PC12 cells was known to be blocked by Mn 2+, Co 2+ and veraparail, but not by tetrodotoxin 23,26. If nicardipine would enhance the Na+/Ca z+ exchange, the observed decrease in Ca2+ influx would result. But then we might expect a decrease of basal (K+-unstimulated) trans-
mitter release by nicardipine, which was not the case. The second explanation is that nicardipine would affect the open Ca2+ channels only, or that nicardipine's action would be facilitated under depolarized condition of high K +. But repetitive stimulation in a nicardipine-containing medium did not produce an inhibition of Ca2+-dependent action potential in PC12 cell (our unpublished observation, see also ref. 17). And Ca 2+ influx into synaptosomes was not inhibited, even by high K+ stimulation. The third and most plausible explanation might be that PC12 cells have two independent pathways for voltage-dependent Ca2+ incorporation; one is a dihydropyridine-insensitive spike-generating channel, the other is a dihydropyridine-sensitive channel with sustained activation state over 30 s. Coexistence of two populations of voltage-sensitive Ca 2+ pathways is also suggested by previous authors.
329 From the analyses on the high. K÷-stimulated Ca 2÷
Are there entirely different molecules? Alternative-
influx in brain synaptosomes, Nachsen and Blau-
ly, a molecule of Ca 2÷ channel consists of several
stein 19 showed two successive phases of the Ca 2÷ influx, fast-inactivating and slowly-inactivating phases.
functional sites (or subunits) including voltage sen-
From differences in La3+-sensitivity and p e r m e a n t cation selectivity, they suggested the presence of two types of voltage-dependent Ca 2+ channels in synap-
sor/gate, ionophore and drug receptor. A n d a pharmacological variety might owe to a difference in the drug receptor site. Recently a single channel current analysis3 showed a striking similarity in gating kinet-
tosomes. It is known that the Ca 2÷ channels in n e u r o n s are
ics of the voltage-dependent Ca 2÷ channels from var-
less sensitive than those in smooth and cardiac muscle cells to verapamil, another class of Ca 2÷ channel
ron, PC12), suggesting that the model of entirely dif-
blocker II. Thus dihydropyridine derivatives might be another tool for pharmacological classification and
ious sources (snail n e u r o n , vertebrate sensory neuferent molecules is less plausible. ACKNOWLEDGEMENTS
characterization of Ca 2÷ channels from various sources. We are still far from explaining what determines the difference in drug sensitivity of the Ca 2+ channel.
The authors are grateful to Dr. H. H a t a n a k a of this institute for providing PC12h ceils and to Dr. T. m m a n o for encouragement.
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