(Hypertension. 1998;31:1195-1199.)
© 1998 American Heart Association, Inc.
Effects of a Novel Antihypertensive Drug, Cilnidipine, on Catecholamine Secretion From Differentiated PC12 Cells
Hisayuki Uneyama;
Hirohisa Uchida;
Ryota Yoshimoto;
Shinya Ueno;
Kazuhide Inoue;
; Norio Akaike
From the Life Science Laboratories, Central Research Laboratories,
Ajinomoto Co, Inc, Yokohama (H. Uneyama, H. Uchida, R.Y.); the Division of
Pharmacology, National Institute of Health Sciences, Tokyo (S.U., K.I.); and
the Department of Physiology, Faculty of Medicine, Kyushu University, Fukuoka
(N.A.), Japan.
Correspondence to Hisayuki Uneyama, PhD, Life Science Laboratories, Central Research Laboratories, Ajinomoto Co, Ltd, Maeda-Cho 214, Totsuka, Yokohama 244, Japan. E-mail LL-UNEYAMA{at}te3.ajinomoto co.jp
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Abstract
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Abstract—Effects of a novel
dihydropyridine type of antihypertensive drug,
cilnidipine, on the regulation of the catecholamine
secretion closely linked to the intracellular Ca2+ were
examined using nerve growth factor (NGF)–differentiated rat
pheochromocytoma PC12 cells. By measuring catecholamine
secretion with high-performance liquid
chromatography coupled with an electrochemical
detector, we showed that high K+ stimulation evoked
dopamine release from PC12 cells both before and after NGF treatments.
Cilnidipine depressed dopamine release both from NGF-treated and
untreated PC12 cells in a concentration-dependent manner. In contrast,
inhibition by nifedipine was markedly decreased in the
differentiated PC12 cells. With intracellular Ca2+
concentration ([Ca2+]i) measurements using
fura 2, the elevation of high K+–evoked
[Ca2+]i was separated into
nifedipine-sensitive and -resistant components. The
nifedipine-resistant
[Ca2+]i increase was also blocked by
cilnidipine, as well as 
-conotoxin-GVIA. By the use of the
conventional whole-cell patch-clamp technique, the compositions of the
high-voltage–activated Ca2+ channel currents in
the NGF-treated PC12 cells were divided into types: L-type, N-type, and
residual current components. It was also estimated that cilnidipine at
1 and 3 µmol/L strongly blocked the N-type current without
affecting the residual current. These results suggest that cilnidipine
inhibits catecholamine secretion from differentiated PC12
cells by blocking Ca2+ influx through the N-type
Ca2+ channel, in addition to its well-known action on the
L-type Ca2+ channel.
Key Words: cilnidipine • calcium channels • calcium, cytoplasmic • dopamine
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Introduction
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Cilnidipine
(FRC-8653) is a newly synthesized DHP type of organic
Ca2+ channel blocker that has been developed as a
slow-onset and long-lasting antihypertensive drug in
Japan.1 2 Recent
electrophysiological data indicate that
cilnidipine might be a dual-channel antagonist for
peripheral neuronal N-type and vascular L-type
Ca2+ channels.3 4 5 In
humans and rodents, cilnidipine depressed the pressor response to acute
cold stress but failed to induce tachycardia evoked by
hypotensive baroreflexes.6 7 In spontaneously
hypertensive rats, vasoconstriction induced by electrical sympathetic
nerve stimulation was also blocked by
cilnidipine.8 In in vitro experiments,
cilnidipine also inhibited
[3H]norepinephrine release evoked
by electrical stimulation in the rabbit mesenteric
artery.9
PC12 cells are derived from a rat pheochromocytoma cell line that
is very popular for investigating neuronal differentiation. In response
to externally applied NGF, PC12 cells acquire sympathetic neuronal
characteristics such as neurite extension, increased CA synthesis, and
expression of neuronal types of voltage-dependent N-type
Ca2+ channels.10 11 12
Therefore, the differentiated PC12 cells are widely used as model cells
for studying the intracellular mechanisms of the stimulus-secretion
coupling, including the activation of the N-type
Ca2+ channel.13 14 With
this model cell, it has been revealed that endogenous
substances such as neuropeptide Y and proadrenomedullin N-terminal 20
peptide inhibit CA release by inhibiting Ca2+
influx through N-type Ca2+
channels.15 16 In the present study, we
examined the effects of cilnidipine on the cellular functions of the
NGF-treated PC12 cells, including CA secretion and intracellular
Ca2+ mobilization triggered by membrane
depolarizations. Consequently, in addition to its well-known L-type
Ca2+ channel blockade,3 it
was clarified that cilnidipine had a potent inhibitory
effect for CA secretion from differentiated PC12 cells via the blockade
of extracellular Ca2+ influx through the N-type
Ca2+ channel.
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Methods
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Cell Culture
PC12 cells were prepared as described
previously.17 In brief, cells were cultured in
Dulbecco's modified Eagle's medium containing 7% fetal bovine serum
(Gibco), 7% heat-inactivated (56°C, 40 minutes) horse
serum (Cell Culture Laboratories), 2 mmol/L
L-glutamine, and 50 µg/mL gentamicin sulfate
(Boehringer Mannheim GmbH) in a humidified atmosphere of 90%
air and 10% CO2 at 37°C. Cells were plated
onto poly-L-lysine–coated glass coverslips
(24x60x0.15 mm), placed in silicone rubber walls (Flexiperm,
W.C. GmbH) at a density of 2.5x105 cells per
well (8x11 mm), and cultured for an additional 2 days. For
measurement of [Ca2+]i in
single cells, cells were plated onto poly-L-lysine–coated
glass coverslips at a density of 2.5x105 cells
per well (8x11 mm).
Dopamine Release
The released DA was measured as described by Ohara-Imaizumi et
al.18 All the procedures, including incubation,
washing, and drug application, were performed at room temperature using
1 mL per dish of balanced salt solution (BSS) with the following
composition (mmol/L): NaCl 150, KCl 5, CaCl2 1.2,
MgCl2 1.2, D-glucose 10, and HEPES 25
(pH adjusted to 7.4 with NaOH). Cells were incubated with BSS for 1
hour, and then the BSS containing 80 mmol/L KCl was added to the
dishes and incubated for 1 minute. At the end of the incubation period,
solutions were transferred to tubes containing 0.25 mL of 1 mol/L
HClO4. Cells were then sonicated, and DA residing
in cells was extracted with 0.2 mol/L HClO4.
After centrifugation (at 4°C for 5 minutes,
10 000g), supernatants of both incubation solutions and the
sonicated cellular solutions were collected for the measurement of DA
content. DA content was determined with high-performance liquid
chromatography coupled with an electrochemical detector
(HPLC/ECD). Each supernatant (20 µL) was applied to the HPLC/ECD
system, which consisted of a reverse-phase HPLC column (ODS, 0.003
mm; 4.6x150 mm) and an ECD (LC-4B and TL-5A, Bioanalytical
Systems) with the electrode potential set at +0.6 V versus the Ag/AgCl
reference electrode. The mobile phase consisted of a monochloroacetate
buffer (140 mmol/L, pH 3.05) containing 10% methanol, 30 mg/L
sodium 1-octanesulfonate, and 1.8 mmol/L EDTA. Flow rate was 1.3
mL/min. The data were analyzed with a
chromatographic processor (Shimazu C-R4A, Shimazu Corp).
The percentage of DA release was calculated using the values obtained
for the DA content in the incubation solution (A) and the DA content
remaining in the cells (B) using the following equation: % of total DA
contents=100xA/(A+B).
[Ca2+]i Measurements
The increase in
[Ca2+]i in single cells
was measured by the fura 2 method as described by Grynkiewicz et
al19 with minor modifications. The cells were
washed with BSS and incubated with 10 µmol/L fura 2-AM (Dojindo)
at 37°C in BSS. Thirty minutes later, the cells were washed twice
with 0.2 mL BSS. The coverslips were mounted on an Olympus IMT-2
inverted epifluorescence microscope equipped with a 75-W xenon
lamp and band-pass filters of 340-nm wavelength for detection of
Ca2+-dependent signal
(F340) and 360-nm wavelength for detection of
Ca2+-independent signal
(F360). Measurements were carried out at room
temperature. Image data, recorded by a high-sensitivity silicon
intensifier target camera (C-2741-08, Hamamatsu Photonics), were
processed by a personal computer.
Electrophysiological Measurements
Electrical measurements were performed in a whole-cell mode by
using a conventional patch-clamp technique.20 21
The resistance between the recording electrode filled with
internal solution and reference electrode in external solution was 2 to
5 M
. The current and voltage were measured with a patch-clamp
amplifier (List Medical, EPC-7), monitored on a storage oscilloscope
(Iwatsu DS-9121), and then stored on DAT tape with a PCM processor
(TEAC RD-120TE) after being filtered at 1 kHz (NF Instruments). Then,
analog signals were reconverted into digital signals with an AD
converter at a sampling frequency of 3 kHz; the signals were stored
with an IBM-compatible computer using pClamp software (Axon
Instruments). The capacitative and leak currents were subtracted by
hyperpolarizing pulses. All experiments were performed at room
temperature (20°C to 22°C). The ionic composition of
Ba2+ external solution was (in mmol/L)
tetraethylammonium Cl 130, CsCl 4,
MgCl2 1, BaCl2 10, HEPES
10, and glucose 10. The pH was adjusted to 7.4 with Tris-OH. The
patch-pipette solution contained (in mmol/L) CsCl 75,
Cs-methanesulfonate 75, ATP-Mg 2, EGTA 5, and HEPES 10. The pH was
adjusted to 7.2 with Tris-OH.
Drugs
-Cg-GVIA was purchased from the Peptide Institute (Osaka,
Japan). Fura 2-AM and HEPES were from Dojindo. NGF (7S) was from Gibco
BRL. Nifedipine and all other chemicals were obtained from
Sigma Chemical Co. Cilnidipine
[2-methoxyethyl(E)-3-phenyl-2-propen-1-yl(±)-1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)
pyridine-3,5-dicarboxylate] was synthesized at the Ajinomoto central
research laboratories (Kawasaki, Japan). All drugs were dissolved in
external solution just before use.
Data Analysis
Experimental values are presented as mean±SEM.
Statistical differences in values for DA release or
[Ca2+]i increase were
determined using ANOVA followed by Dunnett's test for multiple
comparison; a value of P<.05 was considered to be
significant.
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Results
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Effects of Cilnidipine on CA Secretion From Differentiated
PC12 Cells
Rat PC12 cells exhibited neurite outgrowth in response to
externally applied NGF (100 ng/mL) for 8 days (Fig 1
). Rat PC12 cells used in the
present study were changed to contain norepinephrine in
addition to DA. However, since the amount of norepinephrine
content was less than that of DA (data not shown), we measured the rate
of DA production as an indication of the CA release process in
the following experiments. Depolarization with 80 mmol/L KCl
evoked DA release from the PC12 cells both before and after the NGF
treatments (8.53±0.65% release of a total DA content in
undifferentiated PC12 cells and 7.21±0.49% in the differentiated PC12
cells; each n=12). This stimulation was dependent on extracellular
Ca2+ influx through voltage-dependent
Ca2+ channels because it was completely prevented
by addition of 100 µmol/L Cd2+, a
nonselective inorganic Ca2+ channel
antagonist (n=3). The DHP types of
Ca2+ channel antagonists, cilnidipine
and nifedipine, strongly blocked DA release from the
undifferentiated PC12 cells (Fig 2). The
IC50 values for cilnidipine and
nifedipine were 
6x10-7 and
8x10-8 mol/L, respectively. In contrast, in the
NGF-differentiated PC12 cells, inhibitory potency of
nifedipine was markedly reduced (Fig 2B), whereas the
inhibitory effect of cilnidipine was almost equal (Fig 2A)
to that observed in the PC12 cells. The IC50
values for cilnidipine and nifedipine were
7x10-8 and >10-5 mol/L,
respectively. NGF differentiation also reduced the blocking potency of
L-type Ca2+ channel antagonists other
than nifedipine, such as verapamil
(phenylalkylamine-type) and diltiazem (benzothiazepine-type). The
inhibitory percentages of 10 µmol/L
verapamil and 10 µmol/L diltiazem on 80 mmol/L
KCl–evoked DA release were 82.48±2.06% and 72.27±5.53% (each, n=3)
in the undifferentiated PC12 cells and 37.99±2.29% and 41.02±1.40%
(each, n=3) in the NGF-differentiated PC12 cells, respectively. In
contrast, an N-type Ca2+ channel
antagonist peptide, -Cg-GVIA, had no effect on the high
K+–evoked DA release from undifferentiated cells
(0.84±6.29% inhibition, n=6) but inhibited the DA release from the
differentiated PC12 cells (39±5.1% inhibition, n=6). This observation
strongly indicated that the DA release mediated by the N-type
Ca2+ channel activation was caused by the cell
differentiation.
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Figure 1. Phase-contrast micrographs of the undifferentiated
(A) and differentiated (B) rat PC12 cells used in the present
study. Each culture condition is described in the text. After 8 days of
100 ng/mL NGF treatment, rat PC12 cells were well differentiated enough
to exhibit neurite outgrowth. Scale bar, 60 µm.
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As a next step, we estimated whether the potent blocking action
of cilnidipine on the DA release from the differentiated cells might be
derived from the blockade of channels other than the L-type
Ca2+ channel. We examined the effect of
cilnidipine on the residual releases after abolishing the L-type
Ca2+ channel–dependent DA release by
nifedipine. About 58% of the high
K+–evoked DA releases from the
NGF-differentiated cells remained, even in the presence of 10
µmol/L nifedipine (n=6). Under these conditions, it was
clarified that cilnidipine (1 µmol/L) as well as -Cg-GVIA
(1 µmol/L) inhibited the
nifedipine-resistant DA release (Fig 3).
Effects of Cilnidipine on [Ca2+]i
Elevation in Differentiated Rat PC12 Cells
Because Ca2+ influx through
Ca2+ channels is closely related to the CA
secretion in rat PC12 cells, we measured
[Ca2+]i in differentiated
PC12 cells. Eight days after NGF treatment,
[Ca2+]i elevated by
80 mmol/L KCl was blocked by 25±2% with nifedipine
(n=18). As shown in Fig 4, 80 mmol/L KCl evoked a rapid
[Ca2+]i elevation even in
the presence of a high concentration of nifedipine (10
µmol/L), and the nifedipine-resistant
[Ca2+]i elevation was
successfully inhibited by the further administration of -Cg-GVIA
(Fig 4A, a) or cilnidipine in a
concentration-dependent manner (Fig 4A, b). Under this experimental
condition, we confirmed that the initial high K+
response was reproducible at least for four stimulations (data not
shown, n=3). The quantitative results are summarized in Fig 4B. The
nifedipine-resistant
[Ca2+]i elevation was
significantly blocked by cilnidipine as well as -Cg-GVIA. In
addition, the nifedipine- and
-Cg-GVIA–resistant
[Ca2+]i elevation was not
influenced by the further application of 10 µmol/L cilnidipine
(72±9% inhibition, nifedipine plus -Cg-GVIA; 68±11%
inhibition, nifedipine plus -Cg-GVIA plus cilnidipine,
each n=10). These results suggest that cilnidipine blocks
Ca2+ influx through
nifedipine-resistant Ca2+
channels (possibly N-type).
Cilnidipine Blockade of HVA Ca2+ Current in
Differentiated Rat PC12 Cells
Finally, the direct effect of cilnidipine on the
Ca2+ channels in the differentiated rat PC12
cells was investigated by using the conventional whole-cell patch-clamp
technique. To suppress the Ca2+-dependent
inactivation, we used 10 mmol/L Ba2+ instead
of Ca2+ as a charge carrier through the HVA
Ca2+ channels. The typical current traces are
shown in Fig 5A, a. In this experiment,
1 µmol/L -Cg-GVIA was first applied to the cell, then 10
µmol/L nifedipine was applied, and finally 100
µmol/L Cd2+ was applied. The composition of the
HVA IBa was classified into current
compounds that were -Cg-GVIA–sensitive (N-type: 40±12% of total
HVA IBa, n=6),
nifedipine-sensitive (L-type: 13±2% of total HVA
IBa, n=6), and insensitive to those two
compounds (possibly R-type: 37±7% of total HVA
IBa, n=6). Cilnidipine blocked the HVA
IBa after the L-type component was
abolished by 10 µmol/L nifedipine (Fig 5A, b and 5B).
Blocking potencies of 3 and 10 µmol/L cilnidipine for the
-Cg-GVIA–sensitive IBa component were
53±8% and 75±10%, respectively (n=4). Finally, the effect of
cilnidipine on the residual non–L-type and N-type component was also
examined (Fig 6). In this experiment, L- and N-type components were
completely abolished by treatment with 10 µmol/L
nifedipine and 1 µmol/L -Cg-GVIA (Fig 6A). Consequently, cilnidipine failed to
inhibit the -Cg-GVIA–and nifedipine-resistant
current up to 3 µmol/L. These results suggest that cilnidipine
blocks -Cg-GVIA–sensitive N-type current component in
differentiated rat PC12 cells.
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Discussion
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In the present experiment, we investigated the effects of
cilnidipine on DA release,
[Ca2+]i elevation, and
Ca2+ channel currents in differentiated rat PC12
cells. The PC12 cells used in this study clearly showed neurite
outgrowth, and 50% of HVA Ca2+ channel
currents were composed of -Cg-GVIA–sensitive N-type
Ca2+ currents. A typical L-type
Ca2+ channel blocker such as
nifedipine had a small effect on the
Ca2+ channel–operated functions. These
characteristics are consistent with those of differentiated
PC12 cells, which have characteristics similar to those of
peripheral sympathetic neurons, including the development
of N-type Ca2+
channels.10 11 12 22
In the present study, we clearly showed that a DHP type of
antihypertensive drug, cilnidipine, strongly inhibited the high
K+–evoked CA secretion,
[Ca2+]i elevation, and
HVA Ca2+ channel currents in differentiated rat
PC12 cells. In contrast to cilnidipine, the selective L-type
Ca2+ channel antagonist
nifedipine showed a weak inhibition on all three
parameters in the PC12 cells after differentiation by NGF
(Fig 1B
). Cilnidipine directly blocked the isolated N-type channel
current (IC50 value, 1 µmol/L) but had
no effect on the R-type channel current in the differentiated rat PC12
cells (Figs 5 and 6). This observation agreed with previous findings
using rat superior ganglion neurons5 and dorsal
horn ganglion neurons.4 Blockade of N-type
Ca2+ channels by cilnidipine results in reduced
Ca2+ influx through these channels and thereby
reduces CA secretions closely linked with
[Ca2+]i elevation, which
is evoked by various depolarizing stimulations. It is well
characterized that Ca2+ influx through N-type
Ca2+ channels is closely related to the CA
secretion at the nerve endings of the sympathetic neurons as
well.23 The blockade of CA secretion from the
differentiated PC12 cells might well explain why cilnidipine failed to
induce elevation of plasma CA concentration by hypotension-evoked
baroreflexes in vivo7 or why the drug blocked the
norepinephrine release from isolated
vessels.9
Sources of plasma CA were mainly sympathetic nerve endings and adrenal
chromaffin cells, at which N-type Ca2+ channels
were predominantly distributed. It was reported that the blockade of
peripherally distributed N-type Ca2+
channels by -Cg-GVIA led to reduction of plasma CA concentration by
inhibition of its secretion from the vascular
beds24 and adrenal
glands.25 Especially, it is well known that CA
secretion from sympathetic neurons is insensitive to L-type
Ca2+ channel antagonist but sensitive
to -Cg-GVIA.23 Therefore, cilnidipine with
N-type Ca2+ channel blocking action may offer a
new choice in treating hypertension which is refractory to hypotensive
Ca2+ channel antagonists or results
from increased sympathetic nerve activity. In keeping with these
therapeutic implications, recent observations in rats and humans
revealed that the oral administration of cilnidipine successfully
reduced blood pressure elevated by stress
stimulations.6 8 It might also be clinically used
for controlling malignant hypertension resulting in hypersecretion of
CA from human pheochromocytoma, because the PC12 cells used here are
derived from rat pheochromocytoma cells.
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Selected Abbreviations and Acronyms
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| CA |
= |
catecholamine |
| -Cg-GVIA |
= |
-conotoxin GVIA |
| DA |
= |
dopamine |
| DHP |
= |
dihydropyridine |
| HVA IBa |
= |
high-voltage–activated Ba2+ current |
| NGF |
= |
nerve growth factor |
|
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Acknowledgments
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We are grateful to Dr Shuichi Koizumi, National Institute of
Health Sciences (NIHS; Japan) for helpful comments and Hiroko Obama,
NIHS, for technical assistance.
Received August 8, 1997;
first decision September 22, 1997;
accepted January 2, 1998.
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References
|
|---|
1.
Yoshimoto R Dohmoto H, Yamada K, Goto A. Prolonged
inhibition of vascular contraction and calcium influx by the novel
1,4-dihydropyridine calcium antagonist
cinaldipine. Jpn J Pharmacol. 1991;56:225–229.[Medline]
[Order article via Infotrieve]
2.
Hosono M, Iida H, Ikeda K, Hayashi Y, Dohmoto H,
Hashiguchi Y, Yamamoto H, Watanabe N, Yoshimoto R. In vivo and ex vivo
Ca-antagonistic effect of 2-methoxyethyl
(E)-3-phenyl-2-propen-1-yl
(±)-1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)
pyridine-3,5-dicarboxylate (FRC-8653), a new
dihydropyridine derivative. J
Pharmacobiodyn. 1992;15:547–553.[Medline]
[Order article via Infotrieve]
3.
Oike M, Inoue Y, Kitamura K, Kuriyama H. Dual action
of FRC8653, a novel dihydropyridine derivative, on
the Ba2+ current recorded from the rabbit
basilar artery. Circ Res. 1990;1990:993–1006.
4.
Fujii S, Kameyama K, Hosono M, Hayashi Y, Kitamura K.
Effect of cilnidipine, a novel dihydropyridine
Ca++-channel antagonist, on N-type
Ca++ channel in rat dorsal root ganglion neurons.
J Pharmacol Exp Ther. 1997;280:1184–1191.[Abstract/Free Full Text]
5.
Uneyama H, Takahara A, Dohmoto H, Yoshimoto R, Inoue
K, Akaike N. Blockade of N-type Ca2+ current by
cilnidipine (FRC-8653) in acutely dissociated rat sympathetic neurones.
Br J Pharmacol. 1997;122:37–42.[Medline]
[Order article via Infotrieve]
6.
Saihara S, Sumio M, Hosono N, Hayashi Y, Kitamura Y,
Hashimoto S. Effect of FRC-8653 (cilnidipine) on blood pressure changes
during the cold pressor test. Jpn Pharmacol Ther. 1993;21:271–276.
7.
Hosono M, Fujii S, Himura T, Watanabe K, Hayashi Y,
Ohnishi H, Takata Y, Kato H. Inhibitory effect of
cilnidipine on vascular sympathetic neurotransmission and subsequent
vasoconstriction in spontaneously hypertensive rats. Jpn J
Pharmacol. 1995;69:127–134.[Medline]
[Order article via Infotrieve]
8.
Hosono M, Himura T, Watanabe K, Hayashi Y,
Ohnishi H, Takata Y, Kato H. Inhibitory effect of
cilnidipine on pressor response to acute cold stress in spontaneously
hypertensive rats. Jpn J Pharmacol. 1995;69:119–125.[Medline]
[Order article via Infotrieve]
9.
Nakashima M, Kou M, Hashitani H, Chen G, Ono H,
Kuriyama H, Suzuki H. Action of FRC-8653 on smooth muscle cells of the
rabbit mesenteric artery. Jpn J Pharmacol. 1991;57:51–61.[Medline]
[Order article via Infotrieve]
10.
Greene LA, Tischler AS. PC12 pheochromocytoma cultures
in neurobiological research. Adv Cell Neurobiol. 1982;3:373–414.
11.
Takahashi M, Tsukui H, Hamada H. Neuronal
differentiation of Ca2+ channel by nerve growth
factor. Brain Res. 1985;341:381–384.[Medline]
[Order article via Infotrieve]
12.
Usowicz M, Porzig M, Becker C, Reuter H. Differential
expression by nerve growth factor by two types of
Ca2+ channels in rat phaeochromocytoma cell line.
J Physiol (Lond). 1990;426:95–116.[Abstract/Free Full Text]
13.
Passafaro M, Rosa P, Sala C, Clementi F, Sher E. N-type
Ca2+ channels are present in secretory
granules and are transiently translocated to the plasma membrane during
regulated exocytosis. J Biol Chem. 1996;271:30096–30104.[Abstract/Free Full Text]
14.
Desole MS, Kim WK, Rabin RA, Laychock SG. Nitric oxide
reduces depolarization-induced calcium influx in PC12 cells by a cyclic
GMP-mediated mechanism. Neuropharmacology. 1994;33:193–198.[Medline]
[Order article via Infotrieve]
15.
Chen X, Westfall TC. Modulation of intracellular
calcium transients and dopamine release by neuropeptide Y in PC12
cells. Am J Physiol. 1994;266:C784–C793.[Abstract/Free Full Text]
16.
Takano K, Yamashita N, Fujita T. Proadrenomedullin
NH2-terminal 20 peptide inhibits the
voltage-gated Ca2+ channel current through a
pertussis toxin-sensitive G protein in rat pheochromocytoma-derived
PC12 cells. J Clin Invest. 1996;98:14–17.[Medline]
[Order article via Infotrieve]
17.
Inoue K, Kenimer JG. Muscarinic stimulation of calcium
influx and norepinephrine release in PC12 cells.
J Biol Chem. 1988;263:8157–8161.[Abstract/Free Full Text]
18.
Ohara-Imaizumi M, Nakazawa K, Obama T, Fujimori K,
Takanaka A, Inoue K. Inhibitory action of
peripheral-type benzothiazepines on dopamine release from
PC12 pheochromocytoma cells. J Pharmacol Exp Ther. 1991;259:484–489.[Abstract/Free Full Text]
19.
Grynkiewicz G, Poenie M, Tsien RY. A new generation of
Ca2+ indicators with greatly improved
fluorescence properties. J Biol Chem. 1985;260:3440–3450.[Abstract/Free Full Text]
20.
Hamill OP, Marty A, Neher E, Sakmann B, Sigworth
FJ. Improved patch-clamp technique for high resolution current
recording from cells and cell-free membrane patch.
Pflugers Arch Eur J Physiol. 1981;391:85–100.[Medline]
[Order article via Infotrieve]
21.
Uneyama H, Ueno S, Akaike NA.
Serotonin-operated potassium current in CA1 neurons
dissociated from rat hippocampus. J Neurophysiology. 1993;69:1044–1052.[Abstract/Free Full Text]
22.
Mullikin-Kilpatrick D, Treistman SN. Inhibition of
dihydropyridine-sensitive
Ca2+ channels by ethanol in undifferentiated and
nerve growth factor-treated PC12 cells: interaction with the
inactivated state. J Pharmacol Exp Ther. 1995;272:489–497.[Abstract/Free Full Text]
23.
Hirning LD, Fox AP, McCleskey EW, Olivera BM, Thayer
SA, Miller RJ, Tsien RW. Dominant role of N-type
Ca2+ channels in evoked release of
norepinephrine from sympathetic neurons.
Science. 1988;239:57–61.[Abstract/Free Full Text]
24.
Friedman DJ, Duckles SP. Influence of age on control of
norepinephrine release: Ca2+ channels
and dopamine D2 receptors. Eur J
Pharmacol. 1994;252:1–9.[Medline]
[Order article via Infotrieve]
25.
Kimura T, Takeuchi A, Satoh S. Inhibition by
omega-conotoxin GVIA of adrenal catecholamine release in
response to endogenous and exogenous acetylcholine.
Eur J Pharmacol. 1994;264:169–175.[Medline]
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Abstract
Introduction
) and differentiated (
) rat PC12 cells.
Cells were preincubated with various concentrations of Ca2+
channel antagonists for 4 minutes. After preincubation,
cells were stimulated with 80 mmol/L KCl for 1 minute in the
presence of the drugs. Data are shown as percentages of DA amounts
released by high K+. Each point and vertical bar
represent mean±SEM from three distinct determinations.
