Department of Life Science, Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang 790-784, Korea
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ABSTRACT |
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Extracellular ATP elevates cytosolic Ca2+ by activating P2X and P2Y purinoceptors and voltage-sensitive Ca2+ channels (VCCCs) in PC-12 cells, thereby facilitating catecholamine secretion. We investigated the mechanism by which ATP activates VSCCs. 2-Methylthioadenosine 5'-triphosphate (2-MeS-ATP) and UTP were used as preferential activators of P2X and P2Y, respectively. Nifedipine inhibited the ATP- and 2-MeS-ATP-evoked cytosolic Ca2+ concentration increase and [3H]norepinephrine secretion, but not the UTP-evoked responses. Studies with Ca2+ channel blockers indicated that L-type VSCCs were activated after the P2X activation. Mn2+ entry profiles and studies with thapsigargin revealed that Ca2+ entry, rather than Ca2+ release, was sensitive to nifedipine. Although P2X2 and P2X4 receptor mRNAs were detected, studies with pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid revealed that P2X2 was mainly coupled to the L-type VSCCs. The inhibitory effect of nifedipine did not occur in the absence of extracellular Na+, suggesting that Na+ influx, which induces depolarization, was essential for the P2X2-mediated activation of VSCCs. We report that depolarization induced by Na+ entry through the P2X2 purinoceptors effectively activates L-type VSCCs in PC-12 cells.
ATP; nifedipine; depolarization
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INTRODUCTION |
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EXTRACELLULAR NUCLEOTIDES are important signaling molecules that mediate diverse biological processes such as neurotransmitter and hormone secretion and contraction of smooth muscle via cell surface receptors called purinoceptors (for review see Refs. 13 and 39). Purinoceptors have been divided into two types, P1 (also called adenosine receptors) and P2, on the basis of pharmacological, biochemical, and molecular evidence. P2 purinoceptors recognize primarily ATP, ADP, UTP, and UDP. On the basis of differences in molecular structure and signal transduction mechanisms, they are subdivided into P2X and P2Y, which are ligand-gated ion channels and G protein-coupled receptors, respectively (1, 16). P2X and P2Y receptors are known to increase intracellular Ca2+ levels. P2X receptors increase intracellular Ca2+ levels by inducing rapid influx of nonselective cations such as Ca2+, Na+, and K+ across the cell membrane (9, 35), whereas P2Y receptors act by mobilizing Ca2+ from intracellular stores and by allowing store-operated Ca2+ entry (46). Seven mammalian P2X receptors (P2X1-P2X7) and five mammalian P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11) have been cloned, characterized, and accepted as valid members of the P2 receptor family.
PC-12 cells are neuroendocrine cells derived from the pheochromocytoma of a rat adrenal medulla. PC-12 cells have been reported to express P2X and P2Y purinoceptors (3, 7, 10, 28). Extracellular ATP has been shown to elevate cytosolic free Ca2+ concentration ([Ca2+]i) through P2X and P2Y purinoceptors, although there are often marked differences among the results reported by different groups.
Electrophysiological and pharmacological studies have revealed that PC-12 cells express L-type (long-lasting) and N-type (neuronal) voltage-sensitive Ca2+ channels (VSCCs) (22, 29, 34, 37, 44). Because VSCCs are activated by membrane depolarization that is induced via cation entry through channels permeable to Na+, K+, and Ca2+, we looked for possible interaction between VSCCs and P2 purinoceptors in PC-12 cells. Our data show that Ca2+ channel blockers inhibited the P2X2 purinoceptor-mediated [Ca2+]i rise and catecholamine secretion. Here we report that membrane depolarization caused by Na+ entry through P2X2 purinoceptors effectively activates L-type VSCCs, while a similar extent of depolarization induced by 17.5 mM K+ does not evoke any detectable [Ca2+]i increase.
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MATERIALS AND METHODS |
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Materials.
Nifedipine, verapamil, sulfinpyrazone, ATP, UTP, and EGTA were
purchased from Sigma Chemical (St. Louis, MO). SK&F-96365 and pyridoxal
phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) were obtained from
Research Biochemical International (Natick, MA), thapsigargin (TG)
and-conotoxin GVIA from Alomone Laboratories (Jerusalem, Israel),
bis(1,3-diethylthiobarbiturate)-trimethineoxonol (bisoxonol) and fura
2-AM from Molecular Probes (Eugene, OR), 2-methylthioadenosine
5'-triphosphate (2-MeS-ATP) from Tocris Cookson (Bristol, UK), and
[3H]norepinephrine ([3H]NE; specific
activity 14.68 Ci/mmol) from NEN Life Science Products (Boston, MA).
Cell culture. PC-12 cells were grown in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 10% (vol/vol) heat-inactivated bovine calf serum (Hyclone, Logan, UT), 5% heat-inactivated horse serum (Hyclone), and 1% penicillin-streptomycin (Life Technologies) in a humidified atmosphere of 5% CO2-95% air at 37°C. The culture medium was changed every 2 days, and the cells were subcultured weekly.
Measurement of [3H]NE secretion. The release of [3H]NE from PC-12 cells was measured as reported previously (26). Briefly, PC-12 cells were transferred to 24-well plates that had been coated with rat tail collagen (17) at a density of 5 × 105 cells/well. After the cells were allowed to stabilize for 1 day, they were loaded with [3H]NE (1 µCi/ml, 68 pmol/ml) during incubation for 1 h at 37°C in serum-free RPMI 1640 medium containing 0.1 mM ascorbic acid. The cells were then washed twice with Locke's solution and incubated in Locke's solution for 15 min for stabilization. The cells then were again incubated with fresh Locke's solution for 10 min to measure basal secretion of [3H]NE. After the basal secretion was measured twice, the cells were incubated for 10 min in Locke's solution containing the drug under test. The medium was then removed from each well and centrifuged at 2,000 g for 30 s to exclude detached PC-12 cells from the supernatant. Residual catecholamine in each well was extracted from the cells by addition of 0.1 N HCl. Scintillation cocktail was added to the medium and the cell extract, and radioactivity was measured in a scintillation counter. The amount of [3H]NE secreted is expressed as a percentage of total [3H]NE content.
Measurement of cytosolic Ca2+.
[Ca2+]i was determined by using the
fluorescent Ca2+ indicator fura 2, as reported previously
(35). Briefly, PC-12 cell suspensions were incubated in
fresh serum-free RPMI 1640 medium containing fura 2-AM (3 µM) for 30 min at 37°C under continuous stirring. The cells were then washed
with Locke's solution and left at room temperature until use.
Sulfinpyrazone (250 µM) was added to all solutions to prevent dye
leakage (12). Fluorescence ratios were measured by using
an alternative wavelength time-scanning method (dual excitation at 340 and 380 nm; emission at 500 nm). Before each experiment, cells were
preincubated at 37°C for 1 min during continuous stirring; then the
fluorescence signal was recorded. Cells were stirred continuously
during the stimulation. The signal was calibrated by addition of 4 mM
CaCl2 and 0.1% Triton X-100 to obtain maximal
fluorescence. Tris-base (30 mM) was used to neutralize acidic pH, and 4 mM EGTA was added for chelation of extracellular Ca2+ to
obtain minimal fluorescence. Calibration of the fluorescent signal in
terms of [Ca2+]i was performed as described
by Grynkiewicz et al. (19), according to the following
formula: [Ca2+]i = [(R Rmin)/(Rmax
R)] × (Sf2/Sb2) × Kd, where R
is fluorescence ratio and a dissociation constant
(Kd) of 224 nM was used for fura 2.
Mn2+ quenching of fura 2 fluorescence. PC-12 cells preloaded with fura 2-AM as described above were stimulated with ATP, 2-MeS-ATP, and UTP in the presence of 500 µM Mn2+, and changes in fluorescence were measured at the excitation wavelength of 360 nm, which is an isosbestic wavelength, and at the emission wavelength of 500 nm.
RT-PCR. Total RNA was extracted from the PC-12 cells by TRI reagent (Molecular Research Center, Cincinnati, OH). Ten micrograms of total RNA were reverse-transcribed with the use of Superscript II reverse transcriptase (GIBCO BRL, Life Technologies). cDNA was amplified with 20 pmol of specific oligonucleotide primers (Bioneer) using Pfu polymerase (Stratagene, La Jolla, CA). Primer sequences used for the seven subtypes and the reaction conditions were as reported previously (43). The PCR products were separated by electrophoresis. The products were subcloned into a pGEM-T Easy Vector (Promega, Madison, WI) and sequenced with a dideoxynucleotide termination method (40a).
Northern blot analysis.
Total RNA was extracted as described above, and Northern blot analysis
was performed as reported previously (8) with minor modifications. Briefly, total RNA (15 µg) was resolved by
electrophoresis through a 1% agarose gel containing 0.66 M
formaldehyde and transferred to nylon membranes (ICN, East Hills, NY).
The blots were reacted with a P2X2 or a P2X4
cDNA probe labeled with [-32P]dCTP by the random
primer extension method. The hybridization proceeded at 65°C in a
solution containing 10% polyethylene glycol, 7% SDS, 10 mM EDTA, 0.25 M NaCl, 0.085 M Na2HPO4 (pH 7.2), denatured salmon sperm DNA (100 µg/ml), and the radiolabeled probe (5 × 105 cpm/ml). After hybridization, the blot was washed
briefly once in 1× saline-sodium citrate (SSC, i.e., 0.3 M NaCl and
0.03 M sodium citrate) containing 0.1% SDS at room temperature, twice in 0.2× SSC containing 0.1% SDS at 65°C, and once in 0.1× SSC at
room temperature. The filter was then reprobed with a rat
-tubulin cDNA as an internal control.
Measurement of membrane potential with bisoxonol. Changes in the membrane potential were monitored using a fluorescent potential-sensitive anionic dye, bisoxonol, as reported previously (10) with minor modifications. Briefly, after PC-12 cells were preincubated for 1 h at 37°C in incubation buffer (145 mM NaCl, 5 mM KCl, 1.25 mM CaCl2, 0.8 mM MgCl2, 20 mM glucose, and 25 mM HEPES, pH 7.4), they were washed and resuspended at a density of 1.5 × 107 cells/ml. The cells (1.5 × 106 cells/ml) were then incubated with 500 nM bisoxonol for 10 min at 37°C before the addition of stimulants. Valinomycin (1 µM) was added at the end of each experiment to make comparison between different experiments possible. Fluorescence was measured at the excitation wavelength of 540 nm and at the emission wavelength of 580 nm.
Statistical analysis. Statistical analysis of the data was done with the unpaired Student's t-test for comparison between two experimental groups. Differences were considered significant when P < 0.05.
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RESULTS |
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Effects of Ca2+ channel inhibitors on P2X-mediated [Ca2+]i increase and [3H]NE secretion. Extracellular ATP increases [Ca2+]i by opening channels in the plasma membrane and by releasing Ca2+ from intracellular Ca2+ stores. Fasolato et al. (15) reported that extracellular ATP induces membrane depolarization and that the [Ca2+]i increase evoked by ATP seemed to depend in part on the indirect activation of VSCCs. To determine which type of P2 purinoceptor was linked to the VSCC-mediated Ca2+ signaling, we used 2-MeS-ATP and UTP as preferential agonists for P2X and P2Y purinoceptors, respectively (35).
PC-12 cells are known to express mainly L- and N-type VSCCs, and we previously reported that 70 mM K+-induced [Ca2+]i increase was inhibited by nifedipine, a specific L-type Ca2+ channel blocker, to 77 ± 3%, and by
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Decrease of P2X-mediated
[Ca2+]i rise in depolarized
cells.
To confirm that VSCCs were activated by stimulating P2X purinoceptors,
VSCCs were desensitized by high-K+ pretreatment of the
cells. When PC-12 cells were pretreated with 70 mM K+,
which causes membrane depolarization, thereby leading to the activation
and subsequent desensitization of VSCCs, the extracellular ATP-evoked
[Ca2+]i increase was reduced to 72 ± 5% (Fig. 2). The 2-MeS-ATP-evoked Ca2+ rise was reduced by the 70 mM K+
pretreatment to 45 ± 6% of the control, whereas the UTP-evoked [Ca2+]i increase was not affected. Together,
these results suggest that VSCCs are selectively coupled to the P2X
purinoceptors in PC-12 cells.
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Inhibition of P2X-mediated cation entry by
Ca2+ channel blockers.
Because P2X purinoceptors increase [Ca2+]i
not by releasing Ca2+ from intracellular stores but by
triggering Ca2+ entry, we investigated whether
Ca2+ channel blockers would inhibit Ca2+ entry.
First, we studied the effect of Ca2+ channel blockers after
depletion of intracellular Ca2+ stores with TG to confirm
that the Ca2+ channel blockers do not affect the
Ca2+ release from intracellular Ca2+ stores but
inhibit Ca2+ influx. When PC-12 cells were treated with TG
before the P2 purinoceptor stimulation, 2-MeS-ATP and ATP increased
[Ca2+]i by a similar amount. No
[Ca2+]i increase was evoked by UTP, which is
known to increase [Ca2+]i by releasing
Ca2+ from intracellular Ca2+ stores and
subsequent Ca2+ influx through Ca2+
release-activated channels (Fig. 3).
Treatment with Ca2+ channel blockers by themselves did not
cause any change in the TG-induced Ca2+ elevation.
Treatment of the cells with -conotoxin GVIA did not have an
inhibitory effect on the P2X-mediated [Ca2+]i
increase. Nifedipine and verapamil, dihydropyridine and
phenylalkylamine class inhibitors of VSCCs, respectively, reduced the
ATP- and 2-MeS-ATP-evoked [Ca2+]i increase by
a comparable amount after intracellular Ca2+ store
depletion. These results provide additional evidence for the coupling
of P2X purinoceptors to L-type VSCCs. We, therefore, used nifedipine
alone in the following experiments to block the VSCCs that are coupled
to P2X purinoceptors in PC-12 cells.
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P2X receptor subtypes coupled to L-type VSCCs.
Expression of the P2X receptor mRNA in PC-12 cells was tested by RT-PCR
using seven pairs of specific primers for
P2X1-P2X7. As shown in Fig.
5A, amplified products of the
expected size for P2X2 and P2X4 were detected.
We then performed Northern blot analysis to quantify the mRNA levels of
P2X2 and P2X4 receptors. As shown in Fig.
5B, the expression level of the P2X2 receptor
mRNA was significantly higher than that of the P2X4
receptor, and the densitometric values revealed that expression of the
P2X2 receptor mRNA was more than threefold higher than that
of the P2X4 receptor mRNA when normalized with the
expression of -tubulin mRNA.
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Activation of VSCCs by membrane depolarization via
Na+ influx through P2X2
purinoceptors.
P2X2 purinoceptors are nonselective cation channels
permeable to Na+ as well as Ca2+. We studied
the relative amount of membrane depolarization induced by the
activation of the P2 purinoceptors using ATP, 2-MeS-ATP, and UTP.
Changes in membrane potential were measured by using the lipophilic
anionic fluorescent dye bisoxonol. Depolarization causes the dye anions
to bind to the membrane, thus increasing net fluorescence
(40). Figure 6 shows the
fluorescence profiles of PC-12 cells in suspensions equilibrated with
500 nM bisoxonol and then stimulated with ATP, 2-MeS-ATP, or UTP. ATP
and 2-MeS-ATP induced a rapid increase in fluorescence indicative of
depolarization. Addition of UTP caused only a weak increase in the
fluorescence intensity, which was substantially lower in magnitude than
that caused by ATP. When the magnitude of depolarization was compared with valinomycin (1 µM)-induced hyperpolarization as control, ATP-,
2-MeS-ATP-, and UTP-induced depolarization was 20.9 ± 1.8, 18.5 ± 2.1, and 6.0 ± 2.2%, respectively, of the
valinomycin-induced change in fluorescence intensity. The values were
obtained from at least four separate experiments, and P < 0.05 for ATP- vs. UTP-induced depolarization presented as percent
control and for 2-MeS-ATP- vs. UTP-induced depolarization. Moreover,
UTP-induced depolarization was transient, whereas ATP- and
2-MeS-ATP-induced depolarization remained sustained. Differences in
profile and extent of depolarization might contribute to the fact that
depolarization induced by ATP or 2-MeS-ATP, but not UTP, leads to the
activation of VSCCs.
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DISCUSSION |
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Many studies have focused on the functional roles of extracellular ATP in interactions with P2X purinoceptors. P2X receptors on neuronal cells have been reported to mediate rapid and nonselective entry of cations such as Ca2+, K+, and Na+ into the cells. As a result of Na+ acting as the predominant charge carrier, opening of the P2X purinoceptors depolarizes the plasma membrane, which can lead to the activation of VSCCs. It has, therefore, been suggested that the activation of ATP-gated channels might increase [Ca2+]i through a combination of direct P2X receptor-gated Ca2+ influx and secondary influx through VSCCs (15).
A number of research groups have studied the effect of ATP-induced P2 purinergic stimulation on the activation of VSCCs, giving rise to extensive and controversial literature. The large heterogeneity in the responses observed overall in PC-12 cell populations is thought to be in part due to the coexistence of multiple clones and, in part, to variable activation of intracellular signal transduction mechanisms (38). Previous studies have reported very little (32) or a significant (15) contribution of voltage-gated Ca2+ entry to the ATP-induced elevation of [Ca2+]i in PC-12 cells. Some studies reported that ATP-induced catecholamine secretion (3, 46) as well as the increase in [Ca2+]i, was insensitive to inhibitors of VSCCs (6), whereas others showed that organic and inorganic inhibitors of VSCCs reduced ATP-evoked [Ca2+]i increase either partially (11, 21, 23) or nearly completely (18). However, as mentioned by Swanson et al. (48), there has been no direct evidence of the opening of VSCCs subsequent to the ATP-mediated depolarization in PC-12 cells (3, 42). There also have been no reports of inhibition of ATP-evoked catecholamine secretion by the blockage of VSCCs.
We reported previously that the pretreatment of PC-12 cells with nifedipine, an L-type Ca2+ channel blocker, caused a reduction in the subsequent rise in [Ca2+]i induced by ATP (35). This added to the possibility that the VSCC-mediated signaling and the P2 purinoceptor-mediated Ca2+ signaling were coupled in PC-12 cells. In this study, we demonstrate that L-type VSCCs are activated subsequent to depolarization caused by Na+ entry through P2X2 purinoceptors. Nifedipine had an inhibitory effect on ATP-evoked [Ca2+]i rise and [3H]NE secretion.
The fluorescence changes in the presence of the anionic dye bisoxonol revealed that 17.5 mM K+ caused a depolarization comparable to that induced by 300 µM 2-MeS-ATP. K+ (17.5 mM), however, failed to increase [Ca2+]i in our experimental conditions. This observation was consistent with our previous report in which a similar amount of K+ did not evoke any detectable catecholamine secretion in PC-12 cells (45). K+ (17.5 mM) might have increased [Ca2+]i, but not by a detectable amount, which might be due to homeostatic mechanism for controlling [Ca2+]i. The depolarization signal detected by bisoxonol is an average fluorescence signal emanating from the whole cell areas of many cells. The extent of depolarization evoked by 17.5 mM K+ may not be large enough to trigger the opening of VSCCs and to increase [Ca2+]i by a detectable amount when the signal is distributed throughout the cell. When P2X2 receptors are activated, however, although the average fluorescence signal would be the same if spread over the whole cell area, a higher depolarization signal could be localized in a certain area of the plasma membrane and, thus, could be strong enough to stimulate VSCCs.
There may be active zones in the plasma membrane where P2X2 purinoceptors and L-type VSCCs are closely located, and thus the stimulation of P2X2 purinoceptors could lead to the activation of VSCCs via localized signals in those areas. Several lines of evidence suggest that many of the functions of Ca2+ and Na+ in signaling are achieved by generating highly localized signals restricted to small regions in a cell (14, 20, 25, 30, 36). Local Ca2+ signals that arise via Ca2+ influx as well as Ca2+ release have been observed in a wide variety of tissues, including cardiac, skeletal, and smooth muscle cells, in addition to nonexcitable cells (4, 27). Such localized signals could theoretically arise, for example, owing to nonhomogeneous spatial distribution of channels, and there is evidence suggesting a highly nonhomogeneous spatial distribution of P2X receptors (20) as well as VSCCs (5, 24).
There is evidence suggesting that P2X receptors are located on nerve
terminals (47, 49), where they function to modulate neurotransmitter release. Thus ligand-gated P2X receptors are not only
located postsynaptically to mediate fast transmission but also
presynaptically to modulate transmitter release. In a recent study,
Barden et al. (2) determined that there was a spatial
relation between P2X receptors and active zones, which are delineated
by N-type Ca2+ channels. They showed that antibodies
against vesicle-associated proteins of the soluble
N-ethylmaleimide-sensitive factor attachment protein target
receptor (SNARE) complex and the 1B-subunit of N-type
Ca2+ channels were located with respect to clusters of P2X
receptor subunits labeled with specific antibodies. P2X receptors were not uniformly distributed, and different P2X subtype clusters had
different spatial distribution. Specifically, most of the terminal
varicosities possessed active zones that were precisely apposed to
clusters of P2X1 receptors, suggesting the possibility of
colocalization of a specific P2X receptor subtype with a specific subtype of VSCC.
Our data showing that the L-type VSCCs were activated by a rather small amount of depolarization caused by P2X2-gated Na+ entry suggest that VSCCs are efficiently coupled to P2X2 purinoceptors in PC-12 cells. We propose a mechanism in which a localized depolarization caused by the Na+ entry through the P2X2 purinoceptors might lead to the activation of L-type VSCCs in PC-12 cells. This might be achieved through spatial colocalization of P2X2 and L-type VSCCs in PC-12 cells, and it will be of great interest to see whether P2X2 purinoceptors and L-type Ca2+ channels are physically in close proximity in PC-12 cell membranes.
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ACKNOWLEDGEMENTS |
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We are also grateful to G. Hoschek for editing the manuscript.
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FOOTNOTES |
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This work was supported by grants from the Korea Research Foundation (1998) and by grants from the Korea Science and Engineering Foundation, the Brain Research and Engineering Program, and the National Research Laboratory Program sponsored by the Ministry of Science and Technology. This work was also supported by Brain Korea 21 Program of the Ministry of Education.
Address for reprint requests and other correspondence: K.-T. Kim, Dept. of Life Science, Pohang University of Science and Technology, San 31, Hyoja Dong, Pohang 790-784, Republic of Korea (E-mail: ktk{at}postech.ac.kr).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 18 October 2000; accepted in final form 22 November 2000.
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