Coupling of L-type voltage-sensitive calcium channels to P2X2 purinoceptors in PC-12 cells

Eun-Mi Hur, Tae-Ju Park, and Kyong-Tai Kim

Department of Life Science, Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang 790-784, Korea


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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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.


    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) andomega -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 [alpha -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 alpha -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.


    RESULTS
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MATERIALS AND METHODS
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 omega -conotoxin GVIA, a specific N-type Ca2+ channel blocker, to 19 ± 1% (33). Therefore, we used nifedipine and omega -conotoxin GVIA to test the involvement of VSCCs in the ATP-mediated [Ca2+]i rise. When PC-12 cells were pretreated with nifedipine, the ATP- and 2-MeS-ATP-evoked [Ca2+]i increase was reduced to 70 ± 2 and 49 ± 4% of the controls, respectively, whereas the UTP-evoked [Ca2+]i increase was not affected (Fig. 1, A and B). Pretreatment with verapamil, another L-type Ca2+ channel blocker, also inhibited the ATP- and 2-MeS-ATP-evoked [Ca2+]i rise to 68 ± 1 and 51 ± 9% of the controls, respectively. Treatment with omega -conotoxin GVIA, however, had no effect on the ATP- and 2-MeS-ATP-induced Ca2+ rise. The results thus suggest that L-type VSCCs are specifically coupled to the P2X-mediated Ca2+ signaling.


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Fig. 1.   Effect of voltage-sensitive Ca2+ channel (VSCC) blockers on ATP-, 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP)-, and UTP-induced responses in PC-12 cells. A: effect of nifedipine on 2-MeS-ATP- and UTP-evoked increase in cytosolic free Ca2+ concentration ([Ca2+]i). Fura 2-loaded PC-12 cells were stimulated with 300 µM 2-MeS-ATP or UTP after 3 min of incubation with (trace b) or without (trace a) 5 µM nifedipine. Experiments were performed 5 times independently, and results were reproducible. Typical Ca2+ transients are presented. B: effect of Ca2+ channel blockers on ATP-, 2-MeS-ATP-, and UTP-evoked [Ca2+]i increase. Fura 2-loaded PC-12 cells were treated with 5 µM nifedipine (Nif), 2 µM omega -conotoxin GVIA (omega -CgTx), or 20 µM verapamil for 3 min and then stimulated with 300 µM ATP, 2-MeS-ATP, or UTP. Peaks of the elevated [Ca2+]i are compared with the control [Ca2+]i rise stimulated only with ATP, 2-MeS-ATP, or UTP, without pretreatment. Values are means ± SE from 3 experiments. *P < 0.05; **P < 0.01 compared with control. C: effect of nifedipine on ATP-, 2-MeS-ATP-, and UTP-induced secretion of [3H]norepinephrine (NE). [3H]NE-loaded PC-12 cells were incubated with or without 5 µM nifedipine for 10 min. Cells were then treated with 300 µM ATP, 2-MeS-ATP, or UTP in the presence (Nif) or absence (control) of 5 µM nifedipine. Secreted [3H]NE was measured as described in MATERIALS AND METHODS and is expressed as a percentage of total [3H]NE. Values are means ± SE of >= 6 independent experiments. *P < 0.05 compared with control.

Many extracellular signals that evoke cytosolic Ca2+ elevation induce the secretion of catecholamine in PC-12 cells. We investigated whether pretreatment of the cells with Ca2+ channel blockers would reduce the P2X purinoceptor-mediated catecholamine secretion. As shown in Fig. 1C, when PC-12 cells were pretreated with nifedipine for 10 min, the ATP-evoked [3H]NE secretion was reduced to 76 ± 4%. Nifedipine reduced the 2-MeS-ATP-evoked [3H]NE secretion to 53 ± 7% of the control, whereas UTP-evoked secretion was not affected. The inhibitory effect of nifedipine on the ATP-, 2-MeS-ATP-, and UTP-evoked catecholamine secretion was similar to its effect on the ATP-, 2-MeS-ATP-, and UTP-evoked [Ca2+]i increase.

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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Fig. 2.   Effect of 70 mM K+ pretreatment on ATP-, 2-MeS-ATP-, and UTP-induced [Ca2+]i increase. Fura 2-loaded PC-12 cells were treated with 70 mM K+ for 3 min and then stimulated with 300 µM ATP, 2-MeS-ATP, or UTP. Peak [Ca2+]i rise after each stimulation is compared with the control [Ca2+]i rise caused by 300 µM ATP, 2-MeS-ATP or UTP. Values are means ± SE of peak heights from 3 experiments. **P < 0.01 compared with control.

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 omega -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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Fig. 3.   Effect of VSCC blockers on ATP-, 2-MeS-ATP-, and UTP-evoked [Ca2+]i increase after treatment with thapsigargin (TG). A: fura 2-loaded PC-12 cells were treated with 1 µM TG for 6 min and then treated with 300 µM ATP (trace a). The inhibitory effect of Ca2+ channel blockers on the ATP-evoked Ca2+ elevation was tested by adding 5 µM nifedipine (trace b) or 2 µM omega -conotoxin GVIA (trace c) to the cells 3 min after TG treatment. Experiments were performed 4 times, and results were reproducible. Typical Ca2+ transients are presented. B: fura 2-loaded PC-12 cells were treated with 1 µM TG. After 3 min, the cells were treated with 5 µM nifedipine, 2 µM omega -conotoxin GVIA, or 20 mM verapamil and then stimulated with 300 µM ATP, 2-MeS-ATP, or UTP. Values are compared with the control Ca2+ increase, which was obtained by treating the cells with 300 µM ATP, 2-MeS-ATP, or UTP after 6 min of incubation with 1 µM TG. Peaks of the ATP-, 2-MeS-ATP-, or UTP-induced [Ca2+]i increases were measured. Values are means ± SE from 4 independent experiments. **P < 0.01 compared with control.

Second, we studied the effect of nifedipine on Mn2+ and Ca2+ entry. Mn2+ can be used as a specific tracer of Ca2+ entry (41), since Mn2+ can enter the cells through many Ca2+-permeable pathways, while there is no intracellular compartment harboring those ions or an out-pumping mechanism for Mn2+. Figure 4A shows the fluorescence quenching due to Mn2+ influx when PC-12 cells were stimulated with ATP, 2-MeS-ATP, or UTP. The Mn2+ entry was accelerated when the cells were stimulated with ATP, 2-MeS-ATP, or UTP. Nifedipine inhibited the ATP-induced Mn2+ entry, which mimics Ca2+ influx through P2X purinoceptors and VSCCs as well as capacitive Ca2+ entry. Nifedipine also inhibited the 2-MeS-ATP-induced Mn2+ entry that includes influx through P2X purinoceptors and VSCCs. Nifedipine, however, did not affect the UTP-induced Mn2+ entry that mimics capacitive Ca2+ entry. These results indicate that nifedipine blocked the ion influx mediated by the stimulation of P2X purinoceptors.


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Fig. 4.   Effect of nifedipine on Mn2+ quenching and Ca2+ influx. A: fura 2-loaded cells were incubated with 500 µM Mn2+ for 4 min before treatment with 300 µM ATP, 2-MeS-ATP, or UTP (trace a). The inhibitory effect of nifedipine was tested by preincubating the cells with 5 µM nifedipine for 3 min in the presence of 500 µM Mn2+ before treatment with ATP, 2-MeS-ATP, or UTP (trace b). Influx of Mn2+ was measured by the quenching of fura 2 fluorescence excited at 360 nm and emitted at 500 nm. Traces are representative of >5 separate experiments. B: effect of nifedipine on Ca2+ entry. Fura 2-loaded cells were treated with 300 µM ATP, 2-MeS-ATP, or UTP in Ca2+-free medium containing 200 µM EGTA. After 2 min, 2 mM Ca2+ was added to the medium (trace a). The inhibitory effect of nifedipine on Ca2+ entry was tested by incubating the cells with 5 µM nifedipine for 3 min before treatment with ATP, 2-MeS-ATP, or UTP (trace b). Traces are representative of 4 separate experiments.

Blockage of the P2X-mediated Ca2+ entry by nifedipine could also be detected directly by a Ca2+ entry experiment. PC-12 cells were stimulated with ATP, 2-MeS-ATP, or UTP in the absence of external Ca2+ for 90 s, and then 2 mM Ca2+ was added to the medium to measure Ca2+ influx. As shown in Fig. 4B, stimulation with ATP and UTP caused an increase in the fluorescence ratio before the addition of Ca2+, that is, in the absence of external Ca2+, whereas 2-MeS-ATP did not. This confirms that ATP and UTP can increase [Ca2+]i in a way that is independent of external Ca2+, while 2-MeS-ATP requires external Ca2+. In the absence of extracellular Ca2+, nifedipine had no effect on the increase in the fluorescence ratio. Pretreatment with nifedipine inhibited the ATP- and 2-MeS-ATP-evoked Ca2+ entry, whereas UTP-evoked Ca2+ entry was not affected. The Mn2+ and the Ca2+ profiles together thus provide evidence that nifedipine inhibits cation entry through P2X purinoceptors and L-type VSCCs triggered by the stimulation of P2X purinoceptors, suggesting that L-type VSCCs are coupled to the P2X purinoceptors.

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 alpha -tubulin mRNA.


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Fig. 5.   P2X receptor subtypes coupled to L-type VSCCs. A: RT-PCR detection of gene expression of P2X receptor subtypes in PC-12 cells. Primer pairs specific for P2X1-P2X7 were used. B: detection of P2X2 and P2X4 receptor mRNAs by Northern blot analysis. Total RNA was probed with the PCR products amplified from P2X2 and P2X4. Top: representative images showing hybridization signals of P2X receptor mRNAs. Bottom: representative images showing hybridization signals of rat alpha -tubulin mRNA as an internal control. C: effect of pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) on nifedipine-pretreated ATP-induced Ca2+ increase. Fura 2-loaded PC-12 cells were treated with 5 µM PPADS, 5 µM nifedipine, or 5 µM PPADS + 5 µM nifedipine for 5 min and then stimulated with 300 µM ATP. Values are means ± SE from 4 experiments. *P < 0.05; **P < 0.01 compared with ATP-induced Ca2+ increase without pretreatment.

Because 2-MeS-ATP can act as an agonist for P2X2 and P2X4, we tested the effect of a well-known antagonist PPADS, which has been reported to be active at P2X2, but not at P2X4, receptors (44) to investigate which P2X receptor subtype was involved in the coupling to L-type VSCCs. Pretreatment with 5 µM PPADS completely blocked the subsequent 2-MeS-ATP-evoked [Ca2+]i increase (data not shown). Moreover, pretreatment with both PPADS and nifedipine did not have any additional inhibitory effect on the subsequent ATP-induced [Ca2+]i increase compared with the treatment with nifedipine alone. Together, the results suggest that it is the P2X2 receptor that is coupled to the L-type VSCCs (Fig. 5C).

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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Fig. 6.   ATP-, 2-MeS-ATP-, and UTP-induced depolarization measured with bisoxonol. Membrane depolarization was measured as described in MATERIALS AND METHODS. Typical fluorescence responses on addition of 300 µM ATP (A), 2-MeS-ATP (B), or UTP (C) in the presence of 500 nM bisoxonol are shown. At the end of each measurement, cells were treated with 1 µM valinomycin (Val) as control. Horizontal bar, time; vertical bar, relative fluorescence intensity in arbitrary units. Typical traces of >= 5 measurements are shown.

It has been previously reported that the depolarization caused by ATP was mainly due to Na+ influx in PC-12 cells (10, 31). De Souza et al. (10) showed that the increase in the fluorescence of bisoxonol evoked by extracellular ATP was completely abolished when Na+ were replaced by N-methyl-D-glucamine (NMDG), whereas the increase in fluorescence intensity evoked by extracellular K+ was unaffected under the same conditions. It, therefore, has been suggested that it is mainly Na+ influx that induces membrane depolarization subsequent to the purinergic activation in PC-12 cells. Theoretically, depolarization induced by purinergic stimulation can lead to the activation of VSCCs. Interestingly, there has been no direct evidence indicating the opening of VSCCs subsequent to ATP-mediated depolarization in PC-12 cells (3, 46). Because the depolarization following P2 receptor activation was mainly due to Na+ influx, we replaced external Na+ with NMDG or with choline to prevent cells from depolarizing. We then investigated whether nifedipine would inhibit the P2X2-mediated [Ca2+]i increase. As shown in Fig. 7, when Na+ were replaced by the impermeable cations of NMDG or choline, the inhibitory effect of nifedipine on the P2X2-mediated [Ca2+]i increase was completely abolished. In the absence of Na+, there can be more Ca2+ entry through P2X2 purinoceptors, because Na+ competes with Ca2+ to enter cells through nonselective P2X2 cation channels in normal conditions. Because there were no Na+ to induce membrane depolarization and, in turn, VSCC activation, the ATP- and 2-MeS-ATP-induced [Ca2+]i increase must have been due to direct influx of extracellular Ca2+ through P2X2 purinoceptors, rather than through a combination of P2X2 purinoceptors and VSCCs. In the absence of extracellular Na+, Ca2+ channels were insensitive to nifedipine. Our results thus provide strong evidence in support of the hypothesis that Na+ entry through the activated P2X2 purinoceptors leads to the activation of L-type VSCCs.


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Fig. 7.   Effect of nifedipine on ATP- and 2-MeS-ATP-induced [Ca2+]i increase under Na+-free conditions. Fura 2-loaded PC-12 cells suspended in Na+-containing Locke's solution (normal) or Na+-free Locke's solution, in which Na+ was replaced by N-methyl-D-glucamine (NMDG) or choline, were incubated with 5 µM nifedipine for 3 min before treatment with 300 µM ATP (A) or 2-MeS-ATP (B). Values are compared with control [Ca2+]i increase, which was obtained by treating the cells with 300 µM ATP (A) or 2-MeS-ATP (B) without nifedipine pretreatment. Peaks of the ATP- or 2-MeS-ATP-induced [Ca2+]i increases were measured. Values are means ± SE from 3 independent experiments. *P < 0.05; **P < 0.01 compared with control.

The extent of depolarization as detected by bisoxonol indicates an average fluorescence signal emanating from the whole cell areas of many cells. The extent of depolarization induced by 2-MeS-ATP was comparable to that induced by 17.5 mM K+ and was substantially lower than that caused by 70 mM K+ (Fig. 8A).


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Fig. 8.   Effect of 2-MeS-ATP and K+ on depolarization and [Ca2+]i rise. A: 2-MeS-ATP- and K+-induced depolarization. Depolarization induced by 300 µM 2-MeS-ATP (trace a), 17.5 mM K+ (trace b), and 70 mM K+ (trace c) was detected with 500 nM bisoxonol. Membrane depolarization was measured as described in MATERIALS AND METHODS. At the end of each measurement, cells were treated with 1 µM valinomycin as control. Horizontal bar, time; vertical bar, relative fluorescence intensity in arbitrary units. Typical traces of >= 5 measurements are shown. B: 2-MeS-ATP- and K+-evoked Ca2+ increase. Fura 2-loaded PC-12 cells were stimulated with 300 µM 2-MeS-ATP (trace a), 17.5 mM K+ (trace b), or 70 mM K+ (trace c). [Ca2+]i was measured as described in MATERIALS AND METHODS, and typical Ca2+ transients are presented. Experiments were performed 4 times, and responses were reproducible.

Interestingly, the extent of depolarization did not exactly correlate with the extent of [Ca2+]i rise. Although 70 mM K+ induced much greater depolarization than 300 µM 2-MeS-ATP, the peak height of the [Ca2+]i increase induced by 70 mM K+ was lower than that induced by 300 µM 2-MeS-ATP (Fig. 8B). However, the 70 mM K+-induced [Ca2+]i increase was persistent, whereas the 2-MeS-ATP-induced increase was transient. Moreover, 17.5 mM K+, which caused depolarization to the same extent as 300 µM 2-MeS-ATP, failed to evoke any detectable rise in [Ca2+]i. Together, our results suggest that L-type VSCCs are activated by a depolarization produced by Na+ influx through P2X2 purinoceptors in PC-12 cells, and the activation of VSCCs might be achieved through a localized depolarization signal.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 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.


    ACKNOWLEDGEMENTS

We are also grateful to G. Hoschek for editing the manuscript.


    FOOTNOTES

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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