Introduction

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Pardaxin is an amphipathic polypeptide neurotoxin composed of 33 amino acids, which was isolated from the Red Sea sole, Pardachirus marmoratus (Lazarovici et al., 1986). PX belongs to a family of five PX isoforms isolated from Pardachirus species living in the Pacific Ocean and the Red Sea (Adermann et al., 1998). In the present study we used mainly Asp-31-PX (P5) due to its lower cytotoxicity compared with that of Gly-31-PX (P4) (Adermann et al., 1998). Pardaxins, which have been shown to form voltage-dependent pores in liposomes (Loew et al., 1985; Lazarovici et al., 1986) and artificial lipid membranes (Lazarovici et al., 1992; Shi et al., 1995), act as polypeptide ionophores. They are considered unique pharmacological tools for studying neurotransmitter release (Lazarovici, 1994; Bloch-Shilderman et al., 1997), based on their ability to stimulate exocytosis in different neuronal systems, including brain slices (Wang and Friedman, 1986), neuromuscular junction (Renner et al., 1987), neurosecretory chromaffin cells (Lazarovici and Lelkes, 1992), and synaptosomes (Arribas et al., 1993), by both Ca²⁺-dependent and Ca²⁺-independent mechanisms (Lazarovici and Lelkes, 1992; Abu-Raya et al., 1999). PX is thought to act by insertion into the neuronal plasma membrane, leading to the opening of poorly selective cation channels, culminating in depolarization, Ca²⁺ entry, and neurotransmitter release (Lazarovici and Lelkes, 1992; Abu-Raya et al., 1999). In an attempt to elucidate the signal transduction pathways involved in PX-induced DA release we recently showed that PX stimulates the arachidonic acid cascade in a Ca²⁺-dependent and Ca²⁺-independent manner in PC12 cells (Abu-Raya et al., 1998). A direct relationship was also proposed between PX-stimulation of DA release and the arachidonic acid cascade (Abu-Raya et al., 1999).

An additional signal transduction circuit that may contribute to PX-induced DA release is the Ras-mitogen-activated protein kinase (MAPK) pathway (Blenis, 1993). MAPKs/ERKs are a family of protein-serine/threonine kinases with a diverse array of cellular targets, suggesting that they are key regulators of many cellular responses. They are activated by a wide variety of extracellular stimuli affecting eukaryotic cells, including hormones, growth factors, radicals, mitogens, UV, and toxins (Force and Bonventre, 1998). These kinases require phosphorylation of both the serine/threonine and tyrosine residues in the catalytic domain for activation and to be translocated into the nucleus (Treisman, 1996). The ERKs are activated by an upstream activator kinase, MEK (MAPK kinase) (Blenis, 1993), which is selectively blocked by PD98059 and UO126 inhibitors (Alessi et al., 1995; Favata et al., 1998). The potential role of ERKs in secretagogue-induced norepinephrine release from bovine adrenal chromaffin cells was recently proposed (Cox et al., 1996; Cox and Parsons, 1997). However, the basic regulatory role of ERKs and their substrates involved in neurotransmitter release have not been fully characterized.

The aim of the present study was to assess the role of ERKs in regulating PX-induced DA release from PC12 cells. Our results indicate that PX stimulates Ca²⁺-dependent ERK activity in PC12 cells. This process is essential to PX-induced DA release in Ca²⁺-containing medium. Our findings support the importance of ERKs in neurotransmitter release.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Materials

[³H]Dopamine (47 Ci/mmol), ⁴⁵Ca²⁺ (5-50 mCi/mg), and [-³²P]ATP were purchased from Amersham, Arlington Heights, IL; HEPES, myelin basic protein (MBP), trypan blue, sorbitol, bovine serum albumin, EGTA, MgCl₂, CaCl₂, NaCl, NaOH, Triton X-100, glycerol, dithiothreitol, aprotinin, leupeptin, rabbit protein kinase inhibitor, sodium vanadate, phenylmethylsulfonyl fluoride, -mercaptoethanol, bromphenol blue, ATP, SDS, Nonidet P-40, choline chloride, anti-synaptotagmin antibody, and poly(L-lysine) were purchased from Sigma Chemical Co., St. Louis, MO; methanol, KCl, and ascorbic acid were purchased from Merck, Darmstadt, Germany; PD98059 was purchased from BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA; UO126 was purchased from Promega, Madison, WI; anti-phospho ERK and anti-pan ERK antibodies were a generous gift from Dr. Erik Schaefer, QCB, Hopkinton, MA; and nerve growth factor (NGF) was kindly provided by Alomone Labs, Jerusalem, Israel. DMEM, horse and calf serum, antibiotics, normal goat serum, and rat tail type 1 collagen were purchased from Beit Ha'emek, Afula, Israel. The ELISA tetramethylbenzidine developing kit was purchased from Clark Laboratories, Inc., Jamestown, NY, and the ELISA blocking reagent was purchased from Sorin Biomedica, Saluggia, Italy; horseradish peroxidase-goat anti-rabbit was purchased from Jackson Immune Research Laboratories, Inc., West Grove, PA. Protein A agarose was purchased from Amersham Pharmacia Biotech, Buckinghamshire, UK; and polyethyleneimine was synthesized and kindly provided by Prof. A. Domb, Department of Medicinal Chemistry, School of Pharmacy, Hebrew University of Jerusalem, Jerusalem, Israel.

Toxins

Native PX (P4) was purified by liquid chromatography from the lyophilized secretion of the flatfish Pardachirus marmoratus (collected in Eilat, Israel) (Lazarovici et al., 1986). Synthetic pardaxins P4 and P5 were prepared on a 433A peptide synthesizer (PerkinElmer-ABI) using standard Fmoc solid phase chemistry, as previously described (Adermann et al., 1998). The toxins were purified by high pressure liquid chromatography, using Vydac C18 column and analyzed by electrospray mass spectrometry (Adermann et al., 1998). Synthetic pardaxins were kindly provided by Knut Adermann, Institute for Peptide Research-PharmaCeuticals GmbH, Hannover, Germany.

Cell Viability

Cytotoxicity was determined by trypan blue exclusion (Abu-Raya et al., 1999). The concentrations of PX were considered subcytotoxic when cell death was <10%.

PC12 Cultures

PC12 cells were grown in DMEM supplemented with 7% fetal calf serum, 7% horse serum, 100 µg/ml streptomycin, and 100 units/ml penicillin. The cultures were maintained in an incubator at 37°C in an atmosphere of 6% CO₂. The medium was changed twice weekly and the cultures were split at a 1:6 ratio once a week. In all experiments, unless specified otherwise, the cells were plated on six-well dishes coated with equal parts of collagen (0.01 mg/ml collagen in 0.1 M acetic acid), poly(L-lysine) (0.01 mg/ml), and polyethyleneimine (0.1 mg/ml). The PC12 cell variants M-M17-26, expressing the dominant-negative mutant Ha-ras Asn-17 gene (Lazarovici et al., 1997), were grown in the presence of 200 µg/ml G418 (Life Technologies, Gaithersburg, MD).

[³H]Dopamine Release

DA release from PC12 cells was determined with slight modifications, as previously described (Abu-Raya et al., 1999). Briefly, fresh DMEM was added and the cells were allowed to equilibrate at 37°C for 30 min. The cells were then loaded with [³H]dopamine (0.3-1 µCi/ml) for 12 h at 37°C. The medium was removed and the cells were washed once with serum-supplemented medium and twice with serum-free medium containing 1 mM ascorbic acid. Fresh medium was added, and the cultures were incubated with PX (4 µM) or KCl (80 mM) in the presence (1.8 mM) or absence of Ca²⁺ (+1 mM EGTA) for 20 min. Basal release was measured in cultures incubated for similar intervals at 37°C and left untreated. Samples of 0.2 ml were removed from the medium, centrifuged for 10 min (1000g) to remove floating cells, and the radioactivity was measured. To measure total radioactivity, the cells were washed with PBS and dissolved in 1 ml of 0.5 N NaOH and 0.2-ml aliquots were measured for radioactivity. The data are presented as [³H]dopamine release, calculated as percentage of control.

Vesicle Fusion-Based ELISA

The protocol followed was essentially as previously described (Parnas and Linial, 1998). Briefly, PC12 cells were grown on 48-well plates. Control (untreated) cells or cells pretreated with PD98059 for 45 min were further exposed to PX or PBS for an additional 20 min in the presence of polyclonal anti-synaptotagmin I antibody, which was raised against the N-terminal luminal 19 amino acids of synaptotagmin I. This soluble N-ethylmaleimide-sensitive fusion factor attachment protein receptor (SNARE) is briefly exposed on the cell surface during the exocytotic process (Parnas and Linial, 1998), enabling detection with the antibody. We optimized the antibody concentration to 6 µg/ml. Upon termination of incubation, the cells were washed three times with PBS at 37°C, with an interval of 15 min between washes, and then fixed with 4% paraformaldehyde in PBS for an additional 20 min at room temperature. The cells were washed once with 0.5 ml of PBS and twice with 0.5 ml of PBS containing 0.1% Triton X-100, and incubated with 3% H₂O₂ in PBS for 5 min. The cells were then incubated for 30 min at 25°C with blocking solution (5% normal goat serum, 2% bovine serum albumin, 0.1% Triton X-100 in PBS) and then with goat anti-rabbit antibody conjugated to peroxidase (0.35 ml, diluted 1:7500) for 1 h at 25°C. Cells were further washed three times with PBS containing 0.1% Triton X-100. After washing, the cells were incubated with 1:1 chromagen and substrate solution (150 µl) for 5 min. Every 10 s, 75-µl samples from each well were removed and mixed with the stop solution (150 µl, 1 N sulfuric acid). The intensity of the blue color formed was monitored at OD₄₅₀ nm with the aid of an ELISA reader-Kinetic analyzer V-MAX with the attached software SOFTmax (Molecular Devices Corporation, Menlo Park, CA). Control experiments with secondary antibody alone were used to subtract the background value. Staining with nonrelevant antibodies was included routinely. Total immunoreactivity was measured by fixing the cells and incubating with synaptotagmin I antibodies in blocking solution (0.2 µg/ml), followed by incubation with secondary antibody as described above.

Calcium Uptake

The procedure is described in detail in Jiang et al. (1997). Briefly, cells were grown in six-well plates in DMEM containing 10% fetal bovine serum. After 24 h at 37°C, cells were washed once with Ca²⁺ and serum/antibiotic-free medium and incubated in the same medium for 30 min. ⁴⁵Ca²⁺ (1 µCi) was then added to each well along with PX (4 µM) or KCl (80 mM) and the cells were incubated at 37°C for an additional 4 min. The medium was rapidly aspirated, and the cells were washed twice with 3 ml of wash buffer, pH 7.2, containing 20 mM HEPES, 50 mM choline chloride, and 2.5 mM EGTA. The cells were lysed in 0.5 ml of NaOH for 2 h. A 0.4-ml sample of the cell lysate was used to estimate the cell-associated radioactivity and 0.05 ml was used for protein determination. The final counts were normalized per milligram of protein (cpm/mg of protein) and the data are presented as percentage of control.

ERK Assays

Detection of Activated ERKs in PC12 Cell Extracts by Western Blotting Using Anti-Phospho ERK Polyclonal Antibodies. Activation of MAPKs/ERKs requires that these enzymes are dually phosphorylated by MEK (Blenis, 1993) on both the Thr and the Tyr residues in the Thr-Glu-Tyr consensus sequence within the catalytic core of the enzyme (Payne et al., 1991). The phospho-ERK antibody (anti-active ERK) used in the present study was developed against a dually phosphorylated synthetic peptide encompassing residues Thr-183 and Tyr-185 of p42/MAPK2/erk2, corresponding to the active form of the ERK enzymes (Khokhlatchev et al., 1997). The antibody was purified, using a negative adsorption step to remove antibody recognizing the nonphosphopeptide, followed by positive selection-affinity purification with the dually phosphorylated peptide to select for antibody preferentially recognizing ERK1 and ERK2. PC12 cells were treated with 50 ng/ml NGF for 5 min, or with 0.5 M sorbitol for 5 min or PX for 15 min, unless otherwise indicated, or left untreated. The cells were lysed and aliquots (50 µg) of each extract were analyzed by SDS-PAGE (10% gel, under reducing conditions) and transferred to a nitrocellulose membrane. The membranes were probed with the indicated anti-phospho antibody, which recognizes the active (phosphorylated) forms of ERKs or with anti-pan ERK antibody, a polyclonal antibody that recognizes the inactive (nonphosphorylated) form of ERKs, at a dilution of 1:10,000 (Lazarovici et al., 1998).

Measurement of ERK Activity, Using MBP as Substrate. In another approach we measured ERK activity by protein phosphorylation assay, using the substrate MBP. MBP serves as a general nonselective substrate for a variety of protein kinases (Chan and Lazarovici, 1987). The first step was the immunoprecipitation of ERKs from the cell lysates, using anti-ERK polyclonal antibody (at a dilution of 1:200 for 2-h incubation at 37°C with continuous agitation). Thereafter, triplicate lysates of the various samples were incubated with washed protein A agarose (50-µl suspension) for additional 2-h incubation at 4°C. The immunoprecipitated ERK, bound to protein A agarose was washed twice with 0.5 ml of kinase assay buffer. In the second step, the washed immunoprecipitated ERK, bound to protein A agarose, was suspended in 30 µl of kinase assay buffer {7.5 mM HEPES, pH 7.5, 10 mM MgCl₂, 1 mM dithiothreitol, 2.5 µM protein kinase A inhibitor peptide PKI(6-22)-amide, 225 µM cold ATP, 25 µCi of [-³²P]ATP, and 500 µg/ml MBP}, and incubated for 30 min at room temperature. The reaction was terminated by the addition of 30 µl of SDS sample buffer, heated for 5 min and electrophoresed on SDS-PAGE. Phosphorylated MBP was visualized by autoradiography (XAR film; Eastman Kodak, Rochester, NY). The bands were quantified with a laser scanner.

Visualization of ERK by Confocal Laser Scanning Fluorescence Microscopy

PC12 cells were plated onto collagen- and poly(lysine)-coated two-well chambers (Nalgene) 1 day before each experiment. The cells were washed with PBS and then incubated for 2 h at 4°C with rabbit anti-phospho ERK antibody diluted 1:10,000 in PBS containing 10% fetal bovine serum and 0.2% Triton X-100. The cultures were then washed three times, 10 min each, at room temperature with 0.2% Triton X-100 in PBS containing 10% fetal bovine serum. The cultures were then incubated with a goat-anti-rabbit IgG-conjugated with Cy3 for 1 h at 37°C. The cells were washed twice with 1 ml of fresh medium and used immediately. Cells were treated with PX (5 µM) for 1 to 30 min or left untreated. The fluorescence of Cy3 was quantified with the aid of confocal laser scanning fluorescence microscopy (Leica TCS-4D; Leica Lasertechnik, Heidelberg, Germany), using excitation and emission wavelengths of 535 and 570 nm, respectively. Gray scale images with 0 to 255 steps were collected at different time points before and up to 30 min after the addition of PX by using a 512 × 512 pixel format and archived as image files for quantitative analysis. The intensity of the fluorescence in the individual cells was measured using Leica quantitation software. Background levels of immunoreactivity were very low in the control cells and this level of nonspecific signal was subtracted from the digitized images, measured in the PX-treated cells.

Statistics

Images obtained on X-ray film following autoradiography or chemiluminescence were scanned and the protein bands were quantified by densitometry, using NIH Image software. Significant statistical differences between band intensities were determined by ANOVA analysis (p  0.05). Also, the significance of the fluorescence value, obtained by confocal microscopy was analyzed by ANOVA (p  0.01). The significance of the statistical differences between the results obtained from DA release and vesicle fusion-based ELISA were analyzed by Kruskal-Wallis and Dunn tests (p  0.05).

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Pardaxin-Induced Activation of ERKs in PC12 Cells. Using anti-phospho ERKs antibody we observed strong stimulation of ERK1 (p44 MAPK) and ERK2 (p42 MAPK) by NGF (Fig. 1A, top) and to a lesser extent by osmotic shock with sorbitol (Fig. 1A, top). The same NGF- and sorbitol-treated cell lysates were also analyzed using immunoblotting with an anti-pan ERK antibody to confirm that the amount of ERK enzyme was the same in each sample (Fig. 1A, bottom). It is evident that in the absence of NGF and sorbitol both ERK1 and ERK2 were not active. It can also be seen that other ERKs besides ERK1 and ERK2 did not cross-react with the anti-phospho ERK antibody. NGF- and sorbitol-treated samples were immunoprecipitated and the activation of the ERKs was evaluated by measuring ERK activity, using MBP as substrate. Results from these experiments were consistent with those obtained by Western blot, and indicated a similar fold increases in phosphorylation (an increase of 13-fold by NGF, and of 6-fold by sorbitol; data not shown). To examine the effect of PX on ERK activity, the cells were treated with NGF (a known stimulator of ERKs), or PX, or were left untreated (CON). The basal activity of ERK1 and ERK2 was very low in untreated cells (Fig. 1B, CON). Treatment of the cells with NGF for 5 min caused about 16-fold stimulation of ERK1 and ERK2 activity (Fig. 1B, NGF). As shown in Fig. 1B, PX stimulated ERK activity in a time-dependent manner. Treatment of the cells with PX for 15 min resulted in a maximal ERK stimulation (5-fold increase over basal, Fig. 1B, PX). Thereafter (30-60 min), PX-stimulated ERK activity gradually decreased, disappearing within 120 min to less than control (Fig. 1B). The total amount of protein detected using anti-pan ERK antibody did not change throughout the incubation period (data not shown).

View larger version (69K): [in this window] [in a new window]   Fig. 1.   Pardaxin stimulation of ERK activity in PC12 cells. A, PC12 cells were treated (+) for 5 min with 50 ng/ml NGF or 0.5 M sorbitol, or left untreated (). The cells were then collected and 10-µg protein aliquots were analyzed by SDS-PAGE (10% gel, reducing conditions) and electrotransferred to nitrocellulose membranes. The membranes were probed with anti-phospho (anti-active) ERK antibody or, after stripping, reprobed with antibody for unphosphorylated ERK. NGF (lane 2) and sorbitol (lane 4). Controls (lanes 1 and 3). Top, anti-phospho ERK antibody; bottom, anti-ERK antibody. The location of ERK1 and ERK2 is indicated by arrows. B, time course of ERK stimulation by 5 µM PX. Cells were collected and treated as described above. CON, control. NGF, PC12 cells were treated for 5 min with 50 ng/ml NGF.

View larger version (97K): [in this window] [in a new window]   Fig. 2.   Effect of extracellular calcium on ERK stimulation. PC12 cells were treated for 5 min with 50 ng/ml NGF or for 15 min with 5 µM PX or left untreated (CON). The cells were then collected and 10-µg protein aliquots were analyzed by SDS-PAGE (10% gel, reducing conditions) and electrotransferred to nitrocellulose membranes. The membranes were probed with anti-phospho ERK antibody (I) or, after stripping, reprobed with antibody for unphosphorylated ERK (II). +Ca2+, calcium (1.8 mM)-containing medium. Ca2+, calcium-free medium. I, anti-phospho ERK antibody; II, anti-ERK antibody; III, phosphorylated MBP was visualized by autoradiography, as described under Experimental Procedures. The location of ERK1 and ERK2 is indicated by arrows.

View larger version (72K): [in this window] [in a new window]   Fig. 3.   PX-induced translocation of active ERKs to the nucleus. Phase contrast micrographs of PC12 cells before treatment (I) and immunofluorescence micrographs of the same cells treated with PX for 1 min (II) or 30 min (III). The cells were stained with anti-phospho ERK antibody and detected by the fluorescent CY3 dye.

Relationship between the Inhibitory Effect of PD98059 and UO126 on ERK Activity and PX-Induced Dopamine Release. Recently, it was found that PX induces in a time-dependent manner DA release from PC12 cells, both in the presence and absence of extracellular Ca²⁺ (Abu-Raya et al., 1999), as well as activation of ERK (Fig. 1). Therefore, we examined the relationship between DA release and ERK activation. PC12 cells were pretreated with the selective MEK inhibitor PD98059 (50 µM) for 45 min. The cells were then stimulated with NGF (50 ng/ml) or PX (4 µM) or left untreated (Fig. 4). PD98095 completely blocked the basal activity of ERK1 and ERK2. Both NGF- and PX-induced ERK activity was inhibited by 60 and 100%, respectively (Fig. 4). In the presence of extracellular Ca²⁺ the complete inhibition of PX-induced ERK activity was accompanied by 83 and 53% inhibition of PX-induced DA release by PD98095 and UO126, respectively (Fig. 5A). However, both MEK inhibitors did not affect significantly PX-induced DA release in Ca²⁺-free medium (Fig. 6). To further investigate the involvement of ERKs in neurotransmitter release, we designed an exocytotic assay, which quantifies vesicular release, reflected by exposure of the vesicular protein synaptotagmin to the extracellular matrix, using vesicle fusion-based ELISA (Parnas and Linial, 1998). As shown in Fig. 5B, PX increased synaptotagmin exposure on the cell surface by 50% over that of the basal. However, PD98095 (PD + PX) completely inhibited PX-induced synaptotagmin exposure (Fig. 5B). Cumulatively, these results suggest the involvement of ERK1 and ERK2 in PX-induced Ca²⁺-dependent DA release.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Effect of the MEK inhibitor PD98059 on NGF and PX stimulation of ERK. To measure ERK activity, cells were pretreated for 45 min with 50 µM PD98059 (PD) or left untreated. The cultures were then exposed to 50 ng/ml NGF or 5 µM PX for an additional 20 min, or left untreated (CON). Cell lysates (25 µg/sample) were separated, blotted, and analyzed, using anti-phospho ERKs antibodies, and further processed, as described in the legend to Fig. 1. The location of ERK1 and ERK2 is indicated by arrows.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Effect of PD98059 and UO126 on pardaxin-induced neurotransmitter release in Ca2+-containing medium. A, PC12 cells (1 × 106 cells/well) preloaded with [3H]dopamine were treated for 45 min with 50 µM PD98059 (PD), or for 30 min with 30 µM UO126, or left untreated. The cells were then exposed to 4 µM PX for an additional 20 min or left untreated (control). DA release was measured as described under Experimental Procedures. The values presented are the mean ± S.E.M. of three independent experiments (n = 3 in each experiment). *p < 0.05 compared with the control (basal release). **p < 0.05 compared with PX alone. B, exocytosis was measured by vesicle fusion-based ELISA, as described under Experimental Procedures. Control (untreated) cells or cells pretreated for 45 min with 50 µM PD98059 (PD) were exposed to 4 µM PX or left untreated for an additional 20 min in the presence of anti-synaptotagmin I antibody. The cells were washed, fixed, and incubated with blocking solution. Blue color formation was monitored at OD450 nm, with the aid of an ELISA reader. The values presented are the mean ± S.E.M. of three independent experiments (n = 6 in each experiment). *p < 0.05 compared with the control (basal release). **p < 0.05 compared with PX alone.

View larger version (37K): [in this window] [in a new window]   Fig. 6.   Effect of PD98059 and UO126 on pardaxin-induced neurotransmitter release in Ca2+-free medium. PC12 cells (1 × 106 cells/well) preloaded with [3H]dopamine were treated for 45 min with 50 µM PD98059 (PD), or for 30 min with 30 µM UO126, or left untreated. The cells were then exposed to 4 µM PX for an additional 20 min or left untreated (control). DA release was measured as described under Experimental Procedures. The values presented are the mean ± S.E.M. of three independent experiments (n = 3 in each experiment). *p < 0.05 compared with the control (basal release).

Effect of Pardaxin on Dopamine Release in Dominant-Negative Ras Cells. One of the important pathways of ERK activation involves a small guanine nucleotide-binding protein, p²¹Ras, a well known mediator in growth factor receptor tyrosine kinase signaling pathways. To determine whether PX-induced DA release via activation of ERKs is Ras-dependent, experiments with a dominant-negative variant PC12 cell line, M-M17-26 (Lazarovici et al., 1997, 1998), were performed. In these cells the endogenous native Ras cannot stimulate the MAPK pathway because of the large excess of inactive dominant-negative Ras protein competing for raf, which is required for ERK activation. Indeed, in these variant cells, NGF-induced ERK activation, as well as differentiation, were completely blocked (Lazarovici et al., 1997). As can be seen in Fig. 7A, depolarization with KCl increased DA release in WT cells by 2.2-fold. This effect was completely blocked in M-M17-26 cells, as was previously obtained in other study with PC12 cells (Rosen et al., 1994). In contrast, PX stimulated by 2.5-fold and by 2.2-fold, DA release from WT and M-M17-26 cells, respectively, compared with the corresponding controls (Fig. 7A). To determine whether dominant-negative Ras cells are defective in their voltage-dependent Ca²⁺ channels, which may explain the inability of KCl to induce dopamine release, Ca²⁺ uptake was measured. KCl and PX induced similar Ca²⁺ uptake in both the WT and the M-M17-26 cells (Fig. 7B). These findings indicate that the inability of KCl to induce DA release was not due to a lack of depolarization-induced Ca²⁺ entry in these cells. In summary, it appears that although depolarization-induced DA release by KCl is fully dependent on Ras, PX-induced DA release is Ras-independent.

View larger version (35K): [in this window] [in a new window]   Fig. 7.   Pardaxin-induced dopamine release and calcium uptake in a dominant-negative Ras cell line, M-M17-26, and in wild type PC12 cells. A, cells (1 × 106 cells/well) preloaded with [3H]dopamine were stimulated with 4 µM PX or 80 mM KCl for 20 min at 37°C. DA release was measured as described under Experimental Procedures. The values presented are the mean ± S.E.M of three independent experiments (n = 3 in each experiment). *p < 0.05 compared with the control (basal release). B, to measure Ca2+ uptake, cultures were washed and incubated with Ca2+-free medium for 30 min. [45Ca] was then added together with 4 µM PX or 80 mM KCl for an additional 4 min at 37°C. The cells were washed, lysed, and cellular Ca2+ was counted as described under Experimental Procedures. Wild type (); M-M17-26 PC12 cells, (). The values presented are the mean ± S.E.M of three independent experiments (n = 3 in each experiment). *p < 0.05 compared with the control (basal release).

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

In the present study we show that ERKs participate in Ca²⁺-dependent DA release from PC12 cells stimulated by PX. This is supported by the following findings: 1) PX induced activation of ERKs in PC12 cells; 2) Ca²⁺-dependent stimulation of ERKs by PX was temporally correlated with PX-induced DA release; and 3) treatment of the cells with the MEK inhibitor PD98059 completely blocked ERK activation and markedly inhibited the induced DA release, which was also inhibited by UO126. To the best of our knowledge, this is the first report of the involvement of MEK/MAPK in PX-induced DA release, supporting the hypothesis that ERKs participate in the catecholamine secretory process (Cox and Parsons, 1997).

Ras-MAPK is well characterized as a major signal transduction pathway activated by tyrosine kinase receptors such as NGF-tyrosine kinase A receptor (Kaplan and Stephens, 1994). Cross talk between MAPK pathways and Ca²⁺ pathways has been demonstrated in numerous cell types (Ely et al., 1990; Chao et al., 1992; Rosen et al., 1994; Clapman, 1995), but the precise mechanism by which Ca²⁺ mediates activation of ERK is unknown. Since PX did not activate protein kinase C (Bloch-Shilderman et al., 1996) and we do not have any evidence that PX directly stimulates tyrosine kinases, we assume that PX stimulation of ERKs reflects cross talk between Ca²⁺ pathway(s) and the MAPK cassette. Indeed, evidence for this concept was provided by molecular studies (Hawes et al., 1995; Lopez-llasaca, 1998) on G protein-coupled receptors. Activation of such receptors induces disassociation of the G protein to - and -subunits. -subunits in turn, stimulate ERK activation, most probably increasing intracellular Ca²⁺ and activating protein kinase C. In other studies with PC12 cells it was reported that the influx of Ca²⁺ ions, resulting from depolarization, leads to Ras-dependent MAPK/ERK activation (Rosen et al., 1994; Rusanescu et al., 1995). It was suggested that the protein tyrosine kinase PYK2, which is activated by an increase in intracellular Ca²⁺ and by protein kinase C, leads to activation of ERK (Lev et al., 1995). The possibility that PX may stimulate PYK2 kinase, and as a result activate ERKs, requires further investigation, since PYK2 may be a candidate kinase that functions upstream of MEK/MAPK. PD98095 is known to selectively inhibit MEK (Alessi et al., 1995), and also blocks PX stimulation of ERKs (Fig. 4). It is reasonable to assume that ERK activation was not due to the direct binding of PX to MEK enzymes. Rather, PX may have acted upstream of the MEK cassette.

An important function of ERKs is to regulate gene expression. Upon activation, ERKs translocate to the nucleus where they phosphorylate transcription factors (Blenis, 1993). In the present study, we found that following PX activation ERKs translocated to the nucleus. Therefore, transcription factors phosphorylated by ERKs may be of significance in the action of PX. Indeed, experiments in our laboratory show that PX stimulates c-fos early gene expression (P. Lazarovici, unpublished data). This probably requires both Ca²⁺ and the MAPK pathway, as recently reported for depolarization-induced c-fos activation in PC12 cells (Lee et al., 2000). Further investigation of whether DA release induced by PX is inhibited by interference with c-fos or with ERK translocation to the nucleus is required.

There are very few studies describing the interaction of toxins with ERKs. Palytoxin, a nonphorbol ester tumor promoter, sodium ionophore, produced by Palythoa tuberculosa, strongly stimulates another member of the MAPK family, c-Jun NH₂ terminal kinase (Kuroki et al., 1997). In addition to MAPK-activating toxins such as PX and palytoxin there is also a MAPK inhibitory toxin, Anthrax lethal factor. This toxin, produced by Bacillus anthracis, is a metalloprotease that cleaves the amino terminus of MEKs, and thus, like PD98059, inhibits MAPK activation (Duesbery et al., 1998). These various studies suggest that toxins, which affect the MAPK cassette, are potential pharmacological tools for investigating the role of ERKs in signal transduction.

To what extent ERKs contribute to neurotransmitter release has not been clearly established. In previous reports on chromaffin cells (Cox et al., 1996; Cox and Parsons, 1997), it was suggested that ERKs are one of the multiple components in the signaling cascades that upon stimulation with nicotine or other secretagogues regulate optimal secretory activity. In the present study, we found a direct temporal correlation between PX stimulation of ERKs and DA release. In addition, the ability of PD98059 to inhibit both PX-induced ERK activation and DA release suggests that ERKs play an important role in DA secretion from PC12 cells, as was also found for norepinephrine secretion from chromaffin cells (Cox and Parsons, 1997). One way of MAPK activation is via Ras (Blenis, 1993). To examine the role of the Ras-MAPK pathway in DA secretion from PC12 cells we used the dominant-negative Ras, PC12 cell line M-M17-26. The lack of Ras activity in these cells resulted in the absence of ERK activity (Lazarovici et al., 1997). Secretagogue-induced DA release was markedly reduced in M-M17-26 cells compared with that in WT cells. This was not due to a lack of Ca²⁺ uptake by the cells (Fig. 7B), indicating that Ca²⁺ itself is not sufficient for release. Furthermore, there was no difference in DA uptake between PC12 and M-M17-26 cells (data not shown). It is reasonable to assume that the reduced DA release observed in M-M17-26 cells is due to the disruption of one or more steps critical to the exocytotic machinery. Although, quantitatively, DA release was low in M-M17-26 cells, PX, but not KCl, still induced DA release (Fig. 7A). Other studies with PC12 cells (Rosen et al., 1994) and chromaffin cells (Cox and Parsons, 1997) have also shown that depolarization-induced neurotransmitter release is blocked in dominant-negative Ras cells. One plausible interpretation of this finding is that PX and KCl induce DA release by different signal transduction pathways: although KCl-induced neurotransmitter release is completely Ras-dependent, PX activates signal transduction component(s) essential to DA release that are Ras-independent but ERK-dependent.

The involvement of intracellular Ca²⁺ in neurotransmitter release has been well established (Burgoyne and Morgan, 1995). However, Ca²⁺-independent neurotransmitter release has also been reported in different systems (Nicholls et al., 1987; Lonart and Zigmond, 1991; Abu-Raya et al., 1999). ERK activation by NGF and PX was markedly and completely abolished, respectively, in Ca²⁺-free medium (Fig. 2I). As seen in Fig. 6, the inhibitors PD98059/UO126 did not significantly affect PX-induced DA release. Therefore, unlike the ERK-dependence of PX-induced DA release in the presence of extracellular Ca²⁺, PX stimulation of DA release in the absence of extracellular Ca²⁺ is ERK-independent. Recently, we showed (Abu-Raya et al., 1998) that PX stimulates the activity of phospholipase A₂ and the release of eicosanoids, independently of Ca²⁺. Inhibitors of phospholipase A₂ and lipoxygenases markedly blocked DA release induced by PX (Abu-Raya et al., 1999). PX stimulated the release of arachidonic acid from M-M17-26 and WT PC12 cells. This process was not affected by PD98059, indicating that arachidonic acid release by PX is Ras/MAPK-independent (E. Bloch-Shilderman, S. Abu-Raya, V. Trembovler, M. Linial, H. Boschwitz, A. Gruzman, S. Sasson, and P. Lazarovici, in preparation). Therefore, it is tempting to suggest that in the absence of ERK activation by PX, as occurred in Ca²⁺-free medium (Fig. 2), the arachidonic acid cascade plays an essential role in PX-induced DA release. This aspect is now under investigation in our laboratory. Further clarification of the interaction of PX with other MAPK members, such as c-Jun NH₂ terminal kinase, and p38 kinases and Ca²⁺-dependent tyrosine kinases such as PYK should provide insights into the mechanism of action of PX and may contribute putative targets for drug development in synaptic transmission.