INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

ENDOTHELIN (ET)-1 exhibits mitogenic activity in vascular smooth muscle cells (VSMCs; Refs. 1, 6, 7), which indicates a possible role for ET-1 in the pathogenesis of clinical conditions such as hyperlipoproteinemia or atherosclerosis (5, 8). However, the molecular mechanisms of the ET-1-induced mitogenic response in VSMCs are still unclear. Recently, a novel aspect of the role of tyrosine kinase signaling in ET-1 action has been revealed by the finding in rat-1 fibroblasts that the ET type A (ET_A) receptor-mediated mitogenic response as well as other G protein-coupled receptor-mediated responses are accompanied by transactivation of the epidermal growth factor receptor (EGFR) protein tyrosine kinase (PTK). Through the formation of Shc/Grb2/Sos complexes, this event leads to activation of the ras/mitogen-activated protein kinase pathway and transcription of early-response genes (2, 10, 12). Moreover, it was shown that vascular growth by ET-1 fully depends on this EGFR transactivation in VSMCs (6).

We have recently shown (7) that ET-1 activates three types of voltage-independent Ca²⁺ channels (VICCs) as well as voltage-operated Ca²⁺ channels (VOCCs) in rabbit internal carotid artery VSMCs. The VICCs include two types of Ca²⁺-permeable nonselective cation channels (NSCC), which are designated NSCC-1 and NSCC-2, and a store-operated Ca²⁺ channel (SOCC). Importantly, we have also shown that these channels can be distinguished by sensitivity to blockers of the receptor-operated Ca²⁺ channel such as SK&F-96365 and LOE-908 (4, 9); NSCC-1 is sensitive to LOE-908 and resistant to SK&F-96365, NSCC-2 is sensitive to both LOE-908 and SK&F-96365, and SOCCs are resistant to LOE-908 and sensitive to SK&F-96365 (7). Moreover, Ca²⁺ influx through NSCC-1, NSCC-2, and SOCCs plays an essential role for ET-1-induced mitogenesis in internal carotid artery VSMCs (7). Previous reports (3, 6, 10, 13) have demonstrated that extracellular Ca²⁺ influx is important in EGFR PTK transactivation by angiotensin II, bradykinin, and ET-1. Extracellular Ca²⁺ influx through VOCCs is involved in EGFR PTK transactivation in PC12 cells (10, 13). However, it is totally unknown whether extracellular Ca²⁺ influx through VICCs is involved in EGFR PTK phosphorylation. In the present study, we attempted to elucidate which Ca²⁺ channels are involved in ET-1-induced EGFR PTK phosphorylation using SK&F-96365 and LOE-908 in internal carotid artery VSMCs.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell culture. Rabbit internal carotid artery VSMCs were prepared as described previously (7). Briefly, male Japan White rabbits (body wt, 2-3 kg) were anesthetized by injection of thiopental sodium (20 mg/kg iv) and killed by exsanguination. The internal carotid artery was removed, cleaned of surrounding tissue, dissected into small (2 × 5 mm) strips, and kept in Ca²⁺-free Krebs-HEPES solution that contained (in mM) 140 NaCl, 3 KCl, 1 MgCl₂, 11 glucose, and 10 HEPES (pH 7.3, adjusted with NaOH). The strips were incubated overnight (12-24 h) at 4°C in Ca²⁺-free Krebs-HEPES solution that contained papain (0.2-0.3 mg/ml) and 0.5 mM dithiothreitol. Thereafter, the strips were resuspended and incubated in Ca²⁺-free Krebs-HEPES solution that contained collagenase (0.25-0.5 mg/ml) at 35°C for 10 min. The digested strips were cut into pieces with fine scissors and triturated with a blunt-tipped pipette until a sufficient number of single cells were released. Cells were routinely maintained in Dulbecco's modified Eagle's medium that contained 10% fetal bovine serum in a humidified atmosphere of 5% CO₂-95% air.

Measurement of EGFR PTK transactivation. Transactivation of EGFR PTK was measured using a universal tyrosine kinase assay kit (Takara; Tokyo, Japan). Extraction buffer and kinase-reacting solution were included with this kit. Cells seeded at 5 × 10⁶ cells/well in six-well plates were starved for 24 h and then stimulated with various concentrations of ET-1 for the indicated times. The reaction was terminated by washing the cells three times with phosphate-buffered saline. After the addition of 1 ml of extraction buffer, the cells were scraped off with a scraper and centrifuged at 14,500 rpm for 10 min at 4°C. The supernatant was incubated with mouse monoclonal anti-EGFR antibody (Takara) for 1 h at room temperature and subsequently incubated with protein A agarose for an additional 20 min. The mixture was centrifuged at 10,000 g for 1 min at 4°C, and the pellets were washed three times with phosphate-buffered saline. The washed pellets were resuspended in 150 µl of kinase-reaction buffer. EGFR PTK transactivation was determined according to the manufacturer's instructions. The absorbance of the lysate at 450 nm was measured using an EL340 microtiter plate reader (Bio-Tek Instruments; Winooski, VT).

Immunoblotting. The samples that were resuspended with kinase-reaction buffer were analyzed on 12% SDS-PAGE and transferred electrophoretically to polyvinylidene difluoride membranes (15 V for 90 min). Membranes were blocked with 5% bovine serum albumin for 1 h and were allowed to react with anti-phosphotyrosine monoclonal antibody (Santa Cruz Biotechnology) or mouse monoclonal anti-EGFR antibody for 1 h. The blots were washed and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h. After the washing phase, immunoreactive proteins were detected via the enhanced chemiluminscence system.

Drugs. LOE-908 was kindly provided by Boehringer Ingelheim (Ingelheim, Germany). Other chemicals were obtained from the following sources: ET-1 from the Peptide Institute (Osaka, Japan), SK&F-96365 from BioMol (Plymouth Meeting, PA), and AG-1478 from Funakoshi (Tokyo, Japan). All other chemicals were of reagent grade and were obtained commercially.

Statistical analysis. All results are expressed as means ± SE. Data were subjected to a two-way ANOVA, and when a significant F-value was encountered, the Newman-Keuls multiple-range test was used to test for significant differences between treatment groups. A probability level of P < 0.05 was considered statistically significant.

	RESULTS

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Effects of ET-1 on EGFR PTK transactivation in VSMCs. The tyrosine-phosphorylated protein (~180 kDa) was assumed to be EGFR because it was recognized by the anti-EGFR antibody (Fig. 1A). ET-1-induced EGFR PTK transactivation was inhibited remarkably by 5 µM BQ-123, which is a specific blocker of ET_A receptors, whereas it was not inhibited significantly by 5 µM BQ-788, which is a specific blocker of ET_B receptors (Fig. 1A). EGFR PTK transactivation by 10 nM ET-1 increased with time and reached a peak value after ~2 min (Fig. 1B). Thus the stimulation time was set at 2 min. ET-1 activated EGFR PTK in a concentration-dependent manner; EC₅₀ values were ~1 nM (Fig. 1C). The stimulation reached a maximum at concentrations 10 nM (Fig. 1C).

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Effects of endothelin-1 (ET-1) on epidermal growth factor receptor protein tyrosine kinase (EGFR PTK) phosphorylation in vascular smooth muscle cells (VSMCs). A: cells were pretreated with or without 5 µM BQ-123 or 5 µM BQ-788 for 30 min and incubated with 10 nM ET-1 for 2 min. B: cells were incubated with 10 nM ET-1 for the indicated times. EGFR was immunoprecipitated from cell lysates and analyzed by immunoblot with either anti-phosphotyrosine (PY; top) or monoclonal anti-EGF antibody (bottom). C: effects of various concentrations of ET-1 on EGFR PTK phosphorylation in VSMCs. Cells were stimulated with increasing concentrations of ET-1 for 2 min. EGFR PTK phosphorylation was determined using a universal tyrosine kinase assay kit as described in MATERIALS AND METHODS. Data presented are means ± SE of three determinations (each done in triplicate).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Effects of AG-1478 on ET-1-induced EGFR PTK activity in VSMCs. A: cells were pretreated with or without 1 µM AG-1478 for 30 min and incubated with 10 nM ET-1 for 2 min. B: cells were pretreated with various concentrations of AG-1478 and stimulated with () or without () 10 nM ET-1. Data are means ± SE of three determinations (each done in triplicate).

Effects of extracellular Ca²⁺ and nifedipine on ET-1-induced EGFR PTK transactivation. In the absence of extracellular Ca²⁺, the magnitude of ET-1-induced EGFR PTK transactivation was near the basal level (Fig. 3). Therefore, extracellular Ca²⁺ influx plays an important role in the ET-1-induced EGFR PTK transactivation.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Effects of extracellular Ca2+ and nifedipine on ET-1-induced EGFR PTK phosphorylation in VSMCs. A: cells were washed five times with Ca2+-free phosphate-buffered saline and incubated for 2 min with either Ca2+-free or Ca2+-containing phosphate-buffered saline that contained 10 nM ET-1. B: cells were incubated for 15 min with nifedipine and then stimulated with 10 nM ET-1. Data are means ± SE of three determinations (each done in triplicate).

Effects of SK&F-96365 and LOE-908 on ET-1-induced EGFR PTK transactivation. Using SK&F-96365 and LOE-908, we attempted to determine the effects of extracellular Ca²⁺ influx through VICCs on the ET-1-induced EGFR PTK transactivation. In these experiments, nifedipine was added to the incubation media at a final concentration of 1 µM to analyze the role of Ca²⁺ channels other than VOCCs. SK&F-96365 inhibited ET-1-induced EGFR PTK transactivation in a concentration-dependent manner, and the IC₅₀ value was ~3 µM (Fig. 4A). Maximal inhibition was observed at concentrations 10 µM (Fig. 4A). The extent of maximal inhibition was ~80% of the nifedipine-resistant part of EGFR PTK transactivation (Fig. 4B). Similarly, the IC₅₀ values of LOE-908 for inhibition of ET-1-induced EGFR PTK transactivation were ~3 µM, and maximal inhibition was observed at concentrations 10 µM (Fig. 4A). The extent of maximal inhibition was ~60% of EGFR PTK transactivation (Fig. 4B). Notably, the combined treatment with the maximally effective concentrations (10 µM) of SK&F-96365 and LOE-908 completely inhibited the nifedipine-resistant part of EGFR PTK transactivation (Fig. 4B).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   A: effects of SK&F-96365 () and LOE-908 () on ET-1-induced EGFR PTK phosphorylation in VSMCs. B: effects of a maximal effective concentration (10 µM) of SK&F-96365 and LOE-908 on ET-1-induced EGFR PTK phosphorylation in VSMCs. Starved cells were incubated for 15 min with various concentrations of SK&F-96365 or LOE-908 in addition to 1 µM nifedipine and were then stimulated with 10 nM ET-1 for 2 min. Data are means ± SE of five determinations (each done in triplicate); # P < 0.05, significantly different from the control values in each experiment; ## P < 0.05, significantly different from the inhibition by SK&F-96365; ### P < 0.05, significantly different from the inhibition by LOE-908.

View larger version (35K): [in this window] [in a new window]   Fig. 5.   A: effects of SK&F-96365 and LOE-908 on EGF-induced EGFR PTK phosphorylation in VSMCs. B: starved cells were incubated for 15 min with 10 µM SK&F-96365 or LOE-908 in addition to 1 µM nifedipine and then stimulated with 5 ng/ml EGF for 3 min. Data are means ± SE of three determinations (each done in triplicate).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

As described in previously (6), ET-1 induces EGFR PTK phosphorylation in rabbit internal carotid artery VSMCs (see Fig. 1A). Based on its sensitivity to BQ-123 and BQ-788, ET_A receptors play essential roles in the ET-1-induced EGFR PTK phosphorylation (Fig. 1A). In the present study, we used a universal tyrosine kinase assay kit and mouse monoclonal anti-EGFR antibody for measurement of EGFR PTK phosphorylation. The ET-1-induced PTK phosphorylation obtained using this method was completely inhibited by AG-1478, which is a specific EGFR PTK inhibitor (Ref. 6; see Fig. 2B). Moreover, the magnitude of ET-1-induced EGFR PTK phosphorylation in internal carotid artery VSMCs treated with AG-1478 was near basal level in the immunoblotting analysis (Fig. 2A). Therefore, we concluded that this method was suitable for measurement of EGFR PTK phosphorylation.

Previous reports (3, 6) demonstrated that extracellular Ca²⁺ influx plays important roles in the EGFR PTK transactivation. We tried to characterize the Ca²⁺ channels involved in the ET-1-induced EGFR PTK transactivation in internal carotid artery VSMCs. The magnitudes of ET-1-induced EGFR PTK transactivation in the absence of extracellular Ca²⁺ were near the basal level (see Fig. 3). These results indicate that extracellular Ca²⁺ influx is also important in ET-1-induced EGFR PTK transactivation in internal carotid artery VSMCs. Our recent study indicated that NSCC-1, NSCC-2, and SOCCs play major parts in ET-1-induced extracellular Ca²⁺ influx in internal carotid artery VSMCs (7). Moreover, extracellular Ca²⁺ influx through these Ca²⁺ channels is essential in ET-1-induced mitogenesis (7). Thus we examined the involvement of NSCC-1, NSCC-2, and SOCCs in ET-1-induced EGFR PTK transactivation using SK&F-96365 and LOE-908. According to the nifedipine sensitivity of ET-1-induced EGFR PTK transactivation, involvement of VOCCs in this response was estimated to be minor at around ~10% (see Fig. 3B). We demonstrated in a recent report (7) that nifedipine suppressed the 10 nM ET-1-induced sustained increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) by a maximum of 10%. Therefore, Ca²⁺ channels that play important roles in ET-1-induced EGFR PTK transactivation in internal carotid artery VSMCs are different from those in PC12 cells (10, 13). Ca²⁺ channels other than VOCCs may be important in ET-1-induced EGFR PTK transactivation in addition to extracellular Ca²⁺ influx in rabbit internal carotid artery VSMCs.

For several reasons, the inhibitory actions of SK&F-96365 and LOE-908 on the ET-1-induced EGFR PTK transactivation are considered to be mediated by blockade of Ca²⁺ entry through VICCs. First, in our recent work using patch-clamp and [Ca²⁺]_i monitoring, ET-1 was found to activate three types of VICCs in VSMCs: NSCC-1, NSCC-2, and SOCCs. In addition, LOE-908 was found to be a blocker of both NSCC-1 and NSCC-2, whereas SK&F-96365 was found to be a blocker of NSCC-2 and SOCCs (7). Second, the IC₅₀ values of these blockers for the ET-1-induced EGFR PTK transactivation (see Fig. 4A) correlated well with those for the ET-1-induced extracellular Ca²⁺ influx (7). Third, these blockers failed to inhibit EGF-induced EGFR PTK activation (Fig. 5). Fourth, these blockers failed to inhibit phorbol 12-myristate 13-acetate-induced EGFR PTK transactivation (data not shown) that was independent of extracellular Ca²⁺ influx (11). Fifth, neither SK&F-96365 nor LOE-908 is considered to exert cytotoxic effects on the quiescent cells (7). Moreover, because SK&F-96365 and LOE-908 failed to inhibit the ET-1-induced transient increase in [Ca²⁺]_i due to the release of intracellular Ca²⁺ stores (7), the release of sarcoplasmic reticulum Ca²⁺ was not sufficient to stimulate EGFR PTK transactivation. Three types of VICCs seem to be involved in the ET-1-induced EGFR PTK transactivation in terms of its sensitivity to SK&F-96365 and LOE-908 (see Figs. 4 and 6). One type of Ca²⁺ channel is sensitive to LOE-908 and resistant to SK&F-96365, another type is sensitive to both LOE-908 and SK&F-96365, and the third type is resistant to LOE-908 and sensitive to SK&F-96365. Based on pharmacological criteria, these channels are considered to be NSCC-1, NSCC-2, and SOCCs, respectively. Moreover, the percent contributions of NSCC-1, NSCC-2, and SOCCs to the ET-1-induced EGFR PTK transactivation are calculated to be ~20, 40, and 40%, respectively, of the nifedipine-resistant part of EGFR PTK transactivation caused by a 10 nM concentration of ET-1 (Fig. 6). The magnitudes of the ET-1-induced EGFR PTK transactivations that were inhibited by the combined treatment with nifedipine, SK&F-96365, and LOE-908 were similar to those in the absence of extracellular Ca²⁺ (see Figs. 3B and 4B). Therefore, extracellular Ca²⁺ influx through NSCC-1, NSCC-2, and SOCCs plays an important role in ET-1-induced EGFR PTK transactivation in rabbit internal carotid artery VSMCs.

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Calculations for contributions of Ca2+ influx through three types of voltage-independent Ca2+ channels to ET-1-induced EGFR PTK transactivation in VSMCs. A, B: ET-1-induced EGFR PTK phosphorylation in the presence of 10 µM LOE-908 or 10 µM SK&F-96365 is represented as a percentage of values in its absence. Contributions of store-operated Ca2+ channel (SOCCs) and Ca2+-permeable nonselective cation channel (NSCC-1) are represented as X and Z, respectively. Contributions of NSCC-2 are represented as W  Z or Y  X.

In conclusion, extracellular Ca²⁺ influx through NSCC-1, NSCC-2, and SOCCs plays an essential role for ET-1-induced EGFR PTK transactivation in rabbit internal carotid artery VSMCs.