Inhibition of α7-nicotinic acetylcholine receptor expression by arsenite in the vascular endothelial cells

Toxicology Letters 159 (2005) 47–59

Inhibition of ␣7-nicotinic acetylcholine receptor expression by arsenite in the vascular endothelial cells Shih-Hsin Hsu a , Tsui-Chun Tsou b , Shu-Jun Chiu c , Jui-I Chao a,∗ a

Molecular Toxicology Laboratory, Institute of Pharmacology and Toxicology, College of Life Sciences, Tzu Chi University, 701 Section 3, Chung-Yang Road, Hualien 970, Taiwan b Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Kaohsiung, Taiwan c Department of Radiological Technology, Tzu Chi College of Technology, Hualien, Taiwan Received 2 February 2005; received in revised form 19 April 2005; accepted 20 April 2005 Available online 14 June 2005

Abstract The ␣7-nicotinic acetylcholine receptor (␣7-nAChR), expressed in the neuronal and non-neuronal cells, has been shown to regulate cell proliferation. However, the expression and function of the ␣7-nAChR in the proliferation of the vascular endothelial cells remain unclear. In this study, we investigated the expression of the ␣7-nAChR in the arsenite-exposed vascular endothelial cells. The vascular endothelial cells SVEC4-10 and porcine aorta endothelial cells (PAEC) expressed the ␣7-nAChR proteins. Moreover, the location of the ␣7-nAChR proteins on cell membrane of the vascular endothelial cells was identified by the ␣7-nAChR binding to a tetramethylrhodamine-labeled ␣-bungarotoxin (␣-BTX). Arsenite (20 ␮M, 24 h) significantly induced the cytotoxicity, cell growth inhibition, and apoptosis in the vascular endothelial cells. The level of ␣7-nAChR proteins was concentration dependently decreased in the arsenite-treated endothelial cells. Furthermore, a specific ␣7-nAChR antagonist, ␣-BTX, inhibited the cell viability in the vascular endothelial cells. Nevertheless, ␣-BTX, and a ␣7-nAChR agonist, nicotine, did not significantly alter the cytotoxicity in the arsenite-treated endothelial cells. In addition, arsenite decreased the level of endothelial nitric oxide synthase proteins but did not alter choline acetyltransferase proteins in the SVEC4-10 endothelial cells. Together, our results indicate that arsenite can inhibit the ␣7-nAChR protein expression and cause the cell injury in the vascular endothelial cells. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: ␣7-nAChR; ␣-Bungarotoxin; Arsenite; Apoptosis; Nicotine; eNOS

1. Introduction

∗ Corresponding author. Tel.: +886 3 8565301x7071; fax: +886 3 8570813. E-mail address: [email protected] (J.-I Chao).

The nicotinic acetylcholine receptors (nAChRs) are a member of the ionotropic receptor proteins formed by five homologous subunits in neuronal and nonneuronal cells (Albuquerque et al., 1997; Conti-Fine

0378-4274/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2005.04.012

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et al., 2000; Conti-Tronconi et al., 1994; Kihara et al., 2001; Shaw et al., 2002). Neuronal nAChRs include ␣ and ␤ subunits (Conti-Fine et al., 2000; Conti-Tronconi et al., 1994; Wang et al., 2001) while non-neuronal cells may express functional nAChRs that are composed of four kinds of subunits ␣, ␤, ␥, and ␦ or ␧ (Conti-Fine et al., 2000; Conti-Tronconi et al., 1994; Wang et al., 2001). It has been shown that nAChRs may regulate the cellular functions in a variety of cell types (ContiFine et al., 2000; Grando et al., 1995, 1996; Nguyen et al., 2000; Wang et al., 2001). The nAChRs mediate fast synaptic transmission in the neuronal cells (Conti-Fine et al., 2000; Conti-Tronconi et al., 1994). However, the nAChRs in non-neuronal tissues may modulate other functions, including cell proliferation, differentiation, and modulating cell shape and motility (Conti-Fine et al., 2000; Grando et al., 1995). The ␣7-nAChR can form a functional homomeric pentamer nAChR that is more permeable to Ca2+ influx than other nAChRs (Albuquerque et al., 1997; Bitner and Nikkel, 2002; Castro and Albuquerque, 1995). The ␣7-nAChR is activated by agonist such as choline and nicotine (Albuquerque et al., 1997; Papke et al., 1996) but is suppressed by antagonist ␣-bungarotoxin (␣-BTX) (Shaw et al., 2002; Wang et al., 2001). Furthermore, the ␣7-nAChR exists in a variety of cell types including neuron (Albuquerque et al., 1997; Si and Lee, 2001), bronchial epithelial cells (Wang et al., 2001), aortic endothelial cells (Wang et al., 2001), and macrophage (Wang et al., 2003). It has been shown that ␣7-nAChRs are involved in the cell proliferation (Trombino et al., 2004), vasodilation (Si and Lee, 2001), neuroprotection (Kihara et al., 2001; Shaw et al., 2002), and anti-inflammation (Wang et al., 2003). However, the expression and function of the ␣7-nAChR in the vascular endothelial cells remain unclear. Arsenic has been shown to be an important factor of Blackfoot disease (Chen et al., 1988), hypertension (Chen et al., 1995), peripheral neuropathy (Mahajan et al., 1992), and cancers (IARC, 1987). The vascular endothelial cells are the key targets of vasculopathy and atherogenicity induced by arsenic exposure (Engel et al., 1994; Lee et al., 2003; Liu and Jan, 2000; Tseng et al., 1997; Tsou et al., 2004). However, the expression and function of the ␣7-nAChR in the arsenic-induced vascular endothelial damages are still unclear. In this study, we investigated the expression and possible roles of the ␣7-nAChR in the vascular endothelial cells fol-

lowing arsenite exposure. We found that ␣7-nAChR existed on the cell membrane and is involved in the cell survival of vascular endothelial cells, and the expression of the ␣7-nAChR proteins was inhibited by arsenite treatment.

2. Materials and methods 2.1. Chemicals and antibodies ␣-Bungarotoxin (␣-BTX), ␣-BTX-tetramethylrhodamine (␣-BTX-TMR), sodium arsenite (NaAsO2 ), nicotine, Hoechst 33258, and 3-(4,5-dimethyl-thiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO). Anti-actin (I-19), anti-␣7-nAChR (H-302), antiextracellular signal-regulated kinase-2 (anti-ERK-2) (C-14), and anti-choline acetyltransferase (anti-ChAT) (H-95) antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-endothelial nitric oxide synthase (anti-eNOS) (PA3-031) antibody was purchased from Affinity Bioreagents Inc. (Golden, CO). BODIPY FL phallacidin was purchased from Molecular Probes Co. (Eugene, OR). The Cy5-labeled goat anti-rabbit IgG was purchased from Amersham Pharmacia Biotech (Little Chalfont Buckinghamshire, UK). 2.2. Cell culture The SVEC4-10 cell line (ATCC, #CRL-2181) was derived from mouse vascular endothelium. The porcine aorta endothelial cells (PAEC) were isolated from the thoracic aorta of 7 months old pig (Tsou et al., 2004). The SVEC4-10 and PAEC were routinely maintained in DMEM and M199 medium (Invitrogen Co., Carlsbad, CA), respectively, and the complete medium was supplemented with 10% fetal bovine serum, 2.2 g/L sodium bicarbonate, l-glutamine (0.03% w/v), 100 unit/ml penicillin, and 100 ␮g/ml streptomycin. The A549 cell line was derived from human lung carcinoma of a 58-year-old Caucasian male (Giard et al., 1973). The H1299 cell line was derived from a non-small cell lung adenocarcinoma tumor (Mitsudomi et al., 1992). The TSGH8301 cell line was derived from a bladder cancer of a male patient in Taiwan. The SVEC4-10, A549, TSGH8301 cell lines were

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purchased from Food Industry Research and Development Institute (Hsinchu, Taiwan). The PC 12 cell line, derived from rat adrenal pheochromocytoma cells, was a gift of Dr. D.I. Yang of Tzu Chi University. The human cancer and PC12 cell lines were cultured in complete RPMI-1640 medium (Invitrogen). The cells were maintained at 37 ◦ C and 5% CO2 in a humidified incubator (310/Thermo, Forma Scientific Inc., Marietta, Ohio). 2.3. Cytotoxicity assay The cells were plated in 96-well plates at a density of 1 × 104 cells/well for 16–20 h. Then the cells were treated with arsenite (5–40 ␮M) for 24 h in serumfree DMEM medium. After drug treatment, the cells were washed twice with phosphate-buffered saline (PBS), and were cultured in complete DMEM medium (containing 10% serum) for 2 days. Subsequently, the cells were incubated with 0.5 mg/ml of 3-(4,5dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide in complete DMEM medium for 4 h. The surviving cells converted MTT to formazan that generates a blue-purple color when dissolved in dimethyl sulfoxide (Plumb et al., 1989). The intensity was measured at 545 nm using a plate reader (Molecular Dynamics, OPTImax) for enzyme-linked immunosorbent assays. The absorbance of each treatment was the average of six wells. The relative percentage of survival was calculated by dividing the absorbance of treated sample by that of the control in each experiment. 2.4. Cell growth assay The SVEC4-10 cells were plated in a p60 Petri dish at a density of 1 × 105 cells/5 ml, and incubated in complete DMEM medium for 18 h. Then the cells were treated with 20 ␮M arsenite for 24 h in serumfree DMEM medium. Subsequently, the cells were re-incubated in complete DMEM medium for various times before they were trypsinized and the total cell number were counted by a hemocytometer. 2.5. Apoptosis assay The adherent cells were cultured on coverslips. After exposure to arsenite (20 ␮M, 24 h), the cells were

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washed twice with isotonic PBS (pH 7.4), and fixed in 4% paraformaldehyde solution in PBS for 1 h at 37 ◦ C. Then the nuclei were stained with 2.5 ␮g/ml Hoechst 33258 for 30 min. The number of apoptotic nuclei was counted by hemocytometer under a fluorescence microscope. A total of 200 cells were examined for the calculation of apoptotic percentage for each treatment. 2.6. Indirect immunofluorescence and confocal microscopy The cells were cultured on coverslips, which was kept in a p60 Petri dish for 16–20 h before treatment. After exposure to arsenite, the cells were washed with isotonic PBS (pH 7.4), fixed in 4% paraformaldehyde solution in PBS for 1 h at 37 ◦ C. Then the coverslips were washed three times with PBS, and nonspecific binding sites were blocked in PBS containing 10% normal goat serum, 0.3% Triton X-100 for 1 h. Subsequently, the cells were incubated with the primary antibody of rabbit anti-␣7-nAChR (1:250) or rabbit anti-ChAT (1:250) in PBS containing 10% normal goat serum overnight at 4 ◦ C, and washed three times with 0.3% Triton X-100 in PBS. Then the cells were incubated with the secondary antibody of goat anti-rabbit Cy5 (1:250) in PBS for 3 h at 37 ◦ C, and washed three times with 0.3% Triton X-100 in PBS. The nuclei were stained with 2.5 ␮g/ml Hoechst 33258 for 30 min. The actin filament (F-actin) was stained with 0.01 u/ml BODIPY FL phallacidin for 30 min. Finally, the samples were examined under a Leica confocal laser scanning microscope (Mannheim, Germany) that was equipped with a UV laser (351/364 nm), an Ar laser (457/488/514 nm), and a HeNe laser (543/633 nm). 2.7. The α7-nAChR binding assay The cells were cultured on coverslips, which was kept in a p60 Petri dish for 16–20 h before treatment. The cells were pretreated with nicotine (50 ␮M, 30 min) at 37 ◦ C and kept in 5% CO2 , and then the cells were washed with isotonic PBS (pH 7.4). Subsequently, the cells were incubated with 50 ␮g/ml ␣-BTX-TMR at 37 ◦ C and 5% CO2 incubator for 30 min. At the end of treatment, the cells were washed with isotonic PBS (pH 7.4), and mounted with 6 ␮l glycerol (80%). Finally, the

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samples were examined under a Leica confocal laser scanning microscope. 2.8. Western blot analysis Western analyses of the ␣7-nAChR, ERK-1/2, eNOS, and ChAT were performed using specific antibodies. Cells were lysed in the ice-cold cell extraction buffer (pH 7.6) containing 0.5 mM DTT, 0.2 mM EDTA, 20 mM HEPES, 2.5 mM MgCl2 , 75 mM NaCl, 0.1 mM Na3 VO4 , 50 mM NaF, 0.1%

Triton X-100, 1 ␮g/ml aprotinin, 0.5 ␮g/ml leupeptin, and 100 ␮g/ml 4-(2-aminoethyl)benzenesulfonyl fluoride. After removing the cell debris, the supernatant protein concentrations were determined by the BCA protein assay kit (Pierce, Rockford, IL). Equal amounts of whole cell extracts (20–60 ␮g/ml) were subjected to electrophoresis using 10%–12% sodium dodecyl sulfate-polyacrylamide gels. Following electrophoretic transfer of proteins onto polyvinylidene difluoride membranes, they were sequentially hybridized with primary antibody and followed with a horseradish

Fig. 1. Expression of the ␣7-nAChR in the vascular endothelial cells. (A) The SVEC4-10 cells were incubated with rabbit anti-␣7-nAChR antibody and then incubated with goat anti-rabbit Cy5. The ␣7-nAChR proteins displayed red fluorescence with goat anti-rabbit Cy5. The nuclei were stained with Hoechst 33258, which displayed blue fluorescence. (B) The PAEC were incubated with rabbit anti-␣7-nAChR antibody and then incubated with goat anti-rabbit Cy5. The ␣7-nAChR protein displayed red fluorescence with goat anti-rabbit Cy5. The nuclei were stained with Hoechst 33258, which displayed blue fluorescence. The F-actin was stained with BODIPY FL phallacidin, which displayed green fluorescence. (C) The total protein extracts in a variety of cell lines were prepared for Western blot analysis using anti-␣7-nAChR and anti-ERK-2 antibodies.

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peroxidase-conjugated second antibody (BioRad Co., Hercules, CA). Finally, the protein bands were visualized on the X-film using the enhanced chemiluminescence detection system (NEN, Boston, MA). For comparing the level of western blot protein between samples, a densitometer (Molecular Dynamics, Personal Densitometer ST), was used for estimating the intensity of each band on the X-film, and each experiment was repeated at least three times.

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test or a multiple comparison ANOVA test, and p-value of <0.05 was considered as statistically significant in the experiments.

3. Results 3.1. The vascular endothelial cells express the α7-nAChR proteins which locate on the cell membrane

2.9. Statistical analysis Data from the population of cells treated with different conditions were analyzed using paired Student’s t-

To examine the expression of ␣7-nAChR on the vascular endothelial cells, the cells were subjected to immunofluorescence staining and immunoblot

Fig. 2. Location of the ␣7-nAChR in the vascular endothelial cells. (A) The SVEC4-10 cells and (B) the PAEC were pretreatment with 50 ␮M nicotine for 30 min. Then the cells were incubated with 50 ␮g/ml ␣-BTX-TMR for 30 min. The ␣-BTX-TMR displayed red fluorescence.

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analysis. The cells were incubated with rabbit anti-␣7nAChR antibody and then incubated with goat antirabbit Cy5. As shown in Fig. 1A and B, both SVEC4-10 and PAEC were heavily stained with goat anti-rabbit Cy5 that displayed a red color, indicating the presence of ␣7-nAChR proteins. The ␣7-nAChR proteins of whole cell extracts in the SVEC4-10 endothelial cells were identified by immunoblot analysis (Fig. 1C). Moreover, it was observed that the ␣7-nAChR proteins were expressed in PC12 cells and the human lung cancer cells (A549 and H1299) but not TSGH8301 human bladder cancer cells (Fig. 1C). ERK-2 (p42) protein has been used as an internal control in several studies (Chao et al., 2004; Kuo et al., 2004). Interestingly, we found that ERK-1 (p44) could also be detected by antiERK-2 antibody in rodent cell lines (mouse SVEC4-10 and rat PC12 cells) but did not present in human cancer cell lines. To further examine the location of the ␣7-nAChR proteins in the vascular endothelial cells, a fluorescence-labeled ␣-BTX, ␣-BTX-TMR, was used in this study. The SVEC4-10 and PAEC were pretreated with or without 50 ␮M nicotine for 30 min, and then incubated with 50 ␮g/ml ␣-BTX-TMR for 30 min. Both SVEC4-10 and PAEC were heavily stained with ␣-BTX-TMR that displayed red color (Fig. 2A and B, left pictures). However, pretreatment with nicotine decreased the red fluorescence of ␣-BTX-TMR on the vascular endothelial cells (Fig. 2A and B, right pictures). 3.2. Arsenite induces the cytotoxicity and cell growth inhibition in the SVEC4-10 endothelial cells To examine the cytotoxicity of arsenite on the endothelial cells, the SVEC4-10 cells were treated with arsenite (5–40 ␮M) for 24 h, and the percentage of cell viability was estimated by MTT assay. As shown in Fig. 3A, arsenite induced the cell death in a concentration-dependent manner, and approximately 20% of cells survived after exposure to 20 ␮M arsenite for 24 h. To further determine the effects of arsenite on the cell growth, the SVEC4-10 cells were plated at a density of 1 × 105 cells per p60 dish, treated with 20 ␮M arsenite for 24 h, and the cell numbers were counted using a hemocytometer. Treatment with 20 ␮M arsenite for 24 h significantly inhibited the cell growth in the SVEC4-10 cells (Fig. 3B).

Fig. 3. Effects of arsenite on the cell growth and cytotoxicity in the SVEC4-10 cells. (A) The cells were treated with 0–40 ␮M arsenite for 24 h. The cell viability was measured by MTT assay. Results were obtained from 6 to12 experiments and the bar represents ±S.E. (B) The cells were treated with 20 ␮M arsenite for 24 h in the SVEC410 cells. After treatment, the cells were washed twice with PBS, and incubated for various times before they were counted by hemocytometer. Results were obtained from four experiments and the bar represents ±S.E. p < 0.05 (*) and p < 0.01 (**), between untreated and arsenite-treated samples.

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Fig. 4. Effects of arsenite on the level of ␣7-nAChR proteins in the vascular endothelial cells. (A) The SVEC4-10 cells were treated with 0–20 ␮M arsenite for 24 h. The total protein extracts were prepared for Western blot analysis using anti-␣7-nAChR and anti-ERK-2 antibodies. The relative protein levels under each treatment were the average of 3–5 independent experiments. (B) The SVEC4-10 cells were treated with 20 ␮M arsenite for 24 h. (C) The PAEC were treated with 60 ␮M arsenite for 24 h. After treatment, the cells were incubated with rabbit anti-␣7nAChR antibody and then incubated with goat anti-rabbit Cy5. The ␣7-nAChR protein displayed red fluorescence with goat anti-rabbit Cy5. The nuclei were stained with Hoechst 33258, which displayed blue fluorescence. The F-actin was stained with BODIPY FL phallacidin, which displayed green fluorescence.

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3.3. Arsenite decreases the level of α7-nAChR proteins and increases apoptosis in the vascular endothelial cells To examine the effect of arsenite on the ␣7-nAChR protein expression, the vascular endothelial cells were treated with arsenite and subjected to immunoblot anal-

ysis and immunofluorescence staining. As shown in Fig. 4A, arsenite (5–20 ␮M, 24 h) concentration dependently decreased the level of ␣7-nAChR proteins in the SVEC4-10 cells. Furthermore, the red fluorescence (Cy5) intensity exhibited by the ␣7-nAChR proteins were significantly decreased when exposed to 20 and 60 ␮M arsenite for 24 h in SVEC4-10 and PAEC,

Fig. 5. Effect of arsenite on apoptosis in the vascular endothelial cells. (A) The SVEC4-10 cells were treated with or without 20 ␮M arsenite for 24 h. After arsenite treatment, the cells were incubated with rabbit anti-␣7-nAChR antibody and then incubated with goat anti-rabbit Cy5. The nuclei were stained with Hoechst 33258. (B) The cells were treated with 20 ␮M arsenite for 24 h. Counting apoptotic nuclei scored the percentage of apoptotic cells. Results were obtained from four to five experiments and the bar represents S.E. p < 0.01 (**) indicates between untreated and arsenite-treated samples.

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respectively (Fig. 4B and C, lower pictures). In addition, arsenite significantly disrupted the cytoskeleton structure of F-actin and induced apoptosis in the vascular endothelial cells (Fig. 4B and C, the arrows of lower pictures). However, the level of actin proteins was not altered in the arsenite-treated SVEC4-10 endothelial cells (data not shown). The apoptotic nuclei (Fig. 5 A, lower pictures) were counted by hemocytometer in a fluorescence microscope. As shown in Fig. 5B, the percentage of apoptosis was about 70% apoptosis following arsenite (20 ␮M, 24 h) treatment in the SVEC4-10 cells. 3.4. α-Bungarotoxin inhibits cell viability but not enhance the arsenite-induced cell death in the SVEC4-10 cells An ␣7-nAChR antagonist, ␣-BTX, was employed for study in the endothelial cells to examine the effect of ␣7-nAChR on the cell viability. The percentage of cell viability was estimated by MTT assay. As shown in Fig. 6A, treatment with 1–2 nM ␣-BTX for 24 h significantly decreased the cell viability in the SVEC410 endothelial cells. To further investigate the role of ␣7-nAChR in the arsenite-induced cytotoxicity, the ␣BTX- or nicotine-pretreated cells were treated with arsenite (10 ␮M, 24 h). Treatment of 10 ␮M arsenite for 24 h was significantly induced the cytotoxicity in the SVEC4-10 cells (Fig. 6B). However, pretreatment with ␣-BTX or nicotine did not significantly alter the cytotoxicity in the arsenite-treated cells (Fig. 6B). 3.5. Arsenite decreases the level of eNOS proteins but not ChAT proteins in the SVEC4-10 cells We have investigated the levels of eNOS and ChAT proteins in the arsenite-treated vascular endothelial cells. The SVEC4-10 cells were treated with arsenite (0–20 ␮M, 24 h) and the levels of eNOS and ChAT proteins were assayed by immunoblot. As shown in Fig. 7A, arsenite at 5–20 ␮M for 24 h significantly decreased the level of eNOS proteins in a concentration-dependent manner. Furthermore, the red fluorescence (Cy5) intensity exhibited by eNOS was significantly decreased when exposed to 20 ␮M arsenite for 24 h in the SVEC4-10 cells (Fig. 7B). However, arsenite did not significantly alter the level of ChAT proteins in the SVEC4-10 cells (Fig. 7C). Also, the red

Fig. 6. Effects of ␣-BTX and nicotine on the cytotoxicity in the arsenite-treated SVEC4-10 cells. (A) The SVEC4-10 cells were treated with 0–2 nM ␣-BTX for 24 h. (B) The SVEC4-10 cells were pretreatment with ␣-BTX (1 nM, 2 h) or nicotine (1 ␮M, 2 h), and then treated with 10 ␮M arsenite for 24 h. The cell viability was measured by MTT assay. Results were obtained from 3 to 12 experiments and the bar represents ±S.E. p < 0.05 (*) indicates between untreated and drugs treated samples.

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Fig. 7. Effects of arsenite on the levels of eNOS and ChAT proteins in the arsenite-treated SVEC4-10 cells. (A) The SVEC4-10 cells were treated with 0–20 ␮M arsenite for 24 h. The total protein extracts were prepared for immunoblot blot analysis. The relative protein levels under each treatment were the average of four independent experiments. (B) The SVEC4-10 cells were incubated with rabbit anti-eNOS antibody and then incubated with goat anti-rabbit Cy5. The eNOS protein displayed red fluorescence with goat anti-rabbit Cy5. The nuclei were stained with Hoechst 33258, which displayed blue fluorescence. The F-actin was stained with BODIPY FL phallacidin, which displayed green fluorescence. (C) The SVEC4-10 cells were treated with 0–20 ␮M arsenite for 24 h. The total protein extracts were prepared for immunoblot blot analysis. The relative protein levels under each treatment were the average of four independent experiments. (D) After arsenite treatment, the SVEC4-10 cells were incubated with rabbit anti-ChAT antibody and then incubated with goat anti-rabbit Cy5. The ChAT protein displayed red fluorescence with goat anti-rabbit Cy5. The nuclei were stained with Hoechst 33258, which displayed blue fluorescence. The F-actin was stained with BODIPY FL phallacidin, which displayed green fluorescence.

fluorescence (Cy5) intensity exhibited by ChAT was not significantly altered by arsenite in the SVEC4-10 cells (Fig. 7D).

4. Discussion Arsenic has been shown to mediate vasculopathy and hypertension (Chen et al., 1995; Engel et al., 1994; Tseng et al., 1997). The vascular endothelial cells are the important targets of cardiovascular diseases induced by arsenic exposure (Engel et al., 1994; Tseng et al., 1997). The mean value of blood arsenic in a population exposed to arsenic in drinking water has been reported to be around 0.5 ␮M (Heydorn, 1970; Pi et al., 2000). Moreover, the long termed low concentration exposure and the short-termed high concentration exposure may produce similar arsenite accu-

mulations in the cells (Yih and Lee, 1999). In this study, we found that treatment with 5–20 ␮M arsenite for 24 h significantly induced the cytotoxicity, cell growth inhibition, apoptosis, and decreased ␣7-nAChR proteins in the vascular endothelial cells. The vascular endothelial cells SVEC4-10 and PAEC expressed the ␣7-nAChR proteins. A fluorescence-labeled antagonist of ␣7-nAChR, ␣-BTX-TMR, bind to the cell membrane of the vascular endothelial cells, and this binding was inhibited by an ␣7-nAChR agonist nicotine. Furthermore, the ␣7-nAChR is expressed in the human aortic endothelial cells (Wang et al., 2001). It has been shown that ␣7-nAChR can mediate cell proliferation and neuroprotection (Kihara et al., 2001; Shaw et al., 2002). Moreover, the activation of ␣7-nAChR by nicotine can activate the survival pathway of phosphoatidylinositol 3-kinase (PI3K)-Akt against cell death (Kihara et al., 2001; Shaw et al., 2002). In addition, a

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specific ␣7-nAChR antagonist, ␣-BTX, decreased the cell viability in the vascular endothelial cells. However, ␣-BTX did not significantly alter the cytotoxicity of the arsenite-treated endothelial cells. Aslo, an ␣7nAChR agonist, nicotine, did not reduce cell death in the arsenite-treated endothelial cells. Accordingly, we suggest that arsenite inhibits the ␣7-nAChR protein expression via an irreversible pathway for the cell death in the vascular endothelial cells. ␣7-nAChR activation can promote the vasodilation (Si and Lee, 2001). The presynaptic ␣7-nAChR can mediate nicotine-induced nitric oxidergic neurogenic vasodilation in porcine basilar arteries (Si and Lee, 2001). Furthermore, the ␣7-nAChR-mediated vasodilation can be inhibited by lead (Si and Lee, 2003). In our experiments, we have found that lead induced the cell growth inhibition and cell death in the vascular endothelial cells (data not shown). Moreover, the level of ␣7-nAChR proteins was decreased in the arsenitetreated vascular endothelial cells. Concomitantly, the level of eNOS proteins was significantly decreased in the arsenite-treated endothelial cells. In a recent study, treatment with arsenite resulted in the inhibition of eNOS of the human aortic endothelial cells (Lee et al., 2003). Endothelium-derived NO is generated from l-arginine by eNOS that mediates smooth muscle relaxation and vasodilation (Schmidt and Walter, 1994; Snyder, 1995). Moreover, arsenite mediates the vasculopathy and hypertension (Chen et al., 1995; Lee et al., 2003; Liu and Jan, 2000). Therefore, the inhibition of the ␣7-nAChR and eNOS may be involved in the arsenite-induced dysfunction in the vasodilation, and the development of cardiovascular diseases. Choline acetyltransferase (ChAT), an enzyme involved in the synthesis of acetylcholine (Klapproth et al., 1997), is expressed in the vascular endothelial cells (Klapproth et al., 1997; Wang et al., 2001). In humans, acetylcholine is detected in a variety of tissues that may function as a local hormone, and modulates cellular functions in non-neuronal cells by acting on specific receptors such as the ␣7-nAChR (Klapproth et al., 1997; Song et al., 2003). Moreover, acetylcholine is synthesized by ChAT and secreted by the human aortic endothelial cells (Wang et al., 2001). The administration of acetylcholine may influence cellular functions in non-neuronal tissues including cell proliferation and differentiation (Grando et al., 1993; Klapproth et al., 1997; Song et al., 2003). However, the ChAT

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proteins were not significantly altered in the arseniteexposed vascular endothelial cells. Thus, we suggest that the ChAT protein expression is not involved in the cell growth inhibition and apoptosis of the arseniteexposed vascular endothelial cells. Nevertheless, one cannot exclude the possibility that ChAT enzyme activity can be inhibited by arsenite in the vascular endothelial cells. Thus, the roles of ChAT enzyme activity in the arsenite-induced apoptosis need further investigation. In summary, the ␣7-nAChR proteins are expressed on the cell membrane and involve the cell survival in the vascular endothelial cells, and arsenite can inhibit the ␣7-nAChR protein expression of the vascular endothelial cells. Moreover, the arsenite-decreased eNOS protein expression may inhibit the vasodilation of the blood vessel. Acknowledgements This work was supported by National Health Research Institutes, Taiwan, Grant EO-093-PP-05, and Tzu Chi University, Grant TCMRC 92009. We also are indebted to Dr. T.H. Chiu (Institute of Pharmacology and Toxicology, Tzu Chi University) for careful reading of the manuscript. References Albuquerque, E.X., Alkondon, M., Pereira, E.F., Castro, N.G., Schrattenholz, A., Barbosa, C.T., Bonfante-Cabarcas, R., Aracava, Y., Eisenberg, H.M., Maelicke, A., 1997. Properties of neuronal nicotinic acetylcholine receptors: pharmacological characterization and modulation of synaptic function. J. Pharmacol. Exp. Ther. 280, 1117–1136. Bitner, R.S., Nikkel, A.L., 2002. ␣7-Nicotinic receptor expression by two distinct cell types in the dorsal raphe nucleus and locus coeruleus of rat. Brain Res. 938, 45–54. Castro, N.G., Albuquerque, E.X., 1995. ␣-Bungarotoxin-sensitive hippocampal nicotinic receptor channel has a high calcium permeability. Biophys. J. 68, 516–524. Chao, J.I., Kuo, P.C., Hsu, T.S., 2004. Down-regulation of survivin in nitric oxide-induced cell growth inhibition and apoptosis of the human lung carcinoma cells. J. Biol. Chem. 279, 20267– 20276. Chen, C.J., Hsueh, Y.M., Lai, M.S., Shyu, M.P., Chen, S.Y., Wu, M.M., Kuo, T.L., Tai, T.Y., 1995. Increased prevalence of hypertension and long-term arsenic exposure. Hypertension 25, 53– 60. Chen, C.J., Wu, M.M., Lee, S.S., Wang, J.D., Cheng, S.H., Wu, H.Y., 1988. Atherogenicity and carcinogenicity of high-arsenic

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