Introduction

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Nitric oxide (NO) plays various important roles, including regulation of vascular tone (Furchgott et al., 1980; Ignaro, 1989), neurotransmission (Bredt et al., 1989), and immunoregulation (Garthwaite et al., 1989). There are two classes of NO synthase (NOS), constitutive and inducible forms (Hibbs et al., 1988). The constitutive enzymes are calcium- and calmodulin dependent and were initially identified in brain (NOS I or nNOS) (Nathan et al., 1994) and endothelial cells (NOS III or eNOS) (Lamas et al., 1992; Marsden et al., 1993). They are regulated by reversible binding of calcium-calmodulin. The inducible NOS isoform (NOS II or iNOS) is typically expressed in cells only after exposure to cytokines and is calcium independent (Lowenstein et al., 1992; Ikeda et al., 1996; Studer et al., 1996). The inducible NOS contains a tightly bound calmodulin subunit and remains active even at resting levels of intracellular calcium. All three classes of NOS catalyze the five electron oxidation of L-arginine to L-citrulline and generate NO. This oxidation requires NADPH and transfers electrons through FAD and FMN to a heme group and to L-arginine. Tetrahydro-biopterin maintains the enzyme in its active homodimeric form, and is essential for NOS activity. In vascular smooth muscle cells and platelets, NO activates soluble guanylate cyclase, which increases intracellular guanosine 3',5'-cyclic monophosphate, thereby inducing vasorelaxation and inhibition of platelet aggregation (Marin et al., 1990; deGraaf et al., 1992).

Previous studies have demonstrated that NO inhibits proliferation of various kinds of cells such as vascular smooth muscle cells (Garg et al., 1989a) and mesangial cells (Garg et al., 1989b). However, those studies relied on specific pharmacological tools such as NO donors at high pharmacological doses in the millimolar range rather than on authentic NO. NO donors contain several NO-related compounds, including peroxinitrate and oxidative products. Thus, the exact effects of authentic NO on cellular proliferation are still unclear. Recently, Sendai virus-liposome- or adenovirus-mediated endothelial NOS gene transfer into arteries has been reported to improve their vasomotor reactivities and inhibit neointimal formation in vivo (von der Lyden et al., 1995; Cable et al., 1997; Kullo et al., 1997a,b; Ooboshi et al., 1997). However, whether these effects are due to direct inhibition of smooth muscle cell proliferation or to inhibition of platelet aggregation or leukocyte adhesion by NO remains obscure.

Endothelin has been extensively investigated over the past years because of its important role in the pathogenesis of hypertension (Kohno et al., 1990), cardiac hypertrophy (Brown et al., 1995), and atherosclerosis (Lerman et al., 1991). Endothelin has three subtypes, endothelin-1, endothelin-2, and endothelin-3. Endothelin-1, the function of which is coupled to phospholipase C-mediated phosphoinositide hydrolysis (Takuwa et al., 1990), modulates vascular contractility and proliferation and is the most potent mammalian vasoconstrictor identified to date. In the present study, we transferred the endothelial NOS gene into 293 cells, a human embryonic kidney cell line, and investigated whether endogenously generated NO inhibited cellular proliferation in response to endothelin-1.

	Materials and Methods

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Cell Culture. The 293 cells, a human embryonic kidney cell line, were maintained in Dulbecco's modified Eagle's medium/nutrient mixture F12 (DMEM/F12) (Life Technologies, Paisley, Scotland) supplemented with 10% fetal calf serum (CSL Limited, Melbourne, Australia), 1% penicillin/streptomycin, and 1% glutamate and incubated at 37°C under 5% CO₂. The 293 cells were plated in 6-well culture dishes (Falcon Plastics, Cockeysville, MD) for Western blotting, luciferase assay, and NOS activity assay, or in 24-well dishes (Falcon Plasics) for thymidine incorporation assay.

Plasmids and Transfection. The expression plasmids of human endothelin type A receptor (pCDM-ETAR) and endothelin type B receptor (pCDM-ETBR) were obtained from Riken Gene Bank (Ibaragi, Japan). The full-length bovine endothelial NOS cDNA, a kind gift from D. G. Harrison (Emory University School of Medicine, Atlanta, GA), was isolated from the Bluescript SK+ vector by restriction enzyme digestion with SalI (4096 base pairs). To generate pCMV-NOS, this SalI fragment was ligated into the SalI cloning site of the pCMV expression plasmid (4050 base pairs). The pCMV vectors were engineered by introducing the cytomegalovirus immediate early promoter, the human growth hormone first intron, and the simian virus 40 polyadenylation signal into pUC 18 vectors.

Mitogen-Activated Protein Kinase and c-fos Promoter Activity Assay. Mitogen-activated protein kinase activity was measured with the PathDetect in vivo signal transduction pathway reporting system (Stratagene, La Jolla, CA) according to the manufacturer's instructions. Briefly, 293 cells were transfected with pFR-luc (GAL4 specific luciferase reporter gene), pFA-Elk (mitogen-activated kinase-specific transactivator GAL4 expression plasmid), pCDM-ETAR, and pCMV or pCMV-NOS with the calcium-phosphate coprecipitation method. After a 12-h incubation in serum-free medium, transfected cells were stimulated with 10⁹ mol/l human endothelin-1 (Peptide Institute, Osaka, Japan) for 3 h, and then luciferase activities were measured. The c-fos promoter activation was measured by cotransfection of pfos/luc plasmids into 293 cells. After 12 h of incubation in serum-free medium, transfected cells were stimulated with 10⁹ mol/l endothelin-1 for 6 h. Luciferase activity was measured with the luciferase assay system (Promega, Msdison, WI), and are expressed as relative light units.

Thymidine Incorporation Assay. DNA synthesis in transfected 293 cells was measured by thymidine incorporation into the cells as reported previously (Ikeda et al., 1991). The cells were rinsed twice with PBS, cultured in serum-free DMEM/F12 for 12 h, and incubated with endothelin-1 for 12 h, followed by addition of [³H]thymidine (100 µCi/ml; Amersham, Arlington Heights, IL) for another 6 h. The cells were washed with PBS twice and then lysed with 1N NaOH and 0.1% SDS. Radioactivity was measured by liquid scintillation counting.

Immunoblot Analysis of Endothelial NOS Protein. Cells were rinsed with ice-cold PBS and resuspended into the lysis buffer (1% Nonidet P-40, 50 mmol/l Tris-HCl, pH 7.4, 150 mmol/l NaCl, 200 U/ml aprotinine, 1 mmol/l phenylmethylsulfonyl fluoride). After incubation on ice for 30 min, cell extracts were centrifuged to remove cell debris. Cell lysates (30 µg) were then separated through 7.5% polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride membrane (Immunobin; Millipore, Bedford, MA). The membrane was incubated for 1 h at room temperature in Tris-buffered saline/Tween 20 (20 mmol/l Tris-HCl, pH 7.4, 150 mmol/l NaCl, 0.05% Tween 20) with 4% nonfat milk. The membrane was then incubated with anti-endothelial NOS antibody (Biomol, Plymouth Meeting, PA) (1:1000) overnight at 4°C in Tris-buffered/Tween 20. Specific binding of the antibody was visualized by the enhanced chemiluminescence detection system (Amersham) according to the manufacturer's instructions.

Measurement of NOS Activity. To measure NOS enzyme activity, L-arginine to L-citrulline conversion was assayed in transfected 293 cells with the NOS detect assay kit (Stratagene) according to the manufacturer's instructions. Briefly, protein extracts prepared as described above were incubated for 60 min at 37°C in a solution of 10 mmol/l L-[³H]arginine (2183 MBq/mmol), 1 mmol/l NADPH, 1 µmol/l FAD, 1 µmol/l FMN, 100 nmol/l calmodulin, 600 µmol/l CaCl₂, and 3 µmol/l tendothelin receptorahydrobiopterin in a final volume of 40 µl. The reaction was stopped by the addition of 400 µl of stop buffer (10 mmol/l EDTA, 50 mmol/l HEPES buffer, pH 5.5) to the reaction mixture. Then 100 µl of equilibrated resin was added to each mixture. Reaction samples were transferred to spin cups and centrifuged at 10,000g for 30 s. The radioactivity of the flowthrough was measured by liquid scintillation counting. Enzyme activity was expressed as citrulline production in picomoles per minute per milligram protein.

Measurement of [Ca²⁺]_i. The [Ca²⁺]_i levels of transfected cells were estimated with fura-2 fluorescence. The cells were rinsed with physiological saline solution containing 140 mmol/l NaCl, 4.6 mmol/l KCl, 1 mmol/l MgCl₂, 2 mmol/l CaCl₂, 10 mmol/l glucose, and 10 mmol/l HEPES, pH 7.4. They were then loaded with 5 µmol/l fura-2 acetoxymethyl ester (fura-2/AM) for 60 min at 37°C. After aspiration of the fura-2/AM solution, the glass slides were rinsed and then placed in a quartz cuvette at 37°C in a fluorescence spectrometer (model CAF-100; Japan Spectrometer, Tokyo, Japan). The fluorescence was monitored at 500 nm with excitation wavelengths of 340 and 380 nm in the ratio mode. From the ratio of fluorescence at 340 and 380 nm, the [Ca²⁺]_i was determined as described by Grynkiewicz et al. (1985), with the following expression: [Ca²⁺]_i (nmol/l) = K_d × [(R  R_min)/(R_max  R)] × , where R is the ratio of fluorescence of the sample at 340 and 380 nm, and R_max and R_min are determined by treating the cells with 50 µmol/l digitonin and 10 mmol/l MnCl₂, respectively. The term  is the ratio of fluorescence of fura-2 at 380 nm at zero and saturating Ca²⁺. K_d is the dissociation constant of fura-2 for Ca²⁺, assumed to be 224 nm at 37°C.

Statistical Analysis. Values are expressed as means ± S.E. of three or four samples, which represented at least three separate experiments. Differences of values were assessed by ANOVA with the least significant difference for multiple comparisons. Values of P < .05 were considered statistically significant.

	Results

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It has been shown that endothelin-1 not only induces vasoconstriction but also promotes proliferation of vascular smooth muscle cells. Endothelin receptor is known to be functionally linked to G proteins. Endothelin-1 stimulation increases intracellular Ca²⁺ levels, activates protein kinase C and mitogen-activated protein kinase, and enhances transcriptional activation of the c-fos and c-myc proto-oncogenes through ETARs. First, we incubated 293 cells with endothelin-1 as a mitogenic stimulant. As shown in Fig. 1, the c-fos/luciferase activity did not change in response to 10⁹ mol/l endothelin-1 in control cells, indicating that parental 293 cells do not express functional endothelin receptors. However, the c-fos/luciferase activity was increased in response to endothelin-1 in type A receptor-transfected cells.

View larger version (42K): [in this window] [in a new window]   Fig. 1.   c-fos/luciferase activity in ETAR gene-transfected cells. The 293 cells were transfected with pfos/luc and type A receptor expression plasmids by the calcium-phosphate coprecipitation method. After a 6-h incubation with 109 mol/l endothelin-1 (ET), luciferase activities were measured. The luciferase activity did not increase in control cells by endothelin-1 stimulation, whereas luciferase activity was significantly increased by endothelin-1 in type A receptor gene-transfected cells (pCDM-ETAR). Data are means ± S.E. of three samples. *P < .05 compared with ET-unstimulated cells.

We then investigated [³H]thymidine incorporation into 293 cells transfected with pCDM-ETAR or -ETBR. As shown in Fig. 2, [³H]thymidine incorporation into type A receptor-expressing cells was increased by endothelin-1 in a dose-dependent manner (10¹⁰~10⁸ mol/l), whereas incorporation into type B receptor-expressing cells was not increased. These findings suggest that functional ETAR was induced and coupled with cellular proliferation.

View larger version (65K): [in this window] [in a new window]   Fig. 2.   [3H]Thymidine incorporation into endothelin type A and type B receptor gene-transfected cells. The 293 cells were transfected with type A or type B receptor gene by the calcium-phosphate coprecipitation method. After 12 h of incubation with endothelin-1, [3H]thymidine was added to culture medium and the cells were further incubated for 6 h. [3H]Thymidine incorporation into type A receptor gene-transfected cells (pCDM-ETAR) was increased with endothelin-1 in a dose-dependent manner (1010~108 mol/l), whereas its incorporation into type B receptor gene-transfected cells (pCDM-ETBR) was not increased significantly with endothelin-1. Data are means ± S.E. of four samples. *P < .05 compared with endothelin-1-unstimulated cells.

To determine the functional role of endogenous NO, we transfected the endothelial NOS gene into 293 cells by the calcium-phosphate coprecipitation method. As shown in Fig. 3A, the 140-kD endothelial NOS protein was clearly present in transfected cells, whereas the cell lysate from pCMV (control plasmid)-transfected cells did not express the endothelial NOS protein. We also measured NOS activity in pCMV-NOS-transfected cells by the citrulline production. Citrulline production without Ca²⁺ indicates inducible NOS activity, whereas its production with Ca²⁺ represents Ca²⁺-dependent constitutive NOS activity. As shown in Fig. 3B, control cells showed neither inducible nor constitutive NOS activity, however, endothelial NOS-transfected cells demonstrated high constitutive but no inducible NOS activity. These findings indicate that endothelial NOS gene transfer into 293 cells was successful and functional. Therefore, we cotransfected ETAR and endothelial NOS genes into 293 cells in the following experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   A, immunoblotting of endothelial NOS protein. The 293 cells were transfected with endothelial NOS gene with the calcium-phosphate coprecipitation method. Twenty-four hours after transfection, the cells were lysed. Thirty micrograms of cell lysate was separated through 7.5% polyacrylamide gel electrophoresis and blotted onto membrane. Endothelial NOS protein expression (140-kD) was evident in endothelial NOS-transfected cells (pCMV-NOS), whereas only a small amount of endothelial constitutive NOS protein was expressed in control plasmid (pCMV)-transfected cells. B, NOS activity of transfected cells. The 293 cells were transfected with pCMV or pCMV-NOS with the calcium-phosphate coprecipitation method. Twenty-four hours after transfection, the cells were harvested. NO synthase activities were measured by citrulline formation from cell lysate incubated with or without Ca2+, as described in the text. NOS activity in the absence of Ca2+ indicates inducible NOS activity, whereas its activity in the presence of Ca2+ represents constitutive NOS activity. Control cells (pCMV) show neither inducible nor constitutive NOS activity. Inducible NOS activity was not detected in endothelial NOS-transfected cells (pCMV-NOS), whereas constitutive NOS activity was markedly increased in these cells. Data are means ± S.E. of three samples. *P < .05 compared with control cells.

ETAR mediates the mobilization of intracellular Ca²⁺ via activation of phospholipase C. As shown in Fig. 4, the addition of endothelin-1 (10⁹ mol/l) did not increase [Ca²⁺]_i of parental 293 cells, whereas endothelin-1 stimulation rapidly increased [Ca²⁺]_i of type A receptor-transfected 293 cells. Preincubation of the cells with the type A receptor antagonist BQ-485 (10⁸ mol/l; Calbiochem-Novabiochem Japan Ltd., Tokyo, Japan) for 10 min completely blocked the endothelin-1-induced increase in [Ca²⁺]_i. However, endothelial NOS gene transfer did not influence the rise of [Ca²⁺]_i in type A receptor-transfected cells induced by endothelin-1 (Table 1).

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Effects of endothelial NOS gene transfer on [Ca2+]i of ETAR-transfected cells. The 293 cells grown on glass coverslips were loaded with 5 µmol/l fura-2/AM for 60 min at 37°C. The cells were then placed in a quartz cuvette containing 2 ml of physiological saline solution, followed by addition of endothelin-1 (109 mol/l) with or without preincubation with the type A receptor antagonist BQ-485 (108 mol/l). Endothelin-1 did not increase [Ca2+]i in parental 293 cells (A). Endothelin-1 stimulation rapidly increased [Ca2+]i in type A receptor-transfected 293 cells (B), whereas preincubation of the cells with BQ-485 completely blocked the endothelin-1-induced increase in [Ca2+]i (C). Endothelial NOS gene transfer did not affect the endothelin-1-induced rise of [Ca2+]i in type A receptor-transfected cells (D). Data are representative of three independent experiments.

We then investigated the effects of endothelial NOS gene transfer on cellular proliferation using c-fos and mitogen-activated protein kinase luciferase assays. The luciferase activity that represents c-fos promoter activity in control 293 cells was increased up to 3.1-fold by endothelin-1 (Fig. 5). However, in endothelial NOS-transfected cells, the luciferase activity was significantly depressed under both endothelin-1-stimulated and unstimulated conditions. The luciferase activity that represents mitogen-activated protein kinase activity also was increased up to 12.8-fold with endothelin-1 stimulation (Fig. 6). However, in endothelial NOS-transfected cells, the luciferase activity was significantly depressed under both endothelin-1-stimulated and unstimulated conditions.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   c-fos/Luciferase activity in endothelial NOS gene-transfected cells. The 293 cells were transfected with pfos/luc, pCDM-ETAR, and pCMV or pCMV-NOS with the calcium-phosphate coprecipitation method. After 12 h of incubation in serum-free medium, transfected cells were stimulated with 109 mol/l endothelin-1 (ET) for 6 h, and luciferase activity was measured. In control 293 cells (pCMV), c-fos/luciferase activity was increased by 3.1-fold with ET stimulation. In endothelial NOS gene-transfected cells (pCMV-NOS), the basal level of c-fos/luciferase activity was markedly lower than that in control cells and there was no significant increase in its activity with ET stimulation. Data are means ± S.E. of three samples. *P < .05 compared with ET-unstimulated cells.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Mitogen-activated protein (MAP) kinase/luciferase activity in endothelial NOS gene-transfected cells. The 293 cells were transfected with pFR-luc, pFA-Elk, pCDM-ETAR, and pCMV or pCMV-NOS with the calcium-phosphate coprecipitation method. After 12 h of incubation in serum-free medium, transfected cells were stimulated with 109 mol/l endothelin-1 (ET) for 3 h, and then luciferase activity was measured. In control 293 cells (pCMV), luciferase activity was increased by 12.8-fold with ET stimulation. In endothelial NOS gene-transfected cells (pCMV-NOS), the basal level of luciferase activity was lower than that in control cells and there was no significant increase in its activity with ET. Data are means ± S.E. of three samples. *P < .05 compared with ET-unstimulated cells.

We further investigated DNA synthesis of endothelial NOS-transfected and untransfected 293 cells. As shown in Fig. 7, endothelial NOS gene transfer significantly inhibited [³H]thymidine incorporation into 293 cells stimulated with endothelin-1 by 60.3%, and this inhibition was abolished in the presence of the NOS inhibitor N^G-monomethyl-L-arginine acetate (1 mmol/l).

View larger version (37K): [in this window] [in a new window]   Fig. 7.   [3H]Thymidine incorporation into endothelial NOS gene-transfected 293 cells. The 293 cells were transfected with PCDM-ETAR, and pCMV or pCMV-NOS with the calcium-phosphate coprecipitation method. After 12 h of incubation with 109 mol/l endothelin-1, [3H]thymidine was added to culture medium and the cells were further incubated for 6 h. [3H]Thymidine incorporation into endothelial NOS gene-transfected 293 cells (pCMV-NOS) was significantly lower than that into control cells (pCMV). This inhibitory effect was abolished in the presence of the NOS inhibitor NG-monomethyl-L-arginine acetate (L-NMMA) (1 mmol/l). Data are means ± S.E. of four samples. *P < .05 compared with control cells.

We also investigated the effects of endothelin-1 on cellular proliferation of 293 cells. As shown in Fig. 8, the number of 293 cells was significantly increased in the presence of endothelin-1, which was significantly suppressed by endothelial NOS gene transfer as well as by the mitogen-activated protein kinase inhibitor PD98059 (10⁵ mol/l; Biomol).

View larger version (78K): [in this window] [in a new window]   Fig. 8.   Changes in cell number of ETAR gene-transfected 293 cells. Cells were transfected with PCDM-ETAR with the calcium-phosphate coprecipitation method, and incubated with 109 mol/l endothelin-1 (ET) in serum-free DMEM/F12 for 24 h. The cell number was counted with a hemocytometer. The endothelial NOS gene transfer (PCMV-NOS) as well as the mitogen-activated protein kinase inhibitor PD98059 (105 mol/l) significantly inhibited ET-induced cellular proliferation. Data are means ± S.E. of four samples. *P < .05 compared with ET-unstimulated control cells indicated as ().

	Discussion

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Endothelin-1 activities are mediated by binding to specific cell-surface receptors. Two types of endothelin receptors, type A and type B receptors, have been identified, cloned, and sequenced (Arai et al., 1990; Sakurai et al., 1990). The contraction and proliferation of vascular smooth muscle cells in response to endothelin-1 are mediated via type A receptors (Yanagisawa et al., 1988; Eguchi et al., 1994). In the present study, we demonstrated that c-fos promoter activity and DNA synthesis of 293 cells were increased via type A receptor stimulation. Although parental 293 cells did not exhibit inducible or constitutive NOS activity, pCMV-NOS-transfected 293 cells induced functional constitutive NOS expression. Using these transfected cells, we demonstrated that endogenously generated NO inhibits cellular proliferation in response to endothelin-1.

Antiproliferative effects of NO have been extensively investigated. However, most previous studies relied on specific pharmacological tools such as NO donors at high pharmacological doses. Recently, Kullo et al. (1997c) and Sharma et al. (1999) reported that gene transfer of endothelial NOS to vascular smooth muscle cells resulted in the expression of a functional enzyme and inhibition of cellular proliferation in response to fetal calf serum or platelet-derived growth factor. The gene transfer method is thought to be one of promising new tools for investigating the effects of certain molecules and their signaling pathways. In this study, we investigated whether NO generated from overexpression of endothelial NOS by 293 cells could inhibit cellular proliferative activities and which pathways were involved. These 293 cells are easily transduced by the calcium-phosphate coprecipitation method, and because parental 293 cells have no functional endothelin receptors, we reconstructed an endothelin-sensitive cell system.

ETAR is known to be functionally linked to G proteins. Activation of G proteins results in activation of the phospholipase C cascade and increases diasylglycerol and [Ca²⁺]_i, which activates protein kinase C. In the present study, [Ca²⁺]_i was increased in response to endothelin-1 in type A receptor-transfected cells. However, the [Ca²⁺]_i response to endothelin-1 in endothelial NOS-transfected cells was not different from that in the untransfected control cells. These findings suggest that endogenous NO does not modulate the phospholipase C-calcium system. However, exogenous NO has been reported to inhibit protein kinase C gene expression and its activity in a variety of cells. Jun et al. (1994) reported that lipopolysaccharide- or phorbol 12-myristate 13-acetate-induced NO or the NO donor sodium nitroprusside inhibited the expression of the protein kinase C delta gene in murine peritoneal macrophages. Studer et al. (1996) reported that the NO donor S-nitroso-N-acetylpenicillamine inhibited basal protein kinase C activity in mesangial cells. The modulation of protein kinase C activity is important for cellular proliferation and protein synthesis. We investigated whether the expression of the intermediary signaling element mitogen-activated protein kinase, which is downstream of protein kinase C, and the proto-oncogene c-fos were modulated by endothelial NOS gene transfer. Various growth factors, including endothelin-1, are known to activate mitogen-activated protein kinase and c-fos (Komuro et al., 1988; Jones et al., 1995). We observed that mitogen-activated protein kinase and c-fos promoter activities were increased in ETAR-reconstituted cells with endothelin-1 stimulation. However, their activities as well as [³H]thymidine incorporation and cellular proliferation in response to endothelin-1 were significantly suppressed in endothelial NOS-transfected cells. In addition, the mitogen-activated protein kinase inhibitor PD98059 significantly inhibited proliferation of 293 cells induced by endothelin-1. Thus, the antiproliferative effect of authentic NO appears to occur after the phospholipase C-calcium system but before the mitogen-activated protein kinase pathway. However, there are multiple pathways induced by endothelin-1 that could ultimately lead to cellular proliferation, and precise mechanisms of antiproliferative effects of authentic NO remain unclear.

Endothelin-1, a potent vasoconstrictor peptide secreted from endothelial cells, has been implicated in a number of human diseases including hypertension, atherosclerosis, and restenosis after angioplasty. These pathological processes are thought to mediate the cell-proliferative effects of endothelin-1. Azuma et al. (1994) reported that endothelin-1 and endothelin receptor gene expression was enhanced in the neointima of rat carotid artery after balloon injury. There are also several reports that endothelin-1 infusion accelerates and endothelin receptor antagonist administration reduces neointimal formation after balloon injury (Douglas et al., 1993, 1994; Trachtenberg et al., 1993). Therefore, endothelin-1-induced cellular proliferation may contribute to the development of vascular remodeling in response to the vascular injury.

Our results revealed that endogenously generated NO inhibits cellular proliferation in response to endothelin-1 via suppression of the mitogen-activated protein kinase cascade, suggesting that endothelial NOS gene transfer can improve the clinical course of endothelin-1-related proliferative vascular disorders.