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Involvement of endogenous nitric oxide in angiotensin II-induced activation of vascular mitogen-activated protein kinases

¹Department of Pharmacology, ²Life Science Research Center, ³Department of Gastroenterological Surgery, and ⁴Department of Cardiorenal and Cerebrovascular Medicine, Kagawa University Medical School, Kagawa, Japan

Submitted 8 March 2007 ; accepted in final form 6 July 2007

	ABSTRACT

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Angiotensin II (ANG II) is a powerful activator of mitogen-activated protein (MAP) kinase cascades in cardiovascular tissues through a redox-sensitive mechanism. Nitric oxide (NO) is considered to antagonize the vasoconstrictive and proarteriosclerotic actions of ANG II. However, the role of endogenous NO in ANG II-induced redox-sensitive signal transduction is not yet clear. In this study using catheterized, conscious rats, we found that acute intravenous administration of N^G-nitro-L-arginine methyl ester (L-NAME; 5 mg/kg) enhanced phosphorylation of aortic MAP kinases extracellular signal regulated kinase (ERK) 1/2 and p38, which were suppressed only partially by a superoxide dismutase mimetic (Tempol), whereas ANG II-induced MAP kinase phosphorylation was markedly suppressed by Tempol. FK409, a NO donor, had little effect on vascular MAP kinase phosphorylation. On the other hand, acute exposure to a vasoconstrictor dose of ANG II (200 ng·kg^–1·min^–1 iv) failed to enhance phosphorylation of aortic MAP kinases in the chronically L-NAME-treated rats, whereas the vasoconstrictor effect of ANG II was not affected by L-NAME treatment. Furthermore, three different inhibitors of NO synthase suppressed, in a dose-dependent manner, ANG II-induced MAP kinase phosphorylation in rat vascular smooth muscle cells, which was closely linked to superoxide generation in cells. These results indicate the involvement of endogenous NO synthase in ANG II-induced signaling pathways, leading to activation of MAP kinase, and that NO may have dual effects on the vascular MAP kinase activation associated with redox sensitivity.

oxidative stress; N^G-nitro-L-arginine methyl ester; extracellular signal-regulated kinase 1/2; p38

Nitric oxide (NO) is a widespread vasoactive molecule and can stimulate soluble guanylate cyclase, the mechanism that accounts for its biological responses. Several roles of NO, including vascular relaxation and inhibitory actions on leukocyte adhesion to endothelium (9), platelet aggregation (18), and smooth muscle proliferation (6), are considered antagonistic to those of ANG II in a variety of vascular functions. However, the possible role of NO in regulation of vascular MAP kinase is controversial. Kubo et al. (10) have shown that sodium nitroprusside, a NO donor, antagonizes ANG II-induced ERK1/2 activation, and N^G-nitro-L-arginine methyl ester (L-NAME), a nonspecific NO synthase inhibitor, increases ERK1/2 activity in intact rat aortic segments. Palen et al. (13) have recently shown that NO can dephosphorylate basal ERK1/2 in rat vascular smooth muscle cells in a cGMP-dependent fashion. Meanwhile, Gu et al. (5) have demonstrated that NO donor S-nitroso-N-acetylpenicillamine increases p21 protein expression through an ERK activation pathway. Bauer et al. (1) have demonstrated that the antiproliferative effect of NO on vascular smooth muscle cells is mediated by ERK1/2 activation. Furthermore, Mizuno et al. (12) have demonstrated, using human pulmonary vascular smooth muscle cells, that exogenous NO transiently activates ERK via induction of p53 and then suppresses it via inactivation of the Ras/Raf cascade. Pinzar et al. (15) have recently shown that L-NAME suppresses ANG II-stimulated ERK phosphorylation in rat vascular smooth muscle cells. Although these studies indicate a complexity of NO-mediated interaction, through different signal transduction pathways leading to MAP kinase activation, few studies have investigated the possible effect of NO on vascular MAP kinases at the whole-animal level from the viewpoint of ROS sensitivity.

In this study using catheterized, conscious rats, we examined 1) the acute effect of NO synthase inhibition on aortic MAP kinase phosphorylation in the presence or absence of radical scavengers and 2) the involvement of endogenous NO in ANG II-induced aortic MAP kinase phosphorylation. Furthermore, we also examined the effects of NO synthase inhibition on ANG II-induced MAP kinase phosphorylation and superoxide generation using cultured rat vascular smooth muscle cells.

	METHODS

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Animal preparation. Nine-wk-old male Sprague-Dawley rats were used. All animals were allowed to recover overnight after catheterization, and all hemodynamic measurements were performed on conscious rats (23). The femoral arterial catheter was connected to a pressure transducer, and mean arterial blood pressure and heart rate were continuously recorded on a multichannel polygraph recorder. In the acute experiment, L-NAME was administered intravenously at a dose of 5 mg/kg. ANG II and FK409 (Fujisawa Pharmaceutical, Osaka, Japan), a NO donor, were infused at a rate of 200 ng·kg^–1·min^–1 and 0.1 mg·kg^–1·min^–1 for 30 min, respectively. Tempol (Sigma) and 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (3-CP; Sigma), a structurally related and inactive compound of Tempol, were given intravenously at a priming dose of 30 mg/kg, followed by infusion at a rate of 0.5 mg·kg^–1·min^–1. Treatment with Tempol or 3-CP was started 10 min before the administration of L-NAME or ANG II. Thirty min after treatment, the thoracic aorta was removed, quickly frozen in liquid nitrogen, and stored at –80°C.

In a separate experiment, rats received chronic L-NAME treatment (1 g/l in drinking water) for 7 days. ANG II was infused into rats chronically treated with L-NAME for 30 min, with a similar protocol to that of the acute experiment above. All surgical and experimental procedures were approved by the Kagawa University Animal Care and Use Committee (ACUC) and performed according to the Kagawa University ACUC guidelines.

Cell culture. Rat vascular smooth muscle cells prepared by the explant method from the descending thoracic aorta of 4-wk-old male Sprague-Dawley rats were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (vol/vol) fetal bovine serum. All cells were incubated at 37°C in 5% CO₂ in air. Rat vascular smooth muscle cells were authenticated using immunohistochemical staining for smooth muscle -actin (Sigma). Cultures showing 95% staining for -actin between passages five and seven were used. Before the experiments, cells were starved for 48 h. L-NAME, N^G-monomethyl-L-arginine (L-NMMA), and N^G-nitro-L-arginine (L-NNA) were given 30 min before exposure to 100 nM ANG II.

Measurement of phosphorylation of ERK1/2 and p38 MAP kinases. Total and phosphorylated ERK1/2 and p38 MAP kinases were analyzed by Western blot analysis, as previously described (8, 23). Briefly, frozen tissues were homogenized in cold lysis buffer [20 mM Tris·HCl (pH 7.4) 140 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM -glycerophosphate, and 1 mM sodium orthovanadate]. Rat vascular smooth muscle cells were directly lysed in the buffer. The homogenate or lysate was subjected to sonication (20 s, three times) on ice, followed by centrifugation for 30 min at 12,000 g to remove cellular debris. The protein concentration of the supernatant was assessed with a protein assay kit (Bio-Rad). Equal amounts of protein from each sample were resolved by 10% SDS-PAGE. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Hybond TM-ECL; Amersham Pharmacia Biotech). The membranes were blocked for 2 h at room temperature with 5% skimmed milk in PBS and 0.1% Tween 20. The blots were incubated overnight with 1:1,000 diluted primary antibodies, anti-ERK1/2, anti-p38, anti-phospho-ERK1/2, and anti-phospho-p38 (Cell Signaling Tech), followed by incubation for 1 h with a secondary antibody (horseradish peroxidase-conjugated anti-rabbit IgG; 1:2,000). Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech) and quantified by LAS-1000 plus (Fujifilm, Tokyo, Japan).

Detection of superoxide in rat vascular smooth muscle cells. Cells were incubated with 5 µg/ml dihydroethidium (DHE) for 10 min at 37°C. After the cells were washed with PBS, DHE fluorescent images of rat vascular smooth muscle cells were visualized by excitation at 405 nm and emission at 420 nm for detection of DHE-loaded cells, as stained in blue, and by excitation at 488 nm and emission at 610 nm for detection of oxidized DHE product ethidium, as stained in red. L-NAME was given 30 min before the exposure to 100 nM ANG II.

Statistical analysis. Values are expressed as means ± SE. Statistical significance between more than two groups was tested using two-way ANOVA followed by the Newman-Keuls test or unpaired, two-tailed Student's t test. P < 0.05 was considered to indicate statistical significance.

	RESULTS

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Effects of acute L-NAME administration on arterial blood pressure and heart rate in conscious rats. Changes of mean blood pressure and heart rate are summarized in Table 1. Intravenous injection of L-NAME (5 mg/kg) increased mean arterial blood pressure from 103 ± 4 to 141 ± 5 mmHg within 10 min and decreased heart rate from 320 ± 14 to 244 ± 17 beats/min. Tempol or 3-CP alone did not alter mean arterial blood pressure and heart rate during the experimental period (data not shown), and simultaneous administration of Tempol or 3-CP had no significant effects on the hemodynamic changes induced by L-NAME. Intravenous infusion of FK409 (0.1 mg·kg^–1·min^–1) decreased mean blood pressure by 22 mmHg.

View larger version (14K): [in this window] [in a new window]   Fig. 1. Effects of NG-nitro-L-arginine methyl ester (L-NAME), angiotensin II (ANG II), and FK409 on aortic mitogen-activated protein (MAP) kinase phosphorylation. The phosphorylated forms of ERK1/2 and p38 MAP kinases were analyzed in the aorta obtained 30 min after treatment. Top: representative blots. Bottom: densitometric analysis of the phosphorylated forms of MAP kinase. The mean value of each phosphorylated protein in the saline-infused control rats is represented as 1. Data are presented as means ± SE of 4 or 5 rats. *P < 0.05 vs. the control rats. P < 0.05 vs. the L-NAME- or ANG II-treated rats. 3-CP, 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy.

View larger version (11K): [in this window] [in a new window]   Fig. 2. Effects of chronic L-NAME treatment on ANG II-induced hypertension and aortic MAP kinase phosphorylation. A: mean arterial blood pressure was monitored during ANG II infusion. The data used were obtained at the end of a 30-min ANG II (200 ng·kg–1·min–1) or saline infusion. B: phosphorylated ERK1/2 and p38 MAP kinases were analyzed in the aorta obtained 30 min after treatment. B, top: representative blots. B, bottom: densitometric analysis of phosphorylated MAP kinase. The mean value of each phosphorylated protein in the saline-infused control rats is represented as 1. Data are presented as means ± SE of 4 or 5 rats. *P < 0.05 vs. the saline-infused rats in each group. P < 0.05 vs. the rats without L-NAME treatment.

NO synthase inhibition suppresses ANG II-induced MAP kinase activation in rat vascular smooth muscle cells. In rat vascular smooth muscle cells, a 30-min exposure to 100 nM ANG II increased phosphorylation of ERK1/2 and p38 MAP kinases approximately eight and five times more than the basal level, respectively (Fig. 3). L-NAME, L-NMMA, and L-NNA suppressed ANG II-induced phosphorylation at doses >0.1 mM, with the exception of ERK1/2 suppression by L-NAME, which was only significant at a dose of 1 mM. No NO synthase inhibitor alone had any significant effect on the basal phosphorylation of ERK1/2 and p38 MAP kinases in rat vascular smooth muscle cells.

View larger version (16K): [in this window] [in a new window]   Fig. 3. Dose-response effects of nitric oxide (NO) synthase inhibitors, L-NAME, NG-monomethyl-L-arginine (L-NMMA), and NG-nitro-L-arginine (L-NNA), on ANG II-induced ERK1/2 (A) and p38 MAP kinase phosphorylation (B) in rat vascular smooth muscle cells. Different doses of NO synthase inhibitors were given 30 min before exposure to 100 nM ANG II. Thirty min after treatment, cells were harvested, and phosphorylated MAP kinases were analyzed. Top: representative blots. Bottom: densitometric analysis of the phosphorylated MAP kinases. The mean value of each phosphorylated protein in untreated cells is represented as 1. Data are presented as means ± SE of 4 different dishes. *P < 0.05 vs. untreated cells.

View larger version (31K): [in this window] [in a new window]   Fig. 4. Effect of L-NAME on ANG II-induced superoxide generation in rat vascular smooth muscle cells. Superoxide generation of rat vascular smooth muscle cells was visualized by the dihydroethidium (DHE) fluorescence method. L-NAME (1 mM) was given 30 min before exposure to 100 nM ANG II. A: representative fluorescence images after 30 min exposure to ANG II are shown. B: ratio of fluorescence intensity of ethidium (oxidized form of DHE) to DHE at 30 min after ANG II exposure. Data are presented as means ± SE of 4 different cover slides. *P < 0.05 vs. cells without ANG II. P < 0.05 vs. cells without L-NAME treatment.

	DISCUSSION

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Consistent with our previous reports, the increased phosphorylation of vascular ERK1/2 and p38 MAP kinases by acute ANG II challenge was almost completely reversed by simultaneous treatment with Tempol (23). On the other hand, the increase in these phosphorylated MAP kinases by acute L-NAME treatment was not as sensitive to Tempol, indicating that there may be different and complex pathways involved in activation of vascular MAP kinases by acute exposure to L-NAME. Kubo et al. (10) have demonstrated that ERK1/2 phosphorylation in intact rat aortic segments is increased by endothelium denudation or by L-NAME treatment, which indicates a role for endothelial cell-derived NO in vascular ERK1/2 regulation. However, in this study we found that a NO donor, at a large dose that elicited a reduction in arterial blood pressure of 20 mmHg, did not affect the phosphorylation of aortic (and also cardiac; data not shown) MAP kinases in conscious rats. Therefore, despite much in vitro evidence supporting a suppressive role of NO in vascular MAP kinase activation, exogenous NO given intravenously may have little effect on vascular MAP kinase activity under conscious physiological conditions.

Another important finding of this study is that ANG II is no longer effective at inducing aortic MAP kinase phosphorylation under conditions of NO synthase inhibition with chronic L-NAME treatment, indicating that NO synthase potentiates the vascular MAP kinase activation in response to acute ANG II stimulation. Phosphorylated MAP kinase levels in the aorta returned to baseline after 7 days treatment with L-NAME, although arterial blood pressure remained at a significantly elevated level under chronic NO synthase inhibition. Vasoconstrictor activity of ANG II was not affected by chronic NO synthase inhibition. Although Epstein et al. (4) have shown the involvement of ERK1/2 kinase in vasoconstriction, our results indicate that MAP kinase signal transduction and vasoconstrictor mechanisms are regulated differently.

Not only vascular endothelial cells but also vascular smooth muscle cells possess active NO synthase (3). To assess the possibility that endogenous NO synthase in vascular smooth muscle cells contributes to ANG II-induced vascular MAP kinase activation, we examined the effects of NO synthase inhibition on MAP kinase phosphorylation. We found that L-NAME and other NO synthase inhibitors, L-NMMA and L-NNA, could suppress ANG II-induced ERK1/2 and p38 MAP kinase phosphorylation in rat vascular smooth muscle cells in a dose-dependent fashion. Sensitivity to L-NAME treatment was greater for p38 than ERK1/2 MAK kinases, the former of which belongs to the group of stress-activated MAP kinase such as JNK (11). We have previously demonstrated that ROS sensitivity of ANG II-induced phosphorylation is greater in p38 or JNK than in ERK1/2 MAP kinases (8). Therefore, it can be speculated that NO participates in the redox-sensitive signal transduction from AT₁ receptor to MAP kinase activation. We also demonstrated that superoxide-specific fluorescence of ethidium was greatly increased by ANG II stimulation in DHE-loaded rat vascular smooth muscle cells, which was strongly suppressed in the presence of L-NAME. Interestingly, L-NAME treatment alone slightly, but significantly, enhanced ethidium fluorescence compared with the control cells. These results indicate that NO synthase may affect superoxide production differently in basal and stimulated conditions.

Increased vascular production of ROS may reduce the action of endogenous NO, which has been considered as a major mechanism of maintenance of high blood pressure (19). Concomitantly, although endothelium NO synthase may be activated and counteracts ANG II-induced vasoconstriction (17) in response to ANG II stimulation, ANG II stimulates peroxynitrite through the interaction of superoxide and NO in endothelial cells (2, 16). Peroxynitrite stimulation in phosphorylation of MAP kinase in vascular smooth muscle cells has already been demonstrated (22). Although we did not define directly whether ANG II activated vascular MAP kinase via the peroxynitrite pathway in this study, both superoxide and peroxynitrite might be involved in the process of redox-sensitive MAP kinase activation in vasculature. Alternatively, NO synthases themselves may catalyze the uncoupling reduction of oxygen, leading to the generation of superoxide (21, 22). Further studies are necessary to evaluate regulatory mechanisms of MAP kinase activity by NO synthase inhibition in the vasculature.

	GRANTS

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This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan.

	FOOTNOTES

 

Address for reprint requests and other correspondence: S. Kimura, Dept. of Pharmacology, Kagawa Univ. Medical School, 1750-1 Ikenobe, Miki, Kagawa 761-0793, Japan (e-mail: kimura{at}kms.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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This Article Abstract Full Text (PDF) All Versions of this Article: 293/4/H2403    most recent 00288.2007v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhang, G.-X. Articles by Kimura, S. Search for Related Content PubMed PubMed Citation Articles by Zhang, G.-X. Articles by Kimura, S.