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Am J Physiol Heart Circ Physiol 292: H1714-H1721, 2007. First published October 27, 2006; doi:10.1152/ajpheart.00486.2006
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Adrenomedullin inhibits angiotensin II-induced oxidative stress via Csk-mediated inhibition of Src activity

Jing Liu,1 Tatsuo Shimosawa,2 Hiromitsu Matsui,1 Fanyin Meng,3 Scott C. Supowit,3 Donald J. DiPette,3 Katsuyuki Ando,1 and Toshiro Fujita1

Departments of 1Endocrinology and Nephrology and 2Clinical Laboratory Medicine, Faculty of Medicine, The University of Tokyo, Tokyo, Japan; and 3Texas A&M University System Health Science Center College of Medicine and Scott & White Health System, Department of Medicine, Temple, Texas

Submitted 12 May 2006 ; accepted in final form 21 September 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have demonstrated that adrenomedullin (AM) protects against angiotensin II (ANG II)-induced cardiovascular damage through the attenuation of increased oxidative stress observed in AM-deficient mice. However, the mechanism(s) that underlie this activity remain unclear. To address this question, we investigated the effect of AM on ANG II-stimulated reactive oxygen species (ROS) production in cultured rat aortic vascular smooth muscle cells (VSMCs). ANG II markedly increased ROS production through activation of NADPH oxidase. This effect was significantly attenuated by AM in a concentration-dependent manner. This effect was mimicked by dibutyl-cAMP and blocked by pretreatment with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide hydrochloride (H-89), a protein kinase A inhibitor, and CGRP8–37, an AM/CGRP receptor antagonist. This inhibitory effect of AM was also lost following the expression of a constitutively active Src. Moreover, AM intersected ANG II signaling by inducing COOH-terminal Src kinase (Csk) activation that, in turn, inhibits Src activation. These data, for the first time, demonstrate that AM attenuates the ANG II-induced increase in ROS in VSMCs via activation of Csk, thereby inhibiting Src activity.

reactive oxygen species; antioxidant; lucigenin; carboxy-termial Src kinase

ENHANCED PRODUCTION OF reactive oxygen species (ROS) and activation of redox-dependent signaling cascades by vasoactive agents are critical processes underlying a number of cardiovascular and metabolic diseases (37, 43). Virtually all types of vascular cells produce ROS, such as O2– and H2O2. The major source of oxygen intermediates in the vascular wall is nonphagocytic NADPH oxidase (6, 11, 20, 22, 34). Accumulating evidence indicates that NADPH oxidase-derived ROS are essential participants in signaling events in vascular cells (7, 28).

Angiotensin II (ANG II) is an important mediator of vascular smooth muscle cell (VSMC) function, and a number of the effects of ANG II are mediated through the generation of ROS (4, 9, 42). In particular, ANG II-induced hypertrophy is inhibited by overexpression of catalase, which hydrolyzes H2O2, or by inhibition of vascular NADPH oxidases (3, 8, 42).

Adrenomedullin (AM), a potent vasodilator peptide that acts primarily as a protective autocrine/paracrine factor in the cardiovascular system, has antiproliferative and antimigrative effects (32). We previously reported that AM gene transfer prevents the development of cuff-induced vascular injury (31). AM-deficient heterozygous knockout mice also exhibited significant damage in multiple organ systems that was mediated by enhanced oxidative stress in models of ANG II/salt-induced hypertension (30), aging (44), hypoxia (21), and cuff injury (15). In addition, AM expression was stimulated by ROS in vascular endothelial cells (24). These data are consistent with the hypothesis that AM is an endogenous antioxidant that plays a protective role against increased oxidative stress. However, the underlying cellular mechanism(s) and identification of the critical AM-regulated signal transduction pathways remained to be clarified.

Several signaling molecules, such as phospholipase D, protein kinase C, phospholipase A2, and phosphatidylinositol 3-kinase (PI3-kinase) are implicated in ANG II-evoked NADPH oxidase activation (10, 38–40). Whether these pathways function in parallel or in series to stimulate this enzyme is unclear. However, multiple lines of evidence strongly indicate that Src, which influences all of these signaling molecules, is a common upstream mediator (29). Src kinases are a family of nonreceptor tyrosine kinases, of which the prototype c-Src, is the major isoform in the vasculature (23, 35). Src can be switched from an inactive to an active state through control of phosphorylation at two major sites, Tyr416 and Tyr527. Phosphorylation of Tyr416 activates Src, whereas phosphorylation of Tyr527 inactivates this tyrosine kinase. COOH-terminal Src kinase (Csk) is a key enzyme that phosphorylates Src at Tyr527 (19). In VSMCs, c-Src is rapidly activated by ANG II and plays a key role in growth signaling (12). Furthermore, it has been shown that PKA intersects Src signaling in mammalian cells (1). Since cAMP-PKA is the primary postreceptor signal transduction pathway of AM and PKA upregulates Csk activity through phosphorylation, c-Src inactivation via Csk is a likely candidate through which AM intersects ANG II-stimulated ROS signaling.

Therefore, in the present study, we examined whether AM directly inhibits intracellular ROS generation stimulated by ANG II in VSMCs and assessed the role(s) that Src and Csk play in the regulation of the cellular signaling pathway(s) responsible for its antioxidant effect.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials. Rat AM and human CGRP8–37 were purchased from the Peptide Institute (Osaka, Japan). Fetal bovine serum (FBS) was from Trace Scientific (Melbourne, Australia). Anti-Src and anti-phosphotyrosine MAb (4G10) antibody, a dominant-negative mutant of Src (DN-Src), a constitutively active form of avian Src (CA-Src), and Csk-small-interfering (si)RNA kit came from Upstate Biotechnology (Lake Placid, NY). Lipofectin was from Invitrogen (Palo Alto, CA), and Mirus reagent was from Mirusbio (Madison, WI). Anti-Src phosphor-specific antibody Tyr416 was from Biosource International (Camarillo, CA), and anti-Src phosphor-specific antibody Tyr527 was purchased from Cell Signaling Technology (Beverly, MA). Anti-Csk antibody and protein A/G PLUS-Agarose were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Dulbecco's modified Eagle's medium (DMEM), ANG II, rotenone, allopurinol, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide hydrochloride (H-89) and dibutal-cAMP, TNF-{alpha}, and calcium ionophore A-23187 were from Sigma (St. Louis, MO).

Cell culture. All animal studies were approved by the Institutional Animal Care and Use Committee. Rat aortic VSMCs were prepared from the thoracic aorta of 8-wk-old male Sprague-Dawley rats using the explant method (33) and cultured in DMEM containing 10% FBS at 37°C in a humidified atmosphere of 95% air-5% CO2. Subcultured cells (passages 3–12) were deprived of serum for 48 h before experimental use.

Transient transfection. Either a dominant-negative mutant of Src or a constitutive-active mutant of Src was overexpressed in VSMCs by transient transfection. All Src constructs were in the pUSEamp(+) expression vector. Transient transfection was performed with 2 mg of total DNA per 35-mm dish and Lipofectin according to the manufacturer's instructions (DNA:LipofectAMINE, 1:3 ratio). The transfection efficiency was confirmed by immunoblotting for total protein. Pilot studies have documented a transfection efficiency of 15–25% in VSMCs using this approach. Cells transfected with an expression vector containing the same promoter, but without the subcloned transgene cassette (empty expression vector), served as a control. All Csk siRNAs were synthesized and annealed at Dharmacon. VSMCs were plated 2 days before transfections and grown to 60–80% confluence in 12-well plates. SMARTpool Csk or control siRNA (200 nM) was transfected into VSMCs with Mirus reagent (Mirusbio) according to the manufacturer's protocol.

Measurement of intracellular ROS levels. We measured ROS both in living cells and in total VSMC homogenates. ROS production in living cells was measured by the lucigenin method (11) with minor modifications. Briefly, cells were pretreated with or without test compounds for the indicated time periods and then stimulated with or without ANG II (100 nmol/l) for 10 min and AM for 5 min. In some experiments, CGRP8–37 or H-89 was added 30 min before, dibutyl-cAMP 1 h before, TNF-{alpha} 2 h before, and the calcium ionophore A-23187 3 h before AM treatment. The cells were then trypsinized and collected by centrifugation, and the pellet was washed in modified Krebs buffer as described elsewhere (11). After being washed, the cells were resuspended in Krebs buffer with 1 mg/ml BSA, and the cell concentration was adjusted to 1 x 106 in 900 µl buffer. To measure ROS production, the cell suspension was placed in a luminometer (LB 9507; Berthold, Wildblad, Germany). Measurements were started by an injection of 100 µl lucigenin (final concentration, 5 x 10–4 M). Photon emission was counted every 1 min for up to 10 min. Modified Krebs buffer was used as control (blank). For total cell homogenate, cells were treated the same as in living cells and were scraped into ice-cold Hanks' balanced salt solution supplemented with MgCl2 (0.8 mmol/l) and CaCl2 (1.8 mmol/l). Cells were disrupted by rapid freezing in liquid nitrogen followed by sonication. O2– production was measured in the presence of NADPH (100 µmol/l) for 10 min and was expressed in mean arbitrary light units per min.

Western blot analysis for phosphorylated Src. Western blot analysis was performed as described previously (17). Briefly, 30 µg of the cell lysates were subject to 10% SDS-PAGE, and proteins were transferred to nitrocellulose membranes (Genetics, Tokyo, Japan). The membranes were blocked for 1 h at room temperature with 5% skimmed milk. The blots were then incubated overnight at 4°C with anti-Src and anti-Src phosphospecific antibodies, followed by incubation for 1 h with a secondary peroxide-conjugated antibody (Amersham Biosciences UK), and the proteins were detected by enhanced chemiluminescence (ECL) Western blotting detection kit (Amersham Biosciences UK). In figures showing Western blots, the blots shown are representative of at least three independent experiments.

Immunoprecipitation and immunoblot analysis of Csk. Cells were lysed and centrifuged as described elsewhere (36). The supernatant was mixed with the antibody to Csk (2 µg) and rocked at 4°C for 2–16 h, and protein A/G-Agarose was then added for an additional 2 h to overnight. Immunoprecipitates were washed, fractioned by electrophoresis, and transferred to nitrocellulose membranes (Genetics). After being blocked with 5% milk, the membrane was treated with an anti-phosphotyrosine (4G10) antibody conjugated with horseradish peroxidase (Upstate Biotechnology). Immunoreactive proteins were detected by ECL (Amersham Pharmacia Biotech).

Statistical analysis. Data are expressed as means ± SE. Differences between groups were examined for statistical significance using either ANOVA followed by the Dunnett's test or unpaired t-test, where they were appropriate. P values < 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
AM attenuates intracellular ROS production stimulated by ANG II. A 10-min treatment of VSMCs with ANG II evoked a significant increase (~3- to 7-fold from the lowest to the highest concentration of ANG II) in ROS production (Fig. 1A), which was attenuated by AM in a concentration-dependent manner (10–8–10–6 mol/l, Fig. 1B). The highest concentration of AM (10–6 mol/l) inhibited ROS generation to almost the same degree as the NADPH-specific oxidase inhibitor apocynin (Fig. 1B), and neither inhibition of mitochondrial electron transport by rotenone (10 µmol/l) nor inhibition of xanthine oxidase by allopurinol (10 µmol/l) significantly altered ANG II-induced ROS production (Fig. 1C). Likewise, AM treatment had no effect on the Ca2+ ionophore A-23187 or TNF-{alpha}-induced ROS production (Fig. 1D).


Figure 1
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  		Fig. 1. Adrenomedullin (AM) inhibits intracellular reactive oxygen species (ROS) generation stimulated by ANG II in vascular smooth muscle cells (VSMCs). ROS generation was detected by lucigenin-chemiluminescence method. A: concentration-dependent response of ROS generation by ANG II. VSMCs were stimulated with various concentrations of ANG II (10–8–10–6 mol/l) for 10 min. B: AM inhibits intracellular ROS generation stimulated by ANG II in a concentration-dependent manner (10–8–10–6 mol/l). Cells pretreated with AM (10–8–10–6 mol/l) for 5 min or apocynin (10–6 mol/l) for 2 h were stimulated with ANG II (10–7 mol/l) for 10 min. C: treatment of VSMCs with mitochondrial-derived ROS inhibitor rotenone (10 µmol/l) and the xanthine oxidase inhibitor allopurinol (10 µmol/l) for 1 h. D: treatment with calcium ionophore A-23187 (1 µmol/l) for 3 h or TNF-{alpha} (10 ng/ml) for 2 h. ROS generation is expressed as fold increase compared with control (CTR). The values are representative of 3 independent experiments, each performed in triplicate. MLU, mean light units. *P < 0.05 vs. CTR or ANG II.

 
AM inhibits intracellular ROS generation via an AM receptor-mediated and cAMP-PKA-dependent pathway. To determine whether the antioxidant effect of AM is mediated via the AM receptor/cAMP-PKA-dependent pathway, we tested the effects of an AM receptor antagonist and cAMP-related compounds both in living cells (Fig. 2A) and cell homogenates (Fig. 2B). Pretreatment with 3 x 10–6 mol/l CGRP8–37, an AM/CGRP receptor antagonist (2), completely abolished the AM-induced inhibition of intracellular ROS generation stimulated by ANG II. Since the cAMP-PKA pathway is the most well-documented post-receptor signal transduction pathway of AM (5, 46), we examined the involvement of this pathway on the inhibitory mechanism of AM on ROS generation. Dibutyl-cAMP (10–3 mol/l) significantly inhibited ROS generation by ANG II to a similar extent as AM (10–7 mol/l). In contrast, the inhibitory effect of AM on ROS generation was completely abolished by H-89 (10–5 mol/l), a PKA inhibitor. Treatment with AM, CGRP8–37, dibutyl-cAMP, or H-89 alone did not alter intracellular ROS levels in intact cells (Fig. 2A). Similar results were obtained with cell homogenates (Fig. 2B).


Figure 2
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  		Fig. 2. AM inhibits intracellular ROS generation via a receptor-mediated and cAMP-PKA-dependent pathway. ROS production was examined by lucigenin chemiluminescence, both in living cells (A) and cell homogenates (B). VSMCs treated with or without AM (10–7 mol/l), dibutyl-cAMP (10–3 mol/l), N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide hydrochloride (H-89; 10–5 mol/l), or CGRP8–37 (3 x 10–5 mol/l) for 0.5–1 h were stimulated in the absence or presence of ANG II (10–7 mol/l) for 10 min. In some experiments, CGRP8–37 or H-89 was applied 30 min before the AM pretreatment. Quantification of ROS levels by the lucigenin-chemiluminescence method and the calculation and plotting of data are the same as in Fig. 1. The values are representative of 3 independent experiments, each performed in triplicate. *P < 0.05 vs. CTR; {dagger}P < 0.05 vs. ANG II.

 
AM inhibits phosphorylation of Src (Tyr416). It has been reported that c-Src regulates ANG II-mediated NADPH oxidase-derived O2– in rat VSMCs (29). Based on these data, experiments were performed to determine whether AM inhibits ANG II-induced ROS production specifically through inhibition of Src (Tyr416) phosphorylation. As shown in Fig. 3A, phosphorylation of Src (Tyr416) was significantly increased in VSMCs by ANG II (10–7 mol/l) stimulation, which was, in turn, inhibited by AM in a concentration-dependent manner (10–8–10–6 mol/l). Figure 3B shows the effects of dibutyl-cAMP (10–3 mol/l), CGRP8–37, and H-89 (10–5 mol/l) treatment on phosphorylation. Dibutyl-cAMP significantly (P < 0.05) inhibited the phosphorylation of Src (Tyr416) by ANG II, which was comparable with that of AM (10–7 mol/l). As expected, H-89 and CGRP8–37 completely abolished the inhibitory effect of AM on Src (Tyr416) phosphorylation. In the absence of ANG II stimulation, treatment with AM, CGRP8–37, dibutyl-cAMP, or H-89 alone did not significantly change the state of Src phosphorylation.


Figure 3
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  		Fig. 3. AM inhibits Src (Tyr416) phosphorylation stimulated by ANG II via a receptor-mediated and cAMP-PKA-dependent pathway in VSMCs. A: concentration-dependent response of Src (Tyr416) phosphorylation by ANG II. VSMCs were pretreated with various concentrations of AM (10–8–10–6 mol/l) for 5 min and then stimulated with ANG II (10–7 mol/l) for 10 min. Top: representative immunoblot demonstrating phosphorylation of Src (Tyr416). Bottom: represents averaged data from 3 independent experiments quantified by densitometry of immunoblots and expressed as fold increases in phosphorylation compared with unstimulated cells. B: effect of dibutyl-cAMP, CGRP8–37, and H-89 on phosphorylation of Src (Tyr416). VSMCs treated with or without AM (10–7 mol/l), dibutyl-cAMP (10–3 mol/l), H-89 (10–5 mol/l), or CGRP8–37 (3 x 10–5 mol/l) for 0.5–1 h were stimulated with or without ANG II (10–7 mol/l) for 10 min. In some experiments, CGRP8–37 or H-89 was applied 30 min before the AM pretreatment. Phosphorylation of Src (Tyr416) was evaluated by Western blot analysis. Top: a representative immunoblot of the phosphorylation of Src (Tyr416). Bottom: represents averaged data from 3 independent experiments quantified by densitometry of immunoblots and expressed as fold increases compared with unstimulated cells. p-Src, phospho-Src. *P < 0.05 vs. CTR; {dagger}P < 0.05 vs. ANG II.

 
Effects of DN-Src and CA-Src on the antioxidant activity of AM. To confirm that the attenuation of Src activity is directly attributable to the inhibitory effect of AM on ANG II-induced ROS generation, we undertook experiments to transfect VSMCs with DN- or CA-Src constructs. The transfection efficiency was confirmed by immunoblotting for total Src protein. Figure 4A shows that both of the mutant Src constructs were effectively overexpressed in VSMCs. Using the live transfected cells, we then assessed the effect of AM on ANG II-induced ROS production by the lucigenin chemiluminescence method. As seen in Fig. 4B, the DN-Src-transfected cells that serve as the negative controls for this set of experiments display basal levels of ROS generation that were unchanged following treatment with ANG II alone or ANG II and AM. In contrast, in CA-Src overexpressing cells, ROS generation was significantly enhanced and was not increased further by ANG II treatment (Fig. 4C). Consistent with these results, AM treatment failed to inhibit the elevated ROS generation (Fig. 4C).


Figure 4
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  		Fig. 4. Effects of AM on ANG II-induced ROS production in dominant-negative (DN)- or constitutively active (CA)-Src-transfected cells. A: transfection efficiency for these experiments was confirmed by immunoblotting for total Src protein. ROS production was examined by lucigenin-chemiluminescence method, both in DN-Src- (B) and CA-Src-transfected living cells (C). CTRs are DN-Src- and CA-Src-transfected cells in the absence of stimulation. Values are representative of 3 independent experiments, each performed in triplicate.

 
AM- and ANG II-enhanced Csk phosphorylation increases Src (Tyr527) phosphorylation, and the inhibitory effect of AM on Src activation is lost in Csk knockdown VSMCs. Inactivation of Src kinase involves phosphorylation of Tyr527 by Csk (19). Therefore, we examined whether AM inhibition of ANG II-induced Src activation was associated with changes in Csk activity. Csk immunoprecipitates were probed with an anti-phosphotyrosine antibody. Phosphorylation of Csk was significantly increased approximately twofold by ANG II but not by treatment with AM alone. A much more robust increase (approximately fourfold) was observed when the VSCMs were treated with the combinations of ANG II and either AM (10–7 mol/l) or dibutyl-cAMP (10–3 mol/l) (Fig. 5A). In contrast, treatment of the cells with ANG II did not increase the phosphorylation of Src (Tyr527) as shown in Fig. 5C. Treatment with AM produced an increase Src (Tyr527) phosphorylation, but it did not achieve statistical significance (data not shown). However, the combination of ANG II and AM evoked a significant increase in Src (Tyr527) phophorylation, again in the absence of any changes in total Src protein (Fig. 5C). In addition, Csk knockdown studies using siRNA also reversed the inhibitory effects of AM on Src activation (Fig. 5C).


Figure 5
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  		Fig. 5. AM enhances ANG II-induced COOH-termial Src kinase (Csk) and, in turn, Src (Tyr527) phosphorylation; the inhibitory effect of AM on Src activation is lost in Csk knockdown VSMCs. VSMCs were pretreated with AM (10–7 mol/l) for 5 min or dibutyl-cAMP (10–3 mol/l) for 1 h, followed by addition of ANG II (10–7 mol/l) for 10 min. A: Csk was immunoprecipitated from cell lysates with anti-Csk antibody. Immunoprecipitated proteins were subjected to SDS-PAGE and immunoblotted with anti-phosphotyrosine antibody. Top: a representative immunoblot from 3 independent experiments assessing phosphorylated Csk. Bottom: averaged data from 3 independent experiments quantified by densitometry of immunoblots and expressed as fold increases in phosphorylation compared with unstimulated cells. *P < 0.05 vs. CTR; {dagger}P < 0.01, ANG II + AM and ANG II + dibutyl cAMP vs. CTR. IP, immunoprecipitation; IB, immunoblotting. B: VSMCs were transfected with SMARTpool Csk or CTR small-interfering (si)RNA and incubated for 48 h. The expression of Csk protein was selectively inhibited by siRNA (top) without reducing beta-actin CTR protein (bottom). Each experiment was performed 3 times in triplicate. C: immunoblots and quantification of total Src and phosphorylated Tyr416 and Tyr527 with and without Csk siRNA (averaged data from 3 independent experiments quantified by densitometry of immunoblots). *P < 0.05 vs. CTR.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Accumulating evidence strongly supports the hypothesis that AM possesses significant protective properties against end-organ damage, produced by a number of pathophysiological conditions, through inhibition of oxidative stress (15, 21, 30, 44). The mechanisms underlying this activity vary depending on cell types and experimental conditions. In mesangial cells, AM suppresses ROS production via the cAMP-PKA pathway (5), whereas, in the rat ventricle, increased oxidative stress caused by ischemia-reperfusion injury can be attenuated by AM-mediated inhibition of NADPH oxidase via the nitric oxide-cGMP signaling pathway (14). In contrast, enhanced hypoxia-induced generation of ROS in human alveolar epithelial cells is attenuated by a marked AM-stimulated increase in the potent ROS scavenger glutathione (16).

In light of the aforementioned reports, the significant findings of this study are as follows. First, in using rat VSMCs in conjunction with a lucigenin chemiluminescence methodology, we have confirmed that NADPH oxidase is the source of the marked increase in ROS following treatment with ANG II and that the inhibitory effects of AM in this setting are mediated through the cAMP/PKA signal transduction pathway (46). Second, the data presented herein clearly support the hypothesis that, in VSMCs, Src is the primary upstream signaling molecule that mediates the ANG II increase in NADPH-generated ROS (29). Third, we have shown for the first time that the attenuation of Src activity in ANG II-treated VSMCs is regulated by the phosphorylation and subsequent activation of Csk, which is dependent on AM via the cAMP-PKA signaling pathway, and may also require ANG II, which could be playing an autoregulatory role by initiating a potential negative feedback loop.

Before we initiated the series of experiments on the roles that Src and Csk play in AM-mediated attenuation of ANG II-evoked ROS production, it was necessary to demonstrate that ANG II treatment produced, in a concentration-dependent manner, a rapid and robust enhancement in VSMC ROS production that was significantly inhibited to near-basal levels by AM, again in a concentration-dependent fashion. The results presented herein are consistent with those reported by Yoshimoto et al. in both rat aortic VSMCs (46) and endothelial cells (47). Intracellular ROS generation stimulated by ANG II in these cell types has been shown to be mainly derived from the activation of NADPH oxidase (11, 22). This is clearly supported by our findings that inhibitors of xanthine oxidase (allopurinol) and mitochondrial-derived ROS (rotenone) have no significant effect on ANG II-stimulated ROS production. In contrast, treatment of the VSMCs with apocynin, a specific NADPH oxidase inhibitor, reduced ANG II-stimulated ROS generation to levels observed in the untreated control cells, which were similar to those observed following AM treatment. However, apocynin was somewhat more potent than AM for reasons that remain to be clarified. Furthermore, AM was without effect on Ca2+ ionophore A-23187 or TNF-{alpha}-induced ROS production, indicating that its ability to attenuate ANG II-stimulated ROS production in cultured VSMCs is regulated by specific signaling pathway(s) that inhibit, primarily, the activity of NADPH oxidase rather than acting as a general antioxidant.

Although the cAMP-PKA pathway is the primary signal transduction pathway stimulated by AM, this peptide can also trigger other signaling cascades, including NF-{kappa}B (27), tyrosine kinase (13), protein phosphatase 2A (26), and PI3K/Akt (25, 45), depending on the cell type or experimental conditions. To confirm that the antioxidant effects of AM observed in this study were in fact mediated primarily by the AM receptor-mediated upregulation of the cAMP-PKA signal transduction pathway, we demonstrated that the inhibitory effect of AM on ANG II-stimulated ROS generation was completely abolished by pretreatment of the VSMCs with CGRP8–37, an AM receptor antagonist. As expected, dibutyl-cAMP significantly inhibited the ANG II-evoked increase in ROS generation to a similar extent as AM. Likewise, the AM-mediated inhibition of ANG II-evoked ROS production was abolished by H-89, a PKA inhibitor. These data, therefore, provide additional evidence that AM inhibits ANG II-stimulated NADPH oxidase-dependent ROS production through the AM receptor-mediated activation of the cAMP-PKA pathway.

A central role for Src in the regulation of the redox status of ANG II-treated VSCMs was indicated by the observation that phosphorylation of Src (Tyr416), a key step in the activation of this protein kinase, was markedly increased following 10 min of treatment with ANG II. The link between the ANG II type 1 receptor and Src, however, is not clear. It has been reported that in VSCMs, ANG II signaling is regulated, in part, by the transactivation of the epidermal growth factor receptor, which serves as a scaffolding for preactivated c-Src and downstream adaptors. Indeed, interactions between Gbeta{gamma} subunits, their associated kinases, and kinase substrates may form the signaling complex that binds c-Src (18, 48). This phosphorylation of Src was significantly attenuated in a concentration-dependent manner by AM in the absence of any change in total Src protein. Moreover, this attenuation of Src phosphorylation by AM was consistent with the concentration-dependent inhibitory effect of AM on ANG II-enhanced ROS generation. Likewise, the degree of Src Tyr416 phosphorylation, after normalization to total Src protein, following treatment with agents that activated or blocked the AM receptor/cAMP-PKA pathway, was in line with the ability of these agents to inhibit ANG II-evoked ROS production. Indeed, ANG II was unable to stimulate ROS production above baseline levels in DN-Src-transfected VSCMs, and the inhibitory effect of AM on ANG II-induced ROS generation was lost in CA-Src transfected cells, indicating that c-Src is the target molecule through which AM inhibits ROS generation induced by ANG II in VSMCs.

As described previously, c-Src is activated by autophosphorylation of Tyr416 and inactivated by Csk that phosphorylates Tyr527 (1, 19). Indeed, we observed that the knockdown of Csk by siRNA blocked the phosphorylation of Src527, thereby indicating a direct relation between Csk and Src inactivation. It was, however, surprising that treatment of the cells with ANG II under the same conditions that enhance ROS generation produced a significant increase in Csk phosphorylation, one mechanism by which this tyrosine kinase is activated, whereas AM alone did not. However, addition of ANG II in combination with either AM or dibutyl-cAMP resulted in a marked increase in Csk phosphorylation without a change in total Csk protein. In contrast, when Src Tyr527 phosphorylation was assessed, neither ANG II nor AM alone was able to significantly stimulate posphorylation at this site, whereas the combination of the two did result in a statistically significant upregulation of Try527 phosphorylation.

Although we do not have any direct evidence to support this hypothesis, it is tempting to speculate that to maintain a balance between active and inactive Src, which clearly participate in the modulation of the redox status of this cell type, ANG II treatment simultaneously activates Src and also sets the stage for the downregulation of this protein by phosphorylation of Csk. This change in the activation status of Csk is such that it cannot deactivate Src via Tyr527 phosphorylation, except in the presence of a negative regulatory agent like AM that is able to trigger Csk up to a level of activity that is sufficient to downregulate Src, thereby reestablishing homeostasis of the redox state of the cell. Indeed, a report from Touyz et al. (41) provides indirect evidence for this argument. In this study they demonstrated that ANG II-stimulated VSMC growth and remodeling was mediated by the activation of c-Src and that this effect was significantly greater in VSMCs from spontaneously hypertensive rats (SHR) compared with those from normotensive Wistar-Kyoto (WKY) rats. ANG II-mediated c-Src phosporylation (Tyr527) was approximately fourfold greater in SHR than WKY rats. However, ANG II increased Csk phosphorylation two- to threefold in WKY but not in SHR. From these data it was concluded that c-Src phosphorylation and Src-dependent cell growth following ANG II treatment are increased in VSMCs from SHR and that these processes are associated with blunted ANG II-induced phosphorylation of Csk. Since the VSMCs used in our study came from normal rats, our tentative conclusion regarding the mechanism underlying the Csk-mediated inhibition of Src activity and ROS production is consistent with the data reported by Touyz et al. (41). We are, however, well aware that multiple mechanisms for Src and Csk regulation exist, and it ia still unclear as to the exact contribution of the different regulatory mechanisms in a given physiological setting (36).


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Fujita, Dept. of Internal Medicine, Faculty of Medicine, The Univ. of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan (e-mail: fujita-dis{at}h.u-tokyo.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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Abrahamsen H, Vang T, Tasken K. Protein kinase A intersects Src signaling in membrane microdomains. J Biol Chem 278: 17170–17177, 2003.[Abstract/Free Full Text]
  2. Bellibas SE, Guidobono F, Bettica P, Netti C, Pecile A. Effects of pyridoxine neurotoxicity on a distribution of calcitonin gene-related peptide binding sites. Pol J Pharmacol 49: 37–42, 1997.[Web of Science][Medline]
  3. Berk BC, Corson MA. Angiotensin II signal transduction in vascular smooth muscle: role of tyrosine kinases. Circ Res 80: 607–616, 1997.[Abstract/Free Full Text]
  4. Chen XL, Tummala PE, Olbrych MT, Alexander RW, Medford RM. Angiotensin II induces monocyte chemoattractant protein-1 gene expression in rat vascular smooth muscle cells. Circ Res 83: 952–959, 1998.[Abstract/Free Full Text]
  5. Chini EN, Chini CC, Bolliger C. Cytoprotective effects of adrenomedullin in glomerular cell injury: central role of cAMP signaling pathway. Kidney Int 52: 917–925, 1997.[Web of Science][Medline]
  6. De Keulenaer GW, Alexander RW, Ushio-Fukai M, Ishizaka N, Griendling KK. Tumor necrosis factor alpha activates a p22phox-based NADH oxidase in vascular smooth muscle. Biochem J 329: 653–657, 1998.[Web of Science][Medline]
  7. Droge W. Free radicals in the physiological control of cell function. Physiol Rev 82: 47–95, 2002.[Abstract/Free Full Text]
  8. Eguchi S, Inagami T. Signal transduction of angiotensin II type 1 receptor through receptor tyrosine kinase. Regul Pept 91: 13–20, 2000.[CrossRef][Web of Science][Medline]
  9. Frank GD, Eguchi S, Inagami T, Motley ED. N-acetylcysteine inhibits angiotensin II-mediated activation of extracellular signal-regulated kinase and epidermal growth factor receptor. Biochem Biophys Res Commun 280: 1116–1119, 2001.[CrossRef][Web of Science][Medline]
  10. Griendling KK, Sorescu D, Lassègue B, Ushio-Fukai M. Modulation of protein kinase activity and gene expression by reactive oxygen species and their role in vascular physiology and pathophysiology. Arterioscler Thromb Vasc Biol 20: 2175–2183, 2000.[Abstract/Free Full Text]
  11. Griendling KK, Minieri CA, Ollerenshaw JD, Alexander RW. Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res 74: 1141–1148, 1994.[Abstract/Free Full Text]
  12. Ishida M, Ishida T, Thomas S, Berk B. Activation of extracellular signal regulated kinases (ERK1/2) by Ang II is dependent on c-Src in vascular smooth muscle cells. Circ Res 82: 7–12, 1998.[Abstract/Free Full Text]
  13. Iwasaki H, Shichiri M, Marumo F, Hirata Y. Adrenomedullin stimulates proline-rich tyrosine kinase 2 in vascular smooth muscle cells. Endocrinology 142: 564–572, 2001.[Abstract/Free Full Text]
  14. Kato K, Yin H, Agata J, Yoshida H, Chao L, Chao J. Adrenomedullin gene delivery attenuates myocardial infarction and apoptosis after ischemia and reperfusion. Am J Physiol Heart Circ Physiol 285: H1506–H1514, 2003.[Abstract/Free Full Text]
  15. Kawai J, Ando K, Tojo A, Shimosawa T, Takahashi K, Onozato ML, Yamasaki M, Ogita T, Nakaoka T, Fujita T. Endogenous adrenomedullin protects against vascular response to injury in mice. Circulation 109: 1147–1153, 2004.[Abstract/Free Full Text]
  16. Kim JY, Yim JH, Cho JH, Kim JH, Ko JH, Kim SM, Park SJ, Park JH. Adrenomedullin regulates cellular glutathione content via modulation of {gamma}-glutamate-cysteine ligase catalytic subunit expression. Endocrinology 147: 1357–1364, 2006.[Abstract/Free Full Text]
  17. Kyaw M, Yoshizumi M, Tsuchiya K, Kagami S, Izawa Y, Fujita Y, Ali N, Kanematsu Y, Toida K, Ishimura K, Tamaki T. Src and Cas are essentially but differentially involved in angiotensin II-stimulated migration of vascular smooth muscle cells via extracellular signal-regulated kinase 1/2 and c-Jun NH2-terminal kinase activation. Mol Pharmacol 65: 832–841, 2004.[Abstract/Free Full Text]
  18. Luttrell LM, Ferguson SSG, Daaka Y, Miller WE, Maudsley S, Della Rocca GJ, Lin FT, Kawakatsu H, Owada K, Luttrell DK. beta-Arrestin-dependent formation of beta2 adrenergic receptor-Src protein kinase complexes. Science 283: 655–661, 1999.[Abstract/Free Full Text]
  19. Martin GS. The hunting of the Src. Nat Rev Mol Cell Biol 2: 467–475, 2001.[CrossRef][Web of Science][Medline]
  20. Matsubara T, Ziff M. Increased superoxide anion release from human endothelial cells in response to cytokines. J Immunol 137: 3295–3298, 1986.[Abstract]
  21. Matsui H, Shimosawa T, Itakura K, Xing GQ, Ando K, Fujita T. Adrenomedullin can protect against pulmonary vascular remodeling induced by hypoxia. Circulation 109: 2246–2251, 2004.[Abstract/Free Full Text]
  22. Mohazzab KM, Kaminski PM, Wolin MS. NADH oxidoreductase is a major source of superoxide anion in bovine coronary artery endothelium. Am J Physiol Heart Circ Physiol 266: H2568–H2572, 1994.[Abstract/Free Full Text]
  23. Oda Y, Renaux B, Bjorge J, Saifeddine M, Fujita DJ, Hollenberg MD. c-Src is a major cytosolic tyrosine kinase in vascular tissue. Can J Physiol Pharmacol 77: 606–617, 1999.[CrossRef][Web of Science][Medline]
  24. Ogita T, Hashimoto E, Yamasaki M. Hypoxic induction of adrenomedullin in cultured human umbilical vein endothelial cells. J Hypertens 19: 603–608, 2001.[CrossRef][Web of Science][Medline]
  25. Okumura H, Nagaya N, Itoh T, Okano I, Hino J, Mori K, Tsukamoto Y, Ishibashi-Ueda H, Miwa S, Tambara K, Toyokuni S, Yutani C, Kangawa K. Adrenomedullin infusion attenuates myocardial ischemia/reperfusion injury through the phosphatidylinositol 3-kinase/Akt-dependent pathway. Circulation 109: 242–248, 2004.[Abstract/Free Full Text]
  26. Parameswaran N, Nambi P, Hall CS, Brooks DP, Spielman WS. Adrenomedullin decreases extracellular signal-regulated kinase activity through an increase in protein phosphatase-2A activity in mesangial cells. Eur J Pharmacol 388: 133–138, 2000.[CrossRef][Web of Science][Medline]
  27. Pleguezuelos O, Hagi-Pavli E, Crowther G, Kapas S. Adrenomedullin signals through NF-kappaB in epithelial cells. FEBS Lett 577: 249–254, 2004.[CrossRef][Web of Science][Medline]
  28. Sauer H, Wartenberg M, Heschler J. Reactive oxygen species as intracellular messengers during cell growth and differentiation. Cell Physiol Biochem 11: 173–186, 2001.[CrossRef][Web of Science][Medline]
  29. Seshiah PN, Weber DS, Rocic P, Valppu L, Taniyama Y, Griendling KK. Angiotensin II stimulation of NAD(P)H oxidase activity: upstream mediators. Circ Res 91: 406–413, 2002.[Abstract/Free Full Text]
  30. Shimosawa T, Shibagaki Y, Ishibashi K, Kitamura K, Kangawa K, Kato S, Ando K, Fujita T. Adrenomedullin, an endogenous peptide, counteracts cardiovascular damage. Circulation 105: 106–111, 2002.[Abstract/Free Full Text]
  31. Shindo T, Kurihara H, Maemura K, Kurihara Y, Kuwaki T, Izumida T, Minamino N. Hypotension and resistance to lipopolysaccharide-induced shock in transgenic mice overexpressing adrenomedullin in their vasculature. Circulation 101: 2309–2316, 2000.[Abstract/Free Full Text]
  32. Sugo S, Minamino N, Shoji H, Kangawa K, Matsuo H. Effects of vasoactive substances and cAMP related compounds on adrenomedullin production in cultured vascular smooth muscle cells. FEBS Lett 369: 311–314, 1995.[CrossRef][Web of Science][Medline]
  33. Sun J, Marx SO, Chen HJ, Poon M, Marks AR, Rabbani LE. Role for p27Kip1 in vascular smooth muscle cell migration. Circulation 103: 2967–2972, 2001.[Abstract/Free Full Text]
  34. Sundaresan M, Yu ZX, Ferrans VJ, Irani K, Finkel T. Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science 270: 296–299, 1995.[Abstract/Free Full Text]
  35. Thomas SM, Brugge JS. Cellular functions regulated by Src family kinases. Annu Rev Cell Dev Biol 13: 513–609, 1997.[CrossRef][Web of Science][Medline]
  36. Torgersen KM, Vang T, Abrahamsen H, Yaqub S, Horejsi V, Schraven B, Rolstad B, Mustelin T, Tasken K. Release from tonic inhibition of T cell activation through transient displacement of C-terminal Src kinase (Csk) from lipid rafts. J Biol Chem 276: 29313–29318, 2001.[Abstract/Free Full Text]
  37. Touyz RM. Oxidative stress and vascular damage in hypertension. Curr Hypertens Rep 2: 98–105, 2000.[Medline]
  38. Touyz RM, Chen X, Tabet F, Yao G, He G, Quinn MT, Pagano PJ, Schiffrin EL. Expression of a functionally active gp91phox-containing neutrophil-type NAD(P)H oxidase in smooth muscle cells from human resistance arteries: regulation by angiotensin II. Circ Res 90: 1205–1213, 2002.[Abstract/Free Full Text]
  39. Touyz RM, Schiffrin EL. Ang II-stimulated generation of reactive oxygen species in human vascular smooth muscle cells is mediated via PLD dependent pathways. Hypertension 34: 976–982, 1999.[Abstract/Free Full Text]
  40. Touyz RM, Schiffrin EL. Increased generation of superoxide by angiotensin II in smooth muscle cells from resistance arteries of hypertensive patients role of phospholipase D-dependent, NAD(P)H oxidase-sensitive pathways. J Hypertens 19: 1245–1254, 2001.[CrossRef][Web of Science][Medline]
  41. Touyz RM, Wu XH, He G, Salomon S, Schiffrin EL. Increased angiotensin II-mediated Src signaling via epidermal growth factor receptor transactivation is associated with decreased C-terminal Src kinase activity in vascular smooth muscle cells from spontaneously hypertensive rats. Hypertension 39: 479–485, 2002.[Abstract/Free Full Text]
  42. Ushio-Fukai M, Zafari AM, Fukui T, Ishizaka N, Griendling KK. p22phox is a critical component of the superoxide-generating NADH/NADPH oxidase system and regulates angiotensin II-induced hypertrophy in vascular smooth muscle cells. J Biol Chem 271: 23317–23321, 1996.[Abstract/Free Full Text]
  43. Wilcox CS. Reactive oxygen species: role in blood pressure and kidney function. Curr Hypertens Rep 4: 160–166, 2002.[Web of Science][Medline]
  44. Xing G, Shimosawa T, Ogihara T, Matsui H, Itakura K, Qingyou X, Asano T, Ando K, Fujita T. Angiotensin II-induced insulin resistance is enhanced in adrenomedullin-deficient mice. Endocrinology 145: 3647–3651, 2004.[Abstract/Free Full Text]
  45. Yin H, Chao L, Chao J. Adrenomedullin protects against myocardial apoptosis after ischemia/reperfusion through activation of Akt-GSK signaling. Hypertension 43: 109–116, 2004.[Abstract/Free Full Text]
  46. Yoshimoto T, Fukai N, Hirata Y. Anti-oxidant effect of adrenomedullin on angiotensin II-induced ROS generation in vascular smooth muscle cells. Endocrinology 145: 3331–3337, 2004.[Abstract/Free Full Text]
  47. Yoshimoto T, Gochou N, Fukai N, Sugiyama T, Shichiri M, Hirata Y. Adrenomedullin inhibits angiotensin II-induced oxidative stress and gene expression in rat endothelial cells. Hypertens Res 28: 165–172, 2005.[CrossRef][Medline]
  48. Zou YZ, Komuro I, Yamazaki T, Kudoh S, Aikawa R, Zhu WD, Shiojima I, Hiroi Y, Tobe K, Kadowaki T, Yazaki Y. Cell type-specific angiotensin II-evoked signal transduction pathways: critical roles of Gbeta{gamma} subunit, Src family, and Ras in cardiac fibroblasts. Circ Res 82: 337–345, 1998.[Abstract/Free Full Text]




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