Adrenomedullin inhibits angiotensin II-induced contraction in human aortic smooth muscle cells

Adrenomedullin Inhibits Angiotensin II-Induced Oxidative Stress

Via Csk-Mediated Inhibition of Src Activity

Jing Liu1, Tatsuo Shimosawa2, Hiromitsu Matsui1, Fanyin Meng3, Scott C. Supowit3, Donald J.

DiPette3, Katsuyuki Ando1, Toshiro Fujita1

1Department of Endocrinology and Nephrologyዊ�2Department of Clinical Laboratory Medicine,

Faculty of Medicine, The University of Tokyo, Tokyo, Japan. 3Texas A&M University System

Health Science Center College of Medicine and Scott & White Health System, Department of

Medicine, Temple, TX.

Running title: Adrenomedullin inhibits oxidative stress

Keywords: adrenomedullin, reactive oxygen species, antioxidant, Src, lucigenin

Abstract: 168 wordsTotal: 6151 wordsFigures: 5

All correspondence to Toshiro Fujita, MD, PhD

Department of Internal Medicine, Faculty of Medicine, The University of Tokyo,7-3-1 Hongo,

Bunkyo-ku, Tokyo, 113-8655, Japan

Phone: 81-3-5800-9735

Fax: 81-3-5800-9736

E-mail: [email protected]

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Copyright Information

Articles in PresS. Am J Physiol Heart Circ Physiol (October 27, 2006). doi:10.1152/ajpheart.00486.2006

Copyright © 2006 by the American Physiological Society.

1

Abstract

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 miceዊ�However, the mechanism(s) that underlie this activity remains 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)ዊ�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 H-89, a protein kinase A inhibitor, and

CGRP 8-37, an AM /CGRP receptor antagonist. This inhibitory effect of AM was also lost

following expression of a constitutively active Srcዊ�Moreover, AM intersected Ang II signaling

by inducing Csk activation, that in turn, inhibits Src activationዊ� 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.

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Introduction

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 H2O2ዊ�The major source of oxygen intermediates in the vascular wall is non-

phagocytic 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)ዊ�In particular, Ang II-induced hypertrophy is inhibited by over expression of catalase,

which hydrolyzes hydrogen peroxide (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 anti-oxidant 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 clarifiedዊ�

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Several signaling molecules such as phospholipase D (PLD), protein kinase C (PKC),

phospholipase A2, and phosphatidylinositol-3-kinase (PI3K) are implicated in Ang II-evoked

NADPH oxidase activation (10,38,39,40). Whether these pathways function in parallel or in

series to stimulate this enzyme is unclearዊ�However, multiple lines of evidence strongly

indicate that Src, which influences all of these signaling molecules, is a common upstream

mediator (29)ዊ�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

active state through control of phosphorylation at two major sites, Tyr416 and Tyr527.

Phosphorylation of Tyr416 activates Src while phosphorylation of Tyr527 inactivates this

tyrosine kinase. C-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 post-receptor 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 signalingዊ�

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 anti-oxidant effectዊ�

Materials and Methods

Materials

Rat AM and human CGRP (8-37) were purchased from the Peptide Institute (Osaka,

Japan). Fetal bovine serum (FBS) from Trace Scientific LTD (Melbourne, Australia)ዊ�

Anti-Src and anti-phosphotyrosine mAb (4G10) antibody, a dominant-negative

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mutant of Src (DN-Src), a constitutively active form of avian Src (CA-Src), and Csk-siRNA kit

came from Upstate Biotechnology (Lake Placid, NY). Lipofectin was from Invitrogen (Palo

Alto, CA), 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)ዊ�

Anti-Csk antibody and protein A/G PLUS-Agarose were obtained from Santa Cruz

Biotechnology (Santa Cruz, CA)ዊ�Dulbecco’s modified Eagles medium (DMEM), Ang II,

rotenone, allopurinol, N-(2-[p-Bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide

hydrochloride (H-89) and dibutal-cAMP, TNF-ዊ� and calcium ionophore A23187 were from

Sigma (St. Louis, MO)ዊ�

Cell Culture

Rat aortic VSMCs were prepared from the thoracic aorta of 8-week-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% CO2ዊ�Sub cultured cells (passages 3-12) were

deprived of serum for 48 h prior to experimental use.

Transient Transfection

Either a dominant negative mutant of Src, or a constitutive-active mutant of Src was over

expressed 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 ratio,

1:3). The transfection efficiency was confirmed by immunoblotting for total protein. Pilot

studies have documented a transfection efficiency of 15% to 25% in VSMCs using this

approach. Cells transfected with an expression vector containing the same promoter, but without

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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 (200nM) were 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, then stimulated

with or without Ang II (100 nmol/L) for 10 min and AM for 5 minዊ�In some experiments,

CGRP (8-37) or H-89 was added 30 min before, dibutyl-cAMP was included 1 hour before,

TNF-ዊ� 2 hours before and the calcium ionophore A23187 3 hours before AM treatmentዊ�The

cells were then trypsinized, collected by centrifugation, and the pellet washed in modified Krebs

buffer as described elsewhere (11). After washing, the cells were resuspended in Krebs buffer

with 1 mg/ml BSA, and the cell concentration was adjusted to 1x106 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 Hank's balanced salt solution supplemented with

MgCl 2 (0.8 mmol/L) and CaCl 2 (1.8 mmol/L)ዊ�Cells were disrupted by rapid freezing in

liquid nitrogen followed by sonicationዊ�O2- production was measured in the presence of

NADPH (100 µmol/L) for 10 min and was expressed as mean arbitrary light units per minዊ�

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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)ዊ�The membranes were blocked for 1 hour at room

temperature with 5% skimmed milkዊ�The blots were then incubated overnight at 4˚C with anti-

Src and anti-Src phosphospecific antibodies, followed by incubation for 1 hour with a secondary

peroxide-conjugated antibody (Amersham Biosciences UK), the proteins were detected by ECL

Western blotting detection kit (Amersham Biosciences UK)ዊ�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 hours, and then protein A/G-

Agarose was added for an additional 2 hours to overnightዊ�Immunoprecipitates were washed,

fractioned by electrophoresis, and transferred to nitrocellulose membranes (Genetics, Tokyo,

Japan)ዊ�After blocking with 5% milk, the membrane was treated with an anti-phosphotyrosine

(4G10) antibody conjugated with horseradish peroxidase (Upstate Biotechnology)ዊ�

Immunoreactive proteins were detected by enhanced chemiluminescence (Amersham Pharmacia

Biotech)ዊ�

Statistical analysis

Data were expressed as mean ± SEMዊ�Differences between groups were examined for

statistical significance using either ANOVA followed by the Dunnett’s test or unpaired t-test,

where they are appropriateዊ�P values less than 0.05 were considered statistically significantዊ�

Results

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AM Attenuates Intracellular ROS Production Stimulated by Ang II.

A 10 minute treatment of VSMCs with Ang II evoked a significant increase (approximately

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 A23187 or TNF-ዊ�-induced ROS

production (Fig. 1D).

AM Inhibits Intracellular ROS Generation via an AM Receptor-Mediated and cAMP-

PKA-Dependent Pathway.

To determine whether the anti-oxidant 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)ዊ�

Pretreatment with 3x10-6 mol/L CGRP (8-37), an AM/CGRP receptor antagonist (2), completely

abolished the AM-induced inhibition of intracellular ROS generation stimulated by Ang IIዊ�

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 generationዊ�Dibutyl-cAMP (10-3 mol/L) significantly inhibited

ROS generation by Ang II to a similar extent as AM (10-7mol/L)ዊ�In contrast, the inhibitory

effect of AM on ROS generation was completely abolished by H-89 (10-5 mol/L), a PKA

inhibitorዊ�Treatment with AM, CGRP (8-37), dibutyl-cAMP, or H-89 alone did not alter

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intracellular ROS levels in intact cells (Fig. 2A)ዊ�Similar results were obtained with cell

homogenates (Fig. 2B).

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 figure 3A, phosphorylation of Src (Tyr416) was significantly

increased in VSMCs by Ang II (10-7 mol/L) stimulation, that 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), CGRP (8-37) 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 to that of AM (10-7 mol/L). As expected, H-89 and CGRP (8-37) completely

abolished the inhibitory effect of AM on Src (tyr416) phosphorylation. In the absence of Ang II

stimulation, treatment with AM, CGRP(8-37), dibutyl-cAMP, or H-89 alone did not

significantly change the state of Src phosphorylation.

Effects of Dominant Negative and Constituitively-Active Src on the Anti-Oxidant 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

dominant negative or constituitively-active 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 over expressed in VSMCs. Then using the live transfected cells, we

assessed the effect of AM on Ang II-induced ROS production by the lucigenin

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chemiluminescence method. As seen in figure 4B, the dominant-negative Src transfected cells

that serve as the negative controls for this set of experiments, display basal level of ROS

generation that were unchanged following treatment with Ang II alone or Ang II and AM. In

contrast in constituitively-active Src over expressing 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).

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 activityዊ�Csk immunoprecipitates were probed with an anti-phosphotyrosine antibody .

Phosphorylation of Csk was significantly increased approximately 2-fold by Ang II but not by

treatment with AM alone. A much more robust increase (approximately 4-fold 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).

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Discussion

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) while 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: 1) 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). 2) 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). 3) 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 that is dependent on AM via the cAMP/PKA signaling pathway

and may also require Ang II that could be playing an autoregulatory role by initiating a

potential negative feedback loop.

Before initiating 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

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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, the apocynin was somewhat more potent than AM for reasons that

remain to be clarified. Furthermore, AM was without effect on Ca2+ ionophore A23187 or

TNF-ዊ�-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 an 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-kB (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 anti-oxidant effects of AM observed in this study

were in fact mediated primarily by the AM receptor mediated up regulation 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

CGRP (8-37), 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

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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 pathwayዊ�

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 minutes of treatment with Ang II.

The link between the Ang II AT1 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 Gβγ 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 dominant

negative Src transfected VSCMs and the inhibitory effect of AM on Ang II-induced ROS

generation was lost in constitutively-active 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

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by Csk which phosphorylates Tyr527 (1,19). Indeed we observed that the knockdown of Csk

by si RNA 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, while 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 in order to maintain a balance between active and inactive Src, which clearly

participates in 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 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 down regulate 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 to those

from normotensive Wistar-Kyoto (WKY) rats. Ang II mediated C-Src phosporylation (Tyr527)

was approximately 4-fold greater in SHR than WKY. However, Ang II increased Csk

phosphorylation 2-to 3-fold in WKY but not in SHR. From these data it was concluded that c-

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Src phosphorylation and Src dependent cell growth following Ang II treatment is 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 are 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 still

unclear as to the exact contribution of the different regulatory mechanisms in a given

physiological setting (36).

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Figure legends

Figure 1. AM inhibits intracellular ROS generation stimulated by Ang II in VSMCsዊ�

ROS generation was detected by lucigenin chemiluminescence methodዊ�(A) Concentration

dependent response of ROS generation by Ang IIዊ�VSMCs were stimulated with various

concentrations of Ang II (10-8-10-6 mol/L ) for10 minዊ�(B) AM inhibits intracellular ROS

generation stimulated by Ang II in a concentration-dependent manner(10-8-10-6 mol/L )ዊ�Cells

pretreated with AM (10-8 -10-6 mol/L) for 5 min or apocynin (10-6 mol/L) for 2 hours were

stimulated with Ang II (10-7 mol/L ) for 10 minዊ�(C) Treatment of VSMCs with mitochondrial-

derived ROS inhibitor rotenone (10 µmol/L) and the xanthine oxidase inhibitor allopurinol (10

µmol/L) for 1 hourዊ�(D) Treatment with calcium ionophore A23187 (1µmol/L) for 3 hours or

TNF-ዊ� (10 ng/mL) for 2 hours. ROS generation is expressed as fold increase compared with

controlዊ�The values are representative of three independent experiments, each performed in

triplicate. MLU (mean light units and CTR (control). *P<0.05 vs. control; †P<0.05 vs. Ang II.

Figure 2. AM inhibits intracellular ROS generation via a receptor-mediated and

cAMP-PKA-dependent pathwayዊ�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), H-89 (10-5 mol/L) or CGRP(8-37) (3x10-5

mol/L) for 0.5-1 h were stimulated in the absence or presence of Ang II (10-7 mol/L) for10 minዊ�

In some experiments, CGRP(8-37) or H-89 was applied 30 min prior to the AM pretreatmentዊ�

Quantification of ROS levels by the lucigenin chemiluminescence method, and the calculation

and plotting of data are the same as in Fig. 1ዊ�The values are representative of three

independent experiments, each performed in triplicate. *P<0.05 vs. control; †P<0.05 vs. Ang II.

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Figure 3. AM inhibits Src (Tyr416) phosphorylation stimulated by Ang II via a receptor-

mediated and cAMP-PKA-dependent pathway in VSMCsዊ�(A) Concentration dependent

response of Src (Tyr416) phosphorylation by Ang IIዊ�VSMCs were pretreated with various

concentrations of AM (10-8 -10-6 mol/L) for 5 min, then stimulated with Ang II (10-7 mol/L ) for

10 minዊ�The top panel is a representative immunoblot demonstrating phosphorylation of Src

(Tyr416)ዊ�The bottom panel represents averaged data from three independent experiments

quantified by densitometry of immunoblots and expressed as fold increases in phosphorylation

compared with unstimulated cellsዊ�(B) Effect of dibutyl-cAMP, CGRP(8-37) and H-89 on

phosphorylation of Src (Tyr416)ዊ�VSMCs treated with or without AM (10-7 mol/L), dibutyl-

cAMP (10-3 mol/L), H-89 (10-5 mol/L) or CGRP(8-37) (3x10-5 mol/L) for 0.5-1 h were

stimulated with or without Ang II (10-7 mol/L) for 10 minዊ�In some experiments, CGRP(8-37)

or H-89 were applied 30 min prior to the AM pretreatmentዊ�Phosphorylation of Src (Tyr416)

was evaluated by Western blottingዊ�The top panel is a representative immunoblot of the

phosphorylation of Src (Tyr416)ዊ�The bottom panel represents averaged data from three

independent experiments quantified by densitometry of immunoblots and expressed as fold

increases compared to unstimulated cellsዊ�*P<0.05 vs. control; †P<0.05 vs. Ang II

Figure 4. Effects of AM on Ang II-induced ROS production in dominant negative or

constitutively-active Src transfected cellsዊ�(A) The transfection efficiency for these experiments

was confirmed by immunoblotting for total Src protein. ROS production was examined by

lucigenin chemiluminescence method both in dominant-negative Src (B) and constitutive-active

Src transfected living cells (C). Controls are DN-Src and CA-Src transfected cell in the absence

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of stimulation. The values are representative of three independent experiments, each performed

in triplicate.

Figure 5. AM enhances Ang II-induced 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 hour, followed by

addition of Ang II (10-7 mol/L ) for 10 minዊ�(A) Csk was immunoprecipitated from cell lysates

with anti-Csk antibodyዊ�Immunoprecipitated proteins were subjected to SDS-PAGE and

immunoblotted with antiphosphotyrosine antibodyዊ�The top panel is a representative

immunoblot from three independent experiments assessing phosphorylated Cskዊ�The bottom

panel represents averaged data from three independent experiments quantified by densitometry

of immunoblots and expressed as fold increases in phosphorylation compared to unstimulated

cells. *P<0.05 vs control; †P<0.01 Ang II + AM and Ang II + dibutyl cAMP vs control. IP

indicates immunoprecipitation and IB, immunoblottingዊ�(B) VSMCs were transfected with

SMARTpool™ Csk or control siRNA and incubated for 48 h. The expression of Csk protein

was selectively inhibited by siRNA (top panel) without reducing β-actin control protein (bottom

panel). Each experiment was performed three times in triplicate. (C) Immunoblots and

quantification of total Src and phosphorylated Tyr416 and Tyr527 with and without Csk siRNA.

Each panel represents averaged data from three independent experiments quantified by

densitometry of immunoblots. *P<0.05 vs control.

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Fig. 2A

B

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Fig. 3

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Fig. 4A

B C

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Fig. 5A

Fig. 5B

Fig. 5C

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Adrenomedullin inhibits angiotensin II-induced contraction in human aortic smooth muscle cells

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