help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on June 10, 2004, doi:10.1165/rcmb.2004-0098OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2004-0098OCv1
31/3/365    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, X.
Right arrow Articles by Olson, S. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, X.
Right arrow Articles by Olson, S. C.
American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 365-372, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2004-0098OC

Src Kinase Mediates Angiotensin II–Dependent Increase in Pulmonary Endothelial Nitric Oxide Synthase

Xinmei Li, Kenneth M. Lerea, Jianyu Li and Susan C. Olson

Department of Biochemistry and Molecular Biology and Department of Cell Biology and Anatomy, New York Medical College, Valhalla, New York

Address correspondence to: Susan C. Olson, Ph.D., Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595. E-mail: Susan_Olson{at}nymc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously demonstrated that angiotensin II (Ang II) stimulates nitric oxide (NO) production in bovine pulmonary artery endothelial cells (BPAECs) by increasing NO synthase (NOS) expression via the type 2 receptor. The purpose of this study was to identify the Ang II–dependent signaling pathway that mediates this increase in endothelial NOS (eNOS). The Ang II–dependent increase in eNOS expression is prevented when BPAECs are pretreated with the tyrosine kinase inhibitors, herbimycin A and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine, which also blocked Ang II–dependent mitogen-activated protein kinase (MAPK) kinase/extracellular-regulated protein kinase (MEK)-1 and MAPK phosphorylation, suggesting that Src is upstream of MAPK in this pathway. Transfection of BPAECs with an Src dominant negative mutant cDNA prevented the Ang II–dependent Src activation and increase in eNOS protein expression. PD98059, a MEK-1 inhibitor, prevented the Ang II–dependent phosphorylation of extracellular-regulated protein kinases 1 and 2 and increase in eNOS expression. Neither AG1478, an epidermal growth factor receptor kinase inhibitor, nor AG1295, a platelet derived growth factor receptor kinase inhibitor, had any effect on Ang II–stimulated Src activity, MAPK activation, or eNOS expression. Pertussis toxin prevented the Ang II–dependent increase in Src activity, MAPK activation, and eNOS expression. These data suggest that Ang II stimulates Src tyrosine kinase via a pertussis toxin–sensitive pathway, which in turn activates the MAPK pathway, resulting in increased eNOS protein expression in BPAECs.

Abbreviations: angiotensin II, Ang II • bovine pulmonary artery endothelial cells, BPAECs • Dulbecco's modified Eagle's medium, DMEM • dominant negative, DN • epidermal growth factor, EGF • EGF receptor, EGFR • endothelial nitric oxide synthase, eNOS • extracellular-regulated protein kinase, ERK • fluorescein isothiocyanate, FITC • mitogen-activated protein kinase, MAPK • MAPK/ERK kinase, MEK • nitric oxide, NO • neomycin phosphotransferase, NPTII • phosphate-buffered saline, PBS • platelet-derived growth factor receptor, PDGFR • 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine, PP2 • protein tyrosine phosphatase, PTP • sodium dodecylsulfate–polyacrylamide gel electrophoresis, SDS-PAGE • vascular smooth muscle cells, VSMCs


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Nitric oxide (NO), a potent vasodilator, is synthesized from L-arginine by a family of NADPH-dependent NO synthases (NOS) (1): neuronal, endothelial, and a cytokine-inducible isoform. Although endothelial NOS (eNOS) was originally designated as a constitutively expressed enzyme, stimuli such as shear stress, chronic exercise, hydrogen peroxide, estrogen, and oxidized low-density lipoproteins, can induce the expression of eNOS mRNA, ultimately leading to an increase in NO production (2). In addition, Davis and colleagues (3) recently demonstrated that shear stress not only increases the rate of transcription of eNOS mRNA but also increases the half-life of the message. Furthermore, the activity of eNOS can be regulated by post-translational modification and by its association with other proteins (reviewed in Ref. 1).

Emerging evidence indicates that angiotensin II (Ang II), the main effector peptide of the renin–angiotensin system, can stimulate the production of NO, both by inducing eNOS expression (4–6) and by increasing enzyme activity (7, 8). Ang II exerts its effects through at least two major receptor subtypes (9), type 1 (AT1) and type 2 (AT2), both of which have the seven-transmembrane topology found in G-protein coupled receptors. The AT1 receptor, which mediates vasoconstriction, water and sodium intake, and induction of cell growth, is linked to the activation of many signaling molecules (9), including GTP-binding proteins, phospholipase C, protein kinase C, phosphatidylinositol 3-kinase, mitogen-activated protein kinases (MAPK), and tyrosine kinases. The AT2 receptor has been proposed to play a role in vasodilation, inhibition of cell growth, and induction of apoptosis and cell differentiation (9). However, there are conflicting data in the literature regarding the pathways activated via the AT2 receptor. For example, the AT2 receptor has been linked to both an activation and inhibition of protein phosphatases (10, 11) as well as extracellular-regulated protein kinase (ERK) 1/ERK2 phosphorylation (12, 13). Although the pathways mediated via the AT2 receptor have not been clearly delineated, many studies (reviewed in Ref. 9) suggest that activation of AT2 receptor appears to oppose the effects of the AT1 receptor.

Protein tyrosine kinases activated by Ang II include the epidermal growth factor (EGF) receptor (EGFR) kinase (14–16), platelet-derived growth factor receptor (PDGFR) kinase (17), and the nonreceptor tyrosine kinases, Pyk, FAK, JAK and Src (15, 16, 18, 19). Ang II activation of Src can lead to MAPK activation and the induction of the transcription factors, fos, jun and myc, resulting in an increase in gene transcription (20). Multiple mechanisms have been shown to be involved in the Ang II–dependent activation of Src, including heterotrimeric G proteins (21–23), reactive oxygen species (24), and in some cases, transactivation of the EGFR (23, 25).

To date there are many studies linking Ang II and NO production (4–8); however, the mechanism by which Ang II leads to an increase in NO depends not only on the experimental conditions, but also appears to depend on the cell type and the signaling pathways present in that cell. The ability of Ang II to act as a vasoconstrictor as well as stimulate the production of a vasodilator lends to its important role as a regulator of blood pressure. We recently reported that Ang II stimulates an increase in eNOS mRNA, protein, and NO production via the AT2 receptor (26). The purpose of this study was to investigate the signaling cascade that mediates the Ang II–dependent increase in eNOS protein expression in bovine pulmonary artery endothelial cells (BPAECs). We found that Ang II stimulates Src tyrosine kinase via a pertussis toxin–sensitive pathway, which in turn activates the classical MAPK pathway, resulting in an increase in eNOS protein expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Dulbecco's modified Eagle's medium (DMEM) was purchased from GIBCO Invitrogen (Grand Island, NY). Anti-ERK1/ERK2 antibody, anti-actin antibody, protein G agarose, Ang II, PD123319, PD98059, and protease inhibitors were from Sigma Chemicals (St. Louis, MO). Losartan was from DuPont Merck Pharmaceuticals (Wilmington, DE). Acrylamide, bisacrylamide, TEMED, ammonium persulfate, nitrocellulose membranes, and protein assay kit were purchased from Bio-Rad (Hercules, CA). The eNOS antibody and horseradish peroxidase–conjugated anti-rabbit IgG and anti-mouse IgG secondary antibodies were from Transduction Labs (San Diego, CA). Src kinase assay kit, Src cDNA (dominant negative [DN]) Expression kit, EGF, anti-EGFR antibody, anti-phosphospecific EGFR antibody (Tyr1173), antiphosphotyrosine antibody (4G10), and all G protein {alpha} subunit antibodies were from Upstate Biotechnology Inc. (Lake Placid, NY). 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine (PP2), AG1478, and AG1295 were from Calbiochem (Los Angeles, CA). We obtained anti–phospho-MAPK/ERK kinase (MEK) 1/2 (Ser 217/221), anti-MEK1/2 antibody, anti–phospho-ERK1/ERK2 antibody (Thr202/Tyr204), anti-EGFR antibody, anti–phospho-EGFR antibody (Tyr845), and anti–phospho-Src antibody (Tyr416) from Cell Signaling (Beverly, MA). Anti–v-Src antibody was from Oncogene (San Diego CA). Anti-AT1 and anti-AT2 receptor antibodies were from Santa Cruz Technology (Santa Cruz, CA). The goat fluorescein isothiocyanate (FITC)–conjugated secondary antibody was from Molecular Probes (Eugene, OR).

Cell Culture
BPAECs were prepared and characterized as previously described (4). The cells were cultured in DMEM supplemented with endothelial cell growth factor and passed using trypsin-EDTA. Control and treated cells were matched in each experiment for cell line, passage number (3–6), and time to monolayer confluence. BPAECs were grown to 90% confluent and made quiescent for 24 h before stimulation with Ang II.

Electrophoresis and Western Blot Analysis
Proteins (30 µg) collected in HEPES buffer (50 mM HEPES, pH 7.4, containing 2 µM leupeptin, 2 µM pepstatin, 0.5 mM phenylmethylsulfonylfluoride, and 1 mM sodium orthovanadate) were subjected to either 8 or 10% sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes using a Bio-Rad electrophoretic transfer cell. Membranes were probed with various primary antibodies as indicated, followed by their respective horseradish peroxidase-conjugated secondary antibodies. Peroxidase activity was determined using the enhanced chemiluminescence Western blotting kit from Amersham Biosciences (Piscataway, NJ) and exposing membranes to X-ray film. The relative amounts of proteins were quantitated using the AlphaImager Tm2000 (Alpha Innotech Corp., San Leandro, CA) documentation and analysis system. Results are expressed relative to untreated cells.

Src Kinase Assay
BPAECs were grown to confluence on 60 mm tissue culture plates and treated with 1 µM Ang II for 1–10 min. Cells were then washed with cold phosphate-buffered saline (PBS), lysed in buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 2 mM benzamidine, 1 mM Na3VO4, and 2 µM leupeptin), and then centrifuged for 10 min at 14,000 rpm at 4°C. Protein concentrations of the detergent-soluble fraction (referred to as cell lysate) were measured using a detergent-compatible protein assay kit. Cell lysates (600 µg) were incubated with anti–v-src antibody (4 µg) and protein G agarose overnight at 4°C. The immunocomplexes were isolated by centrifugation and the beads were washed four times with lysis buffer.

Src kinase activity was measured using the Src kinase assay kit. Briefly, 10 µl of the substrate peptide (KVEKIGEGTYGVVYK; 150 µM final concentration), 20 µl Src reaction buffer (100 mM Tris-HCl, pH 7.2, 125 mM MgCl2, 25 mM MnCl2, 2 mM EGTA, 250 µM Na3VO4, 2 mM dithiothreitol) and 10 µl [{gamma}-32P] ATP stock (1 µCi/µl with 75 mM MnCl2, 500 µM ATP) were added to 10 µl washed beads. After incubation for 10 min at 30°C, the reaction was stopped by the addition of 20 µl of 40% acetic acid. The phosphorylated substrate was separated from the residual [{gamma}-32P]ATP using P81 phosphocellulose paper and 32P incorporated into substrate was assayed by liquid scintillation counting.

Transient Transfection of BPAECs with Src DN Mutant
BPAECs were transfected with DN Src cDNA using the Src cDNA DN expression kit (K296R/Y528F). BPAECs were plated at 40–60% confluent in six-well tissue culture plates. One microgram of Src DN cDNA in pUSEamp was mixed with 6 µl PLUS reagent in 100 µl of serum-free DMEM for 15 min at room temperature. Serum-free DMEM was then mixed with LipofectAMINE reagent (4 µl; Invitrogen, Carlsbad CA), which was then added to the DNA mixture and incubated at room temperature for 15 min. The LipofectAMINE PLUS reagent–DNA complexes were added to each well of cells and incubated for 3 h at 37°C in a humidified CO2 incubator. The DNA-containing medium was removed from the cells and fresh media was added, and the cells were incubated for an additional 48–72 h. Src expression was monitored by Western blot analysis using an anti-Src antibody, and Src kinase activity was determined as described previously here. Transfection efficiency was monitored by Western blot analysis using an anti–neomycin phosphotransferase (NPTII) antibody. As a control, BPAECs were transfected with an empty vector (1 µg). The transfected BPAECs were then treated with the desired agonists and/or inhibitors.

Coimmunoprecipitation
BPAEC lysates (250 µg) were incubated with 1 µg anti-AT1, anti-AT2, anti-G{alpha}q/11, anti-G{alpha}i2, or anti-G{alpha}i3 antibodies (buffer: 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% nonidet P-40, 10 mM NaF, 1 mM Na3VO4, 1 mM PMSF, and 10 µg each of leupeptin and aprotinin). The immunocomplexes were isolated using protein G agarose for 2 h at 4°C followed by centrifugation. The beads were washed three times with lysis buffer, separated by 10% SDS-PAGE, and transferred to nitrocellulose membranes. The membranes were then probed with an anti-Src antibody.

Immunofluorescence Cell Staining
BPAECs (80% confluent) were grown on gelatin-coated chamber slides and transfected with either a control vector or the vector containing the Src DN mutant cDNA. The cells were washed in PBS, fixed with 100% methanol for 2 h at –20°C, and then permeabilized with 1% Triton X-100 for 5 min at room temperature. BPAECs were blocked with 0.1% bovine serum albumin/PBS for 1 h at room temperature, washed with PBS, and incubated with anti-NPTII antibody (1:100 diluted in blocking buffer) overnight at 4°C. After washing with PBS, the secondary FITC-conjugated antibody was added (1:100 diluted in blocking buffer) and incubated for 1 h at room temperature in the dark. The cells were washed with PBS and viewed with a Nikon Optiphot-2 fluorescence microscope (40x; Micron Optics, Parsippany, NJ).

Statistical Analysis
Data are expressed as means ± SE, and n refers to the number of experiments performed in a minimum of three different cell preparations. Data from at least three independent experiments were averaged and statistical significance was evaluated by Student's t test (with paired control and conditions). Kinetic experiments were analyzed using repeated measures analysis of variance followed by Bonferroni's post test. Statistical significance was identified at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Src Tyrosine Kinase Mediates Ang II–Dependent Increase in eNOS Protein Expression
We previously demonstrated that Ang II may exert a vasodilatory effect by increasing the expression of eNOS mRNA, protein, and NO in BPAECs via the AT2 receptor (26). In this study, we have used the same cells to investigate the signaling pathways that are involved.

To determine whether tyrosine kinases mediate the Ang II–dependent increase in eNOS expression, BPAECs were pretreated with either the generic tyrosine kinase inhibitor, herbimycin A (0.1 µg/ml), or an inhibitor of the Src family of tyrosine kinases, PP2 (10 µM), before stimulation with 1 µM Ang II. As determined by Western blot analysis, Ang II stimulated an increase in eNOS expression at 8 h (2.75 ± 0.2–fold increase versus basal, n = 7, P < 0.02; Figure 1) that was completely blocked by both herbimycin A and PP2. Under these conditions, total cellular actin levels remained unchanged.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 1. Herbimycin A and PP2 prevented the Ang II–dependent increase in eNOS protein expression at 8 h. BPAECs were treated with either herbimycin A (Herb A; 0.1 µg/ml) for 6 h or 10 µM PP2 for 30 min before stimulation with 1 µM Ang II. After 8 h, eNOS and actin expression were analyzed by Western blot analysis. Bar graphs show densities of autoradiographic bands with the value of the untreated cells represented as 100%. The amount of eNOS protein was normalized to actin. Values are means ± SE (*P < 0.02 versus untreated cells; #P < 0.05 versus Ang II–treated cells; n = 7).

 
The effect of Ang II on Src tyrosine kinase activity in BPAECs was investigated using a Src-specific synthetic peptide substrate (see MATERIALS AND METHODS). In the absence of Ang II treatment, ~ 2.64 pmol phosphate was incorporated into the Src peptide. Ang II stimulated a 2.86 ± 0.24–fold increase in Src activity at 1 min (n = 9, P < 0.01; Figure 2A). This increase in Src kinase activity was prevented when BPAECs were pretreated with PP2 (10 µM for 30 min; Figure 2A). In Figure 2, the top panel shows that equal amounts of Src were immunoprecipitated at each time point, demonstrating that the Ang II–induced Src kinase activity is due to increased enzymatic activity.



View larger version (22K):
[in this window]
[in a new window]
  		Figure 2. (A) Kinetics of Ang II–dependent Src kinase activity. Src tyrosine kinase activity was assayed in BPAECs that were treated with 1 µM Ang II for the times indicated. Upper panel shows Western blot of immunoprecipitated Src (mw, 60 kD) at times indicated. Values are mean ± SE of 9 different experiments (*P < 0.01 versus time 0). Pretreatment of BPAECs with 10 µM PP2 (PP2) for 30 min prevented the Ang II–dependent increase in Src activity at 1 min (#P < 0.05 versus Ang II–treated cells; n = 3). (B) Effects of receptor antagonists on Src kinase activity. BPAECs were pretreated with 10 µM losartan (LOS) and/or PD123319 (PD) for 15 min before stimulation with 1 µM Ang II for 1 min. Src activity was assayed as described in MATERIALS AND METHODS. Data are represented as means ± SE from 6 separate experiments (*P < 0.05 versus untreated control; #P < 0.05 versus Ang II–treated cells). Solid bars, Ang II; open bars, control.

 
To identify the receptor linked to Ang II–induced Src activation, BPAECs were pretreated with 10 µM losartan, an AT1 antagonist, or 10 µM PD123319, an AT2 antagonist, for 15 min before stimulation with 1 µM Ang II. Ang II–stimulated Src tyrosine kinase activity at 1 min was blocked when BPAECs were pretreated with PD123319, whereas pretreatment with losartan had no effect on basal or Ang II–stimulated Src tyrosine kinase activity (Figure 2B).

To further explore the role of Src tyrosine kinase, BPAECs were transfected with an expression vector for an Src DN mutant cDNA. Immunofluorescence with anti-NPTII antibody and an FITC-conjugated secondary antibody demonstrated that >= 85% of the cells transiently expressed the control vector or the catalytically inactive form of Src (data not shown). Transfection of BPAECs with the Src DN mutant cDNA greatly reduced the general levels of tyrosine phosphorylation in Ang II–stimulated BPAECs as determined by Western blotting with an anti-phosphotyrosine antibody (Figure 3A). More importantly, transfection of BPAECs with the Src DN mutant cDNA prevented the Ang II–dependent increase in eNOS protein expression at 8 h (Figure 3B). There was no effect on Ang II–stimulated eNOS protein expression in control vector–transfected BPAECs (Figure 3B). Taken together, these data show that Src tyrosine kinase plays a role in the Ang II–dependent increase in eNOS protein expression.



View larger version (38K):
[in this window]
[in a new window]
  		Figure 3. (A) Ang II stimulated a time-dependent increase in tyrosine phosphorylation that was significantly inhibited in BPAECs transfected with Src DN cDNA. Nontransfected BPAECs or BPAECs transiently expressing a catalytically inactive mutant of Src were left unstimulated or stimulated with 1 µM Ang II from 1–5 min. Western analysis was performed on cell lysate proteins with the antiphosphotyrosine antibody, 4G10 (top panel) and anti-NPTII antibody (bottom panel; mw, 32 kD). (B) Transfection of BPAECs with Src DN cDNA prevents the Ang II–dependent increase in eNOS protein expression at 8 h. Control (Non), vector-only transfected (Mock), or BPAECs transiently expressing a catalytically inactive mutant of Src (Src DN) were stimulated with 1 µM Ang II for 8 h. eNOS and actin levels were determined by Western blot analysis. Bar graphs show densities of autoradiographic bands with the value of the untreated cells represented as 100% (*P < 0.05 versus control cells in the absence of Ang II; #P < 0.05 versus Ang II–treated cells; n = 5). Solid bars, Ang II; open bars, control. Bottom panel shows immunoblot analysis of transfected BPAEC lysates with anti-NPTII.

 
Ang II–Dependent Increase in eNOS Protein Expression Requires the MAPK Pathway
Ang II activation of MAPK has been shown to increase the expression of a number of genes via an AP-1 site in their promoter regions (20). Because the eNOS promoter region contains an AP-1 site (2), it seems reasonable to postulate that activation of MAPK pathway by Ang II could stimulate the increase in eNOS mRNA that ultimately leads to an increase in eNOS protein levels. To test this possibility, the effects of Ang II on MEK-1 and ERK1/ERK2 activation in BPAECs were investigated by Western blot analysis using phosphospecific antibodies. Ang II stimulated a time-dependent increase in MEK-1 activation that was maximal at 30 min (3.9 ± 0.4–fold increase over basal, n = 6, P < 0.005; Figure 4A). MEK-1 was still active at 60 min (3.12 ± 0.93–fold increase over basal, n = 6, P < 0.05) and returned to basal level by 2 h. Total MEK-1 protein levels in the whole-cell extracts were unchanged after Ang II stimulation (Figure 4A). This Ang II–dependent activation of MEK-1 was temporally followed by an increase in ERK1/ERK2 phosphorylation that was maximal at 60 min (3.7 ± 1.1–fold increase over basal, n = 6, P < 0.05; Figure 4B). Ang II did not affect the expression of total ERK1/ERK2 protein (Figure 4B). At a concentration that inhibited Src tyrosine kinase activity, PP2 (10 µM) prevented both the Ang II–dependent increase in MEK-1 phosphorylation at 30 min (Figure 4C) and ERK1/ERK2 phosphorylation at 60 min (Figure 4D), but had no effect on the basal phosphorylation state of these proteins.



View larger version (33K):
[in this window]
[in a new window]
  		Figure 4. (A) Kinetics of Ang II–dependent activation of MEK-1. BPAECs were treated with 1 µM Ang II for the times indicated. Phospho-MEK-1 and total MEK-1 levels were analyzed by Western blot analysis. The amount of phosphorylated MEK-1 was normalized against total MEK-1. Ang II–dependent activation of MEK-1 is statistically significant from 0 time at 30 min (*P < 0.005) and at 60 min (*P < 0.05; n = 6). (B) Kinetics of Ang II–dependent activation of ERK1/ERK2. BPAECs were treated with 1 µM Ang II for the times indicated. Levels of phospho-ERK1/ERK2 and total ERK1/ERK2 were determined by Western blot analysis. The amount of phosphorylated ERK1/ERK2 was normalized against total ERK1 and ERK2 (*P < 0.05 versus 0 time point; n = 6). PP2 blocked the Ang II–dependent phosphorylation of MEK-1 (C) and ERK1/ERK2 (D). BPAECs were pretreated with 10 µM PP2 for 30 min, followed by the addition of 1 µM Ang II. The Ang II–dependent increase in MEK-1 phosphorylation at 30 min (C) and ERK1/ERK2 activation at 60 min (D) were determined by Western blot analysis using their respective anti-phosphospecific antibodies (*P < 0.05 versus untreated control; #P < 0.05 versus Ang II–treated cells; n = 6). (E) PD123319 blocked the Ang II–dependent increase in ERK1/ERK2 phosphorylation. BPAECs were pretreated with 10 µM PD123319 for 30 min before stimulation with 1 µM Ang II. After 60 min, ERK1/ERK2 activation was determined by Western blot analysis with an antiphosphospecific antibody (*P < 0.05 versus untreated control; #P < 0.05 versus Ang II–treated cells; n = 6). Shaded bars, ERK1; hatched bars, ERK2.

 
Because PD123319 prevented the Ang II–dependent activation of Src, we decided to examine the effect of this AT2 receptor antagonist on Ang II activation of MAPK at 60 min. The Ang II–dependent increase in ERK1/ERK2 phosphorylation was prevented when BPAECs were pretreated with PD123319 (Figure 4E).

Treatment of BPAECs with a selective MEK-1 inhibitor, PD98059 (10 µM, 15 min), prevented the Ang II–dependent activation of ERK1/ERK2 at 60 min (Figure 5A) as well as the Ang II–dependent increase in eNOS protein expression at 8 h (Figure 5B). These inhibitor studies provide evidence that the Src-dependent activation of MAPK is involved in the Ang II–dependent increase in eNOS protein expression. Furthermore, they suggest that Src is upstream of MAPK in this Ang II–stimulated pathway in BPAECs.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 5. PD98059 blocked the Ang II–dependent increase in ERK1/ERK2 phosphorylation and prevented the Ang II–dependent increase in eNOS protein expression. BPAECs were pretreated with 10 µM PD98059 and then stimulated with 1 µM Ang II. (A) At 60 min, Ang II–dependent ERK1/ERK2 phosphorylation was determined by Western blot analysis. ERK1/ERK2 phosphorylation was normalized against total ERK1/ERK2 (*P < 0.05 versus untreated control; #P < 0.05 versus Ang II–treated cells; n = 3). Shaded bars, ERK1; hatched bars, ERK2. (B) At 8 h, the levels of eNOS and actin were determined by Western blot analysis. Upper panel is a representative Western blot of eNOS protein expression of 9 separate experiments. Bar graphs show densities of autoradiographic bands, with the value of the untreated cells represented as 100% (*P < 0.02 versus untreated cells; #P < 0.05 versus Ang II–treated cells).

 
EGFR and PDGFR Tyrosine Kinases Are Not Involved in the Pathway by which Ang II Induces eNOS Protein Expression
Previous studies have demonstrated that Ang II activation of Src may occur via EGFR transactivation (25) or that Src-dependent activation of MAPK requires EGFR transactivation (16, 21, 27). To determine whether transactivation of the EGFR is involved in the Ang II–dependent increase in Src kinase activation and/or the increase in eNOS protein expression, we employed the EGFR-specific tyrosine kinase inhibitor, tyrphostin AG1478. In this experiment, Src was immunoprecipitated from BPAEC lysates and activation was analyzed by Western blot analysis using the anti–phospho-Src (Tyr416) antibody. Pretreatment of BPAECs with 200 nM AG1478 for 30 min before the addition of Ang II did not prevent the Ang II–dependent Src autophosphorylation at Tyr416 at 1 min, MEK-1 phosphorylation at 30 min, or ERK1/ERK2 phosphorylation at 60 min, nor did it block the Ang II–dependent increase in eNOS protein expression at 8 h (data not shown). These data strongly suggest that transactivation of the EGFR is not involved in the Ang II–dependent increase in Src kinase activity, nor does the EGFR mediate the Src-dependent increase in eNOS protein expression seen in BPAECs.

Other investigators have found a role for the PDGFR in mediating Ang II mitogenic responses in cells (18). To determine if the PDGFR is involved in Ang II–stimulated MAPK in BPAECs, the cells were pretreated with 200 µM AG1295, a PDGFR tyrosine kinase inhibitor, before stimulation with Ang II. Similar to the studies with the EGFR kinase inhibitor, blockade of PDGFR kinase activity had no effect on Ang II–stimulated MEK-1 activation, ERK1/ERK2 phosphorylation, or eNOS protein expression (data not shown).

Pertussis Toxin–Sensitive GTP-Binding Proteins Are Involved in the Ang II–Dependent Increase in eNOS Protein Expression
To investigate the roles of G proteins in Ang II induction of eNOS protein expression, BPAECs were pretreated with pertussis toxin, which ADP ribosylates and inhibits the activity of members of the G{alpha}i GTP-binding proteins. Pertussis toxin treatment did not affect cell viability, as over 90% of the cells were viable as determined by trypan blue dye exclusion assay. In addition, Ang II–stimulated STAT3 phosphorylation at 30 min was not affected by pertussis toxin (data not shown), demonstrating that pertussis toxin treatment was not lethal to BPAECs. Next, the effect of pertussis toxin on Ang II–dependent tyrosine phosphorylation was investigated. In addition to stimulating a rapid increase in tyrosine phosphorylation (see Figure 3A), Ang II also stimulates an increase in tyrosine phosphorylation in BPAECs at 8 h that was prevented when BPAECs were pretreated for 6 h with 50 ng/ml pertussis toxin (Figure 6A). Pertussis toxin also prevented the Ang II–dependent Src kinase activation (n = 6, Figure 6B) and the increase in ERK1/ERK2 phosphorylation (n = 6; Figure 6C). Finally, pretreatment of BPAECs with 50 ng/ml pertussis toxin for 6 h completely blocked the Ang II–dependent increase in eNOS protein expression at 8 h (Figure 6D). Treatment of BPAECs with the pertussis toxin B oligomer, which does not possess ADP-ribosyltransferase activity, had no effect on eNOS expression (Figure 6D). Western blot analysis demonstrated that BPAECs express the pertussis toxin–sensitive G proteins, G{alpha}{iota}2 and G{alpha}{iota}3 (Figure 7A). Coimmunoprecipitation experiments revealed that Src associates with both G{alpha}i2 and G{alpha}i3 in BPAECs (Figure 7B). Together, these data show that Ang II activation of a pertussis toxin–sensitive G protein leads to activation of Src tyrosine kinase and MAPK in BPAECs, and ultimately to an increase in eNOS protein expression.



View larger version (38K):
[in this window]
[in a new window]
  		Figure 6. Pertussis toxin inhibited Ang II–dependent increase in tyrosine phosphorylation, Src kinase activity, ERK1/ERK2 phosphorylation and eNOS protein expression. BPAEC were pretreated with pertussis toxin (50 ng/ml) for 6 h before the addition of 1 µM Ang II. (A) After 8 h treatment with Ang II, tyrosine phosphorylation was detected by Western blot analysis using the anti-phosphotyrosine antibody, 4G10. Molecular weights of standards are shown on the right. (B) Src tyrosine kinase activity was determined at 1 min (*P < 0.05 versus control cells, #P < 0.05 versus Ang II–treated cells n = 6). Solid bars, Ang II; open bars, control. (C) Ang II–dependent ERK1/ERK2 phosphorylation at 60 min was determined using an anti-phospho-p44/42 MAP kinase antibody. (*P < 0.02 versus untreated cells, #P < 0.05 versus Ang II–treated cells, n = 6). Shaded bars, ERK1; hatched bars, ERK2. (D) Where indicated, BPAECs were pretreated with either 50 ng/ml pertussis toxin (PTX) or pertussis toxin B oligomer (PTXB oligo) before the addition of 1 µM Ang II. At 8 h, eNOS protein expression in control and Ang II–stimulated BPAECs was monitored by Western blot analysis and normalized against actin levels (*P < 0.05 versus untreated control; #P < 0.05 versus Ang II–treated cells; n = 6).

 


View larger version (31K):
[in this window]
[in a new window]
  		Figure 7. (A) Expression of GTP-binding protein {alpha} subunits in BPAECs. Expression of G{alpha} subunits was determined by Western blot analysis with their respective antibodies. (B) Coimmunoprecipitation of Src with G{alpha}i3 and G{alpha}i2. AT1, AT2, G{alpha}q/11, G{alpha}i3, or G{alpha}i2 were immunoprecipitated from BPAEC lysates using their respective antibodies. The presence of Src in the immunocomplex was determined by immunoblot analysis.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we demonstrate that Ang II stimulates an increase in eNOS protein expression via a pertussis toxin–sensitive signaling pathway that requires Src tyrosine kinase and MAPK activation. Numerous reports (4–8, 26) have demonstrated that Ang II stimulates NO production; however, there is a discrepancy in the literature concerning the mechanism of activation. The Ang II–dependent increase in NO has been linked to both the AT1 (7, 8) and the AT2 receptor (6, 23), and has been shown to involve acute activation of the eNOS enzyme (7, 8) as well as an increase in expression of eNOS protein (4–6, 23).

Results obtained in this study, in which BPAECs were either treated with the Src family tyrosine kinase inhibitor, PP2, or transiently transfected with the Src DN mutant cDNA, demonstrated that Src mediates the Ang II–dependent increase in eNOS protein expression. Src has also been shown to be involved in stress-dependent increases in eNOS transcription and stabilization of eNOS message (3) and in the estrogen-dependent increase in eNOS (28). In contrast to published reports linking the AT1 receptor to Src activation, Ang II activation of Src tyrosine kinase activity in BPAECs is blocked by the AT2 receptor antagonist, PD123319. Previously, Bottari and colleagues (10) demonstrated that activation of the AT2 receptor in PC12W cells leads to an increase in protein tyrosine phosphatase (PTP) activity. Therefore, we propose that a possible mechanism by which Ang II activates Src in BPAEC is through an AT2 receptor–dependent activation of PTP, which in turn dephosphorylates the C-terminal Tyr527 on Src (23, 29), resulting in its activation.

Growing evidence support a role for reactive oxygen species in Src-dependent signaling pathways; not only is Src linked to activation of the NADPH oxidase, but it is also a downstream target of the oxidase in vascular smooth muscle cells (VSMCs) (23, 24). Furthermore, both exogenous addition of hydrogen peroxide (30) and AT1 receptor–dependent NAD(P)H oxidase–derived hydrogen peroxide (8) stimulate NO production in endothelial cells. Preliminary studies from our laboratory suggest that superoxide does not play a role in regulating eNOS protein expression as treatment of BPAECs with Tiron, a superoxide scavenger, had no effect on eNOS expression. Conversely, we did find that the flavin oxidase inhibitor, diphenylene iodonium, inhibited both basal and Ang II–dependent stimulated eNOS expression (data not shown) in BPAECs. Thus, further investigation is needed to determine the role of reactive oxygen species in regulating Src activity and eNOS protein expression in BPAECs.

Several reports indicate that Src-dependent transactivation of the EGFR is involved in many of the mitogenic affects of G-protein coupled receptors (reviewed in Ref. 23). For example, Ang II–dependent activation of MAPK in rat liver epithelial cells (14) and VSMCs (15) were shown to be mediated via EGFR tyrosine phosphorylation. In contrast, treatment of VSMCs from spontaneously hypertensive rats with AG1478 did not completely block Ang II–induced c-Src phosphorylation and ERK1/ERK2 phosphorylation, indicating that EGFR-independent pathways also contribute to Src-mediated signaling (25). In COS-7 cells expressing a mutated version of the Ang II receptor that does not activate the EGFR, Ang II activation of ERK is mediated through Src alone (31). Furthermore, Andresen and colleagues (32) found that transactivation of the EGFR is not primarily responsible for Ang II–mediated activation of ERK in preglomerular smooth muscle cells. All of these studies obviously reflect different molecular mechanisms operating in the specific cell types.

Our studies using the EGFR kinase inhibitor, AG1478, suggest that the EGFR is not involved in the Ang II–dependent increase in eNOS protein expression in BPAECs. The lack of involvement of EGFR in Ang II signaling may reflect one of two possibilities: first, the EGFR is not present in BPAECs, or second, Ang II signaling and EGF signaling follow two independent, nonconverging pathways. To determine if EGFR is present in BPAECs, we performed Western blot analysis of the total cell lysate using two anti-EGFR antibodies; neither one of the antibodies detected the EGFR in BPAECs. Because it may be that the expression of the EGFR is too low to detect in total cell lysate, proteins from control and Ang II–stimulated cells were immunoprecipitated with an anti-EGFR antibody or with the anti-phosphotyrosine antibody, 4G10. The proteins were separated by SDS-PAGE and subjected to Western blot analysis using antibodies that recognize total EGFR, EGFR phosphorylated on Tyr1173 (the autophosphorylation site), or EGFR phosphorylated on Tyr845 (the Src-targeted residue). Neither the presence of total nor phosphorylated EGFR could be confirmed in BPAECs. In addition, Western blot analysis using the anti-phosphotyrosine antibody, 4G10, demonstrated that EGF (50 nM) did not stimulate a time-dependent increase in tyrosine phosphorylation in our cells. Taken together, these data imply that the EGFR is not present and/or not functional in BPAECs and that EGFR transactivation is not involved in Ang II–dependent Src or MAPK activation.

In a hepatocyte cell line, which does not express the EGFR, Weng and Shukla (18) found that the PDGFR is involved in Ang II activation of the MAPK pathway. To investigate a role for the PDGFR in mediating Ang II increase in eNOS protein expression, BPAECs were pretreated with the PDGFR tyrosine kinase inhibitor, AG1295. Inhibiting PDGFR kinase activity had no effect on Ang II–stimulated MEK-1 phosphorylation, ERK1/ERK2 phosphorylation, or eNOS protein expression, demonstrating that the PDGFR does not mediate the Ang II–dependent increase in eNOS protein expression.

The Ang II–dependent increase in eNOS protein expression is blocked by pertussis toxin, implicating the involvement of the G{alpha}i family of GTP-binding proteins. Although the AT2 receptor has the seven-transmembrane topology typical of the G protein family of receptors, there are conflicting data concerning its coupling to GTP-binding proteins. In rat adrenal glomeruli and human myometrium (33), addition of guanine nucleotides had no effect on AT2 receptor ligand binding, suggesting that the receptor is not coupled to a G protein. Conversely, other studies support our finding implicating a linkage between the AT2 receptor and members of the G{alpha}i family of GTP-binding proteins (34–36). In neurons, pertussis toxin prevented the AT2 receptor–dependent stimulation of delayed rectified K+ current (34). Furthermore, Zhang and Pratt (35) demonstrated that immunoprecipitated G proteins (G{alpha}i2 and G{alpha}i3) were able to bind an AT2-specific receptor ligand in rat fetus membranes, suggesting an association between the G proteins and AT2 receptor. Finally, in membranes from AT2-transfected COS-7 cells, ligand binding to the AT2 receptor stimulated GTP{gamma}S binding to G{alpha}i and G{alpha}o, inferring that AT2 receptors directly activate these G proteins (36). There is also evidence supporting an association between GTP-binding proteins and NO production. Members of the pertussis toxin–sensitive family of G proteins are involved in mediating 5-hydroxytryptamine stimulation of endothelial NO production (37) and in the shear stress–dependent increase in eNOS mRNA levels (38).

In BPAECs, Ang II–dependent Src activation is pertussis toxin–sensitive, suggesting that G{alpha}i GTP-binding proteins are linked to Src tyrosine kinase activity. Published studies (22, 23) have demonstrated that G protein–coupled receptors can activate Src via both pertussis toxin–sensitive (G{alpha}i) and –insensitive (G{alpha}s, G{alpha}q) heterotrimeric GTP-binding proteins. In an in vitro reconstitution assay, Ma and colleagues (22) found that G{alpha}i and G{alpha}s, but not G{alpha}q, G{alpha}12, or Gß{gamma} could directly interact with the catalytic domain of Src, thereby activating it. In addition, the ß{gamma} subunits of the G proteins have been shown to play a crucial role in Src-linked activation of Ras, a monomeric GTP-binding protein, and MAPK (27, 39). Using coimmunoprecipitation experiments, we found that Src can associate with G{alpha}i2 and G{alpha}i3 in BPAECs, suggesting that these G protein {alpha} subunits may interact with Src and induce a conformation change that could directly activate Src, or stimulate its association with another protein that, in turn, regulates its activity (e.g., PTP). Although there is increasing evidence that activation of Src is linked to Ang II, the biochemical mechanisms used by this G protein–coupled receptor to activate Src are unclear.

Ang II can activate three major subfamilies of MAPK: ERK, c-Jun amino-terminal kinase/stress-activated protein kinase, and p38 MAPK by various intracellular mechanisms (reviewed in Ref. 19). Activated ERK1/ERK2 can translocate to the nucleus, where they can then phosphorylate and activate transcription factors, such as fos and jun. These transcription factors could, in turn, bind to one of the AP-1 sites located in the promoter region of eNOS gene (2), thus leading to an increase in its expression. The underlying mechanisms linking Ang II to mitogenic signaling are only partially understood and may include GTP-binding proteins, ß-arrestin, receptor tyrosine kinases, Src, Pyks, phosphatidylinositol-3-kinase, phospholipase C, and calcium (9, 15, 19). In BPAECs, Ang II stimulated an increase in MEK-1 and ERK1/ERK2 phosphorylations that are blocked when the cells are pretreated with PP2, suggesting that Src is upstream of MAPK activation. In addition, the AT2 receptor antagonist, PD123319, blocked the Ang II–dependent ERK1/ERK2 phosphorylation. Linking the AT2 receptor to activation of MAPK has been observed by other investigators. Gendron and colleagues (40) demonstrated, in NG108–15 cells, that the AT2 receptor–dependent increase in ERK1/ERK2 mediates the Ang II–dependent increase in neurite outgrowth. Similarly, Stroth and colleagues (13) found that ERK activation by Ang II in quiescent PC12W cells could be inhibited by the AT2 receptor antagonist PD123177. Moreover, in BPAECs, Ang II activation of MAPK is sensitive to pertussis toxin and is blocked by PD98059. Thus, the data from this study clearly indicate that a pertussis toxin–sensitive G protein–dependent activation of Src, leading to an increase in MEK/MAPK, is involved in the Ang II stimulation of eNOS protein expression in BPAECs. Ang II–induced pathways appear to differ among cell types, as Zou and colleagues (39) found that Ang II–induced ERK activation was abolished by pretreatment with pertussis toxin in cardiac fibroblasts but not in cardiac myocytes. Additionally, a pertussis toxin–sensitive MAPK-dependent pathway has been shown to mediate lipopolysaccharide-dependent increase in eNOS expression (41).

Ang II stimulates a significant increase in NO production in the pulmonary endothelium, consistent with that seen with many stimuli (2) where there is a two- to three-fold increase over basal levels. We propose that this increase in NO in response to Ang II could be important in modulating pulmonary adaptation to systemic hypertension. Other investigators have also reported that an Ang II–dependent increase in NO modulates the constrictive effects of Ang II (5, 42). For example, Navar and colleagues (42) reported that in rats with Ang II–infused hypertension there are renoprotective effects mediated by increased levels of NO. In addition, Hennington and colleagues (5) proposed that Ang II–stimulated NO production could protect renal vessels from Ang II–stimulated vasoconstriction. In summary, the present study demonstrates that Ang II stimulates an increase in eNOS protein expression through a pathway that includes a pertussis toxin–sensitive GTP-binding protein and activation of Src tyrosine kinase, MEK-1, and MAPK.


   Acknowledgments
 
This research was supported by the American Heart Association and the National Heart, Lung, and Blood Institute Grant HL-63182.


   Footnotes
 
Conflict of Interest Statement: X.L. has no declared conflicts of interest; K.M.L. has no declared conflicts of interest; J.L. has no declared conflicts of interest; and S.C.O. has no declared conflicts of interest.

Received in original form March 19, 2004

Received in final form June 9, 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Alderton, W., C. Cooper, and R. Knowles. 2000. Nitric oxide synthases: structure, function and inhibition. Biochem. J. 357:593–615.
  2. Fleming, I., and R. Busse. 2003. Molecular mechanism involved in regulation of endothelial nitric oxide synthase. Am. J. Physiol. Regul. Integr. Comp. Physiol. 284:R1–R12.[Abstract/Free Full Text]
  3. Davis, M. E., H. Cai, G. R. Drummond, and D. G. Harrison. 2001. Shear stress regulates endothelial nitric oxide synthase expression through Src by divergent signaling pathways. Circ. Res. 89:1073–1080.[Abstract/Free Full Text]
  4. Olson, S. C., T. A. Dowds, P. A. Pino, M. T. Barry, and T. Burke-Wolin. 1997. Angiotensin II stimulates endothelial nitric oxide synthase expression in bovine pulmonary artery endothelium. Am. J. Physiol. Lung Cell. Mol. Physiol. 273:L315–L321.[Abstract/Free Full Text]
  5. Hennington, B. S., H. Zhang, M. T. Miller, J. P. Granger, and J. F. Reckelhoff. 1998. Angiotensin II stimulates synthesis of endothelial nitric oxide synthase. Hypertension 31:283–288.[Abstract/Free Full Text]
  6. Ritter, O., K. Schuh, M. Brede, N. Rö, N. Burkard, L. Hein, and L. Neyes. 2003. AT2-activation regulates myocardial eNOS expression via calcineurin-NF-AT pathway. FASEB J. 17:283–285.[Abstract/Free Full Text]
  7. Saito, S., Y. Hirata, T. Emori, T. Imai, and F. Marumo. 1996. Angiotensin II activates endothelial constitutive nitric oxide synthase via AT1 receptors. Hypertens. Res. 19:201–206.[Medline]
  8. Cai, H., Z. Li, S. Dikalov, S. M. Holland, J. Hwang, H. Jo, S. D. Dudley, and D. G. Harrison. 2002. NAD(P)H oxidase–derived hydrogen peroxide mediates endothelial nitric oxide production in response to angiotensin II. J. Biol. Chem. 277:48311–48317.[Abstract/Free Full Text]
  9. De Gasparo, M., R. J. Catt, T. Inagami, J. Wright, and T. Unger. 2000. International Union of Pharmacology XXIII: the angiotensin II receptors. Pharmacol. Rev. 52:415–472.[Abstract/Free Full Text]
  10. Bottari, S., I. King, S. Reichlin, I. Dahlstroem, N. Lydon, and M. de Gasparo. 1992. The angiotensin AT2 receptor stimulates protein tyrosine phosphatase activity and mediates inhibition of particulate guanylate cyclase. Biochem. Biophys. Res. Commun. 183:206–211.[CrossRef][Medline]
  11. Kambayashi, Y., S. Bardhan, K. Tahahashi, S. Tsuzuki, H. Inui, T. Hamakubo, and T. Inagami. 1993. Molecular cloning of a novel angiotensin II receptor isoform involved in phosphotyrosine phosphatase inhibition. J. Biol. Chem. 268:24543–24546.[Abstract/Free Full Text]
  12. Huang, X., E. Richards, and C. Sumners. 1996. Mitogen-activated protein kinases in rat brain neuronal cultures are activated by angiotensin II type 1 receptor and are inhibited by angiotensin II type 2 receptors. J. Biol. Chem. 271:15635–15641.[Abstract/Free Full Text]
  13. Stroth, U., A. Blume, K. Mielke, and T. Unger. 2000. Angiotensin AT2 receptor stimulates ERK1 and ERK2 in quiescent but inhibits ERK in NGF-stimulated PC12 Cells. Brain Res. Mol. Brain Res. 78:175–180.[Medline]
  14. Li, X., J. W. Lee, L. M. Graves, and H. S. Earp. 1998. Angiotensin II stimulates ERK via two pathways in epithelial cells: protein kinase C suppresses a G protein coupled receptor EGF receptor transactivation pathway. EMBO J. 17:2574–2583.[CrossRef][Medline]
  15. Eguchi, S., H. Iwasaki, T. Inagami, K. Numaguchi, T. Yamakawa, E. Motley, K. Owada, F. Marumo, and Y. Hirata. 1999. Involvement of PYK2 in angiotensin II signaling of vascular smooth muscle cells. Hypertension 33:201–206.[Abstract/Free Full Text]
  16. Bokemeyer, D., U. Schmitz, and H. J. Kramer. 2000. Angiotensin II–induced growth of vascular smooth muscle cells requires Src-dependent activation of epidermal growth factor receptor. Kidney Int. 58:549–558.[Medline]
  17. Heeneman, S., J. Haendeler, Y. Saito, M. Ishida, and B. Berk. 2000. Angiotensin II induces transactivation of two different populations of the platelet-derived growth factor ß receptor: key roles for the p66 adaptor protein Shc. J. Biol. Chem. 275:15926–15932.[Abstract/Free Full Text]
  18. Weng, Y. I., and S. D. Shukla. 2002. Angiotensin II activation of focal adhesion kinase and pp60c-Src in relation to mitogen-activated protein kinases in hepatocytes. Biochim. Biophys. Acta 1589:285–297.[Medline]
  19. Touyz, R. M., and C. Berry. 2002. Recent advances in angiotensin II signaling. Braz. J. Med. Biol. Res. 35:1001–1015.[Medline]
  20. Blume, A., T. Herdegen, and T. Unger. 1999. Angiotensin peptides and inducible transcription factors. J. Mol. Med. 77:339–357.[CrossRef][Medline]
  21. Sadoshima, J., and S. Izumo. 1996. The heterotrimeric Gq protein–coupled angiotensin II receptor activates p21ras via the tyrosine kinase–Shc-Grb2-SOS pathway in cardiac myocytes. EMBO J. 15:775–787.[Medline]
  22. Ma, Y.-C., J. Huang, S. Ali, W. Lowry, and X. Y. Huang. 2000. Src tyrosine kinase is a novel direct effect of G proteins. Cell 102:635–646.[CrossRef][Medline]
  23. Ma, Y.-C., and X. Y. Huang. 2002. Novel regulation and function of Src tyrosine kinase. Cell. Mol. Life Sci. 59:456–462.[CrossRef][Medline]
  24. Seshiah, P. N., D. S. Weber, P. Rocic, L. Valppu, Y. Taniyama, and K. K. Griendling. 2002. Angiotensin II stimulation of NAD(P)H oxidase activity: upstream mediators. Circ. Res. 91:406–413.[Abstract/Free Full Text]
  25. Touyz, R. M., X. H. Wu, G. He, S. Salomon, and E. Schiffrin. 2002. 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.[Abstract/Free Full Text]
  26. Olson, S., R. Oeckler, X. Li, L. Du, F. Traganos, X. Zhao, and T. Burke-Wolin. 2004. Angiotensin II stimulates nitric oxide production in pulmonary endothelium via the type 2 receptor. Am. J. Physiol. Lung Cell Mol. Physiol. (in press)
  27. Luttrell, L. M., B. E. Hawes, T. van Biesen, D. K. Luttrell, T. J. Lansing, and R. J. Lefkowitz. 1996. Role of c-Src tyrosine kinase in G protein–coupled receptor and Gß{gamma} subunit–mediated activation of mitogen-activated protein kinase. J. Biol. Chem. 271:19443–19450.[Abstract/Free Full Text]
  28. Haynes, M. P., L. Li, D. Sinha, K. S. Russel, K. Hisamoto, R. Baron, M. Collinge, W. C. Sessa, and J. R. Bender. 2003. Src kinase mediates phosphatidylinositol 3-kinase/Akt–dependent rapid endothelial nitric oxide synthase activation by estrogen. J. Biol. Chem. 278:2118–2123.[Abstract/Free Full Text]
  29. Bjorge, J. D., A. Pang, and D. J. Fujita. 2000. Identification of protein–tyrosine phosphatase 1B as the major tyrosine phosphatase activity capable of dephosphorylating and activating c-Src in several human breast cancer cell lines. J. Biol. Chem. 275:41439–41446.[Abstract/Free Full Text]
  30. Thomas, S. R., K. Chen, and J. F. Keany. 2002. Hydrogen peroxide activates endothelial nitric-oxide synthase through coordinated phosphorylation and dephosphorylation via a phosphoinositide-3-kinase–dependent signaling pathway. J. Biol. Chem. 277:6017–6024.[Abstract/Free Full Text]
  31. Seta, K., and J. Sadoshima. 2003. Phosphorylation of tyrosine 319 of the angiotensin type 1 receptor mediates angiotensin II–induced trans-activation of the epidermal growth factor receptor. J. Biol. Chem. 278:9019–9026.[Abstract/Free Full Text]
  32. Andresen, B. T., J. J. Linnoila, E. K. Jackson, and G. G. Romero. 2003. Role of EGFR transactivation in angiotensin II signaling to extracellular regulated kinase in preglomerular smooth muscle cells. Hypertension 41:781–786.[Abstract/Free Full Text]
  33. Bottari, S. P., V. Taylor, I. N. King, Y. Bogdal, S. E. Whitebread, and M. de Gasparo. 1991. Angiotensin II AT2 receptors do not interact with guanine nucleotide binding proteins. Eur. J. Pharmacol. 207:157–163.[CrossRef][Medline]
  34. Kang, J., E. M. Richards, P. Posner, and C. Sumners. 1995. Modulation of the delayed K+ current in neurons by angiotensin II type 2 receptor fragment. Am. J. Physiol. Cell Physiol. 268:C278–C282.[Abstract/Free Full Text]
  35. Zhang, J., and R. E. Pratt. 1996. The AT2 receptor selectively associates with Gi{alpha}2 and Gi{alpha}3 in the rat fetus. J. Biol. Chem. 271:15026–15033.[Abstract/Free Full Text]
  36. Hansen, J. L., G. Servant, T. J. Baranski, T. Fujita, T. Iiri, and S. P. Sheikh. 2000. Functional reconstitution of the angiotensin II type 2 receptor and G(i) activation. Circ. Res. 87:753–759.[Abstract/Free Full Text]
  37. McDuffie, J. E., S. D. Coaxum, and M. A. Maleque. 1999. 5-Hydroxytryptamine evokes endothelial nitric oxide synthase activation in bovine aortic endothelial cell cultures. Proc. Soc. Exp. Biol. Med. 221:386–390.[CrossRef][Medline]
  38. Malek, A. M., L. Jiang, I. Lee, W. C. Sessa, S. Izumo, and S. L. Alper. 1999. Induction of nitric oxide synthase mRNA by shear stress requires intracellular calcium, G-protein signals and is modulated by PI3 kinase. Biochem. Biophys. Res. Commun. 254:231–242.[CrossRef][Medline]
  39. Zou, Y. Z., I. Komuro, T. Yamazaki, S. Kudoh, R. Aikawa, W. D. Zhu, I. Shiojima, Y. Hiroi, K. Tobe, T. Kadowaki, and Y. Yazaki. 1998. Cell type–specific angiotensin II–evoked signal transduction pathways: critical role for Gß{gamma} subunit, Src family, and Ras in cardiac fibroblasts. Circ. Res. 82:337–345.[Abstract/Free Full Text]
  40. Gendron, L., N. Laflamme, C. Rivard, C. Asselin, M. D. Payet, and N. Gallo-Payet. 1999. Signals from the AT2 (angiotensin type 2) receptor of angiotensin II inhibit p21ras and activate MAPK (mitogen-activated protein kinase) to induce morphological neuronal differentiation in NG108–15 cells. Mol. Endocrinol. 13:1615–1626.[Abstract/Free Full Text]
  41. Cieslik, K., C. S. Abrams, and K. K. Wu. 2001. Upregulation of endothelial nitric oxide synthase promoter by the phosphatidylinositol 3-kinase {gamma}/Janus kinase 2/MEK-1–dependent pathway. J. Biol. Chem. 276:1211–1219.[Abstract/Free Full Text]
  42. Navar, I. G., A. Jehihara, S. Y. Chin, and J. D. Imig. 2000. NO-Ang II interactions in Ang II–dependent hypertension. Acta Physiol. Scand. 168:139–147.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
R. Chang, L. G. Chicoine, H. Cui, N. L. Kanagy, B. R. Walker, Y. Liu, B. K. English, and L. D. Nelin
Cytokine-induced arginase activity in pulmonary endothelial cells is dependent on Src family tyrosine kinase activity
Am J Physiol Lung Cell Mol Physiol, October 1, 2008; 295(4): L688 - L697.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
J. Li, X. Zhao, X. Li, K. M. Lerea, and S. C. Olson
Angiotensin II type 2 receptor-dependent increases in nitric oxide synthase expression in the pulmonary endothelium is mediated via a G{alpha}i3/Ras/Raf/MAPK pathway
Am J Physiol Cell Physiol, June 1, 2007; 292(6): C2185 - C2196.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
W. K. P. Wong, J. A. Knowles, and J. H. Morse
Bone Morphogenetic Protein Receptor Type II C-Terminus Interacts with c-Src: Implication for a Role in Pulmonary Arterial Hypertension
Am. J. Respir. Cell Mol. Biol., November 1, 2005; 33(5): 438 - 446.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2004-0098OCv1
31/3/365    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, X.
Right arrow Articles by Olson, S. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, X.
Right arrow Articles by Olson, S. C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Crit. Care Med.
Copyright © 2004 American Thoracic Society.