c-Src Induces Phosphorylation and Translocation of p47phox
Role in Superoxide Generation by Angiotensin II in Human Vascular Smooth Muscle Cells
Objectives— The aim of this study was to determine molecular mechanisms whereby c-Src regulates angiotensin II (Ang II)-mediated NAD(P)H oxidase-derived ·O2− in human vascular smooth muscle cells (VSMCs).
Methods and Results— VSMCs from human small arteries were studied. Ang II increased NAD(P)H oxidase-mediated generation of ·O2− and H2O2 (P<0.01). PP2, c-Src inhibitor, attenuated these effects by 70% to 80%. Immunoprecipitation of p47phox, followed by immunoblotting with antiphosphoserine antibody, demonstrated a rapid increase (1.5- to 2-fold) in p47phox phosphorylation in Ang II-stimulated cells. This was associated with p47phox translocation from cytosol to membrane, as assessed by immunoblotting and immunofluorescence. PP2 abrogated these effects. Long-term Ang II stimulation (6 to 24 hours) increased NAD(P)H oxidase subunit expression. c-Src inhibition decreased abundance of gp91phox, p22phox, and p47phox. Confirmation of c-Src-dependent regulation of NAD(P)H oxidase was tested in VSMCs from c-Src−/− mice. Ang II-induced ·O2− generation was lower in c-Src−/− than c-Src+/+ counterparts. This was associated with decreased p47phox phosphorylation, blunted Ang II-stimulated NAD(P)H oxidase activation, and failure of Ang II to increase subunit expression.
Conclusions— c-Src regulates NAD(P)H oxidase-derived ·O2− generation acutely by stimulating p47phox phosphorylation and translocation and chronically by increasing protein content of gp91phox, p22phox, and p47phox in Ang II-stimulated cells. These novel findings identify NAD(P)H oxidase subunits, particularly p47phox, as downstream targets of c-Src.
Angiotensin II (Ang II) elicits diverse cellular responses in the vasculature, such as contraction, growth, migration, and inflammation.1 Many of these processes are mediated via generation of reactive oxygen species (ROS), such as superoxide anion (·O2−) and hydrogen peroxide (H2O2), which act as signaling molecules.2–4⇓⇓ Emerging evidence indicates that vascular smooth muscle cell (VSMC) NAD(P)H oxidase is a major source of vascular ROS.5–7⇓⇓ NAD(P)H oxidase is a multi-subunit enzymatic complex responsible for the monoelectronic reduction of oxygen to produce ·O2− at the expense of NAD(P)H.8 Similar to the neutrophil oxidase, vascular NAD(P)H oxidase comprises membrane-bound flavocytochrome b558 (formed by gp91phox [nox 2] or gp91phox homologues [nox 1 and nox 4] and p22phox) and 3 cytoplasmic subunits, p47phox, p67phox, and p40phox.8,9⇓
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Activation of phagocytic NAD(P)H oxidase is a multistep process, initiated by serine phosphorylation of p47phox, which triggers complex formation of cytoplasmic subunits followed by translocation to the membrane, where it associates with cytochrome b558 to assemble the active oxidase.9,10⇓ Of the many factors that stimulate this process, Ang II seems to be one of the most important in the vasculature.11–17⇓⇓⇓⇓⇓⇓ Mechanisms linking Ang II to the enzyme and upstream signaling molecules modulating NAD(P)H oxidase in VSMCs have not been fully elucidated. We showed that phospholipase D (PLD) and protein kinase C (PKC) play a role.16,18⇓ Other studies reported that epidermal growth factor (EGF) receptor transactivation, phosphatidylinositol-3-kinase (PI3K), and Rac may be important.19 Because many of these signaling molecules are downstream of c-Src, we questioned the role of this tyrosine kinase in the activation of NAD(P)H oxidase and investigated mechanisms whereby c-Src regulates the oxidase in Ang II-stimulated VSMCs.
Src kinases are a family of nonreceptor tyrosine kinases that are widely expressed. To date, at least 14 members have been identified, of which 60-kDa c-Src is the prototype.20 Of the many Src kinases, c-Src is highly expressed in VSMCs.21 It is rapidly activated by Ang II and plays a key role in signaling events associated with VSMC contraction, growth, and migration.22–24⇓⇓ We propose here that c-Src is upstream from NAD(P)H oxidase. This is supported by studies demonstrating that PP1, a Src-specific inhibitor, decreased Ang II-stimulated H2O2 production in rat aortic VSMCs19 and that in cells treated with the tyrosine kinase inhibitor herbimycin A, ·O2− formation was attenuated.25 To our knowledge, specific mechanisms whereby c-Src regulates NAD(P)H oxidase activity, particularly in human VSMCs, have not been investigated. To address this, we tested the hypothesis that Ang II-mediated activation of c-Src regulates NAD(P)H oxidase-driven generation of ROS by stimulating phosphorylation of p47phox, which initiates translocation of the p47phox-associated cytoplasmic complex and subsequent activation of NAD(P)H oxidase. Using VSMCs derived from human resistance arteries and VSMCs from mice in which the c-Src gene has been disrupted (c-Src−/− mice), we demonstrate that c-Src regulates NAD(P)H oxidase-mediated ·O2− generation acutely by influencing p47phox activity and chronically by increasing abundance of p47phox, gp91phox, and p22phox. These findings provide novel insights into the Ang II signaling network proximal of NAD(P)H oxidase in human VSMCs and demonstrate that p47phox is an important downstream target of c-Src.
The study was approved by the Ethics Committee of the Clinical Research Institute of Montreal (IRCM). Healthy volunteers (30 to 65 years of age) were recruited at the IRCM Hypertension Clinic. VSMCs from small arteries dissected from gluteal subcutaneous biopsies were cultured and characterized as described.18
VSMCs from c-Src−/− mice, homozygous for a disruption in the c-Src gene, and VSMCs from wild-type c-Src+/+ mice were also studied. c-Src−/− mice were generated from a c-Src+/− F1 cross26 and were genotyped by polymerase chain reaction (PCR). VSMCs were obtained from mesenteric vascular beds of 10- to 12-week-old mice (n=6 to 8/group). VSMCs were isolated by enzymatic digestion, cultured, and characterized as described.17,18⇓
Quiescent VSMCs were stimulated with Ang II (10−7 mol/L). In some experiments, cells were preexposed to PP2 (selective Src inhibitor) (10−5 mol/L, 20 minutes). Cells were washed, scraped, and differentially centrifuged to obtain cell homogenate, membrane, and cytoplasmic fractions.17 Total cell homogenate was used to measure cortactin phosphorylation and NAD(P)H oxidase activity. NAD(P)H oxidase subunit expression and p47phox phosphorylation were assessed in membrane and cytosolic fractions.
Measurement of ROS in Intact Cells
Intracellular ·O2− and H2O2 generation were measured with the fluoroprobes tempo-9-AC and CM-H2DCFDA, respectively (4 μmol/L) (Molecular Probes).27 Cells were stimulated with Ang II (10−9-10−6 mol/L) in the absence and presence of 10−5 mol/L PP2 (20-minute preincubation). In some experiments, cells were pretreated (20 minutes) with DPI (10−6 mol/L), a flavoprotein inhibitor that targets cytochrome b558, and apocynin (3×10−5 mol/L), which blocks NAD(P)H oxidase subunit assembly.28 To confirm that the tempo-9-AC fluorescence signal derived from ·O2−, cells were pretreated with tempol (superoxide dismutase mimetic) (10−4 mol/L).
Measurement of NAD(P)H Oxidase Activity
VSMCs were stimulated with Ang II for 5 to 15 minutes. In some experiments, cells were preexposed to PP2 or PP3 (inactive analogue). Mouse VSMCs were also exposed to paraquat (10−4 mol/L), a potent inducer of ·O2− generation.29 The lucigenin-derived chemiluminescence assay was used to determine NADPH oxidase activity in cell homogenates as described.17,30⇓
Immunoprecipitation of p47phox
For p47phox immunoprecipitation, the cytosolic fraction (100 μg protein) from cells stimulated with Ang II in the absence and presence of PP2 was transferred to microcentrifuge tubes, and anti-p47phox antibody (12 μg) was added and incubated for 60 minutes at 4°C. Agarose conjugate 20 μL (Protein G PLUS-Agarose) was added and incubated (60 minutes, 4°C). Samples were centrifuged, and the supernatant was subjected to immunoblotting, as below.
Immunoblotting of Cortactin and NADPH Oxidase Subunits
Immunoblotting was performed as described.17 Phosphorylation of cortactin, a Src-specific downstream substrate, was measured using a phospho-specific cortactin antibody (Biosource). Membrane preparations were used to detect gp91phox and p22phox, whereas cytoplasmic fractions were used for p47phox and p67phox. To determine p47phox translocation after Ang II stimulation (5 to 10 minutes), p47phox abundance was also assessed in membrane fractions. Previously characterized antibodies10,17,31⇓⇓ recognizing p22phox, gp91phox, 47phox, and p67phox were used. p47phox phosphorylation was assessed in p47phox immunoprecipitates using an anti-phosphoserine antibody (Zymed Labs, South San Francisco, Calif) (1:1000). AT1 receptor expression was assessed in c-Src+/+ and c-Src−/− cells using an AT1 receptor-specific antibody (1:500) (Alpha Diagnostics, San Antonio, Tex). Immunoreactive proteins were detected by chemiluminescence, and blots were analyzed densitometrically (Image-Quant software, Molecular Dynamics).
For immunocytochemistry, VSMCs plated onto glass coverslips were stimulated with Ang II (10−7 mol/L, 3 to 10 minutes) in the absence and presence of PP2 (10−5 mol/L). Fixed cells were incubated with primary antibodies (anti-p47phox, 1:1000; anti-α actin, 1:1000). After washing, cells were incubated with secondary antibody (Alexa 488, Alexa 647, Molecular Probes) and then mounted. Confocal microscopy was performed with a Zeiss LSM 510 system.
Reverse Transcription-PCR of AT1 Receptors in Mouse VSMCs
AT1 receptor mRNA expression was measured by reverse transcription-PCR (RT-PCR), as described in the online supplement.
A detailed Methods section is available in the online supplement, available at http://atvb.ahajournals.org.
Experiments were repeated 3 to 7 times. Results are presented as mean±SEM and compared by ANOVA or by Student’s t test, where appropriate. Tukey-Kramer’s correction was used to compensate for multiple testing. P<0.05 was significant.
Cortactin Phosphorylation by Ang II is Blocked by PP2
We previously reported that c-Src is rapidly activated by Ang II and that c-Src kinase activity is inhibited by PP2 in VSMCs.23,24,32⇓⇓ To additionally confirm the functional significance of c-Src activation by Ang II and to demonstrate that PP2 is an effective Src inhibitor in our model, we assessed Ang II effects on phosphorylation of cortactin, a Src-specific substrate, in the absence and presence of PP2. Ang II stimulation increased cortactin phosphorylation almost 2-fold (Figure I, available online). Responses, which were maximal within 5 to 10 minutes, were sustained for up to 30 minutes. In PP2-treated cells, Ang II-mediated cortactin phosphorylation was inhibited. These findings, together with our previous results,22,23,32⇓⇓ indicate that c-Src is an early signaling event in response to Ang II and that PP2, at the concentration used here, inhibits Src actions.
Src Inhibition Reduces Ang II-Mediated Generation of ·O2− in Human VSMCs
To evaluate whether Ang II-induced generation of ROS is Src-dependent, we measured intracellular ·O2− and H2O2 formation in the presence of PP2. Ang II dose-dependently increased tempo-9-AC fluorescence (Figure 1). Tempol abolished the tempo-9-AC signal, confirming that the fluorescence signal derives primarily from ·O2−. Maximal ·O2− responses were evident within 10 to 15 minutes of Ang II addition. Apocynin and DPI inhibited Ang II-induced actions. In the presence of PP2, Ang II-mediated ·O2− generation was reduced by 70% to 80%. Chronic Ang II stimulation (4 to 6 hours) resulted in a sustained ·O2− increase (30% to 40% above baseline), which was attenuated in the presence of PP2. Ang II also increased CMH2-DCFDA fluorescence (Emax=78±5 arbitrary units). In PP2-pretreated cells, Ang II-induced responses were significantly reduced (Emax=42±8 U, P<0.05). We previously demonstrated that CMH2-DCFDA fluorescence derives mainly from H2O2.18
PP2 Inhibits NADPH-Driven Generation of ·O2−
Exposure of Ang II-stimulated cells to NADPH resulted in a significant increase in lucigenin chemiluminescence (Figure 2). Responses were evident within 5 minutes of stimulation and maintained for up to 20 minutes. In cells pretreated with PP2, Ang II-induced activation of NAD(P)H oxidase was significantly reduced but not completely abolished. PP3 did not influence basal or Ang II-stimulated NAD(P)H oxidase activity. SOD and tiron inhibited the lucigenin signal in Ang II-stimulated cells by >90%, indicating that the chemiluminescence response derived primarily from ·O2−.
c-Src Activation by Ang II Induces p47phox Phosphorylation
Because serine phosphorylation of p47phox is the initiating step in NAD(P)H oxidase activation, we investigated whether this process is influenced by c-Src. As shown in Figure 3A, phosphorylation of p47phox was significantly increased in human VSMCs by Ang II. This effect was rapid, occurring within minutes of Ang II stimulation. In PP2-pretreated VSMCs, Ang II-induced phosphorylation was reduced. In c-Src−/− cells, p47phox phosphorylation was not significantly altered by Ang II compared with controls. However, in c-Src+/+ VSMCs, Ang II significantly increased p47phox phosphorylation (Figure 3B).
p47phox Translocation Is c-Src-Dependent
The role of c-Src in p47phox translocation was assessed using 2 methods; first by determining p47phox abundance in cytoplasmic and membrane fractions from cells stimulated with Ang II (5 minutes) in the presence of PP2, and second by tracking p47phox migration by immunofluorescence in PP2-pretreated Ang II-stimulated cells. As shown in Figure 4, p47phox content was greater in the cytoplasmic than in the membrane fraction in basal conditions. On Ang II stimulation, p47phox abundance was reduced in the cytoplasmic fraction and increased in the membrane fraction. In PP2-pretreated cells, there was no change in p47phox content in either fraction after Ang II stimulation compared with controls.
Immunofluorescence images demonstrated that in basal conditions, p47phox is essentially cytosolic, localizing mainly in the perinuclear area (Figure 5). On Ang II addition, p47phox migrates toward the periphery. After 10 minutes of stimulation, p47phox translocates almost entirely to the membrane region. In PP2-treated cells, Ang II failed to induce p47phox translocation.
PP2 Reduces Abundance of NAD(P)H Oxidase Subunits in Ang II-Stimulated Cells
In addition to influencing NAD(P)H oxidase activity acutely, Ang II regulates the enzyme chronically by stimulating de novo synthesis of NAD(P)H oxidase subunits.17 Because c-Src is critical in protein synthesis,24,33⇓ we investigated whether this tyrosine kinase also influences content of gp91phox, p22phox, p47phox, and p67phox. Exposure of cells to Ang II (6 to 24 hours) resulted in a significant increase in expression of all subunits (Figure 6). In PP2-treated cells, abundance of gp91phox, p22phox, and p47phox, but not p67phox, was significantly reduced (P<0.05).
Generation of Reactive Oxygen Species by Ang II is Decreased in c-Src−/− VSMCs
To additionally examine c-Src involvement in NAD(P)H oxidase-derived ROS by Ang II, we studied VSMCs from c-Src−/− and c-Src+/+ mice. To confirm the presence of Ang II receptors in cells from c-Src−/− and c-Src+/+ mice, AT1 mRNA expression and protein content were determined. RT-PCR analysis and immunoblotting demonstrated that AT1 mRNA and protein were equally expressed in cells from c-Src−/− and c-Src+/+ (Figure II, available online).
Ang II dose-dependently increased tempo-9-AC fluorescence in VSMCs from c-Src+/+ and c-Src−/− mice (Figure IIIA, available online), with maximal effects occurring at 10−7 mol/L. Ang II-induced responses were significantly greater in c-Src+/+ cells than c-Src−/− cells (4- to 5-fold increase in fluorescence versus 1- to 2-fold increase). To evaluate whether NAD(P)H oxidase activity is altered in cells from c-Src−/−, effects of paraquat, which activates NA(D)PH oxidase directly, were examined. Paraquat elicited a potent increase in lucigenin chemiluminescence in both c-Src+/+ (15 000×103 cpm/mg per min) and c-Src−/− VSMCs (15 000×103 cpm/mg per min). Ang II significantly increased NAD(P)H oxidase activity in c-Src+/+ cells (10 000×103 cpm/mg per min) but induced only a modest effect in c-Src−/− cells (45 000×103 cpm/mg per min) (Figure IIIB, available online). Ang II (2- to 24-hour stimulation) did not significantly modify content of p22phox, p47phox, and p67phox in c-Src−/− VSMCs (Figure IV, available online)
Findings from our study demonstrate for the first time that p47phox is a downstream target of c-Src in human VSMCs. In particular, we show that Ang II-mediated activation of c-Src induces p47phox phosphorylation and translocation, critical steps in the initiation of NAD(P)H oxidase activation. These conclusions are based on several observations. First, Src inhibition by PP2 attenuated Ang II-stimulated ·O2− production by NAD(P)H oxidase. Second, PP2 inhibited Ang II-induced p47phox phosphorylation. Third, translocation of activated p47phox was blocked in PP2-treated cells. Finally, VSMCs derived from c-Src−/− mice exhibited blunted Ang II-mediated ROS formation, decreased NAD(P)H oxidase-driven ·O2− generation, and failure of Ang II to stimulate p47phox phosphorylation. In addition to these rapid processes, we found that c-Src increases protein abundance of gp91phox, p22phox, and p47phox, which may be important in long-term regulation of NAD(P)H oxidase. Thus, our data clearly implicate c-Src as a proximal regulator of NAD(P)H oxidase, particularly p47phox, in Ang II-stimulated VSMCs. These findings identify a novel signaling cascade for Ang II in VSMCs.
Ang II rapidly increased phosphorylation of cortactin, a Src-specific substrate, confirming the functional activity of Src in our model. PP2, at 10−5 mol/L, inhibited these effects, indicating that at this concentration, Src-mediated actions are effectively blocked. Src inhibition attenuated ROS generation by Ang II, as measured by tempo-9-AC and CMH2-DCFDA. Abrogation of the Ang II-induced tempo 9-AC response by tempol confirmed the ·O2− specificity of the fluoroprobe.27 PP2 did not completely inhibit tempo-9-AC fluorescence, suggesting that c-Src-independent pathways also contribute to Ang II-mediated ·O2− production in VSMCs.
Activation of NAD(P)H oxidase is a major source of Ang II-elicited ROS production in VSMCs.16,17⇓ This is additionally supported by our findings that Ang II stimulates NAD(P)H-driven ·O2− formation and that DPI and apocynin, two structurally unrelated NAD(P)H oxidase inhibitors, abolished Ang II-induced increase in tempo-9-AC fluorescence. Although signaling pathways whereby Ang II regulates NAD(P)H oxidase have not been fully clarified, PLD, PKC, phospholipase A2, and PI3K are implicated.16,18,19,34⇓⇓⇓ Whether these pathways function in parallel or in series to activate the oxidase is unclear. However, it is possible that c-Src, which influences all of these signaling molecules, could be a common upstream mediator. Previous studies demonstrated that c-Src is a redox-sensitive downstream target of NAD(P)H oxidase.19,35⇓ However, our data together with those reported by others in osteoclasts, leukocytes, eosinophils, and macrophages clearly demonstrate that c-Src is upstream of NAD(P)H oxidase.25,26,36⇓⇓ This may be explained by the feed-forward mechanism whereby low levels of H2O2 activate c-Src, which in turn initiates a signaling cascade leading to NAD(P)H oxidase activation, generation of ROS, and additional c-Src activation.19
Mechanisms underlying c-Src regulation of NAD(P)H oxidase remain obscure. We demonstrate that c-Src inhibition prevents p47phox phosphorylation and translocation after Ang II stimulation. This effect was evident within minutes of Ang II addition, indicating that c-Src actions are rapid. Because activated p47phox functions as a signal-receiving adaptor protein that initiates assembly of the active oxidase, inhibition of its phosphorylation and disruption of its translocation will block enzyme activation. This was evidenced by blunted NAD(P)H oxidase responses to Ang II in PP2-pretreated VSMCs and additionally confirmed in VSMCs from c-Src−/− mice. Exact molecular processes whereby c-Src induces phosphorylation of p47phox are unclear, but intermediate signaling molecules, probably serine kinases, are involved. This is based on the fact that c-Src is a tyrosine kinase, whereas p47phox is serine phosphorylated in response to Ang II, as demonstrated. Potential intermediary kinases include PI3K/AKT, PKC, p21-activated kinases, and Raf-1,37–39⇓⇓ all of which are serine/threonine kinases, and all of which are tyrosine phosphorylated by c-Src in Ang II-stimulated cells.20,37–39⇓⇓⇓ PKC is a highly likely candidate, as we previously suggested,18 because p47phox is directly phosphorylated in response to PKC.38,40⇓ Fontayne et al38 demonstrated in human neutrophils that serines 303, 304, 315, 320, 328, 359, 370, and 379 of p47phox are targets of PKCα, PKCβ, and PKCδ. Serine 328 seems to be the most phosphorylated serine. PKC-induced phosphorylation of p47phox induces a conformational alteration, resulting in the appearance of a binding site through which p47phox interacts with cytochrome b558 during the NAD(P)H oxidase activation process.40,41⇓ Most of these studies were performed in neutrophils. Little is known about the role of PKC and p47phox in nonphagocytic cells. Another Src-sensitive signaling molecule that could activate p47phox is PLD-derived phosphatidic acid. This is supported by our findings demonstrating that phosphatidic acid potently activates NAD(P)H oxidase16 and that p47phox is serine phosphorylated in response to phosphatidic acid.42 Identification of specific molecules or adaptor proteins linking c-Src and p47phox in VSMCs awaits additional clarification.
Ang II regulates NAD(P)H oxidase by modifying de novo protein synthesis of NAD(P)H oxidase subunits.17,36,43⇓⇓ Because c-Src is important in growth signaling, we questioned whether c-Src could also influence NAD(P)H oxidase by modulating availability of membrane and cytosolic subunits. Long-term Ang II stimulation significantly increased content of NAD(P)H oxidase subunits. This was associated with sustained generation of NAD(P)H oxidase-driven generation of ·O2−. In the presence of PP2, Ang II-mediated increase in gp91phox, p22phox, and p47phox was reduced and ·O2− was only modestly increased. These findings indicate that c-Src is also important in long-term regulation of NAD(P)H oxidase by Ang II. p67phox abundance was unaffected by PP2. This could relate to the fact that synthesis of this subunit may be c-Src-independent and that other growth signaling cascades regulate p67phox. Thus, our data suggest that c-Src modulates NAD(P)H oxidase on least at two levels, acutely by influencing p47phox activation and chronically by modulating NAD(P)H oxidase subunit abundance. EGFR transactivation by c-Src has also been shown to control NAD(P)H oxidase.19 From the present study, we cannot exclude this possibility, especially because we previously showed that Ang II signaling in VSMCs is mediated partially via receptor tyrosine kinase transactivation.32
Our findings implicating c-Src as a critical upstream activator of p47phox and NAD(P)H oxidase were confirmed in c-Src−/− VSMCs. Ang II failed to induce significant serine phosphorylation of p47phox, and NAD(P)H oxidase-mediated ·O2− production was blunted in c-Src-deficient cells. Attenuated Ang II-mediated responses in c-Src−/− VSMCs could be a consequence of a generalized reduction of responsiveness independent of c-Src regulation of NAD(P)H oxidase. Paraquat, which generates intracellular ·O2− independently of G-protein-coupled receptor activation,29 induced similar ·O2− production in c-Src+/+ and c-Src−/− cells. These data indicate that when c-Src is bypassed, NAD(P)H oxidase in c-Src−/− VSMCs responds normally. Thus, the reduced response of c-Src−/− cells to Ang II critically implicates c-Src in the activation of NAD(P)H oxidase by Ang II. Normal expression of AT1 receptors, both at mRNA and protein levels in c-Src−/− cells, rules out the possibility that a receptor defect could be responsible.
It is now evident that ·O2− and H2O2 constitute important signaling molecules for Ang II in VSMCs and that a major source of these ROS is NAD(P)H oxidase. What has not been clear is how Ang II regulates NAD(P)H oxidase to generate intracellular ·O2−. Findings from the present study unambiguously demonstrate that Ang II influences VSMC oxidase via c-Src-sensitive pathways. We have also identified at least 2 new mechanisms by which c-Src could modulate NAD(P)H oxidase, first by stimulating p47phox activation and second by increasing abundance of NAD(P)H oxidase subunits. These findings provide novel insights into the Ang II signaling network upstream of NAD(P)H oxidase in human VSMCs and demonstrate that in addition to the classical growth signaling molecules, such as Ras-Raf-ERK1/2, NAD(P)H oxidase, and particularly p47phox, is another downstream target of c-Src.
This study was supported by grants 44018 and 13570 and a group grant to the Multidisciplinary Research Group on Hypertension, all from the Canadian Institute of Health Research (CIHR). Antibodies to NAD(P)H oxidase subunits were kindly provided by Dr M.T. Quinn, Montana State University, Bozeman, Mt.
- Received February 5, 2003.
- Accepted March 13, 2003.
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