Mechanism of Endothelial Cell NADPH Oxidase Activation by Angiotensin II

Endothelial cells express a constitutively active phagocyte-type NADPH oxidase whose activity is augmented by agonists such as angiotensin II. We recently reported (Li, J.-M., and Shah, A. M. (2002) J. Biol. Chem. 277, 19952–19960) that in contrast to neutrophils a substantial proportion of the NADPH oxidase in unstimulated endothelial cells exists as preassembled intracellular complexes. Here, we investigate the mechanism of angiotensin II-induced endothelial NADPH oxidase activation. Angiotensin II (100 nmol/liter)-induced reactive oxygen species production (as measured by dichlorohydrofluorescein fluorescence or lucigenin chemiluminescence) was completely absent in coronary microvascular endothelial cells isolated from p47phoxknockout mice. Transfection of p47phoxcDNA into p47phox−/− cells restored the angiotensin II response, whereas transfection of antisense p47phox cDNA into wild-type cells depleted p47phox and inhibited the angiotensin II response. In unstimulated human microvascular endothelial cells, there was significant p47phox-p22phox complex formation but minimal detectable p47phoxphosphorylation. Angiotensin II induced rapid serine phosphorylation of p47phox (within 1 min, peaking at ∼15 min), a 1.9 ± 0.1-fold increase in p47phox-p22phox complex formation and a 1.6 ± 0.2-fold increase in NADPH-dependent O2⨪ production (p< 0.05). p47phox was redistributed to 舠nuclear舡 and membrane-enriched cell fractions. These data indicate that angiotensin II-stimulated endothelial NADPH oxidase activity is regulated through serine phosphorylation of p47phox and its enhanced binding to p22phox. Endothelial cells express a constitutively active phagocyte-type NADPH oxidase whose activity is augmented by agonists such as angiotensin II. We recently reported (Li, J.-M., and Shah, A. M. (2002) J. Biol. Chem. 277, 19952–19960) that in contrast to neutrophils a substantial proportion of the NADPH oxidase in unstimulated endothelial cells exists as preassembled intracellular complexes. Here, we investigate the mechanism of angiotensin II-induced endothelial NADPH oxidase activation. Angiotensin II (100 nmol/liter)-induced reactive oxygen species production (as measured by dichlorohydrofluorescein fluorescence or lucigenin chemiluminescence) was completely absent in coronary microvascular endothelial cells isolated from p47phoxknockout mice. Transfection of p47phoxcDNA into p47phox−/− cells restored the angiotensin II response, whereas transfection of antisense p47phox cDNA into wild-type cells depleted p47phox and inhibited the angiotensin II response. In unstimulated human microvascular endothelial cells, there was significant p47phox-p22phox complex formation but minimal detectable p47phoxphosphorylation. Angiotensin II induced rapid serine phosphorylation of p47phox (within 1 min, peaking at ∼15 min), a 1.9 ± 0.1-fold increase in p47phox-p22phox complex formation and a 1.6 ± 0.2-fold increase in NADPH-dependent O2⨪ production (p< 0.05). p47phox was redistributed to 舠nuclear舡 and membrane-enriched cell fractions. These data indicate that angiotensin II-stimulated endothelial NADPH oxidase activity is regulated through serine phosphorylation of p47phox and its enhanced binding to p22phox. angiotensin II reactive oxygen species NADPH oxidase phorbol 12-myristate 13-acetate 5-(and 6)-chloromethyl-2′,7′-dichlorohydrofluorescein diacetate endothelial cell coronary microvascular EC human microvascular EC diphenyleneiodonium NG-nitro-L-arginine methyl ester Angiotensin II (AngII)1has pleiotropic acute and chronic effects on many cell types and plays an important role in the pathophysiology of cardiovascular diseases, including hypertension, atherosclerosis, and heart failure (1Kim S. Iwao H. Pharmacol. Rev. 2000; 52: 11-34Google Scholar). A significant body of evidence supports a role for the intracellular production of reactive oxygen species (ROS) in the signal transduction of AngII-dependent cellular responses via activation of redox-sensitive signaling cascades (2Ushio-Fukai M. Alexander R.W. Akers M. Yin Q. Fujio Y. Walsh K. Griendling K.K. J. Biol. Chem. 1999; 274: 22699-22704Google Scholar, 3Ushio-Fukai M. Griendling K.K. Becker P.L. Hilenski L. Halleran S. Alexander R.W. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 489-495Google Scholar, 4Pueyo M.E. Gonzalez W. Nicoletti A. Savoie F. Arnal J.-F. Michel J.-B. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 645-651Google Scholar). Recent studies (5Griendling K.K. Sorescu D. Ushio-Fukai M. Circ. Res. 2000; 86: 494-501Google Scholar) suggest that a significant source of intracellular ROS in cardiovascular cells is a phagocyte-type NADPH oxidase. AngII activates NADPH oxidases in vascular smooth muscle (5Griendling K.K. Sorescu D. Ushio-Fukai M. Circ. Res. 2000; 86: 494-501Google Scholar, 6Schieffer B. Luchtefeld M. Braun S. Hilfiker A. Hilfiker-Kleiner D. Drexler H. Circ. Res. 2000; 87: 1195-1201Google Scholar), fibroblasts (7Cifuentes M.E. Rey F.E. Carretero O.A. Pagano P.J. Am. J. Physiol. 2000; 279: H2234-H2240Google Scholar), endothelial cells (EC) (8Lang D. Mosfer S.I. Shakesby A. Donaldson F. Lewis M.J. Circ. Res. 2000; 86: 463-469Google Scholar, 9Zhang H. Schmeisser A. Garlichs C.D. Plotze K. Damme U. Mugge A. Daniel W.G. Cardiovasc. Res. 1999; 44: 215-222Google Scholar), and cardiomyocytes (10Bendall J.K. Cave A.C. Heymes C. Gall N. Shah A.M. Circulation. 2002; 105: 293-296Google Scholar). Furthermore, NADPH oxidase activation and increased ROS production are implicated in AngII-stimulated vascular smooth muscle hypertrophy (3Ushio-Fukai M. Griendling K.K. Becker P.L. Hilenski L. Halleran S. Alexander R.W. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 489-495Google Scholar, 5Griendling K.K. Sorescu D. Ushio-Fukai M. Circ. Res. 2000; 86: 494-501Google Scholar) and in AngII-dependent hypertension and the associated endothelial dysfunction (11Rajagopalan S. Kurz S. Munzel T. Tarpey M. Freeman B.A. Griendling K.K. Harrison D.G. J. Clin. Invest. 1996; 97: 1916-1923Google Scholar, 12Wang H.D. Xu S. Johns D.G. Du Y. Quinn M.T. Cayatte A.J. Cohen R.A. Circ. Res. 2001; 88: 947-953Google Scholar, 13Rey F.E. Cifuentes M.E. Kiarash A. Quinn M.T. Pagano P.J. Circ. Res. 2001; 89: 408-414Google Scholar). The mechanisms through which AngII activates NADPH oxidase are therefore of interest. To date, NADPH oxidase has been best characterized in neutrophils where it is integral to nonspecific host defense. The neutrophil oxidase comprises a membrane-bound cytochrome b558composed of a p22phox-gp91phoxheterodimer and several cytosolic subunits (p47phox, p40phox, p67phox, and Rac) (14Babior B.M. Blood. 1999; 93: 1464-1476Google Scholar). The enzyme is normally dormant but upon neutrophil stimulation, the cytosolic subunits translocate to the membrane and associate with cytochromeb558, resulting in rapid activation of the oxidase. Phosphorylation of the regulatory subunit p47phox plays a key role in this process. The kinetics of NADPH oxidase activation parallel the kinetics of p47phox phosphorylation (15Rotrosen D. Leto T.L. J. Biol. Chem. 1990; 265: 19910-19915Google Scholar). Phosphorylation of p47phox is thought to induce conformational changes that allow subsequent binding of phospho-p47phox to cytochromeb558 and the other cytosolic subunits (14Babior B.M. Blood. 1999; 93: 1464-1476Google Scholar, 15Rotrosen D. Leto T.L. J. Biol. Chem. 1990; 265: 19910-19915Google Scholar, 16Huang J. Kleinberg M.E. J. Biol. Chem. 1999; 274: 19731-19737Google Scholar). NADPH oxidase in non-phagocytic cells such as EC and vascular smooth muscle exhibits significant differences from the neutrophil enzyme. In particular, in contrast to the neutrophil enzyme, the oxidase in non-phagocytic cells is constitutively active at a low level even in unstimulated cells, yet it can be further stimulated acutely by agonists such as AngII and cytokines (5Griendling K.K. Sorescu D. Ushio-Fukai M. Circ. Res. 2000; 86: 494-501Google Scholar). Recent studies (17Suh Y.-A. Arnold R.S. Lassegue B. Shi J. Xu X. Sorescu D. Chung A.B. Griendling K.K. Lambeth J.D. Nature. 1999; 40: 79-82Google Scholar, 18Lassegue B. Sorescu D. Szocs K. Yin Q. Akers M. Zhang Y. Grant S.L. Lambeth J.D. Griendling K.K. Circ. Res. 2001; 88: 888-894Google Scholar) have indicated the presence of a number of isoforms of gp91phox, termed Noxs (for NADPH oxidase), and it has been suggested that the substitution of gp91phox (also known as Nox2) by Nox1 or Nox4 may account for the different behavior of non-phagocytic enzymes. For example, it has been shown in rat aortic smooth muscle cells that the predominant Nox isoforms are Nox4 and Nox1 with very low to undetectable levels of gp91phox expressed (18Lassegue B. Sorescu D. Szocs K. Yin Q. Akers M. Zhang Y. Grant S.L. Lambeth J.D. Griendling K.K. Circ. Res. 2001; 88: 888-894Google Scholar). In EC, however, all the classic NADPH oxidase subunits, including gp91phox, are expressed (19Li J.-M. Shah A.M. Cardiovasc. Res. 2001; 52: 477-486Google Scholar, 20Li J.-M. Shah A.M. J. Biol. Chem. 2002; 277: 19952-19960Google Scholar). Recently, we have reported that, in contrast to neutrophils, in unstimulated quiescent cultured EC a substantial proportion of the NADPH oxidase is present as already fully preassembled complexes in a predominantly perinuclear location associated with the intracellular cytoskeleton (20Li J.-M. Shah A.M. J. Biol. Chem. 2002; 277: 19952-19960Google Scholar). Thus, the 舠cytosolic舡 subunits p47phox, p40phox, p67phox, and Rac1 could be co-immunoprecipitated down with p22phox and gp91phox in unstimulated EC. The presence of these preassembled complexes is likely to account for the constitutive NADPH oxidase activity of unstimulated EC. However, if the oxidase is already preassembled in unstimulated EC, what is the precise nature of the mechanism(s) underlying the increase in oxidase activity following addition of agonists such as AngII? Recently, we reported that the stimulation of EC NADPH oxidase activity by TNFα or phorbol ester (PMA) required the presence of p47phox, although the mechanisms involved were not defined in that study (21Li J.-M. Mullen A.M. Yun S. Wientjes F. Brouns G.Y. Thrasher A.J. Shah A.M. Circ. Res. 2002; 90: 143-150Google Scholar). Likewise, in vascular smooth muscle cells, the p47phoxsubunit has been shown to be essential for ROS production in response to PMA, AngII, thrombin, and platelet-derived growth factor (22Lavigne M.C. Malech H.L. Holland S.M. Leto T.L. Circulation. 2001; 104: 79-84Google Scholar, 23Barry-Lane P.A. Patterson C. van der Merwe M. Hu Z. Holland S.M. Yeh E.T.H. Runge M.S. J. Clin. Invest. 2001; 108: 1513-1522Google Scholar), and compelling evidence has been provided for its involvement in atherosclerosis (23Barry-Lane P.A. Patterson C. van der Merwe M. Hu Z. Holland S.M. Yeh E.T.H. Runge M.S. J. Clin. Invest. 2001; 108: 1513-1522Google Scholar). Potential mechanisms that could underlie the increase in NADPH oxidase activity induced by AngII may include (a) an increase in the number of fully assembled oxidase complexes, (b) the translocation of p47phox to partially assembled complexes, and/or (c) the phosphorylation of p47phox(or other subunits) that are already part of the assembled oxidase complex in EC. In the present study, we have therefore investigated the mechanisms of AngII-induced EC NADPH oxidase activation, focusing on the role of p47phox, its phosphorylation, and its possible translocation and association with cytochromeb558. Our results provide an insight into the fundamental mechanisms of AngII-induced EC NADPH oxidase activation and ROS production. 5-(and 6)-chloromethyl-2′,7′-dichlorohydrofluorescein diacetate (DCF) was purchased from Molecular Probes. Goat polyclonal antibodies to p22phox and p47phox and the corresponding blocking peptides were from Santa Cruz Biotechnology. Affinity-purified rabbit polyclonal antibodies to p47phox and p22phox were a kind gift from Dr. F. Wientjes (University College London, UK). The anti-phosphoserine-specific monoclonal antibody was from Sigma. All other reagents were from Sigma except where specified. p47phox null mice (p47phox−/−) on a 129 sv background were generated as J. M. M. J. 2000; Scholar) and were provided by Dr. (University College London, UK). All studies with the on the of London, UK). microvascular EC were isolated from the of p47phox−/− and wild-type mice as J.-M. Mullen A.M. Shah A.M. J. 2001; Scholar). were at microvascular EC R.A. S. J. Invest. Scholar) were from the for and cells were from the were to neutrophils by with for and stimulated with (100 for to NADPH oxidase (21Li J.-M. Mullen A.M. Yun S. Wientjes F. Brouns G.Y. Thrasher A.J. Shah A.M. Circ. Res. 2002; 90: 143-150Google Scholar). A number of and were for the of ROS O2⨪ or For of ROS EC were cultured in and to AngII (100 or for were with in for at and fluorescence was as (21Li J.-M. Mullen A.M. Yun S. Wientjes F. Brouns G.Y. Thrasher A.J. Shah A.M. Circ. Res. 2002; 90: 143-150Google Scholar). was from at cells In ROS in cells was measured by on a For these EC were by and stimulated with AngII (100 fluorescence was by the of data NADPH-dependent O2⨪ production was by lucigenin as (19Li J.-M. Shah A.M. Cardiovasc. Res. 2001; 52: 477-486Google Scholar, J.-M. Mullen A.M. Yun S. Wientjes F. Brouns G.Y. Thrasher A.J. Shah A.M. Circ. Res. 2002; 90: 143-150Google Scholar). We measured this in cells and in EC For the cells stimulated with AngII (100 or in were into with and were by rapid in by O2⨪ production was measured in the presence of NADPH (100 for and was expressed as (19Li J.-M. Shah A.M. Cardiovasc. Res. 2001; 52: 477-486Google Scholar, J.-M. Mullen A.M. Yun S. Wientjes F. Brouns G.Y. Thrasher A.J. Shah A.M. Circ. Res. 2002; 90: 143-150Google Scholar). In with an or and these were to was as in with (20Li J.-M. Shah A.M. J. Biol. Chem. 2002; 277: 19952-19960Google Scholar). p22phox or p47phox was down with polyclonal antibodies to of was with rabbit polyclonal antibodies to p47phox and p22phox or an anti-phosphoserine-specific monoclonal isolated from cells was as a was as (20Li J.-M. Shah A.M. J. Biol. Chem. 2002; 277: 19952-19960Google Scholar). The following were (a) at (b) and other at (c) and at and membrane at and The of was by enzyme as (20Li J.-M. Shah A.M. J. Biol. Chem. 2002; 277: 19952-19960Google Scholar). neutrophil and antisense p47phox were generated and were as (21Li J.-M. Mullen A.M. Yun S. Wientjes F. Brouns G.Y. Thrasher A.J. Shah A.M. Circ. Res. 2002; 90: 143-150Google Scholar). of cells were by for the of p47phox a were at and were and for are as ± of at different for mice were for and at were were by of or as was To investigate p47phox is for AngII-stimulated in NADPH oxidase wild-type and p47phox−/− were in parallel in cell ROS production was in unstimulated EC of p47phox−/− fluorescence wild-type cells ± ± EC with AngII for a significant increase in fluorescence signal ± ± In AngII induced increase in fluorescence in p47phox−/− on the fluorescence was AngII ± ± results were in on cultured and by fluorescence and A of fluorescence was in a perinuclear in unstimulated EC from with a level in AngII increased fluorescence in wild-type EC. However, increase was AngII in p47phox−/− The addition of the O2⨪ fluorescence in that the source of by was likely to be O2⨪ an NADPH-dependent O2⨪ production with and AngII was by lucigenin in cells and cell p47phox−/− cells but NADPH-dependent O2⨪ production wild-type AngII increased NADPH-dependent O2⨪ production in wild-type cells ± whereas this response was completely in p47phox−/− NADPH-dependent O2⨪ production was fully inhibited by in but was by or not results were with cell O2⨪ production was in p47phox−/− cells with AngII an increase in O2⨪ production in wild-type cells, whereas increase was in p47phox−/− AngII-induced NADPH-dependent O2⨪ production by wild-type cell was inhibited by or but was by or and with NADPH oxidase as the these results suggest that the p47phox subunit is essential for the AngII-induced increase in NADPH oxidase activity in EC. To that the of ROS response to AngII stimulation was to the of p47phox, we to acutely or p47phox levels in EC by transfection of antisense or p47phox a of the of antisense p47phox cDNA transfection on p47phox in wild-type by was an ± in p47phox fluorescence data from cells from In wild-type EC with AngII increased NADPH-dependent O2⨪ this was in antisense cDNA cells the other transfection of with p47phox cDNA in significant of p47phox In parallel NADPH-dependent O2⨪ production in response to AngII was restored p47phox cDNA transfection These indicate an essential role of p47phox in AngII-induced EC NADPH oxidase activation and ROS production. To the mechanism through which p47phox is involved in AngII-induced NADPH oxidase activation, we p47phox phosphorylation and the binding of p47phox to p22phox. These studies were in which were in the EC cells were stimulated with AngII for min, p47phox was from cell and serine phosphorylation of p47phox was with a In unstimulated cells, significant serine phosphorylation of p47phox was AngII induced rapid 1 min, peaking at ∼15 and The membrane was with an polyclonal antibody to the p47phox phosphorylation and p47phox-p22phox complex formation p22phox was detectable even in the p47phox of unstimulated EC, complex formation in these cells in the of significant p47phox However, the of p22phox co-immunoprecipitated with p47phox increased in AngII-stimulated EC, peaking at ∼15 the data from different and data for NADPH oxidase activity measured in parallel in EC is that AngII-induced in p47phox phosphorylation and complex formation were by significant in oxidase and are expressed on EC and could different (8Lang D. Mosfer S.I. Shakesby A. Donaldson F. Lewis M.J. Circ. Res. 2000; 86: 463-469Google Scholar, 9Zhang H. Schmeisser A. Garlichs C.D. Plotze K. Damme U. Mugge A. Daniel W.G. Cardiovasc. Res. 1999; 44: 215-222Google Scholar, H. Circ. Res. Scholar), we role in AngII-induced p47phox were for with the or the with p47phox was and for serine phosphorylation or inhibited AngII-induced p47phox phosphorylation, although the was To complex we p22phox and for the of p47phox of AngII the of p47phox co-immunoprecipitated with p22phox increased p47phox was as a the may phospho-p47phox it was not in unstimulated EC and was in cells with or and inhibited AngII-induced p47phox-p22phox complex with was by of AngII-induced NADPH oxidase which to a with with we the of AngII on the of p47phox in In unstimulated cells, by the of p47phox ± was in the and with a significant proportion ± in the AngII stimulation, ± of p47phox was in the and was as a In with these NADPH-dependent O2⨪ production was increased in the in AngII-stimulated EC. A significant increase was in the To a in could be by a we and in with and AngII stimulation In unstimulated cells, p47phox a perinuclear and AngII stimulation, the perinuclear p47phox in a and there was of the cell with a of AngII is known to increase EC ROS and studies have implicated the stimulation of NADPH oxidase as an important of this response M.E. Gonzalez W. Nicoletti A. Savoie F. Arnal J.-F. Michel J.-B. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 645-651Google Scholar, 5Griendling K.K. Sorescu D. Ushio-Fukai M. Circ. Res. 2000; 86: 494-501Google Scholar, D. Mosfer S.I. Shakesby A. Donaldson F. Lewis M.J. Circ. Res. 2000; 86: 463-469Google Scholar, 9Zhang H. Schmeisser A. Garlichs C.D. Plotze K. Damme U. Mugge A. Daniel W.G. Cardiovasc. Res. 1999; 44: 215-222Google Scholar). In neutrophils, the activation of NADPH oxidase the translocation and binding of cytosolic subunits (p47phox, p67phox, p40phox, and to cytochrome the phosphorylation of p47phox is to be a key in this (14Babior B.M. Blood. 1999; 93: 1464-1476Google Scholar). In EC, however, we have recently shown that a significant proportion of the oxidase is present as fully preassembled intracellular complexes, even in unstimulated cells (20Li J.-M. Shah A.M. J. Biol. Chem. 2002; 277: 19952-19960Google Scholar). The of the present study are that (a) AngII-stimulated in EC NADPH oxidase activity and ROS production are on the p47phox (b) the phosphorylation of p47phox and its binding to the cytochrome to complexes is required for the AngII and (c) in unstimulated cells, the p47phox that is to cytochromeb558 to be in an or The for p47phox in the AngII response was in on EC isolated from p47phox−/− and wild-type mice. transfection of p47phoxcDNA into p47phox−/− EC restored the ROS response to AngII, whereas in of p47phox from wild-type EC by antisense cDNA transfection in of the ROS response. In the present study, we lucigenin or fluorescence for ROS in cells, cell and/or cell intracellular and O2⨪ whereas fluorescence intracellular T. Harrison D.G. Arterioscler. Thromb. Vasc. Biol. 2002; Scholar). that the species to the by fluorescence in this study was O2⨪ was provided by the that the O2⨪ inhibited the signal The data from these different were in a for p47phox in the AngII the differences are likely to the cells and cells cell The oxidase subunit p47phox several serine the that phosphorylation R.A. U. S. A. 86: Scholar). p47phox is a that with phosphorylation R.A. U. S. A. 86: Scholar, Y. J. Biol. Chem. Scholar). In neutrophils, p47phox phosphorylation is thought to be a for its with p22phox and the formation of a oxidase complex (15Rotrosen D. Leto T.L. J. Biol. Chem. 1990; 265: 19910-19915Google Scholar, 16Huang J. Kleinberg M.E. J. Biol. Chem. 1999; 274: 19731-19737Google Scholar). In EC, however, a significant proportion of the NADPH oxidase is present as fully preassembled intracellular complexes even in unstimulated cells (20Li J.-M. Shah A.M. J. Biol. Chem. 2002; 277: 19952-19960Google Scholar). the oxidase can be further stimulated by agonists such as In the present study, we that p47phox could be co-immunoprecipitated with p22phox in the of AngII and was that the with p22phox in unstimulated EC to be it is p47phox may to p22phox in EC, it has been reported that p47phox can to p22phox in in in the presence of but that this binding supports low O2⨪ production A. H. J. Biol. Chem. 2000; Scholar). The binding of p47phox to cytochromeb558 may therefore account for the that oxidase activity in unstimulated EC is low the presence of fully assembled complexes. The mechanism of further in oxidase activity in a where there are already preassembled complexes could the phosphorylation of already p47phox or the formation of complexes through the association of other oxidase In neutrophils, p47phox phosphorylation is thought to in the p47phox phosphorylation upon with membrane which to and binding of p47phox to cytochrome (15Rotrosen D. Leto T.L. J. Biol. Chem. 1990; 265: 19910-19915Google Scholar). In the present study, we that AngII very induced min), which was by an increase in p47phox-p22phox complex formation and O2⨪ production. These data are with a mechanism where p47phox phosphorylation binding of p47phox to p22phox oxidase by conformational changes in p47phox J. Kleinberg M.E. J. Biol. Chem. 1999; 274: 19731-19737Google Scholar, A. H. J. Biol. Chem. 2000; Scholar), that p47phox phosphorylation the formation of oxidase complexes. with we that AngII stimulation was associated with an translocation of p47phox from other to the where we have shown that the of oxidase are in EC (20Li J.-M. Shah A.M. J. Biol. Chem. 2002; 277: 19952-19960Google Scholar). AngII induced a but significant translocation of p47phox to the membrane-enriched with of the oxidase present on the and p47phox of the membrane in cells as as a of a in the perinuclear The p47phox phosphorylation, binding of phospho-p47phox to and the increase in NADPH oxidase activity that, as in neutrophils (14Babior B.M. Blood. 1999; 93: 1464-1476Google Scholar, 15Rotrosen D. Leto T.L. J. Biol. Chem. 1990; 265: 19910-19915Google Scholar, 16Huang J. Kleinberg M.E. J. Biol. Chem. 1999; 274: 19731-19737Google Scholar), the serine phosphorylation of p47phox is the key for oxidase activation by AngII its effects through the activation of of and and are known to be expressed in endothelial cells H. Schmeisser A. Garlichs C.D. Plotze K. Damme U. Mugge A. Daniel W.G. Cardiovasc. Res. 1999; 44: 215-222Google Scholar, H. Circ. Res. Scholar, M.E. Michel J.-B. Pharmacol. Scholar). of the effects of AngII on cardiovascular cells are by whereas there is data on the which in can be The results of the present study suggested that and were involved in AngII-induced p47phox phosphorylation and NADPH oxidase activation the could be inhibited by or which are known to be of the and However, the effects of to be of in with a study in EC H. Schmeisser A. Garlichs C.D. Plotze K. Damme U. Mugge A. Daniel W.G. Cardiovasc. Res. 1999; 44: 215-222Google Scholar). in the present study was that unstimulated p47phox−/− cells a significant level of NADPH-dependent O2⨪ production in but ROS production wild-type cells, as we have reported (21Li J.-M. Mullen A.M. Yun S. Wientjes F. Brouns G.Y. Thrasher A.J. Shah A.M. Circ. Res. 2002; 90: 143-150Google Scholar). The underlying for this ROS production to be of these results be that the p47phox subunit is not essential for NADPH oxidase activity in EC. with this it has been reported that in NADPH oxidase can be in the of p47phox if of and Rac1 are present Lambeth J.D. J. Biol. Chem. 1996; Scholar, J. Biol. Chem. 1996; Scholar). However, an be that the background ROS production from a completely different The of the present and (21Li J.-M. Mullen A.M. Yun S. Wientjes F. Brouns G.Y. Thrasher A.J. Shah A.M. Circ. Res. 2002; 90: 143-150Google Scholar) studies that the background NADPH-dependent O2⨪ production is by but is by or that such source have to be an NADPH-dependent enzyme. is not a of NADPH of the underlying source of this background ROS production in EC may studies in which NADPH oxidase subunits are In this study that AngII-induced ROS production by EC is on the NADPH oxidase subunit phosphorylation of p47phox, by p47phox translocation and the binding of phospho-p47phox to p22phox are the key that AngII-induced NADPH oxidase activation. to the formation of oxidase complexes. ROS generated in the perinuclear may as signaling for the activation of redox-sensitive and (1Kim S. Iwao H. Pharmacol. Rev. 2000; 52: 11-34Google Scholar, 5Griendling K.K. Sorescu D. Ushio-Fukai M. Circ. Res. 2000; 86: 494-501Google Scholar), whereas ROS may to endothelial dysfunction (11Rajagopalan S. Kurz S. Munzel T. Tarpey M. Freeman B.A. Griendling K.K. Harrison D.G. J. Clin. Invest. 1996; 97: 1916-1923Google Scholar, 12Wang H.D. Xu S. Johns D.G. Du Y. Quinn M.T. Cayatte A.J. Cohen R.A. Circ. Res. 2001; 88: 947-953Google Scholar, 13Rey F.E. Cifuentes M.E. Kiarash A. Quinn M.T. Pagano P.J. Circ. Res. 2001; 89: 408-414Google Scholar).