Nitric Oxide and N-Acetylcysteine Inhibit the Activation of Mitogen-activated Protein Kinases by Angiotensin II in Rat Cardiac Fibroblasts
1998; Elsevier BV; Volume: 273; Issue: 49 Linguagem: Inglês
10.1074/jbc.273.49.33027
ISSN1083-351X
AutoresDeming Wang, Xin Yu, Peter Brecher,
Tópico(s)Redox biology and oxidative stress
ResumoAngiotensin II acts on the cardiac fibroblast to produce a mitogenic response. Nitric oxide and N-acetylcysteine have been used to determine if oxidative stress influenced the effects of angiotensin II on the cardiac fibroblast. Angiotensin II activated the mitogen-activated protein kinases designated extracellular signal-regulated kinases within 5 min by interacting with the AT1 receptor. This activation was completely independent of protein kinase C and was inhibited when farnesylation was blocked, implicating Ras involvement. Pretreatment of cardiac fibroblasts with eitherN-acetylcysteine for 8 h or nitric oxide for 10 min suppressed this activation by angiotensin II in a dose-dependent manner. However, when both agents were added, inhibition was essentially complete. This combined effect of N-acetylcysteine and nitric oxide to block ERKs activation also was found if the activity was stimulated by either another growth factor (platelet-derived growth factor) or by the addition of phorbol ester, suggesting the effect was not limited to the receptor site alone. The results are consistent with the hypothesis that hormonal activation of mitogenic steps such as ERKs is influenced by increased oxidative stress, which is reduced by the combined effects of N-acetylcysteine and nitric oxide. Angiotensin II acts on the cardiac fibroblast to produce a mitogenic response. Nitric oxide and N-acetylcysteine have been used to determine if oxidative stress influenced the effects of angiotensin II on the cardiac fibroblast. Angiotensin II activated the mitogen-activated protein kinases designated extracellular signal-regulated kinases within 5 min by interacting with the AT1 receptor. This activation was completely independent of protein kinase C and was inhibited when farnesylation was blocked, implicating Ras involvement. Pretreatment of cardiac fibroblasts with eitherN-acetylcysteine for 8 h or nitric oxide for 10 min suppressed this activation by angiotensin II in a dose-dependent manner. However, when both agents were added, inhibition was essentially complete. This combined effect of N-acetylcysteine and nitric oxide to block ERKs activation also was found if the activity was stimulated by either another growth factor (platelet-derived growth factor) or by the addition of phorbol ester, suggesting the effect was not limited to the receptor site alone. The results are consistent with the hypothesis that hormonal activation of mitogenic steps such as ERKs is influenced by increased oxidative stress, which is reduced by the combined effects of N-acetylcysteine and nitric oxide. Nitric oxide and N-acetylcysteine inhibit the activation of mitogen-activated protein kinases by angiotensin II in rat cardiac fibroblasts.Journal of Biological ChemistryVol. 274Issue 2PreviewPage 33031, Fig. 5 A: The plus (+) designation for AII in the second bar from theleft is incorrect and should be a minus sign, so that the bar represents an experiment lacking AII(−), containing Cys(+), and lacking SNAP(−). A corrected version of Fig. 5 is shown below: Full-Text PDF Open AccessHypoxia-inducible factor 1α (HIF-1α) is a non-heme iron protein. Implications for oxygen sensing.Journal of Biological ChemistryVol. 274Issue 2PreviewWe have been unable to reproduce the studies on the iron content of the recombinantly expressed HIF-1α protein, as represented in Table I of the above cited article. It appears that the iron was due to a contaminated reagent. Consequently, we wish to retract those data. The results concerning the effect of CO and heme synthesis inhibitors on the hypoxia response of B-1 cells are not in doubt. We apologize for the inconvenience caused by our mistake. Full-Text PDF Open Access angiotensin II mitogen-activated protein kinases extracellular signal-regulated kinase c-Jun N-terminal kinase p38 mitogen-activated protein kinases MAP or ERK kinase platelet-derived growth factor l-buthionine-(S, R)-sulfoximine N-acetylcysteine phorbol 12-myristoyl 13-acetate S-nitroso-N-acetylpenicillamine 1H-[1,2,4]oxadiazolo-[4,3-a]quinozalin-1-one glutathione S-transferase polyacrylamide gel electrophoresis. Angiotensin II (Ang II)1has pleiotrophic effects on several cell types, leading to diverse responses including the regulation of cell growth, programmed cell death, cell migration, and modification of the extracellular matrix (1Sadoshima J. Izumo S. Circ. Res. 1993; 73: 413-423Crossref PubMed Scopus (1323) Google Scholar, 2Kajstura J. Cigola E. Malhotra A. Li P. 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NO acts by stimulating soluble guanylate cyclase, leading to enhanced production of intracellular cyclic GMP, an intracellular second messenger that can activate cyclic GMP-dependent protein kinases (16Schmidt H.H.H.W. Walter U. Cell. 1994; 78: 919-925Abstract Full Text PDF PubMed Scopus (1511) Google Scholar). NO also is capable of reacting with oxygen radicals such as superoxide anion (17Akaike T. Suga M. Maeda H. Proc. Soc. Exp. Biol. Med. 1998; 217: 64-73Crossref PubMed Scopus (151) Google Scholar) as well as directly modulating the activity of signaling molecules (18Kim H. Shim J. Han P.L. Choi E.J. Biochemistry. 1997; 36: 13677-13681Crossref PubMed Scopus (50) Google Scholar, 19Lander H.M. Hajjar D.P. Hempstead B.L. Mirza U.A. Chait B.T. Campbell S. Quilliam L.A.A. J. Biol. Chem. 1997; 272: 4323-4326Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). The interaction with superoxide anion was suggested to be important in mechanisms where NO was implicated in modulating cytotoxic mechanisms, presumably by influencing oxidative stress (19Lander H.M. Hajjar D.P. Hempstead B.L. Mirza U.A. Chait B.T. Campbell S. Quilliam L.A.A. J. Biol. Chem. 1997; 272: 4323-4326Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). Oxidative stress, the term used to encompass changes in the cellular redox state, has been implicated in inflammatory processes such as fibrosis. The cardiac fibroblast, which is the cell type known to proliferate during cardiac fibrosis and produce the excess matrix proteins characteristic of that condition, is a target cell for Ang II. Ang II has been shown to cause proliferation of cardiac fibroblasts in culture (20Shorb W. Booz G.W. Dotal D.E. Conrad K.M. Chang K.C. Baker K.M. Circ. Res. 1993; 72: 1245-1254Crossref PubMed Scopus (374) Google Scholar). We have recently shown that nitric oxide can modulate this proliferative effect (21Takizawa T. Gu M. Chobanian A.V. Brecher P. Hypertension. 1997; 30: 1035-1040Crossref PubMed Scopus (36) Google Scholar), consistent with the known ability of NO to antagonize the actions of Ang II in many cell types. To better understand the role that NO might have in influencing Ang II action in cardiac fibroblasts, we have characterized the activation of ERKs in these cells by Ang II and have examined the effects of N-acetylcysteine and NO on this pathway. We have found novel effects of both NAC and NO on Ang II-induced ERKs activation, suggesting that both NO and oxidative stress, which accompany the development of cardiac fibrosis, could modulate the effects of Ang II on the cardiac fibroblasts. Dulbecco's modified Eagle's medium/F-12, fetal calf serum, PDGF-BB, and tissue culture reagents were from Life Technologies, Inc.S-Nitroso-N-acetylpenicillamine (SNAP) was from Alexis Corp. (San Diego, CA); PD123319, bisindolylmaleimide I (GF109203X), Gö6983, ODQ, genistein, and farnesyltransferase inhibitor-3 were from Calbiochem. [γ-32P]ATP (10 mCi/ml), p42/p44 MAP kinase enzyme assay kit, and the ECL detection system were from Amersham Pharmacia Biotech. MEK1 antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). PD98059 and phosphospecific ERK antibodies were from New England Biolabs (Beverly, MA). Losartan was generously provided by DuPont. Ang II, PMA, NAC, 5,5′-dithiobis(nitrobenzioc acid),l-buthionine-(S, R)-sulfoximine (BSO),dl-cysteine, glutathione monoethylester, and all other chemicals were purchased from Sigma. Cardiac fibroblasts were obtained from 8-day-old rats following an isolation procedure described previously by us (22Fariver R.S. Chobanian A.V. Brecher P. Circ. Res. 1996; 78: 759-768Crossref PubMed Google Scholar). Cells in the 3rd to 4th passage were grown to 80% confluence in either 60- or 100-mm culture dishes and then maintained for 24 h in 0.4% fetal calf serum/Dulbecco's modified Eagle's medium/F-12. Fresh medium was routinely added 2 h before the experiment. Ang II was routinely added to the cells for 5 min at a concentration of 0.1 μm. SNAP was routinely added 10 min prior to the addition of agonists. NAC pretreatment was typically for 8 h, and the medium containing NAC was removed and replaced with Dulbecco's modified Eagle's medium/F-12 lacking NAC for 2 h prior to adding other agonists. Care was taken to adjust the pH of medium containing NAC prior to adding it to the cells. Cell viability was monitored routinely using either trypan blue exclusion or by a measurement of lactate dehydrogenase activity into the culture medium using a commercially available kit. This radioassay was essentially that provided in a kit purchased from Amersham Pharmacia Biotech (catalog no. RPN 84) in which a cell lysate is used to generate a radiolabeled product using a synthetic peptide substrate specific for ERKs. The instructions provided with the kit were followed except for the initial procedures used to obtain a cell lysate. Following treatment of the cells with hormone or drugs, the cells were washed twice with ice-cold PBS, and then cell lysis was accomplished by adding a lysis buffer containing 0.1% Triton X-100, 10 mm Tris, pH 7.4, 50 mm NaCl, 2 mm EGTA, 1 mmdithiothreitol, 1 mm orthovanadate, 1 mmphenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 10 μg/ml aprotinin. The cells were briefly frozen at −70 °C for 10 min and allowed to thaw at 4 °C, and then the lysed cells were transferred to a 1.5-ml Eppendorf tube for subsequent centrifugation at 10,000 × g for 10 min. Fifteen-μl aliquots were then used for the radioassay, following the instructions provided in the kit. Following incubation of the extract with labeled ATP and the synthetic substrate, the radiolabeled product was adsorbed to phosphocellulose paper and washed extensively with 1% acetic acid, and then the radioactivity was measured in a scintillation counter. Protein was determined by using the Bio-Rad protein assay system and bovine serum albumin as a standard. All data were expressed relative to the control values. The procedures followed were essentially those described in the instructions for kits provided by New England Biolabs (ERKs, catalog no. 9800; JNKs, catalog no. 9810; p38 MAPK, catalog no. 9820). Cell lysates were obtained as described above for the radioimmunoassay. In the ERK assays, 200 μg of protein was immunoprecipitated with 1 μg of a monoclonal antibody directed against phosphorylated ERKs. Following the addition of protein A-Sepharose beads (Amersham Pharmacia Biotech), the immunoprecipitated enzymes were subsequently incubated with 1 μg of GST- Elk1 fusion protein for 30 min at 30 °C in the presence of 100 μmATP. The phosphorylated products were solubilized, resolved by 10% SDS-PAGE, and subjected to immunoblotting using an antibody against phosphorylated Elk1 as described in the kit. In the p38 MAPK assay, 200 μg of protein was immunoprecipitated with 1 μg of a polyclonal antibody against phosphorylated p38 MAPK. The immune complexes, adsorbed to protein A-Sepharose beads, were incubated with 1 μg of GST-ATF2 fusion protein and 100 μm ATP for 15 min at 30 °C. The phosphorylated products were determined by Western blot analysis using an antibody against phosphorylated ATF2 as described in the kit. In the JNKs assay, 300 μg of protein was incubated with 2 μg of GST-c-Jun-(1–89) fusion protein bound to glutathione-Sepharose beads to selectively precipitate JNKs. The kinase reaction was performed by the addition of 100 μm ATP for 30 min at 30 °C. c-Jun phosphorylation was selectively measured using a phosphospecific c-Jun antibody as described in the kit. Immune complexes on nitrocellulose membrane were treated with an appropriate secondary antibody conjugated with horseradish peroxidase and visualized with ECL (Amersham Pharmacia Biotech). Densitometric analysis of immunoblots from these assays and all other immunoassays used in this study were performed using a PDI scanner (model 420oe), and the data were reported as a -fold increase over unstimulated cells (control), which arbitrarily were set at 1. Following treatment with Ang II for 5 min, cells were washed twice with ice-cold PBS and then lysed with a concentrated sample buffer containing 250 mm Tris, pH 6.8, 8% SDS, 40% glycerol, 200 mm dithiothreitol, and 0.04% bromphenol blue. Following boiling for 5 min, the suspension was centrifuged at 10,000 × g for 10 min at 4 °C, and the supernatant was used for direct application onto 10% SDS-PAGE. Following transfer to nitrocellulose membrane and blockage with 5% nonfat milk in a Tris-buffered saline solution containing 0.1% Tween 20, the blot was incubated with antibody (1:1000) specific for phospho-ERK44 and -42 (New England Biolabs). After extensive washing, the blot was incubated with a second antibody conjugated with horseradish peroxidase and visualized with ECL (Amersham Pharmacia Biotech). 500 μg of protein in cell lysates obtained as described above for the ERKs radioimmunoassay were incubated 3 h at 4 °C with 5 μg of a polyclonal antibody against MEK-1 (Santa Cruz Biotechnology, catalog no. sc-436). Protein A-Sepharose beads were added, and the immunoprecipitated enzyme was incubated in 30 μl of kinase buffer (25 mm Tris, pH 7.5, 2 mm dithiothreitol, 0.1 mm orthovanadate, 10 mm MgCl2, 5 μm ATP, 10 μCi of [γ-32P]ATP) with a recombinant GST-[K71A]Erk44 lacking enzymatic activity as a substrate (Upstate Biotechnology, Inc., catalog no. 14-135). The labeled substrate was detected by autoradiography following 10% SDS-PAGE. Intracellular glutathione was measured spectrophotometrically following a minor modification of the method of Tietze (23Tietze F. Anal. Biochem. 1969; 27: 502-522Crossref PubMed Scopus (5653) Google Scholar). Cells were lysed with 0.2 ml of 0.35 nperchloric acid. Following centrifugation at 10,000 ×g for 10 min at 4 °C, different aliquots of the lysate were incubated with 0.1 m sodium phosphate, pH 7.5, 5 mm EDTA, 0.21 mm NADPH, 1 unit of glutathione reductase (Boehringer Mannheim), and 0.6 mm5,5′-dithiobis(nitrobenzioc acid) in a total volume of 1 ml. The rate of change in absorbency at 412 nm was measured over a 5-min period to reflect the formation of reaction product and correlated with a standard curve using reduced glutathione. Data are expressed as nmol of glutathione/mg of protein in the lysate. Data are presented as the mean ± S.E. of at least three experiments unless designated otherwise. Statistical analysis was performed using analysis of variance and Student'st test as appropriate. A value of p < 0.05 was considered to be statistically significant. Fig. 1 A shows the rapid and transient increase in the activity of ERKs following the addition of 0.1 μm Ang II in quiescent rat cardiac fibroblasts, where maximal activation occurred 2–5 min after hormone addition and then gradually decreased to basal levels. Fig. 1 B shows that the 5-min response to Ang II was dose-dependent with peak activity occurring at concentrations between 0.01–0.1 μmAng II and a clear increase even at concentrations less than 1 nm. In 35 separate experiments using five different preparations of cardiac fibroblasts in the 3rd or 4th passage, 0.1 μm Ang II increased ERK activity an average of 8-fold over control levels, with a range of 5–15-fold. Fig. 1 Cshows that the response to Ang II was mediated through AT1but not AT2 receptors, based on the almost complete blockade of activation if cells were pretreated with losartan (an AT1 receptor antagonist), whereas pretreatment with PD123319 (an AT2 receptor antagonist) was without effect. The experiments shown in Fig. 1, A–C, were performed using the radioassay for ERKs. To further establish the response to Ang II, experiments using an immunoprecipitation assay for ERKs were performed (Fig. 2, A–C). This assay detects immunoreactive phosphorylated Elk1, a specific substrate for the immunoprecipitated ERKs. In Fig. 2, A–C, the effects of genistein, farnesyltransferase inhibitor-3, and PD98059, respectively, were studied. In each case, pretreatment with the respective inhibitor almost completely abolished the response to Ang II, suggesting that the activation of ERKs by Ang II involved tyrosine kinase activity, the presence of an activated form of Ras, and the subsequent activation of MEK1. It is noted that in the immunoassay procedure we used, the lower and upper bands for phosphorylated Elk1 reflect different degrees of phosphorylation. When activity is relatively low in the immunoprecipitated sample, the lower, less phosphorylated band predominates, whereas when activity is relatively high, the hyperphosphorylated, upper band is most obvious.Figure 2Effect of inhibitors of tyrosine kinase (A), protein farnesylation (B), and MEK1 (C) on the activation of ERKs by Ang II. All experiments were performed using the immunoassay for ERKs as described under “Experimental Procedures.” A, following pretreatment with 50 μm genistein for 15 min, the cells were incubated with 0.1 μm Ang II or 20 ng/ml PDGF-BB for 5 min, and activated ERKs were immunoprecipitated using a phospho-ERK-specific antibody and then used in kinase reactions to phosphorylate GST-Elk1 fusion protein as substrate. Elk1 phosphorylation was then detected by immunoblotting using a phospho-Elk1-specific antibody. B, following pretreatment with 10 μm farnesyltransferase inhibitor-3 (FT-3), a commercially available enzyme inhibitor (Calbiochem) for 1 h, the cells were incubated with either 0.1 μm Ang II or 20 ng/ml PDGF-BB for 5 min, and then extracts were prepared for immunoprecipitation. C, following pretreatment with 50 μm PD98059 for 45 min, the cells were incubated for 5 min with 0.1 μm Ang II, 1 μm PMA, or 20 ng/ml PDGF-BB, and then extracts were prepared for immunoprecipitation. Each immunoblot is representative of at least two separate experiments. pElk1, phosphorylated Elk1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Fig. 3 shows experiments examining the role of protein kinase C in the Ang II-induced activation of ERKs. In Fig. 3 A, using the radioassay for ERKs, cells were pretreated for only 15 min with protein kinase C inhibitors GF109203X (1 μm) or Gö6983 (1 μm), and the subsequent activation by PMA was abolished, whereas no effect on Ang II activation was found. Similar results were shown in Fig. 3 Bby using the immunoassay for ERKs. Cells were preincubated with GF109203X. Responses to PMA were blocked with no effect on Ang II activation. In Fig. 3 C, cells were pretreated for 24 h with 1 μm PMA to down-regulate protein kinase C, and then either Ang II (0.1 μm) or PMA (1 μm) was added. After 5 min, the cells were assayed for ERK activity. Again, pretreatment with PMA for 24 h completely prevented subsequent activation of ERKs by additional PMA but had almost no effect on the increased activity in response to Ang II. Thus, it appears that Ang II activates ERKs through a mechanism that is predominantly independent of protein kinase C. To assess the role of intracellular GSH, a major determinant of the redox state of the cell, on the activation of ERKs by Ang II, cells were pretreated with BSO, an inhibitor of glutathione biosynthesis that decreases intracellular GSH levels. Cells also were treated with NAC, an agent known to increase GSH levels, and additionally with a combination of both drugs. Fig. 4 A shows the effects of these drugs on GSH levels in the cardiac fibroblasts. BSO (50 μm) dramatically decreased cellular GSH levels when pretreatment of the cells was performed for 24 h. NAC (8 mm) increased GSH approximately 3-fold if pretreatment was for at least 8 h. Higher concentrations of NAC (20–30 mm) caused cell damage within 30 min of treatment as measured by the release of lactate dehydrogenase into the culture medium. The combination of BSO and NAC pretreatment blocked the increase in GSH levels generated by exposure to NAC, indicating that the effects of BSO override those of NAC. However, as shown in Fig. 4 B, activation of ERKs by Ang II was essentially unaffected by BSO treatment, whereas NAC pretreatment caused a decrease in ERK activity. This effect of NAC was clearly independent of GSH levels, since ERK activity was lowered even when BSO was added in the presence of NAC. Fig. 4 C shows that the inhibition of ERK activation by NAC was dose-dependent, but NAC did not completely inhibit the response. Since a low GSH concentration presumably makes the cell more sensitive to oxidative stress, and Ang II-mediated effects may be mediated by reactive oxygen species (24Wilhelm D. Bender K. Knebel A. Angel P. Mol. Cell. Biol. 1997; 17: 4792-4800Crossref PubMed Scopus (225) Google Scholar, 25Ushio-Fukai M. Zafari A.M. Fukui T. Ishizaka N. Griendling K.K. J. Biol. Chem. 1996; 271: 23317-23321Abstract Full Text Full Text PDF PubMed Scopus (701) Google Scholar), we performed a series of experiments with BSO-pretreated cells to determine if the response to suboptimal levels of hormone would be influenced in the presence of lower GSH levels. Fig. 4 Dshows that dose-response curves to Ang II activation were similar in the absence or presence of BSO, indicating that intracellular GSH apparently does not affect the signaling pathways used by Ang II. In separate experiments, not shown, we also found that BSO pretreatment had no effect on PMA activation of ERKs throughout a concentration range of 10−10 to 10−6m. In additional experiments, pretreatment with glutathione monoethylester (0.5 mm), which increased intracellular GSH levels about 2-fold, did not affect Ang II-activated ERK activity, whereas relatively brief pretreatment (60 min) with either β-mercaptoethanol (1 mm) or dithiothreitol (0.5 mm) reduced Ang II activation of ERKs by 50–75%. These latter findings suggested that it was the efficacy of NAC as a reducing agent that might account for its inhibitory effect. In all experiments with BSO and NAC pretreatments, cell viability was monitored visually by trypan blue exclusion and in selected experiments by measuring the release of lactate dehydrogenase into the culture medium. No evidence of cell damage to the cardiac fibroblasts was found during the pretreatment protocols. To assess the role of NO, we pretreated the cells with varying concentrations of the NO donor SNAP. Fig. 5 A shows that pretreatment for 10 min with varying doses of SNAP resulted in a dose-dependent inhibition of ERK activation, but complete inhibition was not obtained at SNAP concentrations that maintained cell viability. In the above experiments, equimolar amounts of cysteine were added with SNAP to increase the efficacy of NO release. Fig. 5 B shows that in the absence of cysteine, 50 μm SNAP was only slightly effective in inhibiting ERK activation, whereas a statistically significant reduction in activity was found when cysteine was included. Using another NO donor,S-nitrosoglutathione, a similar effect of cysteine addition was observed. We routinely checked to see if the SNAP addition, either in the presence or absence of cysteine, influenced the basal levels of ERK activity and found no change whatsoever in the control levels throughout the concentration range of SNAP used. To confirm that the inhibitory action of SNAP was due to the presence of NO in the medium, we pretreated cells with 10 μm ODQ, an inhibitor of soluble guanylate cyclase, or 20 μm oxymyoglobin, which sequesters NO; the inhibition of ERK activation by SNAP was prevented (Fig. 5 C). In an additional experiment (not shown), we added 10 μm LY83583, another inhibitor of soluble guanylate cyclase, and found a similar reversal of the inhibition produced by SNAP. With each new agent added to the cells, viability was checked both visually and by lactate dehydrogenase release into the medium, and no adverse effects were noted due to SNAP, cysteine, or the other agents added. In Fig. 6,A–C, we compared the effects of NAC and SNAP treatment on ERK activation using Ang II, PDGF-BB, or PMA as the agonist. The most striking finding with all agonists used was that in combination, SNAP and NAC produced greater inhibition than when either drug was added alone. Since both a radioassay and an immunoassay have been used in experiments described above to document the effects of NAC and NO either alone or in combination, and some quantitative discrepancies sometimes were found, we performed an additional assay for ERK activation, which directly measures the phosphorylated ERK44 and ERK42 in a whole cell extract. Fig. 7 shows representative results and densitometric analysis from such assays using either SNAP or NAC, alone or in combination, prior to the Ang II addition. Consistent with the other assays, there was a greater inhibition of activity when both agents were added together. In this procedure, we found it necessary to obtain concentrated extracts of the cells prior to SDS-PAGE in order to have enough activated enzyme to detect by Western blot analysis. To obtain insight into the possible upstream sites involved in the above effects of NAC and NO, we measured the effects of these agents on the activation of MEK1, an upstream kinase of ERKs, by Ang II. Fig. 8 shows that MEK1 activation by Ang II is marked and that pretreatment with either NAC or SNAP caused significant inhibition. However, combined treatment with both NAC and SNAP inhibited MEK1 activation by Ang II completely. In an additional set of experiments,
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