Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Direct Activation of AMP-activated Protein Kinase Stimulates Nitric-oxide Synthesis in Human Aortic Endothelial Cells

2003; Elsevier BV; Volume: 278; Issue: 34 Linguagem: Inglês

10.1074/jbc.m212831200

ISSN

1083-351X

Autores

Valerie Morrow, Fabienne Foufelle, John Connell, John R. Petrie, Gwyn W. Gould, Ian P. Salt,

Tópico(s)

Pancreatic function and diabetes

Resumo

Recent studies have indicated that endothelial nitric-oxide synthase (eNOS) is regulated by reversible phosphorylation in intact endothelial cells. AMP-activated protein kinase (AMPK) has previously been demonstrated to phosphorylate and activate eNOS at Ser-1177 in vitro, yet the function of AMPK in endothelium is poorly characterized. We therefore determined whether activation of AMPK with 5′-aminoimidazole-4-carboxamide ribonucleoside (AICAR) stimulated NO production in human aortic endothelial cells. AICAR caused the time- and dose-dependent stimulation of AMPK activity, with a concomitant increase in eNOS Ser-1177 phosphorylation and NO production. AMPK was associated with immunoprecipitates of eNOS, yet this was unaffected by increasing concentrations of AICAR. AICAR also caused the time- and dose-dependent stimulation of protein kinase B phosphorylation. To confirm that the effects of AICAR were indeed mediated by AMPK, we utilized adenovirus-mediated expression of a dominant negative AMPK mutant. Expression of dominant negative AMPK attenuated AICAR-stimulated AMPK activity, eNOS Ser-1177 phosphorylation and NO production and was without effect on AICAR-stimulated protein kinase B Ser-473 phosphorylation or NO production stimulated by insulin or A23187. These data suggest that AICAR-stimulated NO production is mediated by AMPK as a consequence of increased Ser-1177 phosphorylation of eNOS. We propose that stimuli that result in the acute activation of AMPK activity in endothelial cells stimulate NO production, at least in part due to phosphorylation and activation of eNOS. Regulation of endothelial AMPK therefore provides an additional mechanism by which local vascular tone may be controlled. Recent studies have indicated that endothelial nitric-oxide synthase (eNOS) is regulated by reversible phosphorylation in intact endothelial cells. AMP-activated protein kinase (AMPK) has previously been demonstrated to phosphorylate and activate eNOS at Ser-1177 in vitro, yet the function of AMPK in endothelium is poorly characterized. We therefore determined whether activation of AMPK with 5′-aminoimidazole-4-carboxamide ribonucleoside (AICAR) stimulated NO production in human aortic endothelial cells. AICAR caused the time- and dose-dependent stimulation of AMPK activity, with a concomitant increase in eNOS Ser-1177 phosphorylation and NO production. AMPK was associated with immunoprecipitates of eNOS, yet this was unaffected by increasing concentrations of AICAR. AICAR also caused the time- and dose-dependent stimulation of protein kinase B phosphorylation. To confirm that the effects of AICAR were indeed mediated by AMPK, we utilized adenovirus-mediated expression of a dominant negative AMPK mutant. Expression of dominant negative AMPK attenuated AICAR-stimulated AMPK activity, eNOS Ser-1177 phosphorylation and NO production and was without effect on AICAR-stimulated protein kinase B Ser-473 phosphorylation or NO production stimulated by insulin or A23187. These data suggest that AICAR-stimulated NO production is mediated by AMPK as a consequence of increased Ser-1177 phosphorylation of eNOS. We propose that stimuli that result in the acute activation of AMPK activity in endothelial cells stimulate NO production, at least in part due to phosphorylation and activation of eNOS. Regulation of endothelial AMPK therefore provides an additional mechanism by which local vascular tone may be controlled. Mammalian AMP-activated protein kinase (AMPK) 1The abbreviations used are: AMPK, 5′-AMP-activated protein kinase; AICAR, 5′-aminoimidazole-4-carboxamide ribonucleoside; DAF-2, 4,5-dianimofluoroscein; DAF-2DA, DAF-2-diacetate; eNOS, endothelial nitric-oxide synthase; HAECs, human aortic endothelial cells; HU-VECs, human umbilical vein endothelial cells; l-NAME, Nω-nitro-l-arginine methyl ester; PEG, poly(ethylene glycol) 6000; PKB, protein kinase B; DN, dominant negative; Pfu, plaque-forming unit; KRH, Krebs Ringer HEPES. is a heterotrimeric member of a protein kinase family highly conserved in animals, plants, and yeast consisting of one catalytic (α) subunit and two regulatory (β and γ) subunits (1Hardie D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 821-855Crossref PubMed Scopus (1276) Google Scholar, 2Mitchelhill K.I. Stapleton D. Gao G. House C. Michell B. Katsis F. Witters L.A. Kemp B.E. J. Biol. Chem. 1994; 269: 2361-2364Abstract Full Text PDF PubMed Google Scholar, 3Cheung P.C.F. Salt I.P. Davies S.P. Hardie D.G. Carling D. Biochem. J. 2000; 346: 659-669Crossref PubMed Scopus (533) Google Scholar). AMPK is the downstream component of a protein kinase cascade activated by a rise in the cellular AMP:ATP ratio and is thought to act as a cellular energy charge sensor (1Hardie D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 821-855Crossref PubMed Scopus (1276) Google Scholar, 4Hardie D.G. Hawley S.A. Bioessays. 2001; 23: 1112-1119Crossref PubMed Scopus (676) Google Scholar). AMPK is therefore activated by treatments that deplete ATP, and once activated, switches off ATP-consuming anabolic pathways via both the direct phosphorylation of and regulation of expression of metabolic enzymes (1Hardie D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 821-855Crossref PubMed Scopus (1276) Google Scholar, 4Hardie D.G. Hawley S.A. Bioessays. 2001; 23: 1112-1119Crossref PubMed Scopus (676) Google Scholar). AMPK can be artificially activated in intact cells by treatment with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) (5Corton J.M. Gillespie J.G. Hawley S.A. Hardie D.G. Eur. J. Biochem. 1995; 229: 558-565Crossref PubMed Scopus (1029) Google Scholar). In most cell types, this nucleoside is taken up and accumulates in the cytoplasm as the monophosphorylated nucleotide, ZMP (5′-aminoimidazole-4-carboxamide ribonucleotide), which activates AMPK without disturbing cellular adenine nucleotide ratios (5Corton J.M. Gillespie J.G. Hawley S.A. Hardie D.G. Eur. J. Biochem. 1995; 229: 558-565Crossref PubMed Scopus (1029) Google Scholar) such that any effects observed are not merely due to depletion of ATP. Studies with AICAR and recent studies in cells expressing constitutively active and dominant negative mutant forms of AMPK have confirmed the role of AMPK in the regulation of metabolism (4Hardie D.G. Hawley S.A. Bioessays. 2001; 23: 1112-1119Crossref PubMed Scopus (676) Google Scholar, 6Woods A. Azzout-Marniche D. Foretz M. Stein S.C. Lemarchand P. Ferre P. Foufelle F. Carling D. Mol. Cell. Biol. 2000; 20: 6704-6711Crossref PubMed Scopus (358) Google Scholar, 7Mu J. Brozinick Jr., J.T. Valladares O. Bucan M. Birnbaum M.J. Mol. Cell. 2001; 7: 1085-1094Abstract Full Text Full Text PDF PubMed Scopus (800) Google Scholar). Additionally, it has recently been proposed that the effects of a number of physiological and pharmacological stimuli are mediated by AMPK, including the antidiabetic drugs metformin and rosiglitazone (8Zhou G. Myers R. Li Y. Chen Y. Shen X. Fenyk-Melody J. Wu M. Ventre J. Doebber T. Fujii N. Musi N. Hirshman M.F. Goodyear L.J. Moller D.E. J. Clin. Invest. 2001; 108: 1167-1174Crossref PubMed Scopus (4432) Google Scholar, 9Fryer L.G.D. Parbu-Patel A. Carling D. J. Biol. Chem. 2002; 272: 25226-25232Abstract Full Text Full Text PDF Scopus (912) Google Scholar) and the hormones, leptin, and adiponectin (10Minokoshi Y. Kim Y.-B. Peroni O.D. Fryer L.G.D. Muller C. Carling D. Kahn B.B. Nature. 2002; 415: 339-343Crossref PubMed Scopus (1679) Google Scholar, 11Yamauchi T. Kamon J. Minokoshi Y. Ito Y. Uchida S. Yamashita S. Noda M. Kita S. Ueki K. Eto K. Akanuma Y. Froguel P. Foufelle F. Ferre P. Carling D. Kimura S. Nagai R. Kahn B.B. Kadowaki T. Nat. Med. 2002; 8: 1288-1295Crossref PubMed Scopus (3460) Google Scholar). Metformin, however, appears to stimulate AMPK without altering adenine nucleotide ratios (9Fryer L.G.D. Parbu-Patel A. Carling D. J. Biol. Chem. 2002; 272: 25226-25232Abstract Full Text Full Text PDF Scopus (912) Google Scholar, 12Hawley S.A. Gadalla A.E. Skytte Olsen G. Hardie D.G. Diabetes. 2002; 51: 2420-2425Crossref PubMed Scopus (578) Google Scholar). Nitric oxide, synthesized by endothelial nitric-oxide synthase (eNOS), is an important regulator of cardiovascular homeostasis. Endothelium-derived NO promotes vasodilation and inhibits platelet aggregation, leukocyte adherence, and vascular smooth muscle proliferation (13Moncada S. Palmer R.M.J. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142PubMed Google Scholar). These effects exert a profound influence on blood flow, vascular remodeling, and angiogenesis. It has recently been demonstrated that eNOS is regulated by reversible phosphorylation. AMPK has been demonstrated to activate cardiac and endothelial cell eNOS by phosphorylation at Ser-1177 (human sequence) in vitro (14Chen Z.-P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (717) Google Scholar). Phosphorylation at this site appears to lower the sensitivity of eNOS to Ca2+ (14Chen Z.-P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (717) Google Scholar, 15Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3047) Google Scholar, 16Michell B.J. Griffiths J.E. Mitchelhill K.I. Rodriguez-Crespo I. Tiganis T. Bozinovski S. Ortiz de Montellano P.R. Kemp B.E. Pearson R.B. Curr. Biol. 1999; 9: 845-848Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar, 17Montagnani M. Chen H. Barr V.A. Quon M.J. J. Biol. Chem. 2001; 276: 30392-30398Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar). Furthermore, ischemia was demonstrated to activate AMPK, with concomitant phosphorylation and activation of eNOS at Ser-1177 in heart (14Chen Z.-P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (717) Google Scholar). Additionally, protein kinase B (PKB) has been demonstrated to phosphorylate and activate eNOS in intact cells at Ser-1177 (15Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3047) Google Scholar, 16Michell B.J. Griffiths J.E. Mitchelhill K.I. Rodriguez-Crespo I. Tiganis T. Bozinovski S. Ortiz de Montellano P.R. Kemp B.E. Pearson R.B. Curr. Biol. 1999; 9: 845-848Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar, 18Fulton D. Gratton J.-P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2232) Google Scholar, 19Gallis B. Corthals G.L. Goodlett D.R. Ueba H. Kim F. Presnell S.R. Figeys D. Harrison D.G. Berk B.C. Aebersold R. Corson M.A. J. Biol. Chem. 1999; 274: 30101-30108Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). Phosphorylation of eNOS at Ser-1177 has been demonstrated to occur in endothelial cells subjected to fluid shear-stress, vascular endothelial growth factor, insulin-like growth factor-I, and insulin (15Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3047) Google Scholar, 16Michell B.J. Griffiths J.E. Mitchelhill K.I. Rodriguez-Crespo I. Tiganis T. Bozinovski S. Ortiz de Montellano P.R. Kemp B.E. Pearson R.B. Curr. Biol. 1999; 9: 845-848Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar, 18Fulton D. Gratton J.-P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2232) Google Scholar, 19Gallis B. Corthals G.L. Goodlett D.R. Ueba H. Kim F. Presnell S.R. Figeys D. Harrison D.G. Berk B.C. Aebersold R. Corson M.A. J. Biol. Chem. 1999; 274: 30101-30108Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 20Kim F. Gallis B. Corson M.A. Am. J. Physiol. 2001; 280: C1057-C1065Crossref PubMed Google Scholar). Expression of dominant negative PKB in endothelial cells inhibits NO production and eNOS Ser-1177 phosphorylation in response to vascular endothelial growth factor and insulin (16Michell B.J. Griffiths J.E. Mitchelhill K.I. Rodriguez-Crespo I. Tiganis T. Bozinovski S. Ortiz de Montellano P.R. Kemp B.E. Pearson R.B. Curr. Biol. 1999; 9: 845-848Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar, 17Montagnani M. Chen H. Barr V.A. Quon M.J. J. Biol. Chem. 2001; 276: 30392-30398Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar, 21Boo Y.C. Sorescu G. Boyd N. Shiojima I. Walsh K. Du J. Jo H. J. Biol. Chem. 2002; 277: 3388-3396Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar) but not fluid shear stress (21Boo Y.C. Sorescu G. Boyd N. Shiojima I. Walsh K. Du J. Jo H. J. Biol. Chem. 2002; 277: 3388-3396Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar). Fluid shear stress-stimulated eNOS phosphorylation is wortmannin-sensitive (15Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3047) Google Scholar, 18Fulton D. Gratton J.-P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2232) Google Scholar, 19Gallis B. Corthals G.L. Goodlett D.R. Ueba H. Kim F. Presnell S.R. Figeys D. Harrison D.G. Berk B.C. Aebersold R. Corson M.A. J. Biol. Chem. 1999; 274: 30101-30108Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar), yet the kinase responsible remains uncertain. Although ischemia has been demonstrated to stimulate AMPK activity, eNOS activity, and eNOS phosphorylation at Ser-1177 in rat heart (14Chen Z.-P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (717) Google Scholar), a number of important questions remain. Ischemia has been demonstrated to deplete cellular ATP and affect a host of signaling pathways (22Steenbergen C. Basic Res. Cardiol. 2002; 97: 276-285Crossref PubMed Scopus (91) Google Scholar, 23Lounsbury K.M. Hu Q. Ziegelstein R.C. Free Radic. Biol. Med. 2000; 28: 1362-1369Crossref PubMed Scopus (125) Google Scholar, 24Burnstock G. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 364-373Crossref PubMed Scopus (360) Google Scholar), and it has yet to be demonstrated that activation of AMPK regulates eNOS activity directly in intact endothelial cells in the absence of disturbed adenine nucleotide ratios. Additionally, there have, to date, been no studies of the effects of AMPK activation on NO synthesis in human cells. There is substantial interest in the AMPK cascade as a possible therapeutic target for the treatment of metabolic disorders such as diabetes (4Hardie D.G. Hawley S.A. Bioessays. 2001; 23: 1112-1119Crossref PubMed Scopus (676) Google Scholar, 25Winder W.W. Hardie D.G. Am. J. Physiol. 1999; 277: E1-E10PubMed Google Scholar, 26Viollet B. Andreelli F. Jørgensen S.B. Perrin C. Geloen A. Flamez D. Mu J. Lenzer C. Baud O. Bennoun M. Gomas E. Nicolas G. Wojtaszewski J.F.P. Kahn A. Carling D. Schuit F.C. Birnbaum M.J. Richter E.A. Burcelin R. Vaulont S. J. Clin. Invest. 2003; 111: 91-98Crossref PubMed Scopus (442) Google Scholar). Administration of AICAR in mice and rats has highlighted the therapeutic potential of AMPK activation in glucose homeostasis (27Bergeron R. Russell III, R.R. Young L.H. Ren J.M. Marcucci M. Lee A. Shulman G.I. Am. J. Physiol. 1999; 276: E938-E944Crossref PubMed Google Scholar, 28Fiedler M. Zierath J.R. Selen G. Wallberg-Henriksson H. Liang Y. Sakariassen K.S. Diabetologia. 2001; 44: 2180-2186Crossref PubMed Scopus (53) Google Scholar, 29Song X.M. Fiedler M. Galuska D. Ryder J.W. Fernstrom M. Chibalin A.V. Wallberg-Henriksson H. Zierath J.R. Diabetologia. 2002; 45: 56-65Crossref PubMed Scopus (173) Google Scholar, 30Iglesias M.A. Ye J.M. Frangioudakis G. Saha A.K. Tomas E. Ruderman N.B. Cooney G.J. Kraegen E.W. Diabetes. 2002; 51: 2886-2894Crossref PubMed Scopus (253) Google Scholar) and has been reported to lower blood pressure in hypertensive, obese Zucker rats (31Buhl, E. S., Jessen, N., Pold, R., Ledet, T., Flyvbjerg, A., Pedersen, S. B., Pedersen, O., Schmitz, O., and Lund, S. Diabetes, 51, 2199–2206Google Scholar), yet the mechanism involved has yet to be characterized. It is, therefore, important that the effects of AMPK activation in tissues such as endothelium, which would be subjected to such agents in vivo, are fully investigated. Incubation of human umbilical vein endothelial cells (HUVECs) with AICAR has been demonstrated to stimulate AMPK (32Dagher Z. Ruderman N. Tornheim K. Ido Y. Biochem. Biophys. Res. Commun. 1999; 265: 112-115Crossref PubMed Scopus (83) Google Scholar, 33Ido Y. Carling D. Ruderman N. Diabetes. 2002; 51: 159-167Crossref PubMed Scopus (296) Google Scholar, 34Dagher Z. Ruderman N. Tornheim K. Ido Y. Circ. Res. 2001; 88: 1276-1282Crossref PubMed Scopus (166) Google Scholar). It should be pointed out, however, that the specificity of AICAR remains uncertain. Therefore, any implicated role of AMPK should be confirmed using another method of manipulating AMPK activity such as the use of dominant negative AMPK in this study. In this study we have expanded the studies of Chen et al. (14Chen Z.-P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (717) Google Scholar) and have investigated the effects of activation of AMPK cascade on NO synthesis in human aortic endothelial cells (HAECs) using the AMPK activator, AICAR. We report that acute incubation of HAECs with AICAR stimulates AMPK activity, Ser-1177 phosphorylation, and NO production, effects inhibited by the expression of a dominant-negative AMPK mutant. We propose that stimuli that result in the acute activation of AMPK activity in endothelial cells stimulate NO production, at least in part due to phosphorylation and activation of eNOS. Therefore, regulation of endothelial AMPK may provide a mechanism that controls NO production and thereby local vascular tone. Materials—Cryopreserved HAECs and cell culture media were obtained from TCS Cellworks (Botolph Claydon, Bucks, UK). AICAR, metformin, soybean trypsin inhibitor, benzamidine, AMP, ATP, and rabbit anti-eNOS antibody were supplied by Sigma. [γ-32P]ATP and horseradish peroxidase-conjugated secondary antibodies were from Amersham Biosciences. l-NAME, A23187, DAF-2DA, PD90859, and mouse anti-eNOS antibodies were from Calbiochem. Insulin (Actrapid) was obtained from Novo-Nordisk (Copenhagen, Denmark). Protein G-Sepharose was from Amersham Biosciences. Sheep anti-PKB antibody was supplied by Upstate Biotechnology, Inc. (Lake Placid, NY). Rabbit anti-p42/44MAPK, anti-phospho-p42/44MAPK, anti-phospho-eNOSSer-1177, and anti-phospho-PKBSer-473 antibodies were obtained from Cell Signaling Technology (Beverly, MA). AMARA peptide (AMARAASAAALARRR) was synthesized by Dr. G. Bloomberg, University of Bristol, UK. Isoform-specific sheep anti-AMPK antibodies have been described elsewhere (6Woods A. Azzout-Marniche D. Foretz M. Stein S.C. Lemarchand P. Ferre P. Foufelle F. Carling D. Mol. Cell. Biol. 2000; 20: 6704-6711Crossref PubMed Scopus (358) Google Scholar, 35Woods A. Salt I. Scott J. Hardie D.G. Carling D. FEBS Lett. 1996; 397: 347-351Crossref PubMed Scopus (230) Google Scholar) and were a generous gift from Prof. D. G. Hardie, University of Dundee, Dundee, UK. All other reagents were from sources described previously (36Salt I.P. Connell J.M.C. Gould G.W. Diabetes. 2000; 49: 1649-1656Crossref PubMed Scopus (107) Google Scholar). HAEC Cell Culture—HAECs were grown in large vessel endothelial cell medium at 37 °C in 5% CO2 and passaged when at 80% confluence. Cells were used for experiments between passages 3 and 6. Evaluation of Nitric Oxide Release Using a Sievers NO Meter—Cells cultured in 24-well plates were preincubated for 1 h at 37 °C in 0.5 ml/well Krebs Ringer HEPES (KRH) buffer (119 mm NaCl, 20 mm Na-HEPES, pH 7.4, 5 mm NaHCO3, 4.7 mm KCl, 1.3 mm CaCl2, 1.2 mm MgSO4, 1 mm KH2PO4, 100 μm l-arginine, 5 mm glucose) at 37 °C. The medium was removed and replaced with 0.2 ml of fresh KRH buffer in the presence or absence of AICAR for 1 h. The medium was removed and refluxed in glacial acetic acid containing NaI. Under these conditions, NO2- , a stable breakdown product of NO, is quantitatively reduced to NO. NO-specific chemiluminescence was then analyzed using a Sievers 280A NO meter. Values were corrected for NO2- present in media in the absence of cells, and the appropriate control experiments were performed in the presence of the eNOS inhibitor, l-NAME. NO production (adjusted for l-NAME-insensitive production) is expressed per hour per well. Evaluation of Nitric Oxide Release by DAF-2 Fluorescence—To further determine NO production in HAECs, a membrane-permeable fluorescent indicator DAF-2DA was used that allows a quantitative determination of NO production (37Nakatsubo N. Kojima H. Kikuchi K. Nagoshi H. Hirata Y. Maeda D. Imai Y. Irimura T. Nagano T. FEBS Lett. 1998; 427: 263-266Crossref PubMed Scopus (338) Google Scholar). Cells grown on coverslips in 6-well plates were preincubated for 30 min at 37 °C in 1 ml/well KRH buffer in the presence or absence of 0.1 mm l-NAME. DAF-2DA (5 μm final) was added, and the coverslips were incubated for a further 15 min at 37 °C. The medium was removed and replaced with 1 ml of fresh KRH buffer in the presence or absence of l-NAME and test substances. After incubation for 30 min at 37 °C, the medium was removed, and the cells fixed in 4% (w/v) paraformaldehyde in phosphate-buffered saline, washed, and mounted on slides. Specimens were observed under a Zeiss LSM5 Pascal laser-scanning confocal microscope with a 40× objective, excitation wavelength of 488 nm, and a LP515-nm filter. Triplicate coverslips were prepared for each experimental condition, and 10 random images of HAECs were collected from each and quantified using MetaMorph (Universal Imaging, West Chester, PA) software. Preparation of HAEC Lysates—Cells grown in 100-mm diameter cell culture dishes were preincubated for 1 h at 37 °C in 5 ml of KRH buffer. The medium was replaced with 5 ml of fresh KRH buffer containing test substances and incubated for various durations at 37 °C. The medium was removed, and 0.5 ml of ice-cold lysis buffer (50 mm Tris-HCl, pH 7.4, at 4 °C, 50 mm NaF, 5 mm Na4P2O7, 2 mm Na3VO4, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 0.1 mm benzamidine, 0.1 mm phenylmethylsulfonyl fluoride, 5 μg/ml soybean trypsin inhibitor, 1% (w/v) Thesit, 250 mm mannitol) added. The cell extract was scraped off and transferred to a microcentrifuge tube. Extracts were vortex-mixed and centrifuged (14,000 × g, 3 min, 4 °C). Supernatants were snap-frozen in liquid N2 and stored at –80 °C before use. Sequential PEG precipitation was used to prepare 2.5–6.25% PEG precipitates from some lysates, which were snap-frozen in liquid N2 and stored at –80 °C before AMPK assay (38Hardie D.G. Haystead T.A.J. Salt I.P. Davies S.P. Protein Phosphorylation: A Practical Approach. 2nd Ed. Oxford University Press, Oxford1999: 201-219Google Scholar). Adenoviruses—Dominant negative AMPK adenovirus (Ad.α1-DN) was constructed from AMPKα1 containing a mutation altering Asp-157 to alanine (D157A) as described previously (6Woods A. Azzout-Marniche D. Foretz M. Stein S.C. Lemarchand P. Ferre P. Foufelle F. Carling D. Mol. Cell. Biol. 2000; 20: 6704-6711Crossref PubMed Scopus (358) Google Scholar). Recombinant adenoviruses were propagated in HEK293 cells, purified by cesium chloride density centrifugation, and stored as described previously (6Woods A. Azzout-Marniche D. Foretz M. Stein S.C. Lemarchand P. Ferre P. Foufelle F. Carling D. Mol. Cell. Biol. 2000; 20: 6704-6711Crossref PubMed Scopus (358) Google Scholar). Infection of HAECs—HAECs were infected with 10 Pfu/cell adenovirus in complete medium. After 2 h, 1 further volume of complete medium was added, and the cells were cultured for 48 h before experimentation. Preliminary studies revealed that within 48 h of infection with a green fluorescent protein-expressing virus, the majority (>95%) of HAECs expressed green fluorescent protein. Immunoprecipitation of eNOS—HAEC lysate (200 μg) was added to 20 μl of 25% (v/v) protein G-Sepharose prebound to 5 μg of mouse anti-eNOS, and the volume was made up to 300 μl with lysis buffer and mixed for 2 h at 4 °C on a rotating mixer. The mixture was then centrifuged (14,000 × g, 30 s, 4 °C), and the pellet was washed 3 times with 1 ml of lysis buffer at 4 °C. Immunoprecipitation and Assay of AMPK—AMPK was immunoprecipitated from lysates and assayed using the AMARA substrate peptide as described previously (36Salt I.P. Connell J.M.C. Gould G.W. Diabetes. 2000; 49: 1649-1656Crossref PubMed Scopus (107) Google Scholar). An AMARA peptide kinase could also be detected in PEG precipitates resuspended in HEPES-Brij buffer (50 mm HEPES, pH 7.4, 1 mm dithiothreitol, 0.02% (v/v) Brij-35) as described previously (38Hardie D.G. Haystead T.A.J. Salt I.P. Davies S.P. Protein Phosphorylation: A Practical Approach. 2nd Ed. Oxford University Press, Oxford1999: 201-219Google Scholar). Protein concentration was determined by the method of Bradford (39Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar). Statistics—Unless stated otherwise, results are expressed as the means ± S.D. Statistically significant differences were determined using a 2-tailed independent-samples Student's t test, with p < 0.05 as significant using Statview software. The expression of two distinct isoforms of the catalytic α subunit of AMPK (termed α1 and α2) has been demonstrated in a number of tissues (3Cheung P.C.F. Salt I.P. Davies S.P. Hardie D.G. Carling D. Biochem. J. 2000; 346: 659-669Crossref PubMed Scopus (533) Google Scholar, 40Stapleton D. Mitchelhill K.I. Gao G. Widmer J. Michell B.J. Teh T. House C.M. Fernandez C.S. Cox T. Witters L.A. Kemp B.E. J. Biol. Chem. 1996; 271: 611-614Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar). To determine the effects of AICAR and metformin on AMPK activity in HAECs, total AMPK was assayed in HAEC lysates prepared from cells incubated for various duration in 0.5 mm AICAR or 2 mm metformin after immunoprecipitation with a mixture of anti-α1 and anti-α2 antibodies. Treatment of HAECs with AICAR elicited a transient activation of AMPK. The effect reached a maximum 2.3-fold stimulation at 30 min, which was sustained for a further 30 min (Fig. 1A). However, treatment of HAECs with metformin had no significant effect on AMPK activity over the time course used in this study (Fig. 1A). Activation of AMPK by AICAR was dose-dependent (Fig. 1B) such that AMPK was stimulated maximally (∼3-fold) by 2 mm AICAR, a concentration at which all further experiments were performed. To determine the relative activities of AMPK complexes containing either catalytic subunit isoform in HAECs, AMPK was immunoprecipitated using anti-α1 or -α2 antibodies and assayed for AMPK activity under basal and AICAR-stimulated conditions. The majority (∼90%) of the AMPK activity in both basal and AICAR-stimulated conditions was in complexes containing the α1 catalytic subunit isoform (Table I).Table IThe effect of 2 mm AICAR on catalytic α subunit isoform-specific AMPK activity in HAECsImmunoprecipitating antibodyBasal2 mm AICARAMPKα10.30 ± 0.060.78 ± 0.15AMPKα20.02 ± 0.010.06 ± 0.04AMPKα1 and AMPKα20.36 ± 0.080.87 ± 0.11 Open table in a new tab Next we examined the ability of AICAR to modulate NO production in HAECs. AICAR-stimulated NO production was measured using a Sievers 280A NO meter. AICAR stimulated l-NAME-sensitive NO production in a dose-dependent manner and was stimulated maximally by 2 mm AICAR (Fig. 2A). AICAR-stimulated NO production was only marginally less than that elicited by A23187, which directly increases intracellular Ca2+ concentrations or insulin, which has been demonstrated to stimulate NO production via PKB-mediated phosphorylation and activation of eNOS at Ser-1177 (17Montagnani M. Chen H. Barr V.A. Quon M.J. J. Biol. Chem. 2001; 276: 30392-30398Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar, 20Kim F. Gallis B. Corson M.A. Am. J. Physiol. 2001; 280: C1057-C1065Crossref PubMed Google Scholar) (Fig. 2A). To confirm the stimulation of NO production by AICAR, HAECs were loaded with DAF-2, a dye that upon binding to an oxidized species of NO results in fluorescence. Quantification of confocal images of DAF-2 fluorescence demonstrated the dose-dependent stimulation of NO production in cells stimulated with AICAR for 30 min (Fig. 2B). AICAR-stimulated DAF-2 fluorescence was completely inhibited in cells pretreated with the NOS inhibitor l-NAME (Fig. 2B). AMPK has been demonstrated to be associated with eNOS immunoprecipitated from rat heart homogenates and also to phosphorylate eNOS at Ser-1177 in vitro (14Chen Z.-P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (717) Google Scholar). As shown in Fig. 3, AMPKα1 was present in eNOS immunoprecipitates, yet increasing concentrations of AICAR had no effect on the level of AMPK associated with eNOS. We next used an anti-phospho-Ser-1177 antibody to determine the phosphorylation of eNOS at Ser-1177 in HAEC lysates from cells stimulated with A

Referência(s)