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• W2017009595 abstract "The plasma copper protein ceruloplasmin (CP) was found to inhibit endothelial nitric-oxide synthase activation in cultured endothelial cells, in line with previous evidence showing that the endothelium-dependent vasorelaxation of the aorta is impaired by physiological concentrations of ceruloplasmin. The data presented here indicate a direct relationship between the extent of inhibition of agonist-triggered endothelial nitric oxide synthase activation and CP-induced enrichment of the copper content of endothelial cells. Copper discharged by CP was mainly localized in the soluble fraction of cells. The subcellular distribution of the metal seems to be of relevance to the inhibitory effect of CP, because it was mimicked by copper chelates, like copper-histidine, able to selectively enrich the cytosolic fraction of cells, but not by copper salts, which preferentially located the metal to the particulate fraction. The plasma copper protein ceruloplasmin (CP) was found to inhibit endothelial nitric-oxide synthase activation in cultured endothelial cells, in line with previous evidence showing that the endothelium-dependent vasorelaxation of the aorta is impaired by physiological concentrations of ceruloplasmin. The data presented here indicate a direct relationship between the extent of inhibition of agonist-triggered endothelial nitric oxide synthase activation and CP-induced enrichment of the copper content of endothelial cells. Copper discharged by CP was mainly localized in the soluble fraction of cells. The subcellular distribution of the metal seems to be of relevance to the inhibitory effect of CP, because it was mimicked by copper chelates, like copper-histidine, able to selectively enrich the cytosolic fraction of cells, but not by copper salts, which preferentially located the metal to the particulate fraction. Ceruloplasmin (CP) 1The abbreviations CPceruloplasminapoCPapoceruloplasminBkbradykininDMEMDulbecco's modified Eagle's mediumeNOSendothelial nitric-oxide synthaseFBSfetal bovine serumm-HBSSmodified Hank's balanced salt solution (136 mm NaCl, 5 mm KCl, 4 mmNaHCO3, 1 mm KH2PO4, and 3 mm Na2HPO4)l-NAMENω-nitro-l-arginine methyl esterNOnitric oxideOAECovine aortic endothelial cellPBSphosphate-buffered salineCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid 1The abbreviations CPceruloplasminapoCPapoceruloplasminBkbradykininDMEMDulbecco's modified Eagle's mediumeNOSendothelial nitric-oxide synthaseFBSfetal bovine serumm-HBSSmodified Hank's balanced salt solution (136 mm NaCl, 5 mm KCl, 4 mmNaHCO3, 1 mm KH2PO4, and 3 mm Na2HPO4)l-NAMENω-nitro-l-arginine methyl esterNOnitric oxideOAECovine aortic endothelial cellPBSphosphate-buffered salineCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidis a copper-containing glycoprotein that is found in the plasma of all vertebrates, in which it carries approximately 90% of plasma copper (1Rydèn L. Lontie R. Copper Proteins and Copper Enzymes. 3. CRC Press Inc., Boca Raton, FL1984: 37-100Google Scholar). Each CP molecule tightly binds six copper atoms to the three different copper binding sites that characterize blue oxidases (2Musci G. Bonaccorsi di Patti M.C. Fagiolo U. Calabrese L. J. Biol. Chem. 1993; 268: 13388-13392Abstract Full Text PDF PubMed Google Scholar, 3Zaitseva I. Zaitsev V. Card G. Moshkov K. Bax B. Ralph A. Lindley P. J. Biol. Inorg. Chem. 1996; 1: 15-23Crossref Scopus (351) Google Scholar). Nevertheless, as indicated by considerable experimental evidence, it is particularly prone to transfer its copper atoms to tissues (4Marceau N. Aspin N. Biochim. Biophys. Acta. 1973; 328: 338-350Crossref PubMed Scopus (68) Google Scholar, 5Campbell C.H. Brown R. Linder M.C. Biochim. Biophys. Acta. 1981; 678: 27-38Crossref PubMed Scopus (74) Google Scholar, 6Harris E.D. Prog. Clin. Biol. Res. 1993; 380: 163-179PubMed Google Scholar) delivering copper to intracellular copper proteins (7Dameron C.T. Harris E.D. Biochem. J. 1987; 248: 669-675Crossref PubMed Scopus (57) Google Scholar, 8Hsieh H.S. Frieden E. Biochem. Biophys. Res. Commun. 1975; 67: 1326-1331Crossref PubMed Scopus (71) Google Scholar). However, recent studies on aceruloplasminemic patients (9Harris Z.L. Takahashi Y. Miyajima H. Serizawa M. MacGillivray R.T.A. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2539-2543Crossref PubMed Scopus (506) Google Scholar, 10Morita H. Ikeda S. Yamamoto K. Morita S. Yoshida K. Nomoto S. Kato M. Yanagisawa N. Ann. Neurol. 1995; 37: 646-656Crossref PubMed Scopus (229) Google Scholar, 11Yoshida K. Furihata K. Takeda S. Nakamura A. Yamamoto K.K. Morita H. Hiyamuta S. Ikeda S. Shimizu N. Yanagisawa N. Nat. Genet. 1995; 9: 267-272Crossref PubMed Scopus (420) Google Scholar) indicate that this protein has no essential role in copper transport, whereas it plays a primary role in iron homeostasis, possibly through its ferroxidase activity (12Frieden E. Hsieh H.S. Adv. Enzymol. 1976; 44: 187-236PubMed Google Scholar). ceruloplasmin apoceruloplasmin bradykinin Dulbecco's modified Eagle's medium endothelial nitric-oxide synthase fetal bovine serum modified Hank's balanced salt solution (136 mm NaCl, 5 mm KCl, 4 mmNaHCO3, 1 mm KH2PO4, and 3 mm Na2HPO4) Nω-nitro-l-arginine methyl ester nitric oxide ovine aortic endothelial cell phosphate-buffered saline 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid ceruloplasmin apoceruloplasmin bradykinin Dulbecco's modified Eagle's medium endothelial nitric-oxide synthase fetal bovine serum modified Hank's balanced salt solution (136 mm NaCl, 5 mm KCl, 4 mmNaHCO3, 1 mm KH2PO4, and 3 mm Na2HPO4) Nω-nitro-l-arginine methyl ester nitric oxide ovine aortic endothelial cell phosphate-buffered saline 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid Ceruloplasmin is an acute-phase reactant; its concentration in the plasma increases up to 3-fold during pregnancy and during multiple pathological processes including trauma and inflammation (13Cousins R.J. Physiol. Rev. 1985; 65: 238-309Crossref PubMed Scopus (1099) Google Scholar). Recent attention has focused on the role that this protein may have in the function of the vascular system in health and disease. CP has been detected in human atherosclerotic lesions (14Hollander W. Colombo M.A. Kirkpatrick B. Paddock J. Atherosclerosis. 1979; 34: 391-405Abstract Full Text PDF PubMed Scopus (119) Google Scholar, 15Swain J. Gutteridge J.M.C. FEBS Lett. 1995; 368: 513-515Crossref PubMed Scopus (135) Google Scholar), and it has been shown to oxidize low density lipoproteins in the presence of vascular cells (16Mukhopadhyay C.K. Fox P.L. Biochemistry. 1998; 37: 14222-14229Crossref PubMed Scopus (53) Google Scholar). A copper binding site labile to Chelex treatment has been proposed to be responsible for the oxidative damage to low density lipoproteins (17Mukhopadhyay C.K. Mazumder B. Lindley P.F. Fox P.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11546-11551Crossref PubMed Scopus (115) Google Scholar). On the other hand, we have shown that CP, at physiological concentrations, inhibits the endothelium-dependent relaxation of rabbit aorta induced by agonists and that this effect is not due to a trapping of NO by the copper sites (18Cappelli-Bigazzi M. Ambrosio G. Musci G. Battaglia C. Bonaccorsi di Patti M.C. Golino P. Ragni M. Chiariello M. Calabrese L. Am. J. Physiol. 1997; 273: H2843-H2849PubMed Google Scholar). Vasodilation requires a (NO)-cGMP transduction pathway between endothelium and smooth muscle cells (19Furchgott R.F. Zawadzki J.V. Nature. 1980; 288: 373-376Crossref PubMed Scopus (9872) Google Scholar, 20Rees D.D. Palmer R.M.J. Moncada S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3375-3378Crossref PubMed Scopus (1626) Google Scholar). Endothelial NO synthase (eNOS) is a constitutive enzyme that converts l-arginine into NO and citrulline (21Palmer R.M.J. Ashton D.S. Moncada S. Nature. 1988; 333: 664-666Crossref PubMed Scopus (4086) Google Scholar) with a relatively low basal activity (22Förstermann U. Pollock J.S. Schmidt H.H.H.W. Heller M. Murad F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1788-1792Crossref PubMed Scopus (545) Google Scholar,23Wang J. Stuehr D.J. Ikeda-Saito M. Rousseau D.L. J. Biol. Chem. 1993; 268: 22255-22258Abstract Full Text PDF PubMed Google Scholar). After agonist stimulation evoking an increase in the [Ca2+]i concentration, Ca2+-bound calmodulin disrupts the inhibitory eNOS-caveolin-1 interactions (24Ju H. Zou R. Venema V.J. Venema R.C. J. Biol. Chem. 1997; 272: 18522-18525Abstract Full Text Full Text PDF PubMed Scopus (524) Google Scholar, 25Garcia-Cardena G. Martasek P. Masters B.S.S. Skidd P.M. Couet J. Li S. Lisanti M.P. Sessa W.C. J. Biol. Chem. 1997; 272: 25437-25440Abstract Full Text Full Text PDF PubMed Scopus (689) Google Scholar), thereby allowing conformational changes within eNOS, leading to the activated form that produces NO (26Michel J.B. Feron O. Sacks D. Michel T. J. Biol. Chem. 1997; 272: 15583-15586Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar, 27Michel J.B. Feron O. Sase K. Prabhakar P. Michel T. J. Biol. Chem. 1997; 272: 25907-25912Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). The agonist bradykinin (Bk) has a distinct role among vasodilators because it has also been shown that its receptor physically associates with eNOS (28Ju H. Venema V.J. Marrero M.B. Venema R.C. J. Biol. Chem. 1998; 273: 24025-24029Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). In this context, it should be recalled that a regulatory role for dietary copper in the control of vascular functions has been assessed by numerous studies focused mainly on copper deficiency-induced defects of vessels and on the impairment of NO-mediated vasodilation under copper restriction (reviewed in Ref. 29Schuschke D.A. J. Nutr. 1997; 127: 2274-2281Crossref PubMed Scopus (43) Google Scholar). On the contrary, an ability of copper ions (Cu2+) to relax vessels seems well established. It has been shown that copper enhances the relaxation of precontracted aortic rings evoked by the calcium ionophore A23187 and sodium nitroprusside (30Plane F. Wigmore S. Angelini G.D. Jeremy J.Y. Br. J. Pharmacol. 1997; 121: 345-350Crossref PubMed Scopus (36) Google Scholar), and it also elevates intracellular cGMP levels and induces relaxation of pulmonary arterial rings (31Ohnishi T. Ishizaki T. Sasaki F. Ameshima S. Nakai T. Miyabo S. Matsukawa S. Eur. J. Pharmacol. 1997; 319: 49-55Crossref PubMed Scopus (19) Google Scholar). More recently, it has been demonstrated that copper induces the activation of eNOS in cultured endothelial cells (32Demura Y. Ameshima S. Ishizaki T. Okamura S. Miyamori I. Matsukawa S. Free Radical Biol. Med. 1998; 25: 314-320Crossref PubMed Scopus (27) Google Scholar), an event supposed to occurin vivo in hypercupremic states induced by Cu2+released by ceruloplasmin. This result is apparently in contrast with our previous findings (18Cappelli-Bigazzi M. Ambrosio G. Musci G. Battaglia C. Bonaccorsi di Patti M.C. Golino P. Ragni M. Chiariello M. Calabrese L. Am. J. Physiol. 1997; 273: H2843-H2849PubMed Google Scholar), which are in better accordance with the inhibitory effects exerted by divalent metal ions on eNOS (32Demura Y. Ameshima S. Ishizaki T. Okamura S. Miyamori I. Matsukawa S. Free Radical Biol. Med. 1998; 25: 314-320Crossref PubMed Scopus (27) Google Scholar, 33Howard A.B. Alexander R.W. Taylor W.R. Am. J. Physiol. 1995; 269: C612-C618Crossref PubMed Google Scholar) as well as on neuronal nitric oxide synthase (34Quinn M.R. Harris C.L. Neurosci. Lett. 1995; 196: 65-68Crossref PubMed Scopus (34) Google Scholar), another constitutive enzyme, when activity measurements are performed on crude cell extracts or on the purified enzyme (35Persechini A. McMillan K. Siler Master B.S. Biochemistry. 1995; 34: 15091Crossref PubMed Scopus (62) Google Scholar). To clarify the mechanism underlying the inhibitory effect of CP on the relaxation of rabbit aortic vessels (18Cappelli-Bigazzi M. Ambrosio G. Musci G. Battaglia C. Bonaccorsi di Patti M.C. Golino P. Ragni M. Chiariello M. Calabrese L. Am. J. Physiol. 1997; 273: H2843-H2849PubMed Google Scholar), we tested whether this protein could affect the agonist-induced activation of eNOS in cultured OAECs. Here we show that exogenously added CP reversibly reduces the formation of cGMP, nitrite, and citrulline but not Ca2+mobilization in endothelial cells stimulated with agonists, with a time course consistent with that of copper delivery to cells, and we show that copper bound to histidine, but not free ionic copper, mimics the effect of CP. Altogether, the results are consistent with a mechanism whereby intracellular, CP-derived copper inhibits eNOS activation by agonists. All reagents were purchased from Sigma Italia (Milan, Italy) unless otherwise noted and were used without further purification. Radioactive chemicals were from Amersham Pharmacia Biotech (Milan, Italy). OAECs were harvested from the internal surface of aortas according to a previously described procedure (36Gospodarowicz D. Moran J. Braun D. Birdwell C. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 4120-4124Crossref PubMed Scopus (349) Google Scholar) using collagenase XI and grown in a culture medium containing DMEM (Life Technologies, Inc.) supplemented with 10% FBS (Life Technologies, Inc.), 30 μg/ml endothelial cell growth supplement (Sigma Italia), 100 units/ml penicillin, 100 μg/ml streptomycin, and 50 μg/ml gentamicin (Life Technologies, Inc.) at 37 °C under an atmosphere of 5% CO2 in air. Their identity was verified by their morphological features and immunofluorescence staining with antibodies to factor VIII. The confluent monolayers were subcultured by conventional trypsinization. For the present study, cells were used at the third to sixth passages. Stimulation by agonists was carried out with confluent cells in either modified Hanks' balanced salt solution (m-HBSS), serum-free DMEM, or DMEM supplemented with 10% FBS (DMEM/FBS). To remove possible traces of thrombin, all CP samples were treated with benzamidine-Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden) immediately before addition to cells. Sheep and human CP were purified as described previously (2Musci G. Bonaccorsi di Patti M.C. Fagiolo U. Calabrese L. J. Biol. Chem. 1993; 268: 13388-13392Abstract Full Text PDF PubMed Google Scholar, 37Calabrese L. Carbonaro M. Musci G. J. Biol. Chem. 1989; 264: 6183-6187Abstract Full Text PDF PubMed Google Scholar) and further purified by mono Q fast protein liquid chromatography to remove traces of prothrombin. The resulting proteins were >99% pure as judged by spectroscopic and electrophoretic analyses. In some experiments, purified CP samples were treated with Chelex 100 (38Ehrenwald E. Chisolm G.M. Fox P.L. J. Clin. Invest. 1994; 93: 1493-1501Crossref PubMed Scopus (237) Google Scholar). Apoceruloplasmin (ApoCP) was prepared as described previously (39Musci G. Di Marco S. Bellenchi G.C. Calabrese L. J. Biol. Chem. 1996; 271: 1972-1978Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). ApoCP had a residual oxidase activity of ∼10% with respect to the native protein, consistent with the presence of ∼10% unremoved copper. Neutralization of CP with anti-CP antibody was carried out by preincubating CP on ice for 30 min with a 2-fold excess of a polyclonal specific anti-sheep ceruloplasmin antibody (generous gift of Dr. Marmocchi) before the treatment of cells. To determine [cGMP]ilevels in cultured OAECs, cells were preincubated in complete DMEM for 30 min at 37 °C under 5% CO2 with 1 mmisobutylmethylxanthine, a phosphodiesterase inhibitor, before treatment with effectors and/or agonists. At the end of the stimulation with the agonist (10 min), cells were washed with PBS and lysed in 10 mm Tris/HCl, pH 7.4, with 0.5% Triton X-100. The lysate was collected in 1% perchloric acid and centrifuged at 10,000 ×g for 10 min at 4 °C. Precipitated proteins were determined with either the biuret method (43Goa J. Scand. J. Clin. Lab. Invest. 1953; 37: 218-223Crossref Scopus (794) Google Scholar) or the bicinchoninic acid reagent (Pierce), whereas the supernatants were lyophilized and assayed for cGMP by radioimmunoassay (Amersham Pharmacia Biotech). Each experiment was performed in duplicate. Formation of nitrites in the medium was quantitated fluorimetrically with 2,3-diaminonaphthalene according to Misko (40Misko P.T. Schilling R.J. Salvemini D. Morre W.M. Currie M.G. Anal. Biochem. 1993; 214: 11-16Crossref PubMed Scopus (954) Google Scholar). Briefly, confluent cells in 100-mm dishes were stimulated in DMEM/FBS without phenol red, and the medium (2 ml) was collected and centrifuged at 10,000 × g for 10 min. 250 μl of a solution of 2,3-diaminonaphthalene (5 mg/100 ml in 0.62n HCl) were added to the supernatant, and the mixture was incubated for 10 min at 20 °C in the dark. After the addition of 250 μl of 1.4 m NaOH, samples were filtered through a 0.45-μm cellulose acetate filter (Iwaki, Japan), and the fluorescence was measured with λex = 375 nm and λem = 426 nm on a Perkin Elmer LS50-B spectrofluorimeter. A standard curve was constructed with a known concentration of sodium nitrite in the same medium. To measure total NOx (nitrite + nitrate), nitrates were first converted to nitrite by incubation for 15 min at 37 °C with 50 milliunits/ml nitrate reductase fromAspergillus sp. in the presence of 30 μmNADPH. Residual NADPH was oxidized by incubation for 5 min at 37 °C with lactic dehydrogenase (5 milliunits/ml) in the presence of 0.3 mm pyruvate. Measurements of total NOx were carried out using m-HBSS as the cell medium because DMEM contains micromolar levels of nitrate. eNOS activity in intact OAECs was assayed by measuring the intracellular formation of [3H]citrulline using the method of Hu and El-Fakahany (41Hu J. El-Fakahany E.E. J. Neurochem. 1995; 65: 117-124Crossref PubMed Scopus (9) Google Scholar), modified as follows. Confluent cells grown in 6-well dishes were loaded with [3H]arginine by a 15-min incubation at 37 °C with DMEM/FBS containing 2 mm glutamine, 1 mm citrulline, and [3H]arginine (10 μCi/ml, 5 μCi/well, and 1 μCi/40 nmol total arginine). After an additional 15-min incubation with or without CP, cells were treated with agonists, washed twice with PBS containing 1 mm arginine, and lysed in cold absolute methanol. Proteins were assayed after quantitative recovery from the wells with 0.1 m NaOH. The methanol extracts were dried in a Jouan RC 10.22 Speedvac, and pellets were redissolved in 500 μl of H2O. Chromatographic separation of [3H]citrulline from [3H]arginine was achieved by batch incubation of the mixture for 10 min with 100 μl of a Dowex 50WX8 slurry under continuous mild agitation. Supernatants were counted for radioactivity to evaluate intracellular formation of [3H]citrulline. Values were normalized for the protein content of each well. The conversion of [3H]arginine into [3H]citrulline was also used to measure eNOS activity in OAEC homogenates, essentially as described by Hecker et al.(42Hecker M. Mulsch A. Bassenge E. Forstermann U. Busse R. Biochem. J. 1994; 299: 247-252Crossref PubMed Scopus (117) Google Scholar). Cells were detached by gently scraping the culture dishes and resuspended in 50 mm Tris/HCl, pH 7.4, containing 120 mm NaCl, 15 mm KCl, 5 mmMgCl2, 1 mm EDTA, 1 mmdithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 10 μg/liter leupeptin, 10 μg/liter pepstatin, 10 μg/liter aprotinin, and 20 mm CHAPS. The suspension was homogenized in ice with a mini-potter apparatus and then centrifuged at 10,000 ×g for 10 min at 4 °C. 20–50-μl aliquots of the supernatants were assayed for eNOS activity by the addition of 50 mm Tris/HCl, pH 7.4, containing 1 mm NADPH, 1.25 mm CaCl2, 1 mm dithiothreitol, 1 mm EDTA, 15 μm6R-tetrahydrobiopterin, 1 μm FAD, 1 μm FMN, 0.1 μm calmodulin, 10 μm arginine, and 5 μCi/ml [3H]arginine (total volume, 150 μl). After incubation at 37 °C for 60 min, the reaction was stopped with 5 volumes of 20 mm Hepes, pH 5.5, containing 10 mm EDTA. The mixture was loaded on a 1-ml column of Dowex 50WX8 pre-equilibrated in the latter buffer. The eluate was counted for radioactivity to evaluate the extent of [3H]citrulline formation. The effect of copper was assessed by adding increasing concentrations of CuCl2 to the homogenate 5 min before adding the cofactors. Cell lysates were separated by SDS-polyacrylamide gel electrophoresis. The proteins were electrophoretically transferred onto polyvinylidene difluoride membranes, immunodetection was carried out with either anti-eNOS (1:1000; Transduction Laboratories) or anti-CP and visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech). Cells were incubated with the indicated amounts of CP or copper-histidine, prepared by adding 1 mol of CuCl2 to 3 mol of histidine in water, or CuCl2, in DMEM/FBS, DMEM or m-HBSS. At the end of the incubation, cells were washed three times with PBS containing 1 mm EDTA and either lysed in 10 mm Tris/HCl buffer, pH 7.4, with 0.5% Triton X-100 to determine total copper content or collected by scraping and homogenized by 30 strokes in a mini-Potter apparatus in ice-cold homogenization buffer (50 mm Tris/HCl buffer, pH 7.4, containing 120 mmNaCl, 15 mm KCl, 5 mm MgCl2, 1 mm EDTA, 10 μg/liter leupeptin, 10 μg/liter pepstatin A, 10 μg/liter aprotinin, and 1 mm phenylmethylsulfonyl fluoride) and centrifuged for 60 min at 100,000 × g. The pellets were resuspended in 50 mm Tris/HCl buffer, pH 7.4, containing 1% Triton X-100. Copper content was determined on aliquots of lysates, homogenates, supernatants, and pellets after digestion with HNO3 by flameless atomic absorption (Perkin Elmer 3030 spectrometer equipped with graphite furnace). The data were normalized by dividing picomoles Cu by mg proteins. Proteins were determined using either the biuret method (43Goa J. Scand. J. Clin. Lab. Invest. 1953; 37: 218-223Crossref Scopus (794) Google Scholar) or the bicinchoninic acid reagent (Pierce). Cells were grown at confluence in chamber slides (Lab-Tek; Nunc, Naperville, IL) and treated with 4 μm fura 2-acetoxymethyl ester for 45 min at 37 °C to monitor variations of cytosolic free Ca2+ concentrations (44Wickham N.W.R. Vercellotti G.M. Moldow C.F. Visser M.R. Jacob H.S. J. Lab. Clin. Med. 1988; 112: 157-167PubMed Google Scholar). Emission fluorescence at 510 nm was measured upon excitation at 340 nm and 380 nm and expressed asF 340/F 380. Nitric-oxide synthase is activated in endothelial cells by several agonists including bradykinin and acetylcholine. Because this leads to NO-dependent activation of endothelial guanylate cyclase (45Mayer B. Schrammel A. Klatt P. Koesling D. Schmidt K. J. Biol. Chem. 1995; 270: 17355-17360Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar), the rise of the intracellular cyclic GMP concentration, [cGMP]i, was used as an index of eNOS activity. Untreated OAECs have a basal level of cGMP that was not affected by CP (Fig. 1 A). Bk induced an approximately 6-fold increase of [cGMP]i that was abolished by inhibition of eNOS by 1 mml-NAME. When added to cells 15 min before stimulation, CP had a strong, dose-dependent inhibitory effect on the agonist-induced increase of cGMP levels, with a maximal effect at 10 μm concentration. The inhibition was already evident at 1 μm CP and required the native form, because copper-free CP had a small effect, the magnitude of which was accounted for by the presence of approximately 10% residual active holoprotein (see “Experimental Procedures”). Treatment of CP samples with Chelex 100 (38Ehrenwald E. Chisolm G.M. Fox P.L. J. Clin. Invest. 1994; 93: 1493-1501Crossref PubMed Scopus (237) Google Scholar) to remove loosely bound copper before the addition to cells had no effects on the results shown. Neutralization by a specific antibody substantially relieved the effect of 10 μm CP. By itself, the antibody affected neither the basal nor the bradykinin-stimulated levels of cGMP. To examine whether CP altered [cGMP]i through NO-independent routes rather than affecting eNOS catalytic activity, the eNOS activity was monitored using the conversion of [3H]arginine to [3H]citrulline as a measure of NO synthase activity (Fig. 1 B). Stimulation of untreated cells by Bk produced a 3-fold enhancement of citrulline production, which was abolished by l-NAME. Preincubation of cells with CP before stimulation substantially reduced Bk-stimulated citrulline production in the same manner as that observed for cGMP production, indicating that the lower levels of [cGMP]iattained by cells stimulated in the presence of extracellular CP were indeed related to an inhibition of eNOS activity. Consistent with this finding, CP strongly hindered the release of NO in the medium. OAECs stimulated in m-HBSS for 10 min with 1 μm bradykinin produced ∼130 pmol/mg protein NOx (nitrite + nitrate), with over 80% of NOx accounted for by nitrite. Preincubation of cells with 10 μm CP for 15 min nearly abolished NOx production. Human CP was also effective in inhibiting the agonist-induced eNOS activation, being ∼80% as active as the ovine enzyme. The effect of CP was independent of the signaling pathway responsible for eNOS activation. As revealed by [cGMP]i(Fig. 1 C) and [3H]citrulline (Fig.1 D), CP exerted a strong inhibitory effect on the response of OAECs to acetylcholine or ADP and on the non-receptor-dependent response to the Ca2+ionophore A23187. Altogether, the data reported in Fig. 1,A-D, show that the effect of CP is independent of the agonist used to stimulate cells and of the product of eNOS activation that is monitored. The fact that similar end points are obtained with all tested agonists suggests that the effect of CP is exerted on the common target of all agonists, i.e. on eNOS itself. In agreement with the observation that aortic rings recover the capability to relax after the removal of CP (18Cappelli-Bigazzi M. Ambrosio G. Musci G. Battaglia C. Bonaccorsi di Patti M.C. Golino P. Ragni M. Chiariello M. Calabrese L. Am. J. Physiol. 1997; 273: H2843-H2849PubMed Google Scholar), these cells were again found to be responsive to agonists upon the removal of CP and subsequent incubation in fresh medium. However, the results reported in Fig. 1 E show that cells that had been exposed to 10 μm CP need 30 min to recover full response to the agonist, either Bk or acetylcholine. The extent of inhibition of the agonist-induced activation of eNOS depended on the time of exposure of cells to CP. In Fig.2, the levels of [cGMP]i, intracellular [3H]citrulline, and nitrite in the medium are shown for cells stimulated with bradykinin at different times after the addition of 10 μm CP to the incubation medium. It should be noted that the indicated times actually encompassed the 10-min time period required for stimulation. It is evident that: (i) the first 15-min preincubation with CP caused an essentially complete (nearly 80%) inhibition of eNOS activity that remained at this level for more than ∼60 min, and (ii) a small inhibition occurred when CP was added simultaneously to bradykinin, i.e. zero time of preincubation. Moreover, no significant inhibition was found when CP was added 2 min after the agonist. This experiment was critical because it showed that CP had to be in contact with cells before the agonist in order to exert its effect and was nearly ineffective in suppressing the response of cells once a response had been evoked. It should be noted that the kinetics of formation of citrulline and nitrite (i.e. NO) are similar; however, they differ at short times from that of cGMP. The lag phase observed in the latter case could be explained when we consider that the formation of cGMP requires the secondary activation of guanylate cyclase. Because agonists activate eNOS through enhancing the free cytosolic calcium concentration, the next question was whether CP could interfere with calcium fluxes in endothelial cells. As indicated by a representative experiment (Fig. 3), theF 340/F 380 fluorescence intensity ratio, which increases upon elevation of [Ca2+]i, varied after stimulation of OAECs by bradykinin, independently of prior exposure of cells to CP. Similar results were also obtained with A23187, with the ionophore inducing only a bigger variation of the fluorescence intensity ratio (data not shown). It has been shown repeatedly that CP interacts directly with the membrane surface of cells of many tissues, including heart and aorta (46Stevens M.D. DiSilvestro R.A. Harris E.D. Biochemistry. 1984; 23: 261-266Crossref PubMed Scopus (63) Google Scholar), and that such interactions lead to a transmembrane transport of copper but not of the protein moiety (47Percival S.S. Harris E.D. Am. J. Physiol. 1990; 258: C140-C146Crossref PubMed Google Scholar). An enhanced metal exportation generally follows the increase of the intracellular concentration of copper in normal cells (48Dameron C.T. Harrison M.D. Am. J. Clin. Nutr. 1998; 67 Suppl. 5: 1091S-1097SCrossref Scopus (97) Google Scholar, 49Harris E.D. Qian Y. Tiffany-Castiglioni E. Lacy A.R. Reddy M.C. Am. J. Clin. Nutr. 1998; 67 Suppl. 5: 988S-995SCrossref Scopus (44) Google Scholar). To assess the possible effect on eNOS of a transient increase in copper levels, the delivery of copper to OAECs by CP was studied. CP affected the copper content of OAECs in a time-dependent manner (Fig. 4). OAECs exposed to 10 μm CP accumulated copper for at least 60 min, with an almost 7-fold increase, after 60 min, with respect to that of untreated cells (Fig. 4 A). Copper uptake by a number of cell types has been the object of intensive investigation, which has also shown that the metal does not easily redistribute among cellular components when cells are homogenized (50Waldrop G. Ettinger M.J. Am. J. Physiol. 1990; 259: G212-G218PubMed Google Scholar)." @default.
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• W2017009595 date "1999-07-01" @default.
• W2017009595 modified "2023-10-14" @default.
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