Autocrine Regulation of Inducible Nitric-oxide Synthase in Macrophages by Atrial Natriuretic Peptide
1998; Elsevier BV; Volume: 273; Issue: 22 Linguagem: Inglês
10.1074/jbc.273.22.13444
ISSN1083-351X
AutoresAlexandra K. Kiemer, Angelika M. Vollmar,
Tópico(s)Cardiac Fibrosis and Remodeling
ResumoAtrial natriuretic peptide (ANP), a cardiovascular hormone, has been shown to inhibit synthesis of nitric oxide in lipopolysaccharide (LPS)-activated mouse bone marrow-derived macrophages via activation of its guanylate cyclase-coupled receptor. The goal of the present study was to elucidate the potential sites of inducible nitric-oxide synthase (iNOS) regulation affected by ANP and revealed the following. 1) ANP and dibutyryl-cGMP did not inhibit catalytic iNOS activity measured by the conversion rate ofl-[3H]arginine tol-[3H]citrulline in homogenates of LPS-treated cells. 2) Pretreatment of cells with ANP dose-dependently reduced the LPS-inducedl-[3H]citrulline production that has been shown to be due to reduced iNOS protein levels detected by Western blot. 3) ANP does not alter the ratio of catalytically active iNOS dimer versus inactive iNOS monomer considered to be a major post-translational regulatory mechanism for the enzyme. 4) Macrophages exposed to ANP display decreased LPS-induced iNOS mRNA levels. 5) Importantly, two basic mechanisms seem to be responsible for this observation, i.e. ANP specifically induced acceleration of iNOS mRNA decay and ANP reduced binding activity of NF-κB, the transcription factor predominantly responsible for LPS-induced iNOS expression in murine macrophages. Moreover, 6) ANP acts via an autocrine mechanism since recently ANP was shown to be secreted by LPS-activated macrophages, and we demonstrated here that LPS-induced NO synthesis was increased after blocking the binding of endogenous ANP by a receptor antagonist. These observations suggest ANP as a new autocrine macrophage factor regulating NO synthesis both transcriptionally and post-transcriptionally. ANP may help to balance NO production of activated macrophages and thus may allow successful immune response without adverse effects on host cells. Atrial natriuretic peptide (ANP), a cardiovascular hormone, has been shown to inhibit synthesis of nitric oxide in lipopolysaccharide (LPS)-activated mouse bone marrow-derived macrophages via activation of its guanylate cyclase-coupled receptor. The goal of the present study was to elucidate the potential sites of inducible nitric-oxide synthase (iNOS) regulation affected by ANP and revealed the following. 1) ANP and dibutyryl-cGMP did not inhibit catalytic iNOS activity measured by the conversion rate ofl-[3H]arginine tol-[3H]citrulline in homogenates of LPS-treated cells. 2) Pretreatment of cells with ANP dose-dependently reduced the LPS-inducedl-[3H]citrulline production that has been shown to be due to reduced iNOS protein levels detected by Western blot. 3) ANP does not alter the ratio of catalytically active iNOS dimer versus inactive iNOS monomer considered to be a major post-translational regulatory mechanism for the enzyme. 4) Macrophages exposed to ANP display decreased LPS-induced iNOS mRNA levels. 5) Importantly, two basic mechanisms seem to be responsible for this observation, i.e. ANP specifically induced acceleration of iNOS mRNA decay and ANP reduced binding activity of NF-κB, the transcription factor predominantly responsible for LPS-induced iNOS expression in murine macrophages. Moreover, 6) ANP acts via an autocrine mechanism since recently ANP was shown to be secreted by LPS-activated macrophages, and we demonstrated here that LPS-induced NO synthesis was increased after blocking the binding of endogenous ANP by a receptor antagonist. These observations suggest ANP as a new autocrine macrophage factor regulating NO synthesis both transcriptionally and post-transcriptionally. ANP may help to balance NO production of activated macrophages and thus may allow successful immune response without adverse effects on host cells. Atrial natriuretic peptide (ANP) 1The abbreviations used are: ANP, atrial natriuretic peptide; AP-2, activating protein-2; BMM, bone marrow-derived macrophages; ABAE, adult bovine aortic endothelial cells; COX-2, cyclooxygenase-2; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay; FCS, fetal calf serum; IL, interleukin; INF, interferon; iNOS, inducible nitric-oxide synthase; IRF-1, interferon responding factor-1; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor κB; nNOS, neuronal nitric-oxide synthase; NO, nitric oxide; NOS, nitric-oxide synthase; NPR, natriuretic peptide receptor; PBS, phosphate-buffered saline; PDTC, pyrrolidinedithiocarbamate; PMSF, phenylmethylsulfonyl fluoride; SNP, sodium nitroprusside; TNF-α, tumor necrosis factor-α.1The abbreviations used are: ANP, atrial natriuretic peptide; AP-2, activating protein-2; BMM, bone marrow-derived macrophages; ABAE, adult bovine aortic endothelial cells; COX-2, cyclooxygenase-2; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay; FCS, fetal calf serum; IL, interleukin; INF, interferon; iNOS, inducible nitric-oxide synthase; IRF-1, interferon responding factor-1; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor κB; nNOS, neuronal nitric-oxide synthase; NO, nitric oxide; NOS, nitric-oxide synthase; NPR, natriuretic peptide receptor; PBS, phosphate-buffered saline; PDTC, pyrrolidinedithiocarbamate; PMSF, phenylmethylsulfonyl fluoride; SNP, sodium nitroprusside; TNF-α, tumor necrosis factor-α. is a 28-amino acid polypeptide secreted mainly by the heart atria in response to atrial stretch. The main and best studied actions of ANP are geared toward the regulation of volume pressure homeostasis (for review see Refs. 1Rosenzweig A. Seidman C.E.E. Annu. Rev. Biochem. 1991; 60: 229-255Crossref PubMed Scopus (345) Google Scholar and 2Maack T. Kidney Int. 1996; 49: 1732-1737Abstract Full Text PDF PubMed Scopus (128) Google Scholar). There are two biochemically and functionally distinct classes of natriuretic peptide receptors (NPR). Clearance receptors (NPR-C) are by far the most abundant class of NPR. Besides their well established role in removing ANP from the circulation, the NPR-C elicit biological functions by interacting with G-proteins (2Maack T. Kidney Int. 1996; 49: 1732-1737Abstract Full Text PDF PubMed Scopus (128) Google Scholar, 3Levin E.R. Am. J. Physiol. 1993; 264: E483-E489PubMed Google Scholar). The guanylate cyclase-coupled receptors (NPR-A) are signaling receptors that mediate all known cardiovascular and renal effects of ANP via cGMP (2Maack T. Kidney Int. 1996; 49: 1732-1737Abstract Full Text PDF PubMed Scopus (128) Google Scholar, 4Kishimoto I. Dubois S.K. Garbers D.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6215-6219Crossref PubMed Scopus (113) Google Scholar). The functions of the natriuretic peptides, however, are not restricted to the regulation of volume homeostasis as suggested earlier by demonstration of ANP and its receptors in diverse tissues besides the cardiovascular and renal system (5Gutkowska J. Nemer M. Endocr. Rev. 1989; 10: 519-536Crossref PubMed Scopus (193) Google Scholar). ANP was suggested to play a role in the immune system because thymus (6Vollmar A.M. Schulz R. Endocrinology. 1990; 126: 2277-2280Crossref PubMed Scopus (42) Google Scholar, 7Throsby M. Yang Z. Lee D. Huang W. Copolov D.L. Lim A.T. Endocrinology. 1993; 132: 2184-2190Crossref PubMed Scopus (16) Google Scholar, 8Vollmar A.M. Schmidt K.N. Schulz R. Endocrinology. 1996; 137: 1706-1713Crossref PubMed Scopus (47) Google Scholar) and macrophages (9Vollmar A.M. Schulz R. J. Clin. Invest. 1994; 94: 539-545Crossref PubMed Scopus (46) Google Scholar, 10Kiemer A.K. Vollmar A.M. Endocrinology. 1997; 138: 4282-4290Crossref PubMed Scopus (59) Google Scholar) are sites of synthesis of the natriuretic peptide and its receptors. In the course of functional investigations concerning ANP in the immune system, the peptide was found to inhibit maturation and differentiation of fetal thymus (11Vollmar A.M. J. Neuroimmunol. 1997; 78: 90-96Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) as well as proliferation of thymocytes of adult animals (8Vollmar A.M. Schmidt K.N. Schulz R. Endocrinology. 1996; 137: 1706-1713Crossref PubMed Scopus (47) Google Scholar). Recently, ANP was shown to reduce nitrite accumulation in lipopolysaccharide (LPS)-activated murine macrophages (10Kiemer A.K. Vollmar A.M. Endocrinology. 1997; 138: 4282-4290Crossref PubMed Scopus (59) Google Scholar). Thus, the peptide might interfere with the synthesis of a mediator that plays an important role in inflammation and host defense response (12Bogdan C. Ro¨llinghoff M. Vodovotz Y. Xie Q. Nathan C. Masihi N. Immunotherapy of Infection. Marcel Dekker, Inc., New York1994: 37-54Google Scholar). The enzyme nitric-oxide synthase (NOS), which catalyzes the synthesis of NO froml-arginine, exists in three different isoforms that differ in their tissue distribution, calcium dependence, and in the regulation of their expression (13Fo¨rstermann U. Kleinert H. Naunyn-Schmiedeberg's Arch. Pharmacol. 1995; 352: 351-364Crossref PubMed Scopus (414) Google Scholar, 14Moncada S.R. Palmer M. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-129PubMed Google Scholar). The two constitutive isoforms expressed in neurons (neuronal nitric-oxide synthase; NOS I) and endothelial cells (endothelial nitric-oxide synthase; NOS III) are calcium/calmodulin-dependent. They are mainly involved in neurotransmission and vascular regulation, respectively (15Bredt D.S. Snyder S.H. Annu. Rev. Biochem. 1994; 63: 175-195Crossref PubMed Scopus (2121) Google Scholar). A major function of NO derived from the inducible NO synthase (iNOS; NOS II) is target cell cytotoxicity. Target cells may include tumor cells as well as bacteria, viral particles, and other microorganisms (12Bogdan C. Ro¨llinghoff M. Vodovotz Y. Xie Q. Nathan C. Masihi N. Immunotherapy of Infection. Marcel Dekker, Inc., New York1994: 37-54Google Scholar). However, NO produced by iNOS of macrophages (as well as other cells) also has the potential for adverse activities depending on its concentration and site of release. These include the induction of severe hypotension and cardiovascular shock and cytotoxicity toward host cells such as vascular cells, lymphocytes, or even macrophages themselves (12Bogdan C. Ro¨llinghoff M. Vodovotz Y. Xie Q. Nathan C. Masihi N. Immunotherapy of Infection. Marcel Dekker, Inc., New York1994: 37-54Google Scholar). Therefore, a better understanding of the physiological regulation of iNOS is important. The major activator of iNOS in macrophages has been shown to be bacterial lipopolysaccharide (LPS) (12Bogdan C. Ro¨llinghoff M. Vodovotz Y. Xie Q. Nathan C. Masihi N. Immunotherapy of Infection. Marcel Dekker, Inc., New York1994: 37-54Google Scholar, 16Stuehr D.J. Marletta M.A. Cancer Res. 1987; 47: 5590-5594PubMed Google Scholar). Co-stimulatory effects were demonstrated for INF-γ and a variety of other cytokines such as TNF-α, IL-2, INF-α, and -β (12Bogdan C. Ro¨llinghoff M. Vodovotz Y. Xie Q. Nathan C. Masihi N. Immunotherapy of Infection. Marcel Dekker, Inc., New York1994: 37-54Google Scholar). So far, little is known about what terminates the production of NO by macrophages. However, this is of particular importance regarding the severe pathophysiological effects of sustained NO production such as circulatory failure and tissue damage. Again cytokines, i.e.transforming growth factor-β, IL-4, and IL-10, have been described to suppress NO release of macrophages (12Bogdan C. Ro¨llinghoff M. Vodovotz Y. Xie Q. Nathan C. Masihi N. Immunotherapy of Infection. Marcel Dekker, Inc., New York1994: 37-54Google Scholar, 17Bogdan C. Nathan C. Ann. N. Y. Acad. Sci. 1993; 685: 713-739Crossref PubMed Scopus (388) Google Scholar). The underlying mechanisms appeared to be different for the respective cytokines (12Bogdan C. Ro¨llinghoff M. Vodovotz Y. Xie Q. Nathan C. Masihi N. Immunotherapy of Infection. Marcel Dekker, Inc., New York1994: 37-54Google Scholar, 17Bogdan C. Nathan C. Ann. N. Y. Acad. Sci. 1993; 685: 713-739Crossref PubMed Scopus (388) Google Scholar, 18Vodovotz Y. Bogdan C. Paik J. Xie Q. Nathan C. J. Exp. Med. 1993; 178: 605-613Crossref PubMed Scopus (603) Google Scholar). The observation that ANP, a circulating hormone best known for its vasodilative effects, inhibits NO synthesis in activated macrophages is particularly interesting since ANP concentrations are highly elevated in septic shock (19Aiura K. Ueda M. Endo M. Kitajima M. Crit. Care Med. 1995; 23: 1898-1906Crossref PubMed Scopus (57) Google Scholar), and moreover LPS-exposed macrophages were shown to produce increased ANP (9Vollmar A.M. Schulz R. J. Clin. Invest. 1994; 94: 539-545Crossref PubMed Scopus (46) Google Scholar). Thus, ANP may be a novel autocrine substance modulating NO production. Consecutively, aim of the present study was to clarify the basic mechanisms underlying the inhibition of NO synthesis by ANP. Mouse ANP 99-126 was purchased from Calbiochem (Bad Soden, Germany). HS-142-1 was a gift from Dr. Matsuda, Tokyo Research Laboratories, Tokyo, Japan. iNOS cDNA probe was provided by Dr. Kleinert, University of Mainz, Germany; COX-2 cDNA was a gift from Dr. Herschman, UCLA; TNF-α cDNA was obtained from Dr. Decker, University of Freiburg, Germany; IL-6 cDNA was provided by Dr. Kremer, GSF, Munich, Germany. Monoclonal antibody against macrophage iNOS was obtained from Transduction Laboratories (Lexington, KY); antiserum against the macrophage antigen F4/80 was from Serotec LTD (Wiesbaden, Germany); cell culture media (RPMI 1640, DMEM), fetal calf serum (FCS), penicillin/streptomycin, and TRIzolTMwere from Life Technologies, Inc. (Eggenstein, Germany). cGMP radioimmunoassay kit (cGMP 125I-assay system) and [3H]l-arginine (60 Ci/mmol), ECL detection system, and random primer labeling system (Rediprime®) were from Amersham (Braunschweig, Germany); Dowex 50 WX8 (Na+ form) was obtained from Serva (Heidelberg, Germany); [α-32P]UTP (800 Ci/mmol), [γ-32P]ATP, and [α-32P]dCTP (both 3000 Ci/mmol) were from Hartmann Analytic (Braunschweig, Germany); T3/T7 RNA polymerase transcription system was obtained from Stratagene (Heidelberg, Germany); dexamethasone solution was ordered from Centravet (Bad Bentheim, Germany); NF-κB and AP-2 binding oligonucleotides, SP6 polymerase, and T4 polynucleotide kinase were obtained from Boehringer Ingelheim Bioproducts (Heidelberg, Germany); IRF-1 binding oligonucleotide was from Santa Cruz Biotechnology (Heidelberg, Germany). Bradford protein assay was from Bio-Rad (Munich, Germany). All other materials were purchased from either Sigma (Deisenhofen, Germany) or ICN Biomedicals (Eschwege, Germany). Mouse bone marrow macrophages (BMM) were prepared as described previously (9Vollmar A.M. Schulz R. J. Clin. Invest. 1994; 94: 539-545Crossref PubMed Scopus (46) Google Scholar) and were seeded at a density of 2 × 105 cells/ml in 24-well tissue plates and grown for 5 days (5% CO2, 37 °C) in RPMI 1640 medium supplemented with 20% L-929 cell-conditioned medium, 10% heat-inactivated FCS, and penicillin (100 units/ml)/streptomycin (100 μg/ml). L-cell-conditioned medium was removed at least 12 h before experiments. BMM were found >95% pure as judged by fluorescence-activated cell sorter analysis (FACscan, Becton Dickinson, San Jose, CA) using an antiserum against the macrophage antigen F4/80 (20Szu-Hee L. Starkey P.M. Gordon S. J. Exp. Med. 1985; 161: 475-489Crossref PubMed Scopus (315) Google Scholar). Adult bovine aortic endothelial cells (ABAE, provided by Dr. Plendl, Munich) were cultivated in 24-well tissue plates in DMEM containing 10% FCS (21Obeso J. Weber J. Auerbach R. Lab. Invest. 1990; 63: 259-269PubMed Google Scholar). Confluent BMM or ABAE (24-well tissue plates) were washed three times and pretreated with 3-isobutyl-1-methylxanthine (0.5 mm) in serum-free RPMI 1640 for 10 min at 37 °C. Various stimuli were added for 30 min. Thereafter, medium was aspirated and cGMP was extracted immediately by the addition of HCl (0.1 n). After 10 min of incubation on ice the cell extracts were transferred to fresh tubes, lyophilized, and assayed for cGMP content by radioimmunoassay using a commercially available kit. BMM (24-well plates, 200 μl) were treated with lipopolysaccharide (LPS, E. coli, serotype 055:B5, 1 μg/ml) in the presence or absence of various concentrations of ANP 99-126 and/or HS-142-1. After 20 h the stable metabolite of NO, nitrite, was measured in the medium by the Griess reaction (22Green L.C. Wagner D.A. Glogowski J. Skipper P.L. Wishnok J.S. Tannenbaum S.R. Anal. Biochem. 1982; 126: 131-138Crossref PubMed Scopus (10586) Google Scholar). 100 μl of cell culture supernatant was removed, and 90 μl of 1% sulfanilamide in 5% H3PO4 and 90 μl of 0.1%N-(1-naphthyl)ethylenediamine dihydrochloride in H2O was added, followed by spectrophotometric measurement at 550 nm (reference wavelength 620 nm). BMM (24-well plates) were either untreated, stimulated with LPS (1 μg/ml) only, or co-incubated with ANP 10 nm–1 μm) for 12 h, washed three times with cold PBS, frozen immediately, and stored at −70 °C. iNOS activity was determined by measuring the conversion of [3H]l-arginine to [3H]l-citrulline according to Ref. 23Stevens-Truss R. Marletta M.A. Biochemistry. 1995; 34: 15638-15645Crossref PubMed Scopus (47) Google Scholar. Briefly, cells were homogenized in 50 mm Tris, pH 7.6, containing EDTA (0.1 mm), EGTA (0.1 mm), and phenylmethylsulfonyl fluoride (PMSF, 1 mm) by freezing and thawing. Homogenates of equal protein concentration (Bradford method) (100 μl, 200 μg of protein) were incubated at 37 °C for 30 min in the presence of l-arginine (10 μm), NADPH (1 mm), l-valine (50 mm), FAD (4 μm), tetrahydrobiopterin (4 μm), and [3H]l-arginine (0.2 μCi; 0.033 μm). Reactions were stopped by adding ice-cold sodium acetate, pH 5.5 (20 mm, 1 ml), containing EDTA (2 mm) and l-citrulline (0.1 mm). [3H]l-Citrulline was separated by ion exchange columns (Dowex 50 WX8, Na+ form) and measured by scintillation counting. The effect of test substances on the specific enzyme activity was evaluated using homogenates of cells treated with LPS for 12 h. Substances were incubated with cell homogenate for 10 min and further processed as described above. Extent ofl-[3H]citrulline formation independent of iNOS activity was determined in each experiment by employing homogenates of cells not exposed to LPS. The results are expressed as percentage of iNOS-specific [3H]citrulline formation. BMM (24-well plates) were treated with LPS (1 μg/ml) or a combination of LPS (1 μg/ml) plus ANP (1 μm) with or without HS-142-1 (100 μg/ml) for 12 h. Cells were washed with ice-cold PBS and stored at −70 °C. Western blot analysis was performed according to Ref. 24Xie Q.W. Leung M. Fuortes M. Sassa S. Nathan C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4891-4896Crossref PubMed Scopus (77) Google Scholar except that the lysis buffer (50 mm Tris-HCl, pH 6.8, 1% SDS, 2% mercaptoethanol, 10% glycerol, 0.004% bromphenol blue) was supplemented with a protease inhibitor mixture (Complete®). After sonication lysates were either boiled for 5 min (fully denaturing conditions) or not boiled (partially denaturing conditions) to discriminate between iNOS dimer and monomer (24Xie Q.W. Leung M. Fuortes M. Sassa S. Nathan C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4891-4896Crossref PubMed Scopus (77) Google Scholar). Samples (60 μg of protein) were loaded on an SDS-polyacrylamide gel (7.5%) and electroblotted, and iNOS protein was detected using an anti-iNOS monoclonal antibody and the ECL detection system. Signal intensities were evaluated by densitometric analysis (Herolab, E.A.S.Y. plus system, Wiesloch, Germany). BMM were stimulated with or without LPS (1 μg/ml) in the presence or absence of ANP (1 μm) or dexamethasone (10 μm) for 6 h (24-well plates). RNA was prepared using TRIzolTM reagent and pooled from 6 wells. Northern blot was performed in principle as described previously (6Vollmar A.M. Schulz R. Endocrinology. 1990; 126: 2277-2280Crossref PubMed Scopus (42) Google Scholar, 9Vollmar A.M. Schulz R. J. Clin. Invest. 1994; 94: 539-545Crossref PubMed Scopus (46) Google Scholar). Membranes were hybridized to a32P-labeled murine macrophage iNOS cRNA probe (2 × 106 cpm/ml). iNOS cDNA (558 base pairs) was subcloned in a pBluescript SK(+) vector, linearized (HindIII), and labeled with [32P]UTP (50 μCi) using a T3 RNA polymerase transcription system. Signal intensity was evaluated by densitometric analysis. To control for comparable amounts of intact mRNA loaded on the gel, membranes were rehybridized with a32P-labeled β-actin probe (2 × 106cpm/ml) as described (6Vollmar A.M. Schulz R. Endocrinology. 1990; 126: 2277-2280Crossref PubMed Scopus (42) Google Scholar). BMM in 24-well plates were treated with LPS (1 μg/ml) or a combination of LPS (1 μg/ml) plus ANP 99-126 (1 μm) for 5 h before addition of actinomycin D (5 μg/ml). Total RNA was prepared at the times indicated and further processed for Northern blot hybridization as described above. In addition to hybridization with an iNOS cRNA probe, blots have been rehybridized with probes for TNF-α (25Estler H.C. Grewe M. Gaussling R. Pavlovic M. Decker K. Biol. Chem. Hoppe-Seyler. 1992; 373: 271-281Crossref PubMed Scopus (74) Google Scholar), COX-2 (26Kujubu D.A. Fletcher B.S. Varnum B.C. Lim R.W. Herschman H.R. J. Biol. Chem. 1991; 266: 12866-12872Abstract Full Text PDF PubMed Google Scholar), and IL-6 (27Van-Snick J. Cayphas S. Szikora J.P. Renauld J.C. Van-Roost E. Boon T. Simpson R.J. Eur. J. Immunol. 1988; 18: 193-197Crossref PubMed Scopus (229) Google Scholar), respectively. To normalize mRNA concentrations membranes were hybridized with a β-actin probe. The iNOS, β-actin, TNF-α, and COX-2 probes were radiolabeled by in vitrotranscription and the IL-6 DNA probe by random-primed cDNA synthesis. The signal intensities were analyzed densitometrically, and signal density for iNOS or the other mRNAs was divided by that of β-actin in order to correct the loading differences. mRNA decay was evaluated based on the assumption that change of mRNA concentration at any time is a first-order process depending on the amount of mRNA present at that time (28Ross J. Microbiol. Rev. 1995; 59: 423-450Crossref PubMed Google Scholar). Accordingly, the ratios of the signal intensities of the respective mRNA/β-actin mRNA at each time point were expressed as percentage of the time point 0,i.e. before addition of actinomycin D and plotted against time. BMM were grown in 24-well plates and stimulated with LPS (1 μg/ml) in the presence or absence of ANP 99-126 (1 μm-10 nm) for 2 h. Dibutyryl-cGMP (1 mm) or pyrrolidinedithiocarbamate (PDTC, 50 μm) was added 1 h prior to LPS stimulation. Nuclear extracts were prepared as described (29Schreiber E. Matthias P. Müller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3903) Google Scholar). Briefly, cells were washed with PBS, resuspended in 400 μl of hypotonic buffer A (10 mm HEPES, pH 7.9, 10 mm KCl, 0.1 mmEDTA, 0.1 mm EGTA, 1 mm DTT, 0.5 mmPMSF), and were allowed to swell on ice for 15 min. Nonidet P-40 (10%, 25 μl) was added followed by 10 s of vigorous vortexing and centrifugation at 12,000 × g for 30 s. The supernatant was removed, and the nuclear pellet was extracted with 50 μl of hypertonic buffer B (20 mm HEPES, pH 7.9, 0.4m NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm DTT, 1 mm PMSF) by shaking at 4 °C for 15 min. The extract was centrifuged at 12,000 × g, and the supernatant was frozen at −70 °C. The protein concentration was determined by the Lowry method (30Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-267Abstract Full Text PDF PubMed Google Scholar). Two 22-mer double-stranded oligonucleotide probes containing a consensus binding sequence for either NF-κB (5′-AGTTGAGGGGACTTTCCCAGGC-3′) or IRF-1 (5′-GGAAGCGAAAATGAAATTGACT-3′) were 5′-end-labeled with [γ-32P]-ATP (10 μCi) using T4 polynucleotide kinase. 10 μg of nuclear protein were incubated (20 min at room temperature) in a 15-μl reaction volume containing 10 mmTris-HCl, pH 7.5, 5 × 104 cpm radiolabeled oligonucleotide probe, 2 μg of poly(dI-dC), 4% glycerol, 1 mm MgCl2, 0.5 mm EDTA, 50 mm NaCl, and 0.5 mm DTT. Nucleoprotein-oligonucleotide complexes were resolved by electrophoresis (4.5% non-denaturing polyacrylamide gel, 100 V). The gel was autoradiographed with an intensifying screen at −70 °C overnight. Specificity of the DNA-protein complex was confirmed by competition with a 100-fold excess of unlabeled NF-κB and AP-2 (5′-GATCGAACTGACCGCCCGCGGCCCGT-3′) binding sequences, respectively. Previous data that have been confirmed by a representative experiment shown in Fig.1 A demonstrated that ANP dose-dependently inhibits nitrite accumulation of LPS-activated BMM. The ANP-induced NO inhibition seems to be mediated by the guanylate cyclase-coupled NPR-A receptor, since HS-142-1, an NPR-A antagonist (31Matsuda Y. Morishita Y. Cardiovasc. Drug Rev. 1993; 11: 45-59Crossref Scopus (34) Google Scholar), abrogated the ANP effect. In addition, expression of the mRNA coding for both of the ANP receptors (NPR-A and NPR-C) has been shown before in BMM (10Kiemer A.K. Vollmar A.M. Endocrinology. 1997; 138: 4282-4290Crossref PubMed Scopus (59) Google Scholar). The aim here was to clarify the actual presence of functional NPR-A protein in BMM. BMM were treated with ANP, and cGMP production was determined. Intracellular cGMP levels were significantly elevated in cells exposed to ANP (10 μm-10 nm) for 30 min compared with untreated cells (Fig. 1 B). HS-142-1 (100 μg/ml) was able to antagonize this increase. To exclude that elevated cGMP levels were due to enzymatic activity of soluble guanylate cyclase (sGC) we incubated the cells with sodium nitroprusside (SNP), which is able to release NO, a known activator of sGC (14Moncada S.R. Palmer M. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-129PubMed Google Scholar, 15Bredt D.S. Snyder S.H. Annu. Rev. Biochem. 1994; 63: 175-195Crossref PubMed Scopus (2121) Google Scholar). SNP (100 μg/ml) did not change intracellular cGMP levels in BMM (Fig. 1 C). In contrast, cGMP production of ABAE, known to express sGC (32Ganz P. Davies P.F. Leopold J.A. Gimbrone Jr., M.A. Alexander R.W. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 3552-3556Crossref PubMed Scopus (46) Google Scholar), was dose-dependently elevated by the addition of SNP (10 and 100 μg/ml) under identical conditions. Thus, BMM release NO when activated but do not possess sGC as an important target system for NO action. Suppression of nitrite accumulation in BMM by ANP has to be discussed as the sum of potentially cumulative actions of ANP on the iNOS system and, for instance, does not allow us to assess effects of ANP on specific enzyme activity. Therefore, ANP (1 μm) was added to the homogenate of LPS (1 μg/ml)-stimulated cells, and iNOS enzyme activity was measured as conversion rate of [3H]l-arginine to [3H]l-citrulline (Fig.2 A). Incubation of cell homogenate with ANP did not result in a change of iNOS activity. The cGMP analog dibutyryl-cGMP (1 mm) had no effect either. The known specific inhibitor of NOS enzyme activityN G-monomethyl-l-arginine (3 mm) served as control and was able to inhibit specific iNOS enzyme activity up to 90%. By having excluded a direct effect of ANP on the enzyme activity, we next wanted to examine whether reduction of NO synthesis by ANP is due to factors such as decreased uptake ofl-arginine or availability of other necessary substrates and cofactors for iNOS. By using an indirect approach, measurement of enzyme activity was performed with cell homogenates of ANP-pretreated cells in the presence of optimal concentrations of substrates and cofactors (i.e. NADPH, l-arginine, BH4, FAD, and FMN). ANP pretreatment of cells dose-dependently (10 μm to 10 nm) reduced iNOS activity up to 70% (1 μm) compared with the activity of only LPS-stimulated cells (Fig. 2 B). Thus, ANP induces inhibition of NO synthesis most likely not by causing an intracellular shortage of iNOS substrates. [3H]l-Citrulline formation in the homogenate of ANP-exposed cells co-treated with the ANP-receptor antagonist HS-142-1 (10 μg/ml) was almost restored completely to levels of LPS treatment only (Fig. 2 B). The reduced activity of iNOS after ANP treatment reflected a decrease of iNOS protein as revealed by immunoblot with a monoclonal mouse anti-iNOS antibody (Fig. 3 A). No iNOS was detected in unstimulated BMM. Upon stimulation with LPS (1 μg/ml, 12 h) a single band (130 kDa) corresponding to iNOS appeared when fully denaturing conditions were used for sample preparation. The expression of iNOS protein was significantly reduced by ANP (1 μm), whereas co-incubation with the NPR-A receptor antagonist HS-142-1 (100 μg/ml) attenuated the decrease in iNOS protein by ANP. The next question was to determine whether ANP influenced the ratio of catalytically active iNOS dimerversus inactive monomer. Western blots were performed under partially denaturing conditions that allow detection of iNOS dimer (260 kDa) (24Xie Q.W. Leung M. Fuortes M. Sassa S. Nathan C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4891-4896Crossref PubMed Scopus (77) Google Scholar). Signal intensity of both dimer and monomer diminished in ANP-treated cells, but the proportion of monomer/dimer remained unchanged compared with LPS treatment only (Fig. 3 B). Northern blot analysis was performed to determine whether ANP inhibits iNOS mRNA accumulation when added simultaneously with LPS (1 μg/ml). Time course experiments showed that appearance of iNOS mRNA in BMM was maximal between 4 and 8 h and decreases after 12 h (data not shown). Subsequently mRNA was isolated from cells after 6 h of treatment. In unstimulated cells no iNOS mRNA was detectable (Fig.4). ANP (1 μm) caused a marked reduction of LPS-induced iNOS mRNA steady-state levels. Dexamethasone (10 μm), a known inhibitor of iNOS i
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