Inducible Nitric Oxide Synthase Expression

Qiao‐wen Xie1

1 Kenneth S. Warren Laboratories, Tarrytown, New York
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 10.9
DOI:  10.1002/0471140856.tx1009s04
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit contains three protocols that can be used to determine iNOS expression in mouse macrophage-like cells, RAW 264.7, by measuring end product, protein, and mRNA. A bacterial product, lipopolysaccharide (LPS), stimulates iNOS expression in RAW 264.7 cells.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Detection of iNOS Expression Based on Nitrite Accumulation
  • Basic Protocol 2: Confirming iNOS Expression at Protein Level by Immunoblot
  • Basic Protocol 3: Detection of iNOS Expression by Northern Blot Analysis
  • Support Protocol: 5¢-End Labeling of Oligonucleotide Probes
  • Reagents and Solutions
  • Commentary
  • Bibliography
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Detection of iNOS Expression Based on Nitrite Accumulation

 Materials
  • RAW264.7 cells (ATCC #TIB 71)
  • Complete RPMI medium/10% FBS with antibiotics (Life Technologies; see recipe)
  • 100 µg/ml lipopolysaccharide (LPS) working solution (see recipe)
  • 10 mM sodium nitrite (see recipe)
  • Griess reagent working solution (see recipe)
  • 100-mm-diameter tissue culture dishes
  • Platform shaker
  • Ultrasonic bath (Lab-Line Instruments)
  • 96-well microtiter plate
  • Microplate reader

NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.

NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified.

Basic Protocol 2: Confirming iNOS Expression at Protein Level by Immunoblot

 Materials
  • 10× PBS (see recipe or purchase from Life Technologies)
  • Lysis buffer (see recipe)
  • Bradford protein assay reagent kit (Bio-Rad)
  • NuPAGE Tris-Acetate SDS Buffer Kit (Novex) containing:
    • 4× sample buffer (also see recipe)
    • 20× running buffer (also see recipe)
    • 10× sample reducing agent
  • Precast NuPAGE 3% to 8% Tris-acetate gel, 1.5-mm-thick, 10 wells (Novex)
  • Multicolored protein standards (NEN Life Science Products)
  • 20× NuPAGE transfer buffer (Novex; see recipe)
  • Blocking solution (see recipe)
  • PVDF or nitrocellulose membrane
  • PBST (see recipe)
  • Mouse monoclonal antibody 1E8-B8 (iNOS-specific; R&D Systems)
  • SuperSignal West Dura Mouse IgG Detection Kit (Pierce) containing:
  •     Horseradish peroxidase (HRP)-conjugated anti-mouse IgG
  •     Luminal/enhancer solution
  •     Stable peroxide solution
  • Cell scraper (Falcon)
  • 15-ml centrifuge tube
  • XCell II Mini-Cell (Novex)
  • XCell II Blot Module (Novex)
  • Polyvinylidene difluoride (PVDF) or nitrocellulose membrane: pore size, 0.2 µM
  • Whatman 3MM filter paper or equivalent
  • Kodak X-Omat AR X-ray film
  • Additional reagents and equipment for culturing cells and inducing iNOS expression (see Basic Protocol 1), SDS-PAGE (appendix 3A) and immunoblotting (unit 2.3)

NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.

NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified.

Basic Protocol 3: Detection of iNOS Expression by Northern Blot Analysis

 Materials
  • TRI Reagent for RNA isolation (Molecular Research Center)
  • Chloroform
  • Isopropanol
  • 75% ethanol
  • DEPC-treated H2O (see recipe)
  • 1% (w/v) agarose gel with 2% (v/v) formaldehyde (see recipe)
  • 10× MOPS buffer (see recipe)
  • Loading buffer (see recipe), freshly prepared
  • Formamide
  • 100% glycerol
  • RNA Molecular Size Standard (Life Technologies)
  • 20× SSPE, pH 7.4 (Life Technologies; also see recipe)
  • 50× Denhardt solution (see recipe)
  • 10% (w/v) sodium dodecyl sulfate (SDS)
  • 10 mg/ml herring sperm DNA (Promega)
  • 20× SSC: 3.0 M NaCl/0.3 M sodium citrate, pH 7.0 (Life Technologies)
  • 50-ml Oak Ridge centrifuge tubes with caps, polypropylene (Nalge)
  • Centrifuge (e.g., Sorvall with SS-34 rotor or equivalent)
  • 55° to 60°C and 65° to 70°C water baths
  • Horizontal electrophoresis apparatus, with circulation (OWL Separation Systems)
  • UV lightbox
  • GeneScreenPlus nylon membrane (NEN Life Scientific Products)
  • Turboblotter Rapid Downward Transfer System (Schleicher & Schuell)
  • Stratalinker UV cross-linker (Stratagene)
  • Heat-sealable bags (Kapak)
  • Impulse sealer (American International Electric)
  • Additional reagents and equipment for culturing cells and inducing iNOS expression (see Basic Protocol 1), determining RNA concentration by spectrophotometry (appendix 3A), and preparing oligonucleotide probes specific to mouse iNOS and -actin (see Support Protocol

NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.

NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified.

Support Protocol: 5¢-End Labeling of Oligonucleotide Probes

 Materials
  • Oligonucleotide iNOS-34: 5¢-TCT GTG CTG TCC CAG TGA GGA GCT GCG GGG AGC C-3¢
  • Mouse -actin oligonucleotide (Clontech)
  • 10× kinase buffer (Promega; also see recipe)
  • 10 mCi/ml [-32P]ATP (6000 Ci/mmol; NEN Life Scientific Products)
  • 10 U/µl T4 polynucleotide kinase (Promega)
  • 65°C water bath
  • Quick-Spin columns (TE) for radiolabeled DNA purification, Sephadex G-25 (Boehringer Mannheim)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  •  FigureFigure 10.9.1 Immunoblot for iNOS (130 kDa) in the lysate from RAW264.7 cells treated with (+) or without (–) LPS (100 ng/ml) for 18 hr.
    View Image
  •  FigureFigure 10.9.2 Northern blot of total RNA from RAW264.7 with (+) and without (–) an 18-hr treatment of LPS (100 ng/ml). The hybridization was performed with two oligonucleotide probes: one is a 34-mer specific for iNOS (4.2 kb) and the other is a 30-mer specific for -actin (1.9 kb) as an RNA loading control.
    View Image

Videos

Literature Cited

 Literature Cited
    Cho, H.J., Xie, Q.-W., Calaycay, J., Mumford, R.A., Swiderek, K.M., Lee, T.D., and Nathan, C. 1992. Calmodulin is a subunit of nitric oxide synthase from macrophages. J. Exp. Med. 176:599-604.
    De Vera, M.E., Shapiro, R.A., Kussler, A.K., Mudgett, J.S., Simmons, R.L., Morris, S.M.Jr., Billiar, T.R., and Geller, D.A. 1996. Transcriptional regulation of human inducible nitric oxide synthase (NOS2) gene by cytokines: Initial analysis of the human NOS2 promoter. Proc. Natl. Acad. Sci. U.S.A. 93:1054-1059.
    Ding, M., St. Pierre, B.A., Parkinson, J.F., Medberry, P., Wong, J.L., Rogers, N.E., Ignarro, L.J., and Merrill, J.E. 1997. Inducible nitric-oxide synthase and nitric oxide production in human fetal astrocytes and microglia. J. Biol. Chem. 272:11327-11335.
    Feinstein, D.L., Galea, E., Roberts, S., Berquist, H., Wang, H., and Reis, D.J. 1994. Induction of nitric oxide synthase in rate C6 glioma cells. J. Neurochem. 62:315-321.
    Finder, J.D., Litz, J.L., Blaskovich, M.A., McGuire, T.F., Qian, Y., Hamilton, A.D., Davies, P., and Sebti, S.M. 1997. Inhibition of protein geranylgeranylation causes a superinduction of nitric oxide synthase-2 by interleukin-1 beta in vascular smooth muscle cells. J. Biol. Chem. 272:13484-13488.
    Fujisawa, H., Ogura, T., Hokari, A., Weisz, A., Yamashita, J., and Esumi, H. 1995. Inducible nitric oxide synthase in a human glioblastoma cell line. J. Neurochem. 64:85-91.
    Geller, D.A., Nussler, A.K., Di Silvio, M., Lowenstein, C.J., Shapiro, R.A., Wang, S.C., Simmons, R.L., and Billiar, T.R. 1993. Cytokines endotoxin,and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes. Proc. Natl. Acad. Sci. U.S.A. 90:522-526.
    Grabowski, P.S., Macpherson, H., and Ralston, S.H. 1996. Nitric oxide production in cells derived from the human joint. Br. J. Rheumatol. 35:207-212.
    Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnock, J.S., and Tannenbaum, S.R. 1982. Analysis of nitrate,nitrite and [15N]nitrate in biological fluid. Anal. Biochem. 126:131-138.
    Koide, M., Kawahara, Y., Nakayama, I., Tsuda, T., and Yokoyama, M. 1993. Cyclic AMP-elevating agents induce an inducible type of nitric oxide synthase in cultured vascular smooth muscle cells. Synergism with the induction elicited by inflammatory cytokines. J. Biol.Chem. 268:24959-24966.
    Kolios, G., Brown, Z., Robon, R.L., Robertson, D.A., and Westwick, J. 1995. Inducible nitric oxide synthase activity and expression in a human colonic epithelial cell line, HT-29. Br. J. Pharmacol. 116:2866-2872.
    Kunz, D., Walker, G., Eberhardt, W., Nitsch, D., and Pfeilschifer, J. 1995. Interleukin 1–induced expression of nitric oxide synthase in rat renal mesangial cells is suppressed by cyclosporin A. Biochem. Biophys. Res. Commun. 216:438-446.
    Kwon, G., Corbett, J.A., Rodi, C.P., Sullivan, P., and McDaniel, M.L. 1995. Interleukin 1–induced nitric oxide synthase expression by rat pancreatic cells: Evidence for the involvement of nuclear factor B in the signaling mechanism. Endocrinology 136:4790-4795.
    Lorbach, R.B., Murphy, W.J., Lowenstein, C.J., Snyder, S.H., and Russell, S.W. 1993. Expression of the nitric oxide synthase gene in mouse macrophages activated for tumor cell killing. Molecular basis for the synergy between interferon-gamma and lipopolysaccharide. J. Biol. Chem. 268:1908-1913.
    Malinski, M. and Taha, Z. 1992. Nitric oxide release from a single cell measured in situ by a porphyrinic-based microsensor. Nature 358:676-678.
    Melillo, G., Cox, G.W., Radzioch, D., and Varesio, L. 1993. Picolinic acid, a catabolite of l-tryptophan, is a costimulus for the induction of reactive nitrogen intermediate production in murine macrophages. J. Immunol 150:4031-4040.
    Nicholson, S., Bonecini-Almeida, M.G., Silva, J.R.L., Nathan, C., Xie, Q.-W., Mumford, R., Weidner, J.R., Calaycay, J., Geng, J., Boechat, N., Linhares, C., Rom, W., and Ho, J.L. 1996. Inducible nitric oxide synthase in pulmonary alveolar macrophages from patients with tuberculosis. J. Exp. Med 183:2293-2302.
    Pahan, K., Sheikh, F.G., Namboodiri, A.M.S., and Singh, I. 1997. Lovastatin and phenylacetate inhibit the induction of nitric oxide synthase and cytokines in rat primary astrocytes, microglia, and macrophages. J. Clin. Invest. 100:2671-2679.
    Palmer, R.M., Hickery, M.S., Charles, I.G., Moncada, S., and Bayliss, M.T. 1993. Induction of nitric oxide synthase in human chondrocytes. Biochem. Biophys. Res. Commun. 193:398-405.
    Pendino, K.J., Laskin, J.D., Shuler, R.L., Punjabi, C.J., and Laskin, D.L. 1993. Enhanced production of nitric oxide by rat alveolar macrophages after inhalation of a pulmonary irritant is associated with increased expression of nitric oxide synthase. J. Immunol. 151:7196-7205.
    Robbins, R.A., Barnes, P.J., Springall, D.R., Warren, J.B., Kwon, O.J., Buttery, L.D.K., Wilson, A.J., Geller, D.A., and Polak, J.M. 1994. Expression of inducible nitric oxide in human lung epithelial cells. Biochem. Biophys. Res. Commun. 203:209-218.
    Saura, M., Perez-Sala, D., Canada, F.J., and Lama, S. 1996. Role of tetrahydrobiopterin availability in the regulation of nitric-oxide synthase expression in human mesangial cells. J. Biol. Chem. 271:14290-14295.
    Sherman, P.A., Laubach, V.E., Reep, B.R., and Wood, E.R. 1993. Purification and cDNA sequence of an inducible nitric oxide synthase from a human tumor cell line. Biochemistry 32:11600-11605.
    Simmons, W.W., Ungureanu-Longrois, D., Smith, G.K., Smith, T.W., and Kelly, R.A. 1996. Glucocorticoids regulate inducible nitric oxide synthase by inhibiting tetrahydrobiopterin synthesis and l-arginine transport. J. Biol. Chem. 271:23928-23937.
    Vodovotz, Y., Kwon, N.S., Pospischil, M., Manning, J., Paik, J., and Nathan, C. 1994. Inactivation of nitric oxide synthase after prolonged incubation of mouse macrophages with IFN- and bacterial LPS. J. Immunol. 152:4110-4118.
    Vodovotz, Y., Lucia, M.S., Flanders, K.C., Chesler, L., Xie, Q.-W., Smith, T.S., Weidner, J., Mumford, R., Webber, R., Nathan, C., Roberts, A.B., Lippa, C.F., and Sporn, M.B. 1996. Inducible nitric oxide synthase in tangle-bearing neurons of patients with Alzheimer's disease. J. Exp. Med. 184:1425-1433.
    Xie, Q.-W., Cho, H.J., Calaycay, J., Mumford, R.A., Swiderek, K.M., Lee, T.D., Ding, A., Troso, T., and Nathan, C. 1992. Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science 256:225-228.
    Xie, Q.-W., Whisnant, R., and Nathan, C. 1993. Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon gamma and bacterial lipopolysaccharide. J. Exp. Med. 177:1779-1784.
 Key References
    Green et al., 1982. See 1982.

Describes in detail the reduction of nitrate to nitrite and the measurement of total nitrite in the sample by a stoichiometric diazotization reaction using the Griess reagent to form a purple azo product.

    Nathan, C. and Xie, Q.-W. 1994. Regulation of biosynthesis of nitric oxide. J. Biol. Chem. 269:13725-13728.

Summarizes the induction and regulation of inducible nitric oxide synthase.

GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library