Grisham 1

1
Virtual Free Radical School
Nitric Oxide Methods to Assess its Role in vivo
Matthew B. Grisham, PhD. Molecular Cellular
Physiology LSU Health Sciences Center Shreveport,
LA. 71130
2
How Does One Assess the Role of iNOS-Derived ?NO
in vivo?

• Determine ?NO levels in vivo

• Quantify iNOS message, protein and enzymatic
  activity

• Modulate iNOS activity in vivo

3
Autoxidation of NO Formation of Reactive
Intermediates
t1/2 ? 1/?NO
d?NO
-k?NO2O2

dt
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Nitrate is the Major NO-Derived Metabolite in vivo
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Quantification of ?NO-Derived NO3- and NO2-

• Nitrate Reductase (NR) Converts all NO3- to NO2-
• All unreacted NADPH must be oxidized
• NO2- quantified by Griess Reaction

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Acidification of NO2- Produces the N-Nitrosating
Agent Dinitrogen Trioxide (N2O3)
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Griess Reaction
Nims, R.W. et.al Colormetric assays for nitric
oxide and nitrogen oxide species formed from
nitric oxide stock solutions and donor compounds.
Methods Enzymol 1996 26893-105.
Grisham, M.B. et.al Quantitation of nitrate
and nitrite in extracellular fluids. Methods
Enzymol 1996 268237-246.
8
N-nitrosation of 2,3 diaminonaphthalene (DAN) to
yield the fluorescent product 2,3-naphthotriazole
(NAT)
Wink, D.A. et.al Detection of S-nitrosothiols
by fluorometric and colormetric methods. Methods
Enzymol 1999 301201-211. Miles, A.M. et.al
Determination of nitric oxide using fluorescence
spectroscopy. Methods Enzymol 1996 268105-120.
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?NO-Dependent Formation of S-Nitrosothiols (RSNOs)
RSNOs S-Nitrosocysteine (CysNO) S-Nitrosoglutathio
ne (GSNO) S-Nitrosoalbumin (AlbNO)
Jourdheuil, D. et.al S-nitrosothiol formation
in blood of lipopolysaccharide-treated rats.
Biochem Biophys Res Commun 2000
27322-26. Jourdheuil, D. et.al Dynamic
state of S-nitrosothiols in human plasma and
whole blood. Free Radic Biol Med 2000
28409-417.
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Physiological Roles of Nitrosothiols (RSNOs)
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Fluorometric Determination of Nitrosothiols (RSNO)
Miles, A.M. et.al Determination of nitric
oxide using fluorescence spectroscopy. Methods
Enzymol 1996 268105-120.
12
How Does One Assess the Role of iNOS-Derived ?NO
in vivo?

• Determine ?NO levels in vivo

• Quantify iNOS message, protein and enzymatic
  activity

• Modulate iNOS activity in vivo

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Preparation of RNA
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Quantification of iNOS Message Real-Time RT-PCR
Quantifies message levels during the exponential
phase of the PCR reaction using a dual-labeled
fluorogenic probe and the ABI Prism 7700 sequence
detector. Amplification of contaminating genomic
DNA is avoided by designing primer sets located
in different exons or on intron/exon junctions.
Thus, DNAase treatment of RNA samples prior to
reverse transcription is unnecessary. This
method combines PCR amplification and product
detection in one single step making this
technique very fast and easy to perform compared
to standard nonquantitative or semiquantitative
RT-PCR methods. The absolute number of copies of
message can be determined using cDNA standards.
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Real-time PCR
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Amplification vs Cycle Number
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18s ribosomal RNA
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LPS treated iNOS mRNA
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Determination of iNOS Protein
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Quantification of iNOS Enzymatic Activity
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How Does One Assess the Role of iNOS-Derived ?NO
in vivo?

• Determine ?NO levels in vivo

• Quantify iNOS message, protein and enzymatic
  activity

• Modulate iNOS activity in vivo

22
Modulate iNOS Activity in vivo

• NOS Inhibitors L-Arginine analogs

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?NO Synthase Inhibitors Based on L-Arginine
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L-NIL Inhibits Nitric Oxide Production
in Lipopolysaccharide (LPS)-Treated Mice
Wild type C57Bl/6 mice (wt) were treated with
L-NIL (2mg/kg, iv) 30 min prior to injection
with LPS (10mg/kg ip). Serum nitrate and
nitrite were determined 6 h later using the
Griess Reaction
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iNOS Deficiency Eliminates Nitric Oxide
Production Induced by LPS
Wild type C57Bl/6 mice (wt) or iNOS deficient
(iNOS-/-) mice were treated with LPS (10mg/kg
ip). Serum nitrate and nitrite were determined 6
h later using the Griess Reaction
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References for Methods
Jourdheuil, D. et al. S-nitrosothiol
formation in blood of lipopolysaccharide- treated
rats. Biochem Biophys Res Commun 2000
27322-26. Jourdheuil, D. et al. Dynamic
state of S-nitrosothiols in human plasma
and whole blood. Free Radic Biol Med 2000
28409-417. Wink, D.A. et al. Detection of
S-nitrosothiols by fluorometric and
colormetric methods. Methods Enzymol 1999
301201-211. Miles, A.M. et al. Determination
of nitric oxide using fluorescence
spectroscopy. Methods Enzymol 1996
268105-120. Nims, R.W. et al. Colormetric
assays for nitric oxide and nitrogen oxide
species formed from nitric oxide stock solutions
and donor compounds. Methods Enzymol 1996
26893-105. Grisham, M.B. et al. Quantitation
of nitrate and nitrite in extracellular
fluids. Methods Enzymol 1996 268237-246.