• NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 1 Nitric Oxide and Lipid Peroxidation Valerie B. O’Donnell a , Neil Hogg b and Victor M. Darley Usmar c a Dept of Med. Biochem. University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK. Ph: 0044-2920-748447 Fax: 0044-2920-744905 Email: [email protected] b Biophysics Research Institute, Medical College of Wisconsin, Milwaukee, WI USA. c Department of Pathology, University of Alabama at Birmingham, AL USA
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•NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 1
Nitric Oxide and Lipid Peroxidation
Valerie B. O’Donnella, Neil Hoggb
and Victor M. Darley Usmarc
aDept of Med. Biochem.University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK. Ph: 0044-2920-748447Fax: 0044-2920-744905Email: [email protected]
bBiophysics Research Institute, Medical College of Wisconsin, Milwaukee, WI USA. cDepartment of Pathology, University of Alabama at Birmingham, AL USA
•NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 2
Nitric Oxide is a Free Radical
•N=O
•NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 3
Nitric oxide is not particularly oxidizing.Nitric oxide is (infinitely) stable*.
Nitric oxide does not rapidly react with organic molecules.
*In the absence of dioxygen.
•NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 4
The Nitric Oxide ‘Radical’ and some of its stable end-products
-O-N=O…nitrite
RO-N=O…alkyl nitrite
RS-N=O…nitrosothiol
•N=O…nitric oxide
•NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 5
Reactions of •NO with Other Free Radicals
Oxidation of •NO to nitrite:
4 •NO+ •O=O• +2H2O → 4HNO2
Nitric oxide reaction with organic peroxyl radicals: •NO+ ROO• → ROONO → ??
•NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 6
Lipid Peroxidation
LOO• + LH + O2 → LOO• + LOOH
LOO• is the central and rate-limiting species of lipid peroxidation
•NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 7
Non-enzymatic lipid oxidation
Initiation Propagation
L•
Propagation
LOOH
LOO•
LOOH
Metal orenzyme
LH
AOO•
O2
LOO•
LH
O2
LOO•L•LH
O2
LOOH
LOH
GPx
LOH
GPx
•NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 8
lipid hydroxides, hydroperoxides, aldehydes,epoxides, ketones, ceroid, isoprostanes
Necrotic core
Fibrous cap
Lipid peroxidation products in an atherosclerotic lesion are both enzymatically and non-
enzymatically produced.
Foam cell15-LOX ↑
•NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 9
Monocyte:PGHS-1,25, 15-LOX
Platelet:PGHS-112-LOX
Neutrophil:PGHS-1,25,12-LOX
Location of lipid oxidation enzymes in the vasculature
EndotheliumPGHS-1
Smooth muscle:12-LOX
LOX: lipoxygenasePGHS: prostaglandin H synthase
•NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 10
Lipid oxidation enzymes are upregulated during vascular disease.
* Inhibition of 12-LOX restores blood pressure in Angiotensin-II dependent hypertensive rats.
* Increased products of PGHS in hypertensive rats, and PGHS inhibition restores blood pressure in humans and rats.
* Smooth muscle 12-LOX ↑ in hypertension
* Platelet 12-LOX ↑ in hypertension
* Foam cell/macrophage 15-LOX ↑ in atherosclerosis.
•NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 11
Nitric oxide reaction with lipid peroxyl radicals
•NOLOONOLOO•
H2O [LO• •NO2]
LOOH + NO2
-
LO• •NO2
•NO [L(O)• •NO2]•NO
LONO NO2-
L(O)NO2
•NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 12
Reactions between •NO-derived reactive nitrogen species and unsaturated lipid
LH
•NO2
•NO
O2 O2•-
ONOO-
L• LOO•O2
LOONO
•NO2, ONOO-LNO2
LOOH
HONO
L(O)NO2
•NO
e-, H+
•NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 13
Nitric oxide inhibition of lipid oxidation coincideswith fast rates of •NO uptake during ABAP-dependent
linoleate oxidation.O
xyge
n (µ
M)
ABAP
(A) (B)
[ •NO] = 0.9 µM added at vertical lines to 7.3 mM linoleate in 0.5% cholate, 11 mM ABAP
0.6200
0.2
NO
(µM
)
100
-0.2
0 -0.60 400 1200800
Time/s
•NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 14
Mechanism of Inhibition of LDL Oxidation by •NO
LOO• + •NO → LOONO
LOOH
LO• + •NO2 → LONO2
OLOO• + •NO
OLOONO → OLONO2
Two •NO consumed for each LOO•
4 •NO + 2LOO• + H2O 2HNO2 + LONO2 + LONO
•NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 15
Apolipoprotein B
Phospholipid
α-Tocopherol
Cholesterol
L•
LOO• LNO2
LOOHL(O)NO2
Oxidant
•NO2O2
LH
L•
•NO2
•NO
LH•NO2
•NO
Cholesteryl Ester Triglyceride
O2
•NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 16
0 4 8 12SNN (µM)
0
100
200
300
400
500
leng
th o
f lag
tim
e (m
in)
SNN
SNN + HpODE
B
0 2 4 6 8 10BHT (µM)
0
50
100
150
200
BHT
BHT + HpODE
A
•NO and LDL
Oxidation: The effect of HPODE
Lag time of LDL (125 µg/ml) oxidation by Cu(II) (20 µM) with and without HPODE (5 µM)
•NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 17
Comparison of nitric oxide and tocopherol inhibition of linoleate oxidation.
0
100
200
300
0 200 400 600 800 1000 1200
Time/s
[Oxy
gen]
/µM
Tinh
ABAPα -tocopherol (1 µM)
•NO (1 µM)
Tinh
Oxidation of linoleate (7.3 mM) by ABAP (11 mM)
•NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 18
0
1
2
0 500 1000 1500
Time (secs)
NO
(µM
)
LOX LOXLOX
(i) (ii) (iii)
Nitric oxide is consumed by rabbit 15-lipoxygenaseduring oxidation of linoleate.
Add figure caption
•NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 19
Site of lipoxygenase inhibition by •NO
LOOH
E-Fe3+ LOO
E-Fe3+
E-Fe2+
NO
E-Fe2+ LOO
LE-Fe2+
O2
•NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 20
Activation of LOX inhibits activation of sGC by •NO
0
50
100
cGM
P(%
con
trol
)
control AA, 5 µM LOX AA, 5 µM, LOX
**
•NO Lox
sGC cGMP
•NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 21
Further Reading:
Darley-Usmar, VM et al., (1992) Free Radic. Res. Commun. 17, 9-20Graham, A et al., (1993) FEBS Lett. 330, 181-185Rubbo, H et al., (1994) J. Biol. Chem. 265, 26066-26075Hogg, N et al., (1995) J. Lipid Res. 36, 1756-1762Struck, N et al., (1995) FEBS Lett 361, 291-294O’Donnell, VB et al., (1997) Biochemistry 36, 15216-15223O’Donnell, VB et al., (1999) Chem. Res. Toxicol. 12, 83-92O’Donnell, VB et al., (1999) J. Biol. Chem. 274, 20083-20091Hogg, N & Kalyanaraman, B (1999) Biochim. Biophys. Acta. 1411, 378-384Rubbo, H et al., (2000) J. Biol. Chem. 275, 10812-10818O’Donnell, VB & Freeman, BA (2001) Circ. Res. 88, 16-25
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