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

bBiophysics Research Institute, Medical College of Wisconsin, Milwaukee, WI USA.

cDepartment of Pathology, University of Alabama at Birmingham, AL USA

aDept of Med. Biochem.University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK. Ph: 0044-2920-748447Fax: 0044-2920-744905Email: o-donnellvb@cardiff.ac.uk

NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 2

Nitric Oxide is a Free Radical

N=O

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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.

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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

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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 ??

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Lipid Peroxidation

LOO + LH + O2 LOO + LOOH

LOO is the central and rate-limiting species of lipid peroxidation

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Non-enzymatic lipid oxidation

Initiation Propagation

L

Propagation

LOOH

LOO

LOOH

Metal orenzyme

LH

AOO

O2

LOO

LH

O2

LOOLLH

O2

LOOH

LOH

GPx

LOH

GPx

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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

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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: lipoxygenase

PGHS: prostaglandin H synthase

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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.

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Nitric oxide reaction with lipid peroxyl radicals

LOONO

LOONO

H2O

LOOH + NO2

-

[LO NO2]

L(O)NO2

[L(O) NO2]

LO NO2

NO

LONO

NO

NO2-

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Reactions between NO-derived reactive nitrogen species and unsaturated lipid

LH

NO2

NO

O2 O2-

ONOO-

L LOOO2

LOONO

NO2, ONOO-LNO2

LOOH

HONO

L(O)NO2

NO

e-, H+

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0

100

200

0 400 800 1200Time/s

Oxy

gen

(µM

)

-0.6

-0.2

0.2

0.6

NO

(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

Nitric oxide inhibition of lipid oxidation coincideswith fast rates of NO uptake during ABAP-dependent

linoleate oxidation.

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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

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Apolipoprotein B

Phospholipid

-Tocopherol

Cholesterol

Cholesteryl Ester Triglyceride

L•

LOO• LNO2

LOOHL(O)NO2

Oxidant

•NO2

O2

LH

L•

•NO2

•NO

LH•NO2

O2

•NO

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NO and LDL

Oxidation: The effect of HPODE

0 4 8 12SNN (M )

0

100

200

300

400

500

leng

th o

f lag

tim

e (m

in)

SN N

SN N + H pO D E

B

0 2 4 6 8 10BHT (M )

0

50

100

150

200

BH T

BH T + H pO D E

A

Lag time of LDL (125 g/ml) oxidation by Cu(II) (20 M) with and without HPODE (5 M)

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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

ABAP -tocopherol (1 µM)

NO (1 µM)

Tinh

Oxidation of linoleate (7.3 mM) by ABAP (11 mM)

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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

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Site of lipoxygenase inhibition by NO

LOOH

E-Fe3+ LOO

E-Fe3+

E-Fe2+

NO

E-Fe2+ LOO

LE-Fe2+

O2

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Activation of LOX inhibits activation of sGC by NO

0

50

100

cG

MP

(%

co

ntr

ol)

control AA, 5 µM LOX AA, 5 µM, LOX

**

NO Lox

sGC cGMP

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Further Reading:

Darley-Usmar, VM et al., (1992) Free Radic. Res. Commun. 17, 9-20

Graham, A et al., (1993) FEBS Lett. 330, 181-185

Rubbo, H et al., (1994) J. Biol. Chem. 265, 26066-26075

Hogg, N et al., (1995) J. Lipid Res. 36, 1756-1762

Struck, N et al., (1995) FEBS Lett 361, 291-294

O’Donnell, VB et al., (1997) Biochemistry 36, 15216-15223

O’Donnell, VB et al., (1999) Chem. Res. Toxicol. 12, 83-92

O’Donnell, VB et al., (1999) J. Biol. Chem. 274, 20083-20091

Hogg, N & Kalyanaraman, B (1999) Biochim. Biophys. Acta. 1411, 378-384

Rubbo, H et al., (2000) J. Biol. Chem. 275, 10812-10818

O’Donnell, VB & Freeman, BA (2001) Circ. Res. 88, 16-25