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: 0044-2920-748447 Fax: 0044-2920-744905 Email: o-donnellvb@cardiff.ac.uk 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. 1

Nitric Oxide is a Free Radical N=O NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 2

Nitric oxide is not particularly oxidizing 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. 3

The Nitric Oxide ‘Radical’ and some of its stable end-products •N=O…nitric oxide -O-N=O…nitrite RO-N=O…alkyl nitrite RS-N=O…nitrosothiol NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 4

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. 5

LOO is the central and rate-limiting species 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. 6

Non-enzymatic lipid oxidation Initiation Propagation L LOOH LOO Metal or enzyme LH AOO O2 LOH GPx NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 7

Lipid peroxidation products in an atherosclerotic lesion are both enzymatically and non-enzymatically produced. Foam cell 15-LOX  Necrotic core lipid hydroxides, hydroperoxides, aldehydes, epoxides, ketones, ceroid, isoprostanes Fibrous cap NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 8

Location of lipid oxidation enzymes in the vasculature LOX: lipoxygenase PGHS: prostaglandin H synthase Smooth muscle: 12-LOX Monocyte: PGHS-1,2 5, 15-LOX Platelet: PGHS-1 12-LOX Neutrophil: PGHS-1,2 5,12-LOX Endothelium PGHS-1 NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 9

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. 10

Nitric oxide reaction with lipid peroxyl radicals NO LOO LOONO 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. 11

Reactions between NO-derived reactive nitrogen species and unsaturated lipid LH NO2 NO O2 O2- ONOO- L LOO LOONO NO2, ONOO- LNO2 LOOH HONO L(O)NO2 e-, H+ NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 12

Nitric oxide inhibition of lipid oxidation coincides with fast rates of NO uptake during ABAP-dependent linoleate oxidation. (A) (B) 0.6 200 ABAP 0.2 Oxygen (µM) NO (µM) 100 -0.2 [ NO] = 0.9 µM added at vertical lines to 7.3 mM linoleate in 0.5% cholate, 11 mM ABAP -0.6 400 800 1200 Time/s NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 13

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. 14

•NO2 •NO2 •NO2 •NO •NO Cholesteryl Ester Triglyceride Phospholipid Apolipoprotein B O2 LH Oxidant •NO2 O2 L• •NO2 LH LOO• LNO2 •NO2 L• •NO LOOH -Tocopherol L(O)NO2 Cholesterol •NO NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 15

NO and LDL Oxidation: The effect of HPODE Lag time of LDL (125 g/ml) oxidation by Cu(II) (20 M) with and without HPODE (5 M) NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 16

Comparison of nitric oxide and tocopherol inhibition of linoleate oxidation. 100 200 300 400 600 800 1000 1200 Time/s [Oxygen]/µM T inh ABAP a -tocopherol (1 µM) NO (1 µM) Oxidation of linoleate (7.3 mM) by ABAP (11 mM) NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 17

Nitric oxide is consumed by rabbit 15-lipoxygenase during oxidation of linoleate. Add figure caption NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 18

Site of lipoxygenase inhibition by NO NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 19

Activation of LOX inhibits activation of sGC by NO 50 100 cGMP (% control) 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. 20

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 NO & LPO Society For Free Radical Biology and Medicine O’Donnell et al. 21