Vascular Biology of Nitric Oxide

1. PHM142 Fall 2013 Instructor: Dr. Jeffrey Henderson Vascular Biology of Nitric Oxide Toby Lee Patricia Lu Kevin Sun Hennie Wei September 25, 2013

2. Agenda • Synthesis • Effect of NO on: • Vascular smooth muscle • Platelets • Vascular smooth muscle cell proliferation • Oxidant/antioxidant properties • Endothelial cell proliferation and apoptosis • Regulation • Relevant drugs

3. Synthesis • NO synthase responsible for synthesizing NO • 3 isoforms, endothelial NOs (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS) • High homology in domains • Enzyme structure: • Dimer with 2 identical monomers • Each monomer has 2 major domains: • Arginine converted through intermediate to L-citrulline and NO

4. Synthesis

5. Synthesis • Reaction that converts L-arginine to L-citrulline • The reaction: • Two step oxidation reaction converts L-arginine to L-citrulline • Initial hydroxylation of L-arginine, forms hydroxy-L-arginine (also substrate for NOS) • Oxidation of intermediate using single electron from NADPH to form L-citrulline and NO • Uses O2 and NADPH • O2 is incorporated into both NO and citruilline • Also requires 4 other coenzymes/cofactors • Protoporphyrin IX haem • FMN • FAD • BH­4 (tetrahydrobiopterin)

6. Review of Smooth Muscle Contraction

7. Vascular Tone • Causes relaxation of vascular smooth muscle cells • Shown that inhibition of NO synthesis in vasculature elevates blood pressure • Vasodilation • Basal release of NO critical for vascular tone • NO produced in endothelium diffuses into vascular smooth muscle cells in blood vessels and binds to soluble guanylylcyclase • Activation of GC increases cytosoliccGMP • Signal transduction events lead to a decrease in intracellular calcium

8. Review of Platelets • Small and disk-shaped (2-4 μm in diameter) • Circulate in blood and involved in hemostasis • Platelet adhesion, aggregation, and recruitment

9. The Role of NO in Platelets • NO inhibits platelet adhesion, aggregation, and recruitment • Mechanism of Action

11. The Role of NO in Vascular Smooth Muscle Cell Proliferation • VSMC proliferation is usually involved with atherosclerosis • NO inhibits proliferation of VSMC • Mice study done by Moroi et al., 1998 • Mechanism of Action 1 • Mechanism of Action 2 • NO can upregulate Fas expression on VSMC

12. Other Properties of NO • NO is a free radical which induces superoxide dismutase expression • Catalyzes the dismutation of superoxide into hydrogen peroxide • Hydrogen peroxide increases the expression of nitric oxide synthase • Increased reduction of oxidative stress • Indirect pathways • Upregulation of heme-oxygenase I • Production of bilirubin, which scavenges superoxide • Increased expression of ferritin • Reduces superoxide formation

13. Endothelial Cell Protection From NO • Low NO concentration • cGMP dependent • Inhibition of cytochrome c release • cGMP independent • Upregulation of bcl-2 expression • Inactivation of caspases through s-nitrosation • Antioxidant properties • Removal of reactive oxygen species

14. Endothelial Cell Apoptosis Due To NO • High NO concentration due to oxidative stress, pathogenic NO + O2-ONOO- • DNA damage, protein damage and lipid peroxidation • Prevented by competition with superoxide dismutase • p53 response • Cell cycle arrest by p21 upregulation • Apoptosis, cytochrome c release and caspase activation

15. Reactivity in body • NO reacts with Heme of erythrocyte hemoglobin • Heme of GuanylylCyclase • dephosphorylates GTP to cGMP • This acts to scavenge NO by hemoglobin https://scontent-a-iad.xx.fbcdn.net/hphotos-ash3/1240264_10200342135030525_309408347_n.jpg

16. Regulation of NO • Cofactors: NADPH, flavin adenine nucleotides, etc • eNOS activity is calcium- and calmodulin-dependent • Basally active • flow dependent NO formation • receptor stimulated NO formation • iNOS basal activity is very low • during inflammation by bacterial endotoxins and cytokines

17. Regulation of NOS • eNOSupregulation • NO insufficiency • atherosclerotic burden, endothelial dysfunction • E.g., increased oxidative stress • Target of: Cardiovascular disease drugs (statins, calcium channel blockers) • eNOSdownregulation • Responsible for cardiovascular side effects of glucocorticoids • Notable drug : Viagara • inhibits breakdown of cGMP via inhibiting cGMP dependent phosphodiesterases • antiplatelet and antiaggregatory effects in the blood https://fbcdn-sphotos-e-a.akamaihd.net/hphotos-ak-prn2/1235489_10200342032547963_1348001150_n.jpg

18. Summary • NO synthase responsible for synthesizing NO • Reaction that converts L-arginine to L-citrulline • Two step oxidation reaction • Uses O2 and NADPH • Also requires 4 other coenzymes/cofactors • Protoporphyrin IX haem • FMN • FAD • BH­4 (tetrahydrobiopterin) • Causes relaxation of vascular smooth muscle cells • Vasodilation • NO causes decrease of intracellular Ca2+ through G protein coupled signal transduction which down regulates smooth muscle contraction • NO inhibits platelet adhesion, aggregation, and recruitment • NO inhibits proliferation of VSMCs • High concentrations produce oxidative species that promote cell death • Heme groups has affinity for NO • eNOS activity is calcium- and calmodulin-dependent and is basally active, either flow dependent or receptor stimulated • iNOS is induced during inflammation by toxins and has low basal activity • NO insufficiency and cardiovascular drugs cause eNOSupregulation: to increase NO production • Viagara inhibits cGMPphosphodiesterase to prolong NO effects = helps smooth muscle relaxation

19. References Alderton WK, Cooper CE, Knowles RG. Nitric oxide synthases: Structure, function and inhibition. Biochem J. 2001;357(Pt 3):593-615. Andrew PJ, Mayer B. Enzymatic function of nitric oxide synthases. Cardiovasc Res. 1999;43(3):521-531. Jin RC, Loscalzo J. Vascular nitric oxide: Formation and function. J Blood Med. 2010;2010(1):147-162. Knowles RG, Moncada S. Nitric oxide synthases in mammals. Biochem J. 1994;298 ( Pt 2)(Pt 2):249-258. Harrison P. Progress in the assessment of platelet function. Br J Haematol. 2000;111(3):733-744. Moroi M, Zhang L, Yasuda T, et al. Interaction of genetic deficiency of endothelial nitric oxide, gender, and pregnancy in vascular response to injury in mice. J Clin Invest. 1998;101(6):1225. Simon DI, Stamler JS, Loh E, Loscalzo J, Francis SA, Creager MA. Effect of nitric oxide synthase inhibition on bleeding time in humans. J CardiovascPharmacol. 1995;26(2):339-342. Walford G, Loscalzo J. Nitric oxide in vascular biology. Journal of Thrombosis and Haemostasis. 2003;1(10):2112-2118. Li H, Wallerath T, Munzel T, Forstermann U. Regulation of endothelial-type NO synthase expression in pathophysiology and in response to drugs. Nitric Oxide. 2002;7(3):149-164. Vaziri ND, Ding Y, Ni Z. Compensatory up-regulation of nitric-oxide synthaseisoforms in lead-induced hypertension; reversal by a superoxide dismutase-mimetic drug. J Pharmacol Exp Ther. 2001;298(2):679-685. Chung HT, Pae HO, Choi BM, Billiar TR, Kim YM. Nitric oxide as a bioregulator of apoptosis. BiochemBiophys Res Commun. 2001;282(5):1075-1079. KalyanaramanB. Teaching the basics of redox biology to medical and graduate students: Oxidants, antioxidants and disease mechanisms. Redox Biol. 2013;1(1):244-257. Moncada S, Higgs EA. The discovery of nitric oxide and its role in vascular biology. Br J Pharmacol. 2006;147 Suppl 1:S193-201.