Nitric Oxide updated.pdf

Dr Sarah Trinder [email protected] 30AY04 Outline  What is nitric oxide (NO)?  NO synthesis  Role of soluble guanylate cyclase  Phosphodiesterases What is NO?..  Colourless gas (Priestly, 1774). N=O.  Free radical,...

Dr Sarah Trinder [email protected] 30AY04 Outline  What is nitric oxide (NO)?  NO synthesis  Role of soluble guanylate cyclase  Phosphodiesterases What is NO?..  Colourless gas (Priestly, 1774). N=O.  Free radical, has an unpaired electron. Unpaired electron  ‘Molecule of the Year’ in 1992  1998 Nobel Prize – Furchgott, Ignarro & Murad – ‘for discoveries concerning NO as a signalling molecule in the cardiovascular system (CVS)’. 1980s – NO’s coming of age!  Endothelial cells release – endothelium derived relaxing factor (EDRF) in response to ACh (Furchgott, 1980).  Relaxes underlying smooth muscle.  1987 – EDRF is NO (Ignarro et al., 1987; Palmer et al., 1987). Rang and Dale’s Pharmacology 8th Ed, 2016  NOS – nitric oxide synthase Synthesis of NO NADPH – nicotinamide adenine dinucleotide phosphate FAD – flavin adenine dinucleotide FMN – flavin mononucleotide BH4 – tetrahydrobiopterin CaM - calmodulin Freire et al., 2009. Front. Neurosci. Nitric oxide synthase  3 isoforms  Neuronal NOS – nNOS or NOS1  Inducible/inflammatory NOS – iNOS or NOS2  Endothelial NOS – eNOS or NOS3  Homodimeric proteins  Each subunit - 2 domains  Oxygenase and reductase domain Campbell et al., 2014. PNAS. From Rang & Dale’s Pharmacology 8th Ed. Lecture 2 – role of BH4 Tetrahydrobiopterin – BH4  4 key roles of BH4 for optimal NOS activity: 1) Enables haem catalytic site to function normally 2) ↑ NOS affinity to L-arg 3) Acts as cofactor to L-arg – e- transfer 4) Stabilises NOS dimerisation BH4 DHFR – dihydrofolate reductase DHFR NOS ‘uncoupling’  e- diverted to O2 → O2- → oxidative stress  Loss of BH4  Oxidation  ↓ DHFR Nitric oxide synthase NOS Isoform Molecular Weight (kDa) Tissue Distribution/Function nNOS/NOS1 321 Neurotransmitter in: - penile erection - Ca2+ dependent - sphincter relaxation - skeletal muscle - GI tract Mediates synaptic plasticity Involved with memory Important in stroke iNOS/NOS2 260 Macrophages and mast cells Very important in inflammation - Ca2+ independent eNOS/NOS3 266 Endothelial cells, cardio myocytes, renal cells, platelets etc. - Ca2+ dependent Regulates vascular tone hence blood pressure and blood flow Inhibits platelet aggregation NO and it’s unpaired e-.  NO or NO t1/2 (aq) = 1-2 secs  Why?.  How does NO stabilise it’s unpaired e-? Perioxynitrite (ONOO ) -  Nicotinamide adenine dinucleotide phosphate (NADPH)  Key source of reactive oxygen species (ROS)/superoxide (O2-)  O2- + NO ONOO-  ONOO- oxidant & nitrating agent Soluble guanylate cyclase (sGC) Soluble guanylate cyclase - sGC  Enzyme, coverts GTP → cGMP Endothelium NO Fe sGC GTP PKG cGMP PDE GMP PDE = phosphodiesterase Vasodilatation Smooth muscle cell PKG = protein kinase G sGC  Primary intracellular receptor for NO  Mediates majority of NO’s physiological effects  Found in cytosol of virtually all mammalian cells  Highest concentration – lungs & brain sGC NO heam Heam domain  Two subunits – α (large) and β (small) subunit Dimerisation domain  2 types α1β1 & α2β1  3 domains – heam-binding, dimerisation & catalytic α β Catalytic domain  NO-heam complex  X histidine-heam bond = conformational change in catalytic domain GTP cGMP sGC and ROS  sGC exists within redox equilibrium  Reduced = NO sensitive  Oxidised = NO insensitive  If ↑[ROS] what will be the implication for sGC? sGC KO mice  Mice with a sGC subunit deleted  β1 KO – hypertensive, intestinal dysmotility & loss of α subunit  Megakaryocyte & platelet β1 KO – prolonged tail bleeding times  α1 KO - males hypertensive Phosphodiesterases - PDEs  PDEs hydrolyse cyclic nucleotides PDE Hydrolyses Expressed brain, heart, kidney, liver, skeletal & 1 cGMP & cAMP smooth muscle brain, heart, kidney, liver, skeletal & 2 cGMP & cAMP smooth muscle heart, smooth muscle, adipose tissue 3 cGMP & cAMP & platelets Kidney, brain, lung, mast cells, heart, 4 cAMP skeletal & smooth muscle brain, platelets, skeletal & smooth 5 cGMP muscle 6 cGMP Retina, breast cancer cells (?) Learning objectives  Discuss how NO is synthesised.  Discuss NOS ‘uncoupling’ and the significance for disease.  Discuss the role of sGC modulating the effects of NO and the significance for disease.  Discuss how PDEs modulate NO’s downstream and functional effects. Papers  EL ASSAR, M., ANGULO, J. & RODRÍGUEZ-MAÑAS, L. 2013. Oxidative stress and vascular inflammation in aging. Free Radical Biology and Medicine, 65, 380-401.  KIETADISORN, R., JUNI, R. P. & MOENS, A. L. 2012. Tackling endothelial dysfunction by modulating NOS uncoupling: new insights into its pathogenesis and therapeutic possibilities. American Journal of Physiology - Endocrinology And Metabolism, 302, E481-E495.  LI, H. & FÖRSTERMANN, U. 2013. Uncoupling of endothelial NO synthase in atherosclerosis and vascular disease. Current Opinion in Pharmacology, 13, 161-167.  ROCHETTE, L., LORIN, J., ZELLER, M., GUILLAND, J.-C., LORGIS, L., COTTIN, Y. & VERGELY, C. 2013. Nitric oxide synthase inhibition and oxidative stress in cardiovascular diseases: Possible therapeutic targets? Pharmacology & Therapeutics, 140, 239-257.  ROE, N. D. & REN, J. 2012. Nitric oxide synthase uncoupling: A therapeutic target in cardiovascular diseases. Vascular Pharmacology, 57, 168-172.