NO Story ---- the Discovery of Nitric Oxide

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1 NO Story ---- the Discovery of Nitric Oxide Feng Gao, MD, PhD Department of Physiology Fourth Military Medical University Supported by NSFC(Nos , , , )& National Fund for Outstanding Young Scholars(No )

The Nobel Assembly at the Karolinska Institute in Stockholm, Sweden, has awarded the Nobel

discoveries concerning "the nitric oxide as a signalling molecule in the cardiovascular

of Pharmacol, SUNY Health Science Center New York, USA Louis J Ignarro born 1941 Dept.

3 The Nobel Assembly at the Karolinska Institute in Stockholm, Sweden, has awarded the Nobel Prize in Physiology or Medicine for 1998 to RF Furchgott, LJ Ignarro and F Murad for their discoveries concerning "the nitric oxide as a signalling molecule in the cardiovascular system". Robert F Furchgott born 1916 Dept. of Pharmacol, SUNY Health Science Center New York, USA Louis J Ignarro born 1941 Dept. of Mol And Med Pharmacol, UCLA Sch of Med Los Angeles, USA Ferid Murad born 1936 Dept. of Integ Biol Pharmacol Physiol, Univ of Texas Medical School, Houston, USA

4 Moncada

6 Nitric Oxide (NO) 第四军医大学 --- Hi, do you know me? --- I am NO body Q

7 Bad reputation 第四军医大学 Nitric Oxide Environmental pollutant Smog, cigarette smoking, exhaust gas from cars Destroyer of ozonosphere Precursor of acid rain Suspected carcinogen Neural toxic agent

8 NO' now 第四军医大学 Gas Free radical Simplest Smallest & Lightest Endogenous signaling molecule. *

Signal transmission by a gas, produced by one cell, which penetrates

9 A New Principle NO is a short-lived, endogenously produced gas that acts as a signalling molecule in the body. Signal transmission by a gas, produced by one cell, which penetrates membranes and regulates the function of other cells is an entirely new principle for signaling in the human organism.

10 L-arg-NOS-NO system

11 Isoforms of NOS 第四军医大学 Type Subcellular location Regulation Mol. Wt. Tissue/Cell I Soluble Ca 2+ /CaM 160 kd Brain, heart (nnos) NANC nerves epithelia cells skeletal muscle II Soluble>particulate Inducible by 130 kd Macrophages (inos) cytokines hepatocytes VSMC, heart III Particulate>soluble Ca 2+ /CaM 135 kd Endothelial cells (enos) heart * cnos: nnos & enos, activate rapidly, transient action, release at very low level ( pm )------Physiological * inos: activate slowly, long-term action, produce at comparatively high level ( nm ) Pathological, toxic

Biological Molecule 第四军医大学 Cytotoxicity CARDIOVASCULAR Vasodilation Platelet adhesion & aggression Neutrophil activation & adhesion Central blood pressure control CENTRAL NERVOUS Long-term synaptic

12 Biological Molecule 第四军医大学 Cytotoxicity CARDIOVASCULAR Vasodilation Platelet adhesion & aggression Neutrophil activation & adhesion Central blood pressure control CENTRAL NERVOUS Long-term synaptic potentiation Pain perception Neurodegeneration PERIPHERAL NERVOUS NANC smooth muscle relaxant neurotransmitter INMMUNOLOGICAL RESPIRATORY GASTROINTESTINAL CARDIOVASCULAR Vasoconstriction I / R injury Myocytes apoptosis Hypotension shock CENTRAL NERVOUS Neuronal death Convulsion Parkinson s disease PERIPHERAL NERVOUS Malfunction of penile erection INMMUNOLOGICAL RESPIRATORY GASTROINTESTINAL *

13 NO has many Clinical Applications 第四军医大学 Signalling between nerve cells in the brain Macrophage Defence against bacterial and parasitic infections NO NO can be inhaled to specifically treat BP pressure in the lungs of newborns NO initiates erection of the penis by dilating blood vessels Apoptosis

14 ? NO so simple, so important, but had not been discovered earlier?

15 NO A gas Too active Too simple Too ugly Cinderella By no means of by accident

16 History on recognizing of NO ---- Tracing back to a century ago 第四军医大学 I. Prelude 1850s: * Nitrate Ester was therapeutically useful in IHD NO - 2 * assay method established Griess reaction 1890s: Nobel produced TNT (NTG) 1933: Schretzenmayer --- the first doctor observed 1933: Flow-dependent vasodilation Mechanism unknown at that time

17 Alfred Nobel suffered from angina pectoris and was prescribed nitroglycerine. In a letter to a friend he wrote: It sounds like the irony of fate that I have been prescribed nitroglycerine internally. They have named it Trinitrin in order not to upset pharmacists and the public. Your affectionate friend, A. Nobel

18 History on recognizing of NO ---- Tracing back to a century ago 第四军医大学 I. Prelude 1850s: * Nitrate Ester was therapeutically useful in IHD NO - 2 * assay method established Griess reaction 1890s: Nobel produced TNT (NTG) 1933: Schretzenmayer --- the first doctor observed 1933: Flow-dependent vasodilation Mechanism unknown at that time

19 II. Puzzling period : RF Furchgott ACh Aorta strips Contraction The pharmacology of vascular smooth muscle 1962: Jelliffe, a PhD student Based on his own experiments, (Rabbit aorta) concluded: ACh : NO carcinogen F Murad --- NTG in the treatment of IHD Find Nitrovasodilator L H 第四军医大学 *Unlike in vivo *Repeatable Relaxation Constriction Then, Scientists realized that N-containing substances had very important effects in our body.

21 II. Puzzling period : RF Furchgott ACh Aorta strips Contraction The pharmacology of vascular smooth muscle 1962: Jelliffe, a PhD student Based on his own experiments, (Rabbit aorta) concluded: ACh : NO carcinogen F Murad --- NTG in the treatment of IHD Find Nitrovasodilator L H 第四军医大学 *Unlike in vivo *Repeatable Relaxation Constriction Then, Scientists realized that N-containing substances had very important effects in our body.

22 It all happened from a careless experiment David 1978, CCh contamination Helical or ring puzzle intima? Histology Sandwich idea

23 Furchgott's Sandwich 第四军医大学 Furchgott showed that ACh-induced relaxation of blood vessels was dependent on the endothelium. His "sandwich" experiment set the stage for future scientific development. He used two different pieces of the aorta; one had the endothelial layer intact, in the other it had been removed. Q

24 III. EDRF-NO period 第四军医大学 1980: RF. Furchgott The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by ACh Nature, 288: : BK PNAS EDRF? Q : Evidence was presented, indicating EDRF = NO Furchgott (NY) & Ignarro (CA): Independently found, Pharmacological similarity between EDRF and nitrovasodilator drugs Murad (Texas, 1977): Nitro-Esters/NO cgmp vasodilation Moncada (English-Honduras, ) Using biological assay and ECL, quantitatively demonstrated that NO release accounts for the biological activity of EDRF Nature 1987; 327:524 One yr later, identified that L-arg as the biological precursor to NO Vascular endothelial cells synthesize NO from L-arg Nature 1988; 333:664

25 Moncada's bioassay BK 第四军医大学 NTG ECL Perfusate NTG Nitric oxide release accounts for the biological activity of endothelium -derived relaxing factor. Nature, June 11, 1987; 327(6122): Endothelium-derived relaxing factor (EDRF) is a labile We suggest that EDRF and NO are identical.

26 III. EDRF-NO period 第四军医大学 1980: RF. Furchgott The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by ACh Nature, 288: : BK PNAS EDRF? Q : Evidence was presented, indicating EDRF = NO Furchgott (NY) & Ignarro (CA): Independently found, Pharmacological similarity between EDRF and nitrovasodilator drugs Murad (Texas, 1977): Nitro-Esters/NO cgmp vasodilation Moncada (English-Honduras, ) Using biological assay and ECL, quantitatively demonstrated that NO release accounts for the biological activity of EDRF Nature 1987; 327:524 One yr later, identified that L-arg as the biological precursor to NO Vascular endothelial cells synthesize NO from L-arg Nature 1988; 333:664

Ignarro's spectral analysis 第四军医大学 Ignarro reported that EDRF relaxed blood vessels. He also identified EDRF as a molecule by using spectral analysis of hemoglobin (Hb).

27 Ignarro's spectral analysis 第四军医大学 Ignarro reported that EDRF relaxed blood vessels. He also identified EDRF as a molecule by using spectral analysis of hemoglobin (Hb). When Hb was exposed to EDRF, maximum absorbance moved to a new wave-length; and exposed to NO, exactly the same shift in absorbance occurred! EDRF was identical with NO. A new principle for signalling between human cells was discovered. Spectrophotometry Hb (yellow) exposed to Ecs that were Stimulated to produce EDRF (green) Hb (yellow) directly exposed to NO (green) The shift of absorption curves is identical, hence EDRF is NO

He bubbled NO-gas through tissue containing the enzyme; cgmp increased! A new mode of drug action had been discovered!

28 Murad's enzyme activation 第四军医大学 Murad knew that nitroglycerine caused relaxation of VSMCs. The enzyme, GC, was activated and increased cgmp, causing relaxation of the muscle. Did nitroglycerin act via release of nitric oxide, NO? He bubbled NO-gas through tissue containing the enzyme; cgmp increased! A new mode of drug action had been discovered! NO induces synthesis of cgmp by stimulation of GC leading to relaxation of myosin (muscle protein) ACh stimulates the ECs to produce a factor, NO, which penetrates into and activates the SMCs causing relaxation.

29 Nitroglycerine, a 100 year old explosive and heart medicine 第四军医大学 In atherosclerosis, plaques reduce blood flow in the arteries. This decreases O 2 supply to the heart muscle causing chest pain (angina pectoris) and sometimes even myocardial infarction. Treatment with nitroglycerine provides NO, dilates the vessels, and increases blood b flow. Thanks to this year's Nobel Laureates we now understand how nitroglycerine, an important heart medicine, works. It acts as a NO donor, causes dilation of the blood vessels, increases O 2 supply and protects the heart from damage and cell death. Cinderella

30 nnos was purified and cloned (David S Bredt, Johns Hopkins, 1991). inos, enos 1992, Molecule of the Year L-arg-NOS-NO system

31 What have we done?

32 IF 14.75

33 IF 6.13

35 Enlightenment 1. 科研思路至关重要 Good idea is half done Discovery of ACh (Loewi, 1920) ---- Isolated double hearts perfusion Simple experiments, smart design.

36 Enlightenment 第四军医大学 2. 尊重实验结果, 让实验说话 Furchgott: Repeatable experiments Jellife: Report data as was David: Tell truth Respect, but not worship the authority

37 Enlightenment 第四军医大学 3. 发现 vs. 发明 Translational Medicine Q NO Viagra 基础研究与应用研究双步竟奔? 重要发现常蕴含潜在应用价值转化

38 Enlightenment 第四军医大学 4. 活跃竞争的学术思想是独创精神不可缺少的土壤 EDRF Furchgott 1980 EDCF EDHF Vanhoutte 1982 EDRF = NO 1986 ET Yanagisawa 1988 Endothelial Biology Nitric Oxide Biology and Chemistry NO tool drugs Viagra 缺乏实验条件资金 vs. 缺乏学术思想创新意识? 硬件差, 但绝非我们与科学发现相距遥远! 实验方法选择 : 简洁能说明问题好素质好思路出色工作

39 A prepared mind + = Nature

40 Enlightenments 第四军医大学 4. 活跃竞争的学术思想是独创精神不可缺少的土壤 EDRF Furchgott 1980 EDCF EDHF Vanhoutte 1982 EDRF = NO 1986 ET Yanagisawa 1988 Endothelial Biology Nitric Oxide Biology and Chemistry NO tool drugs Viagra 缺乏实验条件资金 vs. 缺乏学术思想创新意识? 硬件差, 但绝非我们与科学发现相距遥远! 实验方法选择 : 简洁能说明问题好素质好思路出色工作

PCTH 400. Endothelial dysfunction and cardiovascular diseases. Blood vessel LAST LECTURE. Endothelium. High blood pressure

PCTH 400. Endothelial dysfunction and cardiovascular diseases. Blood vessel LAST LECTURE. Endothelium. High blood pressure PCTH 400 LAST LECTURE Endothelial dysfunction and cardiovascular diseases. Classic Vascular pharmacology -chronic -systemic Local Vascular pharmacology -acute -targeted High blood pressure Blood pressure