1. Jacob Poirier, Philip Johnston, Sami Khan
PHM Fall 2017
Instructor: Dr. Jeffrey Henderson
Nitric Oxide
JP introduction
Jacob Poirier, Philip Johnston, Sami Khan

2. Nitric Oxide (NO)
Originally known as endothelium-derived relaxing factor, however in 1987 it was discovered to be NO1
Small, two-atom gaseous molecule
Short half-life and highly reactive 2, 3
Lipid soluble 3 JP
As you can see by the structure, it has a lone electron pair which makes the NO extremely reactive which contributes to its short half life. The half life has been measured at up to 30s in physiological buffers, but expected to be only a few seconds in cells since it is a free radical and there are an abundance of targets to react with in the body.
Lipid soluble  The lipophilicity of NO is a significant factor in its role as a messenger, as a high lipophilicity together with its small size will allow NO to enter and accumulate in hydrophobic membranes and conseuquently enter cells.

3. Synthesis via Dietary Nitrates
In the acidic stomach environment
SAMI
One process by which nitric oxide can be produced is via the metabolism of dietary nitrate
Nitrates are rich in vegetables such as lettuce, beets and carrots. As part of their initial digestion in the oral cavity, facultative anaerobic bacteria can convert the contained nitrates into nitrites using the enzyme nitrate reductase and NADH as energy. These nitrites are then converted non-enzymatically to nitric oxide in the lumen of the stomach, as the conversion is favoured by the low pH of the stomach
In the stomach, nitric oxide can kill enteropathogens, as NO can readily diffuse across cell membrane, bind with high affinity to iron-sulfur containing respiratory enzymes and also damage bacterial DNA
Nitrate Reductase
(Nitrate)
(Nitrite)
Facultative anaerobic bacteria in the oral cavity
Benjamin et al., 1994
Lundberg et al., 2008

4. Synthesis via Nitric Oxide Synthase
L-Arginine JP
The other source of NO is from NO synthase enzymes in the body.
These enzymes use L-Arginine (which is an amino acid) as a substrate. Then using an oxygen molecule combined with NADPH as an energy source the arginine is oxidized to form an unstable intermediate. Then with the addition of more oxygen and NADPH the intermediate is converted to L-Citrulline and NO is released as a byproduct.
Intermediate
L-Citrulline
Knowles & Moncada, 1994

5. Enzymatic Production via Nitric Oxide Synthase (NOS)
Type of NOS
Primary location
Function
Expression
Neuronal NOS
Nervous tissue
Skeletal muscle (Type II)
Long-term potentiation; excitation contraction coupling
Constitutive
Endothelial NOS
Endothelial cells
Modulation of vascular tone
Inducible NOS
Immune cells
Immune defence against pathogens
Inducible JP
nnos in skeletal muscle Skeletal muscle functions regulated by NO or related molecules include force production (excitation-contraction coupling), autoregulation of blood flow, myocyte differentiation, respiration, and glucose homeostasis.
iNOS —> iNOS produces large quantities of NO upon stimulation by proinflammatory cytokines. can cause vasodilation (allowing for increased blood flow during immune response, necessary for increased circulation of wbcs etc. —> redness, heat, swelling assoicated with inflammation), and act as a radical to damage pathogen.
discuss difference between constitutive and inducible in this context.

6. Mechanism of NO in smooth muscle relaxation
PHILIP
I cant find a citation for this source
The binding of acetylcholine causes the release of NO in vascular endothelial cells that causes the relaxation of the vascular smooth muscle (vasodialator).
1) binding of acetylcholine to G protein receptors causes IP3 production.
2) IP3 releases calcium ions from endoplasmic reticulum.
3) ca++ ions and calmodulin form complex which stimulates NO synthase to produce NO.
4) NO (g) diffuses from endothelial cell into adjacent smooth muscle cells.
5) In smooth muscle cell, NO activates guanylyl cyclase to make cyclic GMP (cGMP).
6) cGMP activates protein kinase G which phosphorylates several muscle proteins to induce muscle relaxation.

7. What is Angina?
Ischemic heart disease is the leading cause of death in the United States and angina pectoris is a common symptom of this disease10
“Angina" is used to describe clinical symptoms such as discomfort in the chest, jaw, shoulder, back, or arms that are induced by physical exertion or emotional stress
Angina is due to the mismatch between myocardial oxygen demand and supply, resulting in myocardial ischemia. Symptoms are alleviated with rest or treatment with nitroglycerin.11
SAMI
The leading cause of death in the united states is ischemic heart disease and its most common symptom is angina pectoralis. Affected individuals may experience angina where during times of myocardial ischemia, a time when the myocardial oxygen supply is not meeting the demand.
Chronic stable angina, the initial manifestation of CAD in approximately 50% of all patients,6 is usually caused by the obstruction of at least 1 large epicardial coronary artery by atheromatous plaque. Angina pectoris is characterized by substernal discomfort, heaviness, or a pressure-like feeling, which may radiate to the jaw, shoulder, back, or arm and which typically lasts several minutes. These symptoms are usually brought on by exertion, emotional stress, cold, or a heavy meal and are relieved by rest or nitroglycerin within minutes
I think we should rearrange points 2 and 3, but leave part about nitroglycerin as is.

8. Nitroglycerin Mechanism of Action
Vascular Smooth Muscle Cell NO
XOR
SAMI
For the treatment of angina pectoralis, nitroglycerin is indicated. It is a nitric oxide donor that is taken sublingually, for quick absorption and bypassing of first-pass metabolism.
From the systemic circulation, nitroglycerin goes to act on vascular smooth muscle cells. Within their mitochondrial matrix, mitochondrial aldehyde dehydrogenase catalyzes the reduction of nitroglycerin to 1,2 glyceryl dinitrate and an nitrite anion. This anion is then converted to nitric oxide in the cytoplasm by xanthine oxidoreductase. This NO can then function through the canonical pathway we mentioned to activate the production of cGMP, lowering of the intracellular Ca ion concentration and subsequently relax the vascular smooth muscle, dilation blood vessels and alleviate symptoms.
NO activates Guanylyl Cyclase and cGMP production
MtALDH
cGMP activates downstream signaling to lower [Ca2+]
Mitochondrial Matrix
Lower [Ca2+] causes muscle relaxation and dilation of blood vessels
MtALDH: Mitochondrial Aldehyde Dehydrogenase
XOR: Xanthine Oxidoreductase

9. Erectile Dysfunction
Due to lack of smooth muscle relaxation = reduced of blood flow
Most common causes include: Antidepressants such as SSRI’s, and Nicotine12, 13
Other causes include: neurogenic disorders, cavernosal disorders, and psychological disorders such as stress/anxiety14
PHILIP
cavernosal —> as in corpus cavernosum, penis tissue

10. Sildenafil (Viagra) Mechanism of Action
PHILIP
The signal (nitric oxide) is released from nerve endings or from endothelial cells and activates a cascade reaction, which ultimately leads to an increased cellular concentration of cGMP (cyclic guanosine monophosphate). This second messenger molecule induces a series of events that lead to smooth-muscle relaxation through a reduction in the intracellular calcium ion concentration. The enzyme PDE-5 (phosphodiesterase type 5) reverses this effect by metabolizing the cGMP to GMP rapidly. The clinically important in- hibitors of this enzyme (sildenafil, vardenafil and tadalafil) all act to promote smooth-muscle relaxation by their abil- ity to allow cGMP to accumulate when nitric oxide is released, as is the case when sexual stimulation is present.
(more mechanistic stuff)
Fazio & Brock, 2004

11. Summary
Nitric oxide (NO) can be produced from dietary nitrates via a two-step process. Bacterial nitrate reductases in the oral cavity reduce nitrates to nitrites, which are then non-enzymatically reduced further from nitrite to NO in the acidic stomach.
NO can also be synthesized from L-Arginine via one of three nitric oxide synthase (NOS) isoforms: neuronal NOS (nNOS) which is found in nervous and skeletal tissue, inducible NOS (iNOS) used by immune cells and endothelial NOS (eNOS) used by endothelial cells.
Endothelial is expressed constitutively and used by the endothelial cells primarily as a vasodilator.
Newly formed NO diffuses into the adjacent smooth muscle cell to make cyclic GMP (cGMP) via activation of guanylyl cyclase. cGMP activates protein kinase G which results in muscle relaxation.
Symptoms of Angina pectoris can be treated by Nitroglycerin (sublingually) which dilates the coronary arteries by first entering the smooth muscle cells and entering the mitochondrial matrix where it is converted enzymatically to 1,2-glyceryldinatrate and nitrite. The nitrite molecule is converted to NO enzymatically by Xanthine Oxidereductase which then activates guanylyl cyclase.
Sildenafil and other PDE-5 inhibitors are used to treat erectile dysfunction (ED) by preventing the degradation of cyclic GMP (cGMP) in the corpus cavernosum resulting in decreased intracellular Ca2+ in the smooth muscle cell, causing relaxation.
Benjamin, N., O'Driscoll, F., Dougall, H., Duncan, C., Smith, L., Golden, M., & McKenzie, H. (1994). Stomach NO synthesis. Nature, 368(6471), 502. doi: /368502a0
Lundberg, J. O., Weitzberg, E., & Gladwin, M. T. (2008). The nitrate-nitrite-nitric oxide pathway in physiology and therapeutics. Nat Rev Drug Discov, 7(2), doi: /nrd2466

12. Summary Vascular Smooth Muscle Cell SAMINO
XOR
SAMI
For the treatment of angina pectoralis, nitroglycerin is indicated. It is a nitric oxide donor that is taken sublingually, for quick absorption and bypassing of first-pass metabolism.
From the systemic circulation, nitroglycerin goes to act on vascular smooth muscle cells. Within their mitochondrial matrix, mitochondrial aldehyde dehydrogenase catalyzes the reduction of nitroglycerin to 1,2 glyceryl dinitrate and an nitrite anion. This anion is then converted to nitric oxide in the cytoplasm by xanthine oxidoreductase. This NO can then function through the canonical pathway we mentioned to activate the production of cGMP, lowering of the intracellular Ca ion concentration and subsequently relax the vascular smooth muscle, dilation blood vessels and alleviate symptoms.
NO activates Guanylyl Cyclase and cGMP production
MtALDH
cGMP activates downstream signaling to lower [Ca2+]
Mitochondrial Matrix
Lower [Ca2+] causes muscle relaxation and dilation of blood vessels
MtALDH: Mitochondrial Aldehyde Dehydrogenase
XOR: Xanthine Oxidoreductase

13. References
Villanueva, C., & Giulivi, C. (2010). Subcellular and cellular locations of nitric-oxide synthase isoforms as determinants of health and disease. Free Radical Biology & Medicine, 49(3), 307–316.
Beckman JS. The physiological and pathological chemistry of nitric oxide. In: Lancaster JR Jr, editor. Nitric oxide. Principles and actions. San Diego: Academic Press; pp. 1–82.
Martin N. Hughes, Chapter One - Chemistry of Nitric Oxide and Related Species, Editor(s): Robert K. Poole, In Methods in Enzymology, Academic Press, Volume 436, 2008, Pages 3-19, ISSN , ISBN ,
Lundberg, J. O., Weitzberg, E., & Gladwin, M. T. (2008). The nitrate-nitrite-nitric oxide pathway in physiology and therapeutics. Nat Rev Drug Discov, 7(2), doi: /nrd2466
Benjamin, N., O'Driscoll, F., Dougall, H., Duncan, C., Smith, L., Golden, M., & McKenzie, H. (1994). Stomach NO synthesis. Nature, 368(6471), 502. doi: /368502a0
Knowles, R. G., & Moncada, S. (1994). Nitric oxide synthases in mammals. Biochemical Journal, 298(Pt 2), 249–258.
Förstermann, U., & Sessa, W. C. (2012). Nitric oxide synthases: regulation and function. European Heart Journal, 33(7), 829–837.
Kapur S, Bédard S, Marcotte B, Côté H, Marette A. (1997). Expression of Nitric Oxide Synthase in Skeletal Muscle: A Novel Role for Nitric Oxide as a Modulator of Insulin Action. Diabetes. 46 (11)  ; DOI:  /diab
Stamler JS, Gerhard M. (2001). Physiology of Nitric Oxide in Skeletal Muscle. Physiological Reviews. 81(1)
Zamora, R., Vodovotz, Y., & Billiar, T. R. (2000). Inducible nitric oxide synthase and inflammatory diseases. Molecular Medicine, 6(5), 347–373.
Lloyd-Jones D, Adams R, Carnethon M, et al. American Heart Association Statistics Committee and Stroke Statistics Subcommittee Heart disease and stroke statistics update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee [published correction appears in Circulation. 2009;119(3):e182] Circulation 2009;119(3):
Cassar, A., Holmes, D. R., Rihal, C. S., & Gersh, B. J. (2009). Chronic Coronary Artery Disease: Diagnosis and Management. Mayo Clinic Proceedings, 84(12), 1130–
Delgado PL, Brannan SK, Mallinckrodt CH, Tran PV, McNamara RK, Wang F, Watkin JG, Detke MJ (2005). "Sexual functioning assessed in 4 double-blind placebo- and paroxetine-controlled trials of duloxetine for major depressive disorder". The Journal of Clinical Psychiatry. 66 (6): 686–92. PMID   doi: /JCP.v66n0603.
Harte, C. B., & Meston, C. M. (2008). Acute Effects of Nicotine on Physiological and Subjective Sexual Arousal in Nonsmoking Men: A Randomized, Double-Blind, Placebo-Controlled Trial. The Journal of Sexual Medicine, 5(1), 110–121.
Hedon F. (2003). Anxiety and erectile dysfuntion: a global approach to ED enhances results and quality of life. Nature 15(2), S16-S19. doi: /sj.ijir
Fazio L., & Brock G. (2004). Erectile dysfunction: management update. Canadian Medical Association Journal, 170(9), doi:  /cmaj
Benjamin, N., O'Driscoll, F., Dougall, H., Duncan, C., Smith, L., Golden, M., & McKenzie, H. (1994). Stomach NO synthesis. Nature, 368(6471), 502. doi: /368502a0
Lundberg, J. O., Weitzberg, E., & Gladwin, M. T. (2008). The nitrate-nitrite-nitric oxide pathway in physiology and therapeutics. Nat Rev Drug Discov, 7(2), doi: /nrd2466