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Nitric Oxide

Nitric oxide (NO) is composed of nitrogen (N) and oxygen (O). The human body creates NO from dietary arginine and nitrate. NO plays a major signaling role in vascular relaxation, and elevated levels are associated with better blood flow and lower blood pressure.

Our evidence-based analysis on nitric oxide features 31 unique references to scientific papers.

Research analysis led by and reviewed by the Examine team.
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Research Breakdown on Nitric Oxide

1Biological Significance


Nitric Oxide (NO) is a small signalling molecule synthesized from the amino acid L-arginine by the family of nitric oxide synthases including eNOS (endothelial, NOS-III), iNOS (inducible, NOS-II), and nNOS (neuronal, NOS-I). This family of enzymes work as dimers, along with multiple co-factors including tetrahydrobiopterin, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), iron, and zinc. While the regulation and modulation of each isoform differs significantly, all of the isoforms catalyze the reaction of L-arginine with NADPH and oxygen, to yield NO, citrulline and NADP (Knowles and Moncada (1994); Marletta (1994).

1.2How NO Signals

The elucidation of nitric oxide as a signaling gas molecule resulted in the award of the Nobel prize in physiology/medicine in 1998, as this was the first time a gas molecule was identified to be produced by one cell, readily diffusing to another cell, and then acting as a signaling molecule in it. For example, NO produced by eNOS in endothelial cells diffuses to the adjacent smooth muscle cells, where it initiates a cascade of reactions by activating the soluble guanlyate cyclase, which catalyzes the production of cyclic GMP [10]. Elevation of cGMP leads to the activation of protein kinase G (PKG), which in turn phosphorylates myosin light chain (MLC) phosphatase (thus activating it). In turn, activated MLC phosphatase dephosphorylates MLC, resulting in relaxation of smooth muscle cells, and thus, vascular relaxation.

Nitric oxide signals by stimulating its receptor, the soluble guanylyl cyclase receptor, and increasing cellular levels of the signalling molecule called cyclic guanidine monophosphate (cGMP)

Additional players in the regulation of vascular tone include the phosphodiesterase family (PDE 1 – 11), which catalyze the hydrolysis of cGMP at the 3’ end [11][12], effectively stopping NO-mediated vascular relaxation. Due to the tight regulation of eNOS and NO production, it is difficult to modulate vascular relaxation by influencing eNOS activity.

Due to the physiologic relevance of PDEs in controlling cGMP levels, these have become a popular target when vascular relaxation and blood flow are desired. An example of this include drugs such as Viagra, Cialis and Levitra, all of which inhibit PDE-5, which happens to be specifically expressed in the smooth muscle cells within the corpus cavernosum in the penis [13]. Since inhibition of these enzymes result in accumulation of cGMP, it essentially becomes possible to potentiate the vascular relaxation effects of NO.

Phosphodiesterases are negative regulators of cGMP and cAMP (they hydrolyze these molecules). While not all PDE enzymes can target cGMP induced by NO effects on guanylate cyclase, a few of them have the ability to control NO signaling via degradation of key intermediate signaling molecule (cGMP)

1.3Oxidative Potential

NO can potentially be degraded into a molecule known as peroxynitrate (OONO-) which is the result of NO reacting with superoxide anions (O2-).[14][15] OONO- also acts as a reactive signalling molecule, although the end results tend to be formation of several structures known to be negative to the body; OONO- can nitrosylate (donate a nitrogen group) to amino acids to form compounds such as 3-nitrotyrosine or S-nitrosocysteine,[16] formation of protein carbonyls, or nitrosylation of phospholipids containing polyunsaturated fatty acids (PUFAs).[17][18] In this sense, Nitric Oxide can be used as a substrate by superoxide to form reactive compounds that are negative to health despite NO being relatively benign.[17]

Nitric Oxide can be transformed (by combining with superoxide radicals) to form peroxynitrate, which then can produce a variety of molecules that are associated with a state of unhealth and thought to play a role in the pathology


2.1Supplemental Nitric Oxide

NO that is synthesized in the body and subsequently released into the blood tends to have a half-life of 5 seconds or less,[19][20] and in vitro some complexes can be made to prolong the half-life to 445s or so for research purposes.[21] These short half-lifes indicate quick degradation of the Nitric Oxide molecule into its components (Nitrogen and Oxygen), and propert storage of NO can prolong its shelf-life has only been confirmed for up to 5 days[22] using Mylar balloons which attenuate degradation.[23] Due to this poor stability ex vivo (outside of the body), Nitric Oxide per se is never used as a supplement; but rather compounds that can stay in the blood for long enough to continuatelly aid in the production of new NO.

Nitric Oxide, per se, is unstable and has a short half-life; it exerts its benefits rapidly, but has no role as a supplment in and of itself. NO supplementation required other compounds that influence the internal NO production system



Nitric Oxide is involved in relaxation of vascular smooth muscle, which is a mechanism underlying cardioprotective effects from Nitric Oxide (via reducing blood pressure)[24]


Nitric Oxide tends to modulate ion channels, intrinsic excitability, mediates synaptic plasticity, and can diffuse through cellular membranes.[25]

Neuronal nitric oxide synthase (nNOS) is able to form a dimer with a protein known as PSD95[26][27] and this complex appears to be a positive regulator of depression, as inhibiting the nNOS-PSD95 interaction has antidepressant effects.[28] This complex is activated downstream of NMDA receptor activation.[29]


4.1Nitric Oxide Donors

Some supplements aid NO production merely by being a source of Nitrogen that the NOS enzyme can use to produce NO with. Arginine is the standard NO donor in supplementation, and citrulline is a more bioavailable form of Arginine. Other NO donors that exist are S-Nitrosoglutathione (formed endogenously) or the two classes of N-diazeniumdiolates or S-nitrosothiols, the latter of which contains the endogenous S-Nitrosoglutathione.[30][31]

Some compounds merely provide nitrogen for the enzyme to use to produce NO


  1. ^ J S Stamler, G Meissner. Physiology of nitric oxide in skeletal muscle. Physiol Rev. (2001)
  2. ^ Stephen J Bailey, et al. Dietary nitrate supplementation reduces the O2 cost of low-intensity exercise and enhances tolerance to high-intensity exercise in humans. J Appl Physiol (1985). (2009)
  3. ^ Bescós R, et al. The effect of nitric-oxide-related supplements on human performance. Sports Med. (2012)
  4. ^ Yvette C Luiking, Mariëlle P K J Engelen, Nicolaas E P Deutz. Regulation of nitric oxide production in health and disease. Curr Opin Clin Nutr Metab Care. (2010)
  5. ^ Dimitrios Tsikas. Methods of quantitative analysis of the nitric oxide metabolites nitrite and nitrate in human biological fluids. Free Radic Res. (2005)
  6. ^ William T Clements, Sang-Rok Lee, Richard J Bloomer. Nitrate ingestion: a review of the health and physical performance effects. Nutrients. (2014)
  7. ^ Hord NG, Tang Y, Bryan NS. Food sources of nitrates and nitrites: the physiologic context for potential health benefits. Am J Clin Nutr. (2009)
  8. ^ Jackson J, et al. The role of inorganic nitrate and nitrite in CVD. Nutr Res Rev. (2017)
  9. ^ Chambliss KL, Shaul PW. Estrogen modulation of endothelial nitric oxide synthase. Endocr Rev. (2002)
  10. ^ Feil R, Kleppisch T. NO/cGMP-dependent modulation of synaptic transmission. Handb Exp Pharmacol. (2008)
  11. ^ Thompson WJ, et al. Assay of cyclic nucleotide phosphodiesterase and resolution of multiple molecular forms of the enzyme. Adv Cyclic Nucleotide Res. (1979)
  12. ^ Beavo JA, et al. Identification and properties of cyclic nucleotide phosphodiesterases. Mol Cell Endocrinol. (1982)
  13. ^ Burnett AL. The role of nitric oxide in erectile dysfunction: implications for medical therapy. J Clin Hypertens (Greenwich). (2006)
  14. ^ Pacher P, Beckman JS, Liaudet L. Nitric oxide and peroxynitrite in health and disease. Physiol Rev. (2007)
  15. ^ Peroxynitrite: biochemistry, pathophysiology and development of therapeutics.
  16. ^ Groves JT. Peroxynitrite: reactive, invasive and enigmatic. Curr Opin Chem Biol. (1999)
  17. ^ a b Eiserich JP, Patel RP, O'Donnell VB. Pathophysiology of nitric oxide and related species: free radical reactions and modification of biomolecules. Mol Aspects Med. (1998)
  18. ^ Szabó C. Multiple pathways of peroxynitrite cytotoxicity. Toxicol Lett. (2003)
  19. ^ Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB J. (1992)
  20. ^ Archer S. Measurement of nitric oxide in biological models. FASEB J. (1993)
  21. ^ Hakim TS, et al. Half-life of nitric oxide in aqueous solutions with and without haemoglobin. Physiol Meas. (1996)
  22. ^ Yoda Y, et al. Storage conditions for stability of offline measurement of fractional exhaled nitric oxide after collection for epidemiologic research. BMC Pulm Med. (2012)
  23. ^ Bodini A, et al. Exhaled nitric oxide in mylar balloons: influence of storage time, humidity and temperature. Mediators Inflamm. (2003)
  24. ^ Lewis SJ, et al. Role of voltage-sensitive calcium-channels in nitric oxide-mediated vasodilation in spontaneously hypertensive rats. Eur J Pharmacol. (2005)
  25. ^ Tozer AJ, Forsythe ID, Steinert JR. Nitric oxide signalling augments neuronal voltage-gated L-type (Ca(v)1) and P/q-type (Ca(v)2.1) channels in the mouse medial nucleus of the trapezoid body. PLoS One. (2012)
  26. ^ Brenman JE, et al. Interaction of nitric oxide synthase with the postsynaptic density protein PSD-95 and alpha1-syntrophin mediated by PDZ domains. Cell. (1996)
  27. ^ Tochio H, et al. Formation of nNOS/PSD-95 PDZ dimer requires a preformed beta-finger structure from the nNOS PDZ domain. J Mol Biol. (2000)
  28. ^ Doucet MV, et al. Small-Molecule Inhibitors at the PSD-95/nNOS Interface have Antidepressant-Like Properties in Mice. Neuropsychopharmacology. (2013)
  29. ^ Guix FX, et al. The physiology and pathophysiology of nitric oxide in the brain. Prog Neurobiol. (2005)
  30. ^ Naghavi N, et al. Nitric Oxide Donors for Cardiovascular Implant Applications. Small. (2012)
  31. ^ Beigi F, et al. Dynamic denitrosylation via S-nitrosoglutathione reductase regulates cardiovascular function. Proc Natl Acad Sci U S A. (2012)