Nitric oxide: more than just a vasodilator

Nitric oxide (NO) is a chemical messenger that possesses an ability to freely diffuse across the cell membranes, and unlike other classical neurotransmitters, this molecule
is neither stored in the synaptic vesicles nor released by the process of exocytosis (R). It’s synthesized from the amino acid L-arginine by the enzyme NO synthase (NOS) in the body. There are three isoforms of NOS, namely neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS).

NOS are highly regulated heme-thiolate enzymes that convert arginine to N-hydroxyl-L-arginine and then to L-citrulline and NO. The complex reaction involves the transfer of electrons from NADPH, via the flavins FAD and FMN in the carboxy-terminal reductase domain, to the haem in the amino-terminal oxygenase domain, where the substrate L-arginine is oxidized to L-citrulline and NO. Tetrahydrobiopterin (BH4) is also necessary as a cofactor in NOS.

The end product of NOS, NO, which is a free radical as it has an unpaired electron and can also be written as ·NO.

Apart from that NOS can generate NO, it also has the capacity to generate reactive oxygen species (ROS) such as superoxide anion (O₂) and hydrogen peroxide (H₂O₂) when electron and proton transfer processes are ineffective in promoting oxygen activation. The reason for this alteration in eNOS activity leading to an increase in O₂ production may be related to a decrease in BH₄ levels since two recent studies show that O₂ production by eNOS is inhibited by BH₄ but not L-arginine. Also, both MnSOD (manganese superoxide dismutase) and catalase protect against uncoupled NOS.

So if someone has a supposed condition of low NO, the enzyme that creates NO isn’t necessarily low, but it’s actually uncoupled and is creating O2 and H2O2 instead of NO. So boosting it further will not necessarily help because it might increase NO slightly, but also boost O2 and H2O2 production. This “uncoupling” of eNOS occurs in several pathologies, like diabetes, hypercholesterolaemia and hypertension (R).

Alternatively, NO can also be produced from nitrates (NO3) ⇒ nitrites (NO2) ⇒ NO. Nitrates and nitrites can come from the diet, but also from the oxidation of NO. Low pH and hypoxia increase the conversion of nitrate to nitrite to nitric oxide. NO can then be converted back to nitrite due to its reaction with oxyhemoglobin and ceruloplasmin. Peroxynitrite, which is formed when NO reacts with superoxide, can also easily be converted to nitrate.

It is advised that people with gastritis and high stomach pH (not enough stomach acid) should avoid dietary nitrates as it will be converted to nitrites and contribute to stomach cancer, including the formation of tumors in the liver, lung, and stomach (R, R).

Under normal circumstances, NO is present in low concentrations and acts as a signal in the body to regulate many processes. NO plays a role in memory, tissue oxygenation, vasodilation, killing viruses, the release of releasing hormones, such as corticotropin-releasing hormone (CRH), luteinizing hormone-releasing hormone (LHRH), and other releasing hormones in the hypothalamus, increasing GABA and growth hormone (GH), inhibiting platelet aggregation and vascular smooth muscle cell proliferation, glucose uptake in muscle, mitochondrial biogenesis (R), increasing AMPK (R), is involved in lipolysis (inhibiting NOS promotes lipolysis (R, R)) (R), etc.

So clearly, a little NO is needed. However, in excess contributes to many pathological conditions.

Excess NOS and NO

NO has also been implicated in the pathology of many inflammatory diseases, including arthritis, myocarditis, colitis, and nephritis and a large number of pathologic conditions such as amyotrophic lateral sclerosis, cancer, diabetes (NO damages the beta-cells of the pancreas. iNOS knock-out mice are protected from high‐fat diet‐induced insulin resistance (R)) (R), CFS (R), osteoarthritis (R), IBD (R), and neurodegenerative diseases.

NO, in excess, also promotes migraines (R), vascular leakage, oxidizes cholesterol, is involved in angina pectoris and finally myocardial infarction, varicose veins (estrogen is also involved), hypoxia (NO-modified hemoglobin formation was inversely proportional to oxyhemoglobin saturation (R) and inhibiting NOS increases tissue oxygenation (R)), intraocular pressure, impairs detoxification, increases parathyroid hormone and aldosterone (which can lead to soft tissue calcification, fibrosis, hypertension and other vascular diseases.), promotes lipid peroxidation (by liberating iron from ferritin), causes DNA damage (R), but inhibits DNA synthesis (by binding to the iron of ribonucleotide reductase) (R) and lowers the NAD and ATP by using it to promote DNA repair (R), promotes cell death, decreased nocturnal melatonin levels and finally even calcification of the gland, which occurs with aging, activates cyclooxygenase and lipoxygenase (which generates prostaglandins and lipoxygenase, which are toxic in high concentrations) (R), contributes to skin conditions (such as psoriasis, atopic dermatitis, irritant dermatitis, allergic dermatitis, lupus erythematous, rosacea, sunburn-induced flushing, nerve-mediated flushing and skin swelling) (R), etc.

iNOS is induced by a variety of factors, but mostly by inflammation, endotoxins, viruses and stress and produces about a 1000 fold more NO than eNOS. It is this chronic induction of iNOS that leads to many pathologies. The NO generated by iNOS reacts with superoxide to generate peroxynitrite, a highly reactive molecule. Alternatively, NO could react with O₂ (dioxygen, not superoxide) to yield nitrosyldioxyl radical (ONOO⋅), the presumed first step in the autoxidation reaction. iNOS uses a lot of arginine that should be used by eNOS, thus lowering eNOS activity. iNOS also promotes arginase, thus increasing the conversion of arginine to ornithine, lowering the arginine for eNOS even more. This leads to an uncoupling of eNOS which then generates the superoxide and hydrogen peroxide instead of NO, which contributes to hypertension. Inhibiting iNOS would be essential to restore proper eNOS activity and to lower oxidative stress.

NO inhibits mitochondrial function

NO inhibits mitochondrial function by binding to a variety of enzymes that use a heme group, such as cytochrome c oxidase, complex I & II of the electron transport chain (NADH-ubiquinone oxidoreductase & NADH-succinate oxidoreductase), aconitase (the enzyme that converts citrate to isocitrate. Inhibition of this enzyme will increase fat synthesis), etc. This binding inactivates the enzymes and blocks cellular respiration leading to cell death.

A little bit of NO from iNOS in the short term stimulates glycolysis and inhibits pyruvate dehydrogenase, thus boosting lactate production, instead of producing energy through oxidative phosphorylation. High production of NO through iNOS inhibits glycolysis completely which leads to cell death due to lack of energy production. To make it even worse, the NO then also causes S-nitrosylation of parkin, which disrupts its E3 ubiquitin ligase activity and results in improper fission, mitophagy and autophagy, thus preventing the proper removal of dead cells in the body (R, R, R).

Nitric oxide in the brain and how it affects neurotransmitters

NO is involved in neurodegeneration and contributes to mental disorders and conditions such as Parkinson’s disease (R), Alzheimer’s disease (R), depression, suicide (R), dementia (R), nerve damage of epilepsy, amyotrophic lateral sclerosis, multiple sclerosis (R), Huntington’s chorea (R), etc.

NO increases the release of acetylcholine and glutamate (also increases the ratio of glutamate to GABA) (R), thus promoting alertness and focus, but when in excess, will promote anxiety and paranoia. NO also inhibits the release of glycine (R) and dopamine (R), which promotes motivation and creativity and prevents excess excitation.

Inhibition of nNOS induced a very large increase in extracellular dopamine and a small increase in serotonin.

Androgens

Some people might think that NO is needed for androgen production and that inhibiting it might reduce steroidogenesis. NO increases the release of luteinizing hormone release hormone in the hypothalamus, which will increase the release of LH, which will stimulate steroidogenesis. NO exerts a biphasic effect on testosterone secretion, which is stimulatory at low and inhibitory at high concentrations; the stimulatory effect of NO is mediated by cGMP, the classic second messenger for NO action (R) (methylene blue increases cGMP levels). But NO isn’t as important for steroidogenesis as you might think, because NO boosters and precursors don’t increase testosterone levels and NOS inhibitors don’t lower testosterone production.

Actually, NOS inhibition increases steroidogenesis and testosterone in rats (R), bovine (R) and ducks (R). No human studies, unfortunately. Sildenafil, which increases intracavernosal cyclic guanosine monophosphate (cGMP) slightly increases testosterone levels, but also estrogen, so it still isn’t the NO that is responsible for the boost (R, R). And the T boosting effect happens only in men with low T.

Things that activate NOS

• Estrogen & Phytoestrogen (R, R)
• Endotoxin (iNOS) (R)
• Acetylcholine (R)
• Insulin (R)
• Bradykinin (R)
• Histamine (R)
• Iron (iNOS) (R)
• Parathyroid hormone (R)
• TSH (TSH can also up-regulate the expression of eNOS; however, it is accompanied by a reduced concentration of NO and increased level of superoxide anion, thereby indicating uncoupled eNOS) (R)
• Resveratrol (inhibit iNOS, but increase eNOS (R, R))
• Serotonin (tryptophan depletion diet lowers NO) (R)
• Infection (iNOS) (R)
• Inflammation (iNOS) (R)
• Oxytocin (eNOS) (R)
• Testosterone & DHT (promote eNOS and inhibit nNOS and lowers iNOS expression (R, R, R, R))
• Prolactin (inhibit eNOS but activate nNOS) (R, R)
• Leptin (the more fat you have, the more leptin and thus NO. Leptin potential iNOS and increases nNOS and eNOS (R, R, R)
• SIRT1 (eNOS (R, R))
• Nitrate-rich foods
• Vitamin C + garlic combo
• Insecticides (e.g. organophosphates) (R)
• Hypoxia (iNOS and eNOS (only short term effect on eNOS) (R, R))
• Omega 3 & 6, but not 9 (such as Mead Acid) (R, R) (inhibit eNOS, but increase iNOS)
• Ammonia (activates iNOS (R))
• Glutamine (inhibit eNOS and increase eNOS) (R)
• Lactate (R)
• Glutamate/aspartate (increase nNOS and iNOS) (R)
• NMDA (increase nNOS) (R)
• Homocysteine (inhibit eNOS and increase iNOS) (R)
• Age (iNOS increases with age (R))
• Vitamin C, A, E and folate (increase eNOS) (R)
• Vitamin A and E (increase nNOS) (R)

Things that inhibit NOS

• Lysine (R)
• Glycine (inhibit iNOS, but induce nNOS through NMDA) (R)
• Taurine (inhibit iNOS) (R)
• Glutamine (inhibit eNOS) (R)
• Hyperglycemia (inhibit eNOS) (R)
• Vitamin K and carotenoids (inhibits iNOS) (R)
• Magnesium (promote eNOS, but inhibit iNOS and nNOS (R, R))
• Glucosamine (inhibits NO production by decreasing cellular free NADPH availability; inhibit iNOS (R))
• Methylene blue (inhibit nNOS (R) and iNOS (R))
• Niacinamide (inhibit iNOS) (R)
• Progesterone (increase eNOS, but inhibit iNOS) (R)) 
• Agmatine (increase eNOS (R), but inhibit iNOS (R) and nNOS (R))
• Caffeine (activates eNOS (R), but significantly lowers exhaled NO, which shows that it lowers excess NOS (R))
• Salicylic acid (increase eNOS (R) and inhibits iNOS (R))
• Emodin (inhibits iNOS (R))
• Fructose (lowers iNOS nRNA (R))
• Zinc (inhibit iNOS (R))
• Berberine (reduces iNOS mRNA (R))
• L-canavanine – a non-proteinogenic amino acid found in certain leguminous plants (inhibits iNOS (R))
• Carnosine (inhibits iNOS (R)). Beta-alanine, at 6g doses, is very effective at increasing carnosine levels in the body.

Mop up NO

Apart from a few NOS inhibitors, you can also scavenge NO.

A few NO scavengers are superoxide dismutase (SOD), hydroxocobalamin and activated charcoal (8).

Methylene blue and red light (670nm wavelength, infra-red light or sunlight) displace NO from heme-containing enzymes and complexes.