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.

Nitric oxide

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 (RR).

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 III 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, RR).

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, RR, 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 (Rand 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.

8 Replies to “Nitric oxide: more than just a vasodilator”

  1. Hello Hans! I got to this through the forum. On a discussion regarding increasing Klotho.
    I am wondering what are the best ways to inhibit NO to get the above mentioned inhibitors through dit, and what signs are that you have the necessary beneficial levels of NO?
    Thanks so much!
    John

  2. Hi Hans, great article!

    What is your opinion about tadalafil? I have sleep apnea and I feel good when I use it. I researched the subject and found that in Obstructive Sleep Apnea the nitric oxide is decreased.

    I am currently using Diamox and Niacin before bed and have had great improvements in sleep. I have even remembered dreams, something that is often interrupted in OSA, as apneas usually occur in REM sleep.

    1. Hi Gus,
      Thanks!
      I’m not a fan of NO boosters like that. If someone is in a healthy state, they can get away with it, whereas if someone is already in a compromised state, then it’s not a good idea. Beets, celery, etc, are safer since they also contain natural antioxidants that prevent oxidative stress. So a NO booster supplement from natural products is safer.
      But I’d rather look for why NO is low in the first place. Is it due to reduced synthesis or is it reduced by ROS?

  3. Thanks for the reply Hans. I have difficulties understanding the mechanism, I will post some links if you allow me. I started diamox recently and noticed improvement in several parameters and dream recall (unusual for me as apnea disrupts REM sleep). Maybe with the increase in CO2 via diamox I can also have improvements in NO. My question was based on the quality of sleep I got when using diamox with tadalafil (I only tested 2 times).
    Yesterday I started metergoline 2mg. The goal is to lower serotonin and see if I get better. I have also been testing BCAA + tyrosine (I felt an improvement in daytime sleepiness).

    ____________________________________________

    Nitric oxide (NO) and obstructive sleep apnea (OSA)
    James S J Haight 1, Per Gisle Djupesland
    Affiliations expand
    PMID: 12861485 DOI: 10.1007/s11325-003-0053-4
    Abstract
    Nitric oxide (NO) and obstructive sleep apnea are inseparable. Obstructive sleep apnea could be described as the intermittent failure to transport the full complement of nasal NO to the lung with each breath. There NO matches perfusion to ventilation. NO is utilized by the efferent pathways that control the unequal, inspiratory battle between the pharyngeal dilators and the closing negative pressures induced by the thoracic musculature. Recurrent cortical arousals are a major short-term complication, and the return to sleep after each arousal uses NO. The long-term complications, namely hypertension, myocardial infarction, and stroke, might be due to the repeated temporary dearth of NO in the tissues, secondary to a lack of oxygen, one of NO’s two essential substrates.
    https://pubmed.ncbi.nlm.nih.gov/12861485/

    _________________________________________

    Circulating nitric oxide is suppressed in obstructive sleep apnea and is reversed by nasal continuous positive airway pressure
    M S Ip 1, B Lam, L Y Chan, L Zheng, K W Tsang, P C Fung, W K Lam
    Affiliations expand
    PMID: 11112132 DOI: 10.1164/ajrccm.162.6.2002126
    Abstract
    Epidemiological studies have implicated obstructive sleep apnea (OSA) as an independent comorbid factor in cardiovascular and cerebrovascular diseases. The recurrent episodes of occlusion of upper airways during sleep result in pathophysiological changes that may predispose to vascular diseases, and we postulate that nitric oxide may be one of the mediators involved. This study investigates the levels of circulating nitric oxide (NO), measured as serum nitrites and nitrates, in the early morning in OSA subjects compared with control subjects, and the effect of overnight nasal continuous positive airway pressure (nCPAP) in OSA subjects. Thirty men with moderate to severe OSA (age = 41.9 +/- 9.0; apnea-hypopnea index, AHI = 48.0 +/- 18.1) were compared with 40 healthy men (age = 40.6 +/- 5.4; AHI = 1.4 +/- 1.2). Serum nitrite/nitrate levels were significantly lower in OSA subjects (OSA = 38.9 +/- 22.9 microM, control subjects = 63.1 +/- 47.5 microM, p = 0.015). There was significant negative correlation between serum nitrites/nitrates and the following parameters: AHI (r = -0.389, p = 0.001), oxygen desaturation time (r = -0.346, p = 0.004), and systolic blood pressure (BP) (r = -0.335, p = 0.005). Stepwise multiple linear regression with systolic or diastolic BP as the dependent variable identified serum nitrites/nitrates as the only significant correlate. Twenty-two OSA subjects had measurements of serum NO at baseline and after an overnight application nCPAP. There was significant increase in serum NO after nCPAP (baseline = 30.5 +/- 14.4 microM, after nCPAP = 81.0 +/- 82.1 microM, p = 0.01). This study demonstrates, for the first time, that circulating NO is suppressed in OSA, and this is promptly reversible with the use of nCPAP. The findings offer support for nitric oxide being one of the mediators involved in the acute hemodynamic regulation and long-term vascular remodeling in OSA.
    https://pubmed.ncbi.nlm.nih.gov/11112132/

    _________________________________________

    Effects of CPAP on nitrate and norepinephrine levels in severe and mild-moderate sleep apnea
    Paula Pinto, Cristina Bárbara, Joseph M Montserrat, Rita S Patarrão, Maria P Guarino, Miguel M Carmo, Maria P Macedo, Cristina Martinho, Rita Dias & Maria JM Gomes
    BMC Pulmonary Medicine volume 13, Article number: 13 (2013) Cite this article

    3370 Accesses

    29 Citations

    2 Altmetric

    Metricsdetails

    Abstract
    Background
    Reduced plasma nitrate (NOx) levels and increased urinary norepinephrine (U-NE) levels have been described in severe obstructive sleep apnea (OSA), and are reverted by continuous positive airway pressure (CPAP). The effect of CPAP on these biomarkers in mild-moderate OSA is not well understood.

    The aim of this study was to compare NOx and U-NE levels and blood pressure (BP) between male patients with mild-moderate and severe OSA and determine the impact of 1 month of CPAP therapy on these parameters.

    Methods
    We undertook a prospective study of 67 consecutive OSA patients (36 mild-moderate, 31 severe). Measurements of plasma NOx at 11 pm, 4 am and 7 am, 24-h U-NE and ambulatory BP were obtained at baseline and after 1 month of CPAP.

    Results
    At baseline, NOx levels showed a significant decrease during the night in both groups (p < 0.001). U-NE level and BP were significantly higher in the severe OSA group. After 1 month of CPAP, there was a significant increase in NOx levels and a reduction in U-NE level and BP only in patients with severe OSA.

    Conclusions
    One month of CPAP results in significant improvements in NOx levels, 24-h U-NE level and BP in patients with severe OSA, but not in patients with mild-moderate OSA.
    https://bmcpulmmed.biomedcentral.com/articles/10.1186/1471-2466-13-13

    _________________________________________

    What is the effect of obstructive sleep apnea (OSA) on the production of nitric oxide?
    Updated: Sep 15, 2020
    Author: Himanshu Wickramasinghe, MD, MBBS; Chief Editor: Zab Mosenifar, MD, FACP, FCCP more…
    References
    OSA has been associated with decreased production of nitric oxide. [119] Several studies have shown impaired vasodilator responses, as measured by either flow-mediated dilatation [120] or reactive hyperemic blood flow [121] techniques. Impaired flow-mediated dilatation was found to best correlate with the degree of oxygen desaturation in an epidemiologic cohort study. [122] Recent studies also indicate that cerebrovascular responses are impaired in patients with OSA.

    1. NO is often low because of elevated ROS, due to either low thyroid hormones, excess heavy metals, PUFAs, mitochondrial damage, etc. So lowering ROS almost always helps to increase NO. Boosting NO artificially will only help temporarily, but then it will stop working or even make things worse if ROS isn’t reduced.

  4. Hello Hans,

    This is the best article I have found breaking down the function of NO.

    I am on a journey of gaining the biggest blood vessels in town. That is my main goal.
    A major part of my routine involves sprints, and sometimes steady state jogs as a form of heart stretching.
    Most of my muscle routine revolves around calisthenics, as a supplement for vasobuilding.

    From I have understood in your article, to achieve my goals, it is crucial that I find foods/supplements that remove excess iNOS, but increase eNOS and nNOS.

    For a while, I have been on a keto diet, but quickly realized that it’s only beneficial for eliminating diseases or potential cancer cells, which isn’t a concern for me.

    My diet revolves around pinto beans, olive oil, hazelnuts/macadamia nuts/almonds, as well as vegetables with high nitrates through juicing. Anything that can constrict blood vessels, that normally tend to be promoted as anti-cancerfoods, is avoided
    Eggs used to be part of my diet, but have been eliminated due to its acidic nature, as well as the risk of increasing oxidated cholesterol.

    Many of my foods/supplements come from your recommendations, that fall within the alkaline end of the pH spectrum.

    I have two questions,

    If you have exercises that really make the heart pumping in an efficient manner to help dilate blood vessel walls safely, I’d love to research them.

    Are there periods before or after my training where your food/supplement recommendations would have a better effect?
    Your glycogen article somewhat answered the second question. If there is anything crucial I have missed, please advise.

    All the best,
    Brant

    1. Hey Brant,
      Thanks for the positive feedback.
      Honestly, if you eat right and train right, you’ll get the best vascular benefits. Vascularity has a lot to do with genetics and androgens. Histamine can also increase vascularity a lot.
      But all in all, you want to focus on optimizing your androgens and staying lean. Carbs and salt will also help.

      When it comes to diet, I’d go for easy to digest fat, so meat, milk, eggs, fruit and starches. Nuts, seeds, beans, wheat, etc, are best avoided. These foods are gut irritating and can even lower your thyroid hormones and androgen and promote intestinal inflammation, which will have a negative effect on your vascularity.

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