What is nitric oxide

Nitric oxide also called mononitrogen monoxide or nitrogen monoxide, is a colorless gas with the formula NO. Nitric oxide is an unstable free-radical gas which reacts rapidly with oxygen to form nitrogen oxides. Water soluble, nitric oxide (NO) is produced normally in numerous tissues and is considered to be a mediator of cell-to cell communication; nitric oxide (NO) functions in numerous processes including vasodilation, inflammation, and neurotransmission. Nitric oxide is a free radical gas, i.e., it has an unpaired electron, which is sometimes denoted by a dot in its chemical formula, i.e., ·NO. Although nitric oxide (NO) was identified as a gas in late eighteenth century, its role as a biological agent was confirmed only in 1980 ¹. Nitric oxide (NO) was identified as the known as an endothelium-derived relaxing factor (EDRF) or vasodilator in 1987 ². Nitric oxide (NO) also inhibits platelet aggregation, induces disaggregation of aggregated platelets, and inhibits platelet adhesion to the vascular endothelium. Nitric oxide activates cytosolic guanylate cyclase and thus elevates intracellular levels of cyclic GMP.

In the U.S., the Occupational Safety and Health Administration (OSHA) has set the legal limit (permissible exposure limit) for nitric oxide exposure in the workplace as 25 ppm (30 mg/m³) over an 8-hour workday. The National Institute for Occupational Safety and Health (NIOSH) has set a recommended exposure limit (REL) of 25 ppm (30 mg/m³) over an 8-hour workday. At levels of 100 ppm, nitric oxide is immediately dangerous to life and health ³.

Nitric oxide (NO) is formed from guanidine nitrogen of L-arginine by the action of 3 isoforms of the enzyme nitric oxide synthase (NOS), namely, endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and neuronal nitric oxide synthase (nNOS) ⁴.

This enzymatic conversion of arginine to nitric oxide (NO) by endothelial nitric oxide synthase (eNOS) requires oxygen and the reduced cofactors, tetrahydrobiopterin (BH4) and nicotinamide adenine dinucleotide phosphate (NADPH) ⁵. Increased intracellular calcium in response to vasodilator agonists such as acetylcholine and bradykinin leads to activation of endothelial nitric oxide synthase (eNOS) and increased production of nitric oxide (NO) in endothelial cells. Nitric oxide (NO) diffuses into vascular smooth muscle cells where it activates soluble guanylyl cyclase, in turn forming cyclic guanosine monophosphate (cGMP) to elicit vasorelaxation, as shown in Figure 1. This nitric oxide/ soluble guanylyl cyclase/cGMP pathway has shown a spatial distribution in the kidney, and its importance in this organ is supported by many studies ⁶. The level of cGMP is determined by the balance between the synthesizing enzyme soluble guanylyl cyclase (sGC) and catabolizing enzyme phosphodiestrase (PDE), which metabolizes cGMP to its biologically inactive metabolite, 5′-GMP, as shown in Figure 1. In most cells, the rate of synthesis of cGMP is 10-fold lower than its rate of hydrolysis by phosphodiestrase (PDE) ⁷. Therefore, inhibition of phosphodiestrase (PDE) is considered a therapeutic target in many pathological conditions such as erectile dysfunction ⁸, in which an elevated levels of nitric oxide is desired. Another interaction of cGMP is with protein kinase G (PKG), which is activated upon CGMP stimulation to phosphorylate different target proteins upon cGMP stimulation. These proteins are involved in vasodilation, neutrophil activation, smooth muscle tone modulator, and matrix expansion ⁹ as shown in Figure 1.

Figure 1. Nitric oxide synthesis

Abbreviations: NO = Nitric oxide; endothelial nitric oxide synthase = eNOS; BH4 = tetrahydrobiopterin; NADPH = nicotinamide adenine dinucleotide phosphate; cGMP = cyclic guanosine monophosphate; GMP = guanosine monophosphate; GC = Guanylate cyclase; PDE = phosphodiesterase; PKG = protein kinase G;

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What does nitric oxide do?

Nitric oxide is a vasodilator. The physiologic effect of nitric oxide is by means of vasodilation. A large body of evidence supports that the nitric oxide system plays a critical role in blood pressure regulation ⁴. The data obtained in the cardiovascular system are consistent, and the literature has described in detail the role of L-arginine (nitric oxide donor) in hypertension ¹⁰. Reduced concentrations of nitric oxide in plasma have been observed in patients with essential hypertension ¹¹ and endothelium dependent vasodilation is impaired in patients with essential hypertension ¹². The endothelium (inner lining) of blood vessels uses nitric oxide to signal the surrounding smooth muscle to relax, thus resulting in vasodilation and increasing blood flow. Sildenafil (Viagra) is a common example of a drug that uses the nitric oxide pathway. Sildenafil does not produce nitric oxide, but enhances the signals that are the downstream of the nitric oxide pathway by protecting cyclic guanosine monophosphate (cGMP) from degradation by cGMP-specific phosphodiesterase type 5 (PDE5) in the corpus cavernosum, allowing for the signal to be enhanced, and thus vasodilation ¹³.

Figure 2. Interactions of nitric oxide with different systems and modulation of their functions in cardiovascular and renovascular systems

Abbreivations: AEA = anandamide; 2-AG = 2-arachidonylglycerol; CB1 = cannabinoid receptor 1; BP = blood pressure; MBF = myocardial blood flow; LVH = left ventricular hypertrophy; H₂S = hydrogen sulfide; Ang2 = angiotensin 2; NA = noradrenaline

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Nitric oxide bioavailability can be improved by both nonpharmacological and pharmacological approaches. Physical exercise is a nonpharmacological approach to enhance nitric oxide bioavailability and improve endothelial function in hypertensive patients ¹⁴. Restoration of eNOS function through augmentation of its substrate, L-arginine, or cofactors for its synthesis has been demonstrated to have beneficial effects. For example, chronic oral supplementation of tetrahydrobiopterin prevented the blood pressure increase in rats with 5/6 nephrectomy ¹⁵ and arterial stiffness in fructose-fed insulin-resistant rats ¹⁶. The critical role of eNOS and nitric oxide in the prevention of hypertension is supported by the finding that mice lacking a functional eNOS gene develop hypertension ¹⁷. NG-monomethyl-l-arginine (l-NMMA) has been frequently employed to block the nitric oxide production pathway to study the importance of this mechanism in hypertension. Intra-arterial infusion of this inhibitor in untreated hypertensive patients resulted in an abnormal basal nitric oxide-induced vasodilation in the forearm arteriolar bed ¹⁸. Intravenous injection of a low dose of l-NMMA in Sprague-Dawley rats affected the renal excretions of sodium and water without altering blood pressure, but at a high dose it induced hypertension ¹⁹.

Supplementation with L-arginine has been a common approach for testing the effect of nitric oxide enhancement on hypertension. By providing a source of nitrogen for synthesis of nitric oxide by NOS, L-arginine is thought to lower pressure by enhancing vasodilation of resistance vessels indirectly by augmenting the production of nitric oxide from NOS. This vasodilator action of nitric oxide (endothelium-derived relaxing factor; EDRF) depends on activation of sGC which converts guanosine triphosphate to its product, cyclic 3′, 5′-guanosine monophosphate (cGMP). It has been reported that the impaired nitric oxide production pathway may lead to the onset of essential hypertension ²⁰. L-arginine and L-citrulline, which is converted to L-arginine, increased the production of nitric oxide and prevented the development of salt-sensitive hypertension in Dahl/Rapp rats ²¹. In patients with hypertension, the oral administration of l-arginine was an effective therapeutic option ²². In some studies, vasorelaxation in response to nitroglycerin was also blunted ²³, indicating associated changes in responsiveness to nitric oxide either supplied exogenously or produced endogenously. Despite several lines of evidence, demonstrating antihypertensive actions of L-arginine, there is a paucity of evidence regarding the role of l-arginine in hypertension. It has been reported that newly diagnosed, mild to moderate hypertensive patients given l-arginine (2 g three times per day) had reduced blood pressure and improved vascular function after one week of treatment ²⁴. Similarly in patients with mild hypertension, the infusion of l-arginine (500 mg/kg for 30 min) lowered the mean blood pressure by 8% and reduced the renovascular resistance ²⁵. L-arginine reduced serum endothelin-1 and angiotensin 2 levels ²⁶ which may be contributory factors in the lowering of blood pressure. Overall, these data support the idea that the endothelium plays an important role in the modulation of blood pressure and that nitric oxide is a key component responsible for this modulation and is generally known as endothelium-derived relaxing factor (EDRF). It may be possible to pharmacologically modulate the endogenous production of nitric oxide by exogenous agents to reduce blood pressure.

The upregulation of the nitric oxide/sGC/cGMP pathway in arterial hypertension has been identified as a promising therapeutic goal for lowering blood pressure and reducing associated complications related to heart and kidney function based on experimental models of hypertension and NOS-inhibition without causing tolerance ²⁷.

Summary

A large body of evidence supports nitric oxide roles as a vasodilator, as an antihypertensive, antihypertrophic, and renoprotective agent ⁴. The linkage of a single molecule with such a diverse number of physiological systems in the human body underscores its promising therapeutic potential in cardiovascular and renovascular complications ⁴. As a sympatholytic agent, nitric oxide not only contributes to the regulation of blood pressure, but also plays a renoprotective role in hypertensive patients by antagonizing the deleterious effects of norepinephrine (noradrenaline) and angiotensin and by mediating the release of another promising gasotransmitter, hydrogen sulfide (H₂S). In conclusion, studies have highlighted the beneficial roles of nitric oxide in the cardiovascular system and kidney, but also emphasized the link between nitric oxide and different physiological systems whose effects are either mediated by nitric oxide or antagonized by nitric oxide.

Nitric oxide supplements

Focusing on vasodilation, several sources ²⁸ have suggested that nitric oxide (NO) is a prime solicitor in the process. Nitric oxide (NO) is not ingested directly, rather it is derived from substances endogenously manufactured or ingested. Nitric oxide (NO) is synthesized via at least two physiological pathways: nitric oxide synthase (NOS)-dependent and NOS-independent. Research pertaining to the nitric oxide synthase (NOS)-dependent nitric oxide donors pathway have consistently shown to not affect on VO₂max ²⁹. Thus, further research on the effects of nitric oxide (NO) on VO₂max should shift its focus on investigating the nitric oxide synthase (NOS)-independent pathway instead.

Research on supplementation through the nitric oxide synthase (NOS)-independent pathway involves sodium nitrate and beetroot ³⁰. The nitric oxide synthase (NOS)-independent donors have shown mixed results. Sodium nitrate decreased VO₂ peak in two studies ³¹, ³², while beetroot juice increased VO₂max in one study ³³ and did not affect VO₂peak or VO₂max in three others ³⁴, ³⁵, ³⁶.

Nitrates, like those found in beetroot juice, have garnered interest due to their effects on lowering blood pressure at rest ³⁴, however there is also a line of research investigating their effects on performance. The current literature regarding the ergogenic effects of nitrates is mostly concentrated on four different nitric oxide donors: L-arginine, L-citrulline, sodium nitrate, and beetroot juice. These nitric oxide donors are categorized into two groups, based on whether they are oxidized in the nitric oxideS-dependent physiological pathway, or reduced in the nitric oxideS independent physiological pathway. L-arginine and L-citrulline pertain to the former, while sodium nitrate and beetroot juice pertain to the latter. The purpose of this study ³⁰ was to investigate the effects of beetroot juice compared to placebo on VO2max and blood pressure during submaximal exercise on recreationally trained college-aged males. VO₂max, respiratory exchange ratio, systolic blood pressure, and diastolic blood pressure were analyzed for differences between the supplement and the placebo. There were no significant differences in any of these variables after beetroot juice supplementation compared to placebo ³⁰. At rest, beetroot juice has been shown to decrease resting systolic blood pressure ³⁶ as well as diastolic blood pressure ³³. Systolic and diastolic blood pressure at 70% of max heart rate calculated using the Karvonen formula did not show a significant change after beetroot juice supplementation. A study by Miyai et al. ³⁷ investigated the blood pressure response to heart rate during exercise in 1033 normotensive men. The blood pressure mean values at 70% max heart rate from the present study were comparable to those found the study. The diastolic blood pressure means from the present study fell into the 50th percentile values of those 1033 men at 70% of heart rate reserve, while the systolic blood pressure means fell into the 25th percentile.

In a study by Larsen et al. ³⁸, supplementation with sodium nitrate (0.1 mmol /kg of body mass/day for 3 days) in moderately trained subjects showed a decrease in VO₂ at submaximal intensities, indicating a beneficial reduced oxygen cost during exercise. Gross efficiency, which was defined as work rate divided by energy expenditure, was also significantly improved. A later study by Larsen et al. ³² on moderately trained subjects used the same loading scheme for 2 days and also showed a significantly lower VO₂ peak after sodium nitrate ingestion without any effect on time to exhaustion. A study done on welltrained subjects showed a similar significantly reduced VO₂ peak compared to placebo, but with no effects on VO₂ at submaximal intensities. The dosage in this study was more acute (3 hours pre-exercise) compared to previous studies and used 10 mg/kg of body mass of sodium nitrate ³¹.

In a study done on physically active individuals, beetroot supplementation for 15 days showed a significant increase in peak power and VO₂max. Additionally, steady-state VO₂ at moderate-intensity exercise was significantly reduced 2.5 hours after ingestion ³³. In other studies, it has been shown to increase power output ³⁵, delay time to exhaustion, and increase efficiency not only at the onset of exercise, but also in moderate (80% of gas exchange threshold) and severe intensity exercise (70% of the difference between the power output at gas exchange threshold and VO₂ peak) ³⁶.

Nitric oxide supplements have been shown to increase mitochondrial efficiency by improving the amount of oxygen reduced per ATP produced, otherwise known as the mitochondrial P/O ratio ³⁹. Theoretically, this would explain the suggested benefits that previous studies have shown on the cardiorespiratory components of aerobic exercise. Keeping this in consideration, along with all the aforementioned results, it could be hypothesized that a nitric oxide supplement would help increase VO2max. With respect to the NOS-independent pathway, results are contradicting between two forms of NO donor. There are two studies using sodium nitrate that showed a significantly reduced VO2peak and VO2max following supplementation ³¹, while there is one study using beetroot juice that showed an increase in VO2max ³³. Furthermore, the study that used beetroot juice used a ramp cycle ergometer test to gather VO2 data, while it has been shown that an individualized ramp treadmill protocol elicits higher, more valid values and is more likely to bring an individual to a true VO2max due to the increased volume of muscle mass used ⁴⁰. In addition, it is widely accepted that typical blood pressure responses during incremental cardiorespiratory endurance exercise include an increase in systolic pressure and a maintenance of diastolic pressure. It is also known that NO is effective at reducing blood pressure at rest ³³. However, there appear to be no studies that have investigated the effect of supplementation on blood pressure values during exercise. The purpose of this study was to investigate the effects beetroot juice on VO2max and on blood pressure during submaximal exercise in recreationally trained, college-aged males.

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