The first pathway to be discovered for the endogenous production of NO was that involving L-arginine. For years, scientists and physicians have investigated L-arginine supplementation as a means to enhance NO production. This strategy has been shown to work effectively in young healthy individuals with functional endothelium or in older patients with high levels of asymmetric dimethyl L-arginine (ADMA), where the supplemental L-arginine can outcompete this natural inhibitor of NO production. However, patients with endothelial dysfunction, by definition, are unable to convert L-arginine to NO and, therefore, this strategy has failed in clinical trials. In fact, the Vascular Interaction With Age in Myocardial Infarction (VINTAGE MI) randomized clinical trial, concluded that L-arginine therapy (when added to standard postinfarction therapies) did not improve vascular stiffness measurements or ejection fraction, and was associated with higher postinfarction mortality.7 Thus, L-arginine should not be recommended following acute myocardial infarction (MI). However, there are also a number of studies showing benefit to patients taking L-arginine and just as many showing no benefit, no harm. Collectively, the literature suggests that strategies to enhance NO production through L-arginine supplementation are equivocal at best. Therefore there must be an alternative pathway for repleting NO in patients.
AGE-DEPENDENT DECLINE IN NITRIC OXIDE PRODUCTION
When we are young and healthy, the endothelial production of NO through L-arginine is efficient and sufficient to produce NO; however, as we age we lose our ability to synthesize endothelial derived NO. Most of the works on the activity of NO in cells and tissues agree that the bioavailability or the generation of nitric oxide synthase (NOS) derived NO decreases with aging. It has been proposed that superoxide can scavenge NO to form peroxynitrite and thereby reduce its effective concentrations in cells.8 It has also been reported that there is decreased NOS expression with aging both in constitutive and inducible isoforms.9,10 Berkowitz et al11 observed the upregulation of arginase (an enzyme that degrades the natural substrate for NOS, L-arginine) in aged blood vessels and the corresponding modulation of NOS activity. Taddei et al12 have shown that there is a gradual decline in endothelial function due to aging, with greater than 50% loss in endothelial function in the oldest age group tested as measured by forearm blood flow assays. Egashira et al13 reported more dramatic findings in the coronary circulation of aging adults whereby there was a loss of 75% of endothelium-derived nitric oxide in 70-80 year-old patients compared to young, healthy 20-year-olds. Vita et al14 demonstrated that increasing age was one predictor of abnormal endothelium-dependent vasodilation in atherosclerotic human epicardial coronary arteries. Whilst Gerhard et al15 concluded from their 1996 study that age was the most significant predictor of endothelium-dependent vasodilator responses by multiple stepwise regression analysis. Collectively, these important findings illustrate that endothelium-dependent vasodilation in resistance vessels declines progressively with increasing age. This abnormality is present in healthy adults who have no other cardiovascular risk factors, such as diabetes, hypertension, or hypercholesterolemia. Most of these studies found that impairment of endothelium-dependent vasodilation was clearly evident by the fourth decade. In contrast, endothelium-independent vasodilation does not change significantly with aging, demonstrating that the responsiveness to NO does not change; only the ability to generate it does.
These observations enable us to conclude that reduced availability of endothelium-derived NO occurs as we age and to speculate that this abnormality may create an environment that is conducive to atherogenesis and other vascular disorders. It appears that aging interrupts NO signaling at every conceivable level, from production to inactivation. Given that NO is a necessary molecule for maintenance of health and prevention of disease, restoration of NO homeostasis may provide a new treatment modality for age and age related disease.
The core of what we are dealing with is referred to as endothelial dysfunction, which is by definition the physiological dysfunction of normal biochemical processes carried out by the endothelium, the cells that line the inner surface of all blood vessels, including arteries and veins (as well as the innermost lining of the heart and lymphatics). Loss of endothelial NO function is associated with several cardiovascular disorders, including atherosclerosis, which is due either to decreased production or to increased degradation of NO.16 Experimental and clinical studies provide evidence that defects of endothelial NO function is not only associated with all major cardiovascular risk factors, such as hyperlipidemia, diabetes, hypertension, smoking, and severity of atherosclerosis, but also has a profound predictive value for the future atherosclerotic disease progression.17-20 The dysfunctional endothelial NOS/NO pathway is considered as an early marker or a common mechanism for various cardiovascular disorders.
It appears that the inability to produce sufficient NO under the right preclinical conditions enhances the risk for a number of diseases that plague the older population. If this is true, then there exists an opportunity to intervene early during this process, and to implement strategies to restore NO homeostasis, and, perhaps, delay or prevent the onset and progression of certain diseases. This gradual loss of NO activity with age can be sped up or slowed down based on individual lifestyle and diet. This idea is illustrated in the hypothetical graphical representation in Figure 1. Adopting healthy habits such as a good diet and exercise can prolong the precipitous drop in NO production that occurs with age. To the contrary, a poor diet along with physical inactivity can accelerate the process and lead to a faster decline in NO production at a younger age. Therapeutic strategies directed at improving endothelial function or providing an alternative source of NO should be the primary focus because they may reduce the incidence of atherosclerosis or other diseases that occur with aging, even perhaps Alzheimer's disease.
Figure 1. Hypothetical representation of NO production based on diet and lifestyle
HUMAN NITROGEN CYCLE
Until recently it was thought that NO acted only as an autocrine or paracrine mediator due to its extremely short half-life (<1 sec), meaning that it could only act in the cell in which it was produced (autocrine) or a nearby cell (paracrine). Our lab was able to convincingly demonstrate that NO can serve as an endocrine mediator as well. Our report in the Proceedings of the National Academy of Science demonstrates that NO produced in the heart can be transported as nitrite or nitrosothiols and limit liver injury.21 This seminal discovery then allows us to develop novel strategies to replete NO in humans by increasing the transport and delivery of NO through nitrite and nitrosothiols. We have shown that nitrite can also transiently form nitrosothiols (RSNOs) under both normoxic and hypoxic conditions22 and a recent study by Bryan et al demonstrates that steady state concentrations of tissue nitrite and nitrosothiols are affected by changes in dietary nitrite and nitrate (NOx) intake.23 This occurs through a reductive process involving bacteria and human enzymes. In fact this nitrate-nitrite-nitric oxide pathway may be a redundant system for overcoming the body’s inability to make NO from L-arginine.
It appears that we have at least two systems for affecting NO production/homeostasis. The first is through the classical L-arginine-NO pathway. This is a complex and complicated pathway, and if any of the co-factors become limiting, then NO production from NOS shuts down, and in many cases, NOS then produces superoxide instead. The enzymatic production of NO is, indeed, a very complex and coordinated effort that normally proceeds very efficiently. However, in disease