Primary hypertension (PH) in childhood and adolescence is not a benign disease and causes significant target organ damage (TOD) present in 30-40% of children already at the diagnosis of elevated blood pressure (BP) (1-5). The main intermediate phenotypes of children with PH are metabolic abnormalities typical of metabolic syndrome, oxidative stress and immune activation. Moreover, these abnormalities are also strictly associated with TOD (6-10). Pathogenesis of PH is multifactorial and it seems that different mechanisms are responsible for elevation of blood pressure and development of TOD. However, these mechanisms are interrelated. For instance, elevation of BP may lead to increase of carotid intima-media thickness (cIMT) and arterial stiffness and increased arterial stiffness causes elevation of BP. Moreover, not all hypertensive patients develop TOD. It suggests that some subjects, and in a case of so common diseases as PH, some part of population, is susceptible to development of TOD and as the consequence, to cardiovascular events (11). Heritability studies suggest that interindividual differences of blood pressure (BP) values are, at least in part, explained by genetic factors (40-60%) (12). However, the interaction of age, gender, ethnicity, diet, used medicines and lifestyle behavior complicates these analyses (13). It is evidenced that the functional impact of genetic polymorphisms on cardiovascular disease is greatest among subjects with lower overall risk (14-16). Since children are relatively free of the common environmental and concomitant clinical factors contributing to cardiovascular disease, the genetic associations exerting theirs effects during the long preclinical phase that begins in childhood, are suspected to be more significant (17, 18).

Human and animal studies point to a number of candidate genes, which may be involved in the development of PH and cardiovascular complications but may also interact with environmental parameters. Because PH is a disease of the arterial tree characterized by increased IMT and arterial stiffening, factors influencing endothelial function are potential modulators of susceptibility to develop PH and TOD. Nitric oxide (NO) has been established as a key signaling molecule in vascular homeostasis. Thus, polymorphisms in the gene that encodes endothelial nitric oxide synthase (eNOS) are of interest because of its potential to affect development of PH and TOD (19).

The discovery of NO, previously known as endothelium-derived relaxing factor, was one of the most significant biological achievements of the 20^th century distinguished by the Nobel Prize in Medicine in 1998. Simple NO molecule is a regulator of many physiological processes. It is synthesized by vascular endothelial cells, it is responsible for vasodilatation and is involved in various processes in the nervous, reproductive and immune systems (20). NO is involved in a wide variety of regulatory mechanisms of the cardiovascular system, including vascular tone (i.e. it is the major mediator of endothelium dependent vasodilatation) and vascular structure (e.g. inhibition of smooth muscle cell proliferation), and cell-cell interactions in blood vessels (e.g. inhibition of platelet adhesion and aggregation, inhibition of monocyte adhesion, cytostatic and cytotoxic properties) (20, 21). In addition to its participation in the regulation of vascular smooth muscle tone, NO directly affects mitochondrial respiration and plays important roles in the development of metabolic syndrome (MS) components, such as insulin resistance, endothelial dysfunction, hypertriglyceridemia and chronic adipose tissue inflammation and is involved in different mitochondrial signaling pathways that control respiration and apoptosis (22, 23).

Moreover, impaired NO bioavailability could also be related to a cellular defect in skeletal muscle tissue, where NO regulates metabolic and contractile processes and also basal, insulin-independent glucose transport (24). Experiments with homozygous eNOS knockout mice have definitively proven the relationship between NO and insulin sensitivity because these mice showed increased blood pressure and insulin resistance (25).

Because of these multiple functions, NO is regarded as an endogenous antiatherosclerotic molecule and tight control of NO production is believed to be critically important for the maintenance of cellular and tissue homeostasis (20, 21). There is hardly a disease not associated with altered NO homeostasis and endothelial dysfunction has become synonymous with reduced biological activity of NO. Thus, endothelial- and NO-dysfunction is a hallmark of not only cardiovascular disease and hypertension but also of obesity, diabetes, malnutrition (26).

There are data indicating on the involvement of eNOS in the pathogenesis of PH and the association of a relative or absolute decrease of eNOS activity with various vascular complications in response to hemodynamic workload (27). There are also data indicating that relative or absolute defect in the production of NO by eNOS or an abundant degradation of NO by enhanced oxidative stress (reactive oxygen species) is associated with various vascular complications in response to hemodynamic workload (27, 28).

NO is an essential molecule, nevertheless, its production is not always beneficial, as an excess or diminished NO production can have detrimental effects. Furthermore, cellular effects of NO may depend not only on its concentration, but also on its site of release and duration of action (29). The discrepant beneficial and detrimental effects that have been ascribed to NO may depend on closely regulated levels of NO in the vessel wall (30, 31). Within endothelial cells that line the lumen of all blood vessels eNOS catalyzes calcium-calmodulin-dependent NO synthesis through the conversion of L-arginine to L-citrulline (32). The normal function of eNOS requires dimerization of the enzyme, the presence of the substrate L-arginine and the essential cofactor (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4), one of the most potent naturally occurring reducing agents. Diminished levels of BH4 or L-arginin have been attributed to the failure of eNOS to form dimers. In monomeric form eNOS (referred to as eNOS uncoupling) catalyzes the reduction of molecular oxygen to the free radical superoxide (O2^-) instead of NO. Moreover O2^- reacts avidly with NO and forms peroxynitrite (ONOO^-), a much more powerful oxidant, which in turn also leads to eNOS uncoupling and enzyme dysfunction.

Superoxide is a free radical which rapidly reacts with NO reducing its bioactivity and producing peroxynitrite; a strong oxidant that can nitrosylate cellular proteins and lipoproteins (33-35). Recent evidence suggests that increased superoxide production accounts for a significant proportion of the NO deficit in several animal models of vascular disease, including hypercholesterolemia, hypertension, and heart failure (36). In addition to effects mediated by scavenging NO, superoxide directly stimulates mitogenesis in vascular smooth muscle cells and reduces eNOS expression and activity in endothelial cells (36, 37).

Potential sources of vascular superoxide production include nicotinamide adenine dinucleotide phosphate (NAD(P)H)-dependent oxidases, xanthine oxidase, lipoxygenase, mitochondrial oxidases, and NO synthases (36). NAD(P)H oxidases represent major sources of this reactive oxygen species and have been found upregulated and activated in animal models of hypertension, diabetes, and sedentary lifestyle and in patients with cardiovascular risk factors (38). Peroxynitrite, the direct reaction product of NO· and O2^-, interacts with lipids, DNA, and proteins via direct oxidative reactions or via indirect, radical-mediated mechanisms. These reactions trigger cellular responses ranging from subtle modulations of cell signaling to overwhelming oxidative injury, committing cells to necrosis or apoptosis (20). It is important to note that particularly ONOO^-, is able to oxidize BH4 to the BH3· radical (38).

The plasma membrane invaginations that form caveolae are also critical for modulation of eNOS activity. Indeed, it is within caveolae that eNOS attains maximal activity and interacts with CAV-1, a 21-24 kDa protein that coats the cytoplasmic surface of caveolae. In caveolae, eNOS activation is modulated through direct-steric inhibition of calmodulin binding with caveolin (39, 40). Agonist activation (or stimulation by shear stress) increases intracellular calcium and calcium-calmodulin binding, which displaces caveolin and reverses its inhibitory effect on eNOS. In addition to this tonic inhibition, interaction with CAV-1 contributes to eNOS concentration in caveolae. A substantial proportion of active eNOS resides in the peri-Golgi area, proper caveolar localization is critical for eNOS activation and maximal activity (41). Reduced activity of eNOS observed in arterial hypertension, can be caused by increased bonding between eNOS and CAV-1, which inhibits the activity of eNOS. CAV-1 binds eNOS via both the caveolin scaffolding domain and its carboxy-terminal domain. Via this interaction, CAV-1 inhibits eNOS function and NO generation. On the contrary, loss of CAV-1 leads to eNOS hyperactivation and uncontrolled NO overproduction. Excess NO, secondary to the loss of CAV-1, induces mitochondrial dysfunction and aerobic glycolysis, via NO effects on the electron transport system, and interactions of NO with free radicals what generates peroxynitrites (42).

Under normal, basal conditions in blood vessels, NO is steadily produced by eNOS and determines vascular tonus. The activity of eNOS is calcium- and calmodulin-dependent. There are two basic pathways for the stimulation of eNOS, and both of them involve release of calcium ions from subsarcolemmal storage sites. First, shearing forces acting on the vascular endothelium generated by blood flow causes a release of calcium and subsequent eNOS activation. Therefore, an increase in blood flow stimulates NO formation (flow--dependent NO formation). Second, endothelial receptors for a variety of ligands stimulate calcium release and subsequent NO production (receptor-stimulated NO formation). Included are receptors for acetylcholine, bradykinin, substance-P, adenosine, and many others vasoactive substances.