Pressure-induced expression of vascular neuronal nitric oxide synthase

Nitric oxide (NO), a major free radical, is an important signalling molecule in the cardiovascular system. It influences vascular function through its vasodilatory, anti-growth and anti-inflammatory properties [1]. In addition, NO modulates other important aspects of vascular function, including leukocyte activation, platelet aggregation and interactions between the endothelium and circulating cells [2,3]. Thus, NO, which signals through cGMP-dependent and cGMP-independent pathways [4], plays a major role in maintaining vascular integrity and homeostasis. Because of its importance in the vasculature, NO production and metabolism must be tightly regulated. Three genes encoding three different nitric oxide synthases (NOS) are responsible for enzymatic generation of NO from l-arginine, including endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS) [5,6]. eNOS, found mainly in the endothelium, and nNOS, located primarily in vascular smooth muscle cells, are Ca2+/calmodulin-dependent and are constitutively expressed [7,8]. iNOS is ubiquitous, Ca2+/calmodulin-independent and induced by cytokines, vasoactive agents and endotoxin [9]. Fluid shear stress and pulsatile stretch are major stimuli for eNOS activity, and have been implicated to play an important role in basal production of vascular NO [6–8,10]. Until recently, the role of nNOS and factors regulating its expression and activity in arteries were unclear. In this issue of the journal, Ebrahimian et al. [11] shed light on putative mechanisms that modulate vascular nNOS. In isolated rat carotid arteries exposed for 24 h to high intraluminal pressure, nNOS expression was markedly increased. This was evident both in the absence and the presence of endothelium and adventitia, indicating that vascular nNOS is located primarily in vascular smooth muscle cells of the media. Using pharmacological inhibitors, it was demonstrated that transmural pressure-induced expression of non-endothelial nNOS was dependent on a functionally active ERK1/2 cascade but independent of endogenous activation of the local renin–angiotensin II system. These findings certainly contribute to our understanding of vascular nNOS regulation, but what do they mean from a functional point of view? The major reason for investigating effects of high intraluminal pressure in intact arteries was to simulate as close as possible the situation in hypertension. To achieve this, isolated arteries were mounted in an organ culture system for 24 h and intraluminal pressure was increased to levels as high as 200 mmHg [11]. Previous studies showed that high intraluminal pressure in hypertension is associated with dysfunctional NO generation due, in part, to altered expression and activation of NOS [12,13]. Studies from NOS knockout mice, gene transfer and transgenic models, experimental models of hypertension, as well as human hypertension, demonstrate that vascular NO bioavailability is reduced [14–16]. This relative NO deficiency leads to vasoconstriction, leukocyte activation, increased vascular smooth muscle cell proliferation, inflammation and increased propensity to thrombosis [13,17]. How then can increased expression of NOS and enhanced NO generation induced by increased intraluminal pressure, as shown in the study of Ebrahimian et al. [11], be reconciled with reduced NO bioavailability in hypertension? The answer to this paradox may lie in the enzymatic source of NO production. Over-expression of nNOS with resultant increased vascular smooth muscle cell NO bioactivity induced by high intraluminal pressure contributes to vascular smooth muscle relaxation. This is demonstrated indirectly in the study by Ebrahimian et al. [11], as preferential inhibition of nNOS with S-methyl-l-thiocitrulline significantly enhanced contractile responses to angiotensin II in pressurized arteries. In hypertension, where intraluminal pressure is increased, augmented nNOS-derived NO may offset decreased eNOS-derived NO, thereby acting as an adaptive mechanism. Overerexpression of nNOS has been demonstrated in vascular smooth muscle cells and resistance arteries from hypertensive rats [18,19]. On the other hand, endothelial-derived NO, due to decreased eNOS expression and activity and/or increased quenching by •O2−, is reduced in hypertension [20,21]. The expressional down-regulation of eNOS has been directly implicated in glucocorticoid-induced hypertension [22]. Furthermore, in eNOS knockout mice, endothelium-derived relaxing factor activity is abolished, endothelial function is impaired and mice are hypertensive with mean arterial blood pressure values that are 20–30 mmHg greater than values observed in wild-type counterparts [23]. To further support a role for decreased eNOS activity in hypertension, Xiao et al. [24] reported that circulating endothelial NOS inhibitory factor, which is increased in patients with renal failure and hypertension, inhibits eNOS activity in cultured human endothelial cells. Accordingly, it may be possible that the effects of NO produced by one NOS isoform antagonize the effects of NO produced by another isoform. Pressure-mediated nNOS-derived NO in the presence of blunted eNOS-generated NO may act as a compensatory mechanism to maintain the production of bioactive NO in high pressure situations, such as in hypertension. However, not all models of hypertension exhibit attenuated eNOS activity. By contrast, NO production may be increased in hypertension. Some studies reported increased expression of both eNOS and nNOS in hypertension [25,26]. This occurs in young and aged spontaneously hypertensive rats (SHR), suggesting that development of hypertension may not be due to a primary impairment of NO production in SHR [27]. Furthermore high shear stress itself induces up-regulation of eNOS. Indeed, in the study by Ebrahimian et al. [11], not only was nNOS activity enhanced by pressure, but also eNOS expression was augmented. These phenomena may act to overwhelm increased oxidative stress, a major factor implicated in vascular damage associated with hypertension [28,29]. Unfortunately, this aspect is not addressed in their study [11], and it would have been particularly interesting to know whether increased intraluminal pressure in organ culture preparations does in fact stimulate production of reactive oxygen species. Others have shown that physical factors, such as shear stress, stretch, flow and pressure, are mediators of oxidative stress in hypertensive vessels [30]. Although the study by Ebrahimian et al. [11] contributes to the body of knowledge regarding nNOS regulation, there are a number of limitations that warrant attention. First, an intraluminal pressure of 200 mmHg is very high. It is rare that peripheral resistance arteries, vessels important in blood pressure regulation, would be exposed to such pressures, even in situations of severe and malignant hypertension. Furthermore, the pressure was constant whereas in-vivo intra-arterial pressure is pulsatile and influenced by dynamic extra-arterial pressure fluctuations. Similarly, changes in shear stress, which were not accounted for in the present study, may significantly influence endothelial function and NO production. Accordingly, caution is warrented when extrapolating data from the organ culture system, where carotid arteries from normotensive rats were studied, to in-vivo conditions in hypertension. Second, physical factors, through mechanotransduction signalling pathways, influence many genes and signalling cascades [31]. Of particular interest within the context of the present study is the fact that increased intraluminal pressure up-regulates expression of pre-pro-endothelin-1 gene expression, endothelin-1 production and ETB receptor expression [32,33]. Because of the interdependencies between the endothelin- and NO- systems, it is possible that these are concurrently influenced by an intraluminal pressure of 200 mmHg. It would be interesting to investigate whether there is a differential time course of activation and whether increased nNOS expression is a primary phenomenon in response to increased intraluminal pressure, or whether it is a secondary response to up-regulation of the vascular endothelin system. Finally, the conclusion that pressure increases nNOS expression in an ERK1/2-dependent angiotensin II-independent manner may indeed be true for acute situations, but may not necessarily be the case in chronic conditions. In the study by Ebrahimian et al. [11], the role of ERK1/2 and endogenous angiotensin II were assessed after only 9 h of pressurization. It is unlikely that significant de novo angiotensin II synthesis would occur within this short time period. Without knowing whether angiotensin II concentrations were elevated after pressurization, it is difficult to assess if increased intraluminal pressure maintained in the organ culture system did activate the endogenous renin-angiotensin II system. It is possible that longer periods of pressurization are required to induce production of angiotensin II, which could influence nNOS expression through its effects on ERK1/2 signalling [34]. Indeed, in rats infused with angiotensin II for 3 h, expression of eNOS and nNOS was unchanged [35]. However, after 3 days of infusion, NOS expression was augmented [35]. These findings are in agreement with those of others [36] where it was shown that vascular nNOS expression was enhanced in rats infused with subpressor doses of angiotensin II for 6 days. Nevertheless, the study provides interesting data in an in-vitro model that resembles in-vivo conditions as close as possible. It is now clear that nNOS is located primarily in vascular smooth muscle cells and that it is influenced by pressure, Furthermore, activation of the MEK1/2-ERK1/2 signalling cascade plays an important role in nNOS regulation. Whether these events are adaptive processes to counterbalance increased oxidative stress and upregulation of the endothelin system, or whether overexpression of nNOS is a pathological and maladaptive consequence of increased intraluminal pressure, requires further clarification.