Mechanisms and Consequences of eNOS Dysfunction in Hypertension

Abstract

Reduced nitric oxide (NO) bioavailability contributes to endothelial dysfunction and hypertension. The endothelial isoform of NO synthase (eNOS) is responsible for the production of NO within endothelium. Loss of eNOS cofactor tetrahydrobiopterin to initial increase in oxidative stress leads to uncoupling of eNOS, in which the enzyme produces superoxide anion rather than NO, further substantiating oxidative stress to induce vascular pathogenesis. The current review focuses on recent advances on the molecular mechanisms and consequences of eNOS dysfunction in hypertension, and potential novel therapeutic strategies restoring eNOS function to treat hypertension.

Introduction

There are at least 970 million people worldwide suffering from hypertension [1]. Patients with high blood pressure develop more cardiovascular complications [2]. Globally cardiovascular disease accounts for approximately 17 million deaths a year, nearly one third of the total mortality [3]. Among these, complications of hypertension account for 9.4 million deaths worldwide every year [3, 4]. It has become clear that nitric oxide (NO), produced by the endothelial isoform of NO synthase (eNOS) in the vascular endothelium, plays an important role in regulating blood pressure. Reduced NO bioavailability, which is considered a hallmark of endothelial dysfunction [5], plays an important role in mediating blood pressure elevation. Endothelial dysfunction also predicts atherosclerotic coronary and cerebral artery disease in hypertension. A better understanding of the molecular mechanisms regulating NO signaling under pathophysiological conditions is critically important in designing new therapeutic options. Therefore, in the present review we will discuss the following aspects of NO signaling that are relevant to hypertension: (1) nitric oxide and blood pressure regulation; (2) mechanisms of eNOS dysfunction; (3) consequences of eNOS uncoupling in hypertension; (4) potential novel therapies targeting uncoupled eNOS in hypertension.

1. Nitric oxide and blood pressure regulation

Accumulating evidence demonstrates a critical role of NO in blood pressure regulation. Released from endothelial cells, NO increases 3',5'-cyclic-guanosine monophosphate (cGMP) production and subsequent cGMP-dependent protein kinase (PKG) activation in the underneath vascular smooth muscle cells (VSMCs), resulting in vasodilatation [6, 7]. Pathophysiological regulation of NO signaling in endothelial cells and its relevance to VSMC regulation are summarized in Fig. 1. Previous studies have confirmed an essential role of NO in vasorelaxation of large human arteries [8]. In addition, impairment in NO mediated vasodilatation in brachial, coronary and renal arteries was also observed in patients with essential hypertension [9–12]. Moreover, the relaxation mediated by NO was depressed in mesenteric arteries of hypertensive rats with reduced renal mass, as the result of an arterial endothelial abnormality [13, 14]. In high-salt treated Dahl hypertensive rats, eNOS mRNA expression was down-regulated in mesenteric arterioles [15]. In deoxycorticosterone acetate-salt hypertensive rats, reduced eNOS phosphorylation resulted in reduced NO/cGMP signaling in mesenteric arteries [16]. Taken together, NO signaling plays an important role in both conduit and resistant arteries in the environment of hypertension. Of note, the mean blood pressure is 20 mm Hg higher in the eNOS knockout mice compared to its wild type littermates [17]. Therefore, understanding molecular mechanisms underlying impaired NO bioavailability and eNOS dysfunction in hypertension may prove beneficial in ultimately promoting development of novel therapeutics to treat hypertension.

Figure 1.

Endothelial dysfunction by eNOS uncoupling. Endothelial nitric oxide synthase (eNOS) produces nitric oxide (NO) to mediate vasorelaxation and preservation of vascular function. Tetrahydrobiopterin (H₄B) is the key cofactor responsible for normal electron transfer from the reductase domain of one eNOS monomer to the oxygenase domain of the other monomer to produce NO, and in its deficiency, eNOS produces superoxide rather than NO, a process now referred to as eNOS uncoupling. H₄B can be supplied by both de novo biosynthesis and salvage pathways. It is newly synthesized from GTP by activations of sequential enzymes of GTP hydrocyclolase 1 (GTPCH1), PTP synthase (PTPS), and sepiapterin reductase (SPR), or restored from its oxidized form H₂B by the salvage enzyme dihydrofolate reductase (DHFR). SPR can also catalyze conversion of H₄B precursor sepiapterin to H₂B, prior to its conversion to H₄B by DHFR. Pathological stimuli such as Ang II activates NADPH oxidase isoform 1 (NOX1) to produce superoxide in endothelial cells, which in turn cause peroxynitrite dependent oxidation of H₄B and hydrogen peroxide dependent DHFR deficiency, leading to persistent reduction in H₄B bioavailability. Superoxide production by uncoupled eNOS further sustains oxidative stress in the vasculature, resulting in endothelial dysfunction, impaired endothelium-dependent vasorelaxation, and elevated blood pressure.

Several mechanisms have been found responsible for NO deficiency in hypertension [9, 18–24]. Destruction of NO by superoxide anion leads to NO deficiency, endothelial dysfunction and high blood pressure. Among many enzymatic systems producing reactive oxygen species (ROS), NADPH oxidase (NOX), xanthine oxidase (XO), uncoupled eNOS, mitochondria, and cyclooxygenase (COX) have been extensively studied [18, 25]. Here we take COX for example, and the other ROS systems will be described in the later sections below. During the generation of prostanoids by COX, ROS are formed as by-products [26]. In most tissues, COX-1 is constitutively expressed, while COX-2 is often induced by a number of inflammation or growth factors [27]. In small resistance arteries of essential hypertensive patients, COX-2 is overexpressed and reduces nitric oxide availability, and COX-2 represents a major source of oxidative stress generation [28]. Indeed, endothelium-dependent contractions were triggered by acetylcholine (ACh) were abolished by COX-2 but not COX-1 inhibitors [29]. In addition, COX-2-derived prostaglandin F(2α) plays an important role in mediating endothelial dysfunction in renovascular hypertension [30]. Recently, Liu et al identified that uncoupling protein 2 inhibited oxidative stress and downregulated COX-2 expression to prevent endothelial dysfunction [31]. ROS from one source are able to trigger ROS production by activating other enzyme systems. For example, ROS produced by NOX can upregulate the expression of COX-2 by p38 MAPK-dependent mechanism, and also can induce eNOS uncoupling [32–34]. Oxidation of the eNOS cofactor tetrahydrobiopterin by peroxynitrite, a product of NO/superoxide interaction, induces eNOS uncoupling to produce superoxide rather than NO, further sustaining oxidative stress (see section 2.2).

Moreover, a defective L-arginine/NO pathway has been linked to NO deficiency in hypertension. Recent studies have confirmed that L-arginine transport is impaired in hypertensive and normotensive subjects with a genetic background of essential hypertension [20], and the offspring of essential hypertensive patients are characterized by a reduced response to acetylcholine linked to a defect in the nitric oxide pathway [19]. These data represent the link between L-arginine and the onset of essential hypertension. Furthermore, it has been shown that L-arginine supplementation improved endothelial dysfunction in hypertension [35]. The K_m of eNOS for L-arginine is about 3 μM, but the L-arginine plasma concentration rarely falls below 60 μM in pathological conditions [36]. An elevation in asymmetric dimethylarginine (ADMA) levels may explain this “L-arginine paradox,” since ADMA is an endogenous competitive inhibitor of NO synthase [37]. Oxidative stress dependent increase in circulating ADMA could lead to eNOS uncoupling [38], vasoconstriction [39], and therefore a marked increase in blood pressure [40, 41]. Of note, elevated ADMA levels have been observed in hypertension, hypercholesterolemia, diabetes, and chronic kidney failure (CKD) [42]. Because ADMA is normally cleared by kidney, patients with CKD have more than 3–10 fold higher plasma ADMA levels, which would lead to eNOS dysfunction and hypertension [43, 44]. Regulation of eNOS function by H₄B deficiency will be discussed in part 3.

2. Mechanisms of eNOS dysfunction

2.1. Synthesis of nitric oxide by NOS

Three NOS isoforms, neuronal (nNOS), inducible (iNOS), and endothelial (eNOS), catalyze the reaction of molecular oxygen with the amino acid substrate L-arginine to produce L-citrulline and NO [40, 45, 46]. nNOS is expressed in the central and peripheral nervous system, and produces NO that functions as a neurotransmitter [47]. It has been shown that nNOS is compensatorily up-regulated in hypertension [48]. Different from constitutively expressed nNOS and eNOS, the expression of iNOS is markedly increased by inflammatory cytokines [49]. Large quantities of NO produced by iNOS induce apoptosis to serve as a host defense mechanism, but also contribute to pathogenesis of chronic diseases [50]. eNOS, or NOS3, is constitutively expressed in vascular endothelial cells, and primarily located in the peri-nucleus, Golgi apparatus and caveolae [51, 52]. The NOS enzymes require several cofactors for enzymatic production of NO, including H₄B, nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and flavin mononucleotide (FMN) [53]. During the process of NO synthesis, the electrons donated by NADPH at the C terminal reductase domain are transferred to the heme catalytic center of the N terminal oxidase domain, where activation of molecular oxygen is “coupled” to NO synthesis by two successive monooxygenations of L-arginine [46, 54, 55].

2.2. Reactive oxygen species and eNOS uncouping

Under some disease conditions, NOX activation increases local production of ROS, which oxidize H₄B to induce H₄B deficiency [56]. H₄B promotes assembly of eNOS monomers into an active dimer, while lack of H₄B results in inability of electron transfer to the N terminal oxygenase domain of the other eNOS monomer [57, 58]. Treatment of endothelial cells with peroxynitrite led to reduced eNOS activity and disruption of eNOS dimers [59]. Peroxynitrite is believed to be primarily responsible for oxidation of H₄B to H₂B in vivo, although H₄B is sensitive to oxidation from many other ROS species [60, 61]. The oxidized form H₂B can compete with H₄B for eNOS interaction, and they share the same binding sites in the N terminal oxygenase domain of eNOS [62]. However, H₂B cannot support eNOS cofactor activity, so this displacement results in eNOS uncoupling. Therefore, under oxidative stress conditions, the salvage pathway of H₄B synthesis will become particularly important. Expanded discussions of mechanisms and contributions of eNOS uncoupling in hypertension are included in sections 3.1 and 3.2 below.

2.3. Phosphorylation Regulation of eNOS function

Regulation of eNOS function through phosphorylation has been well established. The activity is regulated through phosphorylation or dephosphorylation [63–66]. While phosphorylation of Ser615, 633, and 1177 results in the activation of eNOS, the phosphorylation of Thr495 reduces eNOS function [67]. Among these phosphorylated sites, Ser1177 and Thr495 are mostly investigated. The kinases taking part in eNOS phosphorylation at Ser1177, include AMPK, Akt, ERK1/2, and CaMK-II [68–71]. Reduced phosphorylation level at this site has been found in various of cardiovascular dieases, such as portal hypertension, diabetes, atherosclerosis, and myocardial infarction [67]. Of note, there seems to be a dynamic regulation of Ser1177 by transient hydrogen peroxide, acutely with ERK and chronically with Akt [33, 69, 71]. And Akt appears to be downstream of AMPK in this pathway [69]. In addition, a deficiency in Akt/eNOS signaling indicates impaired insulin signaling in type 2 diabetes. In aortas isolated from diabetic animals and in type 2 diabetic patients, Akt and eNOS phosphorylation was decreased [72, 73]. Reduced eNOS_s1177 phosphorylation, vascular dysfunction, and elevated blood pressure have been observed in high-fat fed animals [74]. On the other hand, AMPK and PKC can phosphorylate eNOS at Thr495, and its increased phosphorylation may contribute to hypoxia, diabetes and cerebral ischaemia [67]. Modulation of eNOS activity by dynamic changes in its phosphorylation level at different phosphorylation sites appears to be an important mechanism of eNOS regulation under different pathophysiological conditions.

2.4. Other factors and eNOS dysfunction

In addition to L-arginine deficiency as discussed earlier, several other mechanisms have been implicated in causing eNOS dysfunction/uncoupling. These include acetylation of eNOS, S-glutathionylation of eNOS, and protein-protein interactions other than phosphorylations discussed above. Cigarette smoking (CS) induced oxidative stress downregulates silent information regulator protein 1 (SIRT1), leading to acetylation of eNOS and reduced NO production [75]. In addition, eNOS can also be acetylated by histone deacetylase 3 (HDAC3), which decreases NO production by reduced calmodulin association [76]. More recently, S-glutathionylation of eNOS has been proposed as alternative mechanism provoking eNOS uncoupling [77]. Its interrelationship with H₄B deficiency, however, remains to be further investigated. Two highly conserved systeine residues, Cys 689 and Cys 908, in the C terminal reductase domain have been identified as sites of S-glutathionylation, which are important for eNOS function. Of note, eNOS S-glutathionylation in endothelial cells, accompanied by loss of NO and gain of superoxide, is associated with impaired endothelium-dependent vasodilation [77, 78]. Finally, the activity of eNOS can also be regulated by binding to its regulatory binding partners such as CaM, Caveolin-1 (Cav-1), Hsp90, and nitric oxide synthase interacting protein (NOSIP). For example, the association with Cav-1 inhibits eNOS activity and reduces NO production [79]. In Cav-1 knockout mice, both eNOS activity and vasorelaxation are enhanced in blood vessels [80]. In addition, Cav-1 deficiency was associated with attenuated Ang II-induced hypertension by inhibiting AT1a receptor-mediated uptake of Ang II in the renal proximal tubule [81].

3. Consequences/contributions of eNOS uncoupling in hypertension

3.1. Role of eNOS uncoupling in Angiotensin II dependent hypertension

Angiotensin II (Ang II) levels are often elevated in the kidney and plasma of patients with hypertension [82, 83]. AT1 receptor antagonists effectively reduce blood pressure in Ang II dependent hypertension [84, 85]. Our previous study has confirmed that eNOS uncoupling contributes to high blood pressure in Ang II infused mice, where aortic NO production was markedly decreased [86]. Dihydrofolate reductase (DHFR) overexpression or folic acid (FA) restoration of DHFR function effectively recoupled eNOS to reduce blood pressure [86, 87]. Moreover, in Ang II-infused hyperphenylalaninemia (hph)-1 mice, where 79% of the animals developed severe abdominal aortic aneurysm (AAA), oral FA administration completely prevented AAA from occurring [87]. eNOS uncoupling-mediated aneurysm formation was also prevented by FA in Ang II infused apoE null mice, and again this was attributed to targeted restoration of endothelial DHFR expression and activity [88]. The molecular mechanisms of Ang II induced eNOS uncoupling have been characterized by our group, which involves a rapid and transient activation of endothelial NOX, subsequent H₂O₂-dependent down-regulation of DHFR, and persistent H₄B deficiency (Fig. 1) [33, 56, 89]. More specifically, the NOX isoform 1 (NOX1), has been identified as the mediator of Ang II-dependent uncoupling of eNOS in streptozotocin (STZ) induced diabetic mice [89] [56].

DHFR is the rate limiting salvage enzyme of H₄B, and is responsible for maintaining normal H₄B bioavailability by regeneration of H₄B from its oxidized form H₂B [90]. Therefore, impaired DHFR function is anticipated to lead to eNOS dysfunction [33]. Indeed, several studies have confirmed the crucial role of DHFR in maintaining H₄B and NO bioavailability and hence the coupling state of eNOS [33, 86, 91]. RNAi inhibition of DHFR expression increased eNOS-dependent superoxide production, which was accompanied by reduced NO bioavailability, implying uncoupling of eNOS [33, 91]. Moreover, in angiotensin converting enzyme (ACE) knockout mice, where angiotensin II production was diminished, aortic DHFR protein abundance was significantly up-regulated [33]. All of these findings suggest that DHFR is critically involved in preserving eNOS coupling and blood pressure in the model of Ang II-induced hypertension. Of note, additional studies have also confirmed an important role of DHFR in regulating eNOS coupling/uncoupling activity and vascular function. Crabtree and his colleagues showed that DHFR takes part in controlling H₄B/H₂B ratio, which is different from the function of GTPCH1 that regulates total biopterin levels [91]. Although either DHFR or GTPCH1 knockdown reduced VEGF-dependent NO production, only DHFR RNAi led to formation of ROS [92], implying its role in preserving eNOS coupling. Moreover, DHFR expression was found down-regulated in an Ang II-dependent fashion during renal ischemia [93], which is similar to our findings in Ang II-infused hypertensive mice [86]. Furthermore, DHFR expression was decreased in 6 and 12 months old LDLR−/− animals, corresponding to impaired endothelial function [94].

3.2. Role of eNOS uncoupling in low renin, DOCA-salt hypertension

The association between a high salt intake and hypertension has been investigated for a long time [95]. An increase in dietary salt will lead to increased arterial blood pressure in individuals with salt-sensitive hypertension [96]. One commonly used model, namely deoxycorticosterone acetate (DOCA)-salt hypertension, was firstly established by Selye in 1943. Young rats were co-treated by DOCA and 1% NaCl solution for 7 weeks, and the MBP increased to 187/130 mmHg, compared to 110/80 mmHg in the sham controls [97]. Previous studies have confirmed that NOX is the initial source of ROS leading to H₄B oxidation. H₄B treatment prevented eNOS uncoupling, and blunted the blood pressure increase in DOCA-salt induced hypertension [98]. In addition, mitochondria may also contribute to the ET-1-dependent oxidative stress in DOCA-salt rats [99], while it is believed that Ang II levels are low in these animals. Recent research found an endothelial sepiapterin reductase (SPR) deficiency in aortic endothelial cells from DOCA-salt hypertensive mice [100]. SPR takes part in modulating H₄B biosynthesis in both de novo synthetic pathway and salvage pathway, implicating its indispensable role in regulating NO bioavailability [101]. Interestingly, SPR overexpression increased H₄B content, NO production, and NO-dependent vasorelaxation in both cultured cells and mouse models. RNAi of SPR had opposite effects [102]. Because SPR was lost in the endothelium of DOCA-salt induced hypertensive mice, supplementation of sepiapterin, which could be not metabolized to H₂B before its conversion to H₄B, had no effect in recoupling of eNOS. Nonetheless, combined administration of H₄B and a NOX inhibitor apocynin fully restored NO bioavailability [100]. On a separate note, overexpression of the H₄B synthetic enzyme GTP hydrocyclolase 1 (GTPCH1) was partially effective in improving endothelial function in DOCA-salt hypertensive rats [103]. This partial effect may be explainable by the SPR deficiency that prevents maximal biosynthesis of H₄B in the presence of overexpressed GTPCH1.

4. Potential new therapies targeting uncoupled eNOS in hypertension

Given that eNOS uncoupling is one of the central pathogenic mechanisms of hypertension, restoration of adequate NO signaling via restoration of eNOS coupling activity in the blood vessels may serve as an important therapeutic strategy for hypertension. Restoration of cofactor bioavailability and inhibition of upstream pathways could represent promising strategies to recouple eNOS from its uncoupled state.

4.1. Restoration of cofactor bioavailability

H₄B supplementation has a great therapeutic potential of improving endothelial dysfunction in hypertension [46]. It augments endothelium dependent vasodilation in both normotensive and hypertensive patients [22]. Basic experimental data from cultured cells and animal models support its efficacy in recoupling eNOS [92]. In addition, ascorbate (vitamin C) is important in maintaining H₄B levels in the setting of vascular oxidative stress [104], and treatment of BAECs with both H₄B and ascorbate prevented uncoupling of eNOS by ONOO⁻ [105]. There are some evidences demonstrating that ascorbate improved endothelial function through regulation of eNOS in genetic model of hypertension [106], which is mediated by increasing H₄B stability and its intracellular amount [107, 108]. Moreover, H₄B has been used in various experimental models. In spontaneously hypertensive rats (SHR), H₄B supplementation diminished eNOS dependent generation of ROS, while increasing NO production [109]. Oral administration of H₄B reduced vascular ROS production, increased NO production detected by electron spin resonance (ESR), and blunted the increase in blood pressure in DOCA-salt hypertension [98]. However, there is a limitation in scope for the potential clinical use of H₄B as a pharmaceutical drug, largely due to its chemical instability. H₄B can be easily oxidized to 7, 8-H₂B. Nevertheless, sapropterin dihydrochloride (6R-H₄B) is a novel thermo and photostable H₄B derivate that is commercially available for use as a phenylketonuria drug [110].

In addition, sepiapterin administration may be considered as another option to supply H₄B. Sepiapterin is firstly metabolized to H₂B by SPR, and further to H₄B by DHFR [32]. Sepiapterin supplementation has been employed to recouple eNOS in cell culture and animal models. Treatment of BAECs with sepiapterin improved H₄B and NO bioavailabilities [102]. Furthermore, administration of sepiapterin markedly improved endothelium dependent vasodilatation to different agonists [111]. All these data demonstrate that sepiapterin administration has the potential to be developed as an alternative treatment for hypertension.

4.2. Inhibition of upstream pathways

Since NOX has been identified as an initial activator to uncouple eNOS in both Ang II-dependent [86] and DOCA-salt induced hypertension [98, 100], it can be considered as a logical target for drug development for hypertension. Two isoforms, NOX1 and NOX2, have been shown to play crucial roles in hypertension, by promoting uncoupling of eNOS. The Ang II induced increase in blood pressure was found reduced in mice deficient in NOX1 [112], NOX2 [113] or their catalytic subunit p47phox [114]. Of note, p47phox has been shown to interact with NOX1 as well, in addition to well received notion of interacting with NOX2 [56, 115]. The impaired endothelial function in DOCA-salt hypertension was abolished in p47phox knockout mice [98]. Therefore, targeting the subunit or isoforms selectively and specifically might be beneficial to prevent or treat hypertension. Widely used NOX inhibitors include apocynin, an inhibitor of translocation of p47phox; statins, indirect inhibitors of cytosolic activator of NOX Rac1 [116], and Nox2ds-tat [117]. Recently, several inhibitors have been developed and found to be NOX specific [118]. VAS2870 and its derivative VAS3947 function as pan-NOX inhibitors [119, 120]. VAS2870 was found to inhibit NOX activity in smooth muscle cells [121] and human umbilical vein endothelial cells (HUVECs) [122]. Impaired acetylcholine-induced relaxation in SHR aortas was significantly attenuated by VAS2870 [120]. VAS3947 displays an improved solubility compared to VAS2870. It attenuated NOX activity completely in SHR aortas, without affecting either NOS activity or XO activity in PMA stimulated HL-60 cell [119].

Recent studies have identified GKT137831 as a specific inhibitor for NOX1 and NOX4 [123]. In human aortic endothelial cells (HAEC), high glucose induced NOX1 activation was attenuated by GKT137831 and by transfection of NOX1 siRNA. In addition, administration with GKT137831 into diabetic apolipoprotein E-deficient mice resulted in a significant attenuation of lesion formation, which was comparable to that seen in NOX1/apoE double knockout mice [123]. ML171 is a compound that was identified by cell-based high-throughput screening employing ROS detecting chemiluminescence in NOX1 overexpressing cells [124]. This inhibitor blocked the formation of functional invadopodia in human colon cancer cells, which is well established to be a NOX1 specific response [124]. Fulvene-5 efficiently inhibited NOX activity measured by hydrogen peroxide using a homovanillic acid (HVA) assay in 293 cells stably transfected with constitutively active NOX4 and COSphox cells harboring inducible Nox2/p47phox/p67phox complex, showing that Fluvene-5 can act as a NOX2 and NOX4 inhibitor [125]. Indeed NOX4-induced ROS production and arrhythmic phenotype in zebrafish was abolished by Fluvene 5 and dimethylamino Fluvene [126]. However, none of these NOX inhibitors has been tested in patients with hypertension, although they are highly promising drug candidates for hypertension via preservation of eNOS coupling activity. Given that Ang II is a potent NOX activator for subsequent induction of eNOS uncoupling, the attenuation of Ang II signaling is clearly another feasible strategy to inhibit eNOS uncoupling in hypertension.

Summary

In summary, this review gives an overview of a dynamic research field of how eNOS uncoupling contributes to hypertension, and how eNOS recoupling may serve as a novel and effective therapeutic strategy for hypertension. The major regulatory mechanisms of eNOS are listed in Table 1. Supplementation of H₄B, its stable alternatives or precursor sepiapterin, or inhibition of NOX, may represent promising new therapeutics to preserve eNOS coupling activity for the treatment of hypertension.

Table 1.

Mechanisms regulatory of eNOS function

Post translational regulation of eNOS	References
Uncoupling of eNOS
H4B deficiency
Oxidation by ONOO	[60]
Deficiency of GTPCH1	[127, 128]
Deficiency of SPR	[100, 102]
Deficiency of DHFR	[86, 91, 129]
Deficiency of L-arginine	[130, 131]
Disruption of dimerization	[59]
Protein Modification
Phosphorylation
Phosphorylation at T495	[65, 66, 132–134]
Dephosphorylation at S1177	[65, 133–135]
Acetylation
Downregulation of SIRT1	[75, 136]
Inhibition of HDAC3	[76]
S-glutathionylation*	[77]
Protein/protein interaction
Dissociation with HSP90	[137–139]
Binding to Caveolin-1	[80, 138, 140]

^*S-glutathionylation uncouples eNOS