Acknowledgment in Research article at AUKBC

Review
eNOS phosphorylation in health and disease
Gopi Krishna Kolluru, Jamila H. Siamwala, Suvro Chatterjee*
Vascular Biology Lab, AU-KBC Research Centre, MIT Campus, Anna University, Chennai 600 044, TN, India
a r t i c l e i n f o
Article history:
Received 13 February 2010
Accepted 29 March 2010
Available online 2 April 2010
Keywords:
eNOS
Serine phosphorylation
Nitric oxide
a b s t r a c t
Endothelium plays a fundamental role in maintaining the vascular tone by releasing various biochemical
factors that modulate the contractile and relaxatory behavior of the underlying vascular smooth muscle,
regulation of inflammation, immunomodulation, platelet aggregation, and thrombosis. Endothelium
regulates these cellular processes by activating endothelial nitric oxide synthase (eNOS) responsible for
nitric oxide (NO) production. eNOS is constitutively expressed in ECs in response to humoral, mechanical
or pharmacological stimulus. eNOS activity is regulated mainly by protein-protein interactions and
multisite phosphorylations. The phosphorylation state of specific serine, threonine and tyrosine residues
of the enzyme plays a pivotal role in regulation of eNOS activity. Perturbations of eNOS phosphorylation
have been reported in a number of diseases thereby emphasizing the importance of regulation of eNOS
activity. This review summarizes the mechanism of eNOS regulation through multi-site phosphorylation
in different pathologies. Attempts have been made to highlight phosphorylation of eNOS at various
residues, regulation of the enzyme activity via posttranslational modifications and its implications on
health and disease.
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
There has been a drastic transformation from times past when
eNOS regulation was thought to be a very simple process. Many
studies and research groups working on eNOS regulation have
come a long way in discovering the increasing number of inter-
acting protein kinases and the role of eNOS phosphorylation on
specific serine (Ser) and threonine (Thr) residues. The plethora of
studies on eNOS phosphorylation and control of NO synthesis has
furthered the understanding of pathophysiological conditions of
endothelial dysfunctions and rectification. Inspite of several studies
on eNOS phosphorylation over a decade, the complexity of eNOS
regulation still exists and poses to be a topic of interest for
researchers. The changes in eNOS activity in relation to eNOS
phosphorylation pattern will provide information regarding
attenuated NO availability and the pathogenic mechanisms
involved in multiple disease conditions and their potential treat-
ment conditions related to NO deficiency. This review attempts to
summarize the present knowledge of the multi-site phosphoryla-
tion of eNOS under different physiological and pathological
conditions.
1.1. NO in physiology and pathology
Nitric oxide (NO) is one of the simplest gaseous free radicals and
mediates a significant and diverse number of signaling functions in
nearly every organ system in the body. NO produced in the endo-
thelium via the enzyme eNOS is an important vasoactive compound
[1,2]. NO is responsible to regulate a diverse range of physiological
and cellular processes including endothelial cell migration, prolif-
eration, extracellular matrix degradation, and angiogenesis [3]. NO
is a key component in endothelium-dependent regulation of
vascular tone, platelet function, angiogenesis and mitogenesis
that are crucial in cardiovascular physiology [4]. A potent anti-
inflammatory agent, NO inhibits leukocyte interactions with the
vessel wall, thereby reducing pathological inflammation and
thrombosis [5]. eNOS knockout mice studies showed that leukocyte
adherence to the vessel wall is elevated 10-fold [6]. Also NO inhibits
inflammation in various vascular disease models like myocardial
infarction, glomerulonephritis, lung injury, and stroke [7,8].
Thereby NO controls inflammation and thrombosis in part by
regulating vesicle trafficking. NO decreases granule trafficking from
the Golgi to the plasma membrane by targeting a key component of
exocytic machinery [9]. Loss of endothelial function due to phos-
phorylation and sub-cellular localization of eNOS [11,12], has been
implicated in a number of cardiovascular diseases, making regula-
tion of eNOS through posttranslational modifications, a promising
therapeutic target [2,10].
* Corresponding author. Tel.: þ91 44 2223 4885/2711x48; fax: þ91 44 2223 1034.
E-mail address: soovro@yahoo.ca (S. Chatterjee).
Contents lists available at ScienceDirect
Biochimie
journal homepage: www.elsevier.com/locate/biochi
0300-9084/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.biochi.2010.03.020
Biochimie 92 (2010) 1186e1198

1.2. Expression and regulation of eNOS
NO is an important signaling molecule but is also highly
reactive and highly diffusible. It is therefore important that there
is strict control and regulation of NO production. NO is produced
by a group of enzymes called neuronal NOS (nNOS), inducible NOS
(iNOS) and endothelial NOS (eNOS) [13,14]. These enzymes
convert arginine into citrulline, producing NO in the process. The
activity of NOS enzymes is subject to a discreet and multiple
interconnected mechanisms of regulation. There are many ways
by which regulation of NOS occurs, such as the intracellular
distribution, gene expression, protein binding, enzymatic activa-
tion by phosphorylation and cellular inhibitors of NOS activity.
iNOS expression is controlled by inflammatory mediators and
cytokines and produces large, unregulated quantities of NO,
whereas nNOS and eNOS produce low amounts of NO. nNOS and
especially eNOS activity are induced by physiological and patho-
physiological stimuli [15e17]. Various chemical stimuli like VEGF,
estrogen receptor modulator and sphingosine-1-phosphate or
mechanical factors like shear stress activate eNOS [18e20]. eNOS
is regulated by multiple interdependent control mechanisms and
signaling pathways which can be calcium-dependent and/or
-independent [13,21e24].
1.3. Mechansim of eNOS activation
It is well-established that eNOS is a calmodulin dependent
enzyme [6,25]. eNOS is activated by elevation of intracellular
calcium and subsequent activation of CaM-binding domain by
calmodulin [26,27]. eNOS comprises of a C-terminal reductase
domain, which transfers the electron flux via NADPH to the
N-terminal oxygenase domain, which contains a heme, and binding
sites for arginine, tetrahydrobiopterin and CaM. Further the CaM-
binding domain facilitates electron flux from the reductase domain
to the oxygenase domain [28]. The phosphorylation of eNOS allows
active flux of electrons from the reductase domain to NO generation
in the oxygenase domain by guarding the steric hindrance due to
some non-catalytic inserts [29]. eNOS activity can be induced by
several agonists like acetylcholine, bradykinin B2 receptor,
thrombin and ATP, through intracellular elevation of calcium levels
[30]. Studies [1,31] have shown that the contribution of calcium and
substrate/co-factor have been crucial to the activity of eNOS in
intracellular organelles like Golgi, mitochondria, nuclei and plasma
membrane (Figs. 1 and 2).
1.4. Calcium independent eNOS activation
Apart from canonical calmodulin/Ca-dependent eNOS activa-
tion, there is a Ca-independent regulation of eNOS. Substantial
increase in NO release induced by hemodynamic shear stress
appears to be largely independent of intracellular calcium [26].
Shear stress induces eNOS mRNA and protein levels consistent with
eNOS-derived NO, which is important for flow-dependent vasodi-
lation and remodeling of vessels [32,33]. Increase in blood flow
through exercise upregulates eNOS, which is part of the cardio- and
athero-protective phenomenon [34]. It was also reported that
ceramide promotes eNOS activation and enzyme translocation in
endothelial cells [24].
Various studies have reinforced that substantial amount of NO is
produced in unstimulated endothelial cells by a Ca-independent
mechanism [35e37]. Mechanisms like phosphorylation and asso-
ciated regulatory proteins like Hsp90; gp60, Caveolin-1, a major
caveolae-localized protein through caveolae internalization, play
a crucial role in Ca2þ
-independent activation of eNOS and conse-
quent NO production (discussed below).
1.5. eNOS phosphorylation
Protein phosphorylation, a posttranslational modification and
a key regulator of eNOS activity [6,38e41] is modulated by kinases,
phosphatases [42e45] and proteineprotein interactions [21]. The
requisite substrates and cofactors for eNOS function are L-arginine,
tetrahydrobiopterin (BH4), iron (Fe), FMN, FAD and NADPH [45].
Though eNOS activity is coupled to changes in Ca2þ
levels, it is not
the sole factor required for the regulation of the enzyme activity.
The binding of calmodulin (CaM) and the flow of electrons from the
reductase to the oxygenase domain of the enzyme is also depen-
dent on protein phosphorylation and dephosphorylation [1]. The
primary sites where eNOS gets phosphorylated are serine residues
and, to a lesser extent, on tyrosine (Tyr) and threonine residues.
While phosphorylation of Ser617, 635, and 1179 results in the
activation of eNOS, the phosphorylation of Ser116 and Thr497
reduces the eNOS function [44,46e48]. Recently few reports have
shown that tyrosine residues present within oxygenase domain of
eNOS regulate the production of NO, as the modulation of eNOS
activity by tyrosine phosphorylation is supposedly prominent in
primary endothelial cells [49,50].
Tyr81 and Tyr657 are the residues, which gets phosphorylated
due to oxidative-stress and overexpression of v-Src leading to the
regulation of eNOS activity [46]. It appears that Tyr81 mediates
phosphorylation of eNOS through Src mediated mechanism under
different agonists [47]. While the role played by the other Tyr
residue Tyr657 is on the contrary. This particular tyrosine residue,
which is prominent during shear stress works through PYK2/c-Src
in phosphorylating eNOS [48,49,51e53]. It seems to provide
a negative feed back to eNOS phosphorylation for NO production.
However the proper mechanistic role through which the two Tyro-
sine residues target the phosphorylation is an open area to address.
1.6. Kinases involved in eNOS phosphorylation
Calmodulin is one of the very first protein involved in the
regulation of eNOS [54,55]. Increase in intracellular calcium levels
activates CaM that in turn activates CaM kinase II, which phos-
phorylates Ser1179 on eNOS. Ser1177 (human eNOS) and Ser1179
(bovine eNOS) are phosphorylated by the serine/threonine kinase
Akt (protein kinase B) [6,22,23]. Under physiological conditions,
esatcudeResanegyxO Calmodulin
COOH
P T495 /
497
S116 S1177/1179
P
PS615/
617
S633/
635
P P
HDL
Shear stress
Shear stress, VEGF, IGF-1
bradykinin, insulin, estrogen
sphingosine 1-phosphate,
adiponectin, leptin, statins
8-Br-cAMP
Shear stress, VEGF,
bradykinin, statins
8-Br-cAMP
PMA
NH2
Fig. 1. Regulators of eNOS acting on various phosphorylation sites. The eNOS phos-
phorylation sites are numbered according to human and bovine eNOS sequence.
Abbreviations: VEGF, vascular endothelial cell growth factor; 8-Br-cAMP, 8-bromoa-
denosine-30,50-cyclic monophosphate; S-1-P, sphingosine 1-phosphate.
G.K. Kolluru et al. / Biochimie 92 (2010) 1186e1198 1187

shear stress, hormones and autacoids activate eNOS by phosphor-
ylation/dephosphorylation mechanisms [22,39,40,46,56e59].
Humoral factors like Bradykinin stimulate NO synthesis by
increasing phosphorylation of multi-sites at eNOS-Ser1177
[45,47,60], eNOS-Ser633 and eNOS-Ser615 [61], and stimulating
dephosphorylation of eNOS-Thr495 [45,47] as well. The phos-
phorylation of Ser1179 is common for multiple diverse signaling
systems, such as insulin and adipokines [56,61,62]. Apart from
these, kinases like Akt [47], and PKA, phosphatases like PP1 and
calcineurin (PP2B) [45] have been implicated in the regulation of
phosphorylation.
2. eNOS e posttranslational modifications
2.1. Myristoylation and palmitoylation
eNOS comprises two types of fatty acid modifications: irre-
versible acylation i.e., myristoylation and reversible palmitoylation.
Myristoylation provides general membrane association, while
palmitoylation directs proteins specifically to the plasma
membrane (PM). The presence of eNOS at the PM may bring eNOS
to interact with the factors that are required for its proper function,
such as arginine, calcium, and cofactor BH4. Proteins during acyl-
ation get depalmitoylated at the PM and are redistributed to other
intracellular membranes [40]. Repalmitoylation occurs specifically
at the Golgi, which further directs the proteins to the PM [63,64].
Studies have demonstrated that the myristoyl moiety is an absolute
requirement for the membrane localization and activity of eNOS
[65,66]. Without this modification, eNOS is almost completely
cytosolic and lacks palmitoyl moieties [67,68]. While myr-
istoylation is an absolute necessity for eNOS membrane activity,
palmitoylation determines the sub-cellular localization of the
eNOS. Palmitoylation is in turn regulated by eNOS activators like
bradykinin [69]. The cycle of depalmitoylation and repalmitoyla-
tion, results in a constant shuttling of the enzyme between Golgi
and PM, redistributing eNOS to distinct membrane microdomains.
2.2. Other interacting proteins of eNOS
eNOS interacts with the proteins like caveolae coat protein
caveolin-1 (Cav-1) and heat shock protein 90 (Hsp90) during its
active and inactive states which regulates its activity. Caveolae are
specialized invaginations of the PM composed of cholesterol,
glycosphingolipids, and some structural proteins, such as caveolin
[70,71]. eNOS is bound to caveolin-1 in caveolae facilitated by
myristoylation and palmitoylation, due to which the enzyme
activity of eNOS is basally repressed [40,72e75]. Caveolin-1 medi-
ated regulation of eNOS through caveolae-mediated endocytosis
gets initiated once the glycoprotein gp60 is activated in endothelial
cells [76]. Maniatis et al., had shown that NO production in
pulmonary endothelial cells is significantly mediated by caveolae
internalization and is independent of the increase of intracellular
Ca2þ
. Compartmentalization of eNOS in caveolae is necessary for its
interaction with regulatory proteins, and calcium- and phosphor-
ylation-dependent signal transduction events that modify the
response of the enzyme to extracellular stimuli. Stimuli, such as
shear stress, induce calcium increase thereby displacing eNOS from
caveolin-1 and activating eNOS by redistributing it from plasma
membrane caveolae [77,78].
The chaperone protein Hsp90 can interact with eNOS and
positively influence its function. The C-terminal Hsp70-interacting
protein (CHIP) interacts with both Hsp70 and Hsp90, and nega-
tively regulates eNOS trafficking into the Golgi complex. By
contrast, the NOS interacting protein (NOSIP) and the NOS traffic
inducer (NOSTRIN) can negatively regulate eNOS localization in the
plasma membrane [28,79e81].
2.3. eNOS localization
eNOS, the key endothelial isoform has a unique sub-cellular
localization pattern. In addition to phosphorylation, the location
of eNOS is important for its activation [74,82,83]. Functional eNOS
was found to be primarily located in the plasma membrane
regions of endothelial cells [84]. The eNOS expression and the site
of its synthesis as well, have a major influence on the biological
activity of the molecule. Complex mechanisms that include the
cell specificity of eNOS expression, and co- and posttranslational
processing lead to trafficking of the enzyme to plasma membrane
caveolae [21].
In the plasma membrane, eNOS is mainly targeted to the cav-
eolae [72,83], where it is inhibited by binding to caveolin-1 (cav-1)
through a consensus site [11,74]. Calcium-calmodulin and intra-
cellular calcium can dissociate eNOS from cav-1, allowing activation
of the enzyme [74,85]. On external stimulus, eNOS shuttles
between caveolae and sub-cellular compartments such as cytosol,
Golgi, and/or perinuclear structures [86,87e91]. This sub-cellular
T495S116 S1177
Atherosclerosis
Coronary Artery Disease
Hyperhomocysteinaemia
Myocardial infarction
Cerebral Ischaemia
Severe subarachnoid haemorrhage
Liver Ischemia
Hypoxia
Diabetes
Portal hypertension
Erectile dysfunction
Hertwig's epithelial root sheath
Alzheimers Disease
Cancer
esatcudeResanegyxO Calmodulin
Hyperhomocysteinaemia
Cerebral Ischaemia
Hypoxia
Diabetes
Alzheimers Disease
P PP
Atherosclerosis
Coronary Artery Disease
Hertwig's epithelial root sheath
Alzheimers Disease
Fig. 2. Effect of various pathophysiological and pathological conditions on the phosphorylation sites of eNOS.
G.K. Kolluru et al. / Biochimie 92 (2010) 1186e11981188

localization of eNOS plays an important role in the regulation of its
activity possibly along with phosphorylation [86e91].
Lipid rafts, which are rich cholesterol and sphingolipid regions
found in surface invaginations of cell, polymerize the caveolins to
form caveoli, which are implicated in endocytosis [92]. Many
studies implicate a coordinated role played by lipid rafts and
caveolins in eNOS sub-cellular signaling [71,78,93]. Studies provide
evidence that lipid rafts are implicated in oxidative-stress signaling
on endothelial cells [93,94]. This information hints a possible
regulatory mechanism to control selective movement of eNOS
signaling by modifying the signaling activity of lipid raft or caveolae,
to counteract and protect the cellular sensitivity to redox stress.
2.4. eNOS phosphorylation in human pathology
2.4.1. Artherosclerosis e endothelial dysfunction
Atherosclerosis is the major cause of chronic vascular diseases
such as coronary artery disease, cerebrovascular disease and
peripheral arterial occlusive disease [95]. Coronary artery disease is
characterized by accumulation of atheromatous plagues in lumen of
arteries which supply nutrients, oxygen to the myocardium. Causes
of the disease are obesity, diabetes, hypertension and smoking.
Upon progress of the disease, the lumen becomes obstructed
completely, leading to myocardial infarction, chronic coronary
ischemia, angina and flash edema. Coronary artery disease starts
with the formation of atherosclerotic lesions. It is characterized by
decreased eNOS activity and NO bioavailability and increased
expression of cell adhesion molecules such as VCAM-1 and ICAM-1
[96e98]. eNOS inhibition has also been shown to accelerate
atherosclerosis suggesting that NO may inhibit several key steps in
the atherosclerotic process. Thus, eNOS could be a candidate gene,
which is implicated in atherosclerosis. Modulation of eNOS activity
by dynamic changes in phosphorylation of eNOS has been of
considerable interest because of its pathophysiological role.
In failing cardiomyocytes Napp et al. showed beta (3) adrenergic
stimulation seems to deactivate rather than activate eNOS. During
stimulation with beta (3) adrenergic agonist BRL improvises
a further dephosphorylation of eNOS (Ser1177) and Akt, while the
treatment increases the Ser114 phosphorylation of eNOS. In
atherosclerosis caused by cytomegalovirus infection Ser1177
phosphorylation was shown to be decreased dramatically in
infected cells [99e102].
Black tea was found to increase NO bioavailability in patients
with artherosclerosis [103]. ERa (estrogen receptor alpha) plays
a key role in mediating the activation of eNOS in response to black
tea polyphenols. In cultured cells, the black tea polyphenolic frac-
tion promotes both eNOS catalytic activity and NO bioactivity. This
effect is because of activation of phosphoinositol 3-kinase (PI3-K)
and Akt via a p38 MAPK-dependent mechanism [104]. This was
supported by Anter et al. 2004 who showed that black tea poly-
phenols induced time-dependent phosphorylation of ERa on
Ser118, which was inhibited by ER antagonist ICI 182,780. Ser118
phosphorylation was observed in response to black tea polyphenols
and mutation of this residue abrogated polyphenol-induced eNOS
activation in COS cells. In addition to S118 phosphorylation, Ser1177
was also found to be phosphorylated by 300% in CAD after 4 weeks
of daily aerobic exercise training compared with sedentary controls
[105]. Therefore two key residues S118 and Ser1177 phosphoryla-
tion were required for regulation of NO production in patients with
coronary artery disease (CAD).
2.4.2. Hyperhomocysteinaemia
Homocysteine produces endothelial injury and stimulates
platelet aggregation. Hyperhomocysteinaemia has been associated
with increased risk of thrombosis and atherosclerosis.
Hyperhomocysteinemia (HHcy) impairs endothelium-dependent
vasodilation by increasing reactive oxygen species, thereby
reducing NO bioavailability [106]. HHcy impairs endothelial func-
tion and eNOS activity, primarily through PKC activation [107].
Robin et al. showed significantly less basal eNOS and phospho-
Ser1179-eNOS/eNOS in mesenteric arteries from HHcy mice but no
difference in phospho-thr495-eNOS/eNOS [108]. Signerello et al.
has shown that PKC stimulates the eNOS phosphorylation of the
negative regulatory residue thr495 and the dephosphorylation of
the positive regulatory site Ser1177 [109].
2.4.3. Myocardial infarction
Myocardial infarction (MI) or acute myocardial infarction (AMI)
occurs due to blockage in coronary artery following rupture of
a vulnerable atherosclerotic plague. In chronic myocardial infarc-
tion infarct size in eNOSÀ/À mice was unchanged but evident
remodeling with less capillary density and hypertrophy accompa-
nied with subsequent systolic and diastolic dysfunction and
increased mortality at 28 days was observed [110], emphasizing the
beneficial effects of eNOS-derived NO on ventricular remodeling
after myocardial infarction. Recent data showed that blocking
mineralocorticoid receptor improved endothelial dysfunction and
oxidative stress by increasing reduced eNOS phosphorylation at
Ser1177 thereby making NO available in experimental myocardial
infarction [111]. Furthermore, in rats with chronic heart failure after
large myocardial infarction [112] eplerenone and metformin [113]
in combination with an angiotensin-converting enzyme inhibitor
increased myocardial eNOS phosphorylation at Ser1177.
2.4.4. Ischemia
Ischemia occurs due to shortage of oxygen, glucose and other
blood born nutrients and is caused due to constriction or blockage
of blood vessels Myocardial infarction, stroke, organ trans-
plantation, and cardiopulmonary bypass causes reperfusion injury
which is leading cause of tissue damage leading to ischemia. In all
these conditions, the initial trigger of the damage is the transient
disruption of the normal blood supply to target organs followed by
reperfusion. From a clinical viewpoint, no therapy is currently
available to limit reperfusion injury, which emphasizes the
importance of a better understanding of its underlying pathological
mechanisms, to devise potential future therapeutic strategies.
2.4.5. Cerebral ischemia
Prominent roles of eNOS and vascular NO in maintaining cere-
bral blood flow and prevention of neuronal injury have been shown
in by pharmacological and genetical approaches in animal models
of cerebral ischemia [114e117]. Vascular NO production protects
against stroke regulates and cerebrovascular perfusion by
increasing collateral flow to the ischemic area. eNOS knockout mice
show decreased blood flow in the ischemic border zone and
develop larger cerebral infarctions [116].
The vascular endothelial growth factor (VEGF) contributes to
activation of eNOS by Ca2þ
/calmodulin and also stimulates the
protein kinase Akt, which directly phosphorylate eNOS on Ser1177
and increases enzyme activity. Increase in Ser1177 phospho-eNOS
occurs in endothelial cells of microvessels after ischemic episodes
with temporal expression of VEGF [118]. Osuka et al. (2004) showed
0.5e2 h transient increase in phospho-Akt at Ser 437 after reper-
fusion, whereas after 6 h there was an elevation of phospho-eNOS
at Ser1177. Hashiguchi et al. (2005) showed in the gerbil hippo-
campal microvasculature transient ischemia model that Ser1177
phosphorylation was unchanged by 24 h after reperfusion, despite
post-ischemic up-regulation of eNOS protein [119]. However,
Thr495 phosphorylation significantly and persistently decreased by
48 h [119].

Stroke is loss of brain functions due to ischemia caused by
thrombosis or embolism or haemorrhage. Stroke can lead to
permanent neurological damage, complications and death. Risk
factors include advance age, hypertension, transient ischemic
attack, diabetes, high-cholesterol, cigarette smoking and atrial
fibrillation. Stroke can be ischemic stroke or haemorrhagic stroke.
Dmitriy et al. determined the effects of modulation of the Ser1179
phosphorylation site on vascular reactivity, cerebral blood flow, and
outcome in a middle cerebral artery (MCA) occlusion model of
stroke [120]. Their results indicate that modulation of the Ser1179
phosphorylation site affects endothelium-dependent vasodilation
and cerebral blood flow and that these effects determine outcome
of stroke in vivo.
2.4.6. Liver ischemia
Ischemic hepatitis is caused by diffusion ischemia while hepatic
infarction results from hepatic artery disorders. Ischemic hepatitis
is characterized by diffuse liver damage due to an inadequate blood
or O2 supply. Causes are most often systemic e impaired hepatic
perfusion (due to heart failure or acute hypotension), Hypoxemia
(due to respiratory failure or carbon monoxide toxicity) or
increased metabolic demand (due to sepsis).
Recent findings demonstrate that the acute activation of the
serine-threonine kinase Akt is cardioprotective and PI3K/Akt
pathway activation has protective effects on hepatic I/R injury
[121]. Activation of PI3K leads to phosphorylation of membrane
phosphatidylinositol 3,4 biphosphate, which recruits Akt to the
cell membrane leading to phosphorylation and activation of Akt.
Activated Akt in turn promotes eNOS phosphorylation. In the
liver PI3K-Akt-eNOS thus appears to play a central role in pro-
tecting against ischemiaereperfusion (IeR) injury [122,123].
Roviezzo et al. showed activation of the Akt pathway in ischemic
regions of reperfused ileum through an increased S1179 phos-
phorylation in reperfused intestinal tissue coupled to Akt
activation [124].
2.4.7. Severe subarachnoid haemorrhage
Severe subarachnoid haemorrhage (SAH) is a form of stroke that
induces dysfunction of endothelial nitirc oxide synthase (eNOS),
resulting in severe vasospasm. Clinically, however, some portions of
cerebral arteries may show only mild vasospasm. Although severe
vasospastic arteries after SAH have been intensively studied,
activity of eNOS with the mild form of the disease has received less
attention. Osuka et al. 2004 showed that SAH induces a temporary
activation of AMPK alpha, which phosphorylates eNOS at Ser1177 in
endothelial cells of mild vasospastic basilar arteries. This signal
transduction might play an important role in controlling cerebral
blood flow after SAH [118].
2.4.8. Hypoxia
Hypoxia is a pathological condition in which body as a whole or
a region of the body is deprived of adequate oxygen supply. Chronic
hypoxia increases endothelial nitric oxide synthase (eNOS)
production of nitric oxide (NO) and cardioprotection in neonatal
rabbit hearts [125]. The effect of hypoxia on eNOS expression
remains controversial. The presence of inflammatory mediators
and cytokines under hypoxic conditions further decreases expres-
sion of eNOS and production of NO [126].
Ser1177 phosphorylation under hypoxia was observed by
a number of workers. Shi et al. 2002 showed that association of
Hsp90 with eNOS is important for increasing NO production by
Ser1177 phosphorylation and limiting eNOS-dependent superoxide
anion generation. Hsp90 is associated with eNOS and the extent to
which the enzyme is activated is based on phosphorylation of eNOS
at Ser1177 [126].
Additionally, hypoxic conditions attenuate Akt-mediated phos-
phorylation at Ser1177, alter calcium metabolism, and alter the
balance of eNOS proteineprotein interactions with caveolin and
calmodulin [127]. Coulet et al. 2003 showed that human eNOS is
a hypoxia-inducible gene, whose transcription is stimulated
through HIF-2 interaction with two contiguous sites located at
À5375 to À5366 of the human eNOS promoter [128]. Liu J et al.
2009 have shown that phosphorylation of Ser1177 in eNOS
decreased, whereas phosphorylation of Thr495 increased, in the
prenatally hypoxic pulmonary arteries [129]. These data demon-
strate that prenatal hypoxia results in persistent abnormalities in
endothelium-dependent relaxation responses of pulmonary
arteries in adult sheep due to decreased eNOS activity resulting
from altered posttranslational regulation.
2.4.9. Diabetes
In diabetes, the body either fails to properly respond to its own
insulin, does not make enough insulin, or both. This causes glucose
to accumulate in the blood, often leading to various complications
[130,131]. Endothelial dysfunction is a critical initial factor 2 in the
development of diabetic vascular disease [132]. Endothelial
dysfunction, as represented by impaired endothelium functions,
and NO-mediated relaxation, occurs in cellular and experimental
models of diabetes [133e136]. Similarly, many, but not all, clinical
studies have found that endothelium-dependent vasodilation is
abnormal in patients with type 1 or type 2 diabetes [137e140].
Thus, decreased levels of NO in diabetes may underlie its athero-
genic predisposition.
The defect of Akt/eNOS signaling may play a primary role in
endothelial dysfunction in type 2 diabetes mellitus. In aorta from
diabetic animals, as well as in type 2 diabetic patients, Akt/eNOS
phosphorylation has been shown to be decreased [141,142], at least
partly via hyperglycemia-induced GlcNAc modification of the
enzyme, which may explain the development and progression of
diabetes-associated atherosclerosis [141]. Insulin also mediates its
effects through binding to insulin receptors and triggering down-
stream signaling pathways, of which the most important is the
phosphatidylinositol 3-kinase (PI3K)-Akt/protein kinase B (Akt/
PKB) pathway. This pathway is involved in a variety of insulin
responses including transport of glucose through cell membranes,
myocardial survival, and anti-apoptotic effects in endothelial cells
[143,144]. As such, PI3K-Akt pathway activation by eNOS-derived
NO may result in improved endothelial function and rescue of
impaired myocardial cells [145,146]. Recent studies on eNOS gene
disruption studies in mice revealed that, this leads to insulin
resistance, resulting in hypertension and hyperlipidemia
[147e149]. Further biochemical studies in insulin-responsive cells
have uncovered phosphorylation-dependent signaling role in
insulin-stimulated activation of eNOS [150,151].
In diabetic rat model, Biljana Musicki et al. showed that eNOS
dysfunction in the penis in diabetes by O-GlcNAc modification
affecting phosphorylation at Ser1177 residue, and rendering it
incapable of activation by normal fluid shear stress stimuli and
VEGF signaling [152]. Such eNOS impairment could contribute to
erectile dysfunction and affect long-term penile health in diabetes.
In diabetic liver Elrod et al. showed that although there was no
difference in total hepatic eNOS protein between non-diabetic and
diabetic animals, they discovered decreased phosphorylation of
both Thr495 and Ser1177 residues in diabetic mice as compared
with non-diabetic control animals [153]. In another study of dia-
betic animals [141] it has also been shown that this regulatory site
is subject to posttranslational modification, thereby decreasing
eNOS activity.
NO-based therapies have been proved by numerous investiga-
tions in various animal models [154e156]. Evidence also suggest

that NO is involved in the diabetic pathology [157]. In Type II dia-
betes mellitus (DM) where neointimal hyperplasia conditions lead
to vascular interventions, external delivery of NO proved to recover
arterial injury from neointimal hyperplasia [158,159]. Uncoupling
of eNOS has been demonstrated in animal models of diabetes [160].
Together, these data indicate that diabetes and insulin resistance
are characterized, at least in part, by endothelial dysfunction and
potentially by altered eNOS expression and NO production.
Insulin treatments have stimulated phosphorylation of eNOS
and protein kinase B (Akt) in arteries of diabetic mice [161]. We can
interpret from these studies that activation or inhibition of eNOS
would rectify diabetes pathology. We presume that phosphoryla-
tion of eNOS play key role in rectifying the defects under this
diseased condition and further sites involved in eNOS phosphory-
lation under these conditions should be identified and extensively
studied.
2.4.10. Oxidative stress
Oxidative stress, a pathological imbalance due to anomalous
reactive oxygen species (ROS) production, is a common feature of
many of the pathological conditions like cardiovascular anomalies,
hypertension, diabetes, atherosclerosis, cancer, which lead to
endothelial dysfunction [162]. ROS production involves inactivation
of the signaling molecule nitric oxide (NO), leading to endothelial
dysfunction. Uncoupled endothelial nitric oxide synthase (eNOS)
could be another important source of ROS in the endothelium.
Uncoupling of eNOS is now recognized as important in the patho-
physiology of several cardiovascular disorders and a predictor of
future adverse vascular events such as hypertension, atheroscle-
rosis and diabetes [163,164]. Under deficiency of the NOS cofactor
tetrahydrobiopterin (BH4), the enzyme can become uncoupled and
generate superoxide instead of NO. Furthermore, BH4 itself is prone
to degradation by oxidation, which leads to further amplification of
ROS production due to NOS uncoupling [164,165]. (ONOOÀ
)
oxidizes BH4 and reduces the availability of BH4 resulting in eNOS
uncoupling, thereby increased superoxide and decreased NO
production [166,167].
Role of ROS in oxidative stress under pathological conditions
lead by decoupling of eNOS and consequent reduction of NO
production is very much evident from the literature [165,166]. It
has been studied that Thr495 dephosphorylation has been impli-
cated in the uncoupling of eNOS resulting to increased superoxide
(O2
À
) stress instead of NO, which has resulted in atherosclerosis
[168]. But the increased NO production even under Thr495
dephosphorylation under inducers like bradykinin is still a point of
study [44,169,170]. It has to be explored further as such why Thr495
dephosphorylation behaving in a different manner under various
conditions of pathology and physiology, and is there any feed back
mechanism from this event to the NO signaling system?
Tyrosine phosphorylation is a novel mechanism of eNOS regu-
lation, which can affect the activity of eNOS and also influence the
distribution of eNOS in the cellular compartments. Tyrosine may
interact with other intercellular regulatory proteins like caveolin-1
that may influence the activity of eNOS [40]. However, studies have
to be carried out to elucidate the proper mechanism of tyrosine
phosphorylation and its interaction with other proteins in regu-
lating eNOS activity and thereby NO production, which may
provide a valuable information in therapeutic studies related to
vascular anomalies.
2.4.11. Oxidative stress in type II diabetes
Superoxide production contributes to atherogenesis as evi-
denced by increased superoxide anion content in vessels from
animals fed with high-cholesterol diets [171]. Superoxide reduces
expression of eNOS protein, as well as decrease the number of
caveolae in endothelial cells [172]. The most significant source of
superoxide in the vascular wall is NADPH oxidase [173]. The
enzyme superoxide dismutase acts to detoxify superoxide and
releases hydrogen peroxide (H2O2). H2O2 induces phosphorylation
of eNOS-Ser1177 and dephosphorylation of eNOS-Thr495, by
tyrosine kinaseedependent PI3K/Akt mechanisms [174]. Oxidative
stress plays an important role in type 2 diabetes-related endothelial
dysfunction. Zhang H et al. 2009 showed that Resveratrol restored
endothelial function in type 2 diabetes by inhibiting TNF-alpha-
induced activation of NAD(P)H oxidase and preserving eNOS
phosphorylation (Ser1177) [175].
2.4.12. Hypertension
Hypertension is a risk factor for all clinical manifestations of
atherosclerosis since it is a risk factor for atherosclerosis itself
[176e180]. It is an independent predisposing factor for heart failure
[181], coronary artery disease [182e184] stroke, renal disease
[185e187], and peripheral arterial disease [188,189]. Cirrhosis is
one of the main causes of portal hypertension. Adrenal cortical
abnormalities, kidney diseases, neuroendocrin tumors are also
known to cause secondary hypertension. Defect in eNOS phos-
phorylation has been accounted for endothelial dysfunction further
leading to hypertension, hyperlipidemia, and other diseased
conditions. Atochin et al. has shown modulation of S1179 phos-
phorylation as an approach for treating cardiovascular diseases,
particularly influenced by diabetes, obesity, metabolic syndrome,
hyperlipidemia, and hypertension [120]. However the effects of
phosphorylation sites of eNOS apart from S1179 on vascular path-
ogenesis have to be further studied in detail.
2.4.13. Portal hypertension and liver cirrhosis
Normally, blood from the intestines and spleen is carried to the
liver through the portal vein. But cirrhosis slows the normal flow of
blood, which increases the pressure in the portal vein resulting in
the condition called portal hypertension. Overproduction of
vascular NO plays a central role in both systemic and splanchnic
vasodilatation, which is a hallmark of portal hypertension
[190e193]. In the chronic model of portal hypertension increased
eNOS expression and enzyme activity are well-established events
[191e194]. However, the mechanism of the early induction of
excessive NO production by eNOS remains to be elucidated. The
phosphorylation of eNOS by Akt activates the enzyme and may be
the first step in increasing NO production in portal hypertension.
Iwakiri et al. showed that the phosphorylation of eNOS at Ser1176
was significantly increased in the PVL group [193]. Furthermore,
PVL significantly increased Akt phosphorylation (an active form of
Akt). When vessels were treated with wortmannin (10 nM) to block
the PI3K/Akt pathway, NO-induced vasodilatation was significantly
reduced.
2.4.14. Erectile dysfunction
Erectile dysfunction (ED) is defined as consistent inability to
obtain or maintain an erection for satisfactory sexual intercourse.
ED is predominantly the disease of vascular origin. The incidence of
ED dramatically increases in men with diabetes mellitus, hyper-
cholesterolemia, and cardiovascular disease. Loss of the functional
integrity of the endothelium and subsequent endothelial dysfunc-
tion plays an integral role in the occurrence of ED in this cohort of
men [195].
Age related ED is associated with eNOS activation through
dysregulation of its phosphorylation. PI3-K/Aktedependent eNOS
activation has recently been shown in the penis. In rats and mice,
both neuro- and agonist-induced penile erection produced rapid
increase in Akt and eNOS phosphorylation at Ser1177 in the penis,
which remain elevated after the termination of the initial stimulus

[196]. In another study Akt dependent phosphorylation of a posi-
tive regulatory site Ser1177 on eNOS was shown to be decreased
whereas phosphorylation of a negative regulatory site thr495 on
eNOS increased in the aged rat penis. In diabetic rat penis decreased
eNOS phosphorylation on Ser1177 caused by O-linked N-acetyl-
glucoseamine (O-GlcNAc) modification of eNOS.
2.4.15. eNOS and human aging
Hidetaka Ota et al. 2008 showed the phenomenon of human
aging is known to be a critical cardiovascular risk factor [197].
Cellular senescence of endothelial cells has been proposed to be
involved in endothelial dysfunction and atherogenesis [198]. The
lesions of human atherosclerosis have been extensively studied
histologically, and these studies have demonstrated that there are
vascular cells that exhibit the morphological features of cellular
senescence [199]. Although there is substantial evidence demon-
strating an aging-associated development of cardiac and vascular
dysfunction, the mechanisms responsible for this phenomenon
have not yet been clearly established. Aging is associated with
erectile dysfunction (ED) attributed to reduced nitric oxide syn-
thase (NOS) activity and nitric oxide bioavailability. A lot of work
has been carried out on endothelial cellular senescence [200e202].
Endothelial NO can protect against a state of oxidative stress,
and activation of eNOS and subsequent production of NO delay
endothelial cellular senescence [200,201]. A PDE3 inhibitor, cil-
ostazol, used as a vasodilating antiplatelet drug for treating inter-
mittent claudication, and in preclinical studies was shown to have
a protective effect on endothelial cells by increasing eNOS activity
[202]. Cilostazol increases intracellular cAMP content accordingly
and activates protein kinase A (PKA) or PI3K/Akt signaling [203]. In
yeast, Sir2 (silent information regulator-2) has been identified as an
NADþ
-dependent histone deacetylase [204]. Mammalian sirtuin 1
(Sirt1), the closest homolog of Sir2, regulates the cell cycle, senes-
cence, apoptosis, and metabolism, by interacting with a number of
molecules, including p53, PML, and PPAR-&UnknownEntity;
[205e207]. A recent study showed that production of NO by caloric
restriction increases Sirt1 expression and suggested that eNOS may
be involved in regulating the expression of Sirt1 in murine white
adipocytes [208]. HUVECs treated with other cAMP-elevating
agents and DETA-NO showed a reduction of SA-bgal-positive cells
as well [209]. Cilostazol increased phosphorylation of Akt at Ser473
and of endothelial nitric oxide synthase (eNOS) at Ser1177, with
a dose-dependent increase in Sirt1 expression. Moreover, the effect
of cilostazol on premature senescence was abrogated through
inhibition of Sirt1 [210].
2.4.16. eNOS phosphorylation and Hertwig’s epithelial root sheath
Epithelial cell rests of Malassez (ERM) are quiescent epithelial
remnants of Hertwig’s epithelial root sheath (HERS) that are
involved in the formation of tooth roots. After completion of crown
formation, HERS are converted from cervical loop cells, which have
the potential to generate enamel for tooth crown formation.
Korkmaz 2005 has shown that low concentrations of NO produced
by nNOS, and eNOS activate intracellular soluble guanylate cyclase
(sGC) to produce intracellular cyclic guanosine 30:50-mono-
phosphate (cGMP), which triggers rapid cellular responses such as
cell proliferation, cell differentiation, and apoptosis under physio-
logical conditions [211]. He later showed the basal production of NO
by eNOS in the ERM is modulated by phosphorylation of eNOS at
Ser1177 and Ser116 residues, while the basal activity of the eNOS is
not influenced by phosphorylation of eNOS at Thr495 residue.
2.4.17. Alzheimers disease
Alzheimer’s disease (AD) is associated to a cerebral amyloid
angiopathy with dysregulation of cerebral blood flow (CBF).
Vascular risk factors (VRFs) are probably determinants of
incidence and course of AD. b-amyloid peptides (AbP), the main
component of senile plaques typical of AD, circulate in soluble
globular form in bloodstream. Interestingly, AbP is able to induce
endothelial dysfunction, and this effect may represent the
link between vascular and neuronal pathophysiological factors
involved in AD. Gentile et al. 2004 showed that AbP enhances
eNOS phosphorylation on thr495 and Ser116 and reduces
acetylcholine-induced phosphorylation on Ser1177 [212]. Such an
effect depends on a PKC signaling pathway, as suggested by its
phosphorylation on Ser 660.
2.4.18. RBC-eNOS
RBC-NOS-generated NO contributes to the overall NO pool and
also play prominent role in regulation of blood flow and platelet
Table 1
List of publications showing the effect of different diseases on the phosphorylation status of eNOS.
Phosphorylation site Origin Mode of induction/
inhibition
References
Ser1177; Thr495 Hypoxic pulmonary arteries of sheep Chronic hypoxia Liu J, et al./Am. J. Physiol. Lung Cell. Mol. Physiol. 2009;296(3):L547e54.
Ser1177 Diabetic Mice Aortic EC Diabetes Zhang H, et al./Arterioscler. Thromb. Vasc. Biol. 2009;29(8):1164e71.
Ser1177 Diabetic animal’s aorta Telmisartan Wenzel P, et al./Free Radic. Biol. Med. 2008;45(5):619e26.
Ser1177 Aortas from DOCA rats & unineph-
rectomized rats
PugNAc Lima VV, et al./Hypertension 2009;53(2):166e74.
Ser1177 Ischemic myocardium affected rats WY-14643 Bulhak AA, et al./Am. J. Physiol. Heart Circ. Physiol. 2009; 296(3):H719e27.
Ser1177; Thr495 Mouse aorta Tacrolimus Cook LG, et al./Kidney Int. 2009;75(7):719e26.
Ser1177; Ser114 eNOS in failng myocardium BRL37344 Napp A, et al./J Card Fail. 2009;15(1):57e67.
Ser1177 HAECs TNF-alpha-neutralizing
antibody
Yuen DY, et.al/Diabetes 2009 May;58(5):1086e95.
Ser1177; Thr495 Recombinant BH(4)-free eNOS VEGF/PKC Chen CA, et.al/J. Biol. Chem. 2008 Oct 3;283(40):27038e47.
Ser1177; Thr495 Renal vascular -eNOS Radicicol Ramírez V, et.al/Am. J. Physiol. Renal Physiol. 2008;295(4):F1044e51.
Ser1177 ECV-D8eNOSGFP and CVEC PAF Sánchez FA, et.al/Proc. Natl. Acad. Sci. U. S. A. 2009 21;106(16):6849e53.
Ser1179 Cerebral artery Iscemic stroke Dmitriy et al./Clin. Invest. 2007;117:1961e7.
Ser1177 Forebrain Cerebral ischemia Osuka et al./Stroke 2004; (35) 2582
Ser1177 Mice Myocardial infarction NOS3 deficiency Scherrer-Crosbie et al./Circulation 2001;104:1286e1291
Ser1177 Diabetic myocardial tissue Eplerenone and metformin Calvert et al./Diabetes 2008;57:696e705
Ser1177 Human dermal microvascular
endothelial cells
HIF-1, HIF-2 Coulet et al./J. Biol. Chem. 2003;278: 46230e46240.
Ser1177; Ser116;
Thr495
Vessels of cerebral amyloid aorta AbP Gentile et al./J. Biol. Chem. 2004; 279: 48135e48142

aggregation [213,214]. RBCs play a role in the pathogenesis of
hypertension and stroke [215,216]. Recently, functionally active
endothelial type NO synthase was discovered in mature murine
and human red blood cells (RBC-eNOS). Nikolaev et al. found that
the treatment of mouse erythrocytes with rHuEpo resulted in
a time- and dose-dependent up-regulation of NO production,
mediated via activation of the PI3K/Akt pathway and RBC-eNOS
phosphorylation at Ser1177 [217]. Red blood cell (RBC)-derived NOS
has common but also distinct regulatory mechanisms when
compared with eNOS [218], which depends on intracellular Ca2þ
level and phosphorylation at Ser1177, regulated by PI3K [215]. Suhr
et al. 2009 has investigated the influence of intensive exercise on
eNOS content and the phosphorylation states of the eNOS at Ser116,
Ser1177, and Thr495 in human erythrocytes [219].
Evidence from the literature suggests that in vivo flow dynamics
are more affected by RBC aggregation on to endothelial cells leading
to pathological conditions [220]. RBC adhesiveness and aggregation
have proved to be useful markers for detecting vascular inflam-
mation and atherosclerosis progression in patients with coronary
artery disease [221]. As RBC-derived NO significantly contributes to
the intravascular NO pool, a lack of NOS activity and eNOS phos-
phorylation seems to be likely in these diseases. Rosuvastatin has
shown an improved RBC-eNOS activity through phosphorylation of
eNOS at Ser1177 residue. This improvement resembled the in vivo
situation of RBCs passing through capillaries [222]. RBC-NOS will
serve as a key factor for fields such as atherosclerosis, microcircu-
latory diseases, RBC aging and storage and adaptation mechanisms
for high altitude [218].
2.4.19. eNOS in cancer
eNOS has been detected in tumour cells [223], and catalyzes the
synthesis of NO, which can facilitate S-nitrosylation of the thiol
group of cysteines (Cys) in proteins [224], such as that of Cys118 of
HRas, which enhances the dissociation of guanine nucleotides
thereby increasing GTP-bound HRas [225]. Wildtype Ras proteins
can be required for activation of the MAPK pathway by oncogenic
Ras [226], and membrane-targeting of RasGAP, which inhibits
wildtype but not oncogenic Ras, reverts oncogenic Ras trans-
formation of NIH3T3 cells [227], suggesting that wildtype Ras
proteins may facilitate oncogenic signaling. Kian-Huat Lim et al. has
shown that AKT activation of eNOS maintains tumour growth in the
absence of oncogenic Ras by activating wildtype Ras through
S-nitrosylation of Cys118.
Activated KRas and Ser1177 phosphorylated eNOS were also
elevated in the tumour specimens compared to matched and
unmatched normal tissue controls [228], with the caveat that
biopsies also contain stromal tissue that could contribute to
detected eNOS phosphorylation.
3. Therapeutic challenges
Phosphorylation of eNOS is a key mechanism responsible for
eNOS activity and subsequent NO production [22,23,27,39,41].
Anomalies like cardiovascular dysfunctions, erectile dysfunctions,
stroke and several vascular abnormalities have been implicated for
erratic NO production. In the recent years eNOS phosphorylation
has been a potential target as a new therapeutic area of interven-
tion in many pathological situations, to develop a way of promoting
NO production [120,229]. Dmitriy et al. has shown in vivo that
eNOS phosphorylation is an important determinant of vascular
function, blood flow, and cerebral ischemia. This work also provides
proof of concept for the modulation of Ser1179 phosphorylation as
an approach to prevent cardiovascular disease, particularly influ-
enced by risk factors of diabetes, metabolic syndrome, hyperlipid-
emia, and hypertension.
Recent advances have shown that pharmacological agents like
Raloxifene, Fasudil and Y27632 improve cardiovascular function
[231e234]. Studies by Leung et al. shows that the therapeutic
concentrations of raloxifene (1e3 nM) augment endothelial func-
tions through up-regulation of eNOS activity by increased eNOS
phosphorylation in porcine coronary arteries [230]. Y-27632 and
fasudil, are some of the emerging drugs the physiological role of
Rho-Kinases (ROCKs), particularly in cardiovascular disease
[232e234]. Inhibition of RhoA or ROCKs leads to the rapid activa-
tion of PI3K/Akt and phosphorylation of eNOS [235,236], suggest-
ing the potential role of ROCKs in regulating eNOS activation in
addition to eNOS expression. Fasudil and Y-27632 were shown to
be effective Rho-kinase inhibitors for the treatment of a wide range
of cardiovascular disease, including cerebral and coronary vaso-
spasm, angina, hypertension, pulmonary hypertension, and heart
failure. They rapidly increase the endothelial eNOS activity through
phosphorylation and exert cardiovascular protection [234,236].
Rho/Rho-kinase pathway plays an important role in various intra-
cellular functions that are involved in the pathogenesis of cardio-
vascular disease [237].
4. Future challenges
The pattern of eNOS phosphorylation and dephosphorylation
later evolved as exceedingly complex. The challenge for future
studies will be to examine the relative contribution of each regu-
latory site on both the level and the time course of NO production.
Hopefully, this may help to design smarter eNOS constructs that, on
delivering in cardiovascular tissue, would drive NO release where
and when required. RBC-eNOS is another emerging area of
research, which is gaining prominence for fields like atheroscle-
rosis, vasculocirculatory diseases, RBC aging and storage and high
altitude pathophysiology. Novel diagnostic approaches for quanti-
fying RBC-eNOS and identification of phosphorylation dysfunction
under diseased condition help in the development of new diag-
nostic and therapeutic strategies for these diseases. Regulatory
mechanisms of various phosphorylation sites of eNOS, are to be
further elucidated. Interactive studies of these phosphorylation
residues with other regulatory protein of eNOS have to be carried
out extensively. This may provide valuable information in thera-
peutics of vascular anomalies related to eNOS/NO dysfunctions.
Acknowledgement
This work was supported by a grant from KBC-RF. The authors
also acknowledge Ms. Puja Kumari for compilation of the infor-
mation in Table 1.
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Acknowledgment in Research article at AUKBC

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