Molecular mechanisms underlying the activation of eNOS
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- Fleming, I. Pflugers Arch - Eur J Physiol (2010) 459: 793. doi:10.1007/s00424-009-0767-7
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Endothelial cells situated at the interface between blood and the vessel wall play a crucial role in controlling vascular tone and homeostasis, particularly in determining the expression of pro- and anti-atherosclerotic genes. Many of these effects are mediated by changes in the generation and release of the vasodilator nitric oxide (NO) in response to hemodynamic stimuli exerted on the luminal surface of endothelial cells by the streaming blood (shear stress) and the cyclic strain of the vascular wall. The endothelial NO synthase (eNOS) is activated in response to fluid shear stress and numerous agonists via cellular events such as; increased intracellular Ca2+, interaction with substrate and co-factors, as well as adaptor and regulatory proteins, protein phosphorylation, and through shuttling between distinct sub-cellular domains. Dysregulation of these processes leads to attenuated eNOS activity and reduced NO output which is a characteristic feature of numerous patho-physiological disorders such as diabetes and atherosclerosis. This review summarizes some of the recent findings relating to the molecular events regulating eNOS activity.
KeywordsEndotheliumMechanoreceptorNitric oxide synthaseOxidative stressPhosphorylationShear stress
Robert Furchgott elegantly demonstrated that endothelial cells play a pivotal role in relaxations evoked by acetylcholine in isolated arteries  and at the same time became godfather to a whole new area of research. Thereafter, followed the demonstration of nitric oxide (NO) production by the endothelium and the physiological effectiveness of l-arginine analogs  and eventually the isolation of the first NO-generating enzyme or NO synthase (NOS) . It is now general knowledge that NO is synthesized from the amino acid l-arginine by the NOS family of enzymes. The ‘neuronal’ (nNOS, NOS I, or bNOS) and ‘endothelial’ (eNOS or NOS III) NOS isoforms, which were named after the tissues in which they were first identified, are expressed constitutively and are generally regulated by Ca2+/calmodulin (CaM) as well as by phosphorylation. The inducible NOS isoform (iNOS or NOS II), on the other hand, is not generally expressed in unstimulated cells; although exceptions to this rule of course exist. iNOS binds CaM so tightly that it is essentially Ca2+ independent, and it releases NO in larger quantities during inflammatory or immunological defense reactions. There are only a few intracellular mechanisms that regulate iNOS activity which is usually determined by its expression level.
eNOS is constitutively expressed, but numerous physical and chemical stimuli affect eNOS levels in vitro and in vivo. For example, the fluid shear stress generated by the viscous drag of blood flowing over the endothelial cell surface is an important signal regulating eNOS mRNA and eNOS protein expression in cultured endothelial cells as well as in intact arteries. The signaling pathways involved in the regulation of eNOS expression are relatively complex, but important roles have been attributed to the transcription factors NFκB and KLF-2 and eventually also to FoxO1 and specific microRNAs (for review, see ).
Functionally, eNOS was initially classified as a Ca2+/CaM-dependent enzyme with a low but measurable activity at resting levels of [Ca2+]i. It is now evident that eNOS can be activated by certain stimuli without a sustained increase in [Ca2+]i being necessary. Shear stress can elicit Ca2+ transients; however, in both cultured endothelial cells and in isolated arteries, there is a discrepancy in the time course of the Ca2+ response and the time course of the shear stress-induced production of NO. On the basis of such observations, it was concluded that a sustained increase in [Ca2+]i is not essential for the shear stress-induced activation of eNOS and led to the shear stress-induced activation of eNOS being referred to as “Ca2+-independent”. However, this is, strictly speaking, not the case since the chelation of intracellular Ca2+ also abolishes the response to shear stress. Rather, the shear stress-induced increase in NO production is associated with eNOS phosphorylation (see below) and an increase in the sensitivity of the enzyme to Ca2+ so that the enzyme can be activated at resting Ca2+ levels .
Regulation of catalytic activity
Nitric oxide synthases are multi-domain enzymes consisting of an N-terminal oxygenase domain that contains binding sites for heme, l-arginine and tetrahydrobiopterin (BH4), and a reductase domain with binding sites for nicotinamide adenine dinucleotide phosphate (NADPH), flavin mononucleotide (FMN), FAD, and CaM. The active enzyme is part of a multi-protein complex consisting of the NOS dimer each of which binds one CaM, as well as a spectrum of adaptor and regulatory proteins. NOS dimers contain one zinc ion which is tetrahedrally coordinated to pairs of cysteine residues at the dimer interface. The conserved Cys-(X)4-Cys motif and its strategic location were suggested to establish a structural role for the metal center in maintaining the integrity of the BH4-binding site. However, this point remains controversial, as does the report that peroxynitrite (ONOO−) releases zinc from the zinc-thiolate cluster of eNOS resulting in disruption of the dimer and the uncoupling of enzyme activity, decreasing NO synthesis and increasing superoxide anion (O2−) production.
During the synthesis of NO, NADPH-derived electrons pass to flavins in the reductase domain and must then be transferred to the heme located in the oxygenase domain so that the heme iron can bind O2 and catalyze the stepwise synthesis of NO from l-arginine. The association of CaM with its binding site is generally accepted to activate NO synthesis by enabling the reductase domain to transfer electrons to the oxygenase domain.
There are marked differences in the output of NO from each of the isoforms; iNOS> > nNOS > eNOS, that can be attributed to the absence/presence of intrinsic molecular electron flow "breaking" mechanisms which take the form of specific peptide sequences. The first “so-called” auto-inhibitory loop described corresponds to a 45-amino acid insert in the FMN-binding domains of the Ca2+-dependent enzymes which is not present in iNOS. Three-dimensional molecular modeling suggested that the insert originates from a site immediately adjacent to the CaM-binding sequence and synthetic peptides derived from the 45-amino acid insert were found to potently inhibit the binding of CaM to eNOS as well as enzyme activity. Such a control mechanism would imply that the insert must be displaced to facilitate the binding of CaM. A second auto-inhibitory loop common to all three isoforms was described in the C-terminal tail that curls back to interact with the flavin domain and interfere with the interaction between the two flavin moieties and thus attenuate electron transfer in a CaM-independent manner. In eNOS, the phosphorylation of Ser1177 (see below) may disable this inhibitory control element and thus enhance electron flux through the oxygenase domain, increasing NO production two- to threefold above basal levels. A third peptide sequence, described within the eNOS-connecting domain, has also been reported to interfere with CaM binding (for review see ).
At the post-translational level, eNOS activity is highly regulated by substrate and cofactor availability as well as endogenous inhibitors, lipid modification, direct protein-protein interactions, phosphorylation, O-linked glycosylation, and S-nitrosylation. The sections below focus on substrate and co-factor availability, protein-protein interactions, and phosphorylation. For an extensive recent review on other aspects, see reference .
Under physiological conditions, the intracellular l-arginine concentration is generally high enough to be in excess of the Km for eNOS. However, cardiovascular disease and the associated oxidative stress have been linked with reduced l-arginine transport and/or competition with other arginine-utilizing enzymes such as arginase and arginine decarboxylase. There are an increasing number of studies showing that enhanced arginase gene expression and/or activity contribute to endothelial dysfunction in various cardiovascular disorders .
Protein degradation is associated with the generation of “endogenous” NOS inhibitors, i.e., substances such as asymmetric dimethylarginine (ADMA), which are known to correlate with some disease states and can accumulate in concentrations high enough to affect endothelial NO production . The increase in ADMA levels reflects the expression and activity of the enzymes that metabolize it, i.e., the dimethylarginine dimethylaminohydrolases (DDAH-1 and DDAH-2). The importance of this pathway is highlighted by the fact that the transgenic overexpression of DDAH-1 has been shown to increase NO production and reduce blood pressure in vivo  while its deletion is associated with endothelial dysfunction and elevated blood pressure . Interestingly, it seems that although the downregulation of both DDAH enzymes can affect endothelial NO production, only the loss of DDAH-1 was associated with a reduction in the arginine/ADMA ratio  indicating that DDAH-2 may affect NO output by an ADMA-independent mechanism.
Cofactor availability or NO versus O2−: the “uncoupling” phenomenon
All of the NOS isoforms generate O2− and hydrogen peroxide (H2O2) under specific conditions, i.e., lower than optimal concentrations of the essential co-factor BH4 or the substrate l-arginine. The lack of BH4 results in the uncoupling of NOS, which basically means that the transport of electrons to ferrous-heme-O2 species generated during the stepwise activation of O2 by NOS does not occur fast enough to prevent their oxidative decay. The result being the generation of reactive oxygen species, reduced NO bioavailability, and endothelial dysfunction. Replenishment of BH4 levels with sepiapterin or the overexpression of the guanosine triphosphate cyclohydrolase I (GTPCH; the rate-limiting enzyme in BH4 biosynthesis) effectively augment BH4 levels in cultured endothelial cells and improve NO output. Markedly altered BH4 levels have however, been difficult to detect in vivo and to get around this difficulty it has been proposed that the ratio of BH4: dihydrobiopterin (BH2) is decisive as BH2 and BH4 bind eNOS with equal affinity and BH2 can efficiently replace eNOS-bound BH4 resulting in eNOS uncoupling (for review, see ). Another way of decreasing eNOS-derived NO production at the same time as increasing O2− generation in vitro is to incubate the enzyme or intact cells with NO donors, an effect that can also be reproduced in vivo using inhaled NO [10, 112]. However, also in this case, the apparent uncoupling of eNOS does not seem to be linked to a measurable decrease in BH4 levels. In fact, BH4 levels and the expression of GTPCH were increased by NO donors via a mechanism linked to cyclic AMP and the cyclic AMP response element-binding protein . Thus, it seems that mechanisms that regulate the BH4/BH2 ratio independently of overall biopterin levels may play an important role in regulating eNOS. Intriguingly, in in vitro experiments, laminar shear stress was found to increase the enzymatic activity of GTPCH and the subsequent generation of BH4. The latter effect was not related to a change in GTPCH expression but rather to its serine phosphorylation . CK2 was proposed to be the kinase responsible for the effects observed; and although the kinase has been ignored over the last few years, it has been shown to be activated by laminar flow by others .
Another enzyme of potential importance is this respect is dihydrofolate reductase (DHFR) which can reduce BH2 and regenerate BH4. Indeed, DHFR expression is reduced by angiotensin II, a stimulus that also decreases BH4 levels and elicits eNOS uncoupling . The potential importance of the latter enzyme is highlighted by the finding that the downregulation of the DHFR decreased BH4 and increased BH2 levels resulting in increased eNOS-dependent O2−, but reduced NO production. The knockdown of GTPCH, on the other hand, reduced total biopterin levels but failed to alter the BH4/BH2 ratio [27, 100].
Despite the undisputed role of oxidative stress in the etiology of endothelial dysfunction, large clinical trials with antioxidant therapies have failed to show a beneficial effect on cardiovascular outcome . Moreover, there are a large number of reports that are seemingly incompatible with a major role of eNOS uncoupling in vivo. For example, although the rapid oxidation of BH4 can be observed in solution, the phenomenon cannot be reproduced in intact endothelial cells even when exposed to an ONOO− donor . This discrepancy is probably explained at least in part by the formation of other reactive oxidant species from O2− that have more complex roles in intracellular signaling beyond NO scavenging. Several superoxide dismutases convert O2− to the more stable H2O2 that has widespread and more prolonged effects on endothelial cell function (see  for overview). Hydrogen peroxide is in turn eliminated through the actions of catalase and peroxidases. It is, however, important to note that the exogenous application of catalase can ameliorate endothelial dysfunction in some models of hypertension , whereas in models characterized by the uncoupling of eNOS, catalase aggravates the situation . To date, several studies have reported that promoting the conversion of O2− to H2O2 to relieve NO scavenging does not prevent the formation of atherosclerotic lesions and that SOD activity actually correlates with lesion size . Moreover, inducing oxidative stress by overexpressing Nox5 in endothelial cells increased eNOS activity and failed to elicit eNOS-dependent O2− generation . Thus, although NO output has been successfully improved by enhancing cellular levels of BH4, either using sepiapterin or by preventing the oxidation of BH4, circumstantial evidence indicates that the association of Hsp90 with eNOS , as well as eNOS phosphorylation (see below), can also affect the degree of coupling and the balance of NO/O2− production.
The eNOS signaling complex
CaM was the first reported eNOS-associated protein  and its association with the CaM-binding domain within eNOS is determined by multiple molecular interactions as well as by the phosphorylation/dephosphorylation of Thr495 . However, other modifications such as the binding of Hsp90 and the phosphorylation of Ser1177 have also been reported to affect the association of the two proteins .
The binding of Cav-1 to a consensus site (F350-W358) in eNOS was initially proposed to antagonize CaM binding and thereby inhibit enzyme activity as site-directed mutagenesis of the predicted Cav binding motif within eNOS prevents the Cav-1-induced inhibition of NO production [46, 54]. Indeed, co-expression of eNOS and Cav-1 in COS-7 cells leads to a marked inhibition of enzyme activity , while a cell-permeable peptide harboring the scaffolding domain of Cav-1 can inhibit NO-mediated vascular permeability and vasodilator responses in vivo . The caveolin-binding motif initially identified lies between the heme and the CaM-binding domain adjacent to a glutamate residue which is necessary for the binding of l-arginine; a report that suggested that Cav-1 may interfere with heme iron reduction . However, analysis of the crystal structure of eNOS indicates that this site is unlikely to be readily accessible, and other researchers have detected interaction between both the N- and C-terminal domains of Cav-1 and the oxygenase domain of eNOS . In experiments to examine how a Cav-1 scaffolding domain peptide would affect NO synthesis, it was demonstrated that Cav-1 must bind to the reductase domain of eNOS in order to compromise its ability to bind CaM and to donate electrons to the heme subunit .
Although eNOS and Cav-1 are expressed throughout the arterial system , the majority of studies demonstrating eNOS-Cav association were performed in sub-confluent cells. Indeed, using immunohistochemistry, eNOS and Cav-1 appear to be concentrated along the leading edge of proliferating cells . In confluent cultured cells, as well as in native endothelial cells, the majority of cellular eNOS is concentrated at cell-cell contacts in the vicinity of platelet-endothelial cell adhesion molecule 1 (PECAM-1), and within the Golgi apparatus [2, 58]. Moreover, while both Cav-1 and eNOS can also be found in the Golgi apparatus, they are separated into distinct perinuclear compartments that behave differently in the presence of a microtubule-depolymerizing drug, thus indicating that these two proteins are not in direct physical contact and that eNOS activity is not regulated by Cav-1 within the Golgi complex . Despite such observations indicating that different mechanisms regulate eNOS activity in different cellular localizations, the physiological consequences of the loss of caveolar signaling hot spots (which is what is observed in Cav-1−/− mice ) are clear. For example, isolated arterial rings from Cav-1−/− mice fail to establish a steady contractile tone following agonist stimulation and demonstrate exaggerated relaxant responses to acetylcholine. Closer evaluation revealed that the basal release of NO in endothelial cells from Cav-1−/− endothelial cells was approximately 30% higher than from wild-type cells and was accompanied by a much higher intracellular cyclic GMP content . Thus, both in vitro and in vivo eNOS becomes hyperactive in the absence of Cav-1 [32, 91] but whether or not this can be completely explained by the lack of a direct interaction between eNOS and Cav-1 or to more general defects resulting from the disruption of caveolar signaling in general is, however, unclear.
Some of the most interesting data relating to the regulation of eNOS by Cav-1 is related to the vascular effects of estrogen. Indeed, estrogen has been reported to markedly affect the formation of caveolae  as well as the expression of Cav-1 . Chronic changes in estrogen status can differentially affect eNOS and Cav-1 protein levels in native endothelial cells, i.e., eNOS levels go down and Cav-1 levels go up . Manipulating the expression of either eNOS or Cav-1 alone does not restore eNOS function in arterioles from estrogen-depleted rats and only the simultaneous upregulation of eNOS and downregulation of Cav-1 has been associated with the normalization of activity .
Hsp90 is involved in the folding of NOS enzymes and is reported to determine the insertion of heme into the immature protein . In addition to this function, Hsp90 can also act as an integral part of numerous signal transduction cascades by virtue of its function as a scaffolding molecule. Hsp90 can associate with eNOS in resting endothelial cells and endothelial cell stimulation with vascular endothelial growth factor (VEGF), histamine, fluid shear stress, and estrogen all enhance the interaction between Hsp90 and eNOS at the same time as increasing NO production . The association of Hsp90 with eNOS appears to be determined by the agonist-stimulated tyrosine phosphorylation of both proteins and, in conditions of nitrosative stress, Hsp90 can be S-nitrosylated on a residue on its carboxy terminal domain which affects the ability of the protein to interact with others . Such modifications in the activity of Hsp90 would be expected to have wide-reaching consequences on the regulation of eNOS as many of the kinases shown to phosphorylate eNOS on serine or threonine residues physically associate with the enzyme via binding to Hsp90 .
PECAM-1 (CD31) is concentrated at cell-cell contacts and undergoes homophilic binding between adjacent endothelial cells. Initially, it was attributed a function in the regulation of leukocyte transmigration, cell migration, cell adhesion, and angiogenesis. More recently, PECAM-1 was found to contain two intracytoplasmic immunoreceptor tyrosine-based inhibitory motifs, which has led to a reconsideration of its role in cell signaling . PECAM-1 is of particular interest in mechanotransduction as it is rapidly tyrosine-phosphorylated following the application of fluid shear stress to endothelial cells grown under static conditions. As this response could not be mimicked by Ca2+-elevating agonists or growth factors, it was suggested that PECAM-1 may represent the long elusive mechanoreceptor on the endothelial cell surface .
In addition to their co-localization at the endothelial cell plasma membrane, PECAM-1 and eNOS have also been reported to physically associate. However, reports regarding the shear stress-induced alterations in this liaison are contradictory. For example, one study reports that eNOS and PECAM-1 interact in cultured endothelial cells maintained under static conditions and that the application of fluid shear stress elicits the rapid disassociation of the complex . Other reports describe the time-dependent association of eNOS with PECAM-1 . The reasons for these discrepant findings are currently unclear but may be related to the time frame in which the experiments were performed. Indeed, the dissociation was reported to occur between 15 and 60 s after the application of shear stress, i.e., during the initial Ca2+-dependent phase of the response, while the enhanced association of PECAM-1 and eNOS occurred from 5 to 60 min after the application of shear stress, i.e., within the secondary Ca2+-independent phase of the shear stress response.
The role of PECAM-1 in mechanotransduction has been addressed by downregulating PECAM-1 in human endothelial cells using a siRNA approach as well as by studying shear stress-induced eNOS activation in native and cultured endothelial cells from PECAM-1−/− mice. In human endothelial cells, PECAM-1 downregulation significantly attenuated the shear stress-induced phosphorylation of Akt and eNOS and decreased eNOS activity . A similar phenomenon was observed in cultured endothelial cells from PECAM-1−/− mice and was later confirmed in studies assessing flow-dependent vasodilatation in isolated skeletal muscle arterioles from wild-type and PECAM-1−/− mice. Consistent with the role of PECAM-1 in mechanotransduction, NO production was only attenuated in vessels exposed to flow; and agonist-induced NO production was similar in vessels from both strains .
The association of PECAM-1 with vascular endothelial cell cadherin (which functions as an adaptor) and VEGFR2 (which activates phosphatidylinositol 3-kinase; PI3-K) has been proposed to comprise a mechanosensory complex in endothelial cells . However, the fact that a tyrosine kinase must be activated in order for PECAM-1 to be phosphorylated means it is unlikely that PECAM-1 acts as a mechanoreceptor per se. Rather, it is more likely that PECAM-1 modulates endothelial cell activation in response to shear stress by virtue of its function as a scaffold for the binding of signaling molecules such as tyrosine kinases, the tyrosine phosphatase SHP2, and the scaffolding protein Gab1.
Gab1 and SHP2
Gab1 is an adapter protein that belongs to the insulin receptor substrate-1 family. It translocates from the cytoplasm to endothelial cell junctions in response to flow  where it binds PECAM-1 as well as other proteins such as the tyrosine phosphatase SHP2 and the p85 subunit of PI3-K . Specific mutation of the pleckstrin homology domain of Gab1 revealed that it was possible to abrogate the shear stress-induced phosphorylation of Akt without affecting the phosphorylation and activation of eNOS. The shear stress-induced phosphorylation and activation of eNOS was, however, causally linked with the Gab1-dependent activation of the tyrosine phosphatase SHP2 inasmuch as a Gab1 mutant to which SHP2 cannot bind, as well as a dominant negative SHP2 mutant abrogated the shear stress-induced activation of eNOS in cultured as well as in native endothelial cells . Which kinase is then responsible for the shear stress-induced phosphorylation of eNOS? The most likely candidate is protein kinase (PK) A as a specific PKA inhibitor abrogated the shear stress-induced activation of eNOS in cultured and native endothelial cells . At first sight, the latter observations appeared to contradict a report that Gab1 regulates the phosphorylation and activation of eNOS via Akt as the downregulation of Gab1 (using a siRNA approach) and a Gab1 mutant that was unable to bind PI3-K attenuated both processes . However, preventing the shear stress-induced activation of PI3-K would be expected to affect the activation of both Akt and PKA  and the global downregulation of Gab1 would be expected to prevent both the activation of Akt, which requires the translocation of Gab1 to the plasma membrane, as well as the association of SHP2 with Gab1, which is dependent upon the phosphorylation of Tyr627 and which does affect the shear stress-induced phosphorylation of eNOS.
Soluble guanylyl cyclase
It seems that a small proportion of the “soluble” guanylyl cyclase can become membrane-associated in a stimulus-dependent manner . This translocation would bring eNOS and the soluble guanylyl cyclase (sGC) closer together, thereby increasing the effectiveness of NO signaling and reducing the possibility of inactivation of NO by intracellular O2−. Not all the groups that have addressed this aspect of NO signaling have found any evidence suggesting a direct association between the eNOS and the sGC, however, the β subunit of sGC has been reported to associate with Hsp90 after agonist stimulation and therefore form part of the eNOS signalosome .
Given that eNOS is thought to cycle from caveolae to the Golgi apparatus and back , it is logical to assume that the transport complex contains a motor protein that targets to Golgi membranes, such as dynamin-2. Indeed, interfering with the activity of dynamin-2 appears to deplete eNOS from caveolae . What is surprising is that the association of the reductase domain of eNOS with dynamin-2 affects enzyme activity as well as subcellular localization and the association of eNOS with dynamin-2 increases NO production [18, 19] while in cells expressing a dominant negative dynamin-2 bradykinin-induced NO production is almost completely inhibited . The latter observations were difficult to interpret and implied that the translocation of eNOS from the plasma membrane to the Golgi apparatus is essential for enzyme activation as had been previously suggested . It now appears that eNOS-dependent NO production is functionally coupled to caveolae internalization and a dominant-negative dynamin-2 mutant has been shown to inhibit caveolae-mediated endocytosis and NO generation .
Additional proteins reported to associate with eNOS include G protein-coupled receptors such as the angiotensin II AT1 receptor, the endothelin-1 ETB receptor, and the B2 kinin receptor. However, to what extent these reports represent real in vivo phenomena or simply experimental artifact is unclear. The list of eNOS-associated proteins is continually increasing, with recent additions being polymerized actin and the voltage-dependent anion channel 1, VDAC1, or porin. Some eNOS-associated proteins have also been identified using a yeast two hybrid system. However, it remains to be determined whether NOS3 interacting protein or the NOS3 traffic inducer play an active role in the regulation of eNOS activity and/or subcellular localization in native endothelial cells [6, 40].
Regulation of eNOS activity by phosphorylation
In unstimulated cultured endothelial cells, Ser1177 is not phosphorylated but is rapidly phosphorylated after the application of fluid shear stress [30, 51], VEGF [47, 77] or bradykinin . The kinases involved in this process vary with the stimuli applied. For example, while shear stress elicits the phosphorylation of Ser1177 by PKA, insulin, estrogen, and VEGF mainly phosphorylate eNOS in endothelial cells via Akt. The bradykinin-, Ca2+ ionophore-, and thapsigargin-induced phosphorylation of Ser1177, on the other hand, is mediated by CaMKII [42, 94].
There is evidence suggesting that in vitro hyperglycemia  and albumin advanced glycation end products modified by glucose , as well as type 2 diabetes in human subjects  result in the modification of Ser1177 by O-linked N-acetylglycosylation. Proteins modified in this manner tend to be under-phosphorylated relative to unglycosylated proteins and it has been suggested that O-GlcNAc glycosylation may obscure phosphorylation sites and thus interfere with signaling mechanisms and, in the case of eNOS, to attenuate NO production.
Although the activation of eNOS is linked to simultaneous changes in the phosphorylation of Ser1177 and Thr495 (see below), there are certainly additional eNOS phosphorylation sites. Indeed, the eNOS immunoprecipitated from unstimulated cultured endothelial cells is serine phosphorylated [41, 76], however the residue(s) that is constitutively phosphorylated under these conditions is not Ser1177.
Ser633 is located within one of the auto-inhibitory loops thought to be folded in such a way as to physically impede the access of CaM to its binding domain, thus throttling enzyme activity. Although Ser633 can be phosphorylated in vitro by PKA and PKG , the functional relevance of this observation was unclear and the limited experimental studies which initially compared the potential of phosphorylation on Ser1177 versus Ser633 in regulating eNOS activity concluded that Ser1177 played a major role in the regulation of NO production while either no Ser633 phosphorylation could be detected or no consequence of phosphorylation was evident [30, 47]. More recently, it has been shown that Ser633 is most probably phosphorylated in vivo by PKA following cell stimulation by fluid shear stress, VEGF, bradykinin, and 8-bromocAMP albeit with a slower time course of phosphorylation than that detected on Ser1177 and Thr495 [11, 78].
This phosphorylation site was identified by phosphor-peptide mapping and is reported to be phosphorylated by both PKA and Akt. Mimicking phosphorylation at Ser615 significantly increases the Ca2+-calmodulin sensitivity of eNOS but is not reported to alter maximal enzyme activity . However, Ser615 may be important in regulating phosphorylation at other sites as well as protein-protein interactions and the assembly of the eNOS signalosome .
This residue is constitutively phosphorylated and although, bradykinin, lysophosphatidic acid , and fluid shear stress  were initially reported to enhance Ser114 phosphorylation, this modification has more recently been described as a negative regulatory site that may be more important for directly determining agonist-induced rather than basal NO production .
This residue is constitutively phosphorylated in all of the endothelial cells investigated to date and is a negative regulatory site, i.e., phosphorylation is associated with a decrease in enzyme activity [42, 62, 77]. The link between phosphorylation and NO production can be explained by interference with the binding of CaM to the CaM-binding domain. Indeed, in endothelial cells stimulated with agonists such as bradykinin, histamine, or a Ca2+ ionophore, substantially more CaM binds to eNOS when Thr495 is dephosphorylated . Analysis of the crystal structure of the eNOS CaM-binding domain with CaM indicates that the phosphorylation of eNOS Thr495 not only causes electrostatic repulsion of nearby glutamate residues within CaM but may also affect eNOS Glu498 and thus induce a conformational change within eNOS itself . Recently, the dephosphorylation of Thr495 has been linked to eNOS uncoupling (i.e., O2− production by eNOS) ; however, it remains to be determined whether this occurs in vivo and whether or not the actual cause of the uncoupling is a decrease in BH4 and/or l-arginine availability as a consequence of prolonged activation of the enzyme.
The constitutively active kinase which phosphorylates eNOS Thr495 is most probably PKC [42, 74, 77], even though there is some confusion regarding the specific isoform(s) involved. Nevertheless, attributing Thr495 phosphorylation to PKC can account for the fact that PKC inhibitors and the downregulation of the kinase markedly increase endothelial NO production .
Changes in Thr495 phosphorylation are generally associated with stimuli (e.g., bradykinin, histamine, and Ca2+ ionophores) which elevate endothelial [Ca2+]i and increase eNOS activity by ten- to 20-fold over basal levels. In response to such agonists, the activity of eNOS is not simply determined by the formation of a Ca2+/CaM complex and its unregulated association with the enzyme, but rather by simultaneous changes in Ser1177 and Thr495 phosphorylation and resulting changes in the accessibility of the CaM-binding domain to CaM. It may well be that this control mechanism determines endothelial cell responsiveness to different agonists as the phosphorylation of eNOS on Thr495 has been reported to underlie angiopoietin-1-dependent inhibition of VEGF-induced endothelial cell permeability at least in vitro , as well as the diabetes and cigarette smoke-induced inhibition of eNOS .
The AMP-activated protein kinase (AMPK) is worth a separate mention as it is the only kinase identified to date that can potentially phosphorylate eNOS on more than one amino acid, i.e., on serine (Ser1177, Ser633) and threonine (Thr495). There have been numerous reports of the AMPK-dependent phosphorylation of eNOS (on Ser1177) following endothelial cell stimulation with agents such as VEGF, hypoxia, peroxisome proliferator-activated receptor (PPAR)-agonists, and adiponectin (reviewed in ). However, the effects are generally weak and much less impressive than the stimulation seen in response to hypoxia, shear stress, and thrombin; which, also result in AMPK activation . More recently, the AMPK was reported to phosphorylate eNOS on Ser633 following endothelial cell stimulation with atorvastatin and that no such observation could be detected in endothelial cells from AMPKα2−/− mice . Unfortunately, however, the latter observations were made in vitro and not accompanied by relaxation studies. This is relevant as the α2 subunit is generally accepted to be expressed in lesser amounts that the α1 subunit in endothelial cells and in some cases authors have been unable to detect it at all . Moreover, it should be borne in mind that several stimuli, such as pentobarbital and glucose deprivation which are excellent AMPK activators in endothelial cells, have absolutely no effect on eNOS phosphorylation or NO output. Whether or not this apparent conflict can be explained by the specific α subunit activated by a given stimuli, or to crosstalk between the AMPK and other eNOS-activating kinases, remains to be clarified.
Phosphorylation may not be the only way in which AMPK activation can affect eNOS activity as a dominant negative AMPK mutant was found to decrease eNOS activity and NO production despite being unable to prevent estradiol-induced changes in eNOS phosphorylation on Ser1177 or Thr495. Rather the reduction in NO production was attributed to an impaired association of eNOS with heat shock protein 90 . Although a clear link has been established between AMPK and activation of eNOS in endothelial cell cultures the agonist (acetylcholine)-induced relaxation of conductance and resistance arteries is normal in AMPKα1−/−  as well as in AMPKα2−/− mice (author’s unpublished observations). Such findings may indicate that the AMPK is unlikely to play a major role in the regulation of eNOS under normal conditions in which Akt, PKA, and CaMKII would be expected to dominate the regulation of eNOS. Clearly, a more thorough assessment of vascular function in animals lacking the AMPKα1 and α2 subunits is required to determine the consequences for flow-induced vasodilatation and endothelial cell activation by the stimuli (e.g., VEGF, hypoxia, and PPAR-agonists) shown to activate eNOS in vitro.
There are several potentially phosphorylatable tyrosine residues in eNOS and there have been numerous reports showing that tyrosine kinase inhibitors attenuate endothelial NO production and flow-induced vasodilatation [4, 26, 41]. It has also been shown that the enzyme can be tyrosine phosphorylated in endothelial cells treated with tyrosine phosphatase inhibitors [41, 52], H2O2  or exposed to fluid shear stress , as well as in cells overexpressing v-Src . However, it is only relatively recently that tyrosine-phosphorylated residues within eNOS have been identified and allocated a function in the regulation of NO output. One reason that the saga of the tyrosine phosphorylation of eNOS took so long to unfold was that the modification only seemed to be robust in primary cultures of endothelial cells [37, 41], and was difficult to reproduce in multi-passaged cells. However, both of the tyrosine phosphorylation sites identified to date were initially found in cells overexpressing different tyrosine kinases.
Oxidant stress and the overexpression of v-Src lead to the phosphorylation of Tyr81 in the oxygenase domain of eNOS . This modification was associated with an increase in NO production in situ but no differences in maximal eNOS activity were detected between the wild-type and the phenylanine (Tyr81Phe) eNOS mutants in vitro. Thus, it seems that Tyr81 phosphorylation does not modify eNOS activity directly but may modulate the sensitivity of the enzyme to Ca2+, alter protein–protein interactions or change its subcellular localization. More recently, phospho-specific antibodies have been used to demonstrate that the Src-dependent phosphorylation of eNOS on Tyr81 occurs following the stimulation of native and cultured endothelial cells with a number of agonists including; thapsigargin, VEGF, bradykinin, ATP, sphingosine-1-phosphate, estrogen, angiopoietin, and acetylcholine . Thus, although it appears that Tyr81 phosphorylation is a common feature of endothelial stimulation by a variety of different eNOS-activating agonists, what this phosphorylation site actually alters, e.g., signalosome stability and/or composition, or intracellular trafficking, still remains to be determined.
eNOS tyrosine phosphorylation was initially linked to endothelial cell stimulation by fluid shear stress ; and as fluid shear stress was known to result in the activation of Src , as well as the proline-rich tyrosine kinase (PYK2) , preliminary experiments concentrated on the ability of the two kinases to tyrosine phosphorylate eNOS. Tyr657, which is located within the FMN-binding domain of the enzyme was identified with the aid of mass spectrometry and was phosphorylated in cells overexpressing either c-Src or PYK2 . Intriguingly, the consequences of tyrosine kinase overexpression on NO output were distinct and while eNOS activity was unaltered by Src overexpression, NO production was attenuated in PYK2 expressing cells. Mutation of Tyr657 also resulted in the complete loss of the ability of the enzyme to generate NO, O2− or citrulline indicating that the site must have a direct negative regulatory function. Indeed, while carotid arteries expressing either wild-type or a non-phosphorylatable Tyr657, eNOS mutant responded normally to agonists and increased flow, arteries expressing a phospho-mimetic Tyr657 eNOS mutant did not generate any detectable endothelium-derived NO . These results contrasted markedly with the anticipated subtle modification of NO production but a clue as to why the mutation of Tyr657 could have such dramatic effects can be found by considering the mechanisms known to regulate nNOS. The activity of the latter NOS isoform was recently reported to be determined by a large-scale swinging motion of the FMN domain to deliver electrons to the catalytic module in the holoenzyme . From the crystal structure of nNOS, the phosphorylation of a tyrosine residue (Tyr889, rat nNOS sequence), which is in the vicinity of the FMN domain could prevent its movement, essentially locking the FMN domain into its electron-accepting position, thus inhibiting enzyme activity . Since Tyr657 is the equivalent tyrosine residue in the human eNOS sequence, it is highly likely that its phosphorylation would be associated with a loss of NO production. While shear stress elicits the activation of eNOS in endothelial cells, the level of NO output is generally low (two- to fourfold basal levels) compared to that generated following agonist stimulation (approximately 20-fold). The kinetics of the responses are also markedly different as the shear stress-induced production of NO can be demonstrated as long as the stimulus remains constant while that induced by agonists takes the form of a transient burst of NO production rarely lasting more than a few minutes. Given that shear stress elicits the phosphorylation of a tyrosine residue that negatively regulates eNOS activity, it is tempting to speculate that this event plays a key role in negatively modulating enzyme activity, thus keeping NO output low and reducing the risk of co-factor, i.e., BH4 depletion and the uncoupling of the enzyme.
PYK2 was concluded to be the kinase most likely to be responsible for the in vivo phosphorylation of eNOS and a dominant-negative version of the kinase abrogated flow-induced NO-dependent relaxation of carotid arteries . PYK2 is, however, a rather unusual tyrosine kinase in that it contains no Src homology (SH) 2 or SH3 domains and has been implicated in regulating the organization of the actin cytoskeleton and can be activated by integrin stimulation (for review, see reference ). PYK2 was initially characterized as a Ca2+-dependent tyrosine kinase [70, 117], thus at first glance there is a potential conflict between the Ca2+-dependency of PYK2 and the Ca2+-independency of shear stress-induced NO production. However, Ca2+ does not activate PYK2 in in vitro assays and the kinase does not contain any known consensus sequences for Ca2+ binding  indicating that Ca2+ may be indirectly affecting PYK2 activity.
A number of physiologically relevant stimuli lead to the activation of PYK2 in endothelial cells, including insulin , angiotensin II , and oxidative stress . Moreover, at least in human umbilical vein endothelial cells, the expression of the kinase is unstable and levels are rapidly downregulated in culture , an observation which may account for the early reports that the tyrosine phosphorylation of eNOS was previously considered to be convincing only in primary cultures of endothelial cells (see above). The link to insulin is interesting and may explain some of the controversial reports on the ability of the hormone to activate eNOS. For example, while insulin has been reported to elicit vasodilatation in vivo, several groups have repeatedly failed to detect the activation of eNOS and an increase endothelial cyclic GMP levels or relaxation in freshly isolated arteries [44, 90]. This is despite the fact that insulin stimulates the rapid phosphorylation of Akt as well as eNOS on Ser1177. Given that the phosphorylation of Tyr657 inhibits eNOS activity, it is tempting to hypothesize that the insulin-induced activation of PYK2 and tyrosine phosphorylation of Tyr657 may functionally antagonize the effects of the Ser1177 phosphorylation. Indeed, the siRNA-mediated downregulation of PYK2 has been shown to render eNOS sensitive to insulin . Insulin has also been recently reported to stimulate the translocation of eNOS and Cav-1 to the plasma membrane a phenomenon also found to decrease NO output despite the maintained phosphorylation of the enzyme on Ser1177 . Whether or not the interaction between eNOS and Cav-1 is altered by the tyrosine phosphorylation of the latter has not yet been addressed experimentally.
More recently, the AT1 receptor- and NOX2-dependent activation of PYK2 was found to be responsible for the inhibition of eNOS in a model of angiotensin II-induced hypertension. Again, the activation of PYK2 was linked to the tyrosine phosphorylation of eNOS on Tyr657 . In addition to its effects on PYK2, H2O2 is reported to inactivate the protein tyrosine phosphatase SHP-2 , a protein of potential relevance to the regulation of eNOS activity since it is reported to deactivate PYK2  and can be co-precipitated with eNOS from cultured endothelial cells . It is tempting to speculate that the inactivation of eNOS as a consequence of its phosphorylation by PYK2 may play an important role in the setting of atherosclerosis, as in this state PYK2 may be sufficiently activated to influence eNOS.
Our understanding of the processes responsible for the formation of new blood vessels after tissue ischemia has altered over the last decade. For example, while it was previously assumed that the vascularization of ischemic tissues could be attributed to angiogenesis, i.e., the migration and proliferation of mature endothelial cells, it now seems that circulating progenitor cells derived from the bone marrow play a major role. A decreased number or impaired function of different subpopulations of pro-angiogenic cells (in particular, CD34+ and CD133+ cells) has been linked with atherosclerosis and other cardiovascular diseases (for review, see ). Although the characterization of “endothelial progenitor cells” and their role in neovascularization is the subject of current investigation (and controversy), it does seem clear that eNOS also plays a role and influences their recruitment and mobilization . There are apparently a number of NO-related defects that underlie the impaired function of circulating progenitor cells in situations such as diabetes. For example, diabetic CD34+ cells do not migrate in response to VEGF and are structurally rigid. However, incubating these cells with a NO donor corrects the migration defect as well as cell deformability . Pharmacological strategies that increase eNOS expression (eNOS transcription enhancers as well as angiotensin-converting enzyme, or HMG-CoA reductase inhibitors) result in significant increases in the circulating levels of vasculogenic progenitor cells. Additionally, eNOS uncoupling has been linked to a decrease in the mobilization and function of progenitor cells in diabetic patients . It will be interesting to determine whether or not progenitor cell mobilization and function are also regulated by the altered phosphorylation of eNOS and or changes in protein-protein interactions within the eNOS signalosome.
The author acknowledges the help of Dr. Beate Fisslthaler and Dr Annemarieke Loot in the preparation of the manuscript and the work of the many groups that was not possible to cite because of space limitations. The authors own work was supported by EICOSANOX, an integrated project supported by the European Community's sixth Framework Program (Contract N° LSHM-CT-2004-005033) and by the Deutsche Forschungsgemeinschaft (Exzellenzcluster 147 "Cardio-Pulmonary Systems").