INVITED REVIEW
Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase

Institut für Kardiovaskuläre Physiologie, J. W. Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany

	ABSTRACT

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The endothelial nitric oxide synthase (eNOS), the expression of which is regulated by a range of transcriptional and posttranscriptional mechanisms, generates nitric oxide (NO) in response to a number of stimuli. The physiologically most important determinants for the continuous generation of NO and thus the regulation of local blood flow are fluid shear stress and pulsatile stretch. Although eNOS activity is coupled to changes in endothelial cell Ca²⁺ levels, an increase in Ca²⁺ alone is not sufficient to affect enzyme activity because the binding of calmodulin (CaM) and the flow of electrons from the reductase to the oxygenase domain of the enzyme is dependent on protein phosphorylation and dephosphorylation. Two amino acids seem to be particularly important in regulating eNOS activity and these are a serine residue in the reductase domain (Ser¹¹⁷⁷) and a threonine residue (Thr⁴⁹⁵) located within the CaM-binding domain. Simultaneous alterations in the phosphorylation of Ser¹¹⁷⁷ and Thr⁴⁹⁵ in response to a variety of stimuli are regulated by a number of kinases and phosphatases that continuously associate with and dissociate from the eNOS signaling complex. eNOS associated proteins, such as caveolin, heat shock protein 90, eNOS interacting protein, and possibly also motor proteins provide the scaffold for the formation of the protein complex as well as its intracellular localization.

calmodulin; caveolin; mRNA stability; phosphatase; serine phosphorylation; threonine phosphorylation

	INTRODUCTION

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THE ENDOTHELIAL NITRIC OXIDE synthase (eNOS) is a constitutively expressed enzyme that oxidizes L-arginine to generate L-citrulline and nitric oxide (NO). The catalysis of this reaction requires a number of essential cofactors such as calmodulin (CaM), tetrahydrobiopterin (H₄B), flavin mononucleotide, FAD, and NADPH. The NO thus generated exerts a number of functions on the cardiovascular system. Acute activation of eNOS in blood vessels in response to the application of an agonist such as acetylcholine or bradykinin results in the activation of the soluble guanylyl cyclase in    smooth muscle cells and the production of cGMP. An increase in intracellular cGMP levels may affect vascular tone by a number of mechanisms, for example by decreasing the intracellular concentration of free Ca²⁺ ([Ca²⁺]_i; for review, see Ref. 80) as well as by activating protein kinase G and phosphorylating heat shock protein (Hsp) 20, which is reported to regulate force by binding to thin filaments and inhibiting cross-bridge cycling (10, 104, 105). A basal NO production or "vasodilator tone" can also be said to exist in vivo as the fluid shear stress generated by the flowing blood and pulsatile stretch of the vascular wall, as a consequence of the cardiac cycle, continually stimulate endothelial NO production. Although important for the regulation of blood flow, the continuous production of NO also helps to maintain the endothelium in an anti-atherogenic state, in part by preventing the activation of transcription factors that determine the expression of proatherogenic gene products such as the adhesion molecules required for the attachment and sequestration of monocytes through the endothelial cell monolayer.

	REGULATION OF ENOS EXPRESSION

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Elucidation of the mechanisms and factors determining transcription of the eNOS gene under different physiological/pathophysiological conditions has long been considered central for a thorough understanding of alterations in vascular NO production. Although numerous studies have concentrated on the activity of the eNOS promoter and changes in eNOS mRNA expression, the stimuli associated with the most pronounced effects on eNOS protein levels (i.e., estrogen, TNF-, and shear stress) are now appreciated to regulate posttranscriptional processes that mainly determine eNOS mRNA stability.

The eNOS Promoter

The eNOS promoter exhibits proximal elements such as Sp1 and GATA motifs but does not contain a TATA box, characteristics typical of a constitutively expressed gene (for reviews, see Refs. 39, 131). In addition, the human eNOS promoter possesses binding sites for numerous additional transcription factors, including activator protein (AP)-1, AP-2, Ets family members, myc-associated zinc-finger protein (MAZ), nuclear factor (NF)-1, NF-IL6, NF-B, p53, PEA3, and YY1 as well as CACCC-, CCAAT-, heavy metal, acute phase response-, shear stress-, cAMP response-, retinoblastoma control-, interferon- response-, and sterol-regulatory cis elements. Deletion experiments revealed that some binding sites are essential for eNOS promoter activity, in particular Sp1 and GATA. The eNOS promoter also contains several half-palindrome motifs, for the estrogen- and glucocorticoid-responsive element (ERE and GRE), but no bona fide EREs or GREs (20, 48, 72, 78, 96, 123, 127, 132, 137).

Detailed dissection of the functionally important cis-DNA elements and the multiprotein complexes implicated in the cooperative control of the human eNOS gene in the vascular endothelium has highlighted the importance of two tightly clustered cis-regulatory regions (104/95 and 144/115 relative to transcription initiation) in the proximal enhancer portion of the eNOS promoter (72). The nucleoprotein complexes that form on these regions in endothelial cells are reported to contain Ets family members, Sp1, variants of Sp3, MAZ, and YY1. Moreover, evidence has been presented for positive and negative protein-protein cooperativity involving Sp1, variants of Sp3, Ets-1, Elf-1, and MAZ. It therefore appears that the regulation of eNOS promoter activity in the vascular endothelium is highly complex and most probably tissue specific as it is determined by the binding of multiprotein complexes to the activator recognition sites (72).

Given the list of transcription factors that bind to the eNOS promoter it is hardly surprising that eNOS mRNA levels in cultured and native endothelial cells can be modulated by numerous stimuli. Estrogen is a good example of a hormone whose beneficial cardiovascular effects have been linked to an improvement of vascular function, mainly as a consequence of enhanced NO production. Numerous investigations have assessed the effects of estrogen on eNOS expression and, although 17-estradiol has been reported by numerous investigators to upregulate eNOS mRNA and protein levels in cultured endothelial cells (74, 134), an equal number of investigators have found no convincing effect or even a decrease in eNOS expression (86). The best demonstrations of a link between estrogen and eNOS expression have been made using animal models, but even here chronic changes in estrogen levels have been reported to increase as well as decrease eNOS levels (66, 91, 100, 107, 125, 130).

Considerable effort has been made to determine whether or not common eNOS polymorphisms affect eNOS expression and/or activity in specific populations. Variants in the promoter region (none within any of the transcription factor binding sites identified to date) introns and exons have been identified and, although some are common and vary in different ethnic populations, the reported findings are largely inconsistent (for review, see Ref. 131).

The sequence in some important regions of the human and murine eNOS promoters is highly conserved (124), and using promoter-reporter insertional transgenic murine lines containing 5,200 base pairs of the native murine eNOS promoter directing transcription of a nuclear-localized -galactosidase, it has been possible to map eNOS promoter activity in adult animals (125). Examination of -galactosidase expression in the heart, lung, kidney, liver, spleen, and brain of such mice revealed a robust signal in large and medium-sized blood vessels. However, the eNOS promoter was apparently silent in arterioles, capillaries, and venules (125), a finding that may reflect the capability of smaller blood vessels in the microcirculation to regulate tone via NO-independent mechanisms.

Regulation of eNOS mRNA Stability

eNOS levels in endothelial cells can also be efficiently regulated by changes in eNOS mRNA stability. For example, eNOS mRNA levels are approximately four- to sixfold higher in proliferating than in confluent/growth-arrested endothelial cells (2), a difference that cannot be explained by differences in the eNOS transcriptional rate (114). Hypoxia and cytokines, such as TNF, also downregulate eNOS mRNA levels by decreasing the half-life of eNOS mRNA from 48 h under basal conditions to 3 h (92, 97, 135, 136).

Cell confluence, lipopolysaccharide, and cytokines affect eNOS mRNA half-life by a process involving the induction and expression of at least two cytosolic proteins [molecular mass 51 kDa (114) and 60 kDa (111)] that bind to a cytidine-rich region within the 3'-untranslated region (3'-UTR) of the eNOS mRNA. By binding to the 3'-UTR, the protein probably alters its configuration, which renders it susceptible to RNase activity. There is at least preliminary evidence linking an increase in the binding of a 60-kDa protein to the 3'-UTR with a decrease in eNOS expression in hypercholesterolemic rabbits (67), and interference with the binding of the cytosolic proteins to the 3'-UTR of eNOS mRNA may be the mechanism underlying the increase in eNOS expression in endothelial cells treated with hydrogen peroxide (15, 30), ANG II (70, 98), estrogen (122), aspirin (3), and 3-hydroxy-3-methylglutaryl CoA reductase inhibitors (54, 77, 88).

	POSTTRANSLATIONAL REGULATION

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Intracellular Translocation

It is now appreciated that the consequences of enzyme activation or the activation of signal transduction molecules can be determined to a large extent by the intracellular localization of the signaling complex. The monomers that compose the active eNOS dimer are myristoylated and palmitoylated (12, 109, 116) and thus can associate with intracellular membranes. This membrane association is required for the phosphorylation and activation of eNOS in response to stimuli such as vascular endothelial growth factor (VEGF; 40, 117). Functional eNOS can be detected in at least three membrane compartments, the plasma membrane (61), plasmalemmal caveolae (32, 44, 82, 118), and the Golgi apparatus (83, 99, 117). Both Golgi-associated and plasmalemmal eNOS are functional enzymes (40); however, the disruption of the Golgi apparatus does not affect the agonist-induced, endothelium-dependent, and NO-mediated relaxation of porcine coronary arteries, suggesting that the Golgi eNOS pool contributes little to the regulation of vascular tone (5).

The colocalization of the signal transduction molecules and proteins that comprise the "eNOS signaling complex" within the different membrane compartments facilitates enzyme activation, NO production, and the activation of downstream effector pathways. On the other hand, NO signaling in a compartment such as the caveolae can (at least at high concentrations) significantly modify the responses to other stimuli. For example, nitric oxide donors or analogs of cGMP inhibit the oligomerization of caveolin-1 and thus interfere with signaling via G protein-coupled receptors (79).

Although eNOS has been detected in the cytosol of some endothelial cells, it is unclear whether this eNOS is truly soluble or is still attached to a membrane fraction. For example, eNOS has been suggested to dissociate from the membrane (as a consequence of depalmitoylation) and be translocated to the cytosol in response to cell stimulation with high (receptor sequestration stimulating) concentrations of bradykinin (101, 106). However, this finding could not be confirmed by other groups (81) and may be an artifact resulting from the internalization of caveolae containing the B₂ kinin receptor as well as eNOS (6, 68). eNOS has also been reported to translocate to a Triton X-100-insoluble/cytoskeletal cell fraction following stimulation with bradykinin (129) and tyrosine phosphatase inhibitors (35); again, this is not a universal observation. Although agonist-induced changes in the intracellular localization of eNOS remain controversial (121), the enzyme is thought to shuttle between different intracellular compartments in response to cell stimulation. Certainly estrogen activates the estrogen receptor- and increases eNOS activity by a combination of its phosphorylation on Ser¹¹⁷⁷ and its subcellular translocation (18, 52, 60, 119). However, although it is tempting to speculate that each eNOS pool performs a distinct role, it is currently not possible to state definitively that the movement of eNOS from the caveolae to the peri-nuclear Golgi apparatus is particularly prominent after stimulation with agonists that modulate gene expression rather than NO production and vascular function.

Two groups have reported that eNOS can also be detected in the nucleus of endothelial cells (31) and brown adipocytes (50). Although the nuclear localization of eNOS may facilitate the interaction of NO with transcription factors, the conditions associated with a nuclear localization of eNOS and its exact role in this cell compartment remain to be determined.

Ca²+ and eNOS Activity

Classically, eNOS isoforms have been characterized on the basis of whether they are constitutively expressed and whether activation is dependent on an increase in [Ca²⁺]_i and the binding of Ca²⁺/CaM to the enzyme. eNOS is constitutively expressed in most endothelial cells, particularly in conductance arteries and, of the three NOS isoforms, is the isoform that is most sensitive to changes in [Ca²⁺]_i. For example, when agonists such as acetylcholine or bradykinin are used to stimulate endothelial NO production, either the chelation of extracellular Ca²⁺ or the addition of a CaM antagonist abolishes NO production and endothelium-dependent relaxation (13, 85). Mechanistically, CaM binding to the CaM-binding motif is thought to displace an adjacent autoinhibitory loop on eNOS (and nNOS), thus facilitating NADPH-dependent electron flux from the reductase domain of the protein to the oxygenase domain.

eNOS can, however, be activated by certain stimuli without a sustained increase in [Ca²⁺]_i being necessary; the most important of these stimuli is the fluid shear stress generated by the viscous drag of blood flowing over the endothelial cell surface. Shear stress, especially the application of flow to cultured endothelial cells or isolated vessels maintained for a time under no-flow conditions, can elicit Ca²⁺ transients (4, 65, 71). However, there is a discrepancy in the time course of the Ca²⁺ response and the time course of NO production, the former being transient, the latter maintained (4). Such observations led to the suggestion that a sustained increase in [Ca²⁺]_i is not essential for the shear stress-induced activation of eNOS (4). The application of fluid shear stress to endothelial cells results in the activation of the phosphatidylinositol 3-kinase and the subsequent activation of the serine kinases Akt and protein kinase A (PKA), which phosphorylate eNOS on Ser¹¹⁷⁷ and increase eNOS activity (28, 33, 51) (see below). This process has been referred to as the "Ca²⁺-independent activation of eNOS"; however, the chelation of intracellular Ca²⁺ also abolishes the shear stress-induced increase in eNOS activity, suggesting that the increase in NO production is still strictly speaking Ca²⁺ dependent but that the enzyme can now be activated at resting Ca²⁺ levels (28). There are other stimuli (e.g., bradykinin and histamine) that also affect the phosphorylation of eNOS, but the signal transduction cascade activated requires an increase in [Ca²⁺]_i. This apparent contradiction can be explained by the fact that the kinases that phosphorylate eNOS in response to shear stress or the application of a Ca²⁺-elevating agonist are differentially sensitive to Ca²⁺. For example, the agonist-induced activation of the CaM-dependent protein kinase II (CaMKII) is highly dependent on an increase in [Ca²⁺]_i, whereas the shear stress-induced activation of Akt is not affected by Ca²⁺ removal (28, 36).

eNOS Phosphorylation

eNOS can be phosphorylated on serine, threonine, and tyrosine residues (35), findings that highlight the potential role of phosphorylation in regulating eNOS activity. There are numerous potential phosphorylation sites, but most is known about the functional consequences of phosphorylation of a serine residue (human eNOS sequence: Ser¹¹⁷⁷; bovine eNOS sequence: Ser¹¹⁷⁹) in the reductase domain and a threonine residue (human eNOS sequence: Thr⁴⁹⁵; bovine eNOS sequence: Thr⁴⁹⁷) within the CaM-binding domain.

Ser¹¹⁷⁷. In unstimulated, cultured endothelial cells, Ser¹¹⁷⁷ is not phosphorylated but is rapidly phosphorylated after the application of fluid shear stress (28, 43), estrogen (76), VEGF (28, 95), insulin (73), or bradykinin (36). The kinases involved in this process vary with the stimuli applied. For example, shear stress elicits the phosphorylation of Ser¹¹⁷⁷ by activating Akt (28, 33) and PKA (9); estrogen and VEGF mainly phosphorylate eNOS via Akt; insulin seems to activate both Akt and the AMP-activated protein kinase (AMPK), whereas the bradykinin-induced phosphorylation of Ser¹¹⁷⁷ is mediated by CaMKII (36). When Ser¹¹⁷⁷ is phosphorylated, the flux of electrons through the reductase domain and, as a consequence, also NO production are increased two- to threefold above basal levels (90).

Thr⁴⁹⁵. This residue is constitutively phosphorylated in all of the endothelial cells investigated to date (but not in COS cells transfected with a wild-type eNOS) and is a negative regulatory site, i.e., phosphorylation is associated with a decrease in enzyme activity. The link between phosphorylation and NO production can be explained by interference with the binding of CaM to the CaM-binding domain, and in endothelial cells stimulated with a Ca²⁺-elevating agonist, such as bradykinin, histamine, or a Ca²⁺ ionophore, substantially more CaM binds to eNOS when Thr⁴⁹⁵ is dephosphorylated (36). The constitutively active kinase that phosphorylates eNOS Thr⁴⁹⁵ is most probably protein kinase C (PKC) (36, 89, 95), a finding that could account for the fact that protein kinase inhibitors and the downregulation of PKC markedly increase endothelial NO production (24, 64). The phosphatase that dephosphorylates Thr⁴⁹⁵ appears to be PP1 (see below).

Changes in Thr⁴⁹⁵ phosphorylation are generally associated with stimuli (e.g., bradykinin, histamine, and Ca²⁺ ionophores) that elevate endothelial [Ca²⁺]_i and increase eNOS activity by 10- 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 (Fig. 1). Stimulation of endothelial cells with growth factors/hormones such as estrogen does not appear to result in a change in the phosphorylation of Thr⁴⁹⁵; rather these agonists appear to increase NO production by two- to fourfold over basal levels by exclusively increasing the phosphorylation of Ser¹¹⁷⁷.

View larger version (41K): [in this window] [in a new window]   Fig. 1.   Hypothesis for the activation of endothelial nitric oxide synthase (eNOS) activation in response to the Ca2+-elevating agonist bradykinin. A: eNOS dimer showing electron flux between the 2 monomers and the phenomenon of domain swapping. B: under basal (unstimulated) conditions eNOS produces low amounts of NO (see inset) but the binding of CaM to the enzyme is prevented by phosphorylation of Thr495 in the CaM binding domain (CBD). C: in response to cell stimulation with bradykinin (Bk) there is an increase in [Ca2+]i and PP1 is activated to dephosphorylate Thr495. D: CaM can now bind to the CaM-binding domain and NO production is increased markedly over basal levels. E: at the same time, CaMKII is activated and phosphorylates Ser1177, which further enhances electron flux through the reductase domain and enzyme activity reaches a peak (F). G: dephosphorylation of Thr495 is transient and this residue is rapidly rephosphorylated after a decrease in [Ca2+]i, most probably by PKC. CaM dissociates and enzyme activity decreases. PP2A dephosphorylates Ser1177 and NO output returns to basal levels (H). AL, auto-inhibitory loop.

Other Phosphorylation Sites

Although the activation of eNOS is linked to simultaneous changes in the phosphorylation of Ser¹¹⁷⁷ and Thr⁴⁹⁵, there are certainly additional eNOS phosphorylation sites. Indeed, the eNOS immunoprecipitated from unstimulated cultured endothelial cells is serine phosphorylated (35, 94); however, the residue(s) that is constitutively phosphorylated under these conditions is not Ser¹¹⁷⁷.

Ser¹¹⁴ (bovine sequence Ser¹¹⁶). This residue is localized within the oxygenase domain and was thought to be located within a consensus sequence for ERK1/2 phosphorylation, although these MAP kinases do not seem to play a crucial role in the regulation of eNOS activity (see below). Bradykinin, the lipid signaling molecule lysophosphatidic acid (75), and fluid shear stress (43) have been reported to stimulate the enhanced phosphorylation of this amino acid, but the consequences of Ser¹¹⁴ phosphorylation on endothelial NO production remain to be elucidated. However, given the vicinity of Ser¹¹⁴ to the H₄B binding site, it is tempting to speculate that this residue regulates the dimerization of eNOS by determining zinc binding or may act as a phosphoryl switch determining whether eNOS generates NO or superoxide anions (O₂).

Ser⁶³³ (bovine sequence Ser⁶³⁵). Ser⁶³³ is located within the so-called auto-inhibitory loop, a peptide insert present in the Ca²⁺-dependent NOS isoforms that is thought to be folded in such a way as to physically impede the access of CaM to its binding domain, thus throttling enzyme activity. Deletion of the auto-inhibitory loop increases the activity of the two Ca²⁺-dependent NOS isoforms (128) and as this insert contains a number of phosphorylatable amino acid residues [12 of the 45 amino acids are either serine or threonine (110)], it is tempting to speculate that the phosphorylation may alter the conformation of the insert and thus the ability of CaM to access its binding site. Ser⁶³³ can be phosphorylated in vitro by PKA and PKG (14), but otherwise the functional relevance of Ser⁶³³ phosphorylation has not been addressed in detail in an intact cell system. Indeed, the few experimental studies that compared the potential of phosphorylation on Ser¹¹⁷⁷ vs. Ser⁶³³ in regulating eNOS activity concluded that Ser¹¹⁷⁷ played a major role in the regulation of NO production whereas either no Ser⁶³³ phosphorylation could be detected or no consequence of phosphorylation was evident (28, 41).

Tyrosine. Although eNOS can be tyrosine phosphorylated and endothelial NO production can be modulated by inhibitors of tyrosine kinases as well as tyrosine phosphatases (34, 35), almost nothing is known about the residues that are phosphorylated or the kinases that are involved. The nNOS can be activated by Src-homology tyrosine phosphatase-2-mediated dephosphorylation of tyrosine residues (21), but it is currently speculated that the tyrosine phosphorylation of eNOS is not likely to affect eNOS activity directly but more probably determines the docking of associated scaffolding and regulatory proteins (35, 62). Elaborating the functional consequences of eNOS tyrosine phosphorylation is hampered by the fact that this modification is only clearly evident in primary cultured cells (35, 45), and investigators using passaged endothelial cells have generally been unable to detect phosphotyrosine residues on eNOS (22, 94, 129).

eNOS-Associated Proteins

Concepts related to the regulation of intracellular signaling by alterations in the localization and association of distinct protein mediators are continually changing. Whereas the activation of eNOS has long been known to be dependent on protein-protein interactions, especially between CaM and eNOS, numerous additional eNOS-associated proteins have been identified over the last five years. It now seems that endothelial NO production is not simply dependent on the expression of the eNOS enzyme but is determined by an eNOS signaling complex that consists of the enzyme and a conglomerate of adaptor proteins, structural proteins, kinases, phosphatases, and potentially also motor proteins that affect complex association and determine intracellular localization.

Caveolin-1. The binding of caveolin-1 to a consensus site (F³⁵⁰SAAPFSGW³⁵⁸) in eNOS is proposed to antagonize CaM binding and thereby inhibit enzyme activity (for review, see Refs. 42 and 47). Indeed, coexpression of eNOS and caveolin-1 in COS-7 cells leads to a marked inhibition of enzyme activity (93), whereas a cell-permeable peptide harboring the scaffolding domain of caveolin-1 can inhibit NO-mediated vascular permeability and vasodilator responses in vivo (11). In aortas from caveolin knockout mice, on the other hand, the relaxant response to acetylcholine is enhanced and the constrictor response to phenylephrine blunted as a consequence of enhanced basal NO production (29, 103).

The caveolin-binding motif in eNOS lies between the heme and the CaM-binding domain adjacent to a glutamate residue, which is necessary for the binding of L-arginine, suggesting that caveolin-1 may interfere with heme iron reduction, similar to the L-arginine analogs (47). Other groups have, however, detected interaction between both the NH₂- and COOH-terminal domains of caveolin-1 and the oxygenase domain of eNOS (47, 69). In experiments to examine how a caveolin-1 scaffolding domain peptide would affect NO synthesis, it was demonstrated that caveolin-1 must bind to the reductase domain of eNOS to inhibit enzyme activity (49). Therefore, caveolin-1 binding to the reductase domain of eNOS compromises its ability to bind CaM and to donate electrons to the heme subunit, thereby inhibiting NO synthesis (49).

Although eNOS and caveolin are expressed throughout the arterial system (115), the majority of studies demonstrating eNOS-caveolin association were performed in subconfluent cells. Indeed, using immunohistochemistry, eNOS and caveolin appear to be concentrated along the leading edge of proliferating cells (44). In confluent cultured cells as well as in native endothelial cells, the majority of cellular eNOS is not colocalized with caveolin but is concentrated at cell-cell contacts in the vicinity of platelet-endothelial cell adhesion molecule 1 (PECAM-1) and within the Golgi apparatus (1, 55). Although both caveolin 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 caveolin-1 within the Golgi complex (56).

Some of the most interesting data relating to the regulation of eNOS by caveolin is related to the vascular effects of estrogen. Indeed, estrogen has been reported to markedly affect the formation of caveolae (126) as well as the expression of caveolin-1 (66). Chronic changes in estrogen status can differentially affect eNOS and caveolin-1 protein levels in native endothelial cells, i.e., eNOS levels go down and caveolin-1 levels go up (100). Manipulating the expression of either eNOS or caveolin-1 alone does not restore eNOS function in arterioles from estrogen-depleted rats and only the simultaneous upregulation of eNOS and downregulation of caveolin-1 has been associated with the normalization of activity (133).

Dynamin. As the concept that eNOS can shuttle between at least two intracellular pools gains acceptance, attention has begun to turn to the mechanism controlling this shuttling process and in particular to the association of eNOS with potential motor proteins. One motor protein, which affects the sequestration of G protein-coupled receptors, such as the muscarinic acetylcholine receptor (26), via caveolae and which can also modulate signaling pathways by means of direct protein interactions is dynamin (23, 63). Dynamin 2 can associate with eNOS, in the Golgi, and the association of the two proteins can be increased by Ca²⁺ ionophores (16). Moreover, in both in vitro assays as well as cultured endothelial cells, an increase in dynamin levels results in enhanced eNOS catalysis (16). There is currently no information available regarding the potential role of dynamin in the physical transport of eNOS between different intracellular locations.

G protein-coupled receptors. There have been reports that eNOS associates directly with G protein-coupled receptors and specifically with the intracellular domain 4 of the B₂ kinin, the ANG II AT₁, and the endothelin ET B receptors (68, 87). eNOS and G protein-coupled receptors are reported to be associated in unstimulated cells, whereas dissociation of the proteins occurs in response to cell stimulation. Moreover, the association of eNOS with a fusion protein corresponding to intracellular domain 4 of the B₂ receptor is reported to inhibit enzyme activity (68). An inhibitory effect of a synthetic peptide based on intracellular domain 4 has also been observed using a purified nNOS (53). In the latter case, inhibition could not be attributed to an interference of the peptide with L-arginine or H₄B binding or to interference with the cytochrome c reductase activity of the enzyme. Rather, the binding of the B₂ kinin receptor peptide was reported to block flavin to heme electron transfer in nNOS (53). Reports of an inhibitory interaction between G protein-coupled receptors and NOS are, however, controversial, and there is currently no convincing evidence to suggest that this interaction occurs in native cells in situ.

Hsp90. The primary function of Hsp90 is its involvement in a multicomponent chaperone system that is responsible for the folding of proteins such as steroid receptors and cell cycle-dependent kinases (17). Hsp90 is involved in the folding of NOS enzymes and is reported to determine the insertion of heme into the immature protein (8). In addition to this function, Hsp90 can also act as an integral part of numerous signal transduction cascades.

Hsp90 can associate with eNOS in resting endothelial cells and endothelial cell stimulation with VEGF, histamine, fluid shear stress, and estrogen all enhance the interaction between Hsp90 and eNOS at the same time as increasing NO production (46, 108). Exactly how Hsp90 regulates eNOS activity has been relatively well elucidated and the association of Hsp90 with eNOS appears to be determined by the agonist-stimulated tyrosine phosphorylation of Hsp90 (58). In vitro characterization of the domains of Hsp90 required to bind eNOS revealed that the M region of Hsp90 interacts with the amino terminus of eNOS (amino acids 442-600) and Akt. Moreover, the addition of purified Hsp90 to in vitro kinase assays facilitated Akt-driven phosphorylation of recombinant eNOS protein suggesting that Hsp90 may function as a scaffold for eNOS and Akt. Additional effects cannot be ruled out because Akt activity is enhanced following binding to Hsp90, an effect that may be related to the protection against Akt dephosphorylation by the phosphatase PP2A (112). Thus, in response to endothelial cell stimulation, eNOS and Akt appear to be recruited to an adjacent region on the same domain of Hsp90, which facilitates eNOS phosphorylation and enzyme activation (37). Whether Hsp90 also acts as a scaffold for other eNOS-associated proteins remains to be determined. However, a recent study suggested that Hsp90 increases the affinity of nNOS for CaM (120), and experiments using eNOS have shown that Hsp90 facilitates the CaM-induced displacement of caveolin from eNOS (57).

To study the relationship between Hsp90-mediated signaling and NO production, extensive use has been made of the ansamycin antibiotic geldanamycin, which is assumed to be a specific inhibitor of Hsp90. However, recent studies have suggested that some of the observations made using this substance can be attributed to Hsp90-independent effects. For example, treatment of endothelial cells with geldanamycin results in a dramatic increase in O₂ generation, which is independent of eNOS activity (27). Similar effects have also been reported using nNOS (8). Moreover, geldanamycin directly oxidizes ascorbate, consumes oxygen, and decreases the bioavailability of NO generated by 3,4-dihydrodiazete 1,2-dioxide in smooth muscle cells, an effect that can be prevented by superoxide dismutase (27). The finding that geldanamycin can generate O₂ on its own may account for previous reports suggesting that Hsp90 may play a role in regulating eNOS uncoupling, i.e., the phenomenon by which eNOS generates O₂ rather than NO (102).

Kinases. Most of the kinases shown to phosphorylate eNOS on serine or threonine residues physically associate with the enzyme, either directly or via binding to an adaptor protein. Little is known about the mechanisms determining the association of eNOS with PKA (41), AMPK (19), or CaMKII (36), but the binding of Akt to eNOS is thought to be dependent on the association of eNOS with Hsp90 (see above). Other kinases reported to affect eNOS activity are the MAP kinases, ERK1 and ERK2, as well as the cyclic nucleotide-dependent kinases PKA and PKG. Not much detailed information is available regarding the mechanisms by which these kinases regulate NO generation and some of the reports are controversial, because although eNOS in bovine endothelial cells was reported to be inhibited by ERK1/2 (7), inhibitors of ERK1/2 activation failed to affect the NO-mediated relaxation of isolated arteries or NO generation by porcine or human endothelial cells (36, 113). PKA and PKG, on the other hand, phosphorylate a purified recombinant eNOS protein on Ser¹¹⁷⁷ and Ser⁶³³ in an in vitro system (14). Additional mechanisms may also play a role in vivo as PKA may also stimulate the dephosphorylation of Thr⁴⁹⁵ by activating the phosphatase PP1 (95).

eNOS interacting protein. eNOS interacting protein (NOSIP) is a 34-kDa protein with unknown function that was identified using the yeast two-hybrid system. NOSIP binds to the carboxyl-terminal region of the eNOS oxygenase domain, and coimmunoprecipitation studies demonstrated that the interaction between eNOS and NOSIP in vitro as well as in cultured cells can be inhibited by a synthetic peptide corresponding to the caveolin-1 scaffolding domain (25). Functionally, NOSIP is reported to decrease eNOS activity and promote the translocation of eNOS from the plasma membrane to intracellular compartments (25). However, most studies have been performed using nonendothelial cell lines overexpressing both proteins. Therefore, the physiological role, if any, played by NOSIP in the regulation of eNOS activity remains to be determined.

Phosphatases. Because eNOS activity is regulated by phosphorylation, it is logical that alterations in phosphatase activity can also affect NO generation. Indeed, PP1 and PP2A play distinct roles in the regulation of eNOS phosphorylation (33, 36, 95).

PP1 dephosphorylates Thr⁴⁹⁵ and inhibition of PP1 results in the hyperphosphorylation of Thr⁴⁹⁵, which inhibits eNOS activity (36). The mechanism of PP1 activation by receptor-dependent agonists is still unclear, but after the application of bradykinin to endothelial cells, the dephosphorylation of Thr⁴⁹⁵ is a Ca²⁺-dependent process (36), whereas in forskolin-stimulated endothelial cells, the process is dependent on the activation of PKA (95). The fact that the bradykinin-dependent dephosphorylation of Thr⁴⁹⁵ is Ca²⁺-dependent initially suggested that this process should be regulated by a Ca²⁺-dependent phosphatase such as PP2B (calcineurin)(59). However, the calcineurin inhibitors, cyclosporin A and FK506 do not affect bradykinin-induced changes in Thr⁴⁹⁵ phosphorylation or bradykinin-induced NO production (36, 95).

PP2A is the phosphatase that dephosphorylates Ser¹¹⁷⁷ (33, 95) and PP2A inhibitors, such as okadaic acid, increase eNOS activity by two- to fourfold, i.e., to the same extent as stimuli such as fluid shear stress (33). PKC, which phosphorylates Thr⁴⁹⁵, can also promote the dephosphorylation of Ser¹¹⁷⁷ by regulating the activity of PP2A (36, 95). This dual function amplifies the PKC-mediated inhibition of eNOS activity. Both PP1 and PP2 are reported to associate with eNOS but this association is not modified by cell stimulation (95).

Nothing is known about the phosphatase(s) that regulate the tyrosine phosphorylation of eNOS. However, nNOS can be tyrosine phosphorylated and is reported to interact with SHP-1 (38, 84) and in response to cell activation, SHP-1 rapidly recruits nNOS and tyrosine dephosphorylates it, thus increasing enzyme activity.

	CONCLUSION

TOP ABSTRACT INTRODUCTION REGULATION OF ENOS EXPRESSION POSTTRANSLATIONAL REGULATION CONCLUSION REFERENCES

The regulation of eNOS is a process determined by a cascade of events determining eNOS mRNA and protein levels, the formation of the eNOS signaling complex, its intracellular translocation, and eNOS phosphorylation. Although many of these steps have been relatively well elucidated in in vitro models and in cell lines overexpressing one or more components of the signaling complex, much more work is required to determine which modifications play a dominant role in the regulation of eNOS activity in vivo and how these steps are influenced by the pathophysiological changes that ultimately lead to "endothelial dysfunction" and to the development of atherosclerosis.

	ACKNOWLEDGEMENTS

Experiments performed in the authors laboratory were supported by the Deutsche Forschungsgemeinschaft (SFB 553, B1, and B5).

	FOOTNOTES

Address for reprint requests and other correspondence: I. Fleming, Institut für Kardiovaskuläre Physiologie, J.W.Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany (E-mail fleming{at}em.uni-frankfurt.de).

10.1152/ajpregu.00323.2002

	REFERENCES

TOP ABSTRACT INTRODUCTION REGULATION OF ENOS EXPRESSION POSTTRANSLATIONAL REGULATION CONCLUSION REFERENCES

1.   Andries, LJ, Brutsaert DL, and Sys SU. Nonuniformity of endothelial constitutive nitric oxide synthase distribution in cardiac endothelium. Circ Res 82: 195-203, 1998[Abstract/Free Full Text].

2.   Arnal, JF, Yamin J, Dockery S, and Harrison DG. Regulation of endothelial nitric oxide synthase mRNA, protein, and activity during cell growth. Am J Physiol Cell Physiol 267: C1381-C1388, 1994[Abstract/Free Full Text].

3.   Arriero, MM, de la Pinta JC, Escribano M, Celdran A, Munoz-Alameda L, Garcia-Canete J, Jimenez AM, Casado S, Farre J, and Lopez-Farre A. Aspirin prevents Escherichia coli lipopolysaccharide- and Staphylococcus aureus-induced downregulation of endothelial nitric oxide synthase expression in guinea pig pericardial tissue. Circ Res 90: 719-727, 2002[Abstract/Free Full Text].

4.   Ayajiki, K, Kindermann M, Hecker M, Fleming I, and Busse R. Intracellular pH and tyrosine phosphorylation but not calcium determine shear stress-induced nitric oxide production in native endothelial cells. Circ Res 78: 750-758, 1996[Abstract/Free Full Text].

5.   Bauersachs, J, Fleming I, Scholz D, Popp R, and Busse R. Endothelium-derived hyperpolarizing factor but not nitric oxide is reversibly inhibited by brefeldin A. Hypertension 30: 1598-1605, 1997[Abstract/Free Full Text].

6.   Benzing, T, Fleming I, Blaukat A, Müller-Esterl W, and Busse R. The ACE inhibitor ramiprilat prevents the sequestration of the B₂ kinin receptor within the plasma membrane of native endothelial cells. Circulation 99: 2034-2040, 1999[Abstract/Free Full Text].

7.   Bernier, SG, Haldar S, and Michel T. Bradykinin-regulated interactions of the mitogen-activated protein kinase pathway with the endothelial nitric-oxide synthase. J Biol Chem 275: 30707-30715, 2000[Abstract/Free Full Text].

8.   Billecke, SS, Bender AT, Kanelakis KC, Murphy PJ, Lowe ER, Kamada Y, Pratt WB, and Osawa Y. HSP90 is required for heme binding and activation of APO-neuronal nitric-oxide synthase: geldanamycin-mediated oxidant generation is unrelated to any action of HSP90. J Biol Chem 277: 20504-20509, 2002[Abstract/Free Full Text].

9.   Boo, YC, Sorescu G, Boyd N, Shiojima I, Walsh K, Du J, and Jo H. Shear stress stimulates phosphorylation of endothelial nitric-oxide synthase at Ser¹¹⁷⁹ by Akt-independent mechanisms: role of protein kinase A. J Biol Chem 277: 3388-3396, 2002[Abstract/Free Full Text].

10.   Brophy, CM, Woodrum DA, Pollock J, Dickinson M, Komalavilas P, Cornwell TL, and Lincoln TM. cGMP-dependent protein kinase expression restores contractile function in cultured vascular smooth muscle cells. J Vasc Res 39: 95-103, 2002[ISI][Medline].

11.   Bucci, M, Gratton JP, Rudic RD, Acevedo L, Roviezzo F, Cirino G, and Sessa WC. In vivo delivery of the caveolin-1 scaffolding domain inhibits nitric oxide synthesis and reduces inflammation. Nat Med 6: 1362-1367, 2000[ISI][Medline].

12.   Busconi, L, and Michel T. Endothelial nitric oxide synthase: N-terminal myristoylation determines subcellular localization. J Biol Chem 268: 8410-8413, 1993[Abstract/Free Full Text].

13.   Busse, R, and Mülsch A. Calcium-dependent nitric oxide synthesis in endothelial cytosol is mediated by calmodulin. FEBS Lett 265: 133-136, 1990[ISI][Medline].

14.   Butt, E, Bernhardt M, Smolenski A, Kotsonis P, Frohlich LG, Sickmann A, Meyer HE, Lohmann SM, and Schmidt HHHW Endothelial nitric-oxide synthase (type III) is activated and becomes calcium independent upon phosphorylation by cyclic nucleotide-dependent protein kinases. J Biol Chem 275: 5179-5187, 2000[Abstract/Free Full Text].

15.   Cai, H, Davis ME, Drummond GR, and Harrison DG. Induction of endothelial NO synthase by hydrogen peroxide via a Ca²⁺/calmodulin-dependent protein kinase II/janus kinase 2-dependent pathway. Arterioscler Thromb Vasc Biol 21: 1571-1576, 2001[Abstract/Free Full Text].

16.   Cao, S, Yao J, Mccabe TJ, Yao Q, Katusic ZS, Sessa WC, and Shah V. Direct interaction between endothelial nitric-oxide synthase and dynamin-2. Implications for nitric-oxide synthase function. J Biol Chem 276: 14249-14256, 2001[Abstract/Free Full Text].

17.   Caplan, AJ. Hsp90's secrets unfold: new insights from structural and functional studies. Trends Cell Biol 9: 262-268, 1999[ISI][Medline].

18.   Chambliss, KL, Yuhanna IS, Mineo C, Liu P, German Z, Sherman TS, Mendelsohn ME, Anderson RG, and Shaul PW. Estrogen receptor  and endothelial nitric oxide synthase are organized into a functional signaling module in caveolae. Circ Res 87: E44-E52, 2000[ISI][Medline].

19.   Chen, ZP, Mitchelhill KI, Michell BJ, Stapelton D, Rodriguez-Crespo I, Witters LA, Power DA, Ortiz de Montellano PR, and Kemp BE. AMP-activated protein kinase phosphorylation of endothelial NO synthase. FEBS Lett 443: 285-289, 1999[ISI][Medline].

20.   Cieslik, K, Zembowicz A, Tang JL, and Wu KK. Transcriptional regulation of endothelial nitric-oxide synthase by lysophosphatidylcholine. J Biol Chem 273: 14885-14890, 1998[Abstract/Free Full Text].

21.   Cordelier, P, Esteve JP, Rivard N, Marletta M, Vaysse N, Susini C, and Buscail L. The activation of neuronal NO synthase is mediated by G-protein subunit and the tyrosine phosphatase SHP-2. FASEB J 13: 2037-2050, 1999[Abstract/Free Full Text].

22.   Corson, MA, James NL, Latta SE, Nerem RM, Berk BC, and Harrison DG. Phosphorylation of endothelial nitric oxide synthase in response to fluid shear stress. Circ Res 79: 984-991, 1996[Abstract/Free Full Text].

23.   Danino, D, and Hinshaw JE. Dynamin family of mechanoenzymes. Curr Opin Cell Biol 13: 454-460, 2001[ISI][Medline].

24.   Davda, RK, Chandler LJ, and Guzman NJ. Protein kinase C modulates receptor-independent activation of endothelial nitric oxide synthase. Eur J Pharmacol 266: 237-244, 1994[ISI][Medline].

25.   Dedio, J, Konig P, Wohlfart P, Schroeder C, Kummer W, and Müller-Esterl W. NOSIP, a novel modulator of endothelial nitric oxide synthase activity. FASEB J 15: 79-89, 2001[Abstract/Free Full Text].

26.   Dessy, C, Kelly RA, Balligand JL, and Feron O. Dynamin mediates caveolar sequestration of muscarinic cholinergic receptors and alteration in NO signaling. EMBO J 19: 4272-4280, 2000[ISI][Medline].

27.   Dikalov, S, Landmesser U, and Harrison DG. Geldanamycin leads to superoxide formation by enzymatic and non-enzymatic redox cycling: implications for studies of Hsp90 and eNOS. J Biol Chem 277: 25480-25485, 2002[Abstract/Free Full Text].

28.   Dimmeler, S, Fleming I, Fisslthaler B, Hermann C, Busse R, and Zeiher AM. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 399: 601-605, 1999[Medline].

29.   Drab, M, Verkade P, Elger M, Kasper M, Lohn M, Lauterbach B, Menne J, Lindschau C, Mende F, Luft FC, Schedl A, Haller H, and Kurzchalia TV. Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice. Science 293: 2449-2452, 2001[Abstract/Free Full Text].

30.   Drummond, GR, Cai H, Davis ME, Ramasamy S, and Harrison DG. Transcriptional and posttranscriptional regulation of endothelial nitric oxide synthase expression by hydrogen peroxide. Circ Res 86: 347-354, 2000[Abstract/Free Full Text].

31.   Feng, Y, Venema VJ, Venema RC, Tsai N, and Caldwell RB. VEGF induces nuclear translocation of Flk-1/KDR, endothleial nitric oxide synthase, and caveolin-1 in vascular endothelial cells. Biochem Biophys Res Commun 256: 192-197, 1999[ISI][Medline].

32.   Feron, O, Belhassen L, Kobzik L, Smith TW, Kelly RA, and Michel T. Endothelial nitric oxide synthase targeting to caveolaespecific interactions with caveolin isoforms in cardiac myocytes and endothelial cells. J Biol Chem 271: 22810-22814, 1996[Abstract/Free Full Text].

33.   Fisslthaler, B, Dimmeler S, Hermann C, Busse R, and Fleming I. Phosphorylation and activation of the endothelial nitric oxide synthase by fluid shear stress. Acta Physiol Scand 168: 81-88, 2000[ISI][Medline].

34.   Fleming, I, Bara A, and Busse R. Calcium signalling and autacoid production in endothelial cells are modulated by changes in tyrosine kinase and phosphatase activity. J Vasc Res 33: 225-234, 1996[ISI][Medline].

35.   Fleming, I, Bauersachs J, Fisslthaler B, and Busse R. Ca²⁺-independent activation of the endothelial nitric oxide synthase in response to tyrosine phosphatase inhibitors and fluid shear stress. Circ Res 82: 686-695, 1998[Abstract/Free Full Text].

36.   Fleming, I, Fisslthaler B, Dimmeler S, Kemp BE, and Busse R. Phosphorylation of Thr⁴⁹⁵ regulates Ca²⁺/calmodulin-dependent endothelial nitric oxide synthase activity. Circ Res 88: e68-e75, 2001[Abstract/Free Full Text].

37.   Fontana, J, Fulton D, Chen Y, Fairchild TA, Mccabe TJ, Fujita N, Tsuruo T, and Sessa WC. Domain mapping studies reveal that the M domain of Hsp90 serves as a molecular scaffold to regulate Akt-dependent phosphorylation of endothelial nitric oxide synthase and NO release. Circ Res 90: 866-873, 2002[Abstract/Free Full Text].

38.   Forget, G, Siminovitch KA, Brochu S, Rivest S, Radzioch D, and Olivier M. Role of host phosphotyrosine phosphatase SHP-1 in the development of murine leishmaniasis. Eur J Immunol 31: 3185-3196, 2001[ISI][Medline].

39.   Förstermann, U, Boissel JP, and Kleinert H. Expressional control of the "constitutive" isoforms of nitric oxide synthase (NOS I and NOS III). FASEB J 12: 773-790, 1998[Abstract/Free Full Text].

40.   Fulton, D, Fontana J, Sowa G, Gratton JP, Lin M, Li KX, Michell B, Kemp BE, Rodman D, and Sessa WC. Localization of endothelial nitric-oxide synthase phosphorylated on serine 1179 and nitric oxide in Golgi and plasma membrane defines the existence of two pools of active enzyme. J Biol Chem 277: 4277-4284, 2002[Abstract/Free Full Text].

41.   Fulton, D, Gratton JP, Mccabe TJ, Fontana J, Fujio Y, Walsh K, Franke TF, Papapetropoulos A, and Sessa WC. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature 399: 597-601, 1999[Medline].

42.   Fulton, D, Gratton JP, and Sessa WC. Posttranslational control of endothelial nitric oxide synthase: why isn't calcium/calmodulin enough? J Pharmacol Exp Ther 299: 818-824, 2001[Abstract/Free Full Text].

43.   Gallis, B, Corthals GL, Goodlett DR, Ueba H, Kim F, Presnell SR, Figeys D, Harrison DG, Berk BC, Aebersold R, and Corson MA. Identification of flow-dependent endothelial nitric oxide synthase phosphorylation sites by mass spectrometry and regulation of phosphorylation and nitric oxide production by the phosphatidylinositol 3-kinase inhibitor LY294002. J Biol Chem 274: 30101-30108, 1999[Abstract/Free Full Text].

44.   Garcia-Cardena, G, Oh P, Liu J, Schnitzer JE, and Sessa WC. Targeting of nitric oxide synthase to endothelial cell caveolae via palmitoylation: implications for nitric oxide signaling. Proc Natl Acad Sci USA 93: 6448-6453, 1996[Abstract/Free Full Text].

45.   García-Cardena, G, Fan G, Stern DF, Liu J, and Sessa WC. Endothelial nitric oxide synthase is regulated by tyrosine phosphorylation and interacts with caveolin-1. J Biol Chem 271: 27237-27240, 1996[Abstract/Free Full Text].

46.   García-Cardena, G, Fan R, Shah V, Sorrentino R, Cirino G, Papapetropoulos A, and Sessa WC. Dynamic activation of endothelial nitric oxide synthase by Hsp90. Nature 292: 821-824, 1998.

47.   García-Cardena, G, Martasek P, Masters BS, Skidd PM, Couet J, Li SW, Lisanti MP, and Sessa WC. Dissecting the interaction between nitric oxide synthase (NOS) and caveolinfunctional significance of the NOS caveolin binding domain in vivo. J Biol Chem 272: 25437-25440, 1997[Abstract/Free Full Text].

48.   German, Z, Chambliss KL, Pace MC, Arnet UA, Lowenstein CJ, and Shaul PW. Molecular basis of cell-specific endothelial nitric-oxide synthase expression in airway epithelium. J Biol Chem 275: 8183-8189, 2000[Abstract/Free Full Text].

49.   Ghosh, S, Gachhui R, Crooks C, Wu CQ, Lisanti MP, and Stuehr DJ. Interaction between caveolin-1 and the reductase domain of endothelial nitric-oxide synthaseconsequences for catalysis. J Biol Chem 273: 22267-22271, 1998[Abstract/Free Full Text].

50.   Giordano, A, Tonello C, Bulbarelli A, Cozzi V, Cinti S, Carruba MO, and Nisoli E. Evidence for a functional nitric oxide synthase system in brown adipocyte nucleus. FEBS Lett 514: 135-140, 2002[ISI][Medline].

51.   Go, YM, Park H, Maland MC, Darley-Usmar VM, Stoyanov B, Wetzker R, and Jo H. Phosphatidylinositol 3-kinase gamma mediates shear stress-dependent activation of JNK in endothelial cells. Am J Physiol Heart Circ Physiol 275: H1898-H1904, 1998[Abstract/Free Full Text].

52.   Goetz, RM, Thatte HS, Prabhakar P, Cho MR, Michel T, and Golan DE. Estradiol induces the calcium-dependent translocation of endothelial nitric oxide synthase. Proc Natl Acad Sci USA 96: 2788-2793, 1999[Abstract/Free Full Text].

53.   Golser, R, Gorren ACF, Leber A, Andrew P, Habisch HJ, Werner ER, Schmidt K, Venema RC, and Mayer B. Interaction of endothelial and neuronal nitric-OXIDE synthases with the bradykinin B2 receptor. Binding of an inhibitory peptide to the oxygenase domain blocks uncoupled NADPH oxidation. J Biol Chem 275: 5291-5296, 2000[Abstract/Free Full Text].

54.   Gonzalez-Fernandez, F, Jimenez A, Lopez-Blaya A, Velasco S, Arriero MM, Celdran A, Rico L, Farre J, Casado S, and Lopez-Farre A. Cerivastatin prevents tumor necrosis factor-alpha-induced downregulation of endothelial nitric oxide synthase: role of endothelial cytosolic proteins. Atherosclerosis 155: 61-70, 2001[ISI][Medline].

55.   Govers, R, Bevers L, de Bree P, and Rabelink TJ. Endothelial nitric oxide synthase activity is linked to its presence at cell-cell contacts. Biochem J 361: 193-201, 2002[ISI][Medline].

56.   Govers, R, Van Der SP, Van Donselaar E, Slot JW, and Rabelink TJ. Endothelial nitric oxide synthase and its negative regulator caveolin-1 localize to distinct perinuclear organelles. J Histochem Cytochem 50: 779-788, 2002[Abstract/Free Full Text].

57.   Gratton, JP, Fontana J, O'Connor DS, Garcia-Cardena G, Mccabe TJ, and Sessa WC. Reconstitution of an endothelial nitric oxide synthase, Hsp90 and caveolin-1 complex in vitro: evidence that Hsp90 facilitates calmodulin stimulated displacement of eNOS from caveolin-1. J Biol Chem 275: 22268-22272, 2000[Abstract/Free Full Text].

58.   Harris, MB, Ju H, Venema VJ, Blackstone M, and Venema RC. Role of heat shock protein 90 in bradykinin-stimulated endothelial nitric oxide release. Gen Pharmacol 35: 165-170, 2000[Medline].

59.   Harris, MB, Ju H, Venema VJ, Liang H, Zou R, Michell BJ, Chen ZP, Kemp BE, and Venema RC. Reciprocal phosphorylation and regulation of the endothelial nitric oxide synthase in response to bradykinin stimulation (Abstract). J Biol Chem 19: 16587-16591, 2001.

60.   Haynes, MP, Sinha D, Russell KS, Collinge M, Fulton D, Morales-Ruiz M, Sessa WC, and Bender JR. Membrane estrogen receptor engagement activates endothelial nitric oxide synthase via the PI3-kinase-Akt pathway in human endothelial cells. Circ Res 87: 677-682, 2000[Abstract/Free Full Text].

61.   Hecker, M, Mulsch A, Bassenge E, Forstermann U, and Busse R. Subcellular localization and characterization of nitric oxide synthase(s) in endothelial cells: physiological implications. Biochem J 299: 247-252, 1994[ISI][Medline].

62.   Hellermann, GR, Flam BR, Eichler DC, and Solomonson LP. Stimulation of receptor-mediated nitric oxide production by vanadate. Arterioscler Thromb Vasc Biol 20: 2045-2050, 2000[Abstract/Free Full Text].

63.   Hinshaw, JE. Dynamin and its role in membrane fission. Annu Rev Cell Dev Biol 16: 483-519, 2000[ISI][Medline].

64.   Hirata, K, Kuroda R, Sakoda T, Katayama M, Inoue N, Suematsu M, Kawashima S, and Yokoyama M. Inhibition of endothelial nitric oxide synthase activity by protein kinase C. Hypertension 25: 180-185, 1995[Abstract/Free Full Text].

65.   Hoyer, J, Köhler R, and Distler A. Mechanosensitive Ca²⁺ oscillations and STOC activation in endothelial cells. FASEB J 12: 359-366, 1998[Abstract/Free Full Text].

66.   Jayachandran, M, Hayashi T, Sumi D, Iguchi A, and Miller VM. Temporal effects of 17-estradiol on caveolin-1 mRNA and protein in bovine aortic endothelial cells. Am J Physiol Heart Circ Physiol 281: H1327-H1333, 2001[Abstract/Free Full Text].

67.   Jimenez, A, Arriero MM, Lopez-Blaya A, Gonzalez-Fernandez F, Garcia R, Fortes J, Millas I, Velasco S, Sanchez DM, Rico L, Farre J, Casado S, and Lopez-Farre A. Regulation of endothelial nitric oxide synthase expression in the vascular wall and in mononuclear cells from hypercholesterolemic rabbits. Circulation 104: 1822-1830, 2001[Abstract/Free Full Text].

68.   Ju, H, Venema VJ, Marrero MB, and Venema RC. Inhibitory interactions of the bradykinin B₂ receptor with endothelial nitric oxide synthase. J Biol Chem 273: 24025-24029, 1998[Abstract/Free Full Text].

69.   Ju, H, Zou R, Venema VJ, and Venema RC. Direct interaction of endothelial nitric-oxide synthase and caveolin-1 inhibits synthase activity. J Biol Chem 272: 18522-18525, 1997[Abstract/Free Full Text].

70.   Kammerl, MC, Richthammer W, Kurtz A, and Kramer BK. Angiotensin II feedback is a regulator of renocortical renin, COX-2, and nNOS expression. Am J Physiol Regul Integr Comp Physiol 282: R1613-R1617, 2002[Abstract/Free Full Text].

71.   Kanai, AJ, Strauss HC, Truskey GA, Crews AL, Grunfeld S, and Malinski T. Shear stress induces ATP-independent transient nitric oxide release from vascular endothelial cells, measured directly with a porphyrinic microsensor. Circ Res 77: 284-293, 1995[Abstract/Free Full Text].

72.   Karantzoulis-Fegaras, F, Antoniou H, Lai SL, Kulkarni G, D'Abreo C, Wong GK, Miller TL, Chan Y, Atkins J, Wang Y, and Marsden PA. Characterization of the human endothelial nitric-oxide synthase promoter. J Biol Chem 274: 3076-3093, 1999[Abstract/Free Full Text].

73.   Kim, F, Gallis B, and Corson MA. TNF- inhibits flow and insulin signaling leading to NO production in aortic endothelial cells. Am J Physiol Cell Physiol 280: C1057-C1065, 2001[Abstract/Free Full Text].

74.   Kleinert, H, Wallerath T, Euchenhofer C, Ihrig-Biedert I, Li H, and Förstermann U. Estrogens increase transcription of the human endothelial NO synthase gene: analysis of the transcription factors involved. Hypertension 31: 582-588, 1998[Abstract/Free Full Text].

75.   Kou, R, Prabhakar P, and Michel T. Phosphorylation of the endothelial isoform of nitric oxide synthase at serine 116: Identification of a novel path for eNOS regulation by lysophosphatidic acid. Circulation 104: 509, 2001.

76.   Lantinhermoso, RL, Rosenfeld CR, Yuhanna IS, German Z, Chen Z, and Shaul PW. Estrogen acutely stimulates nitric oxide synthase activity in fetal pulmonary artery endothelium. Am J Physiol Lung Cell Mol Physiol 273: L119-L126, 1997[Abstract/Free Full Text].

77.   Laufs, U, La F V, Plutzky J, and Liao JK. Upregulation of endothelial nitric oxide synthase by HMG CoA reductase inhibitors. Circulation 97: 1129-1135, 1998[Abstract/Free Full Text].

78.   Laumonnier, Y, Nadaud S, Agrapart M, and Soubrier F. Characterization of an upstream enhancer region in the promoter of the human endothelial nitric-oxide synthase gene. J Biol Chem 275: 40732-40741, 2000[Abstract/Free Full Text].

79.   Li, H, Brodsky S, Basco M, Romanov V, De Angelis DA, and Goligorsky MS. Nitric oxide attenuates signal transduction: possible role in dissociating caveolin-1 scaffold. Circ Res 88: 229-236, 2001[Abstract/Free Full Text].

80.   Lincoln, TM, Dey N, and Sellak H. Signal transduction in smooth muscle: invited review: cGMP-dependent protein kinase signaling mechanisms in smooth muscle: from the regulation of tone to gene expression. J Appl Physiol 91: 1421-1430, 2001[Abstract/Free Full Text].

81.   Liu, J, García-Cardena G, and Sessa WC. Biosynthesis and palmitoylation of endothelial nitric oxide synthase: mutagenesis of palmitoylation sites, cysteines-15 and/or 26, argues against depalmitoylation-induced translocation of the enzyme. Biochemistry 34: 12333-12340, 1995[Medline].

82.   Liu, J, García-Cardena G, and Sessa WC. Palmitoylation of endothelial nitric oxide synthase is necessary for optimal stimulated release of nitric oxide: implications for caveolae localization. Biochemistry 35: 13277-13281, 1996[Medline].

83.   Liu, J, Hughes TE, and Sessa WC. The first 35 amino acids and fatty acylation sites determine the molecular targeting of endothelial nitric oxide synthase into the Golgi region of cells: a green fluorescent protein study. J Cell Biol 137: 1525-1535, 1997[Abstract/Free Full Text].

84.   Lopez, F, Ferjoux G, Cordelier P, Saint-Laurent N, Esteve JP, Vaysse N, Buscail L, and Susini C. Neuronal nitric oxide synthase: a substrate for SHP-1 involved in sst2 somatostatin receptor growth inhibitory signaling. FASEB J 15: 2300-2302, 2001[Free Full Text].

85.   Luckhoff, A, Pohl U, Mulsch A, and Busse R. Differential role of extra- and intracellular calcium in the release of EDRF and prostacyclin from cultured endothelial cells. Br J Pharmacol 95: 189-196, 1988[ISI][Medline].

86.   Macritchie, AN, Jun SS, Chen Z, German Z, Yuhanna IS, Sherman TS, and Shaul PW. Estrogen upregulates endothelial nitric oxide synthase gene expression in fetal pulmonary artery endothelium. Circ Res 81: 355-362, 1997[Abstract/Free Full Text].

87.   Marrero, MB, Venema VJ, Ju H, He H, Liang H, Caldwell RB, and Venema RC. Endothelial nitric oxide synthase interactions with G-protein-coupled receptors. Biochem J 343: 335-340, 1999[ISI][Medline].

88.   Martinez-Gonzalez, J, Raposo B, Rodriguez C, and Badimon L. 3-Hydroxy-3-methylglutaryl coenzyme a reductase inhibition prevents endothelial NO synthase downregulation by atherogenic levels of native LDLs: balance between transcriptional and posttranscriptional regulation. Arterioscler Thromb Vasc Biol 21: 804-809, 2001[Abstract/Free Full Text].

89.   Matsubara, M, Titani K, and Taniguchi H. Interaction of calmodulin-binding domain peptides of nitric oxide synthase with membrane phospholipids: regulation by protein phosphorylation and Ca²⁺-calmodulin. Biochemistry 35: 14651-14658, 1996[Medline].

90.   Mccabe, TJ, Fulton D, Roman LJ, and Sessa WC. Enhanced electron flux and reduced calmodulin dissociation may explain "calcium-independent" eNOS activation by phosphorylation. J Biol Chem 275: 6123-6128, 2000[Abstract/Free Full Text].

91.   McNeill, AM, Kim N, Duckles SP, Krause DN, and Kontos HA. Chronic estrogen treatment increases levels of endothelial nitric oxide synthase protein in rat cerebral microvessels. Stroke 30: 2186-2190, 1999[Abstract/Free Full Text].

92.   McQuillan, LP, Leung GK, Marsden PA, Kostyk SK, and Kourembanas S. Hypoxia inhibits expression of eNOS via transcriptional and posttranscriptional mechanisms. Am J Physiol Heart Circ Physiol 267: H1921-H1927, 1994[Abstract/Free Full Text].

93.   Michel, JB, Feron O, Sacks D, and Michel T. Reciprocal regulation of endothelial nitric-oxide synthase by Ca²⁺-calmodulin and caveolin. J Biol Chem 272: 15583-15586, 1997[Abstract/Free Full Text].

94.   Michel, T, Li GK, and Busconi L. Phosphorylation and subcellular translocation of endothelial nitric oxide synthase. Proc Natl Acad Sci USA 90: 6252-6256, 1993[Abstract/Free Full Text].

95.   Michell, BJ, Chen Z, Tiganis T, Stapelton D, Katsis F, Power DA, Sim AT, and Kemp BE. Coordinated control of endothelial NO synthase phosphorylation by protein kinase C and the cAMP-dependent protein kinase. J Biol Chem 276: 17625-17628, 2001[Abstract/Free Full Text].

96.   Miyamoto, Y, Saito Y, Nakayama M, Shimasaki Y, Yoshimura T, Yoshimura M, Harada M, Kajiyama N, Kishimoto I, Kuwahara K, Hino J, Ogawa E, Hamanaka I, Kamitani S, Takahashi N, Kawakami R, Kangawa K, Yasue H, and Nakao K. Replication protein A1 reduces transcription of the endothelial nitric oxide synthase gene containing a 786TC mutation associated with coronary spastic angina. Hum Mol Genet 9: 2629-2637, 2000[Abstract/Free Full Text].

97.   Mohamed, F, Monge JC, Gordon A, Cernacek P, Blais D, and Stewart DJ. Lack of role for nitric oxide (NO) in the selective destabilization of endothelial NO synthas