Cellular signaling and NO production
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- Michel, T. & Vanhoutte, P.M. Pflugers Arch - Eur J Physiol (2010) 459: 807. doi:10.1007/s00424-009-0765-9
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The endothelium can evoke relaxations (dilatations) of the underlying vascular smooth muscle, by releasing vasodilator substances. The best characterized endothelium-derived relaxing factor is nitric oxide (NO), which is synthesized by the endothelial isoform of nitric oxide synthase (eNOS). Endothelium-dependent relaxations involve both pertussis-toxin-sensitive Gi (e.g., responses to serotonin, sphingosine 1-phosphate, alpha2-adrenergic agonists, and thrombin) and pertussis-toxin-insensitive Gq (e.g., adenosine diphosphate and bradykinin) coupling proteins. eNOS undergoes a complex pattern of intracellular regulation, including post-translational modifications involving enzyme acylation and phosphorylation. eNOS is reversibly targeted to signal-transducing plasmalemmal caveolae where the enzyme interacts with a number of regulatory proteins, many of which are modified in cardiovascular disease states. The release of nitric oxide by the endothelial cell can be up- (e.g., by estrogens, exercise, and dietary factors) and down-regulated (e.g. oxidative stress, smoking, and oxidized low-density lipoproteins). It is reduced in the course of vascular disease (e.g., diabetes and hypertension). Arteries covered with regenerated endothelium (e.g. following angioplasty) selectively lose the pertussis-toxin-sensitive pathway for NO release which favors vasospasm, thrombosis, penetration of macrophages, cellular growth, and the inflammatory reaction leading to atherosclerosis. The unraveling of the complex interaction of the pathways regulating the presence and the activity of eNOS will enhance the understanding of the perturbations in endothelium-dependent signaling that are seen in cardiovascular disease states, and may lead to the identification of novel targets for therapeutic intervention.
KeywordsDiabetesG-proteinsHypertensionNitric oxideNitric oxide synthaseCaveolaEndothelium-derived relaxing factor (EDRF)Phosphorylation
Robert Furchgott demonstrated that the removal of the endothelial layer from isolated arteries prevents the in vitro dilator response to acetylcholine . Early bioassay studies demonstrated that the endothelial cells cause arterial relaxation by releasing a vasoactive substance(s) which was termed endothelium-derived relaxing factor (EDRF) [28, 59]. The original EDRF  stimulates soluble guanylyl cyclase in the vascular smooth muscle cells and thus increases the production of cyclic guanosine monophosphate (cyclic GMP) [27, 50, 76]. It is rapidly destroyed by superoxide anions . These experimental facts led to the proposal and the demonstration that EDRF is nitric oxide (NO) . Besides NO, a number of endothelium-derived factors (EDHFs) and the opening of gap junctions can cause NO-independent hyperpolarizations of the underlying vascular smooth muscle and thus endothelium-dependent relaxations [4, 16, 18]. When the ability of the endothelial cells to release nitric oxide (NO) [and to cause endothelium-dependent hyperpolarizations] is reduced, this appears to be the first step in the chain of events that leads to atherosclerosis and coronary disease [11, 76]. Thus, endothelial dysfunction has become a hallmark and a predictor of cardiovascular disease . This article focuses on the cellular mechanisms leading to the release of endothelium-derived nitric oxide (EDNO) and how reduction in such release plays a key role in the genesis of vascular disease. For the survey of the original experimental literature, the reader will be referred mainly to more exhaustive previous review articles.
Nitric oxide protector of the vascular wall
The role played by the endothelial cells to protect against thrombin and platelet products by increasing the activity of eNOS has been demonstrated both in vitro and in vivo [35, 76]. Serotonin (5-hydroxytryptamine, 5-HT) and adenosine diphosphate (ADP) are the two mediators released by aggregating platelets that can activate eNOS and thus augment the production of NO. Serotonin is the most important and stimulates 5-HT1D serotonergic receptors of the endothelial cell membrane. ADP is a relatively minor contributor of the response to platelets and acts on P2y purinoceptors (Fig. 3). The serotonergic receptors and those for thrombin are coupled to the activation of eNOS through pertussis-toxin-sensitive Gi-proteins, while the P2y-purinoceptors are linked to the enzyme by Gq-proteins . If the endothelium is absent or dysfunctional such relaxations are no longer observed, and aggregating platelets induce constrictions (vasospasm), because they release the powerful vasoconstrictors thromboxane A2 and serotonin.
The physiological importance of the endothelium-dependent relaxations to platelet products is obvious [17, 76]. Thus, if platelet aggregation occurs in a coronary artery with a healthy endothelium the release by the platelets of serotonin (and ADP) and the local production of thrombin will stimulate the endothelial cells with the resulting release of NO. The endothelial mediator will cause the underlying smooth muscle to relax, thus increasing blood flow and mechanically impeding the progression of the coagulation process. NO also exerts in synergy with prostacyclin an immediate feedback inhibition on the platelets. When the endothelial barrier is interrupted by injury, the aggregating platelets can approach the vascular smooth muscle cells, and cause their contraction by releasing thromboxane A2 and serotonin, initiating the vascular phase of hemostasis. The endothelium-dependent response to aggregating platelets is not present to the same extent in all arteries, but is the most prominent in the coronary and cerebral circulations .
Intracellular regulation of endothelial nitric oxide synthase
Enzymatic function of eNOS
Endothelial nitric oxide synthase is a member of a family of three mammalian nitric oxide synthase (“NOS”) isoforms that catalyze the oxidation of the terminal guanidino group of l-arginine to produce NO and l-citrulline [13, 24, 64]. The three archetypal mammalian NOS isoforms are (a) nNOS (NOS I) or neuronal NOS; (b) iNOS (NOS II), an inducible NOS isoform expressed in a variety of activated tissues; and (c) eNOS (NOS III) or endothelial NOS. They are the products of distinct genes, and share approximately 50–60% sequence identity. Although these isoforms possess similar overall enzymatic and chemical properties, they contain distinctive catalytic and regulatory properties which characterize them. Nitric oxide synthases are bifunctional enzymes: the N terminus exhibits homology principally to the other NOS isoforms, while the C-terminal domain has significant sequence homology to cytochrome P450 reductases. The NOS N terminus binds tetrahydrobiopterin (BH4) and heme (see Fig. 6). l-arginine binds in the enzyme active site near heme, while molecular oxygen is directed to the ferrous heme iron. The binding sites for tetrahydrobiopterin and heme are localized along the interface of the monooxygenase domains of the dimeric, active form of NOS. Binding of heme and tetrahydrobiopterin, especially for iNOS, may be crucial in promoting dimerization and NOS catalytic activity. The C-terminal domain binds nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and Flavin mononucleotide (FMN) cofactors . The N- and C-terminal domains are linked by a short sequence that binds calmodulin, an allosteric effector that is essential for full NOS activity. The intact NOS functions as a mixed function monooxygenase/reductase enzyme, and has the capability to synthesize a number of molecules besides NO, most notably superoxide anions. In all NOS catalysis reactions, electrons are shuttled within the reductase domain sequentially from NADPH, to FAD, and finally to FMN. Calmodulin is believed to function in facilitating the flow of electrons from both the reductase domain to the monooxygenase domain as well as from FAD to FMN. Because electrons appear to flow from the reductase domain of one NOS monomer to the oxygenase domain of another NOS monomer, enzyme dimerization is required for full enzymatic activity [2, 24].
Post-translational modifications of the eNOS protein
Endothelial eNOS undergoes a complex pattern of post-translational regulatory modifications (Fig. 6). These modifications underlie the dynamic regulation of enzymatic activity in response to physiological stimuli and pathophysiological conditions [13, 26, 63].
In quiescent cells, eNOS is targeted to invaginations of the plasmalemma called caveolae, which are membrane microdomains defined by the presence of the scaffolding protein caveolin. The caveolae are enriched in cholesterol and sphingolipids, and sequester diverse receptors and signaling proteins from a variety of signal transduction pathways, including G-protein-coupled receptors, G-proteins, growth factor receptors, calcium regulatory proteins, as well as eNOS. The targeting of eNOS may permit the enzyme to receive signals from these upstream signaling pathways and facilitate communication with downstream activators. The targeting of eNOS to caveolae is dependent on the irreversible co-translational myristoylation of its N-terminal glycine, followed by the dynamically regulated post-translational palmitoylation of the Cys15 and Cys26 residues. Myristoylation and palmitoylation comprise three acyl anchors that attach eNOS to the membrane. N-myristoylation of eNOS is catalyzed by an N-myristoyltransferase that recognizes a specific N-terminal consensus sequence. By contrast, no consensus sequence has been identified for thiopalmitoylation, and a family of palmitoyltransferases with varying substrate specificities and patterns of expression contribute to the process. In particular, palmitoyltransferase protein DHHC-21 may be implicated in the palmitoylation of eNOS. DHHC-21 co-localizes with the enzyme, but its specificity for eNOS palmitoylation has not been established. Depalmitoylation of eNOS is catalyzed by acyl protein thioesterase-1 (APT-1), an enzyme that also depalmitoylates the G protein Gαs. By contrast to the irreversible fatty acyl amide that links myristate to eNOS, the thiopalmitoyl bonds linking palmitate to eNOS are labile. Prolonged agonist stimulation of eNOS leads to depalmitoylation and intracellular translocation, possible serving as a mechanism for modulating eNOS signaling responses [12, 19, 64].
Intracellular calcium: calmodulin binding
The activation of eNOS is critically dependent on the binding of the ubiquitous intracellular calcium regulatory protein calmodulin. Calmodulin binding to eNOS facilitates transfer of electrons between the enzyme’s reductase and oxygenase domains, and simultaneously disrupts the inhibitory caveolin–eNOS interaction. A diverse group of agonists, including bradykinin, sphingosine 1-phosphate, and acetylcholine, activate a G-protein-dependent signaling pathway that ultimately releases intracellular calcium stores. Receptor-dependent activation of phospholipase C cleaves the cell membrane component phosphatidylinositol 4, 5-triphosphate into diacylglycerol (an activator of protein kinase C) and inositol 1, 4, 5-triphosphate (IP3), which binds to IP3 receptors, regulators of the intracellular calcium concentration densely located in caveolae .
Phosphorylation: protein kinases and phosphoprotein phosphatases
Phosphorylation and dephosphorylation networks complement acylation and calmodulin as major post-translational regulatory influences on eNOS activity [12, 64]. Key serine and threonine residues in eNOS constitute regulatory loci: phosphorylations at Ser 1177 (primary sequence numbering corresponding to the more extensively characterized bovine eNOS), Ser 635, and Ser 617 promote increases in enzyme activity, while phosphorylation at Ser 116, Thr 497, and Tyr659 are inhibitory. Ser 1179 phosphorylation is catalyzed by numerous kinases, including kinase Akt (protein kinase B) as well as the cyclic AMP-dependent protein kinase (PKA), AMP-activated protein kinase (AMPK), cyclic GMP-dependent protein kinase, and calcium/calmodulin-dependent protein kinase II (CaM kinase II). The relative contributions of these different kinase pathways are not completely understood, but it is already obvious that different extracellular stimuli activate distinct kinase pathways leading to eNOS phosphorylation. For example, PKA appears to be the principal kinase that phosphorylates eNOS at ser 1179 in response to hemodynamic shear stress , while S1P- and vascular endothelial growth factor (VEGF)-dependent phosphorylation of this same serine residue involves kinase Akt. An additional level of complexity is revealed by the fact that beta3 adrenergic receptor signaling to eNOS in endothelial cells involves both PKA and Akt. Another critical kinase is the AMP-activated protein kinase, which has broad effects on endothelial metabolism and signaling . AMPK can catalyze eNOS phosphorylation at ser 1177, yet AMPK is also the principal kinase that phosphorylates eNOS at ser 635 . Indeed, AMPK plays a key role in multiple phosphorylation pathways involving eNOS modulation, and AMPK is in turn regulated by upstream serine/threonine kinases including the calcium–calmodulin kinase kinase-beta . In addition, tyrosine phosphorylation of eNOS Tyr 83 mediated by v-Src has been identified as another candidate eNOS post-translational modification . However, the extracellular signals that modulate phosphorylation at this site remain incompletely understood. In particular, it is unclear whether Tyr 83 phosphorylation is responsible principally for activating eNOS catalytic activity, or whether this site modulates signaling by serving as a binding site for proteins with Src homology (SH3) domains. Phosphorylation of tyrosine 659 appears to attenuate eNOS activity; modification of this residue may function to modulate eNOS activity under conditions of oxidative stress [21, 22].
The relative contribution of protein phosphatases versus phosphoprotein kinases in eNOS regulation also remains uncertain. Thus, depending on the specific phosphorylation site, dephosphorylation of eNOS can either activate or inhibit enzyme activity. Several phosphatases, including serine–threonine protein phosphatase 1, serine–threonine protein phosphatase 2A, and calcineurin participate in eNOS regulation. A pathway involving the phosphatase calcineurin leads to dephosphorylation of Ser 116 and to the activation of eNOS. The immunosuppressive drug cyclosporine inhibits calcineurin, preventing VEGF-induced Ser 116 dephosphorylation and thus offering a potential mechanism to explain the mechanism of cyclosporine-induced hypertension [13, 23, 31].
Reversible S-nitrosylation  provides an additional level of in vivo dynamic receptor-mediated post-translational control of the eNOS enzyme in endothelial cells. Indeed, eNOS appears to be the source of the NO required for its own S-nitrosylation, implying a spatial mechanism of specificity in nitrosylation of Cys 94 and Cys 99 given the approximately 30 cysteine residues in eNOS. S-nitrosylation of eNOS leads to enzyme inhibition, whereas denitrosylation is associated with an increased enzyme activity . Treatment of cultured endothelial cells or intact blood vessels with eNOS agonists promotes rapid and reversible denitrosylation of eNOS, temporally associated with enzyme activation. The return to basal eNOS enzyme activity following agonist treatment is associated with the re-nitrosylation of eNOS. These findings imply some component of temporal selectivity in nitrosylation reactions given that eNOS is denitrosylated during the period of maximal NO production. Like phosphorylation and acylation, subcellular localization affects eNOS S-nitrosylation and may help generate the apparent temporal selectivity. Membrane targeting is required for S-nitrosylation, as shown by experiments in which eNOS S-nitrosylation is virtually abolished in the myristoylation-deficient mutant (Myr¯) recombinant eNOS  as compared to the hyper-nitrosylation of the membrane-tethered fusion protein CD8-Myr¯ eNOS. The subcellular dependence of nitrosylation may reflect distinct chemical environments that favor or disfavor nitrosylation .
Protein–protein interactions in eNOS regulation
Caveolins are ~22 kDa intrinsic membrane proteins [32, 64, 65]. Caveolin-1 and caveolin-2 are ubiquitously expressed and abundant in endothelial cells, while caveolin-3 is a muscle-specific isoform expressed in cardiomyocytes and skeletal muscle. Robust protein–protein interactions lead to the binding of caveolar-localized eNOS with caveolin-1 , although this protein–protein interaction is apparently not necessary for the localization of eNOS to caveolae . Caveolin tonically inhibits eNOS in quiescent cells both by impeding the signaling of caveolae-targeted receptors that transduce eNOS-stimulatory signals as well as by inhibiting calmodulin binding to eNOS. Diseases in which caveolins have been implicated include atherosclerosis, hypertension, cardiomyopathy, diabetes, and oncogenesis.
Heat shock proteins
Heat shock protein 90 (hsp90), a chaperone involved in protein trafficking and folding, modulates agonist-dependent eNOS activation . Hsp90 binding stimulates eNOS activity, and also affects eNOS specific activity by binding to kinase Akt. This interaction may be dynamically regulated by hsp90 S-nitrosylation  and by statins. Among other effects on eNOS signaling pathways, statins appear to stimulate eNOS phosphorylation by Akt; one Akt-dependent effect of statins involves tyrosine phosphorylation of hsp90, which in turn facilitates hsp90 binding and activation of Akt.
Dynamic structural changes in the actin cytoskeleton impact on eNOS via links to caveolar membrane domains and caveolar-membrane-associated proteins [see reviews in 65, 66, 69]. For example, shear stress may transduce its effect on eNOS via actin-based cellular architecture. Furthermore, caveolin and caveolae may use the actin network to reversibly translocate between plasmalemma and the intracellular vesicles. Connections between plasmalemmal caveolae and the cytoskeleton have been identified in many cellular systems, and may play a key role in the involvement of the cytoskeleton-associated GTPase Rac1 in eNOS activation. Modulation of Rac1 and other members of the Rho GTPase family by statins may represent another important pathway for the control of eNOS signaling pathways by these therapeutic agents.
NOSIP and NOSTRIN
Two eNOS-associated proteins identified using yeast two-hybrid screening include the eNOS Interacting Protein NOSIP , and the eNOS TRafficking INducer protein NOSTRIN [12, 37]. NOSIP is a 34-kDa protein that binds the carboxy-terminal of the eNOS oxygenase domain and assists in the translocation of eNOS from the plasma membrane caveolae to intracellular membranes . Association of eNOS and NOSIP is inhibited by caveolin-1. NOSTRIN is robustly expressed in the endothelium. Overexpression of NOSTRIN can promote the translocation of eNOS from the plasma membrane to intracellular vesicles, with a concomitant reduction in eNOS enzyme activity. NOSTRIN-dependent shuttling of eNOS is caveolin dependent and likely reflects specialized endocytosis of caveolar endosomes involving the actin cytoskeleton.
Modulation of the protective role of nitric oxide
Both acute and chronic increases in flow, and the resulting increasing force of shearing (shear stress) of the blood on the endothelial cells, augment the expression and the activity of eNOS, and thus the release of EDRF/NO (Fig. 2) [2, 5, 9, 53, 58, 60]. The immediate effect of an increase in shear stress on the release of NO explains flow-mediated dilatation, a phenomenon commonly used to estimate endothelial function in people. In the porcine and human coronary circulation, flow-dependent vasodilatation involves the local production of bradykinin that stimulates the release of NO through a Gq-dependent mechanism (Fig. 2) . The chronic effect of shear stress is due to an up-regulation of eNOS and a greater activation (phosphorylation) of the enzyme, leading to a larger release of NO for each given stimulation, explaining the beneficial effects of regular exercise on endothelial function .
Estrogens and gender
The reintroduction of physiological levels of estrogens in ovariectomized animals augments endothelium-dependent relaxations . The potentiating effect of estrogens on endothelium-dependent relaxations involves both genomic (Fig. 2) and non-genomic effects [52, 76]. Phytoestrogens and selective estrogen receptor modulators also potentiate endothelium-dependent relaxations/vasodilatations . In coronary arteries, the potentiating effect of chronic treatment with estrogens is seen only with stimuli that activate Gi-coupled receptors on the endothelial cells and is counteracted by the chronic administration of progesterone . It is likely that this potentiating effect of estrogens on NO release, presumably resulting from lower oxidative stress, helps to explain why endothelium-dependent relaxations are more pronounced in arteries from female than male animals and why pre-menopausal women are protected against coronary disease. The opposing effects of progesterone helps to explain why hormone replacement therapy has not always had the expected beneficial cardiovascular effects .
Sphingolipids and lysophospholipids
A class of serum-borne lipid mediators including sphingosine 1-phosphate and lyosphosphatidic acid activate endothelial G-protein-coupled S1P receptors (formerly called EDG receptors) play key roles in vascular regulation  stimulate AMP-activated protein kinase the small GTP binding protein Rac1, a critical determinant of the activity of eNOS.
The chronic intake of ω3-unsaturated fatty acids potentiates endothelium-dependent relaxations and has antiatherogenic properties. The same holds true for the intake of flavonoids and other polyphenols of vegetal origin .
Oxygen-derived free radicals
Several enzymes in the endothelial cells can produce superoxide anions. They include NADPH oxidase, xanthine oxidase, cyclooxygenase, and eNOS itself, when it is uncoupled by lack of substrate (l-arginine) or shortage of the essential co-factor tetrahydrobiopterin [23, 42]. Superoxide anions can be dismutated by superoxide dismutase to hydrogen peroxide (H2O2) which can act as an EDHF and contribute to endothelium-dependent relaxations [16, 17], or been broken down by catalase. However, superoxide anions also scavenge NO avidly with the resulting formation of peroxynitrite which reduces the bioavailability of NO [7, 42, 57]. Hence, increases in oxidative stress lead to reduced endothelium-dependent relaxations, and antioxidants acutely improve such responses in vitro and in vivo both in animals and humans [46–48]. However, chronic treatment with antioxidants usually fails to improve endothelial function in people .
Both in animals and in humans, increasing age reduces the ability of the endothelium to elicit endothelium-dependent vasodilatations in vitro and in vivo . This has been attributed to increased activity of arginase [competing with eNOS for the common substrate arginine], augmented production of superoxide anions reducing NO bioavailability, reduced expression/presence and/or activity of eNOS and ultimately NO lesser release of .
Both in animals and in humans, high-fat diets reduce endothelium-dependent relaxations/dilatations and the normalization of the cholesterol level with treatment restores endothelial function . The effect of hypercholesterolemia is due to the combination of increased oxidative stress leading to a reduced bioavailability of NO, impairment of the turnover rate of eNOS, and augmented levels of circulating asymmetric dimethyl arginine (ADMA, an endogenous inhibitor of eNOS) . In addition, lipoproteins appear to reduce eNOS targeting to plasmalemmal caveolae as well the interactions of the enzyme with caveolin and other regulatory proteins that influence its activity .
Endothelium-dependent relaxations are reduced in i arteries harvested from hypertensive animals, and responses to endothelium-dependent vasodilators are decreased in hypertensive humans. This blunting, which can be corrected by an appropriate treatment probably reflects the premature aging of the vasculature exposed chronically to the increased arterial blood pressure . In the spontaneously hypertensive rats and in the essential hypertensive human the reduction of endothelium-dependent relaxations/vasodilatations is caused mainly by the concomitant release of endothelium-derived vasoconstrictor prostanoids (endothelium-derived contracting factors) rather than to a reduced release of NO [73, 75, 76].
Insulin resistance and diabetes cause an impairment of arterial endothelium-dependent relaxations in animals and humans, presumably due to the chronic exposure to hyperglycemia [10, 76]. The endothelial dysfunction of diabetes has been attributed to a relative deficiency of reduced tetrahydrobiopterin, associated with increased eNOS “uncoupling” and ROS production [23, 39, 40, 49, 55, 71, 77], increased activity of arginase competing with eNOS for the common substrate, arginine, elevated levels of the endogenous inhibitor ADMA, augmented production of superoxide anions and thus increased formation of peroxynitrite, and quenching of NO by advanced glycosylation products [72, 76].
Individuals at increased risk of coronary heart disease exhibit impaired dilatations in response to endothelium-dependent vasodilators both in the peripheral and coronary circulations. Both in animals and humans, the presence of such endothelial dysfunction predicts the severity of the outcome, in particular the occurrence of myocardial infarction .
In native, healthy endothelial cells a number of stimuli (e.g. serotonin from aggregating platelets, sphingosine 1-phosphate, and thrombin) activate eNOS causing the release of NO, which relaxes the vascular smooth muscle that surrounds them. NO, in synergy with prostacyclin, further inhibits platelet aggregation. It also reduces the endothelial expression of adhesion molecules and thus the adhesion and penetration of leukocytes (macrophages). The endothelial mediator also prevents the proliferation of vascular smooth muscle cells and limits the formation of oxyLDL. Aging and certain lifestyle factors (e.g. lack of exercise, Western diet, smoking), or certain diseases (e.g. diabetes and hypertension) result in a lesser release of NO and an acceleration of the turnover of the apoptotic process in the endothelium. The apoptotic endothelial cells are replaced by regenerated ones. However, such regenerated cells are dysfunctional, senescent, and incapable of producing the required amounts of NO, which facilitates the inflammatory response leading to the formation of atherosclerotic plaques.
The authors thank Mr. Robert R. Lorenz for the expert help in preparing the figures.