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Cardiovascular Research 2001 51(3):429-441; doi:10.1016/S0008-6363(01)00327-3
© 2001 by European Society of Cardiology
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Copyright © 2000, European Society of Cardiology

Molecular biology of natriuretic peptides and nitric oxide synthases

Bruce C Kone*

Departments of Internal Medicine and of Integrative Biology, Pharmacology, and Physiology, Division of Renal Diseases and Hypertension, The University of Texas Medical School at Houston, 6431 Fannin, MSB 4.138, Houston, TX 77030, USA

bruce.c.kone{at}uth.tmc.edu

* Tel.: +1-713-500-6873; fax: +1-713-500-6882

Received 8 January 2001; accepted 20 April 2001


   Abstract
Top
 Abstract
1 Introduction
 2 The natriuretic peptide...
 3 Natriuretic peptide receptors
 4 Regulation of the...
 5 Nitric oxide synthases
 6 NOS gene products
 7 Regulation of NOS...
 8 Conclusions
 References
 
Natriuretic peptides and nitric oxide play important roles in cardiovascular and renal physiology and disease. The natriuretic peptides — atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide — comprise a family of proteins that participate in the integrated control of intravascular volume and arterial blood pressure. The natriuretic peptides differentially bind distinct classes of receptors that signal through different mechanisms. Membrane-bound, guanylyl cyclase-coupled natriuretic peptide receptors (A- and B-types) mediate natriuretic peptide effects through the production of 3',5'-cyclic guanosine monophosphate (cGMP). C-Type natriuretic peptide receptors, which lack the guanylyl cyclase domain, alter target cell function through Gi protein-coupled inhibition of membrane adenylyl cyclase activity, and also serve to clear circulating natriuretic peptides. The expression of the natriuretic peptides and their receptors are subject to complex controls. Similar structural and regulatory diversity exists for the nitric oxide synthases. The three nitric oxide synthase genes are regulated by a variety of mechanisms ranging from alternative splicing and alternative promoter usage to complex post-translational controls. This review highlights the molecular diversity of the natriuretic peptides and nitric oxide synthases and explores recent insights into their regulation.

KEYWORDS Natriuretic peptide; G proteins; Nitric oxide; Endothelial factors; Endothelial function; Signal transduction


   1 Introduction
Top
 Abstract
 1 Introduction
2 The natriuretic peptide...
 3 Natriuretic peptide receptors
 4 Regulation of the...
 5 Nitric oxide synthases
 6 NOS gene products
 7 Regulation of NOS...
 8 Conclusions
 References
 
The discovery of atrial natriuretic peptide in 1981 by de Bold et al. [1] radically expanded the perception of the heart as simply a mechanical pump to that of a neuroendocrine organ. Likewise, the combined discoveries of Ignarro, Murad and Furchgott (cited in Ref. [2]) that a gas, nitric oxide (NO), formed and released by the vascular endothelium, controlled vascular tone, transformed our thinking of the endothelium to its current conception as a vital organ that senses and responds to changes in blood flow. The natriuretic peptides are a group of structurally related but genetically distinct peptides that exert diverse actions on cardiovascular, renal, and endocrine function. The natriuretic peptides have emerged as important contributors to the control of cardiovascular hemodynamics, ventricular remodeling, and sodium and water balance. NO is widely recognized as a critical cell signaling and host defense molecule with diverse actions on cardiovascular, renal, and immune cell function. The natriuretic peptides, their receptors, and the nitric oxide synthases share considerable commonality in that they function in vasodilator, antihypertensive, and guanylate cyclase agonist pathways. As expected from their central and potent roles in physiology (Fig. 1), these gene families have evolved structural and regulatory complexity over time. The present review focuses on the molecular biology of the natriuretic peptide, natriuretic peptide receptor, and nitric oxide synthase genes and gene products and the regulatory controls on their synthesis and actions.


Figure 1
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  		Fig. 1 Natriuretic peptides/nitric oxide synthases.

 

   2 The natriuretic peptide family
Top
 Abstract
 1 Introduction
 2 The natriuretic peptide...
3 Natriuretic peptide receptors
 4 Regulation of the...
 5 Nitric oxide synthases
 6 NOS gene products
 7 Regulation of NOS...
 8 Conclusions
 References
 
The natriuretic peptide family comprises four principal peptides whose prohormones are encoded by at least three separate genes (Table 1). This family includes atrial natriuretic peptide (ANP), B-type natriuretic peptide (brain natriuretic peptide, BNP), C-type natriuretic peptide (CNP), and urodilatin. Each natriuretic peptide family member shares a 17-amino acid disulfide ring structure with a highly conserved sequence (FGXXXDRIGXXSGL). ANP and BNP are primarily synthesized in the cardiac atria and ventricles, respectively, circulate in the bloodstream, and directly modulate blood pressure and body fluid and electrolyte homeostasis. In contrast, CNP is abundantly expressed in brain, but vascular endothelium and other tissues also produce it. CNP circulates at very low levels and lacks potent natriuretic actions, but it possesses vasodilating and growth-inhibiting actions and may function in paracrine or autocrine roles. In distal tubules of the kidney, alternative cleavage of pro-ANP yields a 32-amino acid carboxy-terminal peptide, termed urodilatin [3]. Urodilatin released from these cells binds to luminal receptors in the collecting duct, resulting in cGMP-dependent suppression of sodium reabsorption. More recently, a fifth putative member of the natriuretic peptide family, related to Dendroaspis natriuretic peptide (DNP), has been reported to be expressed in human plasma and atrial myocardium [4], to exert natriuretic actions [5], to increase urinary and plasma cGMP, and to dilate coronary arteries [6]. The 38-amino acid DNP peptide was originally isolated from the venom of the green Mamba snake, Dendroaspis angusticeps, and it contains the conserved disulfide ring structure [7]. The cDNA and gene structure of the human ortholog of DNP remains to be elucidated.


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  		Table 1 Natriuretic peptides

 
ANP was originally identified in atrial myocardial extracts as a substance promoting natriuresis [1]. The isolation and cloning of human ANP soon followed. ANP is principally produced in the atria of normal adults, but it is also expressed in the ventricles during development [8,9] and in hypertrophied ventricles [10]. ANP infusion in normal man promotes natriuresis, diuresis, and increases glomerular filtration rate [11]. ANP has also been shown to decrease plasma renin activity, plasma aldosterone, and plasma endothelin release in normal humans and in heart failure patients [12,13]. The ANP precursor peptide (NPPA) and BNP precursor peptide (NPPB) genes reside in tandem on human chromosome 1p36.2. The NPPA gene comprises three exons and two introns. Its mRNA encodes a precursor peptide, pro-ANP, of 126 amino acids, which is proteolytically cleaved to a 98-amino acid amino-terminal fragment and a 28-amino acid carboxy-terminal fragment [14]. The carboxy-terminal fragment represents mature, biologically active ANP [14]. Recent studies indicate that corin, a cardiac serine protease, cleaves pro-ANP to ANP in a highly sequence-specific manner and may represent a (the) pro-ANP-converting enzyme [15]. Mice with targeted disruption in the Nppa gene develop salt-sensitive hypertension [16].

BNP was first purified from porcine brain [17]. Using the porcine cDNA as a screening probe, Seilhamer et al. isolated and sequenced the BNP cDNA [18]. Its gene consists of two exons and an intron and localizes approximately 8 kb upstream of the NPPA gene on human chromosome 1p36. The NPPB gene encodes a 108-amino acid pro-BNP, which is processed to a 32-amino acid mature peptide. Although expressed in brain, BNP is most abundant in the cardiac ventricles. Its expression is induced in this tissue in decompensated heart failure [19]. BNP exerts many of the same actions as ANP on target organs. Mice with targeted disruption of BNP develop multifocal fibrotic lesions in the cardiac ventricles in the absence of systemic hypertension or ventricular hypertrophy. Accordingly BNP is viewed as a cardiomyocyte-derived antifibrotic factor in vivo that may function as a local regulator of ventricular remodeling [20].

CNP was also originally isolated in porcine brain [21], but immunoreactivity for this peptide has since been found in human vascular endothelial cells, kidney, reproductive organs, and other tissues. Several studies have demonstrated that CNP serves as a potent inhibitor of the proliferation of smooth muscle cells [22]. CNP displays a positive lusitropic effect associated with a negative inotropic effect in rat papillary muscles [23]. CNP infusion in normal man caused dose-dependent increases in natriuresis, with less potency than ANP, and no effect on blood pressure, diuresis, renal plasma flow, or glomerular filtration rate [11]. In another study, CNP injection caused a transient but significant decrease in blood pressure, significant diuretic and natriuretic activities (but weaker than those of ANP), and significantly suppressed aldosterone secretion [24]. The gene encoding C-type natriuretic peptide (NPPC) is localized to human chromosome 2 [25] and contains two exons separated by an intron. The NPPC gene encodes a 126-residue CNP precursor peptide that is processed to generate 22- and 53-amino acid peptides (human CNP-22 and human CNP-53, respectively) [26]. CNP-53 represents an amino-terminal extension of the CNP-22 peptide. CNP-22 is more widely and abundantly expressed and is more potent that the C-53 peptide.


   3 Natriuretic peptide receptors
Top
 Abstract
 1 Introduction
 2 The natriuretic peptide...
 3 Natriuretic peptide receptors
4 Regulation of the...
 5 Nitric oxide synthases
 6 NOS gene products
 7 Regulation of NOS...
 8 Conclusions
 References
 
The natriuretic peptides signal through a group of cell surface natriuretic peptide receptors (NPRs, Table 2). Each of the three receptors contains a single transmembrane domain and an extracellular binding domain. NPR1 (also termed NPR-A) and NPR2 (also termed NPR-B) include cytoplasmic tails that contain guanylate cyclase activity and kinase homology regions. NPR3 (also termed NPR-C) [27] is homologous in its extracellular domain to NPR1 and NPR2, but contains a 37-amino acid cytoplasmic domain that contains a G protein-activating sequence; moreover this receptor is devoid of kinase and guanylyl cyclase activities [28]. Studies in knockout mice have confirmed that NPR3 serves as a ‘clearance’ receptor [29], clearing natriuretic peptides to effect local regulation. Several laboratories have provided evidence that the NPR3 receptor also mediates signal transduction either through inhibition of adenylyl cyclase via G{alpha}i–2 [30] or activation of phospholipase C via the β{gamma}-subunits of Gi-1 and Gi-2 [31].


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  		Table 2 Natriuretic peptide receptors

 
NPR1 responds to ANP and, to a 10-fold lesser degree, BNP. The human NPR1 gene spans approximately 16 kb on human chromosome 1q21–q22. It contains 22 exons that encode a 1061-amino acid peptide. The sequence encoding the single transmembrane domain resides in exon 7. The NPR1 gene product is abundantly expressed in the vasculature, kidneys, and adrenal glands. Its stimulation promotes vasorelaxation and natriuresis, and decreases aldosterone synthesis. Mice lacking a functional NPR1 gene exhibit hypertension and cardiac hypertrophy and dilatation. Male mice additionally exhibit reduced testosterone levels, experience sudden death before 6 months of age [32,33], and are more susceptible to hypoxia-induced pulmonary hypertension [34]. Activation of the NPR1 guanylyl cyclase requires ANP binding to the extracellular domain and phosphorylation at up to six residues (S497, T500, S502, S506, S510, and T513) [35] within its cytoplasmic region kinase homology domain. In addition, recent studies have identified transcriptional mechanisms controlling the expression of NPR1 expressed on the surface of vascular cells. Studies of NPR1 promoter activity demonstrated that it is dominantly regulated through functional, and possibly physical, interactions of NF-Y and Sp1 [36,37].

NPR2 is a 1047-amino acid peptide that responds primarily to CNP. The cDNA and deduced amino acid sequences of human NPR2 have been described [38]. NPR1 and NPR2 share 44% homology in the ligand-binding region of the extracellular domain. The NPR2 gene comprises approximately 16.5 kb on human chromosome 9p21–p12. It is composed of 22 exons. The gene is strongly expressed in the brain, including the pituitary gland, and may have a role in neuroendocrine regulation. Phosphorylation of up to five residues (Ser-513, Thr-516, Ser-518, Ser-523, and Ser-526) within the kinase homology domain appears to be critical for receptor activation [39]. Both NPR1 and NPR2 are extensively phosphorylated in resting cells, and receptor dephosphorylation correlates with ligand-induced homologous desensitization.

NPR3 interacts with all three natriuretic peptides in the order ANP<CNP<BNP [40]. Natriuretic peptides that are bound by the receptor are internalized and degraded. The cDNA encoding human NPR3 was cloned from human placental and kidney cDNA libraries [27]. The NPR3 gene resides on human chromosome 5p14–p13 and encodes a 540-amino acid peptide. The mature NPR3 receptor exists as homodimers linked by disulfide bonds. It is the most widely and abundantly expressed natriuretic peptide receptor, with a tissue distribution that includes most of the major endocrine glands, the lungs, kidney, and the vascular wall. NPR3 null mice exhibit a prolonged half-life of exogenous ANP, mild reductions in blood pressure, increased daily urinary output, and increased basal bone turnover coupled with bony deformities [29].


   4 Regulation of the natriuretic peptide genes
Top
 Abstract
 1 Introduction
 2 The natriuretic peptide...
 3 Natriuretic peptide receptors
 4 Regulation of the...
5 Nitric oxide synthases
 6 NOS gene products
 7 Regulation of NOS...
 8 Conclusions
 References
 
4.1 ANP gene expression
The human ANP promoter contains numerous cis-elements that govern cell specificity of expression and responsiveness to physiological and pharmacological stimuli. Expression of the endogenous gene occurs almost exclusively in myocardial cells. Studies using human ANP promoter–reporter gene constructs have sought to distinguish molecular details of cell-specific expression. Garmai et al. demonstrated repressor activity in the distal (–2593 to –1152) and proximal (–222) promoter that prevents expression in non-myocardial cells. An E-box motif in the upstream repressor appears to be critical for repressor activity [41]. In addition, the cardiac homeobox gene Csx/Nkx-2.5, binding to the Nkx-2.5 response element-2 in the ANP promoter, has been shown to cooperate with GATA-4 in synergistically transactivating the ANP gene [42]. In agreement with these findings, transgenic mice overexpressing Csx/Nkx-2.5 exhibit dramatically enhanced ANP expression in their hearts [43]. Similarly, the transcription factor GATA-5 together with the coactivator p300 transactivates the ANP gene in cardiomyocytes [44].

{alpha}1- and β1-adrenergic agonists are known to upregulate ANP gene expression. Studies of the molecular control of the gene indicate that responsiveness to {alpha}1-adrenergic agonists depends on occupation of the serum response elements at –114 and –406 bp and the Sp1-like sequence at –69 bp [45]. β-Adrenergic receptor-stimulated ANP transcription in cardiac myocytes is predominantly mediated by activation of Akt and subsequent phosphorylation/inhibition of glycogen synthase kinase 3β [46].

Nuclear hormone receptors also regulate ANP gene expression. Glucocorticoids have been shown to upregulate ANP gene expression in target tissues. A glucocorticoid receptor dimer binding to the glucocorticoid responsive element in intron 2 of the ANP gene has been shown to transactivate the ANP gene in vitro. In contrast 1,25-dihydroxyvitamin D3 represses ANP gene transcription [47]. Recruitment of coactivators, including GRIP1, appears to be important for the hormone's suppressive effects. Finally a 120-base pair region within the ANP gene promoter contains hypoxia-responsive elements that might be responsible for the enhancement of ANP gene expression in atrial myocytes during hypoxic exposure [48].

4.2 BNP gene expression
Synthesis and secretion of BNP, like ANP, are increased in patients with congestive heart failure, in the early phase of acute myocardial infarction [49], and in animal models of ventricular hypertrophy. Sustained increases in plasma BNP concentrations are also correlated with enlargement, decreased contractility, and reduced compliance of the left ventricle in patients with congestive heart failure and myocardial infarction [50,51]. The molecular regulation of BNP differs from that of ANP. The regulatory regions in the proximal promoters of the human BNP and human ANP genes share only 50% sequence similarity, although both contain AP-1 binding sites, serum response elements (CArG/SRE), and GATA sites. The full-length human BNP promoter exerts greater activity in ventricular than in atrial myocytes, and it is inactive in fibroblasts. The distal region of the BNP promoter contains both positive and negative regulatory elements, with the region –127 to –40 conferring tissue specificity [52].

A variety of stimuli have been shown to activate the BNP gene promoter in cardiac myocytes in vitro, including mechanical strain and endothelin. The response to mechanical strain depends on p38 MAPK activation and interaction of the transcription factor nuclear factor NF-{kappa}B with three shear stress response elements (SSRE)-like structures in the proximal human BNP promoter [53]. β-Adrenergic regulation of human BNP involves a G{alpha}i-activated pathway, Src, and Rac, targeting an M-CAT-like element in the proximal human BNP promoter [54]. IL-1β has also been shown to be a transcriptional activator of the human BNP promoter. Recent studies suggest that IL1-β acts through multiple signaling pathways, including Ras, Rac, and p38 MAP kinase [55]. Cell-matrix interactions also appear to be important for controlling BNP gene expression in vitro. Liang et al. [56] demonstrated that dominant-negative inhibitors of integrin function suppressed strain-dependent BNP promoter activity in neonatal rat ventricular myocytes. The BNP gene is one of several cardiac-specific genes (but alone among the natriuretic peptide genes [57]) suppressed by the antineoplastic agent doxorubicin [58], which is known for its myocardial toxicity.

4.3 CNP gene expression
CNP gene expression in cultured endothelial cells is enhanced by transforming growth factor-β [59], IL-1{alpha}, IL-1β, tumor necrosis factor-{alpha}, and lipopolysaccharide [60]. In contrast, oxidized-LDL [61], insulin [62], and vascular endothelial growth factor [63] inhibit endothelial CNP production. Studies in transgenic mice indicate that cis-acting regulatory elements localized in the proximal 4-kb 5'-flanking sequence of the mouse CNP gene are necessary and sufficient to direct spatial and temporal expression of the transgene in oligodendrocytes [64]. The human CNP gene contains a GC-rich element, to which a novel transcription factor TSF1 binds, that is required for both constitutive and TGF-β responsive [65] transcription of the gene.


   5 Nitric oxide synthases
Top
 Abstract
 1 Introduction
 2 The natriuretic peptide...
 3 Natriuretic peptide receptors
 4 Regulation of the...
 5 Nitric oxide synthases
6 NOS gene products
 7 Regulation of NOS...
 8 Conclusions
 References
 
The NOS multigene family comprises three principal members (Table 3): neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). A mitochondrial NOS has also been reported [66,67], but its structure and regulation are not defined. The three major enzymes are subject to expressional control. nNOS and eNOS are inactive until intracellular Ca2+ levels increase sufficiently to maintain calmodulin binding. In contrast iNOS tightly binds calmodulin at basal intracellular Ca2+ concentrations, and thus has been viewed as a Ca2+/calmodulin-independent enzyme. The genes and cDNAs encoding the three NOS isoforms have been structurally characterized. Mice bearing targeted deletions of each of the three major NOS isoforms, and an nNOS/eNOS ‘double knockout,’ have been established and have begun to be physiologically characterized. nNOS null mice exhibit hypertrophy of the pyloric sphincter, increased aggressive behavior in males, bladder–urethral sphincter dysfunction, and relative protection from ischemic neurological events compared to wild-type mice [68]. The principal phenotype of iNOS knockout mice is an increased susceptibility to infection with intracellular pathogens and less susceptibility to sepsis-induced hypotension [69]. The mice also exhibit impaired prostaglandin E2 production [70]. Targeted disruption of the iNOS gene completely abrogates the infarct-sparing effect of the late phase of ischemic preconditioning [71], attenuates neointima formation after perivascular arterial injury [72], and protects kidneys against ischemic acute renal failure [73]. Studies in apoE-deficient mice bred with wild-type and iNOS –/– mice indicate that iNOS contributes to the size of atherosclerotic lesions in apoE-deficient mice, perhaps through a direct effect at the site of the lesion [74]. eNOS knockout mice are hypertensive [75], more vulnerable to cerebral and myocardial ischemia than wild-type mice, and exhibit a higher incidence of bicuspid aortic valves [76]. The mice also exhibit pulmonary hypertension with chronic, mild hypoxia [77], impaired wound repair, deficient growth factor-induced angiogenesis [78], and markedly decreased bleeding times [79]. Interestingly, ANP expression is markedly upregulated in the eNOS –/– mice [80].


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  		Table 3 Nitric oxide synthases

 

   6 NOS gene products
Top
 Abstract
 1 Introduction
 2 The natriuretic peptide...
 3 Natriuretic peptide receptors
 4 Regulation of the...
 5 Nitric oxide synthases
 6 NOS gene products
7 Regulation of NOS...
 8 Conclusions
 References
 
6.1 nNOS
The human nNOS gene spans over 200 kb on chromosome 12q24.2–q24.31. The major neuronal transcript comprises 29 exons and encodes a 160-kDa protein. Considerable structural and allelic diversity of nNOS transcripts arises from the use of alternative promoters and by the deletion or insertion of cassette exons via alternative mRNA splicing. These gene products are principally expressed in neurons, skeletal and vascular smooth muscle, the macula densa segment, and bronchial and tracheal epithelium. Two major transcriptional clusters — neuronal- and testis-specific — reside within the human nNOS gene. The neuronal cluster resides upstream of exon 2, in which the translation initiation codon resides. Nine alternative first exons, each expressed from a unique 5'-flanking region, are spliced to a common second exon [81], giving rise to the same protein with different 5'-untranslated regions (UTR). The highly structured nNOS 5'-UTRs contain cis RNA elements that modulate translational efficiency in vitro and in vivo [81]. The testis-specific transcription cluster exists within intron 3 and gives rise to an mRNA transcript comprised of two new 5' exons spliced to exon 4 of nNOS. This testis-specific transcript encodes a smaller, 125-kDa protein with comparable catalytic activity to that of full-length nNOS. Transcripts bearing deletions of exons 9/10, exon 10, exon 2 and exon 2/3 have been detected in human nNOS transcripts [82,83], but it is unknown whether these mRNAs generate functional proteins in vivo. The exon 2 deletion results in the loss of the translation initiation site located within exon 2, and the apparent use of an atypical alternative start codon within exon 3 (UUG). Several cassette exon insertions have also been reported. A 102-bp insertion, encoding 34 amino acids, between exons 16 and 17 (termed -µ) was identified in rat [84,85]. This variant is principally expressed in muscle complexed with dystrophin, and it is coexpressed with nNOS in the pelvic plexus and bladder [84,85]. A human variant of -µ arising from insertion of an additional exon transcribed from intron 16 of the human nNOS gene has since been described [86].

6.2 iNOS
The human iNOS gene residing on chromosome 17cen–q11.2 contains 26 exons. Multiple transcription initiation sites and alternative splicing give rise to several forms of exon 1. In addition, cassette deletions give rise to four distinct alternative splice variants of human iNOS mRNA. These include deletions of exon 5 (resulting in a truncated protein), exons 8 and 9 (encoding amino acids 242–235), exons 9, 10, and 11 (encoding amino acids 289–427), and exons 15 and 16 (encoding amino acids 604–678, including the FMN binding site) [87]. The iNOS lacking exons 8 and 9 exhibits a functional reductase domain but fails to dimerize and to produce NO when heterologously expressed in cultured cells [88]. The translation and functions of these variants in vivo remain to be established.

6.3 eNOS
The eNOS gene is expressed in the endothelium of a variety of tissues, as well as in cardiac and myometrial myocytes, platelets, and in airway epithelium. The human eNOS gene on chromosome 7q36 contains 26 exons spanning approximately 21 kb of genomic DNA and encodes an mRNA of 4052 nucleotides. No alternative splice variants for this isoform have yet been characterized. Several allelic variants of the eNOS gene have been identified and their association with human disease states studied. Several of these genetic polymorphisms have been reported as ‘susceptibility genes’ in various cardiovascular and pulmonary diseases. Yoshimura et al. [89] discovered a GT substitution in exon 7 (at position 894) in codon 298 of the human eNOS gene, which alters the amino acid at this residue from glutamate to aspartate. In vitro studies have shown that this mutation results in susceptibility to cleavage of the full-length protein into N-terminal 35-kDa and C-terminal 100-kDa fragments [90]. The Glu298Asp variant has been correlated with increased coronary spasm, myocardial infarction and essential hypertension in various populations. In the Cambridge Heart Antioxidant Study (CHAOS), Asp298 homozygosity correlated strongly with coronary heart disease [91]. In separate studies of elderly patients in Australia, no difference in the distribution of the E298D alleles in relation to the presence of coronary artery disease was observed [92]. Similarly, in a study of over 750 white Australians undergoing coronary angiography, the mutation was unassociated with coronary artery disease, myocardial infarction, or with the number of significantly stenosed arteries [93]. Finally, the frequency of the Glu298Asp variant appears to be greater in women with severe preeclampsia [94].

A T786C mutation in the 5'-flanking region of eNOS gene, which reduces eNOS promoter activity in vitro, was associated with coronary spasm in a Japanese population [95,96]. Replication protein A1 (RPA1), a single-stranded DNA binding protein, appears to bind to and repress the mutated promoter sequence [97]. High numbers of CA repeats in intron 13 of the human eNOS gene are also associated with an excess risk of coronary artery disease [98], and variable repeats in intron 4 (intron 4b/a polymorphism) have been reported to be involved in smoking-dependent coronary artery disease [95,99].


   7 Regulation of NOS isoforms
Top
 Abstract
 1 Introduction
 2 The natriuretic peptide...
 3 Natriuretic peptide receptors
 4 Regulation of the...
 5 Nitric oxide synthases
 6 NOS gene products
 7 Regulation of NOS...
8 Conclusions
 References
 
7.1 nNOS regulation
Originally, nNOS was thought to be a constitutively expressed enzyme, but it is now known that nNOS is induced by a variety of physiologic and pathological stimuli. The neuronal transcriptional cluster of the human nNOS gene contains potential binding sites for a number of transcription factors, including AP-2, NF-{kappa}B, CREB, and Ets [82]. Studies of the promoter activity of this region in neuronal and fibroblast cell lines revealed an important role for Oct-2 in transactivating the downstream (designated ‘5.1’) promoter [100]. The upstream promoter (designated ‘5'2’) confers inducibility to nerve growth factor [101]. Most recently, two CREB sites within the 5'-UTR of mouse nNOS exon 2 have been shown to be functionally active in cortical neurons [102]. The corollary regulatory region in the human nNOS gene has not been established. The human testis transcriptional cluster also contains potential cis-regulatory elements [103], but no functional characterization of this region has yet been published.

Several serine/threonine kinases have been shown to phosphorylate nNOS. Phosphorylation of purified nNOS by protein kinases A, G, or C, or Ca2+/calmodulin-dependent protein kinase reduces catalytic activity, whereas calcineurin-mediated dephosphorylation enhances catalytic activity [104]. AMP-activated protein kinase increases nNOS phosphorylation in human skeletal muscle, but its effects on nNOS activity are unknown [105]. Further studies are needed to determine the role and regulation of nNOS phosphorylation in intact cells.

Protein–protein interactions help to regulate the spatial distribution and activity of nNOS in various cell types. Calmodulin serves as an allosteric activator for each of the NOS isozymes. The N-terminus of nNOS contains a PDZ domain that associates with {alpha}1-syntrophin [106], post-synaptic density proteins PSD-93 and PSD-95 [107], and the muscle isoform of phosphofructokinase (PFK-M). An additional protein, CAPON (for carboxy-terminal PDZ ligand of nNOS), binds nNOS in neurons and may compete with PDZ-95 for nNOS association in brain [108]. nNOS also interacts with caveolin-3 in skeletal muscle, where it appears to comprise a component of the dystrophin complex [109,110]. Synthetic peptides corresponding to regions of caveolins 1 and -3 inhibited the catalytic activity of recombinant nNOS [109]. An 89-amino acid protein termed PIN (for ‘protein inhibitor of’) binds to the N-terminus of nNOS and inhibits its activity [111,112]. The molecular chaperone Hsp90 has been shown to complex with nNOS and to activate NO production [113]. Inhibition of Hsp90 by geldanamycin causes the loss of protein in cells. Ubiquitination and proteasomal degradation appears to be critical in removing non-functional monomers in vivo [114].

7.2 iNOS regulation
A variety of transcriptional and post-transcriptional mechanisms regulate iNOS expression and activity, including changes in iNOS gene transcription, mRNA stability, translation and degradation, substrate and cofactor binding and availability, and dimerization. The murine iNOS promoter has been most extensively studied. Structure-function studies of the murine iNOS promoter have demonstrated that the region –48 to –209 serves as a core promoter module [115,116]. Several response elements have been shown to be functionally active, including {kappa}B sites [117], a binding site for ESE-1 (a novel member of the ETS transcription factor family) [118], a hypoxia-responsive enhancer element [119], a novel LPS response element to which an Oct-1-like protein binds [120], a C/EBP box [121], an IFN regulatory factor-1 (IRF-1) site [122], two sequential IFN-stimulated response elements, and an IFN-{gamma}-activated site (GAS) [123]. SAPK/JNK and p38{alpha} MAP kinase signaling cascades are necessary for the IL-1β-induced expression of iNOS and production of NO in renal mesangial cells [124]. In contrast to these stimulatory mechanisms, peroxisome proliferator-activated receptor-{gamma} inhibition of the LPS and IFN-{gamma} induction of the iNOS promoter is achieved partially through antagonizing the activities of NF-{kappa}B, AP-1, and STAT1 [125]. Transcriptional regulation of the human iNOS promoter/enhancer differs markedly from its rodent counterparts. In contrast to murine iNOS, cytokine-enhancer elements in the human iNOS promoter/enhancer localize to sites far upstream of the promoter module. An NF-{kappa}B motif at –5.8 kb was required for cytokine-induced promoter activity, while three neighboring NF-{kappa}B sites elicited a synergistic effect. p38- and ERK-dependent pathways, acting through AP-1 elements in the human iNOS promoter, also confer inducibility to cytokines and LPS/IFN-{gamma} [126].

As with nNOS, interactions with heterologous proteins appear to regulate iNOS activity. Murine macrophages express a 110-kDa protein that interacts with the amino terminus of iNOS, termed NOS-associated protein-110 kDa (NAP110). NAP110 was shown to inhibit iNOS catalytic activity by preventing dimerization [127]. Kalirin associates with iNOS in vitro and in vivo and inhibits iNOS activity by preventing the formation of iNOS homodimers [128]. iNOS may also complex with caveolin 3 in skeletal muscle [129].

Changes in mRNA stability of the iNOS gene have also been implicated in the control of iNOS gene expression. The 3'-UTR of the human iNOS mRNA contains four AUUUA motifs and one AUUUUA motif. The embryonic lethal abnormal vision (ELAV)-like protein HuR was found to bind with high affinity to the AU-rich elements of the iNOS 3'-UTR and to stabilize this mRNA [130]. In other examples, IL-1β stimulates iNOS expression in pancreatic β-cells in part by stabilizing iNOS mRNA through a PKC{delta}-dependent mechanism [131]. β-Adrenergic stimulation enhances IL-1β induction of iNOS in cardiac fibroblasts by stabilizing the iNOS message [132]. Tetrahydrobiopterin, an allosteric cofactor of iNOS, stabilizes the transcript in rat vascular smooth muscle cells [133]. In contrast, increased Ca2+ concentrations reduced iNOS mRNA half-life in human articular chondrocytes [134]. Interestingly ANP has potent autocrine effects on NO production in macrophages. ANP specifically induced acceleration of iNOS mRNA decay and reduced binding activity of NF-{kappa}B, abrogating LPS induction of the iNOS gene [135]. Tyrosine kinases and phosphatases appear to be involved in post-translational modification of iNOS as well, and may potentially play a role in modulating the functional activity of the enzyme [136,137].

7.3 eNOS regulation
The eNOS gene on human chromosome 7q36 spans ~21 kb and contains 26 exons. The ‘TATA-less’ promoter/enhancer region includes a CCAAT box, several half-palindrome sequences for estrogen response elements, a shear stress response element, and potential binding sites for Sp1, AP-1, cAMP response element (CRE), GATA, nuclear factor 1 (NF-1), {gamma}-interferon response element ({gamma}-IRE), and NF-{kappa}B. Mutational analysis of the eNOS promoter/enhancer has established that an upstream Sp1 binding site, a GATA site at position –230, and the PEA3 site at –26 are essential for basal promoter activity in transfected endothelial cells [138,139]. In another study, two positive regulatory domains at –104/–95 and –144/–115, to which Ets family members, Sp1, variants of Sp3, MAZ, and YY1 bound, were important for human eNOS promoter activity in endothelial cells [140]. Sp1 binding to these proximal elements and transactivation of eNOS appears to be regulated in a complex manner that involves post-translational Sp1 phosphorylation and dephosphorylation via casein kinase 2 and protein serine/threonine phosphatase 2A [141]. A third positive regulatory domain in the eNOS promoter was identified 4.9 kb upstream from the transcription start site. Several transcription factors, including MZF-like, AP-2, Sp-1-related and Ets-related factors, were critical for the function of this enhancer in endothelial cells [142]. Finally, studies in mice transgenic for a promoter–reporter construct containing 5.2 kb of the native murine eNOS promoter demonstrated transgene expression in large and medium-sized blood vessels of the heart, lung, kidney, liver, spleen, and brain. The microvasculature, with the exception of the vasa recta of the renal medulla, showed no eNOS transgene expression. Similarly cardiac myocytes, skeletal muscle, and smooth muscle of both vascular and nonvascular sources failed to demonstrate transgene expression [143].

A number of stimuli alter eNOS gene expression. Chronic hypoxia [144] and heart failure [145] are associated with changes in eNOS expression in lung and heart, respectively. Shear stress [146], tumor necrosis factor-{alpha}, lysophosphatidylcholine (LPC), oxidized LDL, PDGF, bFGF, VEGF, TGF-β1, oxidative stress, cyclosporine A, and angiotensin II (reviewed in Ref. [147]) have all been shown to influence the level of eNOS gene product in cultured endothelial cells. LPC is a potent activator of eNOS expression. LPC is a major component of oxidized low density lipoproteins and is abundant in atherosclerotic blood vessels. LPC enhances Sp1-dependent eNOS promoter activity by a pertussis toxin-sensitive, Ras-independent pathway that involves a cascade of PI-3K{gamma}/Jak2, MEK-1, and ERK1/2 [148]. Estradiol has been shown to activate human eNOS promoter activity, in part via activation of the transcription factor Sp1, in cultured endothelial cells [137]. NF-1 binding to a putative TGF-β1 promoter response element is important for TGF-β1 transactivation of the bovine eNOS gene in endothelial cells [149]. Similarly, a PDGF response element between –744 and –1600 of the human eNOS gene appears to be important for transactivation of the eNOS gene in endothelial cells [150]. In contrast to these activating stimuli, glucocorticoids reduced the activity of 3.5-kb human eNOS promoter–reporter gene construct to approximately 70% by decreasing GATA binding activity [151].

Changes in mRNA stability affect eNOS mRNA expression in several settings. The 3'-UTR of eNOS mRNA contains an adenine–uridine (A+U)-rich region with two AUUUA pentamers in the 3' end of 3'-UTR and a C-rich region in the 5' half of the 3'-UTR. Studies in TNF{alpha}-treated endothelial cells demonstrated that a 60-kDa endothelial cytosolic protein was induced and bound to the C-rich region of the 3'-UTR. This was associated with destabilization of eNOS mRNA [152]. In other work, a 51-kDa cytoplasmic protein was found to bind a 43-nucleotide sequence in the eNOS 3'-UTR and alter eNOS mRNA stability during endothelial cell growth [153]. Oxidant stress, induced by hydrogen peroxide, increases eNOS expression through transcriptional activation and stabilization of eNOS mRNA in cultured endothelial cells [154]. Similarly, HMG-CoA reductase inhibitors, by blocking the geranylgeranylation of the GTPase Rho, prolong eNOS mRNA half-life [155].

Acylation and phosphorylation of eNOS regulate both activity and subcellular localization of the enzyme. N-Myristoylation and palmitoylation are required for efficient eNOS targeting to caveolae of the endothelial cell membrane [156,157] and appear to facilitate optimal NO release. At resting intracellular calcium concentrations, eNOS is tonically inhibited through a stable interaction with caveolins [110,158,159]. Agonists, such as bradykinin, increased vascular flow, or fluid shear stress, raise intracellular Ca2+ concentrations and cause calmodulin to bind and caveolin to dissociate from eNOS resulting in an activated eNOS–calmodulin complex. The molecular chaperone Hsp90 appears to facilitate the actions of calmodulin on eNOS dissociation from caveolins [160]. Inhibition of the enzyme also occurs through interactions with membrane-proximal regions of intracellular domain 4 of several G protein-coupled receptors (the bradykinin B2, the angiotensin II AT, and the endothelin-1 ETB receptors). Phosphorylation of serine or tyrosine residues in the eNOS-interacting region of the bradykinin B2 receptor reduces binding affinity of the receptor domain for the eNOS enzyme and reverses the inhibitory effect [161]. Additional complexity in the bradykinin induction of eNOS in endothelium was recently described. Members of the MAP kinase pathway appear to associate reversibly with eNOS, and may influence via phosphorylation events, eNOS activation by bradykinin [162]

In rat heart, eNOS is phosphorylated on Ser1177 and stimulated by an AMP-activated protein kinase both in vitro and during ischemia [163]. The enzyme is also phosphorylated in vitro on Ser633, Ser1177 and activated by cAMP-dependent kinase and cGMP-dependent protein kinase II [164]. The protein kinase Akt (protein kinase B) activates eNOS by phosphorylation of Ser1177 [165,166]. Akt phosphorylation of eNOS is provoked by diverse stimuli, including shear stress [165,166], the statin class of cholesterol lowering agents [167], and estrogen [168]. Estrogen stimulates binding of the estrogen receptor isoform, ER{alpha}, to the p85{alpha} regulatory subunit of phosphatidylinositol-3-OH kinase, activating it. ER{alpha}-associated phosphatidylinositol-3-OH kinase activity activates Akt and, thereby, eNOS [168]. Manipulation of the Akt system in animals, through vascular gene transfer of active and dominant-negative forms of Akt, has confirmed its importance in the control of vasomotor tone [169]. Ceramide [170] and sphingosine-1-phosphate [171] activate eNOS via calcium-independent pathways.


   8 Conclusions
Top
 Abstract
 1 Introduction
 2 The natriuretic peptide...
 3 Natriuretic peptide receptors
 4 Regulation of the...
 5 Nitric oxide synthases
 6 NOS gene products
 7 Regulation of NOS...
 8 Conclusions
References
 
The natriuretic peptide system and nitric oxide synthases are important arbiters of cardiovascular and renal function (Fig. 1). Over the past two decades, a heightened awareness of their importance in physiology, disease, prognosis, and therapeutics has occurred. At the same time, increasing complexity of control mechanisms governing their synthesis and action has been discovered. It is reasonable to project that a growing number of examples of the interplay of the natriuretic peptides and nitric oxide in human health and disease will be established and that these molecules will be increasingly exploited in the diagnosis, risk assessment, and therapy of cardiovascular and cardiorenal diseases.

Time for primary review 34 days.


   Acknowledgements
 
Work in the author's laboratory is supported by grants DK47981, DK50745, and GM20529 from the National Institutes of Health and the Department of Defense ‘DREAMS’ Center.


   References
Top
 Abstract
 1 Introduction
 2 The natriuretic peptide...
 3 Natriuretic peptide receptors
 4 Regulation of the...
 5 Nitric oxide synthases
 6 NOS gene products
 7 Regulation of NOS...
 8 Conclusions
 References
 

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