Báo cáo khoa học: The functional genomics of guanylyl cyclase⁄natriuretic peptide receptor-A: Perspectives and paradigms potx

MINIREVIEW The functional genomics of guanylyl cyclase ⁄ natriuretic peptide receptor-A: Perspectives and paradigms Kailash N. Pandey Department of Physiology, Tulane University Health Sciences Center School of Medicine, New Orleans, LA, USA Introduction The initial work by de Bold et al. [1] established that atrial extracts contained natriuretic and diuretic activities, and demonstrated the existence of atrial natri- uretic factor ⁄ atrial natriuretic peptide (ANP). Members of a family of endogenous peptide hormones including atrial natriuretic factor ⁄ ANP, B-type natriuretic peptide (brain natriuretic peptide) (BNP), C-type natriuretic peptide (CNP) and urodilatin are considered to play an integral role in hypertension and cardiovascular regulation via their ability to mediate excretion of sodium and water, reduce blood volume, and elicit a vasorelaxation effect [2–5]. Interestingly, the natriuretic peptide hormones have been suggested not only to regulate blood pressure but also to play a role in a number of additional processes, namely: antimitogenic effects, inhibition of myocardial hypertrophy, endothe- lial cell function, cartilage growth, immunity, and mitochondrial biogenesis [6–9]. ANP and BNP are also increasingly being utilized to screen and diagnose car- diac etiologies for shortness of breath and congestive heart failure (CHF) in emergency situations [10]. One of the principal loci involved in the regulatory action of ANP and BNP is that encoding the receptor guanylyl cyclase (GC)-A, designated GC-A ⁄ natriuretic peptide receptor-A (NPRA). Interaction of ANP and BNP with GC-A ⁄ NPRA produces the intracellular Keywords cardiac hypertrophy; functional genomics; guanylyl cyclase receptor; hypertension; natriuretic peptides Correspondence K. N. Pandey, Department of Physiology, SL 39, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA 70112, USA Fax: +1 504 9882675 Tel: +1 504 988 1628 E-mail: kpandey@tulane.edu (Received 2 September 2010, revised 7 December 2010, accepted 2 March 2011) doi:10.1111/j.1742-4658.2011.08081.x The cardiac hormones atrial natriuretic peptide and B-type natriuretic pep- tide (brain natriuretic peptide) activate guanylyl cyclase (GC)-A ⁄ natriuretic peptide receptor-A (NPRA) and produce the second messenger cGMP. GC-A ⁄ NPRA is a member of the growing family of GC receptors. The recent biochemical, molecular and genomic studies on GC-A ⁄ NPRA have provided important insights into the regulation and functional activity of this receptor protein, with a particular emphasis on cardiac and renal pro- tective roles in hypertension and cardiovascular disease states. The progress in this field of research has significantly strengthened and advanced our knowledge about the critical roles of Npr1 (coding for GC-A ⁄ NPRA) in the control of fluid volume, blood pressure, cardiac remodeling, and other physiological functions and pathological states. Overall, this review attempts to provide insights and to delineate the current concepts in the field of functional genomics and signaling of GC-A ⁄ NPRA in hypertension and cardiovascular disease states at the molecular level. Abbreviations BNP, B-type natriuretic peptide; CHF, congestive heart failure; CNP, C-type natriuretic peptide; GC, guanylyl cyclase; GCD, guanylyl cyclase catalytic domain; IP 3, inositol trisphosphate; KHD, protein kinase-like homology domain; LVH, left ventricular hypertrophy; MAPK, mitogen- activated protein kinase; NPRA, natriuretic peptide receptor-A; NPRB, natriuretic peptide receptor-B; NPRC, natriuretic peptide receptor-C; PDE, cGMP-dependent phosphodiesterase; PKG, cGMP-dependent protein kinase; RAA, renin–angiotensin–aldosterone; VSMC, vascular smooth muscle cell. 1792 FEBS Journal 278 (2011) 1792–1807 ª 2011 The Author Journal compilation ª 2011 FEBS second messenger cGMP, which plays a central role in the pathophysiology of hypertension and cardiovascu- lar disorders [5,11,12]. Gaining insights into the intrica- cies of ANP–NPRA signaling is of pivotal importance for understanding both receptor biology and the disease state arising from abnormal hormone–receptor interac- tions. It has been postulated that the binding of ANP to the extracellular domain of the receptor causes a conformational change, thereby transmitting the signal to the GC catalytic domain (GCD); however, the exact mechanism of receptor activation remains unknown. Recent studies have focused on elucidating, at the molecular level, the nature and mode of functioning of GC-A ⁄ NPRA. Both cultured cells in vitro and gene- targeted mouse models in vivo have been utilized to gain a better understanding of the normal and abnor- mal control of cellular and physiological processes. Although there has been much appreciation of the functional roles of natriuretic peptides and their cog- nate receptors in renal, cardiovascular, endocrine and skeletal homeostasis; in-depth research studies are still needed to fully understand their potential molecular targets in cardiovascular and other disease states. Ultimately, it is expected that studies on the natriuretic peptides and their receptors should yield new therapeu- tic targets and novel loci for the control and treatment of hypertension and cardiovascular disorders. Natriuretic peptide hormone family ANP is the first described member of the natriuretic peptide hormone family. It is primarily synthesized in the heart atria, and elicits natriuretic, diuretic and vaso- relaxant effects, largely directed to the reduction of fluid volume and blood pressure [2,3,5,7,13,14]. Subse- quently, BNP and CNP, with biochemical and func- tional characteristics similar to those of ANP but derived from separate genes, were identified [15]. BNP was initially isolated from the brain; however, it is pri- marily synthesized in the heart, circulates in the plasma, and displays the most variability in primary structure. CNP is mainly present in endothelial cells, and is highly conserved across species. All three types of natriuretic peptide contain a highly conserved 17-residue disulfide ring, which is essential for the hor- monal activities, but they show differences from each other in the N-terminal and C-terminal flanking sequences (Fig. 1). Although ANP has been considered to exert its predominant effects in lowering blood pressure and blood volume, recent evidence indicates that ANP plays a critical role in preventing cardiac load and overgrowth of the heart in pathological con- ditions. Both ANP and BNP are predominantly synthesized in the heart; ANP levels vary from 50-fold to 100-fold higher than those of BNP. After processing of the 151- residue preprohormone to the 126-residue prohor- mone, the secretion of proANP is believed to occur predominantly in response to atrial distension [14]. Upon secretion, the cleavage of proANP to generate the active and mature 28-residue ANP molecule is cat- alyzed by a serine protease, corin [16]. The synthesis and release of ANP from the heart is enhanced in response to various agents and settings, such as argi- nine–vasopressin, endothelin, and vagal stimuli [14,17]. BNP is synthesized as a 134-residue preprohormone, which yields a 108-residue prohormone. Processing of the proBNP yields a 75-residue N-terminal BNP and a 32-residue biologically active circulating BNP [18,19]. The atria are the primary sites of synthesis for both hormones within the heart. Although the ventricles also produce both ANP and BNP, the concentrations are 100-fold to 1000-fold less than those in the atria. The expression of both ANP and BNP increases dra- matically in both the atria and ventricles in cardiac hypertrophy [20,21]. It is believed that, in the ventri- cles, BNP synthesis is regulated by volume overload, which activates ventricular wall stretch, subsequently enhancing hormone synthesis at the transcriptional level [22,23]. Interestingly, higher levels of ventricular ANP are present in the developing embryo and fetus, with both mRNA and peptide levels of ANP declining rapidly during the prenatal period [24]. CNP is mainly present in the central nervous system [25], vascular endothelial cells [26], and chondrocytes [27]. CNP is synthesized as a 103-residue prohormone, cleaved to a 53-residue peptide by the protease furin, Fig. 1. Comparison of amino acid sequences of the natriuretic pep- tide hormone family. Comparison of amino acid sequences of human ANP, BNP and CNP with conserved amino acids, which are represented by red boxes. The lines between two cysteines in ANP, BNP and CNP indicate a 17-residue disulfide bridge, which seems to be essential for the biological activity of these peptide hormones. K. N. Pandey Update on functional aspects of GC-A ⁄ NPRA FEBS Journal 278 (2011) 1792–1807 ª 2011 The Author Journal compilation ª 2011 FEBS 1793 and subsequently processed to yield the biologically active 22-residue molecule [28]. In addition, a 32-resi- due peptide termed urodilatin, which is identical to the C-terminal sequence of proANP, is known to be pres- ent in urine [29,30]. Urodilatin is not detected in the circulation, and appears to be a unique intrarenal natriuretic peptide with unexplored physiological func- tions [31]. D-type natriuretic peptide is an additional member of the natriuretic peptide hormone family [32]. DNP is present in the venom of the green mamba (Dendroaspis angusticeps) as a 38-residue peptide. GC ⁄ Natriuretic peptide receptor family Natriuretic peptides (ANP, BNP, and CNP) bind and activate specific cognate receptors present on the plasma membranes of a wide variety of target cells. Membrane-bound forms of natriuretic peptide recep- tors have been cloned and sequenced from rat brain [33,34], human placenta [35], and mouse testis [36]. Molecular cloning and expression of cDNAs have identified three different forms of natriuretic peptide receptor, including NPRA, natriuretic peptide recep- tor-B (NPRB), and natriuretic peptide receptor- C (NPRC). These constitute the natriuretic peptide receptor family; however, they show variability in terms of their ligand specificity and signal transduction activity. Two of these receptors contain intrinsic GC activity, and have been designated GC-A ⁄ NPRA and GC-B ⁄ NPRB; they are also referred to as GC-A and GC-B, respectively [37–39]. NPRC lacks the GCD, and has been termed a natriuretic peptide clearance receptor; it contains a short (37-residue) cytoplasmic tail, apparently not coupled to GC activation [40]. Both ANP and BNP selectively stimulate NPRA, whereas CNP primarily activates NPRB, and all three natriuretic peptides indiscriminately bind to NPRC [26,39,41]. NPRA is a 135-kDa transmembrane pro- tein, and ligand binding to the receptor generates the second messenger cGMP. It has been suggested that ANP binding to its receptor in vivo requires chloride, which could exert a chloride-dependent feedback-con- trol effect on receptor function [42]. The general topo- logical structure of NPRA is consistent with that seen in the GC receptor family, containing at least four dis- tinct regions: an extracellular ligand-binding domain, a single transmembrane-spanning region, an intracellular protein kinase-like homology domain (KHD), and a GCD [36,37]. NPRB has an overall domain structure similar to that of NPRA, with binding selectivity for CNP [43]. GC-A ⁄ NPRA is the dominant form of natriuretic peptide receptor found in peripheral organs, and mediates most of the known actions of ANP and BNP. By the use of a homology-based cDNA library screening system, additional members of the GC recep- tor family have also been identified; however, their specific ligand(s) and ⁄ or activator(s) are not yet known (Table 1). The other members of the GC receptor family are GC-C [11], GC-D [44], GC-E [45], GC-F [45], GC-G [46], retinal GC [47], and GC-Y-X1 [48]. Table 1. Ligand specificity, tissue distribution and gene-disrupted phenotypes of particulate GCs ⁄ natriuretic peptide receptors. ROS-GC, rod outer segment GC. Receptor Ligand Tissue distribution Gene knockout phenotype in mice GC-A ⁄ NPRA (Npr1) ANP ⁄ BNP (Nppa ⁄ Nppb) Adrenal glands, brain, heart, liver, lung, olfactory glands, ovary, pituitary gland, placenta, testis, thymus, vascular beds, and other tissues High blood pressure, hypertension, cardiac hypertrophy and fibrosis, inflammation, volume overload, reduced testosterone levels [21,103–105,108,125,126] GC-B ⁄ NPRB (Npr2) CNP (Nppc) Adrenal glands, brain, cartilage, fibroblast, heart, lung, ovary, pituitary gland, placenta, testis, thymus, vascular beds, and other tissues Dwarfism, decreased adiposity, female sterility, seizures, vascular complication [142,143] GC-C Guanylyn, uroguanylyn, enterotoxin Colon, intestine, kidney Resistance to intestinal secretion, diarrhea [11] GC-D Orphan Neuroepithelium, olfactory glands Unknown [44] GC-E Orphan Pineal gland, retina Unknown [45] GC-F Orphan Retina Unknown [45] GC-G Orphan Intestine, kidney, lung, skeletal muscle, and other tissues Unknown [46] ROS-GC Orphan Rod outer segment Unknown [47] Retinal GC Orphan Retina Unknown [47] GC-Y-X1 Orphan Sensory neurons Unknown [48] Update on functional aspects of GC-A ⁄ NPRA K. N. Pandey 1794 FEBS Journal 278 (2011) 1792–1807 ª 2011 The Author Journal compilation ª 2011 FEBS The intracellular region of NPRA is divided into two domains: the KHD is the 280-residue region immedi- ately following the transmembrane domain, and distal to this is the GCD, which is at the C-terminal portion of the receptor molecule. More than 80% of the conserved residues that have been found in all protein kinases [49] are considered to be present in NPRA [5,6]. The GCD of NPRA has been suggested to consist of a 250-residue region at the C-terminal end of the molecule. Deletion of the C-terminal region of NPRA results in a protein that binds to ANP but does not contain GC activity [38,50,51]. Modeling studies based on the crystal struc- ture of the adenylyl cyclase II C 2 homodimer [52,53] predicted that the active sites of GCs and adenylyl cyc- lases are closely related [54,55]. On the basis of these predictions, the GC catalytic active site of murine NPRA includes a 31-residue sequence (residues 974– 1004) at the C-terminal end of the receptor molecule. A comprehensive assessment of the structure–function relationship of GC-A ⁄ NPRA has been described in this series [56]. The transmembrane GC-A ⁄ NPRA contains a single cyclase catalytic active site per polypeptide mol- ecule; however, modeling data suggest that two polypep- tide chains are required to activate the functional receptor [57]. Thus the transmembrane GC receptors seem to function as homodimers [58,59]. The dimeriza- tion region of GC-A ⁄ NPRA has been suggested to be located between the KHD and the GCD, and is pre- dicted to form an amphipathic a-helical structure [58]. NPRB is localized mainly in the brain and vascular tissues, although it is thought to mediate the actions of CNP in the vascular beds and in the central nervous system [43]. The third member of the natriuretic peptide receptor family, NPRC, consists of a large extracellular domain of 496 residues, a single transmembrane domain, and a very short 37-residue cytoplasmic tail that has no homology with any other known recep- tor protein domain. The extracellular region of NPRC is  30% identical to those of GC-A ⁄ NPRA and GC-B ⁄ NPRB. Earlier, it was proposed by default that NPRC functions as a clearance receptor to clear natri- uretic peptides from the circulation; however, several studies have also provided evidence that NPRC plays roles in the biological actions of natriuretic peptides [60–62]. Intracellular signal transduction mechanisms of GC-A ⁄ NPRA ANP markedly increases cGMP levels in target tissues in a dose-related manner [63,64]. The production of cGMP is believed to result from ANP binding to the extracellular domain of NPRA, which probably allos- terically regulates an increased specific activity of the cytoplasmic GCD of the receptor molecule [7,51,65,66]. Because the nonhydrolyzable analogs of ATP mimic the effect of ANP, it has been suggested that ATP can allosterically regulate the GC catalytic activity of NPRA [67–70]. In studies with mutant NPRA specifically lacking the KHD, it was found that the mutant receptor was active independently of ANP, which showed that it had the capacity to be bound with ligand, and most importantly, that it had basal GC activity  100-fold greater than that of wild-type NPRA [70]. Those previous findings suggested that, under natural conditions, the KHD acts as a negative regulator of the catalytic moiety of NPRA. Initially, this model was the standard way of explaining the sig- nal transduction mechanism of GC-coupled natriuretic peptide receptors [71]. However, the model has not been supported by the studies of other investigators, which found that deletion of the KHD in NPRA did not cause an elevation of basal GC activity; neverthe- less, ATP seems to be obligatory for the transduction activities of both NPRA and NPRB [65,67,72]. It has been suggested that NPRA exists in the phos- phorylated form in the basal state, and the binding of ANP causes a decrease in phosphate content as well as a reduction of the ANP-dependent GC activity [73]. This apparent mechanism of desensitization of NPRA is in contrast to what is seen with many other cell sur- face receptors, which appear to be desensitized by phosphorylation [74–76]. Some previously reported observations have also suggested that the GC activity may, in fact, be regulated by receptor phosphorylation [77–80]. However, little is known about the molecular regulatory mechanisms of the desensitization and sig- naling pathways of GC-A ⁄ NPRA, which may involve more than one process. Internalization and sequestra- tion of hormone receptors have been suggested to play important roles in the process of receptor desensitiza- tion and downregulation [81]. It is possible that NPRA may undergo homologous desensitization in response to ANP activation that could be mediated by receptor internalization, sequestration, and metabolic degrada- tion, in addition to phosphorylation ⁄ dephosphoryla- tion mechanisms [82,83]. At the mRNA level, NPRA has been shown to be regulated by glucocorticoids [84], transforming growth factor-b [85], chorionic gonadotropin [86], and angio- tensin II [87,88]. Endogenous transcription factors such as Ets-1 and p300 have been shown to exert remark- able stimulating effects on Npr1 transcription and expression [89,90]. At the protein level, angiotensin II has been shown to inhibit the GC activity of NPRA [87,91,92]. Similarly, at the receptor level, NPRA is K. N. Pandey Update on functional aspects of GC-A ⁄ NPRA FEBS Journal 278 (2011) 1792–1807 ª 2011 The Author Journal compilation ª 2011 FEBS 1795 downregulated following exposure to its ligand ANP or 8-bromo-cGMP [51,64,82,93,94]. Ligand-mediated endocytosis of GC-A ⁄ NPRA After binding to ANP and BNP, GC-A ⁄ NPRA is internalized and sequestered into intracellular compart- ments. Therefore, GC-A ⁄ NPRA is a dynamic cellular macromolecule that traverses different subcellular com- partments during its lifetime. Evidence indicates that, after internalization, the ligand–receptor complexes dissociate inside the cell and a population of GC-A ⁄ NPRA recycles back to the plasma membrane. Sub- sequently, the dissociated ligands are degraded in the lysosomes. However a small percentage of the ligand escapes the lysosomal degradative pathway and is released intact into the culture medium. GC-A ⁄ NPRA is internalized into subcellular compartments in a ligand-dependent manner [95–100]. The ligand-depen- dent endocytosis and sequestration of NPRA involves a series of sequential sorting steps, through which ligand–receptor complexes can eventually be degraded. A proportion of receptor is recycled back to the plasma membrane, and a small percentage of intact ligand is released to the cell exterior [51,97,99,100]. The recycling of endocytosed receptor to the plasma membrane and the release of intact ligand to the cell exterior occur simultaneously with processes leading to degradation of the majority of ligand–receptor com- plexes into lysosomes [51,82]. These findings provided direct evidence that treatment of cells with unlabeled ANP accelerates the disappearance of surface recep- tors, indicating that ANP-dependent downregulation of GC-A ⁄ NPRA involves internalization of the recep- tor [82]. All three natriuretic peptides (ANP, BNP, and CNP) are also bind to internalized involving NPRC and ligand-receptor complexes are internalized. The metabolic degradation of natriuretic peptides is further regulated by neprilyisn, as well as by insulin-degrading enzymes, as discussed in this series [101]. The short GDAY motif in the C-terminal domain of GC-A ⁄ NPRA serves as a signal for endocytosis and trafficking [51,82]. Gly920 and Tyr923 are the critical elements in the GDAY motif. It is thought that Asp921 provides an acidic environment for efficient signaling of the GDAY motif in the internalization of GC-A ⁄ NPRA. The mutation of Asp921 to alanine did not have a major effect on internalization, but signifi- cantly attenuated the recycling of internalized receptors to the plasma membrane [82,83]. On the other hand, mutation of Gly920 and Tyr923 to alanines reduced the internalization of receptor, but did not have any discernible effect on receptor recycling. It was sug- gested that Tyr923 in the GDAY motif modulates the early internalization of GC-A ⁄ NPRA, whereas Asp921 seems to mediate recycling or later sorting of the receptor. Increasing evidence indicates that complex arrays of short signals and recognition peptide sequences ensure accurate trafficking and distribution of transmembrane receptors and ⁄ or proteins and their ligands into intracellular compartments [83,94]. The short signals usually consist of small, linear amino acid sequences, which are recognized by adaptor coat pro- teins along the endocytic and sorting pathways. In recent years, much has been learned about the function and mechanisms of endocytic pathways responsible for the trafficking and molecular sorting of membrane receptors and their ligands into intracellular compart- ments; however, the significance and scope of action of the short motifs in these cellular events of GC-A ⁄ NPRA and GC-NPRB are not well understood. Interestingly, GC-B ⁄ NPRB is also internalized and recycled in hippocampal neurons and C6 glioma cell cul- tures [102]. It was suggested that trafficking of GC- B ⁄ NPRB occurs ligand-dependently in response to CNP binding and stimulation of the receptor protein. The internalization and trafficking of GC-B ⁄ NPRB has been suggested to involve a clathrin-dependent mechanism. Our recent work indicates that the internalization of GC-A ⁄ NPRA also involves clathrin-dependent path- ways [103]. Receptor internalization is severely dimin- ished by inhibitors of clathrin proteins, such as chlorpromazine and monodensyl cadaverine. However, interaction of the GDAY motif in GC-A ⁄ NPRA and GC-B ⁄ NPRB with clathrin adaptor proteins remains to be established. Physiological and pathophysiological functions of GC-A ⁄ NPRA The interaction of ANP with GC-A ⁄ NPRA reduces blood volume and lowers blood pressure by enhancing salt and water release through the kidney and inducing vasorelaxation of smooth muscle cells. Both ANP and BNP are implicated in reducing the preload and after- load of the heart in both physiological and pathological conditions. ANP and BNP acting via GC-A ⁄ NPRA antagonize cardiac hypertrophic and fibrotic growth, thus conferring cardioprotective effects in disease states. ANP has been shown to exert an antimitogenic effect in response to various growth-promoting agonist hormones in a number of target cells and tissues. The binding of ANP and BNP to GC-A ⁄ NPRA produces increased levels of the intracellular second messenger cGMP, which stimulates three known cGMP effector molecules, namely: cGMP-dependent protein kinases Update on functional aspects of GC-A ⁄ NPRA K. N. Pandey 1796 FEBS Journal 278 (2011) 1792–1807 ª 2011 The Author Journal compilation ª 2011 FEBS (PKGs), cGMP-dependent phosphodiesterases (PDEs), and cGMP-dependent ion channels. The activation of these effector molecules elicits a number of physiologi- cal and pathophysiological roles of GC-A ⁄ NPRA in several target cells and tissue systems (Fig. 2). Thus, multiple synergistic actions of ANP and BNP and their cognate receptor GC-A ⁄ NPRA make them novel therapeutic targets in renal, cardiac and vascular dis- eases. The critical physiological and pathophysiological functions of GC-A ⁄ NPRA are described below. Protective role of GC-A ⁄ NPRA in blood pressure regulation Genetic mouse models with disruption of both Nppa (coding for proANP) and Npr1 (coding for GC-A ⁄ NPRA) have provided strong support for the central role of the natriuretic peptide hormone–receptor sys- tem in the regulation of arterial pressure [21,104–109]. Therefore, genetic defects that reduce the activity of ANP and its receptor system can be considered as can- didate contributors to essential hypertension [7]. Previ- ous studies with ANP-deficient (Nppa ) ⁄ ) ) mice demonstrated that a defect in proANP synthesis can cause hypertension [107]. The blood pressure of homo- zygous null mutant mice was elevated by 8–23 mmHg when they were fed with standard-salt or intermediate- salt diets. Those previous findings indicated that genetic disruption of ANP production can lead to hypertension. Transgenic mice overexpressing ANP developed sustained hypotension with an arterial pres- sure that was 25–30 mmHg lower than that of their nontransgenic siblings [110,111]. Interestingly, somatic delivery of the ANP gene in spontaneously hyperten- sive rats induced a sustained reduction of systemic blood pressure [112]. Overexpression of ANP in hyper- tensive mice lowered systolic blood pressure, raising the possibility of using ANP gene therapy for the treatment of human hypertension [113]. It has also been shown that functional alterations of the Nppa promoter are linked to cardiac hypertrophy in proge- nies of crosses between Wistar Kyoto and Wistar Kyoto-derived hypertensive rats, and that a single- nucleotide polymorphism can alter the transcriptional activity of the proANP gene promoter [114]. Genetic studies with Npr1 knockout (Npr1 ) ⁄ ) or zero-copy) mice have indicated that disruption of Npr1 increases blood pressure by 35–40 mmHg as compared with wild-type (Npr1 + ⁄ + or two-copy) animals [21,104,109]. It has been demonstrated that complete absence of NPRA causes hypertension in mice and leads to altered renin and angiotensin II levels [21,104,109,115–117]. In contrast, increased expression of NPRA in gene-duplicated mutant mice significantly reduces blood pressure and increases the levels of cGMP, in correspondence with the increasing number of Npr1 copies [106,115,116,118]. Our studies have examined the quantitative contributions and possible mechanisms mediating the responses of varying num- bers of Npr1 copies by determining the renal plasma flow, glomerular filtration rate, urine flow and sodium excretion patterns following blood volume expansion in Npr1-targeted mice in a gene dose-dependent man- ner [105,116]. Our findings demonstrated that the ANP–NPRA axis is primarily responsible for mediat- ing the renal hemodynamic and sodium excretory responses to intravascular blood volume expansion. Interestingly, the ANP–NPRA system inhibits aldoste- rone synthesis and release from adrenal glomerulosa GTP y IP 3 Ligand Receptor NPRA NPRB NPRC ANP BNP CNP TM KHD LBD cAMP GCD DD IP 3 cGMP PKG Phosphorylaon or dephosphorylaon Physiological funcons PDE CNG GTP cGMP Ca 2+ Vasodilataon Sodium excreon PKC MAPK Anproliferaon cAMP TNF -α IL-6 Anhypertrophy Fig. 2. Representation of hormone specificity, ligand-binding domains, transmembrane-spanning regions, intracellular domains and signaling systems of GC-A ⁄ NPRA, GC-B ⁄ NPRB, and NPRC. The arrows indicate the ligand specificity for specific natriuretic peptide receptors. The extracellular ligand-binding domain (LBD), transmembrane region (TM), KHD and GCD of GC-A ⁄ NPRA and GC-B ⁄ NPRB are shown. DD is the dimerization domain of NPRA and NPRB. The LBD, TM and small intracellular tail of NPRC are also indicated. Both NPRA and NPRB have been shown to gener- ate cGMP from the hydrolysis of GTP. An increased level of intra- cellular cGMP stimulates and activates three known cGMP effector molecules, namely: PKGs, PDEs, and cGMP-dependent ion-gated channels (CNGs). The cGMP-dependent signaling may antagonize a number of pathways, including: intracellular Ca 2+ release, IP 3 for- mation, activation of protein kinase C (PKC) and MAPKs, and pro- duction of cytokines such as tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6). The resulting cascade can mimic ANP ⁄ NPRA ⁄ cGMP-dependent responses in both physiological and pathophysio- logical environments. The activation of NPRC may lead to a decrease in cAMP levels and an increase in IP 3 production. K. N. Pandey Update on functional aspects of GC-A ⁄ NPRA FEBS Journal 278 (2011) 1792–1807 ª 2011 The Author Journal compilation ª 2011 FEBS 1797 cells [3,109,115,119], which may account for its renal natriuretic and diuretic effects. Furthermore, studies with Npr1-disrupted (zero-copy) mice demonstrated that, at birth, the absence of NPRA allows higher renin and angiotensin II levels than in wild-type mice, and increased renin mRNA expression [109]. However, at 3–16 weeks of age, the circulating renin and angioten- sin II levels were dramatically decreased in Npr1 homo- zygous null mutant mice as compared with wild-type (two-copy) control mice. The decrease in renin activity in adult Npr1 null mutant mice is probably caused by a progressive elevation in arterial pressure, leading to inhibition of renin synthesis and release from the kid- ney juxtaglomerular cells [116]. On the other hand, the adrenal renin content and renin mRNA level, as well as angiotensin II and aldosterone concentrations, were elevated in adult homozygous null mutant mice as compared with wild-type mice [109,115]. In light of these previous findings, it can be suggested that the ANP–NPRA signaling system may play a key regula- tory role in the maintenance of both systemic and tis- sue levels of the components of the renin–angiotensin– aldosterone (RAA) system in physiological and patho- logical conditions. Indeed, ANP–NPRA signaling appears to oppose almost all actions of angiotensin II in both physiological and disease states (Table 2). Although expression of ANP and BNP is markedly increased in patients with hypertrophic or failing hearts, it is unclear how the natriuretic peptide system is activated to play a protective role. The ANP–NPRA system may act by reducing high blood pressure and inhibiting the RAA system, or by activating new molecular targets as a consequence of the hypertrophic changes occurring in the heart [21,105,120,121]. Functional role of GC-A ⁄ NPRA and salt sensitivity The disruption of Npr1 indicated that the blood pres- sure of homozygous mutant mice remained elevated and unchanged in response to either minimal-salt or high-salt diets [122]. These investigators suggested that NPRA may exert its major effect at the level of the vasculature, and probably does so independently of salt. In contrast, other studies reported that disruption of Npr1 resulted in chronic elevation of blood pressure in mice fed with high-salt diets [115,118]. The findings that adrenal angiotensin II and aldosterone levels are increased in Npr1-disrupted mice may explain the ele- vated systemic blood pressure with decreasing Npr1 copy (zero-copy and one-copy) numbers [115]. How- ever, adrenal angiotensin II and aldosterone levels are decreased in Npr1 gene-duplicated mice. A low-salt diet increased adrenal angiotensin II and aldosterone levels in all Npr1-targeted (gene-disrupted and gene- duplicated) mice, whereas a high-salt diet reduced adrenal angiotensin II and aldosterone levels in Npr1- disrupted mice and wild-type mice, but not in Npr1- duplicated (three-copy and four-copy) mice. Our findings suggest that NPRA signaling has a protective effect against high salt in Npr1-duplicated mice as compared with Npr1-disrupted (four-copy) mice [115]. Indeed, more studies are needed to clarify the relation- ship between salt sensitivity and blood pressures in Npr1-targeted mice. Protective roles of GC-A ⁄ NPRA in cardiac dysfunction It is believed that ANP and BNP concentrations are markedly increased both in cardiac tissues and in the plasma of CHF patients [123–125]. Interestingly, in hypertrophied hearts, ANP and BNP genes are overex- pressed, suggesting that autocrine and ⁄ or paracrine effects of natriuretic peptides predominate, and might serve as an endogenous protective mechanism against maladaptive pathological cardiac hypertrophy [21,120,124,126–128]. Evidence suggests that a high plasma ANP ⁄ BNP level is a prognostic predictor in humans with heart failure [123,129]. In patients with severe CHF, concentrations of both ANP and BNP are higher than control values; however, the increase in BNP concentration is 10-fold to 50-fold higher than the increase in ANP concentration [20]. Interestingly, the half-life of BNP is greater than that of ANP; thus, the diagnostic evaluations of natriuretic peptides have favored BNP [125]. The plasma levels of both ANP and BNP are markedly elevated under the pathophysi- ological conditions of cardiac dysfunction, including Table 2. Typical examples of antagonistic actions of ANP–NPRA on various angiotensin II-stimulated physiological and biochemical effects in target cells and tissues. CNS, central nervous system; PKC, protein kinase C. Parameters Angiotensin II ANP–NPRA Aldosterone release Stimulation Inhibition Renin secretion Inhibition Inhibition Vasopressin release Stimulation Inhibition Blood vessels Contraction Relaxation Water intake Stimulation Inhibition CNS-mediated hypertension Stimulation Inhibition Gonadotropin release Unknown Stimulation Testosterone synthesis Unknown Stimulation Estrodiol synthesis Unknown Stimulation Intracellular Ca 2+ release Stimulation Inhibition MAPKs Stimulation Inhibition PKC Stimulation Inhibition IP 3 production Stimulation Inhibition Update on functional aspects of GC-A ⁄ NPRA K. N. Pandey 1798 FEBS Journal 278 (2011) 1792–1807 ª 2011 The Author Journal compilation ª 2011 FEBS diastolic dysfunction, CHF, pulmonary embolism, and cardiac hypertrophy [21,124,125,130,131]. It has been suggested that ventricular expression of ANP and BNP is more closely associated with local cardiac hypertrophy and fibrosis than with plasma ANP levels and systemic blood pressure [21,127]. BNP can be con- sidered as an important prognostic indicator in CHF patients; however, N-terminal proBNP is considered to be a stronger risk bio-indicator for cardiovascular events [132,133]. The expression of Nppa and Nppb (coding for pro BNP) is greatly stimulated in hypertrophied hearts, suggesting that autocrine and ⁄ or paracrine effects of natriuretic peptides predominate and might serve as an endogenous protective mechanism against maladaptive cardiac hypertrophy [21,120,134]. Disruption of Npr1 in mice increases the cardiac mass and incidence of cardiac hypertrophy to a great extent [21,104,127,135– 137]. Previous studies have demonstrated that Npr1 disruption in mice provokes enhanced expression of hypertrophic marker genes, proinflammatory cyto- kines, and matrix metalloproteinases, and enhanced activation of nuclear factor kappaB, which seem to be associated with cardiac hypertrophy, fibrosis, and extracellular matrix remodeling [21,126,127]. Interest- ingly, the expression of sarcolemmal ⁄ endoplasmic reticulum Ca 2+ -ATPase-2a progressively decreased in the hypertrophied hearts of Npr1 homozygous null mutant mice as compared with wild-type control mice [21]. It has also been demonstrated that expression of angiotensin-converting enzyme and angiotensin II receptor type A is greatly enhanced in Npr1 null mutant (zero-copy) mice as compared with wild-type (two-copy) control mice [127]. Moreover, it has also been suggested that Npr1 antagonizes angiotensin II receptor-mediated and angiotensin II receptor type A-mediated cardiac remodeling, and provides an endogenous protective mechanism in the failing heart [127,138,139]. The arteries of smooth muscle-specific and endothelial cell-specific Npr1 knockout mice exhibited significant arterial hypertension [140]. It has also been suggested that Npr1 represents a potential locus for susceptibility to atherosclerosis [141]. The impact of Npr1 in cardiovascular pathophysiology has also been described in this series [142]. On the other hand, Npr2-deleted mice exhibit dysfunctional endo- chondral ossification and diminished longitudinal growth in limbs and vertebra, and show normal blood pressure, as compared with their wild-type counter- parts [143]. Mutation of Npr2 has been shown to be associated with Maroteaux-type acromesomedic dysplasia [144]. Biological actions of GC-A ⁄ NPRA in renal and vascular cells ANP–NPRA signaling in the kidneys promotes the excretion of salt and water, and enhances glomerular filtration rate and renal plasma flow [3,4,7,116]. Tar- gets of ANP action in the kidney include the inner medullary collecting duct, glomerulus, and mesangial cells [51,145–147]. The increased production of cGMP at ANP concentrations affecting renal function corre- lates with the effects of dibutyryl-cGMP, which pre- vents mesangial cell contraction in response to angiotensin II [148]. ANP markedly lowers renin secre- tion and also plasma renin concentrations [109,149,150]. The role of ANP in mediating the renal and vascular effects was investigated with selective NPRA antagonists to eliminate the effect of ANP [151,152]. ANP–NPRA signaling exerts direct effect on the kidney, to release sodium and water, by inhibiting sodium reabsorption. Npr1 knockout mice exhibit an impaired ability to initiate a natriuretic response to acute blood volume expansion [105]. In Npr1-dupli- cated mice, a low dose of ANP decreased the frac- tional reabsorption of distal sodium, suggesting that the augmented natriuresis was enhanced by ANP infu- sions and is mediated by Npr1 dosage [153]. These findings suggested that ANP–NPRA signaling inhibits distal sodium reabsorption. ANP–NPRA signaling also exerts indirect effects on renal sodium and water excre- tion by inhibiting the RAA system, as previously described [5,7]. ANP, either in intact aortic segments or in cultured vascular smooth muscle cells (VSMCs), has always been shown to increase cGMP levels. The correlative evidence of ANP-induced cGMP accumulation has suggested its role as the second messenger of dilatory responses to ANP in cultured VSMCs [152,154,155]. ANP and cGMP analogs reduced the agonist-depen- dent increases in cytosolic Ca 2+ levels in VSMCs and inositol trisphosphate (IP 3 ) levels in Leydig cells; thus, intracellular cGMP has been suggested to mediate the ANP-induced decrease in cytosolic Ca 2+ and IP 3 levels [156,157]. ANP has also been found to act as a growth suppressor in a variety of cell types, including vascula- ture, kidney and heart cells, and neurons [51,82,154,155,158]. ANP inhibits mitogen activation of fibroblasts [159], and induces cardiac myocyte apop- tosis [160]. However, the mechanisms involved in these effects of ANP are not yet completely understood. Clearly, more studies are warranted to elucidate the molecular mechanisms underlying the antiproliferative effect of ANP–NPRA signaling in various target cells. K. N. Pandey Update on functional aspects of GC-A ⁄ NPRA FEBS Journal 278 (2011) 1792–1807 ª 2011 The Author Journal compilation ª 2011 FEBS 1799 ANP is considered to be a direct smooth muscle relaxant, and a potent regulator of cell growth and proliferation. It is expected that the antigrowth paradigm could potentially operate through the negative regulation of mitogen-activated protein kinase (MAPK) activities. ANP may be one of the key endog- enous hormones that interacts negatively with elements in the MAPK signaling pathway to control cell growth and proliferation. ANP has been reported to antago- nize the growth-promoting effects in target cells; how- ever, the mechanism of the antigrowth paradigm of ANP and the involvement of specific ANP receptor subtypes (NPRA and NPRC) in different target cells are controversial [51,62,161–163]. Association of gene polymorphisms of Nppa, Nppb and Npr1 in hypertension and cardiovascular diseases Recent genetic and clinical studies have indicated an association of Nppa, Nppb and Npr1 polymorphisms with hypertension and cardiovascular events in humans [128,164–166]. An association between an Nppa promoter polymorphism (–C66UG) and left ventricular hypertrophy (LVH) has been demon- strated in Italian hypertensive patients, indicating that individuals carrying a copy of the Nppa variant allele exhibit a marked decrease in proANP levels associ- ated with LVH [165]. Interestingly, an association between a microsatellite marker in the Npr1 promoter and LVH has also been demonstrated, suggesting that the ANP–NPRA system contributes to ventricular remodeling in human essential hypertension [165]. As the relationship between high blood pressure and car- diovascular risk is continuous, in the absence of ANP–NPRA signaling even small increases in blood pressure have excessive and detrimental effects. Epide- miological studies have demonstrated that substantial heritability of blood pressure and cardiovascular risks can occur, suggesting a role for genetic factors [167]. Intriguingly, a common genetic variant at the Nppa–Nppb locus was found to be associated with circulating ANP and BNP concentrations, contribut- ing to interindividual variations in blood pressure and hypertension [164]. These authors demonstrated that a single-nucleotide polymorphism at the Nppa–Nppb locus was associated with increased plasma ANP and BNP concentrations, and lower systolic and diastolic blood pressures. Rare genetic mutations have been suggested for monogenic forms of hypertension and blood pressure in humans [168,169]. However, common variants asso- ciated with blood pressure regulation were not estab- lished. A number of pathways, namely the RAA system and the adrenergic system, are considered to regulate blood pressure and hypertension; nevertheless, the genetic determinants in these pathways contribut- ing to interindividual differences in blood pressure reg- ulation have not been elucidated. Therefore, the findings of those previous studies indicating an associa- tion of common variants in the Nppa–Nppb locus with circulating ANP and BNP concentrations are novel [164]. Interestingly, a ‘four-minus’ haplotype in the 3¢-UTR of Npr1 has been shown to be associated with an increased level of N-terminal-proBNP in humans [166]. The ‘four-minus’ haplotype constitutes 4C repeats at nucleotide position 14 319 and a 4-bp dele- tion of AGAA at nucleotide position 14 649 of Npr1. Individuals with genetic defects in Npr1 caused by the presence of the ‘four-minus’ haplotype exhibit signifi- cantly higher N-terminal proBNP levels. It has been speculated that the causal mechanism for this effect could be Npr1 mRNA instability, leading to decreased translational production of receptor molecules [170]. This could elicit a feedback mechanism, whereby the diminished function of the BNP–NPRA system caused by the defect in Npr1 provokes compensatory enhanced expression and release of BNP. Taken together, these considerations suggest that a positive association exists between Nppa, Nppb and Npr1 poly- morphisms and essential hypertension, high blood pressure and left ventricular mass index in humans. Further studies are needed for the characterization of more functionally significant markers of Nppa, Nppb and Npr1 variants in a larger human population. Conclusion and future perspectives The studies outlined in this review provide a unique perspective for delineating the genetic and molecular basis of GC-A ⁄ NPRA regulation and function. Recent studies have utilized molecular approaches to delineate the physiological functions affected by decreasing or increasing the number of Npr1 copies as achieved by gene targeting, such as gene disruption (gene knock- out) or gene duplication (gene dosage), of Npr1 in mice. The gene-targeting strategies have produced mice that contain zero to four copies of the Npr1 locus. Using gene-targeted mouse models, we have been able to determine the effects of decreasing or increasing the expression levels of Npr1 in intact mice in vivo. Com- parative analyses of the biochemical and physiological phenotypes of Npr1-disrupted and Npr1-duplicated mutant mice will have enormous potential for answer- ing fundamental questions concerning the biological importance of ANP–NPRA signaling in disease states Update on functional aspects of GC-A ⁄ NPRA K. N. Pandey 1800 FEBS Journal 278 (2011) 1792–1807 ª 2011 The Author Journal compilation ª 2011 FEBS by genetically altering Npr1 copy numbers and product levels in vivo in intact animals with otherwise identical genetic backgrounds. The results of these studies have provided important tools for examination of the role of the ANP–NPRA system in hypertension and cardio- vascular disease states. Future studies will lead to a better understanding of the genetic basis of Npr1 func- tion in regulating blood volume and pressure homeo- stasis, and should reveal new possibilities for preventing cardiovascular sequelae such as hyperten- sion, heart attack, and stroke. Nevertheless, the paradigms of the molecular basis of the functional regulation of Npr1 and the mecha- nisms of ANP–NPRA action are not yet clearly under- stood. Currently, natriuretic peptides are considered to be markers of CHF; however, an understanding of their therapeutic potential for the treatment of cardio- vascular diseases such as hypertension, renal insuffi- ciency, cardiac hypertrophy, CHF and stroke is still lacking. The results of future investigation should be of great value in resolving the problems of genetic complexities related to hypertension and heart failure. Overall, future studies should be directed at providing a unique perspective for delineating the genetic and molecular basis of Npr1 expression, regulation and function in both normal and disease states. The result- ing knowledge should yield new therapeutic targets for treating hypertension and preventing hypertension- related cardiovascular diseases and other pathological conditions. Acknowledgements My special thanks go to B. B. Aggarwal, Department of Experimental Therapeutics and Cytokine Research Laboratory, MD Anderson Cancer Center; and to S. L. Hamilton, Department of Molecular Physiology and Biophysics, Baylor College of Medicine, for pro- viding their facilities during our displacement period caused by Hurricane Katrina. I thank my wife Kamala Pandey for her kind help in the preparation of this manuscript. The research work in the author’s labora- tory was supported by National Institutes of Health grants (HL-57531 and HL-62147). References 1 de Bold AJ, Borenstein HB, Veress AT & Sonnenberg H (1981) A rapid and potent natriuretic response to intravenous injection of atrial myocardial extract in rats. Life Sci 28, 89–94. 2 de Bold AJ (1985) Atrial natriuretic factor: a hormone produced by the heart. Science 230, 767–770. 3 Brenner BM, Ballermann BJ, Gunning ME & Zeidel ML (1990) Diverse biological actions of atrial natriuretic peptide. 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The recycling of endocytosed receptor to the plasma membrane and the release of intact ligand to the