The Functional Genomics of Guanylyl Cyclase/Natriuretic Peptide Receptor-A: Perspectives and Paradigms

Abstract

Cardiac hormones atrial and brain natriuretic peptides (ANP and BNP) activate guanylyl cyclase-A/natriuretic peptide receptor-A (GC-A/NPRA) and produce the second messenger cGMP. The GC-A/NPRA is a member of the growing family of GC receptors. The recent biochemical, molecular, and genomic studies of 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 protective 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 gene (coding for GC-A/NPRA) in control of fluid volume, blood pressure, cardiac remodeling, and other physiological functions and pathological states. Overall, this review attempts to provide insight 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.

1. Introduction

The initial discovery by de Bold and colleagues [1] established that atrial extracts contained natriuretic and diuretic activities and demonstrated the existence of “atrial natriuretic factor/peptide (ANF/ANP)”. A family of the endogenous peptide hormones including ANF/ANP, 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 peptides (NPs) hormones have been suggested not only to regulate blood pressure but also to play a role in a number of additional functions, namely: anti-mitogenic effects, inhibition of myocardial hypertrophy, endothelial cell function, cartilage growth, immunity, and mitochondrial biogenesis [6–9]. ANP and BNP are also increasingly utilized to screen and diagnose cardiac etiologies for shortness of breath and congestive heart failure in emergency situations [10].

One of the principal loci involved in the regulatory action of ANP and BNP is the receptor guanylyl cyclase-A (GC-A) designated as GC-A/NPRA. Interaction of ANP and BNP with GC-A/NPRA produces the intracellular second messenger cGMP, which plays a central role in the pathophysiology of hypertension and cardiovascular disorders [5, 11, 12]. Gaining insight into the intricacies of ANP/NPRA signaling is of pivotal importance for understanding both receptor biology and the disease state arising from abnormal hormone-receptor interplay. 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; however, the exact mechanism of receptor activation remains unknown. Recent works 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 abnormal control of cellular and physiological processes. Although there has been a great deal of appreciation of the functional roles of natriuretic peptides and their cognate receptors in renal, cardiovascular, endocrine, and skeletal homeostasis; in-depth research works are still needed to fully understand their potential molecular targets in cardiovascular and other diseases states. Ultimately, it is expected that studies on the natriuretic peptides and their receptors should yield new therapeutic targets and novel loci for the control and treatment of hypertension and cardiovascular disorders.

2. Natriuretic Peptide Hormone Family

ANP is the first described member in the NPs hormone family. It is primarily synthesized in the heart atria and elicits natriuretic, diuretic, and vasorelaxant effects, largely directed to the reduction of fluid volume and blood pressure [2, 3, 5, 7, 13, 14]. Subsequently, BNP and CNP, were identified with biochemical and functional characteristics similar to ANP but derived from separate genes [15]. BNP was initially isolated from the brain; however, it is primarily synthesized in the heart, circulates in the plasma, and displays the most variability in primary structure. CNP is largely present in endothelial cells and is highly conserved across species. All three types of natriuretic peptides contain a highly conserved 17-amino acid disulfide ring which is essential for the hormonal activities but they show variations from each other in the amino- and carboxyl-terminus flanking sequences (Fig. 1). Although ANP has been considered to exert its predominant effects directed towards lowering blood pressure and blood volume, recent evidence indicates that ANP plays a critical role in preventing cardiac load and overgrowth of heart in pathological conditions.

Figure 1. Comparison of amino acid sequence of natriuretic peptide hormone family.

Comparison of amino-acid sequence of human ANP, BNP, and CNP with conserved amino acid residues, which are represented by red boxes. The lines between two cysteine residues in ANP, BNP, and CNP indicate a 17-amino acid disulfide bridge, which seems to be essential for the biological activity of these peptide hormones. ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; and CNP, C-type natriuretic peptide.

Both ANP and BNP are predominantly synthesized in the heart, ANP levels vary from 50- to 100-fold higher than BNP. After processing of the 151-amino acid preprohormone to 126-residue prohormone molecule, the secretion of the 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 catalyzed 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 arginine-vasopressin, endothelin, and vagal stimuli [14, 17]. BNP is synthesized as a 134-amino acid preprohormone, which yields a 108-amino acid prohormone molecule. Processing of the proBNP yields a 75-residue amino-terminal-BNP and a 32-residue biologically active circulating BNP [18, 19]. The atria are the primary site of synthesis for both hormones within the heart. While the ventricles also produce both ANP and BNP, the concentration is 100- to 1000-fold less than the atria. The expression of both ANP and BNP increases dramatically in both the atria and ventricles in cardiac hypertrophy [20, 21]. It is believed that in the ventricles, BNP synthesis is regulated by volume overload, which activates ventricular wall stretch, subsequently enhancing hormone synthesis at the transcriptional level [22, 23]. Interestingly, higher ventricular ANP is present in the developing embryo and fetus, with both mRNA and peptide levels of ANP declining rapidly during the prenatal period [24].

CNP is largely present in the central nervous system [25], vascular endothelial cells [26], and chondrocytes [27]. CNP is synthesized as a 103-amino acid prohormone, is cleaved to a 53-residue peptide by the protease furin, and is subsequently processed to yield the biologically active 22-amino acid molecule [28]. In addition, a 32-amino acid peptide termed urodilatin (URO), identical to the C-terminal sequence of proANP, is known to be present in urine [29, 30]. URO is not detected in the circulation and appears to be a unique intrarenal natriuretic peptide with unexplored physiological functions [31]. D-type natriuretic peptide (DNP) represents an additional member in the NPs hormone family [32]. DNP is present in the venom of the green mamba (Dendroaspis angusticeps) as a 38-amino acid peptide molecule.

3. Guanylyl Cyclase/Natriuretic Peptide Receptor Family

Natriuretic peptides (ANP, BNP, 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 receptors (NPRs) have been cloned and sequenced from rat brain [33, 34], human placenta [35], and mouse testis [36]. The molecular cloning and expression of cDNAs have identified three different forms of NPRs including natriuretic peptide receptor-A, -B, and -C (NPRA, NPRB, and 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 guanylyl cyclase (GC) activity and have been designated as GC-A/NPRA and GC-B/NPRB, also referred to as GC-A and GC-B, respectively [37–39]. NPRC lacks the GC catalytic domain and has been termed as 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 NPs indiscriminately bind to NPRC [26, 39, 41]. NPRA is a 135-kDa transmembrane protein 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-control mechanism on receptor function [42]. The general topological structure of NPRA is consistent with that seen in the GC receptor family, containing at least four distinct regions: an extracellular ligand-binding domain, a single transmembrane spanning region, intracellular protein kinase-like homology domain (protein-KHD), and GC catalytic domain [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 the natriuretic peptide receptors found in peripheral organs and mediates most of the known actions of ANP and BNP. Using homology-based cDNA library screening system, additional members of GC-receptor family have also been identified; however, their specific ligand(s) and/or activator(s) are not yet known (Table 1). The other member of GC receptor family is GC-C receptor [11]. Additional members include 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 guanylyl cyclases/natriuretic peptide receptors.

Receptor	Ligand	Tissue distribution	Gene-knockout phenotype in mice
GC-A/NPRA (Npr1)	ANP/BNP (Nppa/Nppb)	Adrenal glands, brain, heart, liver, lung, olfactory, ovary, pituitary gland, placenta, testis, thymus, vascular beds, and other tissues	High blood pressure, hypertension, cardiac hypertrophy and fibrosis inflammation, volume overload, reduced testosterone (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	Neuro-epithelium, olfactory	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)
Ret-GC	Orphan	Retina	Unknown (47)
GC-Y-X1	Orphan	Sensory neurons	Unknown (48)

GC-A/NPRA, guanylyl cyclase/natriuretic peptide receptor-A; GC-B/NPRB, guanylyl cyclase/natriuretic peptide receptor-B; GC-C, guanylyl cyclase-C; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; CNP, C-type natriuretic peptide; ROS-GC, rod outer segment guanylyl cyclase; GC-D, -E, -F, -G, -E, and -Y-X1.

The intracellular region of NPRA is divided into two domains: the protein-KHD is the 280-amino acid region immediately following the transmembrane domain, and distal to this is the GC catalytic domain, which is at the carboxyl-terminal portion of the receptor molecule. More than 80% of conserved amino acid residues that have been found in all protein kinases [49] are considered to be present in NPRA [5, 6]. The GC catalytic domain of NPRA has been suggested to consist of a 250-amino acid region at the carboxyl-terminal end of the molecule. Deletion of the carboxyl-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 structure of the adenylyl cyclase II C₂ (AC II C₂) homodimer [52, 53] predicted that the active site of guanylyl and adenylyl cyclases are closely related [54, 55]. Based on these predictions, the GC catalytic active site of murine NPRA includes a 31-amino acid sequence (974–1004 residues) at the carboxyl-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 molecule; however, based on modeling data, two polypeptide chains seems to be required to activate the functional receptor [57]. Thus the transmembrane GC receptors seem to function as homodimers [58, 59]. The dimerization region of the GC-A/NPRA has been suggested to be located between the KHD and GC catalytic domain and is predicted to form an amphipathic alpha helix 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-amino acids, a single transmembrane domain, and a very short 37-amino acid cytoplasmic tail that bears no homology to any other known receptor protein domain. The extracellular region of NPRC is approximately 30% identical to GC-A/NPRA and GC-B/NPRB. Earlier, it was proposed by default that NPRC functions as a clearance receptor to clear natriuretic peptides from the circulation; however, several studies have also provided evidence that NPRC plays roles in biological actions of natriuretic peptides [60–62].

3.1 Intracellular Signal Transduction Mechanisms of GC-A/NPRA

ANP markedly increases cGMP 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 allosterically regulates an increased specific activity of the cytoplasmic GC catalytic domain of the receptor molecule [7, 51, 65, 66]. Because the nonhydrolyzable analogs of ATP mimicked ANP effect, it has been suggested that ATP can allosterically regulate the GC catalytic activity of NPRA [67–70]. Based on studies with mutant NPRA specifically lacking the protein-KHD, it was found that the mutant receptor was active independent of ANP, which showed the capacity to be bound with ligand, and most importantly, had basal GC activity approximately 100-fold times greater than wild-type NPRA [70]. Those previous findings suggested that under natural conditions the protein-KHD acts as a negative regulator of the catalytic moiety of NPRA. Initially, this model was the standard mark in explaining the signal 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 protein-KHD in NPRA did not cause an elevation of basal GC activity; regardless, 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 phosphorylated form in the basal state, and the binding of ANP causes a decrease in phosphate content as well as reduction in the ANP-dependent GC activity [73]. This apparent mechanism of desensitization of NPRA is in contrast to many other cell-surface 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 desensitization and signaling pathways of GC-A/NPRA, which may involve more than one process. Internalization and sequestration of hormone receptors have been implicated to play important roles in the process of receptor desensitization and down-regulation [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 degradation in addition to phosphorylation/dephosphorylation mechanisms [82, 83].

At the mRNA level, NPRA has been shown to be regulated by glucocorticoids [84], transforming growth factor-β [85], chorionic gonadotropin [86], and angiotensin II (ANG II) [87, 88]. Endogenous transcription factors such as Ets-1 and p300 have been shown to exert remarkable stimulating effects on Npr1 gene transcription and expression [89, 90]. At the protein level, ANG II has been shown to inhibit the GC activity of NPRA [87, 91, 92]. Similarly, at the receptor level, NPRA is down-regulated following exposure to its ligand ANP or 8-bromo- cGMP [51, 64, 82, 93, 94].

3.2 Ligand-Mediated Endocytosis of GC-A/NPRA

After binding with ANP and BNP, GC-A/NPRA is internalized and sequestered into intracellular compartments. Therefore, GC-A/NPRA is a dynamic cellular macromolecule that traverses different subcellular compartments through 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. Subsequently, the disassociated ligands are degraded in the lysosomes. However a small percentage of the ligand escapes the lysosomal degradative pathway and is released intact into culture medium. The GC-A/NPRA is internalized into subcellular compartments in the ligand-dependent manner [95–100]. The ligand-dependent endocytosis and sequestration of NPRA involves a series of sequential sorting steps through which ligand-receptor complexes can eventually be degraded. A portion of receptor is recycled back to the plasma membrane, and a small percentage of intact ligand in released into the cell exterior [51, 97, 99, 100]. The recycling of endocytosed receptor to the plasma membrane and the release of intact ligand into the cell exterior occur simultaneously with processes leading to degradation of the majority of ligand-receptor complexes into lysosomes [51, 82]. These findings provided direct evidence that treatment of cells with unlabeled ANP accelerates the disappearance of surface receptors, indicating that ANP-dependent down-regulation of GC-A/NPRA involves the internalization of the receptor [82]. All three natriuretic peptides (ANP, BNP, CNP) are also internalized involving NPRC. The metabolic degradation of natriuretic peptides is further regulated by nephrilyisn as well as insulin-degrading enzymes as discussed in this series [101].

The short sequence GDAY motif in the carboxyl terminal-domain of GC-A/NPRA serves as a signal for endocytosis and trafficking [51, 82]. The Gly⁹²⁰ and Tyr⁹²³ residues constitute the critical elements in the GDAY signal sequence motif. It is thought that the residue Asp⁹²¹ provides an acidic environment for efficient signaling of the GDAY sequence in the internalization of GC-A/NPRA. The mutation of Asp⁹²¹ to alanine did not exert a major effect on internalization, but significantly attenuated the recycling of internalized receptors to the plasma membrane [82, 83]. On the other hand, mutation of Gly⁹²⁰ and Tyr⁹²³ residues to alanines reduced the internalization of receptor, but did not show any discernible effect on the receptor recycling. It was suggested that the Tyr⁹²³ in the GDAY motif modulates the early internalization of GC-A/NPRA, whereas Asp⁹²¹ residue 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 proteins 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 compartments, however, the significance and scope of the short sequence motifs in these cellular events of GC-A/NPRA and GC-NPRB is not well understood.

Interestingly, the guanylyl cyclase-B/natriuretic peptide receptor-B (GC-B/NPRB) is also internalized and recycled in hippocampus neurons and C6 glioma cells cultures [102]. These authors suggested that the 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 pathways [103]. Receptor internalization is severely diminished 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.

4. 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 afterload 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 intracellular second messenger cGMP, which stimulates three known cGMP effector molecules, namely: cGMP-dependent protein kinases (PKGs), cGMP-dependent phosphodiesterases (PDEs), and cGMP-dependent ion channels. The activation of these effector molecules elicits a number of physiological and pathophysiological roles of GC-A/NPRA in several target cells and tissues 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 diseases. The critical physiological and pathophysiological functions of GC-A/NPRA are described below in specific sub-sections.

Figure 2. Representation of hormone specificity, ligand binding domain, transmembrane spanning region, intracellular domains, and signaling system of GC-A/NPRA, GC-B/NPRB, and NPRC, respectively.

The arrows indicate the ligand specificity to specific NP receptor. The extracellular ligand binding domain (LBD), transmembrane region (TM), and intracellular protein kinase-like homology domain (KHD) and guanylyl cyclase catalytic domain (GCD) of GC-A/NPRA and GC-B/NPRB are shown. DD, represents the dimerization domain of NPRA and NPRB. The ligand binding domain, transmembrane region, and small intracellular tail of NPRC are also indicated. Both NPRA and NPRB are shown to generate second messenger cGMP from the hydrolysis of GTP. An increased level of intracellular cGMP stimulates and activates three known cGMP effector molecules namely; cGMP-dependent protein kinases (PKGs), cGMP-dependent phosphodiesterases (PDEs), and cGMP-dependent ion-gated channels (CNGs). The cGMP-dependent signaling may antagonize a number of pathways including; intracellular Ca²⁺ release, IP₃ formation, activation of protein kinase C (PKC) and mitogen-activated protein kinases (MAPKs), and production of cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (Il-6). The resulting cascade can mimic ANP/NPRA/cGMP-dependent responses in both physiological and pathophysiological environments. The activation of NPRC may lead to a decrease in cAMP levels and an increase in IP₃ production.

4.1 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) genes have provided strong support for the central role of the natriuretic peptide hormone-receptor system 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 candidate contributors to essential hypertension [7]. Previous studies with ANP-deficient (Nppa^−/−) mice demonstrated that a defect in the proANP synthesis can cause hypertension [107]. The blood pressure of homozygous null mutant mice was elevated by 8–23 mmHg when they were fed with standard or intermediate salt diets. Those previous findings indicated that genetic disruption in ANP production can lead to hypertension. Transgenic mice overexpressing ANP developed sustained hypotension with arterial pressure that was 25–30 mmHg lower than their nontransgenic siblings [110, 111]. Interestingly, somatic delivery of ANP gene in spontaneously hypertensive rats (SHR) induced a sustained reduction of systemic blood pressure [112]. Overexpression of ANP in hypertensive 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 progenies of crosses between Wistar Kyoto (WKY) and Wistar Kyoto-derived hypertensive (WKYH) rats, and a single nucleotide polymorphism can alter the transcriptional activity of the proANP gene promoter [114].

Genetic studies with Npr1 gene-knockout (Npr1^−/− or 0-copy) mice have indicated that disruption of Npr1 gene increases blood pressures by 35–40 mmHg as compared with wild-type (Npr1^+/+ or 2-copy) animals [21, 104, 109]. It has been demonstrated that complete absence of NPRA causes hypertension in mice and leads to altered renin and ANG 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 second messenger cGMP, corresponding to the increasing number of Npr1 gene copies [106, 115, 116, 118]. Our studies have examined the quantitative contributions and possible mechanisms mediating the responses of varying numbers of Npr1 gene copies by determining the renal plasma flow (RPF), glomerular filtration rate (GFR), urine flow, and sodium excretion patterns following blood volume expansion in Npr1 gene-targeted mice in a gene-dose-dependent manner [105, 116]. These findings demonstrated that the ANP/NPRA axis is primarily responsible for mediating the renal hemodynamic and sodium excretory responses to intravascular blood volume expansion. Interestingly, the ANP/NPRA system inhibits aldosterone (ALDO) synthesis and release from adrenal glomerulosa cells [3, 109, 115, 119], which may account for its renal natriuretic and diuretic effects. Furthermore, the studies with Npr1 gene-disrupted (0-copy) mice demonstrated that at birth, the absence of NPRA allows greater renin and ANG II levels and increased renin mRNA expression compared with wild-type mice [109]. However, at 3–16 weeks of age, the circulating renin and ANG II levels were dramatically decreased in Npr1 homozygous null mutant mice as compared with wild-type (2-copy) control mice. The decrease in renin activity in adult Npr1 null mutant mice is likely due to progressive elevation in arterial pressure, leading to inhibition of renin synthesis and release from the kidney juxtaglomerular cells [116]. On the other hand, the adrenal renin content and renin mRNA level as well as ANG II and ALDO concentrations were elevated in adult homozygous null mutant mice as compared with wild-type mice [109, 115]. In light of those previous findings, it can be implicated that ANP/NPRA signaling system may play a key regulatory role in the maintenance of both systemic and tissue levels of the components of renin-angiotensin-aldosterone system (RAAS) in physiological and pathological conditions. Indeed, the ANP/NPRA signaling appears to oppose almost all actions of ANG II in both physiological and disease states (Table 2). Although expression of ANP and BNP is markedly increased in patients with hypertrophic or failing heart, it is unclear how the NP system is activated to play a protective role. The ANP/NPRA system may act by reducing high blood pressure and inhibiting the RAAS, or by activating new molecular targets as consequence of the hypertrophic changes occurring in the heart [21, 105, 120, 121].

Table 2.

Typical examples of antagonistic actions of ANP/NPRA on various angiotensin II - stimulated physiological and biochemical effects in target cells and tissues.

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 Ca2+ release	Stimulation	Inhibition
MAPKs	Stimulation	Inhibition
PKC	Stimulation	Inhibition
IP3 production	Stimulation	Inhibition

CNS, central nervous system; MAPKs, mitogen-activated protein kinases; PKC, protein kinase-C; IP₃, inositol trisphosphate.

4.2 Functional Role of GC-A/NPRA and Salt-Sensitivity

The disruption of Npr1 gene indicated that the blood pressure of homozygous mutant mice remained elevated and unchanged in response to either minimal 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 gene resulted in chronic elevation of blood pressure in mice fed with high salt diets [115, 118]. The findings that adrenal ANG II and ALDO levels are increased in Npr1 gene-disrupted mice may explain the elevated systemic blood pressure with decreasing Npr1 gene copy (0-copy and 1-copy) numbers [115]. However, adrenal ANG II and ALDO levels are decreased in Npr1 gene-duplicated mice. A low-salt diet stimulated adrenal ANG II and ALDO levels in all Npr1 gene-targeted (gene-disrupted and gene-duplicated) mice, whereas a high-salt diet suppressed adrenal ANG II and ALDO levels in Npr1 gene-disrupted mice and wild-type mice, but not in Npr1 gene-duplicated (3-copy and 4-copy) mice. Our findings suggest that NPRA signaling has a protective effect against high salt in Npr1 gene-duplicated mice as compared with Npr1 gene-disrupted (4-copy) mice [115]. Indeed, more studies are needed to clarify the relationship between salt-sensitivity and blood pressures in Npr1 gene-targeted mice.

4.3 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 congestive heart failure (CHF) patients [123–125]. Interestingly, in hypertrophied hearts, ANP and BNP genes are over expressed, 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 congestive heart failure (CHF), concentrations of both ANP and BNP increase higher than control values; however, the increase in BNP concentration is 10- to 50-fold higher than the increase in ANP concentration [20]. Interestingly, the half-life of BNP is greater than 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 pathophysiological conditions of cardiac dysfunction, including diastolic dysfunction, congestive heart failure, pulmonary embolism, and cardiac hypertrophy [21, 124, 125, 130, 131]. It is implicated that ventricular expression of ANP and BNP is more closely associated with local cardiac hypertrophy and fibrosis than plasma ANP levels and systemic blood pressure [21, 127]. BNP can be considered as an important prognostic indicator in CHF patients; however, NT-proBNP is considered to be a stronger risk bio-indicator for cardiovascular events [132, 133].

The expression of Nppa and Nppb (coding for proBNP) genes are greatly stimulated in the hypertrophied hearts, suggesting that autocrine and/or paracrine effects of NPs 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 gene-disruption in mice provokes enhanced expression of hypertrophic marker genes, pro-inflammatory cytokines, matrix metalloproteinases, and enhanced activation of nuclear factor-kappa B(NF-kB), which seem to be associated with cardiac hypertrophy, fibrosis, and extracellular matrix remodeling [21, 126, 127]. Interestingly, the expression of sarcolemal/endoplasmic reticulum Ca²⁺-ATPase-2a (SERCA-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 the expression of angiotensin converting enzyme (ACE) and ANG II receptor type A (AT1a) are greatly enhanced in Npr1 null mutant (0-copy) mice compared with wild-type (2-copy) control mice [127]. Morevover, it has also been suggested that Npr1 antagonizes ANG II and AT1a receptor-mediated cardiac remodeling and provides an endogenous protective mechanism in the failing heart [127, 138, 139]. Smooth muscle and endothelial cell-specific Npr1 knockout mice showed that arteries from these mice exhibited significant arterial hypertension [140]. It has also been indicated that Npr1 gene represents a potential locus for susceptibility to atherosclerosis [141]. The impact of Npr1 gene in cardiovascular pathophysiology has also been described in this series [142]. On the other hand, Npr2 gene-deleted mice exhibit dysfunctional endochondral ossification and diminished longitudnal growth in limbs and vertebra and show normal blood pressure compard with wild-type conunterparts [143]. Mutation in Npr2 gene has been shown to be associated with Maroteaux-type acromesomedic dysplasia [144].

4.4 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 GFR and RPF [3, 4, 7, 116]. ANP action in the kidney includes the inner medullary collecting duct, glomerulus, and mesangial cells [51, 145–147]. The increased production of cGMP at ANP concentrations affecting renal function correlates with the effects of dibutyryl-cGMP, which prevents mesangial cell contraction in response to ANG II [148]. ANP markedly lowers renin secretion and also plasma renin concentrations [109, 149, 150]. The role of ANP in mediating the renal and vascular effects was obtained with selective NPRA antagonists to eliminate the effect of ANP [151, 152]. ANP/NPRA signaling exerts direct effect on kidney to release sodium and water by inhibiting the sodium reabsorption. Npr1 gene-knockout mice exhibit impaired ability to initiate a natriuretic response to acute blood volume expansion [105]. In Npr1 gene-duplicated mice, the low-dose of ANP decreased fractional reabsorption of distal sodium delivery, suggesting that the augmented natriuresis was enhanced by ANP infusions and is mediated by Npr1 gene dosage [153]. These findings suggested that ANP/NPRA signaling inhibits the distal sodium reabsorption. ANP/NPRA signaling also exerts indirect effects on the renal sodium and water excretion by inhibiting the RAAS, as previously described [5, 154].

ANP either in intact aortic segments or in cultured vascular smooth muscle cells (VSMCs) has always been shown to increase cGMP. 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, 155, 156]. ANP as well as cGMP analogs reduced the agonist-dependent increases in cytosolic Ca²⁺ in VSMCs and inositol trisphosphate (IP₃) in Leydig cells; thus, intracellular cGMP has been implicated to mediate the ANP-induced decrease in cytosolic Ca²⁺ and IP₃ [157, 158]. ANP has also been found to act as a growth suppressor in a variety of cell types including vasculature, kidney, heart, and neurons [51, 82, 155, 156, 159]. ANP inhibits mitogen-activation of fibroblasts [160] and induces cardiac myocyte apoptosis [161]. However, the mechanisms involved in these effects of ANP are not yet completely understood. Clearly, more experimentation is warranted to elucidate the molecular mechanisms underlying the antiproliferative effect of ANP/NPRA signaling in various target cells.

ANP is considered a direct smooth muscle relaxant, and a potent regulator of cell growth and proliferation. It is expected that the antigrowth paradigm could potentially work through the negative regulation of mitogen-activated protein kinases (MAPKs) activities. ANP may represent one of the key endogenous hormones that interacts negatively with elements in MAPKs signaling pathway to control cell growth and proliferation. ANP has been reported to antagonize the growth-promoting effects in target cells, however, the mechanism of the anti-growth paradigm of ANP and the involvement of specific ANP receptor subtypes (NPRA and NPRC) in different target cells are controversial [51, 62, 162–164].

5. Association 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 gene polymorphisms with hypertension and cardiovascular events in humans [128, 165–167]. An association between Nppa promoter polymorphism (-C66UG) and left ventricular hypertrophy (LVH) has been demonstrated in Italian hypertensive patients, which indicated that individuals carrying a copy of Nppa variant allele exhibit marked decrease in proANP levels associated with LVH [166]. Interestingly, an association between a microsatellite marker in Npr1 promoter and LVH has also been demonstrated, suggesting that the ANP/NPRA system contributes to ventricular remodeling in human essential hypertension [166]. Since the relationship between high blood pressure and cardiovascular risk is continuous, thus in the absence of ANP/NPRA signaling even small increases in blood pressure confer excessive and detrimental effects. Epidemiological studies have demonstrated that a substantial heritability of blood pressure and cardiovascular risks can occur suggesting a role for genetic factors [168]. Intriguingly, a common genetic variant at the Nppa and Nppb locus was found to be associated with circulating ANP and BNP concentrations, contributing to inter-individual variations in blood pressure and hypertension [165]. These authors demonstrated that a single nucleotide polymorphism (SNP) at Nppa-Nppb loci 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 [169, 170]. However, common variants associated with blood pressure regulation were not established. A number of pathways, namely RAAS and adrenergic system, are considered to regulate blood pressure and hypertension; nevertheless the genetic determinants in these pathways contributing to inter-individual differences in blood pressure regulation have not been elucidated. Therefore, the findings of those previous studies indicating an association of common variants in Nppa-Nppb loci with circulating ANP and BNP concentrations is novel [165]. Interestingly, a “4-minus” haplotype in the 3′-untranslated region of Npr1 gene has been shown to be associated with an increased level of N-terminal-proBNP (NT-proBNP) in humans [167]. The “4-minus” haplotype constitutes 4C repeats at nucleotide position 14,319 and a 4-bp deletion of AGAA at nucleotide position 14,649 of Npr1 gene. Individuals with genetic defects in the Npr1 gene due to the presence of “4-minus” haplotype exhibit significantly higher NT-proBNP levels. It has been speculated that the causal mechanism for this effect could be Npr1 mRNA instability, leading to decreased translational products of receptor molecule [171]. This could elicit a feedback mechanism, whereby diminished function of BNP/NPRA system caused by the defect in the Npr1 gene provokes compensatory enhanced expression and release of BNP. Taken together, a positive association exists between Nppa, Nppb, and Npr1 gene polymorphisms 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.

6. 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 number of Npr1 gene copies as achieved by gene-targeting, such as gene-disruption (gene-knockout) or -duplication (gene dosage), of the Npr1 gene in mice. The gene-targeting strategies have produced mice which contain zero to four copies of the Npr1 locus (0, 1, 2, 3 or 4 copies). Using gene-targeted mouse models, we have been able to determine the effects of decreasing or increasing expression levels of Npr1 gene in intact mice in vivo. Comparative analyses of the biochemical and physiological phenotypes of Npr1 gene-disrupted and gene-duplicated mutant mice will have enormous potential for answering fundamental questions in understanding the biological importance of ANP/NPRA signaling in disease states by genetically altering Npr1 gene copy numbers and product levels in intact animals in vivo with otherwise identical genetic background. The results of these studies have provided important tools in examining the role of ANP/NPRA system in hypertension and cardiovascular disease states. Future studies will lead to a better understanding of the genetic basis of Npr1 function in regulating blood volume and pressure homeostasis, and should reveal new potentials for preventing cardiovascular sequelae such as hypertension, heart attack, and stroke.

Nevertheless, the paradigms of the molecular basis of the functional regulation of Npr1 gene and the mechanisms of ANP/NPRA action are not yet clearly understood. Currently, NPs are considered as markers of congestive heart failure; however, an understanding of their therapeutic potentials for the treatment of cardiovascular diseases such as hypertension, renal insufficiency, cardiac hypertrophy, congestive heart failure, and stroke is still lacking. The results of future investigation should be of great value to resolve the problems of genetic complexities related to hypertension and heart failure. Overall, future studies should be directed to provide a unique perspective for delineating the genetic and molecular basis of Npr1 gene expression, regulation, and function in both normal and disease states. The resulting knowledge should yield new therapeutic targets for treating hypertension and preventing hypertension-related cardiovascular diseases and other pathological conditions.