Natriuretic Peptide Receptor Type C (NPRC)
The natriuretic system constitutes a family of cardiac- and vascular-derived hormones named Atrial Natriuretic Peptide (ANP), Brain Natriuretic Peptide (BNP), C-type Natriuretic Peptide (CNP), and Urodilatin (URO), which play an essential role on the regulation of blood pressure, intravascular volume, and electrolyte homeostasis in all mammals. Binding of natriuretic peptides (NPs) to either Natriuretic Peptide Receptor Type A (NPRA) or type B (NPRB) leads to activation of the particulate guanylate cyclase (pGC) catalytic domain which generates cGMP-dependent second messenger signaling cascade, mediating most of the biological actions of these peptides (Anand-Srivastava and Trachte 1993). NPs bind also to NPRC, which is considered a clearance receptor responsible for receptor-mediated degradation of these peptides. In addition, recent studies have revealed multiple effects of NPRC on different cells and organs, and most of these effects were enhanced by CNP stimulation rather than by ANP and BNP (Anand-Srivastava 2005).
Human NPRC is encoded by Npr3 gene (OMIM 108962) which localizes in chromosome 5(p14-p13). NPRC gene structure is approximately 75 kilobase pairs (kbp) and contains eight exons and seven introns. The exons size ranges from 88 bp (exon 7) to 769 bp (exon 1), and the introns size ranges from 1.5 kbp (intron 7) to 6.5 kbp (introns 2 and 3) (Rahmutula et al. 2002).
NPRC Structure and Localization, Signaling Pathways, and Biological Actions
The binding affinity of the NPs for NPRC is the following: ANP > BNP > CNP (Cantú et al. 2015). NPRC is the most widely and abundantly expressed NPR. It is present in the cardiac fibroblasts and myocytes, vascular smooth muscles cells (VSMC), kidney, cerebral cortex, brain striatum, hypothalamus, gastrointestinal smooth muscle, zona glomerulosa of the adrenal glands, bone, and chondrocytes. Also, it is the most expressed NPR in endothelial cells (Anand-Srivastava 2005).
NPRC first proposed action was the removal of NPs from circulation leading to their internalization and depuration from blood, considering it as a clearance receptor. After ligand binding, the NPRC-ligand complex undergoes endocytosis and then dissociates intracellularly, followed by hydrolysis of ligand in lysosomes and rapid recycling of the receptor back to the cell surface (Cohen et al. 1996; Nussenzveig et al. 1990). However, several studies indicate that NPRC plays additional roles implicated in NPs biological actions. In this way, novel NPs physiological effects have been described, particularly in the heart and vasculature. CNP and BNP acting through NPRC exert antiproliferative effects in cardiac fibroblasts. Also, CNP has been found to act as a NPRC-dependent endothelium-derived hyperpolarizing factor in the resistance vasculature regulating systemic blood pressure by hyperpolarization of VSMC and controlling local blood flow (Rose and Giles 2008). It has been proposed that these effects are mediated, at least in part, by AC inhibition and decreased cAMP levels. AC comprises three components: a receptor, a catalytic subunit, and Gs or Gi guanine nucleotide regulatory protein. The G proteins are transducers that transmit the signal from the hormone-occupied receptor to the catalytic subunit. The hormonal stimulation mediated by Gs results in increased formation of cAMP whereas the hormonal inhibition mediated by Gi results in decreased formation of cAMP (Anand-Srivastava 2005). NPRC has also been involved in the modulation of other signaling pathways. A possible cross-talk may exist between the AC and PLC pathways in VSMC, as the inhibition of AC and decreased levels of cAMP induced by NPRC activation contributed to stimulation of phosphatidylinositol (PI) turnover (Mouawad et al. 2004). It was demonstrated that when CNP binds to NPRC in pancreas, acini cells stimulate the amylase release through PLC pathway (Sabbatini et al. 2007).
Regulation of NPRC Expression
It has been described that several hormonal factors can regulate NPRC expression. Endothelin 1, angiotensin II (Ang II), and arginine vasopressin (AVP) reduce the density and expression of NPRC in VSMC. The effect elicited by AVP has been associated with an attenuation of AC inhibition mediated by NPRC. ANP has also been reported to regulate its own receptors. Experiments carried out in VSMC culture demonstrated that ANP elicited homologous downregulation of NPRC receptors. This effect depends on the degree of previous receptor occupation. Growth factors are also capable to regulate the expression of NPRC in VSMC and other tissues. In this way, fibroblast growth factors (FGF-1 and FGF-2) and PDGF-BB reduce NPRC mRNA expression in VSMC of pulmonary artery. On the other hand, transforming growth factor β1 (TGF-β1) showed to increase NPRC expression in murine thymic stromal cell line. Additionally, noradrenaline decreased ANP binding which was attributed to downregulation of the NPRC in cultured VSMC (Anand-Srivastava 2005). In the kidney, it was observed that dietary salt supplementation downregulates NPRC mRNA levels, suggesting a mechanism by which local ANP would facilitate natriuresis/diuresis and the maintenance of volume homeostasis (Nagase et al. 1997).
NPRC and Cardiovascular Diseases
Several evidences indicate a possible role of NPRC in the pathophysiology of some cardiovascular diseases (Cantú et al. 2015). An antihypertensive role has been proposed on the basis of alterations in NPRC function, amount, and tissue distribution in various models of hypertension. In this way, a dysfunction of NPRC signaling pathway has been found in spontaneously hypertensive rats (SHR), leading to impairment of NOS response to ANP with reduced nitric oxide (NO) availability, which in turn could help to explain the development of hypertension in this model. Another study in SHR revealed that NPRC concentration in the kidney was higher in normotensive rats compared to Wistar-Kyoto (WKY) control rats, indicating a decrease in renal ANP availability and contributing to the hypertensive state (Rubattu et al. 2010). NPRC mRNA in aorta from stroke prone SHR (SP-SHR) but not NPRA was downregulated as compared to WKY control rats. The treatment of SP-SHR rats with an Ang II receptor type 1 (AT1 receptor) antagonist restored NPRC mRNA to levels similar to those found in control rats, suggesting that vascular NPRC downregulation may be mediated by Ang II. The downregulation of NPRC in aorta was also reported in Deoxycorticosterone acetate (DOCA)-salt hypertensive rats, whereas ANP plasmatic levels were elevated. These results suggest that higher plasma ANP levels could be responsible for NPRC downregulation in this model. The ANP-mediated inhibition of AC was significantly enhanced in heart and aorta from SHR compared to WKY rats. This inhibition was also attenuated in platelets from SHR and hypertensive patients (Anand-Srivastava 2005). Preliminary results revealed a potential role of NPRC in the pathogenesis of atherosclerosis. It has been reported an increase in NPRC expression in neointimal smooth muscle cells in patients receiving percutaneous coronary intervention, as a possible result of a complex response of neointima to injury. Molecular imaging technique demonstrated the presence of NPRC near the luminal surface of atherosclerotic plaques and in VSMC. Moreover, NPRC-dependent extracellular signal-regulated kinase (ERK 1/2) phosphorylation activated the vasoprotective effect of CNP, resulting in an augmentation of endothelial cell proliferation and inhibition of VSMC growth (Naruko et al. 2005). After myocardial infarction, NPRC expression is increased in infarcted and non-infarcted regions of the left ventricular wall while appears to be decreased in the kidneys and lungs. This decrease would reflect a mechanism to favor an increase in plasma concentration of NP in this pathological context. NPRC expression is also enhanced in heart failure as well as in platelets from patients with this condition. It has been postulated that this increase may be responsible for a reduction in ANP availability and for the existence of resistance to biological effects of this peptide in these patients (Rubattu et al. 2010). Conversely, both homozygotes and heterozygotes knockout mice for Npr3 exhibit alteration in cardiovascular and renal functions, with reduced ability to concentrate urine, mild diuresis, blood volume depleted, and blood pressure values below normal levels. All these changes show that NPRC would regulate the local availability of NPs, according to specific local needs (Rukavina Mikusic et al. 2014).
There are several evidences linking mutations in Npr3 and cardiovascular diseases. A study in 200,000 European descents showed an association between rs1173771 polymorphism in NPRC with hypertension. Moreover, several evidences indicate a potentially novel single nucleotide polymorphism (SNP) rs700926 in Npr3 associated with coronary artery disease in Han Chinese population. In these studies, 11 SNPs of Npr3 (rs700926, rs1833529, rs2270915, rs17541471, rs3792758, rs1833529, rs2270915, rs17541471, rs3792758, rs696831, and rs696831) were identified to be associated to coronary artery disease (Hu et al. 2016). Fox et al. found that four Npr3 SNPs (rs700923, rs16890196, rs765199, rs700926) were associated to left ventricular dysfunction after coronary artery bypass grafting, and they were able to predict patient outcome when combined with Npr3 SNPs. Although rs700926 near intron 1 of Npr3 may not directly influence the NPRC mRNA expression, Fox et al. also found that mRNA expression level of NPRC in human peripheral blood leukocytes was significantly higher in individuals-patients carrying the polymorphism than those not carrying it. These findings are consistent with established effects of NPRC variant on coronary artery disease (Fox et al. 2009). Another study performed by Saulnier et al. in patients with type 2 diabetes found a consistent and significant association between the rs2270915 polymorphism of the Npr3 and systolic blood pressure (SBP). Patients who did not carry the polymorphism had lower SBP values than carriers. The rs2270915 also influenced the response of SBP to salt reduction, given the fact that patients without carrying the polymorphism showed a greater reduction of SBP after restriction of salt intake compared to carriers. The authors concluded that this genetic variation may affect pressure response to changes in dietary sodium (Saulnier et al. 2011).
NPRC and Hypoxia
NPRC, but not NPRA, gene expression is selectively downregulated in the lungs of rats and mice in response to hypoxia. This phenomenon is mediated by overexpression of tyrosine kinase-activating growth factors, such as acidic FGF-1 and occurs even in the absence of ANP gene expression. Selective downregulation of NPRC expression in lung in the setting of hypoxia may contribute to the increase in circulating ANP levels seen under hypoxic conditions and may enhance the vasodilator effects of ANP in the lung, thus modulating hypoxic pulmonary vasoconstriction/hypertension (Sun et al. 2001).
NPRC and Obesity
Adipocytes and adipose tissue have a high expression of NPRC. It has been described that NPRC expression is regulated positively by a high-fat diet and is suppressed by fasting (Rubattu et al. 2010). NPRC protein levels are markedly elevated during obesity. It is proposed that overexpression of NPRC would increase the clearance of NPs in adipose tissue, which could potentially contribute to reduced circulating levels of these peptides and predispose to hypertension in obese individuals (Collins 2014). Additionally, in obese hypertensive patients, weight reduction induced by fasting reduced blood pressure levels and increased diuresis and natriuresis (Rubattu et al. 2010).
NPRC and Bone Turnover
NPRC knockout mice resulted in skeletal abnormalities, characterized by hunched backs, dome-shaped skulls, decreased weight, and elongated femurs, tibias, metatarsal, digital bones, vertebral bodies, and body length. This model developed a skeletal-overgrowth phenotype, thus implicating a role for NPs in bone growth (Jaubert et al. 1999).
NPRC has traditionally been considered a clearance receptor of NPs responsible for receptor-mediated degradation of these peptides. In the last years, this view has been overcome by several studies showing evidence of NPRC multiple effects on different cells and organs and of the existence of a specific intracellular mechanism of action. These effects would be mediated by CNP stimulation rather than by ANP and BNP. A main role on vascular, cardiac, and metabolic physiology and on bone turnover has been proposed. Many studies indicate that alterations of NPRC function and structure as well as the existence of gene polymorphisms would be implicated in the pathophysiology of several diseases. Further investigation is needed to completely elucidate the physiological role played by NPRC and also to understand how NPRC alterations at gene and protein level could contribute to the development of pathological states as a cause of natriuretic system disruption.
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