Natriuretic Peptide Receptor Type A (NPRA)
Atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and urodilatin (URO) represent a family of cardiac, vascular, and renal-derived hormones that play an essential role on the regulation of blood pressure, intravascular volume, and electrolyte homeostasis in all mammals. Most of the biological actions of ANP, BNP, and URO are mediated by activation of the natriuretic peptide receptor type A (NPRA), also designated as guanylyl cyclase-A/ natriuretic peptide receptor type A (GC-A/NPRA). Binding of these natriuretic peptides to NPRA leads to activation of the particulate guanylate cyclase (pGC) catalytic domain which generates cGMP-dependent second messenger signaling cascade. An increased level of intracellular cGMP activates three different targets: cGMP-dependent protein kinases (PKGs), cGMP-dependent ion-gated channels (CNGs), and cGMP-dependent phosphodiesterases (PDEs).
Human NPRA is encoded by Npr1 gene (OMIM 108960) which is located in chromosome 1 (q21–q22). Human NPRA gene is approximately 16 kilobases (kb), it contains 22 exons and 21 introns, and encodes for a peptide of 1061 amino acids and 135-kDa (Zhang et al. 2014).
NPRA Structure, Activation, Internalization, and Inactivation/Recyclation
ANP, BNP, and URO bind selectively to NPRA (Koller et al. 1993; Choi et al. 2011). NPRA is a 135-kDa transmembrane protein that constitutes a membrane guanylyl cyclase homodimeric receptor consistent with the guanylyl cyclase receptor family and composed by three domains: extracellular, transmembrane, and intracellular. The extracellular ligand binding domain (LBD) has a chloride-binding site near to the dimerization interface (Zhang et al. 2014) and has five glycosylation sites linked to nitrogen and three intramolecular disulfide bonds (Potter 2011). The transmembrane domain is represented by the juxtamembrane domain (JMD). The intracellular domain includes three highly conserved regions ordered as follows (from membrane to cytoplasm): the protein kinase-like homology domain (KHD) of 280 amino acids, the dimerization domain (DD), and the pGC catalytic domain (GCD) which involves 250 amino acids. The binding of two molecules of ANP causes the activation of NPRA, followed by the phosphorylation of five serines and two threonines near to the N-terminal site of the KHD (Potter 2011). Then, the KHD domain activates pGC catalytic domain by translation of ANP/BNP signals, with the consequent conversion of GTP to cGMP, which, in turn, can activate three different proteins: cGMP-dependent protein kinase I and II (GKI and GKII), PDEs, and CNGs. All of these nucleotides are related to the biological responses to the receptor ligands or agonists (Pandey 2015). It is also known that the phosphorylation state of one domain of these receptors via GKI and GKII determines their sensitivity to ligand (Garbers et al. 2006). cGKI and cGKII activation are linked to inflammation, cell growth, proliferation, and apoptosis. The dephosphorylation by PDEs route generates inactive 5′-nucleotide monophosphates through degradation of cyclic nucleotides, representing a way by which PDEs can regulate the intracellular signaling pathway of natriuretic peptides (Rukavina Mikusic et al. 2014). The cGMP-dependent signaling may also antagonize different pathways such as intracellular Ca2+ release, IP3 formation, protein kinase C (PKC) activation and mitogen-activated protein kinases (MAPKs) and cytokine production (tumor necrosis factor-α and interleukin-6) (Pandey 2015). In adipose cells, PKG-mediated phosphorylation activates different target proteins involved in the initiation of lipolysis (Collins 2014 and Moro and Lafontan 2013).
NPRA in Cancer
Recent research suggests that NPRA expression and signaling are present in different types of cancer like lung, prostate, gastric, and ovarian, being an important factor for tumor growth.
Related to the study of carcinogenic mechanism of NPRA, it has been demonstrated that preimplantation embryos and embryonic stem cells express NPRA. Though, using an RNAi directed to NPRA sequence, it could be identified phenotypic changes related to downregulation of pluripotency factors and upregulation of differentiation genes. NPRA expression seems to be necessary during implantation, angiogenesis, proliferation, and metastasis of tumor cells, representing a novel target for cancer therapy (Zhang et al. 2014). In this way, downregulation of NPRA by transfection of siRNA induced apoptosis and autophagy in prostate and gastric cancers, respectively (Li et al. 2016 and Wang et al. 2011). Moreover, NPRA deficiency protects C57BL/6 mice from lung, skin, and ovarian cancers and inactivates the expression of pro-inflammatory transcription factor NF-κB (Zhang et al. 2014). The fact that tumor burden is significantly reduced by therapy with siNPRA in mice open a new horizon in the study of NPRA for therapeutic purposes in cancer (Zhang et al. 2014).
NPRA in Hypertension and Cardiac Homeostasis
ANP through NPRA activation decreases blood volume and blood pressure by increasing water and salt excretion by the kidneys and by vasodilation of vascular smooth muscle. Several data suggest that impairment in the natriuretic peptide system plays a crucial role in the development of essential hypertension (EH) (Zois et al. 2014). In this sense, a Japanese study revealed a functional deletion mutation of the 5′-flanking region of the Npr1 gene that caused a reduction in gene transcriptional activity and conferred increased susceptibility to EH or left ventricular hypertrophy (LVH) (Nakayama et al. 2000). In addition, Rubattu et al. found that hypertensive patients with Npr1 gene promoter allelic variant 11/12, had also significantly higher values of left ventricular mass index (LVMI). Stimulation of NPRA acts as an endogenous protective mechanism that prevents cardiac remodeling in failing hearts antagonizing the effects of ANG II and AT1 stimulation (Kilić et al. 2007). The fact that patients carrying the mutant alleles of Npr1 gene had altered LVMI suggests the functional relevance of this receptor since promoter gene variants could reduce NPRA activity and be involved in ventricular remodeling in human EH (Rubattu et al. 2006).
NPRA activation and signaling are also important in the maintenance of cardiovascular homeostasis (Madhani et al. 2003). In this way, Npr1 knockout mice at endothelial cells and smooth muscle arteries developed significant hypertension (Sabrane et al. 2005). In contrast, Kishimoto et al. have shown that Npr1 knockout animals subject to selective transgenic changes in NPRA have reduced cardiomyocyte size, but not hypertension (Kishimoto et al. 2001). Moreover, Npr1 gene deficiency has been related to atherosclerosis and cardiac hypertrophy (Alexander et al. 2003). Activation of NPRA by BNP showed antifibrotic effects as a consequence of its important role in the control of extracellular matrix production that leads to cardiac fibrosis. Because of these findings, BNP has been proposed as an oral therapy for cardiac diseases (Garbers et al. 2006). All these evidences indicate that genetic alterations that reduce NPRA signaling are directly involved in impairment of cardiovascular homeostasis and development of EH (Pandey 2011).
NPRA in the Kidney
ANP/NPRA complex exerts its intrarenal actions on glomeruli and renal tubules. NPRA activation increases glomerular filtration rate, renal plasma flow, and water and sodium excretion and also stimulates nitric oxide synthesis in proximal tubules (Pandey 2011; Rukavina Mikusic et al. 2014). Additionally, it has been reported indirect actions of NPRA activation through enhancing renal dopaminergic system. Fernández et al. demonstrated that renal ANP stimulation of NPRA results in an increase of dopamine bioavailability in renal tubular cells allowing the dopamine receptor D1 recruitment and the inhibition of Na+-K+-ATPase activity with the consequent reduction of sodium reabsorption and increasing natriuresis (Fernández et al. 2005). Furthermore, it has been reported by Choi et al. that URO acting through NPRA stimulates cGMP signaling cascade and leads to an increase in renal dopamine uptake and to a decrease of Na+-K+-ATPase activity in the outer and juxtamedullary cortex as well as in the medulla, improving natriuresis and therefore reducing sodium reabsorption (Choi et al. 2011). Renal NPRA activation may also act as a renoprotective pathway against inflammation. In this sense, renal activity of pro-inflammatory NF-κB and other cytokines is increased in a knockout mice model for Npr1 gene (Rukavina Mikusic et al. 2014).
NPRA in Central Nervous System
NPRA in mammalian animal models was found in circumventricular organs of the central nervous system such as the vascular organ of the lamina terminalis, the choroid plexus, the area postrema, and the subfornical organ, and it was also found in the hypothalamus and astroglial cells of several central nervous system regions (Mahinrad et al. 2016).
It has been demonstrated that the complex ANP/NPRA is involved in the regulation of neuroinflammation. When ANP binds to NPRA in the macrophages activated by lipopolysaccharide (LPS), it inhibits pro-inflammatory transcription factors such as activator protein-1 and NF-kB causing the inhibition of IL-1β and nitrite production. It was also reported that ANP increased microglia phagocytic activity in the rat hippocampal area and that the administration of human recombinant BNP could reduce inflammatory markers and microglial activation (Mahinrad et al. 2016).
ANP is also involved in the regulation of norepinephrine metabolism in the rat hypothalamus by acting as a negative neuromodulator by reducing the noradrenergic neurotransmission through activation of guanylate cyclase signaling (Vatta et al. 1999).
ANP was found to regulate aqueous humor production in the eye, and the presence of NPRA receptors were identified by immunohistochemistry in different layers of the retina. Kuribayashi et al. demonstrated that in the rats’ retina, ANP acting through NPRA exerted neuroprotective actions against neurotoxicity induced by the intravitreal administration of N-methyl-D-aspartate (NMDA), which reduced dopamine levels. Moreover, the neuroprotective effect of ANP was inhibited by the administration of a dopamine D1 receptor antagonist (Kuribayashi et al. 2006).
NPRA in Fat Cell Metabolism and Skeletal Muscle
NPRA is abundantly expressed in adipose tissue and its activation induces lipolysis. This effect caused by ANP stimulation of NPRA in adipocytes is specific to primates, so it cannot be studied in other models such as rat, mouse, rabbit, or dog (Moro and Lafontan 2013). Through activation of GKI, NPRA promotes hormone-sensitive lipase-mediated triglyceride degradation and increases free fatty acid availability. By this mechanism, the natriuretic peptides enhance lipid oxidation into the β-oxidation pathway in the liver, skeletal muscle, and adipose tissue (Schlueter et al. 2014). The lipolytic response to NPRA is higher in large adipocytes than in small ones located in the same adipose tissue depot. Moreover, ANP and BNP acting through NPRA increase the expression of mRNA of adiponectin in human fat cells. Nonetheless, a high-fat diet in mice induces a downregulation of NPRA in brown and white fat.
In the skeletal muscle tissue, the overexpression of BNP and cGKI by transgenic method in mice under high-fat diet induced a high level of body energy expenditure and fat oxidation with reduction of fat mass and an increased expression of mitochondrial oxidative genes. Using the same mice model, it was identified a protection pathway to insulin resistance induced by diet. Also, in obese patients, it was shown that NPRA expression in skeletal muscle cells was upregulated in response to aerobic exercise. All these data may suggest that intracellular signaling of cGMP triggered by ANP-BNP/NPRA interaction as well as its lipid mobilizing effect could contribute to mitochondrial biogenesis induced by exercise in human skeletal muscles (Moro and Lafontan 2013).
Also, it was recently demonstrated that energy-balanced control exert by NPRA activation resulted in appetite suppression. Furthermore, external administration of BNP to healthy subjects showed to increase satiety feeling, decreasing hunger and total ghrelin levels (Moro and Lafontan 2013).
NPRA in the Pancreas and Liver
ANP stimulation of NPRA in pancreatic beta islet cells increases insulin secretion by blockade of ATP-sensitive K+ channel and intracellular Ca2+ augment. On the other side, knockout mice for Npr1 exhibited a decreased secretion of insulin associated with a lower β-cell mass, resulting in a higher fasting blood glucose level compared to the wild type (Moro and Lafontan 2013).
Therapeutic Implications of NPRA
The effect of the administration of exogenous natriuretic peptides has been evaluated by several clinical trials. Analogous molecules of ANP and BNP have been designed in order to reproduce the beneficial effects of NPRA activation in patients with chronic heart failure. In this context, different randomized and placebo-controlled trials demonstrated that the administration of nesiritide (a recombinant molecule of human BNP) improves the natriuresis and diuresis and elicits a significant decrease in plasma renin activity and aldosterone inhibition. Other natriuretic peptide analogs like M-ANP and cenderitide-NP are more resistant to degradation by metallopeptidase neprilysin, thereby increasing the half-time life to interact with NPRA (Díez 2016).
ANP, BNP, and URO bind to NPRA, a transmembrane receptor encoded in Npr1 gene. NPRA activation leads to a second messenger cascade signaling commanded by cGMP which triggers activation of other enzymes such as GKI. Its mRNA is expressed, among others, in the heart, kidney, vascular smooth muscle, skeletal muscle, endothelial, adipose, and brain tissues, as well as in the adrenal, lung, and testis. NPRA plays critical physiological and pathophysiological roles on several target cells and tissue system mechanisms, such as cell growth, apoptosis, proliferation, and inflammation. It is related to different processes like cancer, hypertension, cardiac remodeling and hypertrophy, atherosclerosis, renal abnormalities, neuroprotection, fat tissue and skeletal muscle metabolism, insulin secretion, and hepatoprotection. Although new knowledge have been updated in the last years, future studies with molecular biology techniques will provide complementary insights about how genetic alterations of NP receptors could be associated to the origin and development of human pathologies.
- Li Z, Wang JW, Wang WZ, Zhi XF, Zhang Q, Li BW, et al. Natriuretic peptide receptor A inhibition suppresses gastric cancer development through reactive oxygen species-mediated G2/M cell cycle arrest and cell death. Free Radic Biol Med. 2016;99:593–607. doi:10.1016/j.freeradbiomed.2016.08.019.PubMedCrossRefGoogle Scholar
- Nakayama T, Soma M, Takahashi Y, Rehemudula D, Kanmatsuse K, Furuya K. Functional deletion mutation of the 5'-flanking region of type A human natriuretic peptide receptor gene and its association with essential hypertension and left ventricular hypertrophy in the Japanese. Circ Res. 2000;86(8):841–5.PubMedCrossRefGoogle Scholar
- Rubattu S, Bigatti G, Evangelista A, Lanzani C, Stanzione R, Zagato L, et al. Association of atrial natriuretic peptide and type a natriuretic peptide receptor gene polymorphisms with left ventricular mass in human essential hypertension. J Am Coll Cardiol. 2006;48(3):499–505.PubMedCrossRefGoogle Scholar