Feature Paper
Review
13 September 2022
1 Department of Pathophysiology of Heart Failure and Therapeutics, National Cerebral and Cardiovascular Center Research Institute, Suita 564-8565, Japan
2 Center for Regenerative Medicine, National Cerebral and Cardiovascular Center Research Institute, Suita 564-8565, Japan
* Author to whom correspondence should be addressed.
Atrial natriuretic peptide (ANP) is a cardiac peptide hormone that was identified by Kangawa and Matsuo in 1984. In Japan, ANP has been used as an intravenous drug for the treatment of acute heart failure since 1995. Because ANP has a hypotensive effect, it is important to avoid excessive lowering of blood pressure when ANP is used. Recently, a compound that inhibits neutral endopeptidase, the enzyme that degrades ANP, has been developed (angiotensin receptor-neprilysin inhibitor (ARNI)). ARNI has been approved worldwide for the treatment of chronic heart failure and has been authorized in Japan as an antihypertensive drug. However, it is not understood exactly how ANP exerts its hypotensive effect. In this review, we discuss the molecular mechanism of the blood pressure-regulating effects of ANP, focusing on our recent findings.
Natriuretic peptides, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), have cardioprotective effects and regulate blood pressure in mammals. ANP and BNP are hormones secreted from the heart into the bloodstream in response to increased preload and afterload. Both hormones act through natriuretic peptide receptor 1 (NPR1). In contrast, CNP acts through natriuretic peptide receptor 2 (NPR2) and was found to be produced by the vascular endothelium, chondrocytes, and cardiac fibroblasts. Based on its relatively low plasma concentration compared with ANP and BNP, CNP is thought to function as both an autocrine and a paracrine factor in the vasculature, bone, and heart. The cytoplasmic domains of both NPR1 and NPR2 display a guanylate cyclase activity that catalyzes the formation of cyclic GMP. NPR3 lacks this guanylate cyclase activity and is reportedly coupled to G i-dependent signaling. Recently, we reported that the continuous infusion of the peptide osteocrin, an endogenous ligand of NPR3 secreted by bone and muscle cells, lowered blood pressure in wild-type mice, suggesting that endogenous natriuretic peptides play major roles in the regulation of blood pressure. Neprilysin is a neutral endopeptidase that degrades several vasoactive peptides, including natriuretic peptides. The increased worldwide clinical use of the angiotensin receptor-neprilysin inhibitor for the treatment of chronic heart failure has brought renewed attention to the physiological effects of natriuretic peptides. In this review, we provide an overview of the discovery of ANP and its translational research. We also highlight our recent findings on the blood pressure regulatory effects of ANP, focusing on its molecular mechanisms.
The natriuretic peptide system controls body fluid and blood pressure homeostasis via three ligands: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) [1,2,3,4,5,6,7,8]. ANP and BNP are cardiac hormones that induce diuresis, natriuresis, and vasodilation via the transmembrane receptor natriuretic peptide receptor 1 (NPR1) [1,2,3,4,5,6,7,8], also known as guanylyl cyclase (GC)-A. CNP is secreted from vascular endothelial cells, chondrocytes, and cardiac fibroblasts [1,2,3,5,6,7]. CNP acts via natriuretic peptide receptor 2 (NPR2) [1,2,3,5,6,7,8], also referred to as GC-B. Based on its relatively low plasma concentration compared with ANP and BNP, CNP is thought to function as both an autocrine and paracrine factor in the vasculature and the heart [9,10,11,12]. The cytoplasmic domains of both GC-A and GC-B display a guanylate cyclase activity that catalyzes the conversion of guanosine-5-triphosphate to cyclic GMP [1,2,3,5,6,8]. NPR3, which lacks the GC domain, is implicated in the metabolic clearance of natriuretic peptides and is reportedly coupled to G i-dependent signaling [12]. NPR3 is also known as NPR-C. Previously, Saito et al. reported that a 30 min infusion of ANP in patients with congestive heart failure reduced systemic vascular resistance, indicating that one acute effect of ANP is dilation of the resistance vessels [13]. Recently, we reported that continuous infusion of the peptide osteocrin, an endogenous ligand of NPR3, which is secreted by bone and muscle cells, lowered blood pressure in wild-type mice, suggesting that endogenous natriuretic peptides play major roles in blood pressure regulation [14]. Originally, it was thought that ANP acted via GC-A-mediated cyclic GMP production in vascular smooth muscle cells [1,15,16]; however, studies in genetically engineered mice with cell type-specific deletion of GC-A have shown that mice with a smooth-muscle-cell-specific GC-A-knockout (SMC-GC-A-KO) do not exhibit hypertension [17], whereas mice with an endothelial-cell-specific GC-A-knockout (EC-GC-A-KO) do [18]. This suggests that the endothelial ANP/BNP–GC-A system is of greater importance than its smooth muscle counterpart in long-term blood pressure regulation. However, the precise molecular mechanisms underlying endothelial-dependent blood pressure regulation and vasodilation via the ANP/BNP–GC-A system have not yet been fully elucidated.
Neprilysin is a neutral endopeptidase that degrades several vasoactive peptides, including natriuretic peptides [19]; its affinity is 3.6-fold greater for ANP than for BNP [20]. The increased worldwide clinical use of angiotensin receptor-neprilysin inhibitor (ARNI) for the treatment of heart failure has brought renewed attention to the physiological effects of ANP. Because ANP has a hypotensive effect because of its vasodilatory property, the blood pressure of patients taking ARNI should be carefully observed so as to avoid excessive hypotension. Thus, it is important from a clinical viewpoint to clarify the detailed molecular mechanisms of acute and long-term blood pressure regulation by ANP.
This review provides an overview of the discovery, translational research, and blood pressure-regulating effects of ANP, including our recent findings.
With the invention of the electron microscope, the presence of secretory granules in mammalian atrial myocytes was reported in 1964 [21]. However, the physiological significance of this finding was not highlighted until over 15 years later. In 1981, de Bold et al. reported that the intravenous administration of a rat atrial extract to another rat produced marked diuresis and hypotension [22]. Furthermore, Currie et al. reported in 1983 that gel filtration chromatography-treated samples of human, rat, and pig atrial extracts relaxed the rabbit aorta and chick rectum, and produced natriuresis in rats [23]. They also reported that boiling atrial extracts for 10 min did not abolish their aortic relaxant activity, whereas trypsin treatment did, which indicated that the intra-atrial natriuretic factor was a small-molecular weight protein [23]. During the same period, Matsuo et al. had been searching for novel, brain-derived peptide hormones. They were interested in the paper by Currie et al. and sought to identify natriuretic factors in the human atria. They successfully determined the amino acid sequence of human mature ANP (α-ANP) and confirmed that α-ANP has a potent diuretic and natriuretic activity, publishing their results in a paper titled “Purification and complete amino acid sequence of alpha-human atrial natriuretic polypeptide (α-hANP)” in 1984 [24]. Although human α-ANP is composed of 28 amino acids, they showed that there are three molecular forms of ANP, specifically α-ANP (molecular weight ~3000 Da), β-ANP (molecular weight ~5000 Da), and γ-ANP (molecular weight ~13,000 Da) [24]. Subsequent studies confirmed that β-ANP is an inverted parallel dimer of α-ANP and that γ-ANP is a precursor of α-ANP. Kangawa et al. published the amino acid sequences of both molecules in 1985 and also showed that the diuretic activities of β-ANP and γ-ANP were approximately 25% and 15%, respectively, of that of α-ANP [25]. Note that Flynn et al. discovered rat ANP (atrial natriuretic factor (ANF)) at about the same time as the discovery of human ANP by Kangawa and Matsuo et al., and reported its 28-amino-acid sequence in their paper [26]. The gene encoding ANP contains three exons, and its transcript is translated to a 151-amino-acid precursor, preproANP. The 25-amino-acid signal peptide is then removed, yielding the 126-amino-acid proANP (γ-ANP), which is the tissue form of ANP [4]. ProANP (γ-ANP) is thought to be proteolytically converted to α-ANP (the mature biologically active form of ANP) by the transmembrane enzyme corin during its secretion from the heart [27,28].
The identification of a receptor for ANP was necessary in order to fully determine its physiological significance. Hirata et al. reported in 1984 that stimulating the cultured vascular smooth muscle cells with ANP increased intracellular cyclic GMP [29]. Then, in 1989, Chinkers et al. cloned the cDNA of GC-A, a receptor for ANP and BNP [30]. They isolated partial-length GC sequences from a human cDNA library using the cDNA of sea urchin membrane-type GC as a probe [30]. This enabled them to identify the site encoding full-length GC from a rat brain cDNA library and to confirm that the gene transfer of the cDNA into COS-7 cells increased intracellular cyclic GMP upon ANP stimulation [30]. Furthermore, Schulz et al. reported in 1989 that GC-B is a membrane-bound GC that differs from GC-A [31]. Although it is now known that GC-B is the receptor for CNP, CNP was not identified until Sudoh et al. reported it in 1990 [32]. Thus, the receptor (GC-B) was found one year earlier than the ligand (CNP). Figure 1 shows the ligands and receptors of the natriuretic peptides. Natriuretic peptides are removed from the blood by binding to NPR3, a clearance receptor; because NPR3 lacks an intracellular GC domain, its binding does not increase the intracellular cyclic GMP concentration [1,5,6].
Figure 1. Natriuretic peptides and their receptors. ANP—atrial natriuretic peptide; BNP—brain natriuretic peptide; CNP—C-type natriuretic peptide; GC-A—guanylyl cyclase-A; GC-B—guanylyl cyclase-B; NPR3—natriuretic peptide receptor 3.
Saito et al. reported in 1987 that the intravenous infusion of ANP markedly improved hemodynamics in patients with heart failure [13]. In their study, the intravenous administration of ANP at 0.1 µg/kg/min for 30 min in patients with New York Heart Association III or IV heart failure decreased pulmonary artery wedge pressure by an average of 13.7 mmHg and increased the stroke volume index by an average of 7.8 mL/m 2 [13]. ANP administration also markedly reduced systemic vascular resistance in heart failure patients [13]. Because ANP has no obvious inotropic effect, the increase in stroke volume index was presumably secondary to a decrease in systemic vascular resistance. The authors provided a very suggestive note in the discussion of their paper: “The decreased total systemic resistance observed in this study indicates that ANP dilates resistance vessels” [13]. Based on these translational studies, an α-human ANP product was approved in Japan in 1995 for the treatment of acute heart failure.
ANP administration was also found to improve the prognosis of patients with acute myocardial infarction. A multicenter, randomized, placebo-controlled, clinical trial named J-WIND enrolled patients with an initial acute myocardial infarction that occurred within 12 h of symptom onset [33,34]. The infusion of ANP for 3 days reduced the infarct size by 14.7% compared with the placebo treatment. Moreover, compared with patients who received the placebo, those treated with ANP had a higher left ventricular ejection fraction at the chronic stage after myocardial infarction. Although ANP administration did not improve the survival rates or the incidence of cardiovascular events, the incidences of cardiac death and hospital readmission due to heart failure were significantly reduced in ANP-treated patients compared with the controls [33,34].
Meanwhile, omapatrilat, which inhibits both neprilysin and the angiotensin-converting enzyme, was developed but was not clinically approved, owing to the appearance of angioedema in the clinical trial phase [35]. Bradykinin is also a substrate of neprilysin [19], and the synergistic effect of angiotensin-converting enzyme inhibition on bradykinin degradation may have caused the angioedema. Because angiotensin II is also a substrate of neprilysin [19], the clinical application of neprilysin inhibitors requires simultaneous inhibition of the renin-angiotensin system (RAS). ARNI inhibit RAS through angiotensin II receptor antagonism, which probably prevents the overproduction of bradykinin. The PARADIGM-HF study evaluating the superiority of ARNI over enalapril in heart failure patients with a reduced left ventricular ejection fraction was terminated early due to the significant prognostic value of the ARNI [36], and this resulted in its approval.
Now that ARNI is applied clinically, elucidating the molecular mechanisms of blood pressure regulation by ANP is an important issue. However, it remains somewhat unclear how ANP exerts its acute and long-term hypotensive effects. Because few reports have examined the tissue expression of GC-A, we performed immunohistochemical staining of GC-A in rat tissues using a mouse monoclonal antibody to GC-A, paying particular attention to blood vessels (Figure 2) [37].
Figure 2. GC-A is abundantly expressed in rat small arteries and arterioles. Localization of GC-A in the aorta (A), mesenterium (B), and skeletal muscle (C) was examined by immunohistochemistry in 4-week-old male Sprague-Dawley rats. The region within the red dotted box in the upper part of each panel is magnified in the bottom part of each panel. This figure is a reconstruction of the figure in our original article [37].
There was minimal expression of GC-A in the aortic e
