1. Overview

ANP was the first reported member of the large family of natriuretic peptide hormones and it is present at high concentrations in the atrial tissue of many species. The ventricle also produces ANP, albeit at levels 1,000-fold lower than the atrium. ANPs have been found in a wide range of tissues where they may have a variety of functions. BNP and CNP were isolated from extracts of porcine brain by Sudoh et al. [105, 106]. In mammals, ANP, BNP, and CNP have been found in various animals, such as the human, bovine, pig, and rat. In non-mammalian species, ANP has been isolated from two species of frogs (Rana catesbeiana and R. ridibunda) and eel (Anguilla japonica), BNP from fowl (Gallus domesticus), and CNP from fowl, bullfrog, newt (Cynopus pyrrogaster), killifish (Fundulus heteroclitus), eel, and four species of sharks (Scyliorhinus canucula, Squalus acanthias, Triakis scyllia, and Lamna ditropis). Furthermore, it has been reported that a peptide toxin (DNP) isolated from the venom of the green mamba (Dendroaspis angusticeps) and ventricular natriuretic peptide (VNP) isolated from eel and trout (Oncorhynchus mykiss) exhibit sequence homology to and share bioactivity with natriuretic peptides. cDNA of eel ANP and VNP [117] and dogfish CNP [100] have been isolated.

The structures of 23 members of the natriuretic peptide family are shown in Figure 1. The amino acid sequences of ANPs and CNPs are highly conserved across species but those of BNP vary. In particular, human, porcine, and rat CNPs are identical. CNPs from the fowl and bullfrog are identical to the mammalian form with the exception of three amino acid substitutions. Furthermore, homology of greater than 80% has been noted between mammalian and fish CNPs despite the large phylogenetic distance between mammals and fish. By contrast, mammalian BNPs are only 50% homologous to one another at the amino acid level. It remains to be determined whether this variation among species in BNP is meaningful.

Fig. 1

Comparison of the amino acid sequences of natriuretic peptides from various classes of vertebrate. Previously published sequences for A-type natriuretic peptides of human [49], rat [30], Rana catesbeiana [96], Rana ridibunda [63] and eel [113]; B-type natriuretic peptide of human [80], bovine [81], pig [105], dog [30], rat [56], and fowl [1, 75]; C-type natriuretic peptides of human [120], fowl [8], Rana catesbeia na I [127], Rana catesbeiana II [58], newt [79], killifish [90], eel [115], Scyliorhinus canucula [109], and Squalus acanthias [100]; the natriuretic peptide of the green mamba [102], and the ventricular natriuretic peptide of eel [114] and rainbow trout [116] are shown in the single-letter amino acid code. Gaps have been introduced to maximize matching. Black boxes enclose identical amino acids. *Disulfied linkages between these cysteine residues are conserved in all the natriuretic peptides.

ANP, like many other biologically active peptides, is synthesized first in a “prepro” form that contains from 149 to 153 amino acids, depending upon the species. Prepro ANP is rapidly converted to a 126-amino-acid peptide, proANP, the predominant storage form of ANP. The bioactive peptide is generated by proteolytic cleavage of proANP just prior to the release of ANP into the circulation by an increase in blood volume. BNP [89] and CNP [57, 119] are also synthesized as large precursors. The half-life of ANP in the circulation is very short; in humans, the half-life of ANP in plasma has been estimated to be about 3 minutes [126]. ANP is metabolized by a neutral endopeptidase [54] and is trapped from the circulation after its binding to NPR-C [71].

2. Structure

The natriuretic peptides form a family of structurally related peptides, as shown in Figure 1. These peptides have in common a ring of 17 amino acids that is formed via a bow between two cysteine residues and they exist as multiple amino- and carboxyl-terminally extended or truncated forms. Figure 2 shows the mature forms of the human peptides; 11 of the amino acids are identical in ANP, BNP, and CNP. BNP-32 is the major form of the hormone in human atrial tissue, and human plasma has been reported to contain both BNP-32 and a high-molecular-weight form of BNP (probably pro-BNP). Two forms of CNP are present in most tissues that express CNP; CNP-53 [74, 119] is identical to CNP-22 except for an extension of 31 amino acids at the aminoterminal end. Urodilatin, first isolated from human urine and synthesized in the kidney, is identical to ANP except for the addition of four amino acids at the amino-terminal end [93].

Fig. 2

Structures of the three major subtypes of natriuretic peptides. Filled circles show amino acids that are identical in the three members of the family of human natriuretic peptides.

3. Biological action

ANP elicits a variety of responses that are directed toward the reduction of both blood pressure and blood volume. In the kidney, ANP has powerful natriuretic effects that result from the direct inhibition of the absorption of sodium ion in the collecting ducts, increased glomerular filtration, and the modulation of renal vascular resistance [23, 128]. Injection of ANP into the rat brain causes changes in diuresis and salt appetite [29, 45], in heart rate and blood pressure [66, 104], and in the secretion of vasopressin from the hypothalamus [97]. ANP also lowers vascular tone that has been induced by various vasoconstrictors. Furthemore, ANP inhibits the secretion of aldosterone from the adrenal gland, the release of vasopressin from the posterior pituitary and the secretion of renin from the kidney [10, 21, 37]. A number of reports on the physiologic effects of ANP have been published. By contrast, the major roles of BNP and CNP remain to be elucidated.

As its name suggests, BNP was originally identified in porcine brain, but studies in the rat [80], pig [73], and human [118] have demonstrated that levels of BNP in the heart are much higher than those in the brain. Resembling ANP, BNP can elicit vasorelaxing, natriuretic, and diuretic responses. It is of particular interest, moreover, that expression and release of BNP from the ventricle increase dramatically in patients with severe congestive heart failure [78].

In mammals, CNP has been detected at very low levels within the circulation. CNP was first identified in porcine brain [106] and its high concentrations in the brain and the central nervous system [57, 61] suggest that CNP acts as a neuropeptide. CNP has also been detected in endothelial cells [108], monocytic cells [44], and chondrocytes [40], suggesting an autocrine/paracrine role for CNP in the regulation of vascular tone, growth, or hormone release. However, the precise functional role of CNP has not yet been elucidated.

In non-mammalian vertebrates, a spectrum of actions of natriuretic peptides involve the cardiovascular system, renal and extrarenal osmoregulatory mechanisms, and the central nervous system [28, 112]. Mammalian as well as homologous ANP are vasodepressor in birds [39] and fish [64, 113] except a transient pressor effect observed in the trout which is mediated by sympathetic activation [84]. Eel ANP inhibits drinking in esophagus cannulated eels in a dose-dependent manner [111]. Mammalian or homologous ANP also inhibits the absorption of Na⁺ ion and water across the intestine of the flounder [82] and eel [5]. ANP inhibits aldosterone secretion in the fowl [38], turtle (Amyda japonica) [19], and frog (Rana ridibunda) [67] as in mammals, but it stimulates cortisol secretion in seawater fish [9]. Teleost fish utilize cortisol as mineralocorticoid. Mammalian ANP is diuretic and natriuretic in the fowl [39] and teleost fish [64, 84], but it is rather antidiuretic in the eel when homologous ANP is administered [111]. Antidiuretic effect of ANP is also reported in a shark, Squalus acanthias [11].

It was, recently, proposed that natriuretic peptides have growth-regulatory properties in a variety of tissues and cultured cells, such as adrenal gland, kidney, brain, bone, smooth muscle cells, and endothelial cells [7, 65]. For example, Levin has reviewed the relationship between NPR-C and the proliferation of cells [65]. Appel [6] and Itoh et al. [46] have described how ANP inhibits mitogenesis of rat mesangial cells and vascular smooth muscle cells, respectively, via the cGMP that is produced by the receptor guanylate cyclase. Furthermore, Pines and Hurwitz [87] showed that the proliferation of chondroprogenitor cells of the avian epiphyseal growth plate is modulated by ANP. This modulation is also probably mediated by cGMP as a second messenger. We have also reported that CNP inhibits the proliferation of rat chondrocytes in an autocrine/paracrine manner [40]. Recent studies by Garg and Hassid support the concept that cGMP is important in the inhibition of mitogenesis in rat mesangial cells [35] and vascular smooth muscle cells [36]. However, the cellular mechanisms involved in these actions of natriuretic peptides are by no means totally understood.

1. Overview

Receptors for natriuretic peptides can be biochemically and functionally divided into two major classes. The NPR-A (GC-A) and NPR-B (GC-B) receptors are members of the receptor guanylate cyclase family and each seems to be present as a tetramer, formed via disulfide bonds, on the plasma membrane [18, 48, 68]. The other type of receptor contains NPR-C which has a single membrane-spanning domain and a very short cytoplasmic tail (37 amino acids). NPR-C exists as a homodimer [103]. The structures of three representatives of the family of natriuretic peptide receptors are illustrated schematically in Figure 3. Yamaguchi et al. [124] characterized the gene for rat NPR-A (17.5 kilobases) and we [94] characterized the gene for bovine NPR-C (>85 kilobases). These genes are very similar in terms of the organization of exons and introns that correspond to the extracellular domain and the membrane-spanning domain. It appears, therefore, that the genes for NPR-A and NPR-C arose from several common ancestral genes by shuffling of exons.

Fig. 3

Structures and properties of members of the family of receptors for natriuretic peptides. Disulfide bonding patterns and domain structures of rat NPR-A [17] and NPR-B [101] and human NPR-C [88] are shown schematically.

Figure 4 shows a comparison of the amino acid sequence of the extracellular and membrane-spanning domains of natriuretic peptide receptors published to date. Natriuretic peptide receptors have been isolated from many mammalian species but their genes have not been cloned from nonmammalian species. As shown in Figure 4, the amino acid sequences of the extracellular domains that contain the ligand-binding sites are not highly conserved among the three receptors. However, using site-directed mutagenesis, we determined that His-Trp residues, which are highly conserved, contribute significantly to the binding of ligands to the receptors [47].

Fig. 4

Sequence comparison of the extracellular and membrane-spanning domains of receptors for natriuretic peptides. Previously published sequences for human NPR-A (hA) [70], rat NPR-A (rA) [17], mouse NPR-A (mA) [86], human NPR-B (hB) [15], rat NPR-B (rB) [101], eel NPR-B (eB) [52], human NPR-C (hC) [88], and bovine NPR-C (bC) [31], and the sequence of eel NPR-C (eC) [110] are shown in the single-letter amino acid code (only the extracellular and membrane-spanning domains are shown in the sequences of NPR-A and NPR-B). Gaps have been inserted to achieve maximum similarity. Black boxes enclose identical amino acids. Underlining shows regions of putative membrane-spanning domains. An asterisk shows the HW residues that contribute to binding of the ligand. The numbers on the right are the numbers of the last amino acids on the respective lines, counted from the first amino acid of the sequence of bovine NPR-C (bC), which was taken as number 1.

2. Membrane-bound guanylate cyclases (NPR-A and NPR-B)

NPR-A was initially cloned from rat brain [70] and human kidney [17] by use of a cDNA that encoded the membrane form of guanylate cyclase from the sea urchin Arbacia punctulata by Garbers and his colleagues. After the discovery of NPR-A, low-stringency hybridization resulted in the discovery of a second form of receptor guanylate cyclase, NPR-B [15, 101]. NPR-A and NPR-B each has a single membrane-spanning domain and large extracellular and cytoplasmic domains. In addition to the guanylate cyclase domain, the cytoplasmic tail contains a protein kinase-like domain that acts as a modulator of the activation of guanylate cyclase [16] (Fig. 3). Ohyama et al. [83] demonstrated the presence of an alternative splicing variant of rat NPR-B that lacks a part of the cytoplasmic regulatory domain encoded by exon 9 and also lacks some biological functions. NPR-A and NPR-B are structurally and functionally very similar but they have quite different ligand specificities; NPR-A responds to ANP and BNP, whereas NPR-B is highly specific for CNP [60]. A selective nonpeptide antagonist, HS-142-1, that is specific for NPR-A and -B has been reported but its mechanism of action is unclear [77]. HS-142-1 abolishes ANP-induced diuresis and natriuresis in animals.

We have reported marked increase in levels of cGMP mediated by the NPR-A and -B and a decrease in the expression of NPR-C in cultured vascular endothelial cells treated with NaCl [51] or exposed to high pH (pH 7.7) [50]. Kato et al. [53] also showed that cGMP down-regulates NPR-C in cultured vascular endothelial cells. Hirata et al. [42] demonstrated the ligand-induced and phorbol esterinduced down-regulation of NPRs. Chabrier et al. [14] demonstrated the induction by angiotensin II of downregulation of NPRs in rat vascular smooth muscle cells.

NPR-A is formed mainly in the kidney, adrenal gland, lung, and vascular bed. The diverse biological activities of ANP are thought to be mediated by the intracellular accumulation of cGMP that is produced by NPR-A.

Autoradiographic studies with ¹²⁵I[Tyr⁰]-CNP [62], Northern blot analysis [20, 101], PCR analysis [13, 83] and in situ hybridization [123] have demonstrated the existence of NPR-B in many tissues of the mammalian body, including the brain, lung, kidney, adrenal gland, intestine, uterus, and oviduct. NPR-B has also been identified in cultured cells, such as rat vascular smooth muscle cells [32], brain endothelial cells [122], glioma cells [26], pheochromocytoma (PC12) cells [92], chondrocytes [40, 41] and human mesangial cells [107]. However, the physiological roles of NPR-B in the target tissues have not yet been elucidated.

Recent studies have suggested a role for the CNP/NPR-B system in specific tissues and cells. For example, porcine seminal plasma contains large amounts of CNP, and NPR-B mRNA has been demonstrated in the uterus and oviduct, suggesting that the CNP/NPR-B system might play a role in fertilization [20]. This system also has been reported as a potent inhibitory regulator of cell proliferation [33, 40]. In the pituitary, CNP is an autocrine regulator of gonadotropes [72]. NPR-B has been cloned from the human retina, and ATP has been shown to be a prerequisite for CNP signaling in the retina [24].

3. C-Type natriuretic peptide receptor (NPR-C)

NPR-C has been purified from bovine lung [103] and cultured rat smooth muscle cells [99]. Molecular cloning has shown that NPR-C lacks a kinase-like domain and a guanylate cyclase domain[31]. We demonstrated the presence of a variant of NPR-C, produced by alternative splicing of RNA, that has an additional cysteine residue between the sixth intron and seventh exon [76]. Binding studies have demonstrated that NPR-C has equal affinity for each of the three types of natriuretic peptide. A specific competitor of NPR-C, designated C-ANF, with the structure des [Gln¹⁸, Ser¹⁹, Gly²⁰, Leu²¹, Gly²²]ANP_4–23-NH₂, has been synthesized [71]. Although NPR-C accounts for most of the NPR in most target tissues (more than 95% of the total population of NPRs), its physiological roles are not entirely clear. Maack and his colleagues [71] postulated that this protein functions as a clearance receptor to remove ANP from the circulation. However, recent evidence suggests that NPR-C inhibits the adenylate cyclase/cyclic AMP system through activation of a pertussis toxin-sensitive Gi protein in some tissues [2, 3]. Tseng et al. [121] showed that ANP inhibits formation of cAMP and secretion of thyroglobulin in cultured human thyroid cells which only have NPR-C. Antimitogenic and antiproliferative effects of ANP, mediated by NPR-C, have been proposed in rat astroglial cells [69], rat aortic smooth muscle cells [12], and hepatoblastoma cells [91]. In these cells, the NPR-C-selective ligand C-ANF inhibited the proliferation of cells in a cGMP-independent manner.