2.1.1. Natriuretic peptide family

There are three members in the natriuretic peptide family, ANP, BNP and C-type natriuretic peptide (CNP) (Wilkins et al. 1997, Levin et al. 1998) (Table 1). De Bold and co-workers (1981) first demonstrated that atrial extracts contain a substance which produced natriuresis and diuresis. Soon after that the ANP molecule was purified and sequenced (Flynn et al. 1983, Atlas et al. 1984). Some years later two other major new peptides of this family were discovered. BNP was originally isolated from porcine brain (Sudoh et al. 1988) but it was reported to be more abundant in cardiac atria and ventricles than in the central nervous system (Minamino et al. 1988, Hosoda et al. 1991, for review

see Davidson & Struthers 1994). CNP was also firstly identified in porcine brain (Sudoh et al. 1990) but it has been reported to be produced by endothelial cells (Suga et al. 1992a, Suga et al. 1993, for review see Chen & Burnett 1998).

It is now well established that natriuretic peptides are polypeptide hormones produced in a regulated manner (for review see Ruskoaho 1992, Yandle 1994, Wilkins et al. 1997, Levin et al. 1998). Each natriuretic peptide is encoded by a specific gene. ANP is mainly synthesized in the atria of normal adult heart, where ANP mRNA levels make up as much as 1-3 % of all mRNA. Ventricular expression is next in abundance, although the level is much lower than in the atrium (Gardner et al. 1986). In addition, a number of extracardiac tissues, including the central nervous system, lung, adrenal, kidney and vasculature contain ANP mRNA or proANP-like immunoreactivity (Gardner et al. 1986, for review see Ruskoaho 1992). BNP is also produced in the heart, although the rations of atrial to ventricular expression are more in the range of 3:1 rather that the 30:1 seen for ANP (Gerbes et al. 1994). Significant levels of CNP gene expression are found in the central nervous system, anterior pituitary gland, kidney and in endothelial cells of the vasculature (Suga et al. 1992a, Suga et al. 1993, Chen & Burnett 1998).

ANP and BNP are stored in atrial myocytes in storage granules that are morphologically similar to those known to store other polypeptide hormones. The translation product of ANP gene is preproANP, from which proANP is formed by cleavage of signal peptide. ProANP is transported unprocessed through the Golgi complex to secretory granules and thus 126-amino acid proANP is the major storage form (Vuolteenaho et al. 1985). On the contrary, the major storage form of BNP in the heart is cleaved mature peptide (Saito et al. 1989). Thus, this part of ANP processing differs from that of most other endocrine peptides, which are stored as bioactive peptide hormones. Finally, ANP-peptide is released by exocytosis to the interstitial fluid (Page et al. 1986). During or soon after its release proANP_1-126 is further split into an amino-terminal fragment (ANP_1-98, N-terminal ANP) and the biologically active hormone, the carboxy-terminal peptide (ANP_99-126, ANP). In some studies proteases from serum were suggested to convert proANP_1-126 to ANP_99-126 (Bloch et al. 1985, Itoh et al. 1987, for review see Ruskoaho 1992). However, ANP_99-126 is also released from isolated perfused hearts (Lang et al. 1985, Ruskoaho et al. 1986a) and atria (Vuolteenaho et al. 1985) in the absence of blood components. Therefore, the activation of proANP_1-126 probably either occurs intracellularly or simultaneously during release from the atrial myocyte, when an extracellular stimulus initiates secretion, possibly by simultaneously changing the activity of the processing enzyme(s) (for review see Ruskoaho 1992). Some researchers suggest that the N-terminal ANP is further processed to release three peptides (proANPs 1-30, 31-67 and 79-98) which may have either renal or vasorelaxant actions, or both (Vesely et al. 1994). Despite extensive study the mechanisms of proteolytic processing of ANP are not well understood.

The biologically active forms of natriuretic peptides share a common structural motif, consisting of a 17-amino acid loop formed by an intramolecular disulphide linkage between two cysteine residues (for review see Ruskoaho 1992, Yandle 1994, Levin et al. 1998). This disulphide loop and varying N- and C- terminal extensions are essential for biological activity of natriuretic peptides. The biologically active form of ANP is a 28-amino acid peptide, which amino acid sequence is highly homologous between species. BNP contains characteristic 17-amino acid structure to the natriuretic peptide family, but the sequence variability between species is large. The carboxy-terminal tail commonly found in ANP and BNP is completely lost in CNP. Like ANP, the amino acid sequence of CNP is highly homologous between species. The biologically active form of BNP is 32-amino acid peptide, whereas CNP is found in two forms, 22- and 53-amino acid peptides (Chen & Burnett 1998).

2.1.2. Natriuretic peptide receptors

Biological effects of natriuretic peptides are mediated by cell membrane receptors. Three subtypes of natriuretic peptide receptors have been described: ANP_A, ANP_B and ANP_C receptors (for review see Maack 1992, Anand-Srivastava & Trachte 1993). ANP_A and ANP_B receptors are guanylyl cyclases through which the ligands induce the production of cyclic guanosine monophosphate (cGMP). The ANP_A receptor is thought to mediate many of the effects of ANP and BNP (Maack 1992, Davidson & Struthers 1994) while CNP acts via ANP_B receptors (Koller et al. 1991, Chen & Burnett 1998). ANP_C receptor is a clearance receptor, which may signal through alternative pathways (Anand-Srivastava et al. 1990, Levin 1993). Besides the capacity to activate guanylyl cyclases, these receptors seem to activate the phosphoinositide second messenger system (Maack 1992). The rank order of potency of binding (and stimulation of cGMP production for types ANP_A and ANP_B receptors) for the receptors is as follows: ANP_A receptor = ANP ≥ BNP > > CNP; ANP_B receptor = CNP >> ANP ≥ BNP; ANP_C receptors = ANP > CNP > BNP (Koller et al. 1991, Suga et al. 1992b). No high-affinity guanylyl-cyclase-linked receptor that is specific for BNP has been identified. The properties of natriuretic peptides are predominantly mediated through increases of cGMP in target cells. These include cGMP-dependent protein kinases (PKG), cGMP-gated ion channels and cGMP-regulated phosphodiesterases (Lincoln & Cornwell 1993, de Bold et al. 1996).

Several studies have revealed natriuretic peptide receptors in many tissues, including blood vessels, kidney, adrenal gland, heart, lung and central nervous system (for review see Anand-Srivastava & Trachte 1993). ANP binding sites in the heart have been found in cardiac myocytes, atrial and ventricular endocardium and cells of conductive system (Anand-Srivastava & Trachte 1993). In the heart, both myocytes and fibroblasts are capable of expressing the natriuretic peptide receptor genes (Lin et al. 1995). There are no differences in distribution of ANP_A and ANP_B receptors in the atrial and ventricular tissues of rat hearts. However, the amount of the ANP_C receptors is greater in the atria than in the ventricles (Nunez et al. 1992).

Since natriuretic peptides and receptors are expressed in cardiac tissue, they may have paracrine/autocrine effects. Especially the role of natriuretic peptide receptors in the regulation of cardiac function, such as feedback regulation of natriuretic peptide release and modulation of contractility of the heart is of interest. The production of cGMP in myocytes is stimulated by ANP and BNP via ANP_A receptors, whereas CNP is ineffective (Lin et al. 1995). In the heart, cGMP has a negative inotropic effect (Lohmann et al. 1991). In addition, ANP inhibits cardiac L-type Ca²⁺ channel activity via cGMP dependent mechanisms (Tohse et al. 1995). Leskinen and co-workers (1997a) have shown that ANP modulates its own release by ANP_A receptors in vivo whereas CNP, the effects of which are mainly mediated by ANP_B receptors, does not act as a feedback regulator of ANP release.

ANP is rapidly eliminated from blood; the half-life of ANP in the circulation is 1/2-5 minutes, depending upon species. The clearance of ANP involves two predominant mechanisms: enzymatic degradation and receptor-mediated uptake (see Maack 1992, Ruskoaho 1992, Levin et al. 1998). The majority of ANP is eliminated by ANP_C receptor-mediated mechanisms in several tissues including lung, liver, kidney and intestine (Maack 1992). The binding of ANP to these receptors leads to the internalization and subsequent intralysosomal hydrolysis of the peptide. Natriuretic peptides are also cleared by neutral endopeptidase (NEP), an ectoenzyme with a broad substrate specificity and a wide tissue distribution, which includes lung and kidney. NEP inactivates all three natriuretic peptides; it has a greater affinity for CNP than for ANP and the affinity for BNP is much lower than that for the other two peptides (Kenny et al. 1993). Also other enzymes, such as angiotensin converting enzyme and kallikreins have been suggested to participate in the metabolism of ANP (for review see Ruskoaho 1992).

2.1.3. Physiological effects of natriuretic peptides

Natriuretic peptides are important regulators of blood pressure and fluid homeostasis. In vivo, ANP lowers blood pressure by decreasing cardiac output and reducing peripheral vascular resistance (Levin et al. 1998). Overexpression of the ANP gene lowers systemic blood pressure (Steinhelper et al. 1990) while the knock-out of ANP gene or ANP_A-receptor gene leads to hypertension (John et al. 1995, Lopez et al. 1995) and cardiac hypertrophy (Oliver et al. 1997). ANP and BNP cause natriuresis and diuresis both by renal hemodynamic and direct tubular actions (Levin et al. 1998). ANP vasodilates afferent arterioles and vasoconstricts efferent arterioles, leading to increased glomerular capillary hydrostatic pressure (Dunn et al. 1986). In consequence, intrarenal infusion of ANP increases sodium, potassium and phosphate excretion (Burnett et al. 1984).

Collectively, the natriuretic peptide family counterbalances the effects of the renin-angiotensin-aldosterone system (Espiner 1994, Wilkins et al. 1997, Levin et al. 1998). ANP and BNP have been shown to be physiological antagonists of the effects of angiotensin II (Ang II) on vascular tone, aldosterone secretion, renal-tubule sodium reabsorption, and vascular cell growth (Harris et al. 1987, Itoh et al. 1990, Wilkins et al. 1997, Levin et al. 1998). In addition, secretion of vasopressin (Obana et al. 1985) and endothelin-1 (ET-1) (Saijonmaa et al. 1990) are decreased by ANP.

In vasculature ANP inhibits the growth of endothelial and vascular smooth muscle cells (Itoh et al. 1990, 1992). CNP, like ANP, has antitrophic effects in cell-culture systems (Furya et al. 1991) which suggest that both peptides can directly inhibit the structural remodeling of blood vessels that occurs in response to hypertension and vascular injury. In addition, ANP acts as an antigrowth factor for cardiac fibroblast thereby modulating growth of the interstitial compartment in the development of cardiac hypertrophy (Cao & Gardner 1995).