Synthesis and biological properties of antiparallel and parallel dimers of alpha-human atrial natriuretic peptide

To obtain antiparallel and parallel dimers of alpha-human atrial natriuretic peptide (alpha-hANP), two fully protected peptides I and II having the same amino acid sequence as alpha-hANP with different protective groups at the cysteinyl residues were synthesized, the former having Acm and Npys and the latter MeBzl and Acm. Equivalent amounts of peptides I and II were mixed and subjected to HF deprotection. Next, the first disulfide bond was linked between the remaining Npys group in I and the liberated SH group in II to form a monodisulfide dimer. The second disulfide bond was formed within the newly formed dimer between the remaining Acm groups by treatment with iodine, giving an antiparallel dimer. The parallel dimer of alpha-hANP was synthesized similarly starting from the protected peptide II. These dimers could be clearly segregated on HPLC. The retention time on HPLC of the antiparallel dimer was identical with that of natural beta-hANP. Both dimers showed biological activities as high as one third to one sixth of alpha-hANP in smooth muscle spasmolytic activity, and almost the same level of natriuretic activity as alpha-hANP at a high dose (10 nmol/kg) but about one fifth the activity at a low dose (1 nmol/kg). In these assay systems, the antiparallel dimer showed a slower onset and a tendency of longer duration than alpha-hANP.     

Relaxation of smooth muscle by cardiodilatin/atrial natriuretic peptide is inhibited by cAMP-dependent phosphorylation

Cardiodilatins/atrial natriuretic peptides (CDD/ANP) exhibit a common amino acid sequence: Arg101-Arg102-Ser103-Ser104. Cyclic AMP-dependent phosphorylation of Ser104 of atrial peptides with [gamma-32P]ATP enables rapid identification of cardiac hormones. The biological activity of in vitro phosphorylated cardiodilatin (CDD-28/alpha-hANP) is dramatically altered compared to the unphosphorylated peptide: the vaso-relaxant effect of cardiodilatin 28 is inhibited upon phosphorylation.     

Purification and complete amino acid sequence of alpha-human atrial natriuretic polypeptide (alpha-hANP)

The present survey for natriuretic factors in human atrial extract was performed by using in vitro assay for the relaxant effect on the contractility of chick rectum. Three distinct components (alpha, beta and gamma) of a potent relaxant activity were found in the chromatographic regions of the crude extract. As alpha-component of Mr 3,000 daltons, a 28-amino acid peptide has been isolated in a pure state and found to elicit potent diuretic and natriuretic activities as well as vasorelaxant activity, when injected into the assay rats. Accordingly, we proposed a name "alpha-human atrial natriuretic polypeptide (alpha-hANP)" for the peptide. The complete amino acid sequence of the peptide has been established by microsequencing as well as synthesis.     

Alpha-human atrial natriuretic polypeptide is released from the heart and circulates in the body

In order to clarify whether or not atrial natriuretic polypeptides are hormones in man, we have measured plasma alpha-human atrial natriuretic polypeptide (alpha-hANP)-like immunoreactivity (alpha-hANP-LI) with or without extraction procedure. alpha-hANP-LI was detected in plasma extracts from all 5 normal subjects and 7 patients with heart diseases. The alpha-hANP-LI concentration in normal peripheral plasma was 37.7 +/- 7.0 pg/ml (mean +/- SE). Plasma concentrations of alpha-hANP-LI in the coronary sinus obtained by cardiac catheterization were 3 to 10 times higher than those in the peripheral vein, inferior vena cava, right atrium, pulmonary artery and aorta. High performance gel permeation chromatography coupled with a radioimmunoassay (RIA) for alpha-hANP revealed that alpha-hANP-LI in normal peripheral plasma eluted at the position corresponding to that of authentic alpha-hANP without detectable amounts of high molecular weight forms. alpha-hANP-LI extracted from plasma taken from the coronary sinus of two patients also showed a single peak of alpha-hANP-LI co-eluting with alpha-hANP. In contrast, not only alpha-hANP but gamma-hANP and beta-hANP, high molecular weight forms, were present in the human atrial tissue. These results indicate that alpha-hANP is the predominant form of alpha-hANP-LI in human plasma and that this form generated in the atrial cardiocytes is preferentially released from these cells and circulates in the body.     

Vasodilatory and diuretic actions of alpha-human atrial natriuretic polypeptide (alpha-hANP)

Vascular and diuretic actions of synthetic alpha-human atrial natriuretic polypeptide (alpha-hANP) were studied using anesthetized dogs and isolated canine arterial strip preparations. alpha-hANP, when given intra-arterially or intravenously, dilated the renal artery more selectively than the vertebral, femoral, common carotid and coronary arteries. alpha-hANP selectively relaxed the high K+-contracted renal artery strip as compared with the basilar, coronary and femoral arterial strips. Intravenous alpha-hANP also increased urine volume and urinary excretion of electrolytes at doses, at which it increased renal blood flow and lowered systemic blood pressure without changing heart rate. It is concluded that alpha-hANP has a vasodilatory property relatively specific for the renal artery, and that it possesses diuretic, natriuretic, kaliuretic, magnesiuretic, calciuretic and chloruretic activities concomitantly with a definite hypotensive activity.     

Natriuretic peptide immunoreactivity in nerve structures and Purkinje fibres of human, pig and sheep hearts

Atrial natriuretic peptide is a well-described peptide in cardiac Purkinje fibres and has been shown to interfere with the autonomic regulation in the heart of various species, including man. Recently, we detected immunoreactivity for the peptide in intracardial ganglionic cells and nerve fibre varicosities of bovine hearts, by the use of a modified immunostaining technique that induced an improved detection of natriuretic peptides. These findings raised the question as to whether natriuretic peptides are detectable in these tissues in man and other species. The conduction system from human, pig and sheep hearts was dissected and processed with antisera against atrial natriuretic peptide and the closely related brain natriuretic peptide. Immunostaining for the brain natriuretic peptide was detected in some Purkinje fibres in all of these species. Interestingly, in pig, sheep and human hearts, some ganglionic cells and nerve fibres showed atrial natriuretic peptide immunoreactivity, particularly in the soma of human ganglionic cells. This is the first study showing immunoreactivity for the atrial natriuretic peptide in nerve structures and for the brain natriuretic peptide in Purkinje fibres of the human heart. The results give a morphological correlate for the documented effects of atrial natriuretic peptide on the heart autonomic nervous system and for the presumable effects of brain natriuretic peptide in the conduction system of man     

Natriuretic peptide immunoreactivity in nerve structures and Purkinje fibres of human, pig and sheep hearts

Atrial natriuretic peptide is a well-described peptide in cardiac Purkinje fibres and has been shown to interfere with the autonomic regulation in the heart of various species, including man. Recently, we detected immunoreactivity for the peptide in intracardial ganglionic cells and nerve fibre varicosities of bovine hearts, by the use of a modified immunostaining technique that induced an improved detection of natriuretic peptides. These findings raised the question as to whether natriuretic peptides are detectable in these tissues in man and other species. The conduction system from human, pig and sheep hearts was dissected and processed with antisera against atrial natriuretic peptide and the closely related brain natriuretic peptide. Immunostaining for the brain natriuretic peptide was detected in some Purkinje fibres in all of these species. Interestingly, in pig, sheep and human hearts, some ganglionic cells and nerve fibres showed atrial natriuretic peptide immunoreactivity, particularly in the soma of human ganglionic cells. This is the first study showing immunoreactivity for the atrial natriuretic peptide in nerve structures and for the brain natriuretic peptide in Purkinje fibres of the human heart. The results give a morphological correlate for the documented effects of atrial natriuretic peptide on the heart autonomic nervous system and for the presumable effects of brain natriuretic peptide in the conduction system of man     

The elucidation of the structure of atrial natriuretic factor, a new peptide hormone

The benchmark experiments of Adolfo de Bold and Harald Sonnenberg revealed that heart atria contained a substance or substances (atrial natriuretic factor) which when injected into rats caused a profound diuresis, natriuresis, and fall in blood pressure. Acid extraction and purification of atrial natriuretic factor resulted initially in the purification of a low molecular weight peptide containing a disulfide bond. This peptide was named cardionatrin I. Amino acid sequencing of less than 1 nmol of cardionatrin I revealed it to be a 28-residue peptide with the following structure: (sequence; see text) The position of the disulfide bond was verified by a radioactive method. From the sequence of complementary DNA for atrial natriuretic factor, the 28-residue peptide was shown to be the C-terminal portion of a larger protein called pro-atrial natriuretic factor. The discovery and characterization of atrial natriuretic factor substantiated the idea that the heart atria serve in an endocrine capacity.     

Presence of the atrial natriuretic peptide in human cerebrospinal fluid

Using a highly sensitive and specific radioimmunoassay (RIA) for detection of the atrial natriuretic peptide (ANP), the presence of alpha-human ANP (alpha-hANP) in human cerebrospinal fluid (CSF) was confirmed. Its concentration in CSF, 3.6 +/- 2.3 pg/ml, n = 16, mean +/- SD, was remarkably lower than that in the plasma (161.8 +/- 157.4, p less than 0.0001). The regression coefficient between these concentrations was 0.320 (p = ns). Gel permeation chromatography conducted in conjunction with RIA indicated ANP in CSF to be eluted at the position of a low molecular weight form corresponding to alpha-hANP. No high molecular weight form could be detected. But in the plasma, both low and high molecular forms were found to be present. It is thus evident that ANP is present in human CSF and its origin may possibly be the brain and not the atrium.     

Augmented synthesis of beta-human atrial natriuretic polypeptide in human failing hearts

To elucidate the synthesis of atrial natriuretic polypeptide (ANP) in the failing heart, eighteen human right auricles obtained at cardiovascular surgery were studied. The concentration of alpha-human ANP-like immunoreactivity (alpha-hANP-LI) in human right auricles ranged from 13.8 to 593.5 micrograms/g, and the tissue alpha-hANP-LI concentration in severe congestive heart failure (CHF) (New York Heart Association (NYHA) functional class III or IV) was much higher than those in mild CHF of NYHA class I and class II. The alpha-hANP-LI in the human auricle consisted of 3 major components of ANP, gamma-human ANP (gamma-hANP), beta-human ANP (beta-hANP) and alpha-human ANP (alpha-hANP). The predominant component of alpha-hANP-LI was gamma-hANP in the mild CHF, whereas beta-hANP and/or alpha-hANP were prevailing in the severe CHF and, especially, beta-hANP was markedly increased in human failing hearts.     

Atrial natriuretic peptides. III. Synthesis and biological activity of alpha-r-ANP, APII, APIII and des-Ser5,Ser6-APII

A series of atrial natriuretic peptides, viz., alpha-r-ANF, APII, APIII, and des-Ser5, Ser6-APII, have been obtained by condensation of earlier synthesized fragments promoted with complex F. Acetylaminomethyl protecting group were removed from cystein residues with simultaneous cyclization of the peptide. Biological activity of the atrial natriuretic peptides was studied.     

Specific receptors for atrial natriuretic factor (ANF) in cultured vascular smooth muscle cells of rat aorta

Specific binding site for atrial natriuretic factor (ANF), a potent natriuretic and vasorelaxant polypeptide recently isolated from mammalian atria, was studied in cultured vascular smooth muscle cells (VSMC) of the rat aorta. Binding studies of 125I-labeled-synthetic alpha-human natriuretic peptide (alpha-hANP) revealed the presence of a non-interacting, single class of high affinity binding sites for alpha-hANP on VSMC in culture: the apparent dissociation constant (Kd) was approximately 1-2 X 10(-9)M and the number of maximal binding sites was approximately 200,000-300,000 sites/cell. A variety of vasoactive substances and other polypeptide hormones did not affect the binding of 125I-labeled-alpha-hANP to its binding sites. alpha-hANP significantly increased the concentrations of intracellular cyclic GMP in VSMC in a dose-dependent manner (3.2 X 10(-9)-1.6 X 10(-7)M). These data indicate that the specific receptor for ANF is present in VSMC and suggest that intracellular cyclic GMP may be involved in its vasorelaxant effect.     

N-terminal amino acid sequence analysis indicates that isolated atrial amyloid is derived from atrial natriuretic peptide

Isolated atrial amyloid, the most frequent senile cardiac amyloid type, was chemically analysed. Amyloid fibrils obtained from a patient (NIP) were extracted and the predominant low-molecular-weight polypeptide (approximately 3.5 kDa, designated ASc2 NIP) was isolated by size exclusion high performance liquid chromatography in 60% formic acid. N-Terminal amino acid sequence analysis of this polypeptide was identical to that of the atrial natriuretic peptide alpha-hANP for the first 12 residues determined.     

Conversion of beta-human atrial natriuretic polypeptide into alpha-human atrial natriuretic polypeptide in human plasma in vitro

Using synthetic beta-human atrial natriuretic polypeptide (beta-hANP), an antiparallel dimer of alpha-hANP, and radioimmunoassay (RIA) for alpha-ANP which also detects beta-hANP, we investigated the disappearance profile and the change in the molecular form of exogenously added beta-hANP in human plasma in vitro, compared with those of alpha-hANP. The ANP-like immunoreactivity (ANP-LI) level in beta-hANP-added human plasma exhibited slower disappearance than that in alpha-hANP-added plasma during the incubation at 37 degrees C. High performance-gel permeation chromatography and reverse phase-high performance liquid chromatography coupled with RIA revealed that beta-hANP (6K) was converted into a smaller peptide with an approximate molecular weight of 3K corresponding to alpha-hANP during the incubation. Amino acid analysis and amino-terminal sequencing confirmed that the converted peptide from beta-hANP in human plasma is authentic alpha-hANP. The demonstrated conversion of beta-hANP into alpha-hANP in human plasma could be relevant to the in vivo natriuretic and diuretic actions with slower onset and longer duration of this unique peptide.     

Specific binding activities and cyclic GMP responses by atrial natriuretic polypeptide in kidney epithelial cell line (LLC-PK1)

Receptor binding activities and cyclic GMP responses by alpha-human atrial natriuretic polypeptide (alpha-hANP) and its fragments were studied in a kidney epithelial cell line (LLC-PK1). Binding of 125I-alpha-hANP to the cells at 0 degrees C was saturable, time-dependent and reversible, indicating the presence of a single class of binding sites. alpha-hANP (7-23)NH2 fragment inhibited most effectively the specific binding of 125I-alpha-hANP to the LLC-PK1 cells, followed by alpha-hANP (17-28) and alpha-hANP (8-22), while alpha-hANP (1-6) and alpha-hANP (24-28) did not. alpha-hANP stimulated the formation of cyclic GMP in the LLC-PK1 cells dose-dependently. Although no fragments of alpha-hANP used were effective for cyclic GMP formation in the LLC-PK1 cells, alpha-hANP (7-23) NH2 antagonized the action of alpha-hANP on cyclic GMP formation. These data suggest that the LLC-PK1 cells retain specific receptors for atrial natriuretic polypeptide (ANP) and respond to ANP by stimulating cyclic GMP formation, and therefore this cell line may be useful for studying the mechanism of action for ANP in renal tubular cells.     

High-level expression of alpha-human atrial natriuretic peptide from multiple joined genes in Escherichia coli

A method is described which allows alpha-human atrial natriuretic peptide to be synthesized in stable form and with high yield in Escherichia coli. In the final expression system, eight copies of the synthetic alpha-hANP gene were linked in tandem, separated by codons specifying a 4-amino-acid (aa) linker with lysine residues flanking the authentic N and C termini of the 28-aa hormone. This sequence was in turn joined to the 3' end of a fragment containing the lac promoter and a leader sequence coding for the first seven N-terminal amino acids of beta-galactosidase. The expressed multidomain protein accumulated intracellularly into stable inclusion bodies and was easily purified by urea extraction of the insoluble cell fraction. The purified protein was cleaved into monomers by digestion with endoproteinase Lys-C, trimmed to expose the authentic C terminus by digestion with carboxypeptidase-B and a single disulfide bond was formed by gentle oxidation with potassium ferricyanide. The fully processed recombinant peptide was shown by reverse phase liquid chromatography to be indistinguishable from the chemically synthesized standard alpha-hANP in both the reduced and in the folded form.     

Brain natriuretic peptide interacts with atrial natriuretic peptide receptor in cultured rat vascular smooth muscle cells

The effect of synthetic porcine brain natriuretic peptide (pBNP), a novel brain peptide with sequence homology to alpha-human atrial natriuretic peptide (hANP), on receptor binding and cGMP generation, was studied in cultured rat vascular smooth muscle cells (VSMC) and compared with that of alpha-hANP. 125I-pBNP bound to the cells in a time-dependent manner similar to that of 125I-alpha-hANP. Scatchard analysis indicated a single class of binding sites for pBNP with affinity and capacity identical to those of alpha-hANP. pBNP and alpha-hANP were almost equipotent in inhibiting the binding of either radioligand and stimulating intracellular cGMP generation. These data indicate that BNP and ANP interact with the same receptor sites to activate guanylate cyclase in rat VSMC.     

Natriuretic peptides in cardiovascular diseases

The natriuretic peptide family comprises atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), dendroaspis natriuretic peptide (DNP), and urodilatin. The activities of natriuretic peptides and endothelins are strictly associated with each other. ANP and BNP inhibit endothelin-1 (ET-1) production. ET-1 stimulates natriuretic peptide synthesis. All natriuretic peptides are synthesized from polypeptide precursors. Changes in natriuretic peptides and endothelin release were observed in many cardiovascular diseases: e.g. chronic heart failure, left ventricular dysfunction and coronary artery disease.     

C-type natriuretic peptide is a growth inhibitor of rat vascular smooth muscle cells.

C-type natriuretic peptide (CNP) which potently stimulates particulate guanylate cyclase activity in cultured rat vascular smooth muscle cells (VSMC) inhibited serum-induced DNA synthesis of the cells 10-fold more effectively than alpha-human atrial natriuretic peptide (alpha-hANP). The inhibitory effect of CNP was mimicked by 8-bromo-cGMP. The proliferation of VSMC was also suppressed by CNP more potently than alpha-hANP, while the peptide was less active for cGMP augmentation and for vasorelaxation than alpha-hANP in isolated rat aorta. These results suggest that CNP may be a growth regulating factor of VSMC rather than a vasodilator.     

Vascular receptor binding activities and cyclic GMP responses by synthetic human and rat atrial natriuretic peptides (ANP) and receptor down-regulation by ANP

Biological activities of a variety of synthetic human (h) and rat (r) atrial natriuretic peptide (ANP) and related peptides as assessed by receptor binding and cyclic GMP response, and regulation of vascular ANP receptors were studied in rat aortic vascular smooth muscle cells (VSMC) in culture. alpha-hANP1-28 and alpha-hANP7-28 equally inhibited the binding of 125I-labeled-alpha-hANP to its vascular receptors, whereas Met(O)12-alpha-hANP1-28 was less potent and reduced and carboxymethylated (RCM)-alpha-hANP1-28 was ineffective. rANP5-27 and rANP5-28 were equipotent in receptor binding, whereas rANP5-25 had somewhat less potent effect and rANP8-28 fragment was ineffective. alpha-hANP1-28, alpha-hANP7-28, rANP5-27 and rANP5-28 similarly stimulated intracellular cyclic GMP formation, whereas rANP5-25 showed less stimulatory effect, and RCM-alpha-hANP1-28, Met12-sulfoxide and rANP fragment were ineffective. Pretreatment with unlabeled alpha-hANP (3.2 X 10(-9) and 3.2 X 10(-8)M) for 24 hrs resulted in a substantial reduction (55 and 75%) of total receptor number without changing the affinity of ANP receptors. These results suggest that the common ring structure formed by the disulfide bond in the molecule is critical for receptor binding and subsequent biological actions, and that a hydrophobic amino acid located at the position of 12, and (24-26) residues at the C-terminal side, but not (1-6) at the N-terminal side, of the disulfide bridge may play a part in modulating receptor binding and/or biological functions. The present study also indicates "down-regulation" of vascular ANP receptors by homologous ligand.