Importance of hydrophobic residues in alpha-human atrial natriuretic peptide (alpha-hANP) for vasorelaxant activity

To investigate the roles of the hydrophobic residues in the ANP molecule on biological activities, we synthesized a series of analogs containing various phenylalanine-homologs in position 8 or methionine-homologs in position 12. Among the analogs [pCl-Phe8]-alpha-hANP(7-28) was 4.8 times as potent as alpha-hANP(7-28) in cGMP accumulation and 3.5 times as potent in vasorelaxant activity. All the analogs showed nanomolar affinity to the receptor. In contrast, vasorelaxation and cGMP accumulation activity of the analogs ranged widely. These results suggest that these hydrophobic residues in the cyclic core are critical for vasorelaxant activity rather than for the "apparent receptor binding", and that these residues may possibly discriminate the "bioactive receptor" which is coupled to guanylate cyclase from the non-coupled receptor.     

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.     

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.     

Protective effect of alpha-human atrial natriuretic polypeptide (alpha-hANP) on chemical-induced pulmonary edema

It has been established that alpha-hANP, the newly discovered peptide extracted from human cardiac atria, has potent natriuretic and hypotensive actions. Our present investigation is the first to demonstrate that alpha-hANP is capable of protecting against pulmonary edema caused by various chemicals, using isolated perfused guinea pig lung system. Lungs were perfused via pulmonary artery with Krebs-Ringer bicarbonate buffer at 5.0 ml/min, and wet weight of lungs and perfusion pressure of pulmonary artery (Pa) were monitored. Bolus injection of Triton-X or CHAPS into cannulated pulmonary artery produced edema as indicated by a massive increase in wet weight and a slight increase in Pa. Constant infusion of alpha-hANP through pulmonary artery at 200 ng/ml was effective in causing decrease in wet weight of lung. Perfusion of lung with paraquat or PGF2 alpha, and repeated bolus injection of arachidonic acid or PGE2 caused elevation in both wet weight of lung and Pa. The treatment with alpha-hANP similar to that described above also protected against edema caused by paraquat or arachidonic acid. Bolus administration of epinephrine induced a slight increase in wet weight and Pa, and alpha-hANP was effective in decreasing the elevated lung wet weight and Pa of lungs. Infusion or bolus administration of alpha-hANP into control lungs increased cGMP level in outflow perfusate as well as in lung tissue significantly. In lungs with edema which were induced by Triton-X or paraquat, there was a slight increase in cGMP level in Triton-X treated and no increase in paraquat treated lung tissues. In either cases, was there any increase in cGMP level in perfusate. The specific binding study of [125I]alpha-hANP revealed that the lack of increase in cGMP was not due to a loss of receptor in Triton-X or paraquat treated lungs. Thus our study demonstrated that alpha-hANP had a direct anti-edematic action(s) in lung which was not secondary to the systemic natriuretic and/or hypotensive action(s).     

Determination of alpha-human atrial natriuretic peptide (alpha-hANP) in urine using combination HPLC with RIA strongly indicates non-immunoreactive metabolites

Employing HPLC coupled with RIA, it was shown that alpha-human atrial natriuretic peptide is excreted in urine. Freshly collected urine had to be acidified to obtain reproducible results. When prepurified urine was subjected to HPLC (ion exchange and reversed phase) the subsequent quantification of alpha-hANP immunoreactive material in the eluate showed 10- to 30-fold greater amounts of alpha-hANP after treatment with HPLC; substances with the same elution parameters as synthetic alpha-hANP were detected, but they gave no response in the RIA.     

Changes in the plasma and urine alpha human atrial natriuretic peptide (alpha hANP) concentration in patients with thyroid disorders

In order to assess the possible involvement of thyroid hormone in alpha human atrial natriuretic peptide (alpha hANP), we investigated the plasma and urine ANP concentration in patients with primary hyperthyroidism and hypothyroidism. Plasma and urine were extracted through Sep-Pak C18 cartridges and the urine ANP concentration was corrected by urine creatinine (cre. mg/dl) and expressed as fmol/mg.cre.. The plasma ANP concentration in patients with untreated hyperthyroidism (32.3 +/- 7.0 fmol/ml; n = 22) was higher than in normal subjects (p less than 0.01 vs control; 6.2 +/- 0.7 fmol/ml). After restoration to euthyroidism, the plasma ANP concentration (patients with treated hyperthyroidism) fell to normal (8.9 +/- 1.9 fmol/ml). The plasma ANP concentration in patients with untreated hypothyroidism (14.1 +/- 3.0 fmol/ml; n = 7) was higher than normal, but in two of them there was mild renal dysfunction and an incomplete right blundle branch block in the electrocardiogram. It was possible that these factors contributed to the observed increase in plasma ANP. However, a significant positive correlation was found between plasma ANP and free thyroxine (n = 40, r = 0.449; p less than 0.01) and free triiodothyronine (n = 40, r = 0.546; p less than 0.01). The urine ANP concentration in patients with untreated hyperthyroidism was markedly higher than in normal subjects (p less than 0.01), but in untreated hypothyroidism not significantly different from normal.     

Alpha-human atrial natriuretic polypeptide (alpha-hANP) in normal volunteers and patients with heart failure or hypertension

The presence of alpha-hANP immunoreactive material in human heart and plasma was investigated with a specific and sensitive radioimmunoassay and immunohistochemical method. It was found that alpha-hANP immunoreactive staining of specific atrial granules was located around the nucleus of atrial cardiocytes. No immunoreactive staining was found in the ventricle. The content of immunoreactive hANP was 0.5 pmol/mg protein in the atria and 0.11 +/- 0.01 pmol/ml in the plasma of 26 normal volunteers. In 16 patients with congestive heart failure and 26 patients with essential hypertension, the plasma level of immunoreactive alpha-hANP was significantly lower than that in normal humans. The above evidence indicate that alpha-hANP is a putative hormone secreted by human atrium. A relative shortage of alpha-hANP in the circulatory system may be involved in the mechanism of heart failure and hypertension.     

Direct radioimmunoassay for human atrial natriuretic peptide (hANP) and its clinical evaluation

A direct radioimmunoassay for the rapid and accurate detection of human ANP from unextracted plasma is described. The sensitivity was approximately 50 pg/ml, respectively 2.5 pg/tube, the intra-assay variation 4%, and the inter-assay variation less than 12%. Rat ANP (1-28, 5-25, 5-27 and 5-28), oxydized and reduced hANP as well as plasma samples from various patients run in parallel to the 1-28 hANP standard curve. These findings imply, that the antibody primarily recognizes the mid-region (amino acids 6-25) of the intact ANP, that the C-terminal portion further increases the immunoreactivity, and that circulating plasma hANP is reliably measured. Plasma hANP ranged from 50-166 pg/ml (mean +/- SD: 98.3 +/- 44.6) in healthy individuals, there was no significant difference between samples were drawn in upright or lying position, the apparent half-life of injected hANP was 5.65 minutes. Patients with liver cirrhosis revealed significantly higher hANP levels of 244.5 +/- 173.5 pg/ml. Patients with various forms of cardiac disease had hANP concentrations ranging from 50 to 1744 pg/ml, depending at least partially on the right atrial pressure. No difference was observed if the samples were drawn from either right or left intracardial locations. Our findings with this system demonstrate that hANP is reliably measured even without prior extraction.     

Chicken atrial natriuretic peptide (chANP) and its secretion

Summary An immunohistochemical study using antiserum raised against synthetic chicken natriuretic polypeptide was used to investigate the distribution of this peptide in the chicken heart. Immunoreactive cells, both in the atrial and ventricular walls, were identified by electron microscopy, and electron-dense granules in the atrial and ventricular cardiocytes were revealed to be storage sites of the peptide. The electron-dense material, thought to be the peptide, was found in the sarcoplasmic reticulum, and it is suggested that a secretory pathway of the peptide through the latter to extracellular space, may be present, in addition to an exocytotic one.     

Superactive analogs of the atrial natriuretic peptide (ANP)

Three analogs of the atrial natriuretic peptide ANP(105-126), lacking the N-terminal exocyclic peptide segment and containing 2-mercaptoacetic acid, 3-mercaptopropionic acid or 4-mercaptobutyric acid in place of the cysteine residue in position 105 of the peptide sequence, were synthesized by the solid-phase method. The resulting des-amino analogs showed 2 to 4 times higher diuretic/natriuretic activity than the most active natural ANP and displayed a potent hypotensive effect as well. All three analogs were relatively less potent in various in vitro bioassays and in a binding assay, indicating that their high activities in vivo may be due to resistance to enzymatic degradation and to reduced non-specific tissue adsorption. These compounds not only will serve as useful pharmacologic tools but also represent prototypes for the development of further reduced-size ANP analogs.     

Degradation of human atrial natriuretic peptide by human brain membranes

Human atrial natriuretic peptide (Ser 99-Tyr 126) was rapidly degraded by both choroid plexus and hypothalamic membranes with a complex pattern of cleavage. The use of protease inhibitors allowed a preliminary characterization of the enzymes involved in the hydrolysis of the Ser-Phe and Phe-Arg bonds of iodine-labelled atrial natriuretic peptide.The C-terminal tripeptide was generated by three different enzymatic activities acting on the Ser-Phe bond: endopeptidase 24.11, a phosphoramidon-insensitive metallopeptidase and a thiol protease. Peptides like substance P, neurotensin, bradykinin inhibited the cleavage of the Ser-Phe bond of atrial natriuretic peptide. The C-terminal tripeptide was further degraded by aminopeptidases. Cleavage of the C-terminal dipeptide was inhibited by aprotinin, suggesting the contribution of brain kallikrein in the formation of this metabolite.These results show that many different proteases were involved in the hydrolysis of the C-terminal sequence of atrial natriuretic peptide, at least in vitro and underline the complexity of neuropeptide catabolism by brain preparations.     

A highly sensitive radioimmunoassay of atrial natriuretic peptide (ANP) in human plasma and urine

A highly sensitive radioimmunoassay has been established for measurement of human plasma and urine concentrations of atrial natriuretic peptide (ANP) and requires no extraction or concentration process such as Sep-Pak C-18 cartridge treatment. An antiserum was prepared from rabbits immunized with alpha-human ANP (alpha-hANP) coupled with bovine-thyroglobulin. The sensitivity of this method was 0.3 pg/tube of synthetic alpha-hANP utilized as authentic standard. Recovery of alpha-hANP spiked to plasma and urine was 97.7 +/- 15.4% and 97.1 +/- 9.5% (mean +/- SD), respectively. Plasma and urinary ANP concentrations versus assay data showed satisfactory linearity. In 124 healthy subjects, the plasma ANP-concentration was 31.7 +/- 12.0 pg/ml. Two different molecular forms of ANP in plasma and a single form in urine were found by gel permeation chromatography.     

Lack of atrial natriuretic peptide receptors in human aldosteronoma

The effect of synthetic alpha-human atrial natriuretic peptide (ANP) on aldosterone secretion was studied in human aldosterone producing adrenocortical adenoma obtained surgically from a patient with primary aldosteronism and in human apparently normal adjacent adrenal cortical tissues obtained from a patient with pheochromocytoma, in vitro. Apparently normal adrenal cortical tissue responded to ANP with the known inhibition of aldosterone secretion. In contrast, the aldosterone producing adenoma did not respond to ANP. When stimulated by either ACTH or angiotensin II, there is no inhibition by ANP in the adenoma tissue, whereas normal tissue was inhibited. Immunohistochemical examination utilizing an ANP-receptor antiserum demonstrated that there was no evidence of binding site in the cortical adenoma, in contrast, zona glomerulosa cells in the cortical tissues adjacent to either aldosterone producing adenoma or pheochromocytoma were densely stained. This apparent lack of ANP-receptors is an associated finding with the hypersecretion of aldosterone in the aldosterone producing adenoma.     

Plasma and atrial levels of atrial natriuretic peptide (ANP) in pulmonary hypertensive rats

Immunoreactive atrial natriuretic peptide (IR-ANP) was measured in plasma and atrium of normal and monocrotaline induced pulmonary hypertensive rats (PH rats). In these animals, there was right ventricular hypertrophy and right ventricular systolic pressure was elevated. Fourteen days after a single dose of monocrotaline (40 mg/kg), plasma IR-ANP concentrations were significantly elevated (964.3 +/- 63.0 pg/ml vs. 521.0 +/- 81.9 pg/ml in controls, p less than 0.001). Tissue levels of IR-ANP in the right atrium in PH rats was significantly lower than those in the controls (45.1 +/- 3.9 ng/mg vs. 240.5 +/- 10.4 ng/mg, p less than 0.001), while there was no significant difference in tissue levels of atrial IR-ANP in the left atrium between the two groups. Thus, development of pulmonary hypertension led to an increase in release of ANP from the right atrium.     

Expression of mRNA for atrial natriuretic peptide receptor guanylate cyclase (ANPRA) in human retina

Summary 1. Guanylate cyclase plays an important role in the visual cycle. Here we report the mRNA expression for the atrial natriuretic peptide receptor type A form of guanylate cyclase (ANPRA) in human retina.2. Polymerase chain reaction using two sets of primers on the cDNAs reverse-transcribed from human retinal poly(A)⁺ RNA amplified two products under two different reaction conditions. The primers used in the reaction were designed from the reported sequence of human placental ANPRA cDNA.3. Sequencing of the amplified products showed 100% sequence homology to the human placental ANPRA gene. Northern blot analysis indicated the presence of a 4.4-kb ANPRA mRNA in human retina, similar to that present in human brain.     

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.     

A new polymorphic restriction site at the human atrial natriuretic peptide (hANP) gene locus

A unique two allele polymorphism for both HpaII and SmaI is described in the second intron of the human atrial natriuretic peptide gene. It should be a useful marker of this candidate gene in familial susceptibility to hypertension.     

Expression of natriuretic peptide receptor mRNA and functional response to atrial natriuretic peptide and C-type natriuretic peptide in rainbow trout (Oncorhynchus mykiss) head kidney leucocytes

The stimulatory effect of vasomodulatory natriuretic peptide hormones on macrophages and peripheral blood leucocytes in mammals is well-established. However, the relationship in lower vertebrates has not been characterised. Expression of atrial natriuretic peptide, ventricular natriuretic peptide and C-type natriuretic peptide-1, and the guanylyl cyclase-linked (GC) natriuretic peptide receptor-A and -B-type receptors (NPR-A and NPR-B, respectively) was determined by PCR from the mRNA of rainbow trout head kidney leucocytes yielding gene fragments with 100% homology to the same respective natriuretic peptide and NPR-A and -B sequences obtained from other rainbow trout tissues. A mixed population of isolated rainbow trout head kidney leucocytes was stimulated in vitro with trout atrial natriuretic peptide (specific NPR-A agonist) and trout C-type natriuretic peptide (NPR-A and -B agonist) as well as the cGMP agonist 8-bromo-cGMP or the GC inhibitor 8-bromo-phenyl-eutheno-cGMP. Respiratory burst was stimulated by trout atrial natriuretic peptide, trout C-type natriuretic peptide-1 and 8-bromo-cGMP in a dose dependant manner with the highest activity as a result of stimulation with trout C-type natriuretic peptide-1 in excess of that achieved by phorbol myristate acetate (PMA). Equimolar concentrations of the inhibitor, inhibited the respiratory burst caused by the natriuretic peptides and 8-bromo-cGMP. The natriuretic peptide receptors on rainbow trout head kidney leucocytes appear to have a stimulatory function with regard to respiratory burst that is activated through a cGMP second messenger pathway and the natriuretic peptides expressed in the head kidney leucocytes may well act in a paracrine/autocrine manner.     

Release of atrial natriuretic peptide by atrial distension

A heterologous radioimmunoassay was used to measure the concentration of immunoreactive atrial natriuretic peptide (iANP) in plasma from the femoral artery of eight chloralose anaesthetized dogs. Mitral obstruction which increased left atrial pressure by 11 cmH2O increased plasma iANP from 97 +/- 10.3 (mean +/- SE) to 135 +/- 14.3 pg/mL. Pulmonary vein distension increased heart rate but did not increase plasma iANP. Bilateral cervical vagotomy and administration of atenolol (2 mg/kg) did not prevent the increase in iANP with mitral obstruction. Samples of blood from the coronary sinus had plasma iANP significantly higher than simultaneous samples from the femoral artery confirming the cardiac origin of the iANP. Release of iANP depends on direct stretch of the atrium rather than on a reflex involving left atrial receptors.