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.     

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.     

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.     

Free and bound forms of atrial natriuretic peptide (ANP) in rat plasma: preferential increase of free ANP in spontaneously hypertensive rats (SHR) and stroke-prone SHR (SHRSP)

Free and bound forms of atrial natriuretic peptide (ANP) in rat plasma were analysed by gel permeation chromatography combined with a radioimmunoassay (RIA) for rat ANP (rANP). Gel permeation chromatography showed two immunoreactive peaks in rat plasma, one corresponding to alpha-rANP, rANP(99-126), and the other eluted at a high molecular weight, clearly different from gamma-rANP, rANP(1-126). The chromatographic profile of rat plasma after incubation with synthetic alpha-rANP demonstrated that the high molecular immunoreactivity had ANP-binding capacity. This bound form of ANP was almost totally excluded following extraction procedure, therefore, the immunoreactive ANP (ir-ANP) measured with the extraction assay was mainly free ANP. On the other hand, direct RIA may detect not only the free but also the bound form of ANP. Using both direct RIA and the extraction method, bound forms of plasma ANP in spontaneously hypertensive rats (SHR) and stroke-prone SHR (SHRSP) were compared to normotensive Wistar Kyoto rats (WKY). Bound forms of plasma ANP in 20-week-old SHR and SHRSP were significantly higher than that in age-matched WKY. The ratio of free/bound form of plasma ANP in SHR and SHRSP also significantly increased compared to WKY, indicating a preferential increase in free ANP in the plasma of these hypertensive rats. These findings suggest that a bound form of ANP may be present in rat plasma and that it may play some pathophysiological role in the hypertension of SHR and SHRSP. Increased free ANP in plasma may indicate a compensatory increase in ANP release in these hypertensive rats.     

Alpha-human atrial natriuretic polypeptide binding sites in human adrenal membrane fractions

In a previous study evidence was presented that synthetic alpha-human atrial natriuretic polypeptide (alpha-hANP) significantly inhibits the secretion of aldosterone, cortisol, and dehydroepiandrosterone (DHEA) from cultured human adrenal cells. In the present work using crude membrane fractions prepared from human adrenal tissues obtained at autopsy, we noted the existence and molecular weight of specific binding sites for [125I]alpha-hANP. The mean maximal binding capacity (Bmax) and dissociation constant (Kd) of 4 human adrenal membrane fractions were 8.0 +/- 1.6 fmol/mg protein and 25.7 +/- 7.4 pM, respectively, as calculated by Scatchard plot analysis. The interaction of [125I]alpha-hANP with the high-affinity binding sites in human adrenal membrane fractions was unaffected by the addition of lysine vasopressin (LVP), somatostatin-14 and angiotensin-II (A-II). When the membrane fractions were incubated with [125I]alpha-hANP and then cross-linked with disuccinimidyl suberate (5 mM), the 67,000-Da protein was specifically radiolabeled. The very high affinity of [125I]alpha-hANP binding sites suggests that human adrenal steroidogenesis may be influenced by plasma levels of hANP, under physiological conditions.     

The lung as a possible target organ for atrial natriuretic polypeptide secreted from the heart

Using a specific radioimmunoassay (RIA) for alpha-rat atrial natriuretic polypeptide (alpha-rANP), we have demonstrated the presence of a considerable amount (6.10 +/- 0.38 ng/g) (mean +/- SE) of alpha-rANP-like immunoreactivity (alpha-rANP-LI) in the rat lung, the first organ through which atrial natriuretic polypeptide (ANP) released from the heart passes. High performance gel permeation chromatography coupled with the RIA revealed that most of alpha-rANP-LI eluted at the position of a low molecular weight form corresponding to synthetic alpha-rANP. In 2- or 5-day water-deprived rats, the concentration and content of alpha-rANP-LI in the lung decreased significantly compared with those of control rats. In addition, water-deprivation induced a significant decrease in the plasma concentration of alpha-rANP-LI simultaneously determined. There was a significant positive correlation between the concentrations of alpha-rANP-LI in the lung and plasma (r = 0.591, P less than 0.01). These results indicate the presence of ANP in the lung and suggest physiological roles of ANP in pulmonary function.     

Atrial and brain natriuretic peptide in adrenal steroidogenesis.

We elucidated the role of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in human and bovine adrenocortical steroidogenesis. The urinary volume, sodium excretion and cyclic GMP (cGMP) excretion and plasma cGMP were markedly increased by the synthetic alpha-human ANP (alpha-hANP) infusion in healthy volunteers. Plasma arginine vasopressin (AVP) and aldosterone levels were significantly suppressed. Both ANP and BNP inhibited aldosterone, 19-OH-androstenedione, cortisol and DHEA secretion dose-dependently and increased the accumulation of intracellular cGMP in cultured human and bovine adrenal cells. alpha-hANP significantly suppressed P450scc-mRNA in cultured bovine adrenal cells stimulated by ACTH. Autoradiography and affinity labeling of [125I]hANP, and Scatchard plot demonstrated a specific ANP receptor in bovine and human adrenal glands. Purified ANP receptor from bovine adrenal glands identified two distinct types of ANP receptors, one is biologically active, the other is silent. A specific BNP receptor was also identified on the human and bovine adrenocortical cell membranes. The binding sites were displaced by unlabelled ANP as well as BNP. BNP showed an effect possibly via a receptor which may be shared with ANP. The mean basal plasma alpha-hANP level was 25 +/- 5 pg/ml in young men. We confirmed the presence of ANP and BNP in bovine and porcine adrenal medulla. Plasma or medullary ANP or BNP may directly modulate the adrenocortical steroidogenesis. We demonstrated that the lack of inhibitory effect of alpha-hANP on cultured aldosterone-producing adenoma (APA) cells was due to the decrease of ANP-specific receptor, which caused the loss of suppression of aldosterone and an increase in intracellular cGMP.     

Molecular forms of atrial natriuretic polypeptides circulating in human plasma

Molecular forms of atrial natriuretic polypeptides circulating in human plasma were analyzed by chromatographic separation, coupled with radioimmunoassay. Analyses were done with human plasma samples taken from 24 human subjects, divided into groups of healthy volunteers, patients with renal disease, and patients with heart disease, with 8 subjects in each group. Although alpha-hANP was found as a major circulating form in most of the plasma specimens (16 cases), beta-hANP accompanied by alpha-hANP was found in 6 cases including 2 healthy volunteers. In addition, hANP-immunoreactive macromolecule, which is likely a bound form of hANP, was found to some extent in all the specimens tested. Diversity of human plasma in ANP molecular distribution was discussed in connection with a radioimmunoassay for plasma ANP.     

Immunoreactive alpha-human atrial natriuretic polypeptide in human plasma

A radioimmunoassay (RIA) has been developed for the determination of alpha-human atrial natriuretic polypeptide (alpha-hANP) in human plasma. Antibodies generated in rabbits recognized alpha-hANP-related peptides containing the subsequence flanked by two cysteine residues at position 7 and 23 equally. Radiolabelled tracer prepared by iodination with chloramine-T method was purified by high performance liquid chromatography. Immunoreactive (ir-) alpha-hANP was extracted from human plasma by Sep-Pak C18 column. The plasma ir-alpha-hANP concentrations in normal, healthy adults were 178 +/- 16 pg/ml in male and 182 +/- 18 pg/ml in female, respectively. Plasma ir-alpha-hANP increased significantly after acute intravenous administration of isotonic saline. Plasma levels were elevated in patients with various disease states accompanying increased body fluid volume, whereas those in patients with idiopathic edema were decreased despite excessive salt and water retention. These results suggest that alpha-hANP plays an important role in the regulation of body fluids and may have primary or secondary pathophysiological significance in various disease states.     

Plasma factors controlling atrial natriuretic peptide (ANP) aggregation: role of lipoproteins

We have previously shown that human plasma atrial alpha-natriuretic peptide (alpha-hANP) sequestering is a protective phenomenon against amyloid aggregation. In the present work, the possible role of lipoproteins as alpha-hANP binding factors has been investigated in vitro using an experimental model, developed in our laboratory, that allows to work at physiological concentrations. This approach consists of gel filtration on Sephacryl S-300 HR of big alpha-[(125)I]hANP generated in phosphate buffered saline or in human normal plasma supplemented or not with lipoproteins. The results of these experiments indicate that high density lipoproteins (HDL) are responsible for the ANP binding phenomenon observed in vitro, while low density lipoproteins and very low density lipoproteins do not directly interact with ANP. Moreover, the HDL remodeling process occurring in vitro has been analyzed during plasma incubation by monitoring the redistribution of lipids and apolipoproteins among the HDL subclasses. The changes in HDL size and composition observed in incubated plasma were compared with the redistribution of endogenous and labeled big ANP. The obtained results revealed that both tend to follow the molecular rearrangement in plasma of apolipoprotein A-I containing particles and suggested that, among HDL species, the small particles are mainly involved in the ANP binding phenomenon. This hypothesis was further demonstrated by ligand blotting experiments that confirmed the existence of differences in the ability of HDL particles to bind alpha-[(125)I]hANP.     

The use of a monoclonal antibody to measure plasma atriopeptins in rat

A monoclonal antibody with specificity for atrial natriuretic peptides (ANP) was produced, that can be used for the radioimmunological determination of ANP-immunoreactivity (ANP-IR) in rat plasma. The antibody recognizes atriopeptin I, II, III, as well as alpha-hANP and alpha-hANP fragment (7-28) and does not crossreact with ANP-fragments (13-28) and (18-28). Plasma levels of ANP-IR in conscious Wistar rats were determined before and after volume-loading. Basal plasma levels of ANP-IR were 108 +/- 12 pg/ml, and after volume-loading increased to 800 +/- 59 pg/ml.     

Defective regulation and action of atrial natriuretic peptide in type 2 diabetes.

Increased plasma atrial natriuretic peptide (ANP) levels and impaired ANP action have been reported in patients with diabetes or insulin resistance. The aim of this study was to assess the interaction between insulin and ANP in type 2 diabetes. In 12 normotensive, normoalbuminuric type 2 diabetics, we infused insulin at a high (6.6 pmol/min/kg) or, on a different day, at a low rate (0.6 pmol/min/kg) during 4 hours of isoglycemia under isovolumic, isoosmolar conditions. The normal response was established in 12 healthy volunteers using an identical protocol. Despite higher baseline ANP levels (17.7 +/- 2.8 vs. 10.8 +/- 1.8 pg/ml, p = 0.04), urinary sodium excretion was similar in diabetics and controls (113 +/- 8.5 vs. 102 +/- 8.8 mEq/24 hours, p = ns). In both groups, hyperinsulinemia caused a decrease in blood volume (0.33 +/- 0.10 l, p < 0.01), diastolic blood pressure (6 %, p < 0.02), and natriuresis. However, plasma ANP decreased in controls (from 12.7 +/- 1.9 to 8.6 +/- 1.4 pg/ml, p = 0.01) but not in type 2 diabetics (15.1 +/- 2.7 vs. 17.2 +/- 3.8 pg/ml, p = ns). We conclude that ANP release is resistant to volume stimulation in type 2 diabetic patients, and natriuresis is resistant to ANP action. This dual disruption of ANP control may play a role in blood pressure regulation in diabetes.     

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.     

Existence of atrial natriuretic polypeptide in kidney

Using a specific radioimmunoassay (RIA) for alpha-atrial natriuretic polypeptide (alpha-ANP), we have demonstrated the presence of alpha-rat ANP-like immunoreactivity (alpha-rANP-LI) in the rat kidney which is considered to be a target organ for atrial natriuretic polypeptides released from the heart. Most of alpha-rANP-LI was localized in the cortex. High performance gel permeation chromatography coupled with the RIA revealed that renal alpha-rANP-LI was eluted at the position of a low molecular weight form corresponding to alpha-rANP without detectable amounts of high molecular weight forms. This is in contrast to the observation that gamma-rANP, a high molecular weight form of 13k daltons, is the dominant form of alpha-rANP-LI in the rat atrium. In water-deprived rats, the concentration and content of alpha-rANP-LI in the kidney showed a significant decrease compared with control rats. In addition, the alpha-rANP-LI concentration and content in this organ revealed a substantial decrease after perfusion with physiological saline. These results indicate the existence of atrial natriuretic polypeptide (ANP) in the kidney and suggest that part of renal ANP may originate from the heart.     

Atrial natriuretic peptide in the sheep

To study atrial natriuretic peptide (ANP) physiology in the chronically catheterized pregnant sheep model we developed a heterologous radioimmunoassay for ovine ANP using an antiserum raised against 1-28 human ANP. This antiserum (Tor I) is specific for the aminoterminus of the human ANP molecule and shows little cross reaction with any carboxyterminus ANP fragments. Ovine ANP immunoreactivity was characterized using this antiserum and a commercially available carboxyterminus ANP antiserum obtained from Peninsula Laboratories. Each antiserum detected 2 peaks of immunoreactivity in ovine atrial extracts chromatographed on a Biogel P-10 column. The minor peak migrated at a position close to 125I-human ANP whereas the major peak represented a larger molecular weight species of ANP. Examination of gel filtration eluates of ovine plasma extracts showed one immunoreactive ANP peak using the Tor I assay system and 2 peaks with the Peninsula Laboratories assay. Plasma immunoreactive ANP levels were determined in 9 sheep using both radioimmunoassay systems. Mean (+/- SEM) levels were similar using the Peninsula Laboratories and the Tor I assay systems (57 +/- 8 pg/ml versus 43 +/- 4 pg/ml, P greater than 0.05). Using the Tor I antiserum, fetal plasma immunoreactive ANP levels were found to be significantly higher than maternal levels (188 +/- 17 versus 48 +/- 8 pg/ml, P less than 0.01) whereas pregnant and nonpregnant adult sheep had similar plasma immunoreactive ANP levels (48 +/- 8 versus 43 +/- 4 pg/ml, P greater than 0.05). Disappearance curves of synthetic human ANP from the plasma of maternal and fetal sheep were assessed using both immunoassay systems and found to be similar.     

Pharmacokinetics of synthetic alpha-human atrial natriuretic polypeptide in normal men; effect of aging

We reported that plasma human atrial natriuretic polypeptide (hANP) in healthy aged men was significantly higher than that in young men, presumably due to a diminished cellular response to endogenous hANP in the aged subjects (J. Clin. Endocrinol. Metab., 64 (1987) 81). To examine the effect of age on the metabolic clearance rate for hANP, synthetic alpha-hANP (2 micrograms/kg) was administered intravenously to healthy young (n=6) and aged (n=4) men. The plasma hANP was measured by a direct radioimmunoassay. The disappearance of alpha-hANP from plasma was characterized by a biexponential decay curve, in both groups. There was no difference of the initial phase between young and aged groups (young vs aged; 1.2 +/- 0.2 min vs 2.9 +/- 1.0 min), while the second phase of alpha-hANP disappearance in the aged group was significantly prolonged compared with that in the young individuals (young vs aged; 17.3 +/- 3.9 min vs 34.3 +/- 3.0 min, P less than 0.05). We tentatively conclude that the reduced metabolic clearance rates for hANP were responsible, in part, for the high plasma concentrations in the aged men.     

Decreased content in left atrium and increased plasma concentration of atrial natriuretic polypeptide in spontaneously hypertensive rats (SHR) and SHR stroke-prone

The atrial contents and concentrations, and the plasma concentrations of atrial natriuretic polypeptide (ANP) in spontaneously hypertensive rats (SHR) and SHR stroke-prone (SHRSP) were measured and compared with those of age-matched Wistar Kyoto rats (WKY) using a specific radioimmunoassay (RIA) for alpha-rat ANP (alpha-rANP). The contents of alpha-rANP-LI in the atria of SHR (19.0 +/- 0.9 micrograms, mean +/- SEM) and SHRSP (19.3 +/- 0.6 micrograms) were significantly lower than that of WKY (22.8 +/- 1.4 micrograms) (p less than 0.05). The atrial concentration of alpha-rANP-LI was also significantly lower in SHR (248.2 +/- 11.3 ng/mg, p less than 0.05) and tended to be lower in SHRSP (272.2 +/- 12.4 ng/mg) than that of WKY (300.0 +/- 14.2 ng/mg). Furthermore, the concentrations in the left auricles of SHR and SHRSP were significantly lower than that of WKY (p less than 0.01 and p less than 0.05, respectively). In contrast, no significant difference was observed in the alpha-rANP-LI concentrations in the right auricles of WKY, SHR and SHRSP. Gel filtration studies coupled with RIA showed that gel filtration profiles of the extracts from the right and left auricles of WKY, SHR and SHRSP were essentially identical. The plasma alpha-rANP-LI levels in SHR (260 +/- 34 pg/ml) and SHRSP (319 +/- 19 pg/ml) were significantly higher than that in WKY (170 +/- 17 pg/ml) (p less than 0.05 and p less than 0.01, respectively). These results suggest that the secretion of ANP from the heart is increased in SHR and SHRSP compared with WKY.     

Effect of synthetic atrial natriuretic polypeptide on hemorrhage-induced adrenocorticotropin secretion of the rat

The effect of synthetic alpha-human atrial natriuretic polypeptide (alpha-hANP) on the in vivo and in vitro release of ACTH and corticosterone was examined. In the in vivo study ACTH and corticosterone responses to rapid 2-ml/rat hemorrhage were measured in sixteen conscious rats after alpha-hANP administration. The hemorrhage increased plasma ACTH and corticosterone concentrations in the control group of rats (p greater than 0.01). ANP inhibited hemorrhage-induced ACTH secretion (p less than 0.05), but the plasma corticosterone response was not affected. In the in vitro study a high concentration of ANP (1 microM) reduced basal corticosterone secretion from the isolated rat adrenal gland (p less than 0.05), but the response to ACTH (10 ng/ml) and dibutyryl cyclic AMP (0.5 mM, 5.0 mM) was not affected. Our data suggest that ANP inhibits hemorrhage-induced ACTH secretion from the anterior pituitary but inhibits corticosterone secretion from the adrenal gland very weakly.     

Presence of endothelin-1-like immunoreactivity in human cerebrospinal fluid

By use of radioimmunoassay (RIA) specific for endothelin-1 (ET-1), we have studied whether ET-1-like immunoreactivity (LI) exists in human cerebrospinal fluid (CSF). Human CSF from patients with old cerebrovascular disease contained far greater amounts of ET-1-LI (28 +/- 1.3 pg/ml, n = 9) than normal human plasma. Reverse-phase HPLC coupled with ET-1 RIA revealed a single major ET-1-LI component in human CSF extract coeluting with standard ET-1. These data suggest ET-1 is a neuropeptide secreted from neural tissues in humans.     

The concentration of atrial natriuretic peptide (ANP) is decreased in plasma but not in atria in hypophysectomised rats

To study the role of the pituitary gland in the release of Atrial Natriuretic Peptide (ANP) plasma and atrial concentrations were measured both in intact and in hypophysectomized rats. The plasma concentration of ANP (pg/ml) was significantly (p less than 0.01) decreased from 143 +/- 35 to 82 +/- 29 (mean +/- SD, n) while the tissue concentration (ng/wet tissue mg) remained unchanged, 192 +/- 46 and 194 +/- 39, respectively. The total atrial amount of ANP (ug) was, however, significantly (p less than 0.01) decreased from 29.7 +/- 7.8 to 17.0 +/- 3.3 after hypophysectomy. In intact animals, a volume load (1.1ml/100 body weight g 0.9% NaCl) resulted in 2-fold (p less than 0.001) increase in the plasma ANP levels whereas similar load had no effects on plasma ANP levels in hypophysectomized animals. In both groups, the right atrial pressure was increased from about 2 to about 6 mmHg. We conclude that in the absence of pituitary gland the right atrial pressure and the atrial ANP concentration do not change but plasma ANP levels and the response to volume stimulus are attenuated.