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.     

Alpha-human natriuretic polypeptide (alpha-hANP) specific binding sites in bovine adrenal gland

The effects of synthetic alpha-human atrial natriuretic polypeptide (alpha-hANP) on steroidogenesis in bovine adrenocortical cells in primary monolayer culture were investigated. alpha-hANP did not inhibit basal aldosterone secretion. alpha-hANP induced a significant dose-dependent inhibition of basal levels of cortisol and dehydroepiandrosterone (DHEA) secretion and also of ACTH (10(-8) M)-stimulated increases in aldosterone, cortisol and DHEA secretion. Visualization of [125I]alpha-hANP binding sites in bovine adrenal gland by an in vitro autoradiographic technique demonstrated that these sites were highly localized in the adrenal cortex, especially the zona glomerulosa. These results suggest that the adrenal cortex may be a target organ for direct receptor-mediated actions of alpha-hANP.     

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.     

Effects of human atrial natriuretic polypeptide on pancreatic exocrine secretion in the dog

We examined the effects of atrial natriuretic polypeptide (hANP) on exocrine function in the isolated and blood-perfused dog pancreas in situ. Intra-arterial injection of hANP (1-10 micrograms) resulted in the dose-dependent increases of the pancreatic juice secretion. The secretory activity of 3 micrograms of hANP was approximately equal to one third of the secretory activity of 0.1 units of secretin. The use of hANP increased the concentration of bicarbonate but not that of sodium and protein in the pancreatic juice as compared with the basal values. These secretory responses to hANP were not inhibited by treatment with haloperidol, sulpiride, phentolamine, propranolol, atropine, cimetidine or ethacrynic acid. These results suggest that hANP acts directly on the pancreatic exocrine gland to stimulate pancreatic secretion; without, however, increasing sodium excretion. The mechanism of this effect remains to be elucidated.     

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.     

Expression of human atrial natriuretic polypeptide gene in Cos 7 cells

Cos 7 cells transfected with human atrial natriuretic polypeptide (hANP) gene with SV40 enhancer and replication origin sequences expressed hANP gene. The expressed RNA was indistinguishable from native hANP mRNA and the transcribed protein seemed to be properly processed to alpha-hANP and beta-hANP. This system provides a useful approach to investigate the processing of hANPs and the structure-function relationship of amino acid sequences of hANPs.     

Internalization of atrial natriuretic peptide by adrenal glomerulosa cells

Internalization of 125I-labelled atrial natriuretic peptide ([ 125I]ANP) by rat adrenal glomerulosa cells in vivo was investigated by means of an ultrastructural autoradiographic approach. One to 30 min after IV injection of [125I]ANP, silver grains were found, at the light microscope level, over all glomerulosa cells; coinjection of 20 micrograms of unlabelled ANP inhibited this binding by 64%. At the electron microscope level, the time-course study indicated maximal silver grain densities in plasma membranes 1 min after IV injection; grains were detected in mitochondria (external membranes and matrix) 2 min after injection, with maximal labelling at 15 min. The cytoplasmic matrix was labelled only 30 min after injection. During the time-course, labelling of nuclei, Golgi apparatus, and lysosomes was minimal. The data suggest that after binding to plasma membranes ANP is rapidly internalized and distributed within glomerulosa cells. The association of radioactivity with mitochondria suggests that ANP may have intracellular sites of action complementary to those on plasma membranes.     

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.     

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.     

Rat atrial natriuretic polypeptide stimulation of adrenal zona glomerulosa cell growth

Rat atrial natriuretic polypeptide (rANP) was found to stimulate [3H] thymidine incorporation into the DNA of bovine adrenal glomerulosa cells in a primary culture in a dose-dependent manner. The minimum effective dose was a very low concentration (10(-12) M of ANP), suggesting that ANP had a physiological effect. These findings are the first indication that ANP possesses growth-stimulating activity with regard to adrenal zona glomerulosa cells.     

Regulated expression of human atrial natriuretic polypeptide gene in mouse L cells

To investigate whether the human atrial natriuretic polypeptide (hANP) gene is responsive to glucocorticoid, we co-introduced the hANP gene (with SV40 enhancer) with HSV-tk gene into mouse tk- L cells. The transformants with hANP gene with SV40 enhancer expressed hANP specific RNAs. The administration of 1 microM dexamethasone reduced the expressed hANP specific RNAs, especially those that had a physiological initiation site. These results suggest that the hANP gene is really a glucocorticoid responsive gene and may be negatively regulated by glucocorticoid.     

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.     

Inhibition of aldosterone secretion by atrial natriuretic peptide in chicken adrenocortical cells

Dispersed chicken adrenocortical cells were preincubated with atrial natriuretic peptide (rANP), sodium nitroprusside (SNP) or 8-bromo cyclic GMP, followed by incubations with ACTH, chicken PTH, cholera toxin or various steroid intermediates of aldosterone production. Cyclic AMP production and aldosterone secretion were evaluated, in order to determine the sites of ANP inhibition in the sequence of events leading to aldosterone secretion. Dose-dependent inhibitory effects on ACTH-stimulated aldosterone secretion by rANP and SNP were observed. Both agents appeared to stimulate cGMP production by the particulate fraction of the avian adrenocortical cells. Aldosterone production, stimulated by cyclic AMP agonists such as ACTH, chicken PTH and cholera toxin, was significantly inhibited by ANP. On the other hand, ANP did not interfere with production or degradation of cAMP. Each of the aldosterone intermediates--pregnenolone, progesterone, 11-deoxycorticosterone and corticosterone--promoted aldosterone production when included in the incubation media. Atrial natriuretic peptide and SNP inhibited aldosterone secretion when enhanced by the intermediates, by about 40-60%, but the ACTH-stimulated secretion was inhibited by over 90%. The results suggest two sites of inhibition by ANP in the pathway of aldosterone synthesis and secretion: synthesis of cholesterol or pregnenolone, and conversion of corticosterone to aldosterone. The inhibition by 8-bromo cGMP of aldosterone secretion and the similar sites of inhibition for ANP and SNP suggest that cyclic GMP mediates the inhibition in both cases.     

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.     

Pancreatic polypeptide inhibits somatostatin secretion

Pancreatic polypeptide (PP) is a major agonist for neuropeptide Y4 receptors (NPY4R). While NPY4R has been identified in various tissues, the cells on which it is expressed and its function in those cells has not been clearly delineated. Here we report that NPY4R is present in all somatostatin-containing cells of tissues that we tested, including pancreatic islets, duodenum, hippocampus, and hypothalamus. Its agonism by PP decreases somatostatin secretion from human islets. Mouse embryonic hippocampal (mHippo E18) cells expressed NPY4Rs and their activation by PP consistently decreased somatostatin secretion. Furthermore, central injection of PP in mice induced c-Fos immunoreactivity in somatostatin-containing cells in the hippocampus compared with PBS-injected mice. In sum, our results identify PP as a pivotal modulator of somatostatin secretion.     

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.     

Alpha-human atrial natriuretic peptide is a coronary vasodilator in the Langendorff-perfused guinea pig heart

The circulating form of atrial natriuretic peptide is now believed to be composed of 28 amino acids (1). Therefore, we studied the coronary vasoactivity of the 28 amino acid, alpha-human atrial natriuretic peptide (alpha-hANP) in five isolated guinea pig hearts Langendorff-perfused at constant pressure (46 mmHg) with Krebs-Henseleit solution. The reactivity of the coronary bed was assured in each heart with bolus injections of norepinephrine, adenosine, and the vasoconstrictor atrial natriuretic peptide, atriopeptin II (APII). APII was a coronary constrictor in each of these five hearts. Nineteen boluses of alpha-hANP were administered to the five hearts, spanning the range 1.6 to 64 nmol/g wet heart weight. alpha-hANP was vasodilator in all five hearts. The equation for the regression of y = flow, % increase, on x = dose, nmol/g, is y = 17.98 logx - 4.11. The correlation coefficient, r, is 0.83, and the coefficient of determination, r2, is 0.69. Analysis of variance of the regression of y on x yields an F statistic of 36.9, P less than 0.00001. These results indicate that coronary vasodilation is correlated with dose of alpha-hANP over much of the range 1.6-64 nmol/g.     

Release of atrial natriuretic polypeptide by graded right atrial distension in anesthetized dogs

In order to verify the contribution of right atrial pressure to atrial natriuretic polypeptides (ANP) release, we measured plasma levels of immunoreactive (ir)-ANP when graded rise of right atrial pressure was executed in anesthetized dogs. Increasing right atrial pressure (RAP) from 2.7 +/- 0.6 to 9.0 +/- 0.7 mmHg, plasma levels of ir-ANP in aorta tended to increase by 33% but not significantly (p greater than 0.05). However, when RAP was increased from 9.0 +/- 0.7 to 17.0 +/- 1.1 mmHg, ir-ANP levels in aorta were significantly (p less than 0.05) increased by 132% of control within 5 min from the start of RAP elevation. The RAP elevation produced a sustained increase in plasma levels of ir-ANP. There was a positive correlation between right atrial pressure and plasma levels of ir-ANP. The plasma levels of ir-ANP were similar between aorta and pulmonary artery. These results demonstrate that increasing atrial pressure is closely correlated with ANP release and ANP is not greatly metabolized by pulmonary circulation.     

Immunohistochemical localization of atrial natriuretic polypeptide (ANP) in human atrial and ventricular myocardiocytes

To date, there have been few immunohistochemical investigations of atrial natriuretic polypeptide (ANP) in human cardiac tissue, especially the ventricles. In this study, myocardial tissue was obtained from two sources: the bilateral atria and ventricles at autopsy; and biopsy tissues from the right auricle and left ventricle of a patient with myocardial infarction undergoing surgery. These tissues were examined by the avidin-biotin immunoperoxidase technique using three kinds of primary ANP-antibodies. ANP-immunoreactivity was observed in the perinuclear region of myocytes of all tissues examined. The intensity of the reaction was stronger in atrial tissue, weaker in ventricular tissue. In the later tissue, the positive-staining myocytes were not part of the pulse-conducting system. Although the tissues we studied were not obtained from normal hearts, our data demonstrates that ANP-reactivity can be detected in ventricular myocytes outside the pulse-conducting system.