Issei Tanaka

Atrial natriuretic factor in rat hypothalamus, atria and plasma: determination by specific radioimmunoassay.

A rapid and reproducible radioimmunoassay method was developed for rat atrial natriuretic factor (ANF)-IV. The method is also applicable to human atrial peptide. ANF was detected in rat hypothalamus (5.03 pmoles/g tissue), right (86.8 pmoles/mg tissue) and left atria (52.5 pmoles/mg tissue), and plasma (156 fmoles/ml). After high salt intake immunoreactive ANF in atria and plasma increased significantly, while a significant decrease was observed in hypothalamus. Gel chromatography revealed high and low molecular weight ANF in atria and hypothalamus while only a low molecular weight form was found in plasma.

Atrial pressure and secretion of atrial natriuretic factor into the human central circulation

Atrial natriuretic factor, a peptide found in mammalian cardiac atria, has natriuretic and vasodilatory properties that may be important in the regulation of intravascular volume. To study factors related to its release in human subjects, intracardiac pressures and plasma atrial natriuretic factor concentrations in the central circulation were measured in 34 patients with a variety of cardiovascular disorders. Plasma atrial natriuretic factor concentration increased from the inferior vena cava to the right atrium (76 +/- 24 to 162 +/- 37 pg/ml, p less than 0.001) and from the vena cava to the aorta (76 +/- 24 to 177 +/- 46 pg/ml, p less than 0.001). Mean right atrial pressure was positively correlated with atrial natriuretic factor concentration in the pulmonary artery (r = 0.58, p less than 0.001), and mean pulmonary capillary wedge pressure was positively correlated with concentration in the aorta (r = 0.64, p less than 0.001). In six patients whose atrial natriuretic factor concentrations were measured at two different levels of atrial pressure, increased atrial pressure was accompanied by increased atrial natriuretic factor concentration in the pulmonary artery (p less than 0.01) and aorta (p less than 0.01). Atrial natriuretic factor levels measured in fresh myocardium from a patient undergoing cardiac transplantation showed tissue concentrations in the atria 500-fold higher than tissue concentrations in the ventricles. These data document that atrial natriuretic factor is found in human atrial myocardium and suggest that it may be released in response to increased atrial pressure. Such a secretory release mechanism is consistent with the hypothesis that atrial natriuretic factor plays a role in the regulation of circulatory volume.

Immunocytochemical localization of atrial natriuretic factor in the kidney, adrenal medulla, pituitary, and atrium of rat.

Mammalian atria have previously been shown to produce a variety of peptides with natriuretic and vasorelaxant activities. Certain of these atrial natriuretic factors (ANF) have been localized immunocytochemically in secretory granules of atrial myocytes. However, the precise sites of action and extra-atrial synthesis or accumulation of ANF have not been identified immunocytochemically. In the present study, immunoreactive ANF was detected in rat atrial myocytes, intercalated cells of the renal collecting ducts, adrenal medullary chromaffin cells, and gonadotrophs of the anterior pituitary using an antibody against synthetic rat ANF-IV (H2N-Arg-Ser-Ser-Cys-Phe-Gly- Gly-Arg-Ile-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe- Arg-Try-COOH). The localization of ANF in specialized cells of the renal collecting tubules and ducts supports suggestions that these structures may be a site of natriuretic action of ANF. In addition, immunocytochemical localization of ANF in the rat adrenal medulla and anterior pituitary suggests the existence of alternate sites of action and/or synthesis. We believe these findings are important for a more complete understanding of the role of ANF in fluid and sodium regulation and of the participation of ANF in the development of sodium-dependent hypertension.

Effects of changes in water-sodium balance on levels of atrial natriuretic factor messenger RNA and peptide in rats

Responses of atrial mRNA, atrial peptide and plasma peptide of atrial natriuretic factor (ANF) to treatments to alter fluid volume were studied in rats using RNA dot hybridization assay and radioimmunoassay. Specific changes in the level of ANF mRNA relative to total atrial RNA were observed in atria from sodium restricted rats and water deprived then sodium loaded rats, demonstrating an association of change in water-sodium balance with the expression of ANF gene. The levels of mRNA and the immunoreactive ANF in plasma decreased to 30% and 15% of controls, respectively, on water-deprivation and then increased again to control levels after administering 1.8% NaCl solution, whereas atrial immunoreactive ANF increased to about twice the control on water-deprivation and decreased again after supplying NaCl solution, in parallel with the level of the hematocrit. These findings suggest that atrial ANF content is dependent more on ANF release than on biosynthesis.

Alterations in atrial and plasma atrial natriuretic factor (ANF) content during development of hypoxia-induced pulmonary hypertension in the rat

Abstract Distension of the atrial wall has been proposed as a signal for the increased release of atrial natriuretic factor (ANF) from atrial myocytes in response to perceived volume overload. To determine whether pressure changes resulting from hypertension in the pulmonary circulation may stimulate release of ANF, rats were exposed to chronic hypobaric hypoxia for 3 or 21 days and the ANF concentration in the atria and plasma were determined by specific radioimmunoassay. Exposure to chronic hypoxia resulted in significant increases in hematocrit at both 3 (p < 0.025) and 21 days (p < 0.005) and in the development of right ventricular hypertrophy (RVH) expressed as the ratio of the weight of the right ventricle to the weight of the left ventricle and septum (RV/LV+S) at both 3 (RV/LV+S = 0.278±0.005) and 21 days (RV/LV+S = 0.536±0.021). After 21 days, left atrial (LA) ANF content was significantly increased in hypoxic rats compared to controls (508±70 ng/mg tissue vs 302±37 ng/mg), while right atrial (RA) ANF content was significantly reduced (440±45 vs 601±58 ng/mg). At this time, plasma ANF concentration was significantly elevated compared to controls (238±107 pg/ml vs 101±10 pg/ml). These results suggest that the development of pulmonary hypertension following chronic hypobaric exposure induces altered atrial ANF content and increased plasma ANF concentration as a result of altered distension of the atrial wall.

Increased concentration of plasma immunoreactive atrial natriuretic factor in Dahl salt sensitive rats with sodium chloride-induced hypertension

In order to determine whether there is a relationship between genetically determined salt-induced hypertension and atrial natriuretic factor (ANF), a radio-immunoassay for ANF was applied to the determination of immunoreactive ANF in plasma, atrium, hypothalamus and pons of Dahl salt-sensitive (S) and -resistant (R) rats which were fed high- or low-salt diet for 7 weeks. A twofold higher concentration of plasma ANF was observed in high-salt S rats, which developed hypertension, compared with low-salt S rats or R rats on high or low salt, which were normotensive. No significant difference was seen in atrial concentrations of ANF between S and R rats. The brain ANF concentration of the high-salt group was lower than that of the low-salt group in both S and R rats. It is proposed that the elevation of plasma ANF in the hypertensive rats may reflect a compensatory mechanism induced by volume expansion in the salt-fed S rats.

Release of immunoreactive atrial natriuretic factor from rat hypothalamus in vitro.

Atrial natriuretic factor (ANF) had been found in brain tissues. Its role and the mechanisms by which it is produced and functions in the brain were not clear. We have initiated in vitro studies to find whether it is released from brain tissue and to elucidate the mechanism of its release. ANF was found to be released from rat hypothalamus by a depolarizing concentration of potassium and by a calcium-dependent mechanism. The ANF released was found to be predominantly a low molecular weight form. A small amount of high molecular weight form was also released. These results suggest that ANF produced in brain tissues is released, by a depolarization-induced and calcium-mediated mechanism, presumably from neuronal cells.

Discovery of atrial natriuretic factor in the brain: its characterization and cardiovascular implication.

Summary1.We have devised a radioimmunoassay for atrial natriueretic factor (ANF). Its application to rat brain extract led to the discovery of ANF in the brain. In addition to the hypothalamus and the pontine medullary region, it was widely distributed.2.ANF in the brain is stored in a low molecular weight form, in contrast to pro-ANF in the atria. Thus, the processing of pro-ANF in the bran neuronal cells is different from that in the atria.3.ANF was found in the anterior and posterior lobes of the pituitary, the peripheral ganglia, adrenergic neurons, and the adrenal medulla.4.Brain ANF suppressed stimulated dipsogenesis, basal and stimulated vasopressin release, and angiotensin II-stimulated pressor effects.5.ANF in the peripheral neuronal system inhibits catecholamine synthesis and release. Thus, central ANF functions to reduce the peripheral fluid volume and vascular tone in concert with the peripheral ANF.

Increased level of atrial natriuretic peptide messenger RNA in the hypothalamus and brainstem of spontaneously hypertensive rats.

Objective: The aim of this study was to investigate atrial natriuretic peptide (ANP) gene expression in the central nervous system (CNS) during hypertension. Methods: We measured and compared immunoreactive atrial natriuretic peptide (irANP) and ANP messenger RNA (mRNA) in the hypothalamus and brainstem of 17-week-old spontaneously hypertensive rats (SHR) with those of age-matched Wistar-Kyoto (WKY) rats using ribonuclease (RNase) protection assay for ANP mRNA and a specific radioimmunoassay for irANP. Results: RNase protection assay revealed that the concentrations of ANP mRNA in the hypothalamus and brainstem of SHR were higher than those of WKY rats. IrANP concentrations in the hypothalamus and brainstem of SHR were determined by a specific radioimmunoassay and found to be higher than those of WKY rats. Elevated mRNA levels in the hypothalamus and brainstem of SHR indicated that increased level of irANP in the CNS resulted from increased synthesis of ANP. Conclusion: We propose that increased synthesis of brain ANP in SHR may reflect a compensatory mechanism induced by hypertension.

Biochemical Studies of Rat Atrial Natriuretic Factor

The natriuretic substances were purified from rat atrium (ANF, atrial natriuretic factor) and were shown to be identical with the inhibitor of norepinephrine-induced contraction of smooth muscle. Their four native forms were isolated. Amino acid sequence analyses showed they are peptides with 35, 31, 30 and 25 amino acid residues respectively and contain a ring structure consisting of 17 amino acid residues and a disulfide bridge. The presence of a high molecular weight prohormone was shown. cDNA coding for the precursor was cloned and used to deduce the amino acid sequence of the preprohormone. Genomic DNA for ANF was cloned and the presence of two introns were found. Several ANF peptides were synthesized. Structure-function studies showed that the ring structure is essential for the activity. Antibodies produced against the synthetic 25 amino acid residue ANF were used to develop radioimmunoassay. The presence of ANF in rat plasma was demonstrated as evidence that ANF is a circulating hormone. ANF was also found in the hypothalamus of rats. The quantitative determination of the synthetic ability of ANF has been determined by the application of ANF cDNA for the quantification of ANF messenger RNA. Immunohistochemical methods localized ANF in cardiac atriocytes, gonadotrophs in anterior pituitary and adrenal medulla (chromaffin cells). A strong immuno-reactivity was found in dark cells of the collecting ducts of the kidney. ANF increases cyclic GMP in target cells suggesting that cyclic GMP may be the intracellular mediator of ANF action.