Plasma atrial natriuretic factor concentrations in essential and renovascular hypertension.

Plasma atrial natriuretic factor concentrations were measured in 44 patients with mild untreated essential hypertension and 48 normotensive controls. Mean venous plasma atrial natriuretic factor concentrations were 13.2 (SEM 1.5) and 13.0 (1.3) ng/l in the hypertensive patients and controls, respectively. Plasma atrial natriuretic factor concentrations were significantly correlated with age in both groups. Plasma atrial natriuretic factor concentrations were also measured during renal vein catheterisation in a group of 15 hypertensive patients; of these, eight had renovascular hypertension, and in all eight cases plasma atrial natriuretic factor concentrations were increased in the aorta and inferior vena cava. It is concluded that mild essential hypertension is not associated with increased plasma atrial natriuretic factor concentrations, whereas an age related increase in concentrations occurs in hypertensive and normotensive people.     

The physiology of atrial natriuretic factor

Following the discovery of the natriuretic effect of atrial extract, our laboratory attempted to dissect the possible physiological role of atrial natriuretic factor. Initial micropuncture experiments demonstrated that the reduction of tubular sodium reabsorption was localized in the medullary collecting duct, a nephron site in which sodium transport was known to be inhibited after acute hypervolemia. Partial removal of the endogenous source of atrial natriuretic factor was associated with a reduced renal response to hypervolemia, confirming that the factor is causally involved in acute sodium balance. In vitro incubation of atrial tissue was used to investigate mechanisms of release of atrial natriuretic factor. It was found that agonists known to activate the intracellular polyphosphoinositide system in other tissues were effective in releasing natriuretic activity from the atria into the incubation medium. To determine whether atrial natriuretic factor might play a role in hypertension, atrial natriuretic content was measured in spontaneously hypertensive rats and their normotensive controls. Hypertension was associated with increased content. Since the renal response to exogenous factor was not impaired in these animals, we suggested that the increased content might play a compensatory role. Our early studies thus indicated that atrial natriuretic factor was a previously unrecognized hormone involved in cardiovascular regulation.     

Effects of atrial appendectomy on circulating atrial natriuretic factor during volume expansion in the rat

This study examined the changes in the circulating level of endogenous atrial natriuretic factor during diuresis and natriuresis produced by acute volume expansion in anesthetized rats with either bilateral atrial appendectomy (n = 9) or sham operation (n = 9). Following control measurements in the sham-operated rats, 1% body weight volume expansion with isotonic saline produced an increment in urinary sodium excretion of over 4 mueq/min (P less than 0.05) while urine volume increased by more than 20 microliter/min (P less than 0.05). These responses were associated with a significant increase in immunoreactive plasma atrial natriuretic factor from a baseline value of 82 +/- 10 pg/ml to a level of 120 +/- 14 pg/ml (P less than 0.05). In contrast, in the group of rats with bilateral atrial appendectomy an identical degree of volume expansion increased urinary sodium excretion and urine volume by only 0.61 mueq/min (P less than 0.05) and 3.07 microliter/min (P less than 0.05), respectively. In this group, immunoreactive plasma atrial natriuretic factor remained statistically unchanged from a control value of 70 +/- 12 pg/ml to a level of 82 +/- 16 pg/ml (P greater than 0.05). Comparison of the two groups indicates that the natriuresis, diuresis, and plasma atrial natriuretic factor levels during volume expansion were significantly reduced in the rats with bilateral atrial appendectomy. No differences in mean arterial pressure and heart rate were observed between the two groups. These data demonstrate that removal of both atrial appendages in the rat attenuated the release of atrial natriuretic factor during volume expansion; and this effect, in turn, was associated with a reduction in the natriuretic and diuretic responses.     

Effects of atrial natriuretic factor on norepinephrine release in the rat hypothalamus.

The effects of atrial natriuretic factor on the mechanisms involved in norepinephrine release were studied 'in vitro' in slices of Wistar rat hypothalamus. Atrial natriuretic factor (10, 50 and 100 nM) decreased spontaneous [3H]norepinephrine secretion in a concentration dependent way. In addition, the peptide (10 nM) also reduced acetylcholine induced output of norepinephrine. The atrial factor (10 nM) was unable to alter the amine secretion when the incubation medium was deprived of calcium or when a calcium channel blocker such as diltiazem (100 microM) was added. In conclusion, atrial natriuretic factor reduced both spontaneous and acetylcholine evoked [3H]norepinephrine release in the rat hypothalamus. These findings suggest that the atrial natriuretic factor may alter catecholamine secretion by modifying the calcium available for the exocytotic process of catecholamine output.     

Involvement of the adrenal glands in the action of the atrial natriuretic factor

Adrenalectomized, medullectomized and sham operated rats were treated with either a chronic infusion or a bolus injection of the synthetic atrial natriuretic factor (ANF). ANF did not enhance natriuresis and diuresis in sham operated conscious animals during chronic infusion, but it had a potent action when injected as a bolus into anesthetized rats. The absence of the whole adrenal glands, but not adrenal medulla profoundly modified the renal response to ANF: a) following chronic administration of ANF, the baseline natriuresis paradoxically decreased in adrenalectomized rats, and b) in response to a bolus injection of ANF the natriuretic and diuretic actions of the peptide were attenuated in these animals. The medullectomy-induced decreased natriuresis and dopamine excretion were corrected by ANF infusion. Furthermore, ANF suppressed the compensatory increase of norepinephrine excretion secondary to adrenalectomy. The data suggest that the presence of the adrenal cortex is necessary for the natriuretic and diuretic actions of ANF. The decrease in urinary DA excretion may reflect diminished dopaminergic activity and contribute to the post-medullectomy antinatriuresis, a phenomenon which can be corrected by ANF infusion. ANF may also have a depressing activity on the increased sympathetic tone.     

Dissociation between right atrial pressure and plasma atrial natriuretic factor following prolonged high salt intake

The influence of prolonged high salt intake on intravascular volume, right atrial pressure, plasma atrial natriuretic factor, and extra-atrial tissue (lung, kidney, and liver) COOH- and NH2-terminal atrial natriuretic factor content was investigated in normotensive rats. Despite prolonged high salt (8% NaCl) intake for 5 weeks, total intravascular volume was not impaired. However, right atrial pressure was increased by 54% (p less than 0.01) after salt loading. Although this increment in right atrial pressure should favor atrial natriuretic factor release after NaCl intake, plasma atrial natriuretic factor (COOH-terminal) concentrations markedly decreased from 97.8 +/- 27 to 38.9 +/- 8 pg/mL. Sodium and circulatory homeostasis was, however, well preserved. The lungs contained the highest levels of COOH- and NH2-terminal atrial natriuretic factor. Salt loading resulted in increased concentrations of low as well as high molecular weight atrial natriuretic factor in the lung but not in the kidney or the liver. Our study indicates a limited role of atrial natriuretic factor in adaptation to prolonged salt consumption in rats. Dissociation between right atrial pressure and plasma atrial natriuretic factor after salt intake implicates other factors regulating circulating peptide levels. Prolonged salt intake increases lung generation of atrial natriuretic factor.     

The effect of atrial natriuretic factor on force development in rat cardiac trabeculae

Studies in single cardiac muscle cells have demonstrated that atrial natriuretic factor decreases the L-type calcium current. Recent investigations in human atrial cells have also demonstrated that atrial natriuretic factor causes a voltage-dependent reduction in sodium channel activity and thus may reduce intracellular calcium via decreased activity of the sodium-calcium exchange mechanism. By reducing intracellular calcium, atrial natriuretic factor may have a negative inotropic effect on cardiac muscle. To characterize the effect of atrial natriuretic factor on the development of force, we studied the force-sarcomere length relationship in 11 right ventricular rat trabeculae, both before and after exposure of the muscles to increasing concentrations of atrial natriuretic factor. Sarcomere length was measured by laser diffraction techniques and controlled by a servomotor system. The addition of atrial natriuretic factor to the superfusion solution, at concentrations of 10(-9)-10(-7) M, increased stimulus threshold, reduced peak twitch force in a dose-dependent manner by 38% (maximum), and reduced time to peak twitch force by 15% (maximum). Incubation of muscle preparations with concentrations of atrial natriuretic factor below 10(-9) M had no effect on force generation. The negative inotropic effect of atrial natriuretic factor was associated with a change in the shape of the force-sarcomere length relationship, similar to a reduction of the extracellular calcium concentration. ANF (10(-7) M) had no effect on the rate of decay of force following post extra-systolic potentiation. These observations are consistent with the assumption that the negative inotropic effect of atrial natriuretic factor is mediated by reduction of calcium entry into the cardiac cell.     

Atrial natriuretic prohormone peptides 1-30, 31-67, and 79-98 vasodilate the aorta

Human prohormone atrial natriuretic peptides 1-30, 31-67, and 79-98 caused vasodilation of porcine aortas which began in 30 seconds and was maximal at 10 minutes. These three peptides were found to be equally potent to atrial natriuretic factor in their vasodilatory activity which was found with or without endothelium present. This vasodilation was associated with a 4 to 5 fold increase in cyclic GMP in the aorta secondary to activation of particulate guanylate cyclase [E.C. 4.6.12]. These data demonstrate that three N-terminal peptide segments of the atrial natriuretic factor prohormone cause vasodilation.     

Plasma atrial natriuretic factor concentrations and renal actions in the domestic fowl

Plasma atrial natriuretic factor concentrations in Rhode Island red hens averaged 72.1±6.9 pg·ml^-1, range 33.4–136.0 pg·ml^-1. The intravenous infusion of isotonic saline containing 3% dextran for 2 h produced no significant changes in plasma osmotic or electrolyte concentrations; however, haematocrit changes indicated vascular expansions of 14.4% after 1 h and 21.3% after 2 h and plasma atrial natriuretic factor concentrations were elevated by 190% and 257%, respectively. The intravenous infusion of chicken atrial natriuretic factor at rates of 10, 25, 50 and 100 ng·kg^-1·min^-1 for 20 min produced levels of plasma atrial natriuretic factor that were directly related to the infusion rate and which, in birds undergoing a steady-state diuresis/natriuresis driven by the intravenous infusion of isotonic saline at 1 ml·min^-1, produced dose-dependent increases of 19, 26, 38 and 55% in urine flow rate and of 8, 30, 49 and 77% in sodium excretion. Potassium excretion was significantly increased only at the two highest atrial natriuretic factor infusion rates. The observed correlation between plasma atrial natriuretic factor concentration and vascular volume together with the atrial natriuretic factor-induced modulation of renal salt and water elimination is consistent with the concept that in the chicken this peptide has a physiological role as a regulatory hormone in volume homeostasis.Abbreviations AII angiotensin II - ANF atrial natriuretic factor - AVT arginine vasotocin - BV blood volume - chANF chicken atrial natriuretic factor - CHE chicken heart extract - ECF extracellular fluid - EDTA ethylenediaminetetra-acetate - Hct haematocrit - i.v. intravenous - PCR plasma clearance rate - PRA plasma renin activity - RIA radioimmunoassay     

Atrial natriuretic factor increases the magnitude of duodenal spontaneous phasic contractions

The present investigation was designed to determine if atrial natriuretic factor relaxes non-vascular smooth muscle. Rather than cause a relaxation, atrial natriuretic factor induced a two-to-four fold enhancement in the amplitude of the spontaneous phasic contractions of duodenal longitudinal muscle. Dose-response curves revealed that ANF enhanced these contractions over a concentration range of 10 picomoles to 100 nanomoles with the ED50 at 1 nanomolar. The increased amplitude of contraction began within 30 seconds and was calcium-dependent. The increased force of contraction was associated with a three-fold increase in cyclic GMP levels and activation of particulate guanylate cyclase [E.C.4.5.1.2.]. Atrial natriuretic factor had its half-maximal [ED50] activation of guanylate cyclase at its 1 nM concentration while maximal enhancement was at its 100 nM concentration in duodenum, jejunum, and ileum. Atrial natriuretic factor did not stimulate adenylate cyclase [E.C.4.6.1.1.]. Thus, atrial natriuretic factor increases the force of the spontaneous phasic contractions of the small intestine which are calcium-dependent and associated with activation of the guanylate cyclase-cyclic GMP system.     

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.     

Atrial granules contain an amino-terminal processing enzyme of atrial natriuretic factor

At least three enzymes have been identified in atrial tissue homogenates that are capable of processing pro-atrial natriuretic factor to active atrial peptides. The atrial peptides possess potent natriuretic, diuretic, vasorelaxant, and hemodynamic properties, and their existence has implicated the mammalian heart as an endocrine organ. We have purified and characterized a serine proteinase (Mr approximately equal to 70,000) associated with atrial granules that preferentially hydrolyzes the Arg-Ser bond in the synthetic substrates Gly-Pro-Arg-Ser-Leu-Arg, benzoyl-Gly-Pro-Arg-Ser-Leu-Arg, and benzoyl-Gly-Pro-Arg-Ser-Leu-Arg-Arg-2-naphthylamide, the Arg-2-naphthylamide bond in the substrate benzoyl-Gly-Pro-Arg-2-naphthylamide, and the Arg-Ser bond in a 31-residue substrate (Gly96-Tyr126 peptide) corresponding to residues Arg98-Ser99 in pro-atrial natriuretic factor. The Gly96-Tyr126 peptide contains the putative processing site in pro-atrial natriuretic factor and the sequence for the bioactive peptides. Our results indicate that the minimum processing site sequence is -Gly-Pro-Arg-Ser-Leu-Arg-Arg- and that the Ser99-Tyr126 natriuretic peptide is the predominant hydrolytic product. After prolonged incubation or at high enzyme concentrations, the Ser103-Tyr126 natriuretic peptide may also be formed. The Ser103-Arg125 natriuretic peptide was only a very minor product. The doublet of basic amino acids is not the primary processing site in pro-atrial natriuretic factor, but their presence may influence cleavage at the single Arg residue "upstream." Our findings are consistent with the idea that the pro-protein and the processing enzymes are packaged into the secretory granule and in response to the proper stimulus, the pro-protein is processed to the active peptides, probably during the process of secretion. The processing pathway of pro-atrial natriuretic factor is discussed.     

Inactivation of atrial natriuretic factor in blood

Tissue extracts of rat heart atria contain a family of peptides with natriuretic and vasorelaxant properties. It has been shown by others that inactivation of this atrial natriuretic factor may involve endogenous peptidases. The present experiments demonstrate that incubation in blood in vitro reduces the natriuretic activity of the factor. Specifically, inactivation was associated with a white cell/platelet fraction, indicating that these blood elements may play a physiological role in the metabolism of this new putative hormone.     

Atrial natriuretic factor and sympathetic nervous output in response to volume shifts in the immature canine circulation.

This study evaluated the immature circulation's response to acute shifts in intravascular volume with respect to atrial natriuretic factor and plasma catecholamines. Serial measurements were performed on thirteen beagle puppies during volume expansion with a saline and albumin solution followed by volume contraction with furosemide. Atrial natriuretic factor correlated with right (r = .73, p less than 0.001) and left (r = .62, p less than 0.001) atrial pressures and increased to much greater levels than previously reported for mature animals. Simultaneously, 10 puppies had a progressive decrease in plasma norepinephrine over the 60-minute infusion (p less than 0.05) while two puppies demonstrated a marked increase between the 30- and 60-minute samples. Furosemide increased urine output and reversed the hormonal changes caused by volume expansion. Thus a greatly augmented output of atrial natriuretic factor occurs in the immature canine circulation in response to increased atrial and pulmonary pressures, while sympathetic output remains unchanged or falls with increasing intravascular volume until a critical decrease in cardiac output triggers a catecholamine surge.     

Atrial natriuretic factor: atrial conversion of high to low molecular weight forms

The atrial natriuretic factor elutes by gel filtration in high and low molecular weight fractions. Extraction and elution of rat atria in 1.0 M acetic acid yielded a predominance of the high molecular weight form(s); whereas when these procedures were carried out in 0.1 M acetic acid, there was a predominance of the low molecular weight forms. When partially purified high molecular weight natriuretic activity was eluted in 0.1 M acetic acid, the high molecular weight form(s) remained intact. When partially purified high molecular weight natriuretic activity was mixed with crude atrial extract in 0.1 M acetic acid, there was an apparent conversion to the low molecular weight forms. Extraction of rat atria in boiling 0.1 M acetic acid blocked this conversion. It is concluded that rat atria contain a heat labile factor that converts high molecular weight natriuretic activity to the low molecular weight forms.     

Effects of atrial natriuretic factor on renal handling of water and electrolytes in rats

The intravenous injection of an extract of atrial myocardium into anesthetized rats during a hypotonic diuresis resulted in an increase in the renal excretion of water, sodium, potassium, calcium, magnesium, and phosphate. There was an increase in urine concentration which was probably a result of the secretion of vasopressin since it did not occur in Brattleboro (di/di) rats. A transient increase in glomerular filtration rate and renal plasma flow occurred during the first five minutes with a more sustained rise in filtration fraction. Injection of atrial extract also caused a partial inhibition of solute-free water formation in Brattleboro rats subjected to water diuresis and a partial inhibition of solute-free water reabsorption in rats subjected to maximal antidiuresis by infusing vasopressin. In neither case was the degree of inhibition as profound as that observed after injecting furosemide in a dose which caused a comparable natriuretic response. A large dose of furosemide blocked the natriuretic response to atrial extracts whereas, when a comparable level of sodium and water output was produced by massive infusions of saline, the natriuretic response to atrial extract was increased. It is suggested that atrial natriuretic factor might inhibit sodium transport in nephron segments beyond the medullary thick ascending limb. Furosemide might also act at the same tubular site or inhibit tubular secretion of the atrial natriuretic factor.     

Atrial natriuretic factor inhibits noradrenaline release in the presence of angiotensin II and III in the rat hypothalamus

1. Atrial natriuretic factor effects on neuronal noradrenaline release evoked by angiotensin II or III and high potassium solution plus angiotensin II and III in the rat hypothalamus were studied.2. Atrial natriuretic factor (10 nM) did not modify spontaneous noradrenaline release. On the other hand, the atrial factor diminished the increase of noradrenaline release induced by both angiotensin II (1 μM) or angiotensin III (1 μM).3. Ten nanomolar ANF reduced the amine output induced by 100 nM KCl. Both angiotensins enhanced the 3H-noradrenaline secretion stimulated by high potassium solutions. When atrial natriuretic factor was added to the medium containing the depolarizing KCl solution plus angiotensin II or III (1 μM), the diminishing effects were greater than when the atrial factor was added to the depolarizing solution alone.4. Our results suggest that atrial natriuretic factor effects on noradrenaline release, evoked by angiotensin II, III and KCl, may be involved in the regulation of the central catecholamine pathways and sympathetic activity.     

Atrial natriuretic factor inhibits the sympathetic nervous activity in one-kidney, one-clip hypertension in the rat

The one-kidney, one-clip model of rat hypertension was found to have an increased natriuresis following chronic infusion of atrial natriuretic factor (ANF). We have now found that this natriuretic effect of ANF is associated with a suppression of the initially elevated urinary excretion of norepinephrine and epinephrine and increase of the excretion of the main dopamine metabolite-dihydroxyphenylacetic acid as well as of the urinary dopamine to norepinephrine ratio. These data are compatible with the hypothesis that ANF suppresses the increased sympathetic activity in this model of hypertension and this action combined with opposite changes of dopamine may contribute to the natriuretic effect of ANF.     

Immuno-electron microscopy of atrial natriuretic factor secretory pathways in atria and ventricles of control and cardiomyopathic hamsters with heart failure

Summary The secretory pathways of atrial natriuretic factor have been investigated in atrial and ventricular cardiocytes of control and cardiomyopathic Syrian hamsters in severe congestive heart failure with four antibodies: a monoclonal antibody (2H2) against rat synthetic atrial natriuretic factor (101–126), which is directed against region 101–103 of rat atrial natriuretic factor (99–126), and polyclonal, affinity-purified antibodies produced in rabbits against synthetic C-terminal atrial natriuretic factor (101–126), synthetic N-terminal atrial natriuretic factor (11–37) or the putative cleavage site of atrial natriuretic factor (98–99): atrial natriuretic factor (94–103). Application of the immunogold technique on thin frozen sections (immunocryoultramicrotomy) revealed an identical picture with the four antibodies. In atria of both control and cardiomyopathic hamsters where atrial natriuretic factor secretion is regulated, the atrial natriuretic factor propeptide travels, uncleaved, from the Golgi complex to immature and mature secretory granules. In ventricles of control hamsters, where secretion is constitutive, the atrial natriuretic factor propeptide travels from the Golgi complex to secretory vesicles. In the ventricles of hamsters with severe congestive heart failure, the Golgi complex is larger, secretory vesicles more abundant and a few secretory granules are present in 20% of cardiocytes. Here again, the peptide travels uncleaved in all these pathways. These results reveal the pathways of secretion of atrial natriuretic factor in atrial and ventricular cardiocytes and indicate that the propeptide is not cleaved intracellularly.Supported by a grant from the Medical Research Council of Canada to the Multidisciplinary Research Group on Hypertension, by the Canadian Heart Foundation and the Pfizer Company (England)     

cis-dominance of rat atrial natriuretic factor gene regulatory sequences in transgenic mice.

We have produced transgenic mice that express the prokaryotic marker protein chloramphenicol acetyltransferase under the control of regulatory sequences derived from the rat atrial natriuretic factor gene. The transgene, which contains 2.4 kilobases of the rat atrial natriuretic factor gene regulatory region, was found to direct 4000-fold more chloramphenicol acetyltransferase expression in adult atria than in ventricles. Low-level activity was also detected in the hypothalamus, demonstrating that these sequences contain the signals necessary for cardiac and central nervous system expression of the hormone atrial natriuretic factor. Developmental analyses showed early, high-level transgene expression in fetal atrial and ventricular tissues but marked reduction of ventricular transgene expression following birth. Further, the developmental expression patterns of the endogenous murine atrial natriuretic factor gene and rat transgene were found to be quite distinct. Although both the rat and mouse atrial natriuretic factor genes are activated early in embryogenesis, perinatal ventricular expression appears to differ in these two rodent species. The transgene is expressed in a pattern analogous to the neonatal rat rather than the endogenous murine gene. These studies demonstrate that the cis-acting signals required for correct tissue specificity and developmental regulation of the rat atrial natriuretic factor gene are encoded in this 2.4-kilobase fragment and that these sequences act in a dominant fashion.