Atrial natriuretic factor (ANF) gene expression in the Brattleboro rat

Atrial natriuretic factor (ANF) is a 28-amino acid peptide hormone of cardiac origin. It has natriuretic, diuretic and vasorelaxant properties and inhibits several cardiovascular modulators. Because of the possible effects of arginine vasopressin (AVP) on ANF secretion, we have investigated ANF gene expression in Brattleboro rats which are genetically deficient in AVP. Our results indicate that cardiac ANF mRNA and ANF content are higher in Brattleboro rats compared to Long-Evans controls, whereas the plasma levels are similar in both groups. Typical secretory granules containing immunoreactive ANF are present in ventricular cardiocytes of Brattleboro but not of Long-Evans rats. These data suggest that ANF release may be uncoupled from its synthesis in the absence of AVP.     

Cloning and expression of the atrial natriuretic factor gene

Atrial natriuretic factor (ANF) is a 28-amino acid peptide hormone with potent natriuretic, diuretic and vasodilator properties. Isolation and DNA sequence analysis of rat and human cDNA clones revealed that ANF is synthesized from a 126-amino acid precursor which is highly conserved in both species. Southern blot analysis indicated that the ANF gene is present in a single copy per haploid genome. Both human and rat ANF genes were isolated and showed a similar structural organization which consisted of three exons and two introns. The ANF gene was localized to the short arm of human chromosome 1 and mouse chromosome 4. While atria are the major site of expression of the ANF gene in adult heart, other tissues like ventricles, lung, anterior pituitary, hypothalamus and adrenal synthesize ANF albeit to a much lower extent. In ventricles, ANF mRNA levels are 150 times lower than in atria. However, in cardiac hypertrophy or in congestive heart failure, ventricular ANF mRNA and peptide levels are dramatically (100-fold) increased both in animal models and in humans. This suggests that ventricles are a major site of ANF gene expression in certain pathophysiological conditions and that ANF is not an exclusively atrial peptide as was originally thought.     

Effects of rat atrial extract on sodium transport and blood pressure in the rat

Atrial cardiocytes contain specific atrial granules ( SAGs ) which are the storage site of atrial natriuretic factor (ANF). The purpose of the present study was to determine whether ANF produces natriuresis by inhibiting Na+-K+ pump activity and whether this factor is similar to the humoral sodium transport inhibiting factor ( HSTIF ) previously demonstrated in acutely volume expanded animals and humans as well as in experimental and human essential hypertension. Our results indicate that, in contrast to the HSTIF , ANF does not inhibit membrane Na+,K+-ATPase, vascular smooth muscle cell Na+-K+ pump activity, or sodium transport in the toad bladder. Intravenous infusion of ANF in the bilaterally nephrectomized, hexamethonium-treated rat produces only a small transient pressor response, probably due to potentiation of endogenous norepinephrine. These findings strongly suggest that the ANF is not the same as the HSTIF detected on acute volume expansion and in some forms of hypertension. They also suggest that the diuretic and natriuretic effects of ANF are due to mechanism(s) other than blood pressure elevation and inhibition of Na+-K+ pump activity.     

Atrial natriuretic factor and urinary kallikrein in the rat: antagonistic factors?

The rat atrium contains a potent natriuretic factor which appears to inhibit the sodium reabsorption in the collecting tubules of the kidneys. We examined the effects of the injection of partially purified atrial natriuretic factor (ANF) and synthetic ANF (8-33) into rats with simultaneous infusions of dextrose or aprotinin. Aprotinin, an inhibitor of serine proteases, increases the natriuretic and diuretic effects of the atrial factor by 50%. Urinary kallikrein excretion is also slightly increased by ANF but is not affected by aprotinin. As a comparison, aprotinin has no effect on the diuretic or natriuretic responses of furosemide, although it inhibits by 50% the kallikrein excretion induced by furosemide. When ANF is incubated with purified rat urinary kallikrein, the natriuretic and diuretic effects are decreased by more than 50%. We conclude that glandular kallikrein or a similar serine protease may be involved in the catabolism of ANF.     

Atrial natriuretic factor as a marker in congestive heart failure

Cardiac overload is associated with an overexpression of the atrial natriuretic-factor (ANF) gene in experimental models and in man. Sites of ANF gene overexpression are the atria but also the ventricular myocardium. This recruitment phenomenon of the ventricle to synthesise and secrete ANF is directly dependent on the increase in stress-stretch relationship in each cardiocyte. Therefore, the levels of plasma ANF and its second messenger, cyclic glycophosphate mutase in plasma and urine appear as markers of congestive heart failure in animal models and in man. Particularly, plasma ANF has been recognized recently as independent prognostic factor in congestive heart failure.     

Atrial natriuretic factor during fetal and postnatal life: a review

This review summarizes current understanding of the role of atrial natriuretic factor (ANF) during fetal and postnatal life. The cardiac localization of ANF synthesis changes during development from ventricular to predominantly atrial cardiocytes. ANF is present as a circulating hormone during fetal life and fetal plasma ANF clearance rates and production rates are higher than in adults. ANF is a natriuretic hormone in fetal and newborn animals. However, unlike the adult, this natriuresis does not appear to be related to suppression of the renin-angiotensin system. During fetal life, ANF levels can be increased through both atrial distension and humoral influences. Hence, mechanisms of ANF release during development appear to be similar to those present at maturity.     

Radioimmunoassay of atrial natriuretic factor (ANF) in rat atria

We describe a solid phase radioimmunoassay for atrial natriuretic factor (ANF) and its application for measurement of this peptide in homogenates of rat atria. The method uses a synthetic 26 amino-acid fragment (8-33 ANF) of the native peptide. Sample (or standard) are incubated with the rabbit anti-8-33 ANF antiserum in peptide (8-33 ANF)-coated wells. Then an excess of I125 goat anti-rabbit IgG is added. The radioactivity bound is directly proportional to the amount of ANF present. The concentration of immunoreactive ANF has been found to be about 4 times higher in the right atrium than in the left atrium of the rat.     

Blood-brain barrier and atrial natriuretic factor

In brain, binding sites for atrial natriuretic factor (ANF) have been characterized in areas such as circumventricular organs that lack the tight capillary endothelial junctions of the blood-brain barrier and therefore are exposed to circulating peptides. Since atrial natriuretic factor acts directly on vascular endothelium and has been proposed to be actively involved in blood pressure regulation and fluid homeostasis, it is interesting to know whether ANF receptors exist on brain capillaries that constitute the blood-brain barrier and participate in the constant fluid exchange between blood and brain. The present paper reports recent evidence of the presence of ANF receptors located on the structure. It assesses the specific binding of 125I-labelled ANF on bovine brain microvessel preparations and its coupling with a guanylate cyclase system. The potential physiological role of ANF on brain microcirculation and blood-brain barrier functions is discussed.     

A Raf-induced, MEK-independent signaling pathway regulates atrial natriuretic factor gene expression in cardiac muscle cells

The atrial natriuretic factor (ANF) gene is activated in cardiac myocytes by Ras and its effector Raf. However, MEK, the best-characterized Raf substrate, cannot efficiently activate ANF suggesting that Raf uses a MEK-independent pathway to activate ANF. By manipulating MEK and Raf activities so that they are equally effective at activating ERK, we now demonstrate that Raf activates at least two signaling pathways in cardiac myocytes that regulate the ANF promoter; the MEK-->ERK pathway inhibits ANF gene expression while a Raf-induced, MEK-independent pathway activates expression. This mechanism may provide increased ability to regulate ANF expression in response to hypertrophic stimuli.     

Characterization, distribution and plasticity of atrial natriuretic factor binding sites in brain

The [125I]iodotyrosyl derivative of atrial natriuretic factor [( 125I])ANF) apparently binds to a single class of high affinity sites in guinea pig brain membrane preparations. Ligand selectivity pattern reveals that the structural requirements of brain [125I]ANF binding sites are similar to those reported in most peripheral tissues. In vitro receptor autoradiographic studies demonstrate that the brain distribution of [125I]ANF binding sites is species dependent. In rat, high levels of binding are found in olfactory bulb, subfornical organ, area postrema, choroid plexus, and ependyma. In guinea pig, these regions are also enriched with [125I]ANF binding in addition to various thalamic nucleic, amygdala, hippocampus, and cerebellum. In monkey, high densities of sites are seen in the cerebellar cortex. This suggests that brain ANF receptor sites could mediate ANF effects related to the central integration of cardiovascular parameters, as well as other actions not associated with these systems. As in the periphery, it appears that brain [125I]ANF binding sites are associated with guanylate cyclase. Moreover, the density of [125I]ANF receptor binding sites is altered in certain brain regions in spontaneously hypertensive rats and in cardiomyopathic hamsters, demonstrating the plasticity of brain ANF receptors. Thus, ANF and ANF receptors are complementary facets of a new neurotransmitter-neuromodulator system present in mammalian brain.     

Morphological characteristics of the secretion of natriuretic factor by atrial cardiomyocytes in spontaneous hypertension in rats

The results of electron microscopic studies of the synthesis and secretion of atrial natriuretic factor (ANF) in right atrial cardiomyocytes of spontaneously hypertensive rats (SHR) and the corresponding normotensive controls are presented. Enhanced secretory activity in cardiomyocytes of SHR has been revealed. The role of enhanced ANF secretion in the origin of arterial hypertension is discussed. It is suggested that enhanced ANF secretion can be attributed to increased ANF demand in BP elevation, changes in the renal function in hypertensive subjects or genetic defect in the excretory renal function in SHR.     

The actions of atrial natriuretic factor on the vascular wall

The actions of atrial natriuretic factor (ANF) on the vascular wall are diverse and show a profound regional heterogeneity. ANF is a potent relaxant of aortic smooth muscle, a response which is associated with activation of particulate guanylate cyclase and elevation in tissue levels of cyclic GMP. However, many large and small muscular arteries and most veins are unresponsive to the peptide. The regional vascular heterogeneity may be due to an altered distribution of high affinity receptors and (or) alterations in the coupling of receptor activation to elevations in cyclic 3',5'-guanosine monophosphate (cGMP). Species differences exist in the structural requirements for receptor activation as well as the effects of infused ANF on peripheral resistance. Although the relaxation to ANF in vitro does not require an intact endothelium, endothelial cells contain multiple receptor subtypes for ANF. Differences amongst tissues and (or) species in the receptor profile for ANF may, in part, explain some of heterogeneity in responsiveness to ANF.     

Developing essential hypertension: a syndrome involving ANF deficiency?

The pathogenesis of essential hypertension may possibly involve a deficiency in, or a decreased response to, endogenous vasodilator and natriuretic factor(s). Searching for hereditary or familial defects, it is plausible to evaluate blood pressure (BP) regulating factors in (yet) normotensive offspring of hypertensive parents (OHyp), some of whom are in fact in a stage of prehypertension. Studies by our group demonstrated that compared with healthy offspring of normotensive parents, OHyp have plasma atrial natriuretic (ANF) factor levels that are unaltered on a low salt intake but often fail to increase normally in response to a high salt intake. Plasma levels of cyclic GMP, the presumed second messenger of ANF, also may tend to be decreased in certain OHyp. On the other hand, renal excretory responses of cyclic GMP and electrolytes to ANF infused in "physiological" dose were unchanged in some OHyp tested so far. In borderline to moderate, uncomplicated essential hypertension, plasma ANF levels are often "normal." This may be inappropriately low relative to the existing BP, although the relationship of circulating ANF to atrial pressures in essential hypertension remains to be clarified. A conversion to higher plasma ANF values may occur with cardiac complications such as left ventricular hypertrophy, enlargement, dysfunction, or overt heart failure. Acute or short-term elevation of circulating ANF within the physiological and pathophysiological range by ANF infusion produces an exaggerated natriuresis and lowers BP in essential hypertensive patients. We postulate a syndrome of ANF deficiency, characterized by an impaired response of circulating ANF to high salt intake and by low cyclic GMP levels in certain yet normotensive offspring of essential hypertensive parents and by inappropriately "normal" plasma ANF in some patients with uncomplicated essential hypertension. At the stage of prehypertension, a disturbance in the ANF - cyclic GMP pathway may be expressed primarily at the circulatory rather than at the renal level. Hypertension-prone humans also tend to have an exaggerated vascular reactivity to norepinephrine. Whether the two disturbances may be interrelated is presently unknown. Both defects may potentially predispose to the development of essential hypertension. Relative ANF deficiency, an enhanced natriuretic response to ANF, and a sustained antihypertensive effect of infused ANF may represent a rational basis for treatment of essential hypertension with agents that activate the ANF system.     

The role of atrial natriuretic factor in dehydration and schedule-induced drinking behaviour

Dehydration-induced drinking (DID) has been defined as a type of homeostatic behaviour controlled by factors related to water balance, whereas schedule-induced polydipsia (SIP) is considered to be a type of nonhomeostatic drinking subsequent to a general increase in motor excitability. In this study, we have attempted to assess the role of atrial natriuretic factor (ANF) in both models to elucidate the mechanisms controlling water intake. Intracerebroventricular injection of ANF (2-8 nmol) caused a dose related suppression of water intake in both DID and SIP, but intravenous injection with a higher dose of ANF (8 nmol) produced a significant suppression of water intake only in DID. Before drinking started, tissue ANF levels increased in atria in both models and decreased in hypothalamus in DID but not in SIP. After 1 hour of drinking, ANF levels decreased in atria in both models and increased in hypothalamus in SIP but not in DID. These results suggest that DID and SIP are different in their thirst regulation, and that the notion that peripheral ANF serves as a humoral factor sending signals to central in the fluid homeostatic control mechanism is questionable.     

An emerging relationship between peripheral sympathetic nervous activity and atrial natriuretic factor

The sympathetic nervous system and atrial natriuretic factor (ANF) are intimately involved in sodium, volume and blood pressure homeostasis, particularly in response to volume and pressure overloads. Although rapid progress in this field indicates several levels of interaction between both systems, the role of sympathetic nervous activity (SNA) in ANF release remains a controversial topic. There is growing evidence that ANF is an inhibitory modulator of sympathetic outflow (which in turn, may contribute to ANF's effect) and, vice-versa, SNA attenuates the target actions of ANF. Compensatory sympathetic reactions to changes induced by ANF may also have an overriding influence on its target actions. Dopamine appears to play a special role in these interactions. It is not only a precursor of norepinephrine but probably fulfills an independent function in the regulation of salt balance, similar in many respects to that of ANF.     

The responses of atrial natriuretic factor concentrations to acute volume changes in conscious rats

A rapid and sensitive radioimmunoassay has been developed for measurements of atrial natriuretic factor (ANF) in rat plasma. The antiserum, raised to rat ANF (99-126), cross-reacts with rat ANF (103-123), ANF (103-125), ANF (103-126) but not with smaller fragments, human ANF (99-126), angiotensin II, bradykinin or vasopressin. The plasma ANF concentration is 181 +/- 24 pg/ml (N = 24) in the unstressed conscious rats (Charles River CD, male). The ANF immunoreactivity in the plasma extracts was verified by HPLC analysis, which displayed one major immunoreactive peak of ANF corresponding to rat ANF (99-126) and some smaller fragments. Intravenous injection of saline elevated circulating ANF, whereas acute volume depletion by hemorrhage, water deprivation and furosemide diuresis greatly lowered plasma ANF. The prompt response of plasma ANF levels to acute changes in volume status is consistent with the proposed role of ANF as a volume-regulatory hormone.     

Identification of protease 3.4.24.11 as the major atrial natriuretic factor degrading enzyme in the rat kidney

We have identified a metalloendoprotease from rat kidney cortex that cleaves the cysteine-phenylalanine bond (Cys7-Phe8) within the 17 amino acid ring structure of atrial natriuretic factor (ANF). Cleavage at this site represents the major ANF degradative activity in rat kidney, and is inhibited by the known metalloendoprotease inhibitors, thiorphan, phosphoramidon and zincov with IC50 values in the nanomolar range. Since these are specific inhibitors of protease 3.4.24.11, both protease 3.4.24.11 and ANF degrading activities were monitored during purification. Both activities copurified at each chromatographic step. Furthermore, purified protease 3.4.24.11 cleaved ANF specifically at the Cys7-Phe8 bond. It is concluded from this work that the major ANF degrading enzyme in rat kidney is protease 3.4.24.11.     

Atrial natriuretic factor mRNA and binding sites in the adrenal gland.

The factor inhibiting aldosterone secretion produced by the adrenal medulla may be atrial natriuretic factor (ANF), since the latter abolishes aldosterone release in response to a number of secretagogues, including angiotensin II and K+. In this study we have shown that cells in the adrenal medulla contain ANF mRNA and therefore have the potential to synthesize this peptide. The presence of binding sites for ANF predominantly in the adrenal zona glomerulosa suggests that, if ANF is synthesized in the medulla and transferred to the cortex, it may affect mineralocorticoid status.     

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.