Thirty years of research on atrial natriuretic factor: historical background and emerging concepts

The discovery of the natriuretic properties of atrial muscle extracts pointed to the existence of an endocrine function of the heart that is now known to be mediated by the polypeptide hormones atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP). On the basis of such a finding, approximately 27 000 publications to date have described a wide variety of biological properties of the heart hormones as well as their application as therapeutic agents and biomarkers of cardiac disease. Stimulation of secretion of ANF and BNP from the atria is mediated through mechanisms involving G proteins of the G(q) or G(o) types. We showed that the latter type underlies the transduction of muscle stretch into stimulated secretion and that it is more highly abundant in atria than in ventricles. The Gα(o)()-1 subunit appears to play a key role in the biogenesis of atrial granules and in the intracellular targeting of their contents. Protein interaction studies using a yeast two-hybrid approach showed interactions between Gα(o)()-1, proANF, and the intermediate conductance, calcium-activated K(+) channel SK4. Pharmacological inhibition of this channel decreases ANF secretion. Unpublished studies using in vitro knockdowns suggest interdependency in granule protein expression levels. These studies suggest previously unknown mechanisms of intracellular targeting and secretion control of the heart hormones that may find an application in the therapeutic manipulation of circulating ANF and BNP.     

Determinants of natriuretic peptide gene expression

The cardiac natriuretic peptides (NP) atrial natriuretic factor or peptide (ANF or ANP) and brain natriuretic peptide (BNP) are polypeptide hormones synthesized, stored and secreted mainly by cardiac muscle cells (cardiocytes) of the atria of the heart. Both ANF and BNP are co-stored in storage granules referred to as specific atrial granules. The biological properties of NP include modulation of intrinsic renal mechanisms, the sympathetic nervous system, the rennin-angiotensin-aldosterone system (RAAS) and other determinants, of fluid volume, vascular tone and renal function. Studies on the control of baseline and stimulated ANF synthesis and secretion indicate at least two types of regulated secretory processes in atrial cardiocytes: one is stretch-stimulated and pertussis toxin (PTX) sensitive and the other is Gq-mediated and is PTX insensitive. Baseline ANF secretion is also PTX insensitive. In vivo, it is conceivable that the first process mediates stimulated ANF secretion brought about by changes in central venous return and subsequent atrial muscle stretch as observed in acute extracellular fluid volume expansion. The second type of stimulation is brought about by sustained hemodynamic and neuroendocrine stimuli such as those observed in congestive heart failure.     

Capsaicin-sensitive nerves influence the release of atrial natriuretic factor by atrial stretch in the rat.

Although many factors may modulate the release of atrial natriuretic factor (ANF), the primary mechanism has been demonstrated to be atrial stretch. Recent studies have led to the suggestion that the peptidergic innervation of the heart, through the release of peptides, may be involved in the control of ANF secretion. We have examined the influence of chronic capsaicin treatment on three models of atrial stretch that release ANF. This treatment inhibited ANF released through in vivo blood volume expansion and through balloon inflation in the right atrium of in vitro isolated perfused hearts. Immunohistochemical and electron microscopical analysis confirmed the absence of innervation of the heart by calcitonin gene related peptide and substance P immunoreactive nerve fibres and apparent lack of effect on atrial granules in capsaicin treated rats. We conclude that capsaicin-sensitive cardiac innervation is a component modulating the release of ANF, stimulated by atrial stretch in the rat.     

Atrial natriuretic factor: an overview

Heart atrial muscle cells in mammals are differentiated for a contractile as well as a secretory function. Through the latter, the heart plays an endocrine role; it synthesizes, stores, and releases a group of peptides collectively referred to as atrial natriuretic factor (ANF). ANF has natriuretic and hypotensive properties as well as an inhibitory effect on aldosterone and renin secretion. Thus ANF intervenes in the short- and long-term regulation of water and electrolyte balance and blood pressure. It is expected that further research in this new field will provide fresh insights into the pathophysiology of several important clinical entities and in the development of new pharmaceutical products.     

T-type calcium currents in rat cardiomyocytes during postnatal development: contribution to hormone secretion

T-type Ca(2+) channels have been suggested to play a role in cardiac automaticity, cell growth, and cardiovascular remodeling. Although three genes encoding for a T-type Ca(2+) channel have been identified, the nature of the isoform(s) supporting the cardiac T-type Ca(2+) current (I(Ca,T)) has not yet been determined. We describe the postnatal evolution of I(Ca,T) density in freshly dissociated rat atrial and ventricular myocytes and its functional properties at peak current density in young atrial myocytes. I(Ca,T) displays a classical low activation threshold, rapid inactivation kinetics, negative steady-state inactivation, slow deactivation, and the presence of a window current. Interestingly, I(Ca,T) is poorly sensitive to Ni(2+) and insensitive to R-type current toxin SNX-482. RT-PCR experiments and comparison of functional properties with recombinant Ca(2+) channel subtypes suggest that neonatal I(Ca,T) is related to the alpha(1G)-subunit. Atrial natriuretic factor (ANF) secretion was measured using peptide radioimmunoassays in atrial tissue. Pharmacological dissection of ANF secretion indicates an important contribution of I(Ca,T) to Ca(2+) signaling during the neonatal period.     

Cardiac secretion of atrial natriuretic factor with exercise in chronic congestive heart failure patients.

We have previously reported a fivefold increase of plasma atrial natriuretic factor (ANF) in patients with congestive heart failure (CHF) compared with normal subjects. However, given the marked increase of ANF under basal conditions, the extent to which ANF secretion can further increase under physiological stress is not been clarified in CHF. We therefore evaluated ANF secretion during ergometric exercise in 11 patients with CHF, with peripheral venous ANF samples obtained at rest and peak exercise. In seven patients, simultaneous peripheral venous and right ventricular ANF samples were obtained to estimate myocardial ANF secretion. Hemodynamic characteristics of exercise included a significant increase of heart rate, mean arterial pressure, and cardiac output (all P < 0.01); reduction of systemic vascular resistance (P < 0.001); and increase of right atrial and pulmonary wedge pressures (P < 0.001). ANF was abnormally elevated at baseline (108 +/- 58 fmol/ml) yet increased further to 183 +/- 86 fmol/ml with exercise (P < 0.003). A step-up of right ventricular ANF, particularly during exercise, was consistent with active myocardial secretion, despite elevated baseline ANF levels.     

Coronary hemodynamics and cardiac beating modulate atrial natriuretic factor release from isolated Langendorff-perfused rat hearts

The role of coronary hemodynamics and cardiac beating on atrial natriuretic factor (ANF) release was studied in the isolated Langendorff-perfused rat heart. ANF release was measured by radioimmunoassay. When the coronary flow rate was changed, ANF release decreased or increased in a flow-dependent manner. When the perfusion pressure was changed, ANF release also increased or decreased, respectively, with concomitant changes in coronary flow rate. Furthermore, perfusion with 50 mM potassium chloride showed immediate cardiac arrest and a decrease of ANF release to an undetectable level with a significant decrease in coronary flow. However, low but readily detectable amounts of ANF were released when coronary flow rate was maintained. These results may suggest that coronary hemodynamics and cardiac beating could be factors modulating ANF secretion from the atrium.     

Atrial natriuretic factor is biologically active also in the absence of vasopressin: studies on Brattleboro-strain diabetes insipidus rats

Since atrial natriuretic factor (ANF) has been shown to inhibit vasopressin secretion, the role of this effect in the acute biological actions of ANF was investigated using Brattleboro-strain diabetes insipidus (DI) rats. Under thiobarbital anesthesia, synthetic rat ANF of a 25 amino acid sequence was administered intravenously as a bolus (8 micrograms/kg) into the jugular vein. The urine volume, urinary sodium and potassium concentration, blood pressure, and heart rate were determined. It was found that ANF administered exogenously can exhibit its diuretic, natriuretic and vasorelaxant activities even in the absence of vasopressin. This indicates that the inhibition of vasopressin secretion is not an indispensible mechanism for acute biological effects of ANF.     

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.     

Revisiting the surgical creation of volume load by aorto-caval shunt in rats

Cardiac hypertrophy is an early landmark during the clinical course of heart failure, and is an important risk factor for subsequent morbidity and mortality. The hypertrophy response to different types of cardiac overload is distinguished both at the molecular and cellular levels. These changes have been extensively characterized for pressure load hypertrophy; however, similar information for volume load hypertrophy is still needed. This study was undertaken to improve the existing method of producing experimental cardiac volume load. Previous investigators have employed surgical aorto-caval shunt (ACS) as a model for volume load hypertrophy (VO) in rats. The procedure is relatively simple and involves glue to seal the aortic hole after ACS. However, it has several limitations mostly related to the use of glue e.g. poor visualization due to hardening of tissues, imperfect sealing of the puncture site and glue seeping through the aortic hole resulting in shunt occlusion. We have modified the procedure using aortic adventitial suture instead of glue and 18G angiocatheter instead of 16G needle, which eliminated the technical difficulties from the former method. The ACS was visually confirmed at sacrifice, and the VO demonstrated by time-related changes in the heart weight/body weight ratio which increased from 78% at 4 weeks to 87% at 10 weeks and increased liver/body weight ratio by 22% at 10 weeks of post aorto-caval shunt. Cardiac expression of atrial natriuretic peptide (ANF) also demonstrated time-related increase in ANF mRNA (+275% increase at 4 weeks, p < 0.05, and +370% increase at 10 weeks, p < 0.001). This modified technique of aorto-caval shunt offers simpler, reproducible and consistent model for VO hypertrophy in rats.     

A decade of atrial natriuretic factor research.

Present views on the biological significance of atrial natriuretic factor (ANF) relate this polypeptide hormone to the regulation of blood pressure and volume through its modulating effects on renal function, on blood vessel tone and permeability, and on the renin-angiotensin-aldosterone system. Although very important advances in the understanding of ANF have been made over the decade since its discovery, some fundamental facts about ANF biosynthesis and release remain to be elucidated. Stretch-induced enhancement of ANF release appears as the most significant mechanism underlying the endocrine response of the atria to acute volume load. This response decays over a period of minutes, indicating that chronic stimulation of ANF release involves mechanisms different from, or in addition to, those acting during acute stretch-stimulated release. In neither acute nor chronic conditions are the cellular or molecular mechanisms underlying ANF release understood. To better understand long-term stimulation of ANF release, we have conducted extensive in vitro testing of several hormones and neurotransmitters to determine their ability to modify ANF release. From these studies, clear-cut evidence of ANF stimulation was obtained with the vasopressor peptide endothelin. Investigations on the cell and molecular biology of cardiac muscle development and hypertrophy have shown that ANF is involved in cardiac growth. The role played by ANF in these processes is now being determined, but this is one line of evidence that suggests that this hormone, together with other natriuretic peptides, may have autocrine or paracrine functions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies of ANF processing and secretion using a primary cardiocyte culture model.

In contrast to most other endocrine peptides ANF is stored in the heart as part of a larger prohormone, often called pro-ANF, yet is found in the circulation as a 28 amino acid peptide, called ANF. It has been shown that the conversion of the 126 amino acid pro-ANF to ANF occurs in the heart. This paper summarizes studies from our laboratory that have used a primary neonatal rat heart cell culture system to investigate the location and mechanism of this relatively unusual processing event. We have found that in culture the maintenance of the cells in a glucocorticoid-containing serum-free medium is required to observe processing as occurs in vivo. The cells contain the prohormone while ANF accumulates in the medium. Various experiments with protease inhibitors, pulse-chase biosynthetic labeling, incubation of cells with ANF-related peptides, and enrichment of cultures for myocytes have resulted in our conclusion that the processing of pro-ANF takes place most likely within the cardiac myocyte just prior to, but in concert with secretion. We have expanded on the use of this processing-competent atrial myocyte culture system to investigate mechanisms of stimulated ANF secretion. It has been shown that the activation of several phospholipase C-coupled receptors (e.g., alpha 1-adrenergic and endothelin receptors) produces a robust release of ANF, but only in cultures that have been maintained under appropriate conditions. Further, it is apparent that the phenylephrine- or endothelin-mediated release of ANF depends in part on influx of extracellular calcium (Ca2+o), while the remaining component of stimulated release may depend on mobilization of intracellular calcium. It also appears that these agonists produce an initial phase of stimulated release, occurring within the first 5 min of agonist exposure, independent of Ca2+o, and a sustained phase that persists as long as the agonists remain on the cells, and depends on the presence of Ca2+o and thus calcium influx. Taken together our studies indicate that the hormonal environment may be an important factor directing the development of differentiated endocrine functions by atrial myocytes and may be involved in the regulation of ANF expression, biosynthesis, and secretion.     

The δA isoform of calmodulin kinase II mediates pathological cardiac hypertrophy by interfering with the HDAC4-MEF2 signaling pathway

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is a new promising target for prevention and treatment of cardiac hypertrophy and heart failure. There are three δ isoforms of CaMKII in the heart and previous studies focused primarily on δB and δC types. Here we report the δA isoform of CaMKII is also critically involved in cardiac hypertrophy. We found that δA was significantly upregulated in pathological cardiac hypertrophy in both neonatal and adult models. Upregulation of δA was accompanied by cell enlargement, sarcomere reorganization and reactivation of various hypertrophic cardiac genes including atrial natriuretic factor (ANF) and β-myocin heavy chain (β-MHC). Studies further indicated the pathological changes were largely blunted by silencing the δA gene and an underlying mechanism indicated selective interference with the HDAC4-MEF2 signaling pathway. These results provide new evidence for selective interfering cardiac hypertrophy and heart failure when CaMKII is considered as a therapeutic target.     

Atrial natriuretic factor stimulates exocrine pancreatic secretion in the rat through NPR-C receptors

Increasing evidence supports the role of atrial natriuretic factor (ANF) in the modulation of gastrointestinal physiology. The effect of ANF on exocrine pancreatic secretion and the possible receptors and pathways involved were studied in vivo. Anesthetized rats were prepared with pancreatic duct cannulation, pyloric ligation, and bile diversion into the duodenum. ANF dose-dependently increased pancreatic secretion of fluid and proteins and enhanced secretin and CCK-evoked response. ANF decreased chloride secretion and increased the pH of the pancreatic juice. Neither cholinergic nor adrenergic blockade affected ANF-stimulated pancreatic secretion. Furthermore, ANF response was not mediated by the release of nitric oxide. ANF-evoked protein secretion was not inhibited by truncal vagotomy, atropine, or Nomega-nitro-l-arginine methyl ester administration. The selective natriuretic peptide receptor-C (NPR-C) receptor agonist cANP-(4-23) mimicked ANF response in a dose-dependent fashion. When the intracellular signaling coupled to NPR-C receptors was investigated in isolated pancreatic acini, results showed that ANF did not modify basal or forskolin-evoked cAMP formation, but it dose-dependently enhanced phosphoinositide hydrolysis, which was blocked by the selective PLC inhibitor U-73122. ANF stimulated exocrine pancreatic secretion in the rat, and its effect was not mediated by nitric oxide or parasympathetic or sympathetic activity. Furthermore, CCK and secretin appear not to be involved in ANF response. Present findings support that ANF exerts a stimulatory effect on pancreatic exocrine secretion mediated by NPR-C receptors coupled to the phosphoinositide pathway.     

Atrial natriuretic factor is a circulating hormone

The radioimmunoassay of atrial natriuretic factor (ANF) has been applied for determination of immunoreactive ANF (IR-ANF) in rat plasma. Immunoreactive ANF has been extracted from rat plasma by immunoaffinity column on Sepharose-4B anti-ANF or by Vycor glass. The mean concentrations of IR-ANF in ether anesthetized rats were found to be 1.61 +/- 0.14 ng/ml in female and 1.25 +/- 0.21 ng/ml in male rats when extracted on Sepharose-4B anti-ANF, and 1.21 +/- 0.10 ng/ml in females and 1.02 +/- 0.11 ng/ml in males when extracted by Vycor glass. A close linear correlation has been observed between the plasma IR-ANF concentrations in aorta and jugular vein. The described results indicate that atrial cardiocytes secrete atrial natriuretic factor into plasma. The heart is, therefore, an endocrine organ.     

Mechanisms of release and renal tubular action of atrial natriuretic factor

Inasmuch as atrial natriuretic factor (ANF) is apparently involved causally in the renal response to acute hypervolemia, it became of interest to study cellular mechanisms of release and renal tubular action. To study release mechanisms, freshly excised rat heart atria were incubated in vitro. Activation of the cellular adenylate cyclase system by either beta-adrenergic stimulation or the vasopressin analog deamino-8-D-arginine vasopressin did not result in ANF release. By contrast, activation of the polyphosphoinositide system by alpha-adrenergic stimulation or stimulation of the V1-type vasopressin receptors, and by a calcium ionophore or active phorbol ester, significantly increased natriuretic activity in the medium and reduced it in tissue. It is concluded, therefore, that activation of this latter system is the mechanism for ANF secretion from atrial myocytes. To test the effect of ANF on tubular transport in the medullary collecting duct, microcatheterization was used in rats before and during i.v. infusion of synthetic atrial peptide (23 amino acids). It was found that tubular delivery of salt to this part of the nephron was increased, and that reabsorption in the duct itself was reduced. In control experiments, increased delivery was associated with proportionately increased reabsorption, which demonstrated glomerulotubular balance in the nephron segment under normal conditions. The natriuretic effect of ANF, therefore, was not caused solely by enhanced tubular load, but included specific inhibition of duct sodium reabsorption as an essential feature of the renal response.     

Cellular signals regulating the release of ANF.

Atrial natriuretic factor (ANF), a peptide hormone that regulates salt and water balance and blood pressure, is synthesized, stored, and secreted from mammalian myocytes. Stretching of atrial myocytes stimulates ANF secretion, but the cellular processes involved in linking mechanical distension to ANF release are unknown. We reported that phorbol esters, which mimic the action of diacylglycerol by acting directly on protein kinase C and the Ca2+ ionophore A23187, which introduces free Ca2+ into the cell, both increase basal ANF secretion in the isolated perfused rat heart. Phorbol ester also increased responsiveness to Ca2+ channel agonists, such as Bay k8644, and to agents that increase cAMP, such as forskolin and membrane-permeable cAMP analogs. In neonatal cultured rat atrial myocytes, protein kinase C activation by 12-O-tetradecanoylphorbol 13-acetate stimulated ANF secretion, whereas the release was unresponsive to changes in intracellular Ca2+. Endothelin, which stimulates phospholipase C mediated hydrolysis of phosphoinositides and activates protein kinase C, increased both basal and atrial stretch-induced ANF secretion from isolated perfused rat hearts. Similarly, phorbol ester enhanced atrial stretch-stimulated ANF secretion, while the increase in intracellular Ca2+ appeared to be negatively coupled to the stretch-induced ANF release. Finally, phorbol ester stimulated ANF release from the severely hypertrophied ventricles of hypertensive animals but not from normal rat myocardium. These results suggest that the protein kinase C activity may play an important role in the regulation of basal ANF secretion both from atria and ventricular cells, and that stretch of atrial myocytes appears to be positively modulated by phorbol esters.