Effect of nitric oxide on basal and stretch-induced release of atrial natriuretic factor (ANF) from isolated perfused rat atria

We investigated the effect of the NO donor SNAP (6.7 nM) on basal and stretch-induced ANF release from isolated perfused rat atria. There was no significant difference in basal ANF secretion between the vehicle- and SNAP-infused atria (SNAP: 388+/-63 pg. 100 microl(-1), n = 13 vs. vehicle: 349+/-26 pg. 100 microl(-1), n = 5). Atrial distention caused an increase in ANF secretion in both the buffer- and SNAP-treated groups. SNAP greatly attenuated the stretch-induced increase in ANF (SNAP: 225+/-7 pg. 100 microl(-1), n = 5 vs. vehicle: 448+/-72 pg. 100 microl(-1), n = 13, P < 0.05). The compliance of atria treated with SNAP was lower than that of the vehicle-perfused atria (P < 0.05). Thus, although SNAP appeared to attenuate stretch-induced ANF secretion, there was in fact no significant difference in the ratio of Delta[ANF] to Deltaintraluminal volume (SNAP: 5.8+/-1.3 pg. 100 microl(-1). microl(-1) vs. vehicle: 8.2+/-1.4 pg. 100 microl(-1). microl(-1).). In conclusion, we found no evidence that NO alters the control of basal or stretch-induced ANF secretion. NO can however reduce ANF release by shifting the pressure-volume curve, so that a given increase in atrial pressure is associated with a smaller increase in intraluminal volume and reduced atrial distention.     

Intra-atrial communication and control of atrial natriuretic factor (ANF) release

Atrial natriuretic factor (ANF) release was studied in isolated perfused atria prepared from rats. When the vein-atrial junction (VAJ) was distended with an inflatable balloon, ANF release into the perfusate was greater in intact atria than in appendectomized atria. It was concluded that distention of the VAJ causes ANF release from the atrial appendage. A cascade experiment was then prepared whereby buffer from one isolated atrium perfused a second atrium. Although the VAJ of the first atrium could be distended by balloon, the atrial appendage was ligated so ANF was not secreted into the perfusate. The second atrium was intact, but no balloon was inserted. Despite the fact that there were no changes in intraluminal pressure, ANF secretion from the second atrium increased when the VAJ of the first atrium was distended. This response was blocked by the endothelin (ET) A receptor antagonist BQ-123. However, no distention-induced changes in ET-1 levels could be found in the perfusate from the first atrium. It is proposed that, in response to changes in distention of the VAJ, ANF is released remotely from the atrial appendage. The mediator does not appear to be ET-1 itself, but rather some factor that stimulates ET-1-induced ANF release within the tissue of the atrial appendage.     

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.     

Dissociation and enrichment of rat atrial myocytes containing atrial natriuretic factor (ANF)

Methodologies developed for the dissociation and subsequent enrichment of muscle and nonmuscle cells from atrial myocardium were used to evaluate the contribution of these cell populations to the natriuretic, diuretic and vasoactive properties of crude atrial tissue extracts. Suspensions of single cells, which contained approximately 34% myocytes, were prepared from atrial tissue blocks with a collagenase-trypsin digestion followed by gentle mechanical disruption. Differential centrifugation and unit gravity sedimentation techniques were employed to enrich the 'muscle' and 'nonmuscle' cell suspensions to a purity of approximately 91 and 95%, respectively. Cell extracts were bioassayed for natriuretic activity in saline-expanded, pentobarbital-anesthetized, female rats. Extracts obtained from 'initial' and 'muscle' cell suspensions significantly enhanced sodium and chloride excretion as well as urine flow while extracts from 'nonmuscle' cell suspensions had no effect on renal function. Sodium excretion was dose-dependent and increased linearly with increasing numbers of extracted and infused myocytes. This simple two-step centrifugation and sedimentation protocol can be utilized to obtain enriched atrial myocyte populations for subsequent physiologic and biochemical studies.     

Dopamine receptor antagonists inhibit the natriuretic response to atrial natriuretic factor (ANF)

The diuretic and natriuretic response of anesthetized rats to low doses of semi-purified atrial extracts or synthetic alpha-hANP was completely blocked by intravenous injection of 50 micrograms of haloperidol or chlorpromazine. Sulpiride or metoclopramide at the same doses did not show this effect. We conclude from these results that dopamine receptors, probably of the D1-type, are involved in the natriuretic effect of the atrial peptides.     

Immunohistochemical localization of atrial natriuretic factor (ANF) in the excretory system of the rabbit parotid gland

The immunohistochemical localization of atrial natriuretic factor (ANF) in the rabbit parotid gland was performed using an antibody against rabbit ANF and avidin-biotin or streptoavidin as detector. Results showed positivity in cuboidal and columnar cells of intralobular ducts and in basal cells of extralobular and main excretory duct. These data support the hypothesis that ANF produced by intralobular ducts could act through a paracrine mechanism; ANF produced by extralobular and main ducts may play a role in the regulation of salivary composition.     

Capsaicin-sensitive neural pathway mediates atrial natriuretic factor (ANF) release in response to physiological stimuli

Increases in intravascular volume are detected by mechanoreceptors situated at the junctions of the great veins with the atria. We had previously shown that localized distension of the superior vena caval/right atrial junction, simulating increased cardiac preload, elicits release of ANF remotely from the atrial appendage. We proposed that ANF secretion is stimulated via intrinsic neural pathways running from the venoatrial junctions to the appendage. We developed a technique whereby non-adrenergic, non-cholinergic sensory nerves could be selectively destroyed in the heart of adult rats by instilling capsaicin into the pericardial space. Four days later, the animals were killed, and isolated perfused atria were prepared with small balloons positioned so that the superior vena caval/right atrial junction could be discretely stretched. Immunoreactive ANF secretion into the perfusate was measured. Although distension of the venoatrial junction increased ANF secretion from the control atria, there was no such response in the denervated atria. We conclude (A) that local application of capsaicin to the heart of adult rats induces selective functional neural deficits and (B) that information regarding distension of the junction of the great veins and the atria is normally transmitted across the atrium via these nerves to stimulate ANF secretion from peptide stores located in the atrial appendage. We propose that these pathways are crucial to ensure appropriate ANF secretion in response to an increase in circulating blood volume.     

Presence of the atrial natriuretic factor (ANF) in human ascitic fluid

Presence of atrial natriuretic factor (ANF)-like material was demonstrated by radioimmunoassay in ascitic fluid of 14 patients with cirrhosis of the liver. Immunoreactive ANF concentrations (M +/- SEM) were 2.4 +/- 0.5 fmol/ml in ascites, significantly lower (p less than 0.001) than the corresponding plasma concentrations of 15.5 +/- 2.6 fmol/ml. High performance gel permeation chromatography and reverse phase high performance chromatography of the ascitic ANF immunoreactivity showed correspondence to the alpha human ANF (99-126). ANF levels in ascites were significantly (p less than 0.01) correlated to levels in plasma (r = 0.66).     

Expression of atrial natriuretic factor (ANF) during Xenopus cardiac development

We have isolated the Xenopus orthologue of the atrial natriuretic factor (ANF) gene. Characterization of embryonic expression indicates that the ANF gene is initially expressed throughout the developing myocardium at the late heart tube stage (about stage 32). This is in contrast to all previously characterized Xenopus cardiac differentiation markers that are first expressed in the cardiogenic plate at approximately stage 27. ANF expression becomes restricted exclusively to the atrium at about stage 47, long after the commencement of beating and the original formation of the atrial and ventricular compartments, but shortly after septation of the single atrium into two distinct atria. Received: 5 May 2000 / Accepted: 3 August 2000     

Localization and characterization of the N-terminal fragment of atrial natriuretic factor (ANF) precursor in the frog heart

The localization of the N-terminal fragment of the atrial natriuretic factor (ANF) precursor in the heart of the frog Rana ridibunda was examined by the indirect immunofluorescence and the immunogold techniques using an antiserum directed against synthetic rat ANF (Asp11-Ala37). At the optic level, positive material was found in most atrial myocytes. Staining of consecutive sections of frog heart with antibodies against N-terminal and C-terminal regions of the proANF molecule showed that both peptides are contained in the same cardiocytes. In the rat atrium, antibodies against the N-terminal ANF region induced a more intense labeling than in the frog atrium. Electron microscopic studies indicated that all secretory granules present in frog atrial cardiocytes contain N-terminal ANF-like immunoreactive material. The positive material localized in frog atrium was characterized by gel filtration and radioimmunological detection. Serial dilutions of frog atrial extracts exhibited displacement curves which were parallel to that obtained with synthetic human ANF (Asn1-Asp30). Sephadex G-50 gel chromatography of the immunoreactive material showed that the N-terminal ANF-like immunoreactivity eluted in a single peak corresponding to high molecular weight material. These results indicate that the N-terminal fragment of frog proANF is immunologically and biochemically related to the homologous mammalian peptide.     

Synthesis and biological activity profiles of atrial natriuretic factor (ANF) analogs

Several analogs of the atrial natriuretic factor (ANF) were synthesized by the solid-phase method using the acetamidomethyl (Acm) group for sulfhydryl protection. The compounds were tested in a receptor binding assay using bovine adrenal zona glomerulosa cell membranes and in the rat diuresis/natriuresis assay. Substitution of tyrosine in position 116 of ANF(101-126) and of the analog [3-Mpr105]ANF(105-126)(3-Mpr = 3-mercaptopropionic acid) did not alter the biological activity profiles and, therefore, these two analogs in radioiodinated form will be useful for enzymatic degradation and clearance studies. Replacement of 3-mercaptopropionic acid with 2-mercaptopropionic acid in [3-Mpr105]ANF(105-126) resulted in an analog with very low potency in both assay systems, presumably as a consequence of the steric bulk and/or local conformational restriction produced by the methyl group attached to the alpha-carbon in position 105. The analog [3-Mpr105,Nva109]ANF(105-126)(Nva = norvaline) showed very low affinity in the receptor binding assay but displayed considerable diuretic/natriuretic activity. The obtained biological activity profiles suggest that in comparison with other ANF peptides the des-amino ANF(105-126) analogs may have a somewhat longer half-life in vivo, or alternatively, may indicate a more complex situation of ANF receptor or binding site heterogeneity.     

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.     

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

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 less than 0.025) and 21 days (p less than 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.     

Pressure overload induces heterologous expression of the atrial natriuretic factor (ANF) gene

Several investigations have demonstrated the regional heterogeneity of myocardial phenotype, and hypertrophy may also induce regionally disparate changes. We have utilized the direct DNA injection technique to study regional variations in overload-induced ANF expression. Pressure overload was induced by stenosis of the ascending aorta in canines. ANF promoter reporters were injected into the left ventricle; in different regions including the base, the midwall region, and the apex. Injections were made at different depths to include the epicardial and endocardial layers. The animals were sacrificed 7 days following surgery and the left ventricle harvested for tissue analysis. Under normotensive conditions, ANF reporter expression was similar throughout the heart. PO increased ANF expression and the increases were greater in the endocardium than in the epicardium. PO also significantly increased expression in the midwall and base regions, but not in the apex. It is unknown from these experiments, whether the greater increases in midwall expression are a function of greater wall stress, metabolic demand, or phenotypic differences in the midwall myocytes. These findings do indicate that regional differences in overload-induced changes in gene expression are evident and may be functionally important in determining myocardial response to increased functional demand.     

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.     

Release of atrial natriuretic factor (ANF) induced by acute airway obstruction

The aim of this study was to measure the effects of an increase in negative intrathoracic pressure on the release of ANF. With the subjects seated comfortably, 3 control blood samples were obtained over 30 minutes. Eight subjects then breathed for 30 min. through an inspiratory resistance in such a way that maximal inspiratory pleural pressures were between -30 to -40 cmH2O. Three blood samples were withdrawn after 20, 25, and 30 min., with the subject still breathing against the artificial resistance. Plasma concentrations of ANF were analysed by RIA. They measured: control value 24.6 +/- 3.7 pg ANF/mL (X +/- SE); with resistance 37.1 +/- 8.1 pg/mL (p less than or equal to .05). These results suggest that ANF could be released during an asthma attack.     

Localization and identification of immunoreactive atrial natriuretic factor (ANF) in the frog ventricle

The localization of ANF-like immunoreactivity in the ventricle of the frog Rana ridibunda was examined by the indirect immunofluorescence and the immunogold techniques, using an antiserum against synthetic ANF (Arg 101-Tyr 126). At the optic level, an appreciable number of positive cardiocytes was observed in the frog ventricle. Electron microscopic studies showed that all secretory granules present in ventricular cardiocytes contain immunoreactive ANF. The immunoreactive material has been characterized by Sephadex G-50 gel chromatography and reverse phase high performance liquid chromatography (RP-HPLC). After gel filtration, ANF-like immunoreactivity eluted in 3 peaks. The major immunoreactive peak corresponded to high molecular weight material, while one peak co-eluted with synthetic ANF (Arg 101-Tyr 126). Further analysis of frog ventricular extracts by RP-HPLC revealed that the low molecular weight material has the same retention time as synthetic ANF, suggesting a high degree of sequence homology between amphibian and mammalian ANF. These results indicate that in amphibians, ventricular cardiocytes synthesize a peptide immunologically and chemically related to mammalian ANF.     

Effect of changes in sodium intake on atrial natriuretic factor (ANF) and peptides derived from the N terminus of the ANF prohormone in the rat.

The purpose of the present study was to determine whether variations in salt intake would alter the plasma concentrations of atrial natriuretic factor and the N-terminal atrial natriuretic factor prohormone peptides proANF 1-98 and proANF 31-67. Two groups of rats were placed on different salt intakes for 1 week. The low salt group of rats was fed a diet providing less than 0.1 mM NaCl/day and given deionized water to drink. The normal salt group of rats was fed regular rat chow with deionized water to drink, providing them with approximately 2 mM NaCl/day. Plasma atrial natriuretic factor was 204 +/- 60 pg/ml (mean +/- SE) in normal salt rats and was significantly lower in the low salt group (44 +/- 13 pg/ml, P less than 0.01). ProANF 1-98 was also significantly higher in the normal salt group (635 +/- 47 pg/ml) compared with the low salt group (353 +/- 33 pg/ml, P less than 0.01). ProANF 31-67 was 123 +/- 21 pg/ml in the normal salt group and 59 +/- 12 pg/ml in the low salt group (P less than 0.05). Plasma renin activity in ng angiotensin l/ml/hr averaged 1.80 +/- 0.15 in the normal salt group of rats and was significantly higher in the low salt group of rats (5.66 +/- 1.07, P less than 0.05). These results suggest that atrial natriuretic factor and the atrial natriuretic factor prohormones may play a role in the physiological adjustments to low salt intake.     

Degradation and clearance of atrial natriuretic factors (ANF)

Atrial natriuretic factor, the first well defined natriuretic hormone is synthesized in the human heart as 151 aminoacid (AA) preprohormone and stored as 126 AA prohormone in atrial granules. Upon appropriate stimulation, the prohormone is cleaved into a 98 AA N-terminal fragment and a 28 AA C-terminal fragment, the biological active ANF(99-126), both circulating in plasma. Circulating ANF(99-126) is cleared by various organs, such as lung, liver and intestine, kidney and upper and lower limbs. Reported arterial-venous extraction ratios vary greatly, but are not much different between organs, the average extraction ratio being about 35%. Due to marked differences of organ blood flow, the contribution of various organs to total body ANF clearance differs considerably. Major mechanisms for ANF clearance are uptake by clearance receptors and degradation by an endoprotease (EC 3.4.24.11.). Clearance receptors, distinct from the receptors mediating the biological actions of ANF, have been demonstrated in various organs. Characterization of the ANF degrading enzyme activity has been performed in kidney tissue. Whether and how pathophysiological states affect ANF clearance is still poorly understood. Inhibition of clearance by ANF analogues binding to clearance receptors and by inhibitors of degrading peptidase can increase the biological action of circulating ANF. This may prove to be a therapeutic approach in diseases with smooth muscle contraction or volume overload.