Atrial dynamics and release of atrial natriuretic factor in vivo.

In anaesthetized rabbits blood volume was altered by infusion and withdrawal of donor blood over the range of +60 to -40% of the blood volume. Right and left atrial pressures were measured and it was shown that sonomicrometry allowed adequate measurement of phasic changes in atrial dimensions. Plasma immunoreactive atrial natriuretic peptide concentration changed in a nonlinear fashion with changes in blood volume, and was linearly related to both peak systolic and peak diastolic right and left atrial wall stress. It was not possible to make the distinction between distension (diastolic stress) or tension (systolic stress) as the major determinant of ANF release in response to changes in blood volume.     

The effect of isoproterenol on right and left atrial dynamics and plasma atrial natriuretic factor in anesthetized rabbits.

The effect of isoproterenol on mean right and left atrial pressures (RAP, LAP) and dimensions (RAD, LAD), and plasma immunoreactive atrial natriuretic factor (IR-ANF) was investigated in anesthetized rabbits. Infusion of isoproterenol (10 micrograms.kg-1.min-1 for 10 min) significantly increased plasma IR-ANF and heart rate. There were no significant changes in mean RAP or LAP following isoproterenol. Neither mean RAD, systolic RAD and diastolic RAD nor mean LAD, systolic LAD or diastolic LAD changed significantly. Systolic right and left atrial wall stress and diastolic right and left atrial wall stress did not change significantly during the infusion of isoproterenol. Since atrial dimensions did not increase, it is unlikely that the release of IR-ANF in response to isoproterenol is mediated by atrial stretch. These results suggest that the release of IR-ANF in response to this dose of isoproterenol is mediated by factors other than stretch or changes in atrial dynamics.     

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.     

Synergism of atrial pressure and adrenergic stimulation for ANF release in the rat.

The effect of adrenergic stimulation on atrial natriuretic factor (ANF) release was studied in conscious rats. 60 min i.v. infusion of 15 micrograms/kg/min phenylephrine produced an elevation in mean arterial pressure of about 50 mmHg that was associated with an immediate, transitory increase in both central venous (CVP) and left ventricular end-diastolic (LVEDP) pressures. Heart rate was not changed. The elevation in CVP, but not LVEDP, persisted until 5 min, together with a 50-fold increase in plasma C-terminal ANF concentrations (from 19 +/- 5 to 1010 +/- 339 fmol/ml); at 30 min, in the absence of any increases in CVP or LVEDP, plasma ANF was still elevated by 5-fold (114 +/- 35 fmol/ml). It is suggested that adrenergic stimulation 'per se' can induce ANF release, independently of atrial pressure. Furthermore, combined adrenergic stimulation and increased atrial wall tension could result in a potentiation of the ANF secretory response 'in vivo', in the case where both stimuli are present concomitantly.     

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.     

Determinants of atrial natriuretic factor secretion in dogs expanded with isotonic saline or colloid solutions.

Through increments in blood volume and atrial pressure are thought to be the primary stimuli for ANF secretion, plasma levels of this peptide do not always behave as a simple function of volume status. To outline the relationship between the latter and cardiac ANF release, we used five different volume-expansion protocols in anesthetized dogs. A stepwise expansion of plasma volume (PV) was achieved by two consecutive infusions: 0.9% saline followed or preceded by 4 or 25% bovine serum albumin (BSA), 4 or 25% dextran (Dx), or homologous plasma. Saline expansion led to a two- to four-fold increase in arterial plasma ANF level in all five protocols. Both 4 and 25% BSA caused no or very modest increase in plasma ANF, while all other colloid expanders caused the expected ANF release. In all protocols, plasma ANF closely correlated with central venous pressure (CVP). BSA expansion was the only protocol with no correlation between PV and ANF release. Changes in serum Ca2+ could not explain this finding. During BSA expansion, the lack of atrial response was related to the absence of increment (or even fall) in CVP despite the expanded PV. Similarly, urinary Na+ excretion was correlated both with CVP and ANF level but not with PV in BSA expansion. When the dogs were depleted of histamine before BSA infusion, the atrial secretory response was restored, suggesting that this colloid was associated with augmented capillary leakiness and vascular fluid efflux. These results show that the expansion of PV leads neither to ANF release nor to Na+ excretion if it is not accompanied by an expanded central blood volume with elevated atrial pressure.     

Hypoxia-induced release of atrial natriuretic factor (ANF) from the isolated rat and rabbit heart

The effect of hypoxia on the release of atrial natriuretic factor (ANF) was studied in isolated, constant-flow perfused hearts of rats and rabbits. Effluent samples were frozen pending extraction and radioimmunoassay of ANF. Hypoxia (10 min) caused a 3.9-fold (rats) and 4.6-fold (rabbits) increase of ANF release over control values. ANF release returned to control levels within 8-11 min of reoxygenation. Prolonged (20 min) hypoxia evoked further ANF release. The increase in ANF release and decrease in ventricular pressure, heart rate and coronary perfusion pressure were fully reversible, suggesting that tissues were not damaged. These results demonstrate that hypoxia induces a massive release of ANF by an as yet unexplained mechanism.     

Quantification of messenger ribonucleic acid for atrial natriuretic factor in atria and ventricles of Dahl salt-sensitive and salt-resistant rats

The levels of atrial natriuretic factor (ANF) and the mRNA for ANF were measured in the left ventricles of Dahl salt-sensitive (S) and salt-resistant (R) rats. ANF and ANF mRNA were both much higher in ventricular tissue of newborn rats of both strains compared to young adults, which represents the normal developmental pattern. There was no strain difference between S and R when the rats were young (1.5 months of age), but in older animals (8.5 months of age), when S rats were markedly hypertensive, there was a 5- to 10-fold increase in both left ventricular ANF and left ventricular ANF mRNA in S, but not R, rats. Atrial ANF mRNA was not similarly increased in hypertensive S rats. The ANF levels present in ventricles could not be accounted for by contamination with plasma ANF. Moreover, HPLC analysis of the forms of ANF in ventricles of newborn and hypertensive S rats showed that immunoreactive ANF in ventricles was present mainly in the same precursor form found in atria and not the shorter peptide form found in plasma. Northern blot analysis showed that ANF mRNA for atria and ventricles were the same size. It is concluded that in the S rat the heart left ventricle responds to hypertension by increasing production and storage of ANF.     

Biochemically stimulated release of atrial natriuretic factor from heart-lung preparation of Dahl rats

We investigated the effect of sodium chloride and adrenergic agents on the release of atrial natriuretic factor (ANF) using working heart-lung preparations from Dahl salt-hypertension sensitive (S) and Dahl salt-hypertension resistant (R) rats. High concentrations of NaCl moderately increased ANF release, but this was attributed to small increases in left atrial pressure rather than to a direct effect of NaCl on ANF release; S and R rats responded similarly. Neither isoproterenol (beta 1 + beta 2 agonist) nor clonidine (alpha 2 agonist) had any effect on ANF release in the heart-lung preparation. In contrast, phenylephrine (alpha 1 agonist) stimulated ANF release. This could not be accounted for by change in atrial pressure and appeared to be a direct effect. S and R rats both released ANF in response to phenylephrine, but there was a modest tendency for hypertensive S rats to release more ANF than normotensive R rats, which is consistent with previous data on mechanically induced (atrial stretch) ANF release in these strains.     

Correlation between cardiac hypertrophy and plasma levels of atrial natriuretic factor in non-spontaneous models of hypertension in the rat

We have compared atrial and plasma concentration of atrial natriuretic factor (ANF) in 4 models of non spontaneous experimental hypertension with different pathogenic mechanisms in the rat: two-kidney, one-clip (2-K, 1-C), one-kidney, one-clip (1-K, 1-C), DOCA-NaCl and adrenal regeneration hypertension (ARH) and their respective normotensive controls. All hypertensive groups developed cardiac hypertrophy. In all hypertensive groups plasma ANF was higher than in controls. Atrial ANF concentration was lower in the right and left atrium of 1-K, 1-C rats and in the left atrium of ARH. A good correlation was found between systolic BP and plasma ANF in 2-K, 1-C (r = 0.82; p less than 0.01) and 1-K, 1-C animals (r = 0.70; p less than 0.01). This correlation was less good in DOCA-NaCl (r = 0.41; p less than 0.05) and non existent in ARH (r = 0.28; NS). A negative correlation between plasma ANF and atrial ANF concentrations was found only in the 1-K, 1-C group (r = 0.41; p less than 0.05). A good correlation between plasma ANF levels and cardiac weight was found in all groups: 2-K, 1-C (r = 0.83; p less than 0.01), 1-K, 1-C (r = 0.73; p less than 0.01), DOCA-NaCl (r = 0.69; p less than 0.01) and ARH (r = 0.71; p less than 0.01). We suggest that the release of ANF in experimental hypertension depends of the pathogenesis and could be related either to the level of BP (hence the magnitude of the left ventricular end-diastolic pressure) or to the existence of an expanded blood volume. The correlation between plasma ANF levels and cardiac hypertrophy suggests that ANF could be partially released by the ventricles.     

Specific binding of atrial natriuretic factor to renal glomeruli in Doca- and Doca-salt-treated rats correlation with atrial and plasma levels

Since volume expansion and high blood pressure (BP) are known stimuli of atrial natriuretic factor (ANF) release, and since this peptide may be involved in mineralocorticoid escape, we investigated the effects of chronic deoxycorticosterone (DOCA) and DOCA-NaC1 treatment on renal glomerular ANF receptor density and affinity in relation to atrial and plasma ANF levels. An increase in plasma immunoreactive ANF (IR-ANF) was observed both after two and four weeks of treatment. IR-ANF concentrations were elevated in the left atrium only in four-week DOCA treated rats. Administration of the mineralocorticoid alone resulted in a decreased density of glomerular ANF receptors in both time periods investigated. DOCA-NaC1-treated animals presented an increased receptor density during the pre-hypertensive stage (2 weeks) and a reduced density in the later hypertensive period (4 weeks). Receptor affinity in both groups was identical to that in the controls after 2 weeks and was augmented after 4 weeks of treatment. Our data suggest that the down-regulation of renal glomerular ANF receptors during chronic DOCA-NaC1 administration may play a role in the maintenance of high BP in this model of volume-expanded hypertension.     

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.     

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.     

Atrial natriuretic factor receptors are negatively coupled to adenylate cyclase in cultured atrial and ventricular cardiocytes

We have studied the effect of synthetic rat atrial natriuretic factor (ANF) on adenylate cyclase activity in cultured cardiocytes from atria (left and right) and ventricles from neonatal rats. ANF (Arg 101-Tyr 126) inhibited adenylate cyclase activity in a concentration dependent manner in cultured atrial (right and left atria) and ventricular cells. However the inhibition was greater in atrial cells as compared to ventricular cells. The maximal inhibition observed in ventricular cells was about 35% with an apparent Ki of about 10(-10) M, whereas about 55% inhibition with an apparent Ki between 5 X 10(-10) M and 65% inhibition with an apparent Ki of 10(-9) M were observed in right and left atrial cardiocytes respectively. The inhibitory effect of ANF was dependent on the presence of guanine nucleotides. Various hormones and agents such as isoproterenol, prostaglandins, adenosine, forskolin and sodium fluoride stimulated adenylate cyclase activities to various degrees in these atrial and ventricular cardiocytes. ANF inhibited the stimulatory responses of all these agonists, however the degree of inhibition varied for each agent. In addition ANF also inhibited cAMP levels in these cells. These data indicate that ANF receptors are present in cardiocytes and are negatively coupled to adenylate cyclase.     

Natriuretic peptide gene expression in DOCA-salt hypertension after blockade of type B endothelin receptor

We investigated the effect of long-term in vivo blockade of the ET-1 receptor subtype B (ET(B)) with A-192621, a selective ET(B) antagonist, on atrial and ventricular natriuretic peptide (NP) gene expression in deoxycorticosterone acetate (DOCA)-salt hypertension. In this model, stimulation of the cardiac natriuretic peptide (NP) and the endothelin system and suppression of the renin-angiotensin system is observed. DOCA-salt induced significant hypertension, cardiac hypertrophy and increased NP plasma and left atrial and right and left ventricular NP gene expression. ET(B) blockade per se produced hypertension and left ventricular hypertrophy but induced little change on the levels of ventricular NP and only increased left atrial natriuretic factor (ANF) mRNA levels. Combined ET(B) blockade/DOCA-salt treatment worsened hypertension, increased left ventricular hypertrophy and induced right ventricular hypertrophy. All animals so treated had increased ventricular NP gene expression. Collagen III and beta-myosin heavy chain gene expression were enhanced in both the right and the left ventricle of DOCA-salt hypertensive rats. The results of this study suggest that the ET(B) receptor does not participate directly in the modulation of atrial or ventricular NP gene expression and that this receptor mediates a protective cardiovascular function. ET(B) blockade can induce significant ventricular hypertrophy without an increase in ANF or brain NP gene expression.     

Left ventricular hypertrophy in obese hypertensives: is it really eccentric? (An echocardiographic study)

In order to study left ventricular hypertrophy patterns in obese hypertensives, we examined 132 patients with essential hypertension by 2D, M-mode and Doppler echocardiography. The patients were classified in four comparable groups, corresponding to the values of Quetelet's body mass index (BMI) and grades of obesity. More obese hypertensives had on average larger left ventricles with thicker walls and larger left atria than less obese, or lean ones. Left ventricular mass increased significantly and progressively with advancing grades of obesity, but relative wall thickness (wall thickness/cavity size ratio) did not diminish. Doppler echocardiography revealed significantly higher prevalence of left ventricular diastolic dysfunction among obese than among lean hypertensives. In the second part of our study, we analyzed the subgroups defined by the severity of hypertension and the age of the patients. The correlation of the indices of left ventricular and left atrial hypertrophy with the BMI values was considerably better in the group of moderate than in the group of mild hypertension. The r values were 0.62 vs. 0.22 for left ventricular mass and 0.64 vs. 0.26 for left atrial dimension. The group of patients with severe hypertension was characterized by left ventricular cavity enlargement in correlation with increasing BMI values, but without corresponding left ventricular wall thickening. So called left ventricular "eccentricity index", as the reverse value of relative wall thickness, correlated well (r = 0.76) with the BMI values. The indices of left ventricular hypertrophy correlated with the BMI values slightly better in middle age groups than in the groups of the youngest (< or = 30 years) or the eldest (> or = 61 years) hypertensives. In conclusion, eccentric left ventricular hypertrophy does not seem to be a distinctive feature of hypertensive heart disease in obesity. There is only some tendency toward the "eccentricity" of left ventricular geometry which becomes more apparent in more severe forms of hypertension, especially in very obese persons.     

Calcium dependence of phenylephrine-, endothelin-, and potassium chloride-stimulated atrial natriuretic factor secretion from long term primary neonatal rat atrial cardiocytes

Since calcium is involved in both excitation-secretion and excitation-contraction coupling, it was of interest to evaluate its involvement in atrial natriuretic factor (ANF) release from atrial cardiocytes. In medium containing physiological levels of calcium (1.4 mM), the secretion of ANF from primary atrial cells was stimulated from 3- to 6-fold by a variety of agents including KCl, phenylephrine, and endothelium (ET). However, in medium containing 2 nM calcium, KCl was incapable of increasing ANF secretion above basal levels, while the stimulatory effects of phenylephrine and ET were only partially diminished. Nifedipine or verapamil could mimic the effects of the 2 nM calcium medium on KCl-, phenylephrine-, and ET-stimulated ANF secretion. Kinetic studies indicated that during the initial 5 min of ET-stimulated secretion the cells exhibited little requirement for extracellular calcium; however, the requirement was more apparent during the sustained secretion observed between 10 min and 2 h of secretagogue exposure. Additionally, the stimulation of ANF secretion by ET increased to a maximum of about 15-fold over basal by 10-min after ET application; subsequent to this time there was an apparent functional desensitization wherein the rate of secretion decreased by approximately 3-4-fold and remained at this level for the duration of secretagogue exposure up to 2 h. All forms of stimulated secretion could be inhibited through ionomycin-mediated depletion of intracellular calcium pools. Taken together, these results indicate that atrial cardiocytes require both extracellular and intracellular calcium to support maximal rates of stimulated ANF secretion, and that intracellular calcium pools may be used during the early phase of secretion, while the extracellular source of calcium may be important for the sustained phase of secretion.     

Atrial natriuretic factor in essential hypertension

We measured circulating levels of immunoreactive atrial natriuretic factor (ANF) in 10 patients with untreated, uncomplicated mild to moderate essential hypertension and in 15 normotensive controls. ANF concentrations were significantly higher in the hypertensive group than in the control group (38.4 +/- 6.9 pg/ml versus 18.3 +/- 1.8 pg/ml, p less than 0.02). A positive correlation between ANF levels and systolic, diastolic and mean blood pressure was noted in the total study population (p less than 0.008, r = 0.52; p less than 0.005, r = 0.55; p less than 0.02, r = 0.46, respectively). Thus, plasma ANF concentrations are elevated in essential hypertension and may result from increased intraarterial pressure.     

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

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

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.