Atrial natriuretic factor (ANF) inhibits the growth and the secretory activity of rat adrenal zona glomerulosa in vivo

A prolonged infusion with ANF induced atrophy of zona glomerulosa cells of rat adrenals and lowering of plasma concentration of aldosterone, without provoking significant changes in PRA. It also notably reduced the rise in the aldosterone plasma level caused by the acute stimulation with angiotensin II. Zona fasciculata cells and the blood concentration of corticosterone did not display any significant change. These findings are interpreted to indicate that ANF exerts an inhibitory effect on the growth and secretory activity of rat zona glomerulosa.     

Inhibitory effect of somatostatin on the growth and steroidogenic capacity of rat adrenal zona glomerulosa

Chronic administration of somatostatin induced atrophy of zona glomerulosa cells of rat adrenals and lowering of plasma aldosterone concentration. Zona fasciculata cells did not display any significant change and corticosterone plasma concentration was not significantly affected. These findings are interpreted to indicate that somatostatin exerts a direct inhibitory effect on the growth and steroidogenic capacity of rat adrenal zona glomerulosa.     

Evidence that prolonged alpha-MSH infusion enhances the steroidogenic capacity of rat adrenal zona glomerulosa in vivo

Prolonged infusion with 120 micrograms/kg/day alpha-MSH significantly increased basal plasma level of aldosterone in the rat, as well as raised the acute aldosterone response to a bolus administration of a high dose of ACTH or angiotensin II. These findings suggest that chronic alpha-MSH treatment stimulates the steroidogenic capacity of rat zona glomerulosa.     

Rat atrial natriuretic polypeptide stimulation of adrenal zona glomerulosa cell growth

Rat atrial natriuretic polypeptide (rANP) was found to stimulate [3H] thymidine incorporation into the DNA of bovine adrenal glomerulosa cells in a primary culture in a dose-dependent manner. The minimum effective dose was a very low concentration (10(-12) M of ANP), suggesting that ANP had a physiological effect. These findings are the first indication that ANP possesses growth-stimulating activity with regard to adrenal zona glomerulosa cells.     

Characterization of specific receptors for atrial natriuretic factor in bovine adrenal zona glomerulosa

We have recently shown that synthetic rat atrial natriuretic factor (ANF) directly inhibits mineralocorticoid and glucocorticoid secretion in cultured bovine adrenal cells with a potency of 100 pM. [125I]iodo-ANF was used in the present study to characterize potential receptor sites in bovine zona glomerulosa membranes. ANF binds to a class of high affinity binding sites with a pK of 10.2 and a density of 1.3 pmol/mg protein. Detailed competition curves with ANF document a class of high affinity sites with a pK of 10.2 and also a second class of lower affinity sites with a pK of 8.5. Nonspecific binding amounts to less than 10% of [125I]iodo-ANF binding at concentrations less than 100 pM. High affinity binding of [125I]iodo-ANF is reversible with a half-time of association of 15 minutes at 25 pM and a half-time of dissociation of 140 minutes. Monovalent cations Na, Li and K equipotently enhance [125I]iodo-ANF specific binding. Divalent cations Mg, Ca and Mn also increase [125I]iodo-ANF specific binding, with Mn being the most active cation. No effect of guanine nucleotide could be detected on ANF binding. The binding of [125I]iodo-ANF is very specific and is not inhibited by 1 microM angiotensin II, ACTH, VIP, somatostatin, Leu-enkephalin, dynorphin or by the N-terminal of POMC. The N-terminal fragment ANF-(1-16) is also completely inactive. Reduction of the disulfide bridge of ANF inactivates the peptide. This enabled the development of a highly specific radio-receptor assay for ANF with a minimum detectable dose of 2 femtomoles. The results document the specific receptor involved in the potent inhibitory effect of ANF on adrenal steroidogenesis and indicate that bovine adrenal zonal glomerulosa provide a highly sensitive system for studying the recently discovered atrial natriuretic factor.     

Affinity cross-linking of atrial natriuretic factor to its receptor in bovine adrenal zona glomerulosa

125I-Labeled atrial natriuretic factor (ANF) was covalently cross-linked to its binding sites in bovine adrenal zona glomerulosa membranes using the hydrophilic cross-linker bis(sulfosuccinimidyl) suberate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol revealed that two protein bands with apparent Mr 68,000 and 114,000 were specifically labeled. The labeling of the two bands was prevented in a dose-dependent fashion by unlabeled ANF with a significant inhibition observed at 10(-10) M. High concentrations of angiotensin II and adrenocorticotropic hormone did not compete with 125I-ANF for binding and cross-linking. The dose-response curve for inhibition of covalent cross-linking of 125I-ANF by unlabeled ANF coincided with the dose-response curve for inhibition of binding to the receptor. No radioactive bands were observed in liver membranes. Experiments in which adrenal membranes were prepared and incubated in the presence of protease inhibitors showed no difference in the labeling pattern. Electrophoresis in the absence of reductant showed that the affinity-labeled species are not derived from larger disulfide-linked components. The apparent molecular weight of the two labeled species was not affected by a 100-fold variation in cross-linker concentration, and the labeling of both species increased in parallel. Possible relationships between the two labeled species are discussed.     

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.     

Amiloride potentiates atrial natriuretic factor inhibitory action by increasing receptor binding in bovine adrenal zona glomerulosa

The interaction of atrial natriuretic factor (ANF) with the diuretic amiloride was studied in bovine adrenal zona glomerulosa. Amiloride enhances 2 to 3-fold high affinity binding of [125I] ANF to zona glomerulosa membrane receptor with an ED50 of 10 microM. This effect is due to a recruitement of high affinity receptor sites and to an increase of their affinity from a Kd of 23 to 8 pM. This enhancing effect is almost equipotently elicited by guanabenz, while clonidine is 20-fold less potent and arginine is inactive. ATP reduces by 30 to 50% [125I] ANF binding with an IC50 of 50 microM. Amiloride and ATP opposite effects on [125I] ANF binding are mutually competitive. Low concentrations of amiloride (less than 100 microM) potentiate the inhibitory effect of ANF in hormone-stimulated steroid secretion with a 3-fold decrease in ANF IC50 at 10 microM amiloride. Higher concentrations of amiloride (greater than 100 microM) directly inhibit aldosterone secretion with an IC50 of 500 microM and a maximum of 80 to 100% reversal of stimulation by various secretagogues. These results indicate that amiloride synergistically potentiates ANF inhibitory action by altering ANF receptor binding properties. They also suggest a role for sodium transport and for phosphorylation-dephosphorylation mechanisms in the mode of action of ANF.     

Actions of synthetic atrial natriuretic factor on bovine adrenal glomerulosa

Synthetic atrial natriuretic factor (ANF) inhibited aldosterone production by suspensions of bovine adrenal glomerulosa cells. Inhibition by ANF was most pronounced when basal aldosterone production was measured. The effects of angiotensin II (AII), N6,O2'-dibutyryl-adenosine 3':5'-cyclic monophosphate (dibutyryl cyclic AMP), and elevated potassium were also inhibited by ANF. Inhibition could be partially overcome by high doses of agonist. Inhibition was localized to the early pathway of aldosteronogenesis, to a step before cholesterol side-chain cleavage. ANF had no effect on binding of AII to receptors, on the stimulation by AII of phospholipid turnover, or on the alteration by AII of calcium fluxes.     

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.     

Effect of atrial natriuretic factor (ANF)-related peptides on aldosterone secretion by adrenal glomerulosa cells: critical role of the intramolecular disulphide bond

We previously demonstrated that synthetic 48-73 atrial natriuretic factor (ANF) (previously called 8-33 ANF) blocked the response of rat adrenal glomerulosa cells to angiotensin II, ACTH and potassium. We have now investigated the effects of natural 43-73 ANF, oxidised synthetic 48-73 ANF and the natural 1-73 ANF on aldosterone output by rat glomerulosa cells. The natural 43-73 ANF and the natural 1-73 ANF were equipotent to 48-73 ANF in inhibiting the stimulation of aldosterone secretion produced by angiotensin II with an IC50 of 2 X 10(-9)M. Similar results were obtained with ACTH and potassium. After oxidation with performic acid, 48-73 ANF was completely devoid of activity on the response of aldosterone to angiotensin II, ACTH and potassium. We conclude that the intramolecular disulphide bond in 48-73 ANF is critical for maintaining the active conformation of ANF.     

Structure-activity relationships of atrial natriuretic factor (ANF). III. Correlation of receptor affinity with relative potency on aldosterone production in zona glomerulosa cells

The activity of various fragments of ANF as inhibitors of aldosterone secretion and as competitors of [125I] ANF (Arg101-Tyr126) binding to specific receptors was studied in bovine zona glomerulosa. Shortening or lengthening the N-terminal segment of ANF does not alter its biological activity while minimally altering affinity for its receptor. Removal of the C-terminal to Cys121 or expansion up to Arg128 leads to 1000-fold decrease in receptor affinity and activity. The results indicate the importance of the C-terminal segment of ANF in determining its active conformation.     

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.     

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.     

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).     

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.     

Binding sites for atrial natriuretic factor (ANF) in kidneys and adrenal glands of spontaneously hypertensive (SHR) rats

Binding sites for atrial natriuretic factor (ANF) were studied in kidneys and adrenal glands of 17 week old male spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) normotensive rats by quantitative autoradiography using 125I-ANF-28. In kidney, 125I-ANF-28 binding sites were found in high concentrations in glomeruli and in much lower concentrations in the renal papilla. In adrenal gland, 125I-ANF-28 binding sites were highly localized to the zona glomerulosa and were of moderate density in the inner cortical regions. ANF binding sites did not occur in the adrenal medulla. The maximum binding capacity (Bmax) of 125I-ANF-28 was reduced by 50% in the kidney glomeruli of SHRs compared to WKY controls. In contrast, the affinity constant (Ka) for 125I-ANF-28 was elevated by 100% in kidney glomeruli of SHRs. There were no significant strain differences in values for Bmax or Ka for 125I-ANF-28 binding in the adrenal zona glomerulosa. These findings suggest that the natriuretic and diuretic actions of ANF within kidney glomeruli may be compromised in adult SHR rats and these alterations may contribute to the development and maintenance of hypertension in rats of this strain.     

Effects of prolonged cysteamine administration on the rat adrenal cortex: evidence that endogenous somatostatin is involved in the control of the growth and steroidogenic capacity of zona glomerulosa.

A week daily administration of cysteamine (CYS, 300 mg kg-1) lowered plasma aldosterone concentration in rats, without affecting PRA, kalaemia and the plasma levels of ACTH and corticosterone. Prolonged CYS treatment caused a notable hypertrophy of adrenal zona glomerulosa (ZG) and its parenchymal cells, without inducing any apparent change in zona fasciculata morphology. Isolated ZG cells from CYS-treated rats evidenced a notable enhancement in their basal and maximally-stimulated productions of aldosterone and corticosterone. All these effects of chronic CYS administration were completely reversed by the simultaneous infusion of rats with somatostatin (SRIF, 12 micrograms kg-1 h-1). CYS exposure was not found to directly affect the secretory activity of isolated ZG cells from normal rats. Since CYS is known to be a specific depletor of SRIF in different organs of rats, these findings suggest that endogenous SRIF may be involved in the modulation of ZG function.     

Functional heterogeneity of atrial natriuretic factor receptor in bovine adrenal zona glomerulosa is explained by an amiloride-sensitive high affinity molecular complex

The effects of amiloride on the molecular characteristics of the atrial natriuretic factor (ANF) receptor from bovine adrenal zona glomerulosa were studied by computer modeling of competitive binding data, by affinity labeling experiments, and by steric exclusion high performance liquid chromatography of solubilized receptor. The order of potency of a series of truncated ANF analogs in competing for 125I-ANF binding to bovine adrenal zona glomerulosa membranes was the same as that obtained for inhibition of aldosterone secretion. Deletion of amino acids at the COOH-terminal end drastically reduced the affinities of the peptides. Computer analysis of competition curves revealed that all ANF analogs tested show similar binding characteristics: shallow competition curves, discrimination of varying proportions of high and low affinity binding states, and sensitivity to amiloride which increases the proportion of the high affinity binding component. These results from binding studies are suggestive of potential heterogeneity of ANF binding sites. In contrast, results from affinity cross-linking experiments are consistent with the notion of a single receptor protein. Incubation of membranes with increasing concentrations of 125I-ANF-(99-126) up to 3 nM resulted in the labeling of a single band of Mr 130,000. The ability of ANF analogs to compete for the labeling of the Mr 130,000 band by 125I-ANF-(99-126) agreed well with their potency as inhibitors of 125I-ANF binding to intact membranes. Addition of amiloride caused a dose-dependent increase in the labeling of the Mr 130,000 band. A single Mr 130,000 band was also labeled in bovine aorta and LLC-PK1 cell membranes. In order to further investigate the molecular basis for the apparent heterogeneity of ANF binding we have prelabeled the membrane receptor with 125I-ANF-(99-126) prior to solubilization with octyl-beta-D-glucoside and chromatography on a Superose 6 steric exclusion column. The elution profile of the prelabeled receptor consistently showed two peaks of radioactivity with mean Stokes radii of 70 and 50 A. When amiloride was added to the incubation medium, the elution profile consisted almost exclusively of the 70-A peak. Quantitative analysis of the chromatographic profiles revealed that amiloride increases by 2-3 times the area of the 70-A peak. We conclude that the 70-A form represents a ternary complex of the receptor with an amiloride-sensitive effector protein.     

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.