Bovine adrenal chromaffin granules are a site of synthesis of atrial natriuretic factor

We have previously reported the existence of a peptide factor in the adrenal medulla which inhibits aldosterone secretion in cultured bovine zona glomerulosa cells. The acid extracts of chromaffin granules from bovine adrenal medulla were purified by a four step high performance liquid chromatography procedure. Two active fractions exhibited sequence homology with bovine atrial natriuretic factor ANF (Ser99-Tyr126) and its polypeptide precursor (Asn1-Tyr126). The occurrence of both precursor and mature forms of ANF within chromaffin granules indicates the endogenous character of ANF in the adrenal medulla and suggests the potential usefulness of cultured adrenal chromaffin cells for investigating the synthesis, maturation and secretion of atrial peptides.     

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.     

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.     

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.     

Purification and primary structure of pro-aldosterone secretion inhibitory factor from bovine adrenal chromaffin cells

This report documents the purification and the complete primary structure of bovine aldosterone secretion inhibitory factor precursor (pro-ASIF). ASIF-(1-103) contains at position 69-103 of its carboxy-terminal end the formely identified 35-amino acid biologically active form, hence confirming the endogenous character of ASIF in the adrenal medulla. Compared to atrial natriuretic factor (ANF)-related peptide precursors, bovine ASIF displays 65% homology at the carboxy-terminal while the remaining amino-terminal part shows much more variability. Bovine pro-ASIF exhibits 73% homology with porcine pro-brain natriuretic peptide (BNP), a situation reminiscent of the relationship of pro-ANF in various species. When ANF- and BNP-related COOH-termini of bovine, porcine, human, rat, and chicken are compared, it appears that bovine ASIF and porcine BNP are closely related and belong to the same family which however appears to be much more heterogenous than the ANF-related family. These results strongly suggest that bovine ASIF is encoded by a precursor gene similar to the gene of BNP but different from the one encoding ANF.     

Identification of aldosterone secretion inhibitory factor in bovine adrenal medulla

We report the first demonstration of an Aldosterone Secretion Inhibitory Factor (ASIF) in acid extracts of bovine adrenal medulla. Following separation from catecholamines and enkephalins, this factor leads to an 80% inhibition of PGE1-stimulated secretion of aldosterone from bovine adrenal zona glomerulosa. ASIF is retained on cation exchange gels and behaves as a small 5K-dalton peptide on Sephadex G-50. This factor cross-reacts in a radio-receptor assay for [125I] atrial natriuretic factor (ANF). ASIF is distinct from all neuropeptides formerly detected in the adrenal medulla, e.g. somatostatin, enkephalin, neuropeptide Y, dynorphin, neurotensin. In the adrenal gland, this ANF-like factor is predominantly found in the medulla (4 pmol/mg protein), with only trace amounts in the cortex (0.1 pmol/mg protein). ASIF might perhaps correspond to the endogenous ligand for the receptor sites that we have previously identified with [125I]ANF in bovine adrenal cortex and could contribute to the formerly reported attenuating influence of the adrenal medulla on mineralocorticoid production.     

Blood-brain barrier and atrial natriuretic factor

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

Natriuretic Peptides Inhibit Nicotine-Induced Whole-Cell Currents and Catecholamine Secretion in Bovine Chromaffin Cells: Evidence for the Involvement of the Atrial Natriuretic Factor R2 Receptors

Abstract: There is increasing evidence that members of the natriuretic peptide family display sympathoinhibitory activity, but it remains uncertain which receptor pathway is implicated. We performed cyclic GMP production studies with chromaffin cells treated with either atrial natriuretic factor (ANF) or C-type natriuretic peptide (CNP) and found that these cells specifically express the ANF-R_1C but not the ANF-R_1A receptor subtype. Evidence for the existence of ANF-R₂ receptors was obtained from patch-clamp experiments where C-ANF, an ANF-R₂-specific agonist, inhibited nicotinic currents in single isolated chromaffin cells. Involvement of ANF-R₂ receptors in the modulation of nicotinic currents was further supported by the significant loss of this inhibitory activity after the cleavage of the disulfide-bridged structure of C-ANF. This linearized form of C-ANF also displayed a lower binding affinity for ANF-R₂ receptors. Like the patch-clamp studies, secretion experiments demonstrated that both CNP and C-ANF are equally effective in reducing nicotine-evoked catecholamine secretion by cultured chromaffin cells, raising the possibility that this effect of CNP is predominantly mediated by the ANF-R₂ and not the ANF-R_1C receptors. Finally, this response appears to be specific to nicotinic agonists because neither histamine- nor KCI-induced secretions were affected by natriuretic peptides. In the present study, we report (1) the presence of ANF-R_1C and ANF-R₂ receptor subtypes in bovine chromaffin cells, (2) the inhibition by natriuretic peptides of nicotinic whole-cell currents as well as nicotine-induced catecholamine secretion, (3) the possible mediation of these effects by the ANF-R₂ class of receptors, and (4) the specificity of this inhibition to nicotinic agonists. Because bovine chromaffin cells release ANF, BNP, and CNP together with catecholamines, all three peptides might exert negative feedback regulation of catecholamine secretion in an autocrine manner by interacting with the nondiscriminating ANF-R₂ receptor subtype.     

Rat atrial natriuretic factor suppresses proopiomelanocortin-derived peptides secretion from both anterior and intermediate lobe cells and growth hormone release from anterior lobe cells of rat pituitary in vitro

Synthetic rat atrial natriuretic factor (ANF) was found to attenuate, in a dose-dependent manner, basal and corticotropin-releasing factor-induced secretion of proopiomelanocortin-derived peptides from cultured anterior and intermediate lobe cells of rat pituitary. ANF was also found to suppress basal and growth hormone-releasing factor-stimulated secretion of growth hormone from anterior lobe cells of rat pituitary. These results, together with reports of the existence of ANF-positive neurons in the hypothalamus and ANF-positive fibers in the median eminence, suggest that hypothalamic ANF is probably involved in the regulation of pituitary hormone secretion, especially that of proopiomelanocortin-derived peptides and growth hormone.     

Centrally applied atrial natriuretic factor diminishes bile secretion in the rat.

Little is known about the role of centrally applied peptides in the regulation of bile secretion. We previously reported that the intravenous injection of atrial natriuretic factor (ANF) reduces bile acid dependent flow without affecting portal venous pressure in the rat. In the present work, we studied the effects of centrally applied ANF on bile secretion and the possible pathways involved. Rats were cannulated in the brain lateral ventricle for the administration of 1, 10 and 100 ng/microl ANF. After 1 week, the common bile duct was cannulated and bile samples were collected every 15 min for 60 min after the administration of ANF. The excretion rate of various biliary components was assessed. Bile secretion experiments were also performed after bilateral truncal vagotomy or atropine administration to evaluate the participation of a vagal pathway. In addition, the role of the sympathetic system was addressed by combined administration of propranolol and phentolamine. Centrally applied ANF did not modify blood pressure but diminished bile flow and bile acid output. It also reduced sodium and potassium secretion but did not modify protein or phospholipid excretion. Neither bilateral truncal vagotomy nor atropine administration abolished ANF response. Furthermore, combined administration of adrenergic antagonists did not alter ANF inhibitory effect on bile flow. In conclusion, centrally applied ANF reduced bile acid dependent flow not through a vagal or adrenergic pathway in the rat, suggesting the involvement of a peptidergic pathway.     

Proatrial natriuretic factor is phosphorylated by rat cardiocytes in culture

Proatrial natriuretic factor (proANF) is phosphorylated in primary cultures of neonatal rat cardiocytes. Rittenhouse et al. (Rittenhouse, J., Moberly, L., O'Donnell, M. E., Owen, N. E., and Marcus, F. (1986) J. Biol. Chem. 261, 7607-7610) observed that cyclic AMP-dependent protein kinase phosphorylated synthetic peptides related to atrial natriuretic factor (ANF) and that phosphorylated ANF peptides were more effective in stimulating Na/K/Cl cotransport in smooth muscle cells than nonphosphorylated forms. In our studies, rat cardiocytes in culture were incubated with [32P]orthophosphoric acid, and ANF-related peptides in cell extracts and culture media were isolated using antisera to ANF. Both atrial and ventricular cardiocytes contained and secreted phosphorylated proANF, a 126-amino acid precursor of ANF. Phosphorylated and nonphosphorylated isoforms of proANF were resolved by isoelectric focusing; approximately 35% of the proANF secreted by cardiocytes was phosphorylated. proANF is phosphorylated on a serine residue localized to a 42-amino acid tryptic fragment (proANF residues 26-67). We conclude that proANF is phosphorylated by rat cardiocytes but not within the portion of the molecule destined to become ANF (proANF residues 99-126). Phosphorylation may have a role in the cellular mechanisms of proANF storage and secretion or in the modulation of potential biological activities of the circulating amino-terminal portion of proANF.     

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.     

Basal and stimulated ANF secretion: role of tissue preparation

Our previous results showed that addition of agonists, such as vasopressin and angiotensin, added to incubation medium with freshly excised rat atria caused marked release of atrial natriuretic factor (ANF). This release was in the form of prohormone rather than active peptide. Since others had difficulty reproducing these findings, in the present study we investigated ANF release with and without angiotensin addition in two sets of atrial tissue. In the first, tissue was blotted and carefully cleaned as previously described; in the second, atrial tissue was placed into incubation medium without prior preparation. ANF activity in the medium was measured by radioimmunoassay and receptor assay. Using the immunoassay, basal release of ANF was threefold greater from prepared vs. nonprepared atrial tissue; significant stimulation by angiotensin was seen only in the prepared atria. ANF release measured by radioreceptor assay was 1/5-1/10 of that measured by immunoassay. Taking the difference between the two measurements as an index of prohormone secretion, the results confirm that both basal and stimulated release was primarily in the form of proANF. Scanning electron microscopy revealed that cleaning of the atria had removed the endocardial lining of the tissue. The results thus indicate that an intact endocardium can prevent agonist-induced proANF secretion, suggesting that this tissue may be an important modulator of plasma ANF levels.     

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.     

Protein kinase C and calmodulin kinase are required for endothelin-stimulated atrial natriuretic factor secretion from primary atrial myocytes.

Endothelin (ET), a potent stimulator of atrial natriuretic factor (ANF) secretion in atrial myocyte cultures, has been hypothesized to act via the stimulation of protein kinase C (PKC). This study was carried out in order to determine if ET activates PKC in atrial cultures and whether this activation fully accounts for the effects of ET on ANF secretion. By monitoring the phosphorylation of p80 upon exposure to phorbol ester or ET, it was shown that ET activated PKC in atrial cultures, but to a lesser extent than phorbol ester. In contrast, ET stimulated ANF secretion to a level five times greater than phorbol ester, indicating that PKC activation alone does not fully account for the effects of ET on ANF secretion. Down-regulation of PKC or exposure to the PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) resulted in a 50% decrease in ET-stimulated ANF secretion. Interestingly, increasing calcium influx with BAY K 8644 stimulated ANF secretion but did not effect the phosphorylation of p80, indicating a PKC-independent pathway of ANF secretion. Similarly, a component of ET-stimulated secretion that required calcium influx was independent of PKC activation but was sensitive to the Ca2+/calmodulin kinase (CaMK) inhibitor KN-62. Complete inhibition of ET-mediated ANF secretion was obtained only in the presence of both H7 and KN-62. These results demonstrate that ET activates PKC in atrial myocyte cultures and that the full effects of ET on ANF secretion require both PKC and Ca2+/calmodulin kinase activities.     

Stimulatory effects of atrial natriuretic factor on phosphoinositide hydrolysis in cultured bovine aortic smooth muscle cells

The effects of atrial natriuretic factor (ANF) on phosphoinositide hydrolysis were examined in preparations of cultured bovine aortic smooth muscle cells. In homogenates or particulate fractions from cultured bovine aortic smooth muscle cells, ANF and atriopeptin I increased the formation of inositol phosphates and GTPase activity. The effects on inositol phosphates were markedly enhanced with guanosine 5'[gamma-thio]triphosphate. Both atrial peptides also stimulated the formation of diacylglycerol in intact cultured cells. In these experiments, atriopeptin I was about 10-fold more potent than ANF. These studies indicate that atrial peptides have stimulatory effects on phosphoinositide hydrolysis which are mediated through a guanine nucleotide regulatory protein. The greater potency of atriopeptin I on GTPase activity and the accumulation of inositol phosphates suggests that the nonguanylate cyclase-coupled receptor for ANF (ANF-R2) mediates the stimulatory effects of ANF on phosphoinositide hydrolysis through a guanine nucleotide regulatory protein.     

Regulation of atrial natriuretic factor-(99-126) secretion from neonatal rat primary atrial cultures by activators of protein kinases A and C

Atrial natriuretic factor (ANF) is stored in atrial myocytes as a prohormone (ANF-(1-126] and is cosecretionally processed to the circulating ANF-related peptides, ANF-(1-98) and ANF-(99-126). Recently, we have shown that the cosecretional processing of ANF can be replicated in primary cultures of neonatal rat atrial myocytes maintained under serum-free conditions and that glucocorticoids are responsible for supporting this processing activity. Activators of protein kinase C (phorbol esters and alpha-adrenergic agonists) and of protein kinase A (cAMP analogs, forskolin, and beta-adrenergic agonists) were tested for their abilities to alter the rate of ANF secretion from the primary cultures. ANF secretion was stimulated approximately 4-fold after a 1-h incubation of the cultures with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA); maximal release occurred at about 100 nM TPA. Reversed-phase high performance liquid chromatography analysis of secreted material indicated that the cells efficiently cosecretionally processed ANF under both basal and TPA-stimulated conditions. However, incubating the cultures for more than 1 h with TPA resulted in a blunted secretory response to further TPA challenge and a 40-50% decrease in the quantity of ANF in the cells. The alpha-adrenergic receptor agonist phenylephrine was also capable of stimulating ANF secretion by about 4-fold at a half-maximal dose of about 1 microM. Phenylephrine-stimulated ANF secretion was inhibited by the alpha 1-adrenergic antagonist prazosin with half-maximal inhibition occurring at approximately 1 nM. Forskolin, 8-bromoadenosine 3':5'-cyclic monophosphate, and N6-2(1)-O-dibutyryladenosine 3':5'-cyclic monophosphate inhibited basal, TPA- and phenylephrine-stimulated ANF secretion. The beta-adrenergic agonist isoproterenol partially inhibited phenylephrine-stimulated ANF secretion with the maximal effect occurring at 1 nM. These results indicate that ANF secretion from the neonatal rat atrial cultures is enhanced by activators of protein kinase C, and decreased by activators of protein kinase A, and that these secretory effects may be mediated through the actions of alpha- and beta-adrenergic receptors, respectively.     

Distribution and regulation of natriuretic factor-R1C receptor subtypes in mammalian cell lines

The differential distribution of natriuretic peptide receptor subtypes and their distinct properties were assessed in mammalian cellular models which were screened for their ability to produce cGMP upon stimulation by different natriuretic peptides. The ANF-R_1A receptor subtype was distinguished by its selective activation by atrial natriuretic factor (ANF) while the ANF-R_1C was characterized by preferential stimulation by C-type natriuretic peptide (CNP). AT-t20 pituitary cells, bovine adrenal chromaffin cells, and NIH-3T3 fibroblasts mainly express the ANF-R_1C receptor subtype. Other cell lines such as PC12, RASM and GH3 express significant but varying amounts of both ANF-R_1A and ANF-R_1C subtypes. A10 and NIH cells which express high density of ANF-R₂ receptor subtype, also demonstrate a higher sensitivity to CNP over ANF suggesting that they express significant amounts of ANF-R_1C. Studies of the regulation by ATP of guanylyl cyclase activity indicate that both ANF-R_1A and ANF-R_1C subtypes are modulated in the same manner. In the presence of Mn²⁺, ATP inhibits the CNP-stimulated guanylyl cyclase activity while in the presence of Mg²⁺ adenine nucleotides potentiate the stimulation by CNP. In addition, we show that like the ANF-R_1A, the ANF-R_1C guanylyl cyclase activity can be regulated by phosphorylation since preincubation with TPA or FKL attenuates the subsequent stimulation by CNP in cultured cells. The results presented demonstrate that specific cell types express distinct natriuretic peptide receptor subtypes and also that the newly characterized ANF-R_1C subtype is regulated by ATP and serine/threonine kinases in the same way as the ANF-R_1A subtype.Abbreviation ANF atrial natriuretic factor - BNP brain natriuretic peptide - CNP C-type natriuretic peptide - ATP adenosine-5-triphosphate - IBMX 3-isobutyl-1-methylxanthine - TPA 12-O-tetradecanoyl-phorbol-13-acetate - FKL forskolin - PKC calcium-phospholipid-dependent protein kinase - PKA cAMP-dependent protein kinase - PKG cGMP-dependent protein kinase - C-ANF [Cys¹¹⁶]-ANF-(102-116)-NH₂ - CC chromaffin cells     

Atrial peptides induce mast cell histamine release.

Human atrial natriuretic peptide [ANF(1-28)] contains five arginine residues and carries an overall positive change of four. It was hypothesized that atrial peptides may induce mast cell histamine release. In vitro, three atrial peptides [ANF(1-28), (3-28) and (5-28)] were demonstrated to induce dose-dependent histamine release from isolated rat peritoneal mast cells. In vivo, ANF(3-28) produced a dose-dependent increase in rat skin permeability which was blocked by antagonists of histamine and serotonin. The results indicate atrial peptides are capable of inducing mast cell degranulation in a manner similar to that described for other positively charged peptides.