Atrial Natriuretic Factor Processing Research Articles

Altered regulation of natriuretic peptides in the rat heart by prenatal exposure to morphine.

1. Both endogenous and exogenous opioids modulate blood pressure and cardiac function by stimulating cardiac synthesis of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP). Since morphine crosses the placental barrier, it could alter the ANF-BNP system in the fetal heart. The aim of this study was to characterize cardiac natriuretic peptides in normal rat development and in rats prenatally exposed to morphine. 2. Female rats received either saline or morphine (10 or 20 mg kg-1 day-1) via osmotic minipumps during gestation. The effects of this treatment were investigated in offspring at 1, 4 and 22 days of age. 3. During maturation, atrial ANF and ANF mRNA increased by 3-fold from birth to 3 weeks of age, but BNP and BNP mRNA tended to decrease. In the ventricles, both ANF and BNP content decreased at 3 weeks after birth, from 25.11 +/- 3.6 to 0.81 +/- 0.1 ng (mg protein)-1 (P < 0.001), and from 3.36 +/- 0.33 to 0.19 +/- 0.01 ng (mg protein)-1 (P < 0.001), respectively. However, whereas ventricular ANF mRNA decreased, BNP mRNA levels did not change during maturation. Prenatal exposure to morphine significantly increased ANF content in the left atria of 22-day-old rats, and in the right atria of 1-, 4- and 22-day-old rats compared with age-matched saline controls. In contrast, prenatal exposure to 20 mg kg-1 day-1 morphine significantly inhibited BNP and BNP mRNA in the ventricles at all ages studied. 4. These observations suggest that alterations in mRNA synthesis or stability and/or post-translational processing of ANF and BNP occur in the heart during maturation, and that prenatal exposure to morphine alters cardiac production, and possibly release, of both peptides.

Mechanical and neuroendocrine regulation of the endocrine heart

The cardiac natriuretic peptides (NP) -- atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) -- are polypeptide hormones produced by cardiocytes in the atria of mammals. ANF and BNP are continuously released from the heart, but appropriate mechanical or neuroendocrine stimuli increase their rate of release with or without a concomitant increase in synthesis. The results of our investigations lead us to propose that the endocrine response of the heart to pressure or volume load varies in relation to whether the challenge is acute, subacute or chronic. The acute response to stretch is based on a phenomenon referred to as "stretch-secretion coupling" which results in enhanced secretion of NP stored in the atria. NP release following stretch is made at the expense of a depletable NP pool with no apparent effect on synthesis. The stimulation of NP production that is seen during mineralcorticoid escape is referred to as "subacute" and is characterized by stimulation of atrial ANF and BNP gene transcription secondary to volume overload in which plasma ANF, but not plasma BNP, is significantly elevated. With chronic stimulation, as seen in DOCA-salt treatment at the hypertensive stage, activation of the cardiac fetal program in ventricle is seen together with a stimulation of ANF and BNP production in both atria and ventricles. However, the activation of NP gene expression in the atria is not necessarily associated with fetal isogene expression even though the ventricular hypertrophic process is characterized by the expression of fetal isogenes, including ANF and BNP, that are normally expressed in the fetal ventricle. It seems likely that the acute stimulation of NP release is based on an electromechanical coupling. However, protracted stimulation of release is seen in situations in which profound neuroendocrine changes have taken place, thus suggesting that the primary stimulus for chronically enhanced NP gene expression and NP release is based on changes in the hormonal environment of the atrial cardiocyte. It is concluded that the endocrine heart responds to changes in hemodynamic load with specific changes in translational, post-translational and storage processes for ANF and BNP following acute or chronic stimulation. As a result, plasma levels of ANF and BNP may be used as indicators of the degree of atrial hemodynamic overload and ventricular hypertrophy, respectively. It may be advanced that the endocrine heart differentiates and responds to different hemodynamic challenges in either acute or chronic conditions with specific changes in transcription, translation, post-translational processing, storage, and release of ANF and BNP. We propose that this differentiation is part of the reason for the heart to produce two hormones with similar spectra of activity. This paradigm warrants further investigation.

Brefeldin A defines distinct pathways for atrial natriuretic factor secretion in neonatal rat atrial and ventricular myocytes.

The intracellular pathways for basal atrial natriuretic factor (ANF) secretion from the heart and their correlation with ANF processing to the active form were characterized in cultured neonatal rat atrial and ventricular myocytes. Brefeldin A, a fungal antimetabolite that blocks transport of newly synthesized proteins from the endoplasmic reticulum, was used to inhibit nascent protein trafficking. Thus, release of newly synthesized hormone was blocked, but release of stored hormone was unaffected. Whereas brefeldin A inhibited basal ventricular ANF release to 10% of the control value, basal ANF release from atrial cells was enhanced. Furthermore, basal atrial ANF secretion was inhibited by agents preventing myocyte depolarization, Ca2+ influx, release of Ca2+ from intracellular stores, or activation of protein kinase C, whereas ventricular ANF secretion was unaffected by these agents. Brefeldin A did not alter maturational processing of pro-ANF to ANF-(99-126) in either atrial or ventricular cultures. These findings indicate that (1) basal secretion of ANF from ventricular cells relies largely on newly synthesized hormone and is probably constitutive, (2) basal secretion of ANF from atrial cells is independent of transport of newly synthesized protein and occurs via a regulated pathway controlled at least in part by signaling changes associated with myocyte beating, and (3) processing of pro-ANF occurs either with constitutive or regulated secretion of hormone, which may indicate multiple cellular locations for the processing enzyme.

Post-translational processing of atrial natriuretic factor by adult rat atrial cardiocytes in culture

Post-translational processing of the cardiac polypeptide hormone atrial natriuretic factor (ANF) was studied using primary cultures of cardiocytes derived from adult rat atria. Atrial cardiocytes attached to microcarrier beads were maintained for up to 15 days under continuous superfusion in minichromatographic columns. The cultures were characterized for their ability to store, process, and release ANF and by immunofluorescence microscopy for ANF, desmin, and myosin. Nuclear staining using the fluorescent DNA stain Hoechst 33258 was carried out to determine the total number of cells in culture. Column eluates were assayed for ANF by radioimmunoassay and analyzed by reverse phase high-performance liquid chromatography. For comparison purposes, superfusion experiments using freshly isolated cardiocytes supported in Bio-Gel P2 were carried out. Freshly isolated atrial cardiocytes stored high molecular weight ANF (5.2 +/- 1.9 pmol/micrograms DNA) and released mostly (83.3 +/- 6.7%) low molecular weight ANF, at an average rate of 97 +/- 18 fmol.min-1 x micrograms-1 DNA. The cell content and the rate of release of ANF after 15 days in culture were 1.3 +/- 0.4 pmol/micrograms DNA and 1.7 +/- 0.4 fmol.min-1 x micrograms-1 DNA, respectively, and 62.7 +/- 6.3% of the released peptide was of a low molecular weight. There was no correlation between changes in cell population and the extent of processing. Cultures of noncardiocytes, superfused with exogenous proANF, did not significantly process proANF to a lower molecular weight peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene expression and atrial natriuretic factor processing and secretion in cultured AT-1 cardiac myocytes.

Studies were carried out to characterize several biochemical features of cultured AT-1 cells. These cells were obtained from a transplantable atrial cardiomyocyte tumor lineage. Reverse transcriptase-polymerase chain reaction-based analyses demonstrated that the pattern of gene expression of cultured AT-1 cells was similar to that of adult atrial myocytes. AT-1 cells expressed atrial natriuretic factor (ANF), alpha-cardiac myosin heavy chain, alpha-cardiac actin, and connexin43. Radioimmunoassays verified that the cells synthesized, stored, and secreted ANF. Through size-exclusion, reversed-phase, and carboxymethyl-ion-exchange high-performance liquid chromatography, it was shown that cultured AT-1 cells stored ANF as pro-ANF (ANF-[1-126]), which was cosecretionally processed quantitatively to ANF-(1-98) and the bioactive 28-amino-acid ANF-(99-126). In addition, cultured AT-1 cells secreted ANF at almost a sixfold greater rate in response to endothelin-1, a potent secretagogue of ANF. KCl, metenkephalinamide, isoproterenol, phenylephrine, and 12-O-tetradecanoyl-phorbol-13-acetate also stimulated ANF release. These studies, in combination with previous findings, demonstrated that cultured AT-1 cells, while maintaining the ability to proliferate, have retained functional, biochemical, and ultrastructural features that are characteristic of adult atrial myocytes.

Changes in atrial natriuretic factor processing and secretion with age.

The effect of age on atrial natriuretic factor (ANF) metabolism in the rat was studied. Plasma ANF levels rose with age. A positive correlation was found between age and plasma ANF levels. Molecular forms of ANF were also examined. alpha-ANF and gamma-ANF were present in the circulation. The ratio of gamma-ANF/alpha-ANF increased with age. The atria were isolated and perfused to determine the ANF secretion rate. The spontaneous release of ANF decreased with age. The released ANF mainly consisted of alpha-ANF with a small amount of gamma-ANF. The ratio of gamma-ANF/alpha-ANF in the effluent increased with age. ANF concentrations in the atria varied with age, but the molecular forms did not. gamma-ANF was the main form found in the atria. These results suggest that ANF secretion as well as post-transcriptional processing is altered with age.

Proteinase activities in bovine atrium and the possible role of mast cell tryptase in the processing of Atrial Natriuretic Factor (ANF)

1. 1. Using low salt, Triton X-100 and high salt extracts of bovine atria, two main proteinases were identified by means of fluorogenic oligopeptide substrates. 2. 2. An acidic proteinase, extracted in low salt and Triton X-100 was identified as cathepsin B, but it caused little hydrolysis of the Z-Gly-Pro-Arg-containing substrate that resembles the cleavage site for activation of pro-ANF. 3. 3. An alkaline proteinase was extracted only with high salt and had characteristics of the serine proteinase tryptase. It cleaved Z-Gly-Pro-Arg-containing substrates more efficiently than others tested and was localized in and around mast cells histochemically. Previously, Imada et al., 1988 ( J. biol. Chem. 263, 9515–9519) found an identical enzyme would cleave ANF from pro-ANF. 4. 4. These results suggest therefore that mast cell typtase may be involved in the activation of ANF from pro-ANF.

Effect of manipulations of Ca2+ environment on atrial natriuretic factor release

The effects of Ca2+ on the kinetics of atrial natriuretic factor (ANF) release [measured as immunoreactive cardionatrin (IRC)] were studied on an in vitro, spontaneously beating rat atrial preparation. It was found that ethylene glycolbis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and Ca2+-free media induced a significant increase in the rate of basal IRC release. Reintroduction of Ca2+ reversed the augmented basal IRC release induced by EGTA and restored mechanical activity. It was also found that the stretch-induced IRC release was independent of extracellular Ca2+ and took place even in the presence of EGTA. The presence of absence of Ca2+ had no apparent effect on ANF processing. In all instances, cardionatrin I (ANF 99-126) was found to be the most abundant peptide released. Morphologically, no obvious differences were observed during either basal or stretch-induced IRC release. These results suggest that, unlike most other endocrine secretory systems, a reduction of cytosolic Ca2+ stimulates basal IRC release. These findings suggest an adaptation of atrial cardiocytes to accomplish their dual role as secretory and contractile cells.

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.

Ultrastructural aspects of atrium development: demonstration of endocardial discontinuities and immunolabeling of atrial natriuretic factor in the Syrian hamster

The endocardium ultrastructure of 13 embryonic day old hamsters was examined, especially in relationship with the atrial myocytes. The endothelial morphology was described, including the junctional attachments and their relationships with subjacent atrial myocytes. Characteristic atrial myocytes organelles were identified: myofibrils, atrial granules, lipidic inclusions, and polysomes. Immunogold labeling demonstrated that atrial natriuretic factor (ANF)-containing granules were already present in the differentiating cardiomyocytes, even before the myofibrils were completely organized. At this stage of development, while the endothelium was a narrow barrier between the blood and the cardiomyocytes, it displayed fenestrations, but also epithelial discontinuities. In addition it also contains immunoreactive-ANF products. In light of the current knowledge about ANF processing it was proposed that the endocardium lining could be an obligated passageway for transport or activating proANF into ANF before its release into the blood stream. In addition the endocardial gaps could suggest that, until about 13 to 14 days of fetal development, heart atrial tissue could be more susceptible to the effects of pathogenetic compounds than in a later state of development.

The secretion of atrial natriuretic factor-(99-126) by cultured cardiac myocytes is regulated by glucocorticoids.

Atrial natriuretic factor (ANF) is stored in mammalian atria primarily as ANF-(1-126), the precursor to the known circulating form of the hormone ANF-(99-126). When primary cultures of atrial myocytes were maintained in a complete serum-free medium, they contained and secreted an ANF-(1-126)-like peptide. The addition of dexamethasone to the culture medium, however, resulted in the secretion of a molecule with chromatographic characteristics identical to ANF-(99-126), although the intracellular storage form of ANF was unchanged. Radiosequencing and amino acid analysis confirmed that the cultures maintained in dexamethasone secreted authentic ANF-(99-126). Chronic exposure of the cells to dexamethasone also resulted in a significant increase in the quantity of immunoreactive ANF both contained and secreted by the cultures. Dexamethasone stimulated ANF processing and secretion by atrial cultures in a dose-dependent manner, with an approximate EC50 of 10 nM. This stimulation could be reversed by removing the glucocorticoid from the culture medium. ANF processing was also stimulated by the specific glucocorticoid receptor agonist RU 28362, and both DEX- and RU 28362-stimulated ANF processing was inhibited by the specific glucocorticoid receptor antagonist RU 38486. Ventricular cells, which possess few granules and release ANF in a constitutive fashion, were also capable of processing ANF in a glucocorticoid-dependent fashion. Medium freshly removed from atrial cultures did not convert ANF-(1-126) to ANF-(99-126) nor was exogenous ANF-(1-126) efficiently processed when added to the medium of actively processing cultures. These results indicate that the post-translational processing of ANF-(1-126) to ANF-(99-126) likely occurs within or in close association with the cardiac myocytes and is not dependent on the presence of large quantities of secretory granules. Furthermore, it is apparent that both the expression and the post-translational processing of ANF by cultured cardiac myocytes is specifically regulated by glucocorticoids.

The post-translational processing of rat pro-atrial natriuretic factor by primary atrial myocyte cultures.

Primary cultures of neonatal rat atrial myocytes were maintained in two different serum-free media for up to 25 days. Reversed-phase high performance liquid chromatography coupled with atrial natriuretic factor (ANF)-specific radioimmunoassay demonstrated that the cultures maintained in our previously described serum-free medium (Glembotski, C.C., and Gibson, T. R. (1985) Biochem. Biophys. Res. Commun. 132, 1008-1017) secreted primarily ANF-(1-126)-like material, whereas those cultures maintained in a different formulation of medium secreted mostly ANF-(99-126)-like material. Cultures that secreted ANF(99-126)-like material were biosynthetically labeled with [35S]cysteine followed by immunoprecipitation of secreted ANF and analysis by reversed-phase, size exclusion, and ion-exchange high performance liquid chromatography. The labeled ANF-(99-126)-like peptide was shown to be chromatographically indistinguishable from other synthetic peptides related to ANF-(99-126). Labeled ANF purified from extracts of the cultured cells was chromatographically indistinguishable from authentic ANF-(1-126), and could be cleaved specifically by thrombin into labeled ANF-(99-126)-like material. These results indicate that primary atrial myocytes maintained under certain serum-free conditions are capable of secreting ANF-related material that is chromatographically indistinguishable from ANF-(99-126), the known circulating form of the hormone. Additional preliminary studies suggest that the presence of glucocorticoids in the culture medium may confer ANF processing ability on cultured myocytes.

Analysis of atrial natriuretic factor biosynthesis and secretion in adult and neonatal rat atrial cardiocytes

Atrial natriuretic factor (ANF) is stored in atrial cardiocytes as the 126 amino acid polypeptide, proANF, which is later cleaved to the 24–28 amino acid carboxyterminal peptides, the major circulating forms. Earlier studies have demonstrated that isolated, cultured neonatal rat cardiocytes both store and secrete proANF, which can be cleaved to the smaller circulating form(s) by a serum protease. Since differences may exist between neonatal and adult cardiocytes with respect to ANF synthesis and processing, we compared the forms of ANF stored and secreted by neonatal rat cardiocytes with those of adult cells. Using four to five day cultures of isolated atrial cardiocytes prepared from the hearts of neonatal and adult rats, pulse-chase studies were performed with 35S-cysteine and 35S-methionine. Analysis of ANF stored and secreted by these cells was performed by immunoprecipitation of cell extracts and culture media using antibodies directed to either the carboxyterminus or aminoterminus of proANF followed by SDS-PAGE and autoradiogarphy. Cell extracts from both adult and neonatal cultures were found to contain only a 17-kDa polypeptide, previously identified as proANF. The predominant form found in the culture media was also the 17-kDa peptide, with smaller quantities of its 3-kDa carboxyterminal and 14-kDa aminoterminal cleavage products. We conclude from these studies that proANF is the major form stored and secreted by both adult and neonatal cardiocytes in culture; the activity of the protease that cleaves proANF to the smaller forms found in the circulation is either attenuated or is overwhelmed by high ANF-secretory rates in these cultures. Alternatively, the ANF processing and secretory pathways may be somehow altered in culture such that proANF escapes protease cleavage. Further studies will elucidate the nature and location of this protease.

Processing of Atrial Natriuretic Factor by Atrial Thiol Dependent Protease

Processing of Atrial Natriuretic Factor by Atrial Thiol Dependent Protease