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.     

Atrial natriuretic factor in the vena cava and sinus node

We investigated the localization of atrial natriuretic factor (ANF) mRNA and of immunoreactive ANF in the vena cava and sinus node of rat and, for comparative purposes, in atria and ventricles. In situ hybridization with an ANF cRNA probe revealed that the supradiaphragmatic portion of the inferior vena cava contains almost as much mRNA as the atria, whereas the levels were less in the superior vena cava and higher than in ventricles in the sinus node. Immunoreactive ANF (high Mr form) was found to be 22 times less abundant in the supradiaphragmatic vena cava and 148 times less abundant in the superior vena cava than in atrial cardiocytes. The wall of the supradiaphragmatic portion of the vena cava and the valve (eustachian valve) that separates the atrial cavity from that of the vein are made up of atrial-like cardiocytes containing secretory granules. The subendothelial area of the superior vena cava also contains atrial-like cardiocytes with secretory granules, whereas the outer portion of the vein is made up of "transitional cells" without or with only a few secretory granules. Secretory granules in the vena cava and nodal cells, as well as transitional cells, contain immunoreactive ANF. With immunocryoultramicrotomy, virtually all cells, whether atrial-like, transitional, or nodal, and even those without secretory granules, were found to contain immunoreactive ANF in their Golgi complex and in secretory vesicles in the vena cava and in the sinus node.     

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 autoradiography. 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.     

Effects of rat atrial extract on sodium transport and blood pressure in the rat

Atrial cardiocytes contain specific atrial granules ( SAGs ) which are the storage site of atrial natriuretic factor (ANF). The purpose of the present study was to determine whether ANF produces natriuresis by inhibiting Na+-K+ pump activity and whether this factor is similar to the humoral sodium transport inhibiting factor ( HSTIF ) previously demonstrated in acutely volume expanded animals and humans as well as in experimental and human essential hypertension. Our results indicate that, in contrast to the HSTIF , ANF does not inhibit membrane Na+,K+-ATPase, vascular smooth muscle cell Na+-K+ pump activity, or sodium transport in the toad bladder. Intravenous infusion of ANF in the bilaterally nephrectomized, hexamethonium-treated rat produces only a small transient pressor response, probably due to potentiation of endogenous norepinephrine. These findings strongly suggest that the ANF is not the same as the HSTIF detected on acute volume expansion and in some forms of hypertension. They also suggest that the diuretic and natriuretic effects of ANF are due to mechanism(s) other than blood pressure elevation and inhibition of Na+-K+ pump activity.     

Localization and characterization of atrial natriuretic factor (ANF)-like peptide in the frog atrium

The distribution of ANF was studied in the heart of the frog (Rana ridibunda) using indirect immunofluorescence. ANF-like immunoreactivity was localized mainly in the right and left atrium, most of cardiocytes being intensively labelled. At the electron microscopic level, all secretory granules present in atrial cardiocytes contained ANF immunoreactive material. Using a specific radioimmunoassay, we found higher concentrations of ANF in the left atrium (208 +/- 25 ng/mg protein) than in the right atrium (120 +/- 16 ng/mg protein) whilst in the rat, the right atrium contains the highest ANF concentration. The concentration of ANF in the ventricle was 10 times lower than in the whole atrium (32 +/- 4 ng/mg protein). Sephadex G-50 gel filtration of atrial extracts showed that ANF-like immunoreactivity eluted in three peaks. Most of the immunoreactivity corresponded to high molecular weight material eluting at the void volume while 20% of the material co-eluted with synthetic (Arg 101-Tyr 126) ANF. These results indicate that frog cardiocytes synthetize a peptide which is immunologically and biochemically related to mammalian ANF.     

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

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

Immunohistochemical identification of Purkinje fibers and transitional cells in a terminal portion of the impulse-conducting system of porcine heart

Summary The ultrastructure of porcine ventricular tissue was studied by electron microscopy and immunocytochemical techniques. Electron-dense specific granules were found in both Purkinje fibers and transitional cells in the ventricular walls, and were positively stained by the immunogold staining method using an antiserum against atrial natriuretic polypeptide (ANP). This suggests that both the Purkinje fibers and transitional cells display the same specific granules as atrial cardiocytes containing ANP. These results demonstrate that Purkinje fibers and two types of transitional cells, in addition to the ordinary ventricular cardiocytes, can be identified in porcine ventricular wall tissue.     

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.     

Neonatal atria and ventricles secrete atrial natriuretic factor via tissue-specific secretory pathways

The cellular mechanisms regulating secretion of the peptide hormone atrial natriuretic factor (ANF) differ in neonatal atrial and ventricular cardiocytes. We demonstrate that although both cell types synthesize and secrete ANF, only atrial cells store peptide in abundant secretory granules. Neonatal ventricular cells secrete ANF rapidly after synthesis and lack secretory granules. We propose that ventricular ANF is released by a constitutive secretory pathway whereas atrial ANF is stored and released by a regulated pathway. Furthermore, ventricular ANF mRNA and hormone concentrations decrease during the first week of life. Developmental variation in the use of ANF secretory pathways may reflect changing requirements for maintenance of intravascular volume and pressure. Tissue-specific modulation of hormone secretory pathways appears to be a novel response to developmentally induced changes in the requirements for a peptide hormone.     

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

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

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.     

Immunohistochemistry and immunocytochemistry of atrial natriuretic polypeptide in porcine heart

Specific granules in porcine hearts were observed in atrial cardiocytes, Purkinje fibers, and transitional cells of the ventricle. These granule-containing cells were immunohistochemically stained by applying the avidin-biotin-peroxidase complex method using an antiserum against alpha-human atrial natriuretic polypeptide (ANP). Immunoelectron microscopy of sections stained using the immunogold method indicated that these specific granules are storage sites of ANP. Furthermore, an impulse-conducting system consisting of immunoreactive cells was clearly distinguishable from nonimmunoreactive ventricular cardiocytes. We conclude that specific-granule-containing cells, i.e., ANP-producing cells, are located in both the atrial walls and the ventricular impulse-conducting system. The presence of ANP may be correlated with impulse conduction.     

Atrial-specific granules in the hearts of normal and water-deprived rats

Summary The morphology of atrial-specific granules, which contain atrial natriuretic polypeptide (ANP), was studied in the cardiac tissue of untreated controls and water-deprived rats by means of conventional and immunoelectron microscopy. Immature secretory vesicles or granules appeared to become buded off from the Golgi cisternae and then fused to form specific A-granules. An electron-dense plate with a fuzzy coat was frequently found on the limiting membrane at the end of such fusion. Pale specific B-granules, which were less electron-dense, larger, and more granular than A-granules, were found in small numbers in the left atrial cardiocytes, but rarely in the right ones. Very pale granules with a less granular matrix, considered to be B-type granules which had lost their electron-density, and which had less immunoreactivity for ANP, were numerous in the cardiac tissue after water deprivation. This morphological change, which is interpreted as an indication of granule degradation, was in agreement with the noted increase of natriuretic activity in the atrial tissue of water-deprived specimens.     

Thirty years of research on atrial natriuretic factor: historical background and emerging concepts

The discovery of the natriuretic properties of atrial muscle extracts pointed to the existence of an endocrine function of the heart that is now known to be mediated by the polypeptide hormones atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP). On the basis of such a finding, approximately 27 000 publications to date have described a wide variety of biological properties of the heart hormones as well as their application as therapeutic agents and biomarkers of cardiac disease. Stimulation of secretion of ANF and BNP from the atria is mediated through mechanisms involving G proteins of the G(q) or G(o) types. We showed that the latter type underlies the transduction of muscle stretch into stimulated secretion and that it is more highly abundant in atria than in ventricles. The Gα(o)()-1 subunit appears to play a key role in the biogenesis of atrial granules and in the intracellular targeting of their contents. Protein interaction studies using a yeast two-hybrid approach showed interactions between Gα(o)()-1, proANF, and the intermediate conductance, calcium-activated K(+) channel SK4. Pharmacological inhibition of this channel decreases ANF secretion. Unpublished studies using in vitro knockdowns suggest interdependency in granule protein expression levels. These studies suggest previously unknown mechanisms of intracellular targeting and secretion control of the heart hormones that may find an application in the therapeutic manipulation of circulating ANF and BNP.     

Effect of native and synthetic atrial natriuretic factor on cyclic GMP

Mammalian atrial cardiocyte granules contain a potent natriuretic and diuretic peptide. Since cGMP appears to be involved in the modulation of cholinergic and toxin-induced sodium transport, we examined the effect of atrial natriuretic factor (ANF) on this nucleotide. Atrial but not ventricular extracts elicited approximately a 28-fold increase of urinary cGMP excretion parallel to the natriuresis and diuresis. The atrial extracts also elevated cGMP levels in kidney slices and primary cultures of renal tubular cells. The effect of ANF on cGMP appeared to be specific since antibodies which were capable of inhibiting the ANF-induced diuresis also suppressed cGMP excretion. Furthermore, during the course of ANF purification, the ANF-induced increase of cGMP production by kidney cells paralleled the heightened specific natriuretic activity of the atrial factor. A synthetic peptide (8-33)-ANF similarly increased urinary plasma and kidney tubular cGMP levels. The exact mechanism of action of ANF on cGMP remains to be elucidated, but indirect inhibition of cGMP phosphodiesterase appears to participate in its effect.     

Atrial natriuretic factor in the impulse-conduction system of rat cardiac ventricles

Summary A complex network of atrial natriuretic factor-producing cells has been delineated by biochemical and morphological techniques in the rat ventricular myocardium. The chordae tendineae spuriae (CTS; false tendons) contain ANF mRNA and the ANF propeptide (Asn 1-Tyr 126) as assessed by Northern blot analysis, high-pressure liquid chromatography and immunohisto- and -cytochemistry, using three different affinity-purified antibodies: monoclonal and polyclonal antibodies against C-terminal ANF (Arg 101-Tyr 126) and polyclonal antibodies against N-terminal ANF (Asp 11-Ala 37). Two types of cells harboring ANF-containing secretory granules constitute the CTS: the majority (Purkinje type I) have ultrastructural similarities with both atrial and classical Purkinje fibers. Purkinje type-II fibers resemble working ventricular cardiocytes. Both cell types harbor a large paranuclear Golgi complex. The subendocardial Purkinje network is also made up of these two cell types. In this location, Purkinje type-I fibers form cable-like structures while Purkinje type-II fibers are either located beneath the former or abut directly on the endocardium. The latter are not separated from adjacent working ventricular cardiocytes by connective tissue septa. Coronary arteries and arterioles, as in birds, are surrounded by a cushion of Purkinje type-II fibers which blend with the surrounding myocardium. These results indicate that, in the rat, the entire intraventricular conduction system is constituted of endocrine cells producing ANF.Supported by a Medical Research Council of Canada Group Grant to the Multidisciplinary Research Group on Hypertension, by the National Research Council of Canada, the Pfizer Company (England), Bio-Méga Inc. and the Canadian Heart Foundation     

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.     

Immunocytochemical localization of atrial natriuretic factor in the heart and salivary glands

Antibodies produced in the mouse by repeated intraperitoneal injections of partly purified atrial natriuretic factor (low molecular weight peptide (LMWP) and high molecular weight peptide (HMWP)) have been used to localize these factors by immunohistochemistry (immunofluorescence and immunoperoxidase method) and by immunocytochemistry (protein A-gold technique) in the heart of rats and of a variety of animal species including man and in the rat salivary glands. Immunofluorescence and the immunoperoxidase method gave identical results; in the rat, atrial cardiocytes gave a positive reaction at both nuclear poles while ventricular cardiocytes were consistently negative. The cardiocytes of the right atrial appendage were more intensely reactive than those localized in the left appendage. A decreasing gradient of intensity was observed from the subpericardial to the subendocardial cardiocytes. The cardiocytes of the interatrial septum were only lightly granulated. Sodium deficiency and thirst (deprivation of drinking water for 5 days) produced, as already shown at the ultrastructural level, a marked increase in the reactivity of all cardiocytes from both atria with the same gradient of intensity as in control animals. Cross-reactivity of intragranular peptides with the rat antibodies allowed visualization of specific granules in a variety of animal species (mouse, guinea pig, rabbit, rat, dog) and in human atrial appendages. No reaction could be elicited in the frog atrium and ventricle although, in this species, specific granules have been shown to be present by electron microscopy in all cardiac chambers. With the protein A-gold technique, at the ultrastructural level, single labeling (use of one antibody on one face of a fine section) or double labeling (use of two antibodies on the two faces of a fine section) showed that the two peptides are localized simultaneously in all three types (A, B and D) of specific granules. In the rat salivary glands, immunofluorescence and the immunoperoxidase method showed reactivity exclusively in the acinar cells. The reaction was most intense in the acinar cells of the parotid gland. In the sublingual gland, only the serous cells, sometimes forming abortive "demi-lunes", were reactive. In the submaxillary gland, the reaction was weaker and distributed seemingly haphazardly in the gland. The most constantly reactive cells were localized near the capsule while many cells did not contain visible reaction product.     

Immunohistochemistry and immunocytochemistry of atrial natriuretic polypeptide in porcine heart

Summary Specific granules in porcine hearts were observed in atrial cardiocytes, Purkinje fibers, and transitional cells of the ventricle. These granule-containing cells were immunohistochemically stained by applying the avidin-biotin-peroxidase complex method using an antiserum against -human atrial natriuretic polypeptide (ANP). Immunoelectron microscopy of sections stained using the immunogold method indicated that these specific granules are storage sites of ANP. Furthermore, an impulse-conducting system consisting of immunoreactive cells was clearly distinguishable from nonimmunoreactive ventricular cardiocytes. We conclude that specific-granule-containing cells, i.e., ANP-producing cells, are located in both the atrial walls and the ventricular impulse-conducting system. The presence of ANP may be correlated with impulse conduction.     

Characterization and localization of atriopeptin in rat atrium

An antiserum specific for atriopeptin was used to characterize and localize atriopeptin-like immunoreactive material in rat atrium by radioimmunoassay and immunohistochemical techniques. The antiserum recognizes atriopeptin I, atriopeptin III, and -human atrial natriuretic polypeptide, but does not recognize met-enkephalin, cholecystokinin, dynorphin A (1–13), bradykinin, substance P, or β-endorphin. A high content of atriopeptin was found in crude extracts of rat atria, as compared to ventricles, and the atriopeptin-like immunoreactive material was found to be located exclusively in granules within atrial cardiocytes. Fractionation of the immunoreactive material by gel filtration and reverse-phase HPLC revealed the presence of multiple atriopeptins.