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.     

Molecular forms of immunoactive atrial natriuretic peptide released from cultured rat atrial myocytes

Primary cultures of atrial myocytes were prepared from newborn rats and maintained for 8 days in complete serum-free medium. The culture content of immunoactive atrial natriuretic peptide (ANP) increased from 10 to 25 ng/culture during this time. The cells released immunoactive ANP at a rate of 2 to 3% of culture content per hour in a linear fashion for at least 6 hours. When analyzed by gel filtration the major immunoactive material released by and contained within the cells displayed a molecular weight of approximately 15,000 daltons. The medium and cellular ANP-related peptides were further shown to be indistinguishable by reversed-phase HPLC. When the 15,000 dalton material was incubated with rat serum it was converted to ANP-related material possessing a molecular weight of approximately 3,000 daltons. These results suggest that under basal conditions, atrial myocytes release a large molecular weight form of ANP that is converted in the circulation to a low molecular weight form of ANP, which has been previously identified in plasma.     

Ventricular and atrial myocytes of newborn rats synthesise and secrete atrial natriuretic peptide in culture: Light-and electron-microscopical localisation and chromatographic examination of stored and secreted molecular forms

Summary We have demonstrated that atrial natriuretic peptide-like immunoreactivity is stored and secreted by ventricular and atrial myocytes in dissociated cell culture preparations from the heart of newborn rat. Culture preparations were maintained in either foetal calf serum-supplemented medium 199 or in hormone-supplemented, serum-free medium 199. The presence of atrial natriuretic peptidelike immunoreactivity in the cultured myocytes was demonstrated at both light-and electron-microscopical levels. Release of atrial natriuretic peptide-like immunoreactivity into the culture medium was measured by radioimmunoassay; molecular forms of the stored and secreted peptide were determined by gel column chromatography. The atrial natriuretic peptide-like immunoreactivity of cultured atrial and ventricular myocytes was concentrated in the perinuclear cytoplasm and was localised to electron-dense secretory granules. The number of immunoreactive ventricular myocytes and the intensity of their immunofluorescence changed with time in culture and was higher in cultures in foetal calf serum-supplemented medium than in serum-free medium. Gamma-atrial natriuretic peptide was stored and released by cultured atrial and ventricular myocytes, but was broken down to alpha-atrial natriuretic peptide in the growth medium. This process was foetal calf serum-independent, since it occurred in both the media used, indicating that cardiac myocytes in culture may release a factor that cleaves gamma-atrial natriuretic peptide to form alphaatrial natriuretic peptide.     

Immunohistochemical localization of atrial natriuretic polypeptide (ANP) in human atrial and ventricular myocardiocytes

To date, there have been few immunohistochemical investigations of atrial natriuretic polypeptide (ANP) in human cardiac tissue, especially the ventricles. In this study, myocardial tissue was obtained from two sources: the bilateral atria and ventricles at autopsy; and biopsy tissues from the right auricle and left ventricle of a patient with myocardial infarction undergoing surgery. These tissues were examined by the avidin-biotin immunoperoxidase technique using three kinds of primary ANP-antibodies. ANP-immunoreactivity was observed in the perinuclear region of myocytes of all tissues examined. The intensity of the reaction was stronger in atrial tissue, weaker in ventricular tissue. In the later tissue, the positive-staining myocytes were not part of the pulse-conducting system. Although the tissues we studied were not obtained from normal hearts, our data demonstrates that ANP-reactivity can be detected in ventricular myocytes outside the pulse-conducting system.     

Immunohistochemical localization of atrial natriuretic polypeptide (ANP) in human atrial and ventricular myocardiocytes

Summary To date, there have been few immunohistochemical investigations of atrial natriuretic polypeptide (ANP) in human cardiac tissue, especially the ventricles. In this study, myocardial tissue was obtained from two sources: the bilateral atria and ventricles at autopsy; and biopsy tissues from the right auricle and left ventricle of a patient with myocardial infarction undergoing surgery. These tissues were examined by the avidin-biotin immunoperoxidase technique using three kinds of primary ANP-antibodies. ANP-immunoreactivity was observed in the perinuclear region of myocytes of all tissues examined. The intensity of the reaction was stronger in atrial tissue, weaker in ventricular tissue. In the later tissue, the positive-staining myocytes were not part of the pulse-conducting system. Although the tissues we studied were not obtained from normal hearts, our data demonstrates that ANP-reactivity can be detected in ventricular myocytes outside the pulse-conducting system.     

Secretion of atrial natriuretic peptide (ANP) from fish atrial and ventricular myocytes in tissue culture

Primary cultures of atrial and ventricular myocytes (approx. 1 x 10(5) cells/culture) were prepared from adult teleost fish Gila atraria and maintained for 10 days. Immunoreactive atrial natriuretic peptide (ir-ANP) from fish atrial and ventricular cells was 3.9 and 2.8 ng/culture respectively, values not significantly different. Atriocytes from rat and mouse secreted comparable amounts of ANP which were not significantly different from atrial fish cultures (5.2 and 4.3 ng/culture). In contrast, their ventricular myocytes secreted only small quantities of ANP (0.8 and 0.3 ng/culture). When analyzed by reversed-phase HPLC, the media of both fish atrial and ventricular myocytes contained a peptide which exhibited properties similar to authentic human ANP (Ser 99-Tyr 126), suggesting a significant degree of sequence homology between fish and mammalian ANP. Fish ventricular cells, unlike normal mammalian ventricular cells, secrete substantial quantities of immunoreactive-ANP.     

Endothelial cells stimulate ANF secretion from atrial myocytes in co-culture

Using a novel in vitro co-culture system, we investigated the possible influence of vascular endothelial cells on the secretion of atrial natriuretic factor (ANF) from atrial myocytes. Co-culture of bovine aortic endothelial cells grown on Cytodex-3 microcarrier beads with primary monolayer cultures of neonatal rat myocytes induced a 2.1-fold increase in immunoreactive ANF (irANF) in the medium, compared with irANF in medium from atrial cultures alone. This increase did not appear to be the result of processing of prohormone to more immunoreactive species, and could be inhibited by 47% with 10 microM acetylcholine. The endothelium-derived vasoconstrictor peptide, endothelin, elicited a dose-dependent increase in ANF secretion from atrial cultures, but, contrary to vasopressin, was incapable of further stimulating release from atrial-endothelial co-cultures. These experiments suggest that endothelium stimulates the release of ANF from myocytes, possibly by the action of the peptide endothelin.     

Interstitial Cajal-like cells (ICLC) in atrial myocardium: ultrastructural and immunohistochemical characterization

We have previously reported (Hinescu & Popescu, 2005) the existence of interstitial Cajal-like cells (ICLC), by transmission electron microscopy, in human atrial myocardium. In the present study, ICLC were identified with non-conventional light microscopy (NCLM) on semi-thin sections stained with toluidine blue and immunohistochemistry (IHC) for CD117/c-kit, CD34, vimentin and other additional antigens for differential diagnosis. Quantitatively, on semi-thin sections, ICLC represent about 1-1.5% of the atrial myocardial volume (vs. approximately 45% working myocytes, approximately 2% endothelial cells, 3-4% for other interstitial cells, and the remaining percentage: extracellular matrix). Roughly, there is one ICLC for 8-10 working atrial myocytes in the intercellular space, beneath the epicardium, with a characteristic (pyriform, spindle or triangular) shape. These ICLC usually have 2-3 definitory processes, emerging from cell body, which usually embrace atrial myocytes (260 nm average distance plasmalemma/sarcolemma) or establish close contact with nerve fibers or capillaries (approximately 420 nm average distance to endothelial cells). Cell prolongations are characteristic: very thin (mean thickness = 0.15+/-0.1 microm), very long for a non-nervous cell (several tens of microm) and moniliform (uneven caliber). Stromal synapses between ICLC and other interstitial cells (macrophages) were found (e.g. in a multicontact type synapse, the average synaptic cleft was approximately 65 nm). Naturally, the usual cell organelles (mitochondria, smooth and rough endoplasmic reticulum, intermediate filaments) are relatively well developed. Caveolae were also visible on cell prolongations. No thick filaments were detected. IHC showed that ICLC were slightly and inconsistently positive for CD117/c-kit, variously co-expressed CD34 and EGF receptor, but appeared strongly positive for vimentin, along their prolongations. Some ICLC seemed positive for a-smooth muscle actin and tau protein, but were negative for nestin, desmin, CD13 and S-100. In conclusion, we provide further evidence of the existence of ICLC in human atrial myocardium, supporting the possible ICLC role in pacemaking, secretion (juxta- and/or paracrine), intercellular signaling (neurons and myocytes). For pathology, ICLC might as well be 'players' in arrhythmogenesis and atrial remodeling.     

Bidimensional reversed-phase high-pressure liquid chromatography analysis of cultured cell neuropeptides: application to atrial natriuretic factor

We report here a one-step procedure for extraction and analysis of neuropeptides in chromaffin cell culture media and acid extracts using reversed-phase HPLC. The bidimensional HPLC system consists of a precolumn connected to a six-port switching valve which is on-line with an analytical column. The direct injection of the biological samples onto the precolumn previously equilibrated with 15% acetonitrile allows the elimination of interfering substances. The samples purified on the precolumn can then be eluted onto the analytical column via the switching valve for neuropeptide separation. This trace-enrichment system allows a minimum of sample handling, both saving time and reducing possibilities of loss and contamination. This method has been applied to monitor the precursor and mature forms of atrial natriuretic factor from chromaffin cell secretion media and cell content extracts. The recovery of atrial natriuretic factor is in the range of 80-100%. This procedure could be applied to the study of the precursor-product relationship of any neuropeptide, e.g., from radiolabeled extracts of pulse-chase experiments performed on cultured chromaffin cells.     

Bilateral acute heart atrial auriectomy reduced diuresis and natriuresis following hypertonic sodium load in anaesthetized dogs

Anaesthetized dogs were deprived of a portion of the atrial natriuretic factor producing tissue by bilateral acute heart atrial auriectomy. Their ability to respond by diuresis and natriuresis either to the expansion of extracellular fluid volume with isotonic saline (3% b.w.) or to hyperosmolality induced by hypertonic saline loading (0.13% b.w. of 20% NaCl solution) was subsequently reduced by about 50%. It is thus suggested that atrial natriuretic system may also play a role in osmoregulation by taking part in the promotion of renal sodium excretion.     

DNA synthesis in cultures of atrial and ventricular cardiomyocytes in the rat

By means of 3H-thymidine autoradiography DNA replicative activity has been studied in cultured atrial and ventricular myocytes, and non-muscle cells from hearts of 2-week-old rats (age when cell proliferation in the myocardium is already significantly depressed). PAS-reaction was used as a cytochemical marker of cardiomyocytes: atrial myocytes are richer in glycogen than ventricular cells. Labeling indices of atrial myocytes after a 24 hour exposure to 3H-thymidine were higher than ventricular ones: on day 6 of culturing--47 and 5%, and on day 11-34 and 8%, respectively. After 10 days of culturing the number of binucleated atrial myocytes, non-typical for atrial myocardium in vivo, increased by 25-40% as compared with 8-13% on days 2-3 in culture. In 10-day cultures, 3- and 4-nucleated atrial myocytes were observed. Both mononucleated and binucleated atrial and ventricular myocytes incorporated 3H-thymidine. To find out whether the deeper inhibition of replicative activity in ventricular myocytes influences fibroblasts and endothelial cells from ventricles, the proliferative activity of non-muscle cells was studied. Non-muscle cells, both in atrial and ventricular cultures, behaved as a totally proliferating population (labeling indices on the 6th day are about 75-90%) and their growth rate decreased during the formation of the contact-inhibited monolayer. These cells, contrary to myocytes, are predominantly mononucleated in all the periods studied. The deeper depression of replication in ventricular myocytes appears to be related with their higher level of differentiation as compared to myocytes of the atrial myocardium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular forms of immunoactive atrial natriuretic peptide in the rat hypothalamus and atrium

Acid extracts of rat hypothalamus and atrium were prepared by a procedure previously shown to minimize proteolytic degradation of peptides. The majority of the immunoactive material in the atrial extracts had a molecular weight of approximately 9,000 to 15,000 daltons, while that in the hypothalamic extracts had a molecular weight of about 1,500 to 1,800 daltons. The major molecular weight forms of atrial natriuretic peptide from each extract were further distinguishable when analyzed by RP-HPLC. These results suggest that small peptides such as atriopeptins I, II, and III, may not be authentic post-translational processing products in the atrium, and that the hypothalamus and atrium may differentially cleave pro-atrial natriuretic peptide to form tissue-specific products.     

Heart ventricular cells of neonatal rats in a culture: DNA synthesis, ultrastructure and localization of atrial natriuretic peptide

We determined the optimal conditions suitable for expanding cardiac cells in vitro for their future use in experimental transplantation into injured myocardium of adult animals. Ventricular cardiac cells were isolated enzymatically from 2-3 day-old rats and cultured at different cell densities within 5-7 days to 4 weeks. Mixed cultures of muscle and non-muscle cells were examined by light autoradiography, electron microscopy, and immunogold method. The best results were obtained at a density of 3 x 10(5) cells/ml in the medium, consisting of 90% DMEM and 10% fetal calf serum, during 5-7 days of cultivation. In such cultures myocytes made 62.5 +/- 7.9%. After a 24 h incubation with 3H-thymidine, 22.0 +/- 2.2% of myocytes were labeled. Muscle cells contact with each other and with non-muscle cells, contain myofibrils, contract and display atrial natriuretic peptide (ANP)-like immunoreactivity.     

Inhibition of water-sodium intestinal absorption by an atrial extract

The rat atrium contains a diuretic and natriuretic factor which appears to inhibit the sodium reabsorption in the kidney tubules. We observed, in rats, that our atrial extract possesses a potent diuretic and natriuretic effect that was accompanied by an increased dextrose excretion. Similarly, extracts of rat atria, but not of ventricles, reduced intestinal absorption of water, sodium, and dextrose. The omission of sodium or dextrose in the perfusion fluid annulled this effect. These data suggest that the substance inhibiting the intestinal absorption of water and solutes is probably atrial natriuretic factor and that it acts on the sodium-dextrose cotransport mechanism.     

Culture and characterization of fetal human atrial and ventricular cardiac muscle cells

Summary Atrial and ventricular cardiac muscle cells isolated from 14- to 18-wk old fetal human hearts were grown in culture and characterized. Once established in culture the flattened cells contracted spontaneously and possessed differentiated ultrastructural characteristics including organized sarcomeres, intercalated discs, and transverse tubules with couplings. Atrial granules were present in the cultured atrial cells. Some cultured ventricular myocytes also contained electron-dense granules associated with Golgi cisternae, which were similar in size and appearance to atrial granules. The cultured ventricular myocytes divided and expressed the genes for thymidine kinase, histone H4, myosin heavy chain, muscle-specific creatine kinase, atrial natriuretic factor, and insulin-like growth factor II. These results establish that differentiated fetal human heart muscle cells can be cultured in sufficient quantities for biochemical, molecular, and morphological analyses. This work was supported by a postdoctoral fellowship from the American Heart Association, Louisiana Affiliate (JBD) and the National Institutes of Health, Bethesda, MD (HL-35632) (WCC).     

Inhibition of atrial natriuretic peptide secretion by forskolin in noncontracting cultured atrial myocytes

Secretory rates for immunoreactive atrial natriuretic peptide (ANP) by 7 - 8 day-old primary cultures of atrial myocytes from adult rats (with myocyte contraction inhibited by tetrodotoxin (TTX)) were (a) constant for at least two hours, and (b) significantly slowed by forskolin (1, 5, and 25 microM), dibutyryl cyclic adenosine monophosphate (1 mM), or isobutylmethylxanthine (100 microM). The substantial rates of ANP secretion which persisted in cells rendered noncontracting either by inhibiting Ca2+ influx via reduction of external [Ca2+] to less than 10(-7) M or by inhibiting sarcoplasmic reticulum Ca2+ release with 100 microM ryanodine were significantly slowed by 25 microM forskolin, but forskolin sensitivity was lost by cells exposed simultaneously to external Ca2+ concentration of less than 10(-7) M and 100 microM ryanodine. Quiescent myocytes whose ANP secretory rate was depressed by forskolin remained responsive to secretory stimulation by phorbol ester.     

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.     

An immunocytochemical study of the sinuatrial node and atrioventricular conducting system of the rat for atrial natriuretic peptide distribution

Summary Immunocytochemical studies of the adult rat heart show that specific heart granules and atrial natriuretic peptide immunoreactivity are absent from the majority of the myocytes of the specialized nodes, atrioventricular bundle and bundle branches. Immunoreactive granules are present in a small proportion of the transitional sinuatrial and atrioventricular nodal myocytes but, in these regions, they are smaller than their counterparts in the general atrial myocytes. A rarer type of cell profile, identical to general atrial myocytes but lacking immunoreactive granules, is also present at the periphery of the sinuatrial node. A very small proportion of myocytes in the ventricular myocardium, generally in the subendocardial layers subjacent to the terminal ramifications of the bundle branches, contain a few immunoreactive granules.     

Microtubule-associated distribution of specific granules and secretion of atrial natriuretic factor in primary cultures of rat cardiomyocytes

A close spatial relationship between specific granules containing atrial natriuretic factor (ANF) and microtubules was demonstrated in primary cultures of neonatal rat cardiac myocytes. For the detection of specific granules and microtubules, the myocytes were double immunolabelled with antibodies against -ANF and -tubulin and examined by conventional fluorescence or laser scanning confocal microscopy. In addition, the ultrastructural distribution of specific granules was demonstrated by electron microscopy. In the atrial myocytes, ANF was stored in numerous specific granules that were mainly localized in the perinuclear sarcoplasm. In the ventricular myocytes, however, a minority of the cells (10%) exhibited limited ANF immunoreactivity after 4 days in culture. Microtubules were present throughout the sarcoplasm of the myocytes. They were most densely packed in the perinuclear regions. Depolymerization of the microtubules with nocodazole was followed by dispersal of ANF immunostaining both in the atrial myocytes and in the ventricular myocytes exhibiting ANF immunoreactivity. When the microtubules were allowed to recover, the perinuclear distribution of specific granules, as seen in non-treated myocytes, reappeared. Measurements of secreted immunoreactive ANF by radioimmunoassay revealed that the secretion of ANF from atrial myocytes into the medium was significantly reduced following nocodazole treatment, whereas a similar decrease in secretion from ventricular myocytes was not observed. These findings indicate that ANF-containing specific granules are closely associated with microtubules within the myocytes. It is suggested that secretion of ANF from the atrial myocytes, in contrast to the ventricular myocytes, is microtubule-dependent.     

Plasma atrial natriuretic factor concentrations and renal actions in the domestic fowl

Plasma atrial natriuretic factor concentrations in Rhode Island red hens averaged 72.1±6.9 pg·ml^-1, range 33.4–136.0 pg·ml^-1. The intravenous infusion of isotonic saline containing 3% dextran for 2 h produced no significant changes in plasma osmotic or electrolyte concentrations; however, haematocrit changes indicated vascular expansions of 14.4% after 1 h and 21.3% after 2 h and plasma atrial natriuretic factor concentrations were elevated by 190% and 257%, respectively. The intravenous infusion of chicken atrial natriuretic factor at rates of 10, 25, 50 and 100 ng·kg^-1·min^-1 for 20 min produced levels of plasma atrial natriuretic factor that were directly related to the infusion rate and which, in birds undergoing a steady-state diuresis/natriuresis driven by the intravenous infusion of isotonic saline at 1 ml·min^-1, produced dose-dependent increases of 19, 26, 38 and 55% in urine flow rate and of 8, 30, 49 and 77% in sodium excretion. Potassium excretion was significantly increased only at the two highest atrial natriuretic factor infusion rates. The observed correlation between plasma atrial natriuretic factor concentration and vascular volume together with the atrial natriuretic factor-induced modulation of renal salt and water elimination is consistent with the concept that in the chicken this peptide has a physiological role as a regulatory hormone in volume homeostasis.Abbreviations AII angiotensin II - ANF atrial natriuretic factor - AVT arginine vasotocin - BV blood volume - chANF chicken atrial natriuretic factor - CHE chicken heart extract - ECF extracellular fluid - EDTA ethylenediaminetetra-acetate - Hct haematocrit - i.v. intravenous - PCR plasma clearance rate - PRA plasma renin activity - RIA radioimmunoassay