Fine structure of atrial natriuretic peptide (ANP)-granules in the atrial cardiocytes of the mouse, rat and Mongolian gerbil.

Mouse, rat and Mongolian gerbil atrial and ventricular cardiocytes were examined by immunohistochemistry, and the right atrium including the auricle was examined by transmission electron microscopy. In addition the ANP granules of both right atrial and auricular cardiocytes were analyzed by ultrastructural morphometry. ANP immunoreactivity was detected in the atria of all three species, and the most intensely reacting cardiocytes were localized in the right auricular part of the atrium. These reactions were more prominent in the mouse and rat than in the Mongolian gerbil. ANP immunoreactivity was not detected in the ventricular myocardium of any of the three species, but was occasionally seen in the subendocardium of the ventricular septum. Ultrastructurally, the ANP granules in the auricular and atrial cardiocytes were observed to be variable in size and number, and these granules were located principally in the paranuclear region in association with the Golgi apparatus, and found throughout the sarcoplasmic layers in all three species. The ANP granules were classified into two types: A-granules containing a conspicuous electron-dense core possessing a membrane, and B-granules having profiles with a fibrillogranular, less electron-dense core than the A-granules and an indistinct membrane. The features of these granules were similar in all three species. When examined by ultrastructural morphometry, the number of each type granule and the total number of granules in the right auricular and atrial cardiocytes of the mouse and rat were significantly greater than in the Mongolian gerbil. The total number of granules in the right auricular cardiocytes was significantly greater than in the cardiocytes of the right atrium exclusive of the auricle, however, there was no significant difference between the number of A-granules and B-granules in the three species. The diameter of each type of granule in the right auricular and atrial cardiocytes of the mouse and rat was significantly greater than in the Mongolian gerbil, and the diameter of the A-granules was significantly greater than the diameter of the B-granules in all three species.     

Atrial natriuretic peptide detected by immunocytochemistry in peripheral organs of Tupaia belangeri

ANP (atrial natriuretic peptide), a peptide found in granules of mammalian atrial cardiac myocytes, has been shown to be active in regulation of blood pressure and body water homeostasis. The existence of ANP in atrium, pituitary, adrenal gland, and kidney of the rat had been immunocytochemically demonstrated with an antibody against rat ANP (102-126). We used the same antibody in immunocytochemical studies for the detection of ANP in peripheral organs of the tree shrew (Tupaia belangeri). The antibody stained granules in myocytes of cardiac atria which indicated that it reacted with tree shrew ANP. In contrast to the rat, no immunoreactive cells were found in pituitaries and adrenal glands. However, in the kidneys distal tubules in outer medulla and cortex were labeled. Ascending limbs of distal tubules were intensely stained when either the peroxidase-antiperoxidase (PAP) or the indirect immunofluorescence method were used. Collecting ducts and convoluted distal tubules in the outer cortex showed a granular type of staining when the immunofluorescence method was used. These data indicate that ANP is present in epithelial cells of distal tubules and collecting ducts, where it may be involved in the regulation of renal salt excretion.     

Atrial natriuretic peptide detected by immunocytochemistry in peripheral organs of Tupaia belangeri

Summary ANP (atrial natriuretic peptide), a peptide found in granules of mammalian atrial cardiac myocytes, has been shown to be active in regulation of blood pressure and body water homeostasis. The existence of ANP in atrium, pituitary, adrenal gland, and kidney of the rat had been immunocytochemically demonstrated with an antibody against rat ANP (102–126). We used the same antibody in immunocytochemical studies for the detection of ANP in peripheral organs of the tree shrew (Tupaia belangeri). The antibody stained granules in myocytes of cardiac atria which indicated that it reacted with tree shrew ANP. In contrast to the rat, no immunoreactive cells were found in pituitaries and adrenal glands. However, in the kidneys distal tubules in outer medulla and cortex were labeled Ascending limbs of distal tubules were intensely stained when either the peroxidase-antiperoxidase (PAP) or the indirect immunofluorescence method were used. Collecting ducts and convoluted distal tubules in the outer cortex showed a granular type of staining when the immunofluorescence method was used. These data indicate that ANP is present in epithelial cells of distal tubules and collecting ducts, where it may be involved in the regulation of renal salt excretion.     

Atrial natriuretic peptide in the granular cells of the snail heart

Cardiomyocytes of vertebrates combine contractile and endocrine functions. They synthesize and secrete atrial natriuretic peptide (ANP), which is localized in their specific granules. The presence of ANP has been shown in some tissues of invertebrates, including the heart of molluscs. We have studied localization of ANP in cells of the snail heart. METHOD: The atrial and ventricular tissues of the snail Helix pomatia were studied by electron microscope immunocytochemistry, using anti-ANP antibodies. ANP-immunoreactivity has been detected in granules of granular cells located on the luminal surface of the snail myocardium. These cells are abundant in the atrium being very rare in the ventricle. Granular cells at different stages of maturation were revealed. Immature granular cells have light granules of moderate size with homogeneous tight content, while mature granular cells are huge in size and all their granules are fused together. The material of these granules loosens up and almost completely fills up the cytoplasm. No ANP-immunoreactivity was observed in muscle cells or nerve fibers. A possible origin of granular cells from the cardiac endothelial cells is discussed. The molluscan heart, similar to that of vertebrates, is a bifunctional organ. However, contrary to the heart of vertebrates, in the molluscan heart contractile and endocrine functions are separated between different types of cells.     

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.     

Regional appearance of atrial natriuretic peptide in the ventricles of infarcted rat hearts

The appearance of atrial natriuretic peptide (ANP) in the ventricular myocardium was investigated in rat hearts subjected to severe left ventricular infarction. The left coronary artery was ligated for 1, 2, 3, 4 and 6 days and for 3 weeks, and the tissue was prepared for microscopic examination of immunoreactive ANP and for electron microscopy. In the normal and sham-operated hearts, and in hearts subjected to 1 day of coronary ligation, ANP immunoreactivity was restricted to a few ventricular myocytes of the conduction system. Following 2–3 days of coronary ligation, ANP immunoreactivity was detected in the viable myocardium of the lateral border of the infarct and in a few layers of viable cardiac myocytes located in the subendocardial areas of the ischemic left free ventricular wall. Further, during the following days and after 3 weeks of coronary ligation, a gradient of specific labeling was commonly seen across the lateral border area of the infarct. Thus, the strongest immunoreactivities were present in the cardiac myocytes located adjacent to the non-contracting myocardium. Electron microscopic examination of the immunoreactive cardiac myocytes confirmed the presence of electron-dense specific granules within these cells. The present findings suggest that the increased regional production of ANP within the ventricular myocardium is induced by increased mechanical stretch of the cardiac myocytes, and that this might contribute to the increased release of ANP in myocardial infarction.     

Fine structure of the atrial cardiocytes in the house musk shrew (Suncus murinus).

The atrial and ventricular cardiocytes of the house musk shrew were examined by immunohistochemistry, and the right atrium containing the auricle was studied by transmission electron microscopy. The atrial natriuretic peptide (ANP)-granules of the cardiocytes in the auricle and the rest of the atrium were also analyzed by ultrastructural morphometry. On immunohistochemistry, ANP immunoreactivity was detected in the atria, with the most intensely reacting cardiocytes being localized in the right auricular part of the atrium. ANP immunoreactivity was not detected in the ventricular muscles. On ultrastructure, in most of the atrial cardiocytes including the auricle, ANP-granules, well-developed Golgi apparatus and rough endoplasmic reticulum were observed, and the nuclei were characteristically situated in the periphery of the cardiocytes, being different from many other mammalian hearts. The ANP-granules were classified into two types (A and B), with most of these granules being located in the paranuclear region in association with the Golgi apparatus, and a few ANP granules being observed throughout the sarcoplasmic layers intervening between the myofibrilar bundles. On ultrastructural morphometry, the total number of granules in the right auricular cardiocytes was significantly greater than those in the atrial cardiocytes, and the diameter of the A-granules was significantly greater than that of the B-granules in both the atrial and auricular cardiocytes.     

Atrial natriuretic polypeptide in human adrenal pheochromocytoma: immunohistochemical and immunoelectron microscopical localization

Using the immunoperoxidase and immunogold methods with specific antibody, we studied the atrial natriuretic polypeptide (ANP) in seven tumor tissues of six patients with adrenal pheochromocytoma. Light microscopically, the reaction product for ANP was observed in all seven tumor tissues. Intracytoplasmic immunoreaction product for ANP was finely granular. In four cases studied with the electron microscope, the immunogold stain for ANP was demonstrated in secretory granules of the tumor cells. A considerable amount of alpha-hANP immunoreactive substance was also extracted from two tumor tissues (67.2 and 28.7 pg/mg wet tissue). This is the first report of the human adrenal pheochromocytoma that contains immunoreactive ANP. These findings provide additional evidence for the multisecretory APUD cells of neural crest origin.     

Co-localization of a kallikrein-like serine protease (arginine esterase A) and atrial natriuretic peptide in rat atrium

Atrial natriuretic peptide (ANP) is stored in atrial granules primarily as a larger molecular weight precursor (pro-ANP), which is believed to be rapidly converted to an active peptide of 28 amino acids during or shortly after secretion. A tissue kallikrein-like serine protease has been suggested as a potential processing enzyme. In the present immunocytochemical study, using specific monoclonal antibodies, we found that esterase A, a kallikrein-like serine protease, was demonstrable in rat atrial myocytes and in ventricular myocytes, and was capable of cleaving pro-ANP to yield a low molecular weight product. Using colloidal gold immunocytochemistry at the electron microscopic level, we have found esterase A in atrial myocytes, both in granules and in another subcellular site that corresponds to sarcoplasmic reticulum. Double-label electron microscopic immunocytochemical results indicated that esterase A can co-localize with ANP in granules of atrial myocytes.     

The effect of water deprivation on the expression of atrial natriuretic peptide and its receptors in the spinifex hopping mouse,Notomys alexis

This study investigated the mRNA expression of the atrial natriuretic peptide (ANP) system (peptide and receptors) during water deprivation in the spinifex hopping mouse, Notomys alexis, a native of central and western Australia that is well adapted to survive in arid environments. Initially, ANP, NPR-A and NPR-C cDNAs (partial for receptors) were cloned and sequenced, and were shown to have high homology with those of rat and mouse. Using a semi-quantitative multiplex PCR technique, the expression of cardiac ANP mRNA and renal ANP, NPR-A, and NPR-C mRNA was determined in 7- and 14-day water-deprived hopping mice, in parallel with control mice (access to water). The levels of ANP mRNA expression in the heart remained unchanged, but in the kidney ANP mRNA levels were increased in the 7-day water-deprived mice, and were significantly decreased in the 14-day water-deprived mice. NPR-A mRNA levels were significantly higher in 7-day water-deprived mice while no change for NPR-A mRNA expression was observed in 14-day water-deprived mice. No variation in NPR-C mRNA levels was observed. This study shows that water deprivation differentially affects the expression of the ANP system, and that renal ANP expression is more important than cardiac ANP in the physiological adjustment to water deprivation.     

Cardiac atrial natriuretic peptide concentrations in experimental obese mice

Obesity is usually associated with expansion of blood volume. Therefore, we studied whether obesity affects cardiac and plasma atrial natriuretic peptide (ANP) levels in experimental animal model. Mice made obese with gold thioglucose developed cardiac hypertrophy associated with increases in ANP in atrial tissue and plasma. There were significant (p less than 0.01) correlations between the cardiac ANP concentration and body weight or cardiac weight. These data suggest that enhanced synthesis of atrial ANP in obese mice can be mainly ascribed to increased blood volume associated with cardiac hypertrophy.     

An immunocytochemical study on co-localization of cathepsin B and atrial natriuretic peptides in secretory granules of atrial myoendocrine cells of rat heart

To examine localization of cathepsin B, a representative lysosomal cysteine protease, in atrial myoendocrine cells of the rat heart, immunohistochemistry at the light and electron microscopic level was applied to the atrial tissue, using a monospecific antibody for rat liver cathepsin B. In serial semi-thin sections, immunoreactivity for cathepsin B and atrial natriuretic peptides (ANP) was detected in the para-nuclear region of atrial myoendocrine cells. Several large granules and many fine granules in the region of the cells were positively stained by the cathepsin B antibody. Gold particles indicating cathepsin B antigenicity labeled secretory granules in the cells, which were also labeled by those indicating ANP, using thin sections of the Lowicryl K4M-embedded material. Moreover, some granules labeled densely by immunogold particles for cathepsin B seemed to be lysosomes. By double immunostaining using thin sections of the Epon-embedded material, gold particles indicating cathepsin B and ANP antigenicities were co-localized in secretory granules of the cells. By enzyme assay, activity of cathepsin B was three times higher in atrial tissue than ventricular tissue. The results suggest that co-localization of cathepsin B and ANP in secretory granules is compatible with the possibility that cathepsin B participates in the maturation process of ANP.     

自发性高血压大鼠右心耳肌细胞特殊颗粒的形态分析和ANP免疫反应强度的观察

本研究运用透射电镜及形态计量学方法结合免疫组织化学技术对成年自发性高血压大鼠（ＳＨＲ）的右心耳肌细胞心房特殊颗粒（ＡＳＧ）和心房利钠肽（ＡＮＰ）的免疫反应强度进行了观察和定量研究。成年自发性高血压大鼠的心肌细胞内,ＡＳＧ数目增加,直径增大,高尔基复合体发达;线粒体轻度肿胀,部分嵴溶解断裂,部分内质网扩张,糖原颗粒增多。ＡＮＰ免疫反应增强与ＡＳＧ数目的增加一致。提示自发性高血压大鼠ＡＮＰ的合成和释放均增加,以维持机体在高血压状态下血压的平衡和内环境的稳定。     

Increased plasma brain natriuretic peptide levels in DOCA-salt hypertensive rats: relation to blood pressure and cardiac concentration

Four experimental groups of rats treated with (1) DOCA-salt, (2) DOCA or (3) salt, and (4) controls were used to study the participation of brain natriuretic peptide (BNP) in the development of hypertension. Plasma and cardiac tissue concentrations of BNP as well as atrial natriuretic peptide (ANP) were measured in each group by using radioimmunoassays specific to rat BNP or ANP. Plasma BNP levels in DOCA-salt hypertensive group were higher than those in control (p less than 0.01), salt (p less than 0.01) and DOCA (p less than 0.01) groups. A positive correlation was observed between plasma BNP levels and blood pressure (r = 0.70, p less than 0.001) and between plasma ANP levels and blood pressure (r = 0.62, p less than 0.001). Plasma BNP/ANP ratio increased parallel with elevation of blood pressure. Plasma BNP levels correlated negatively with atrial BNP concentration (r = -0.33, p less than 0.05), but positively with ventricular BNP (r = 0.76, p less than 0.001). Compared with controls, tissue BNP-45/gamma-BNP ratio in the DOCA-salt rats was lower in atrium, but higher in ventricle. Thus, in DOCA-salt hypertension atrial BNP decreased with exhaustion of stored BNP-45, while ventricular BNP increased as BNP-45 accumulated. These results suggest that BNP is a novel cardiac hormone, synthesized, processed and secreted in response to changes in blood pressure. BNP may play different roles in controlling blood pressure than those assumed by ANP.     

The lung as a possible target organ for atrial natriuretic polypeptide secreted from the heart

Using a specific radioimmunoassay (RIA) for alpha-rat atrial natriuretic polypeptide (alpha-rANP), we have demonstrated the presence of a considerable amount (6.10 +/- 0.38 ng/g) (mean +/- SE) of alpha-rANP-like immunoreactivity (alpha-rANP-LI) in the rat lung, the first organ through which atrial natriuretic polypeptide (ANP) released from the heart passes. High performance gel permeation chromatography coupled with the RIA revealed that most of alpha-rANP-LI eluted at the position of a low molecular weight form corresponding to synthetic alpha-rANP. In 2- or 5-day water-deprived rats, the concentration and content of alpha-rANP-LI in the lung decreased significantly compared with those of control rats. In addition, water-deprivation induced a significant decrease in the plasma concentration of alpha-rANP-LI simultaneously determined. There was a significant positive correlation between the concentrations of alpha-rANP-LI in the lung and plasma (r = 0.591, P less than 0.01). These results indicate the presence of ANP in the lung and suggest physiological roles of ANP in pulmonary function.     

Fine structural and immunohistochemical localization of cardiac hormones (ANP) in the right atrium and hypothalamus of the white rat

Atrial natriuretic peptide (ANP) is a polypeptide hormone secreted primarily by atrial myoendocrine cells. It has diuretic, natriuretic and vasorelaxant effects. ANP has been characterized by non-morphological methods in a number of extra-atrial tissues, particularly the hypothalamus, but little is known of the immunohistochemistry of hypothalamic ANP cells in comparison to atrial ones. Although the presence of ANP-producing cells has previously been confirmed in the right atrium of the rat and other vertebrate species, to our knowledge, this is the first study to demonstrate the presence of these cells in the hypothalamus using a purely morphological method such as electron microscopy. The fine structural and immunohistochemical characteristics of right atrial and hypothalamic ANP positive cells were investigated using immunogold labeling with goat anti-alpha-human ANP (1-28) as primary antibody. Atrial ANP cells were characterized by the presence of membrane-bound electrondense spherical or oval granules with a diameter of about 250 nm. The opaque content of the granules is separated from the limiting membrane by a thin electron translucent band about 20 nm wide. Electron dense crystalloid inclusions were evident within the granule matrix of some atrial ANP granules. Hypothalamic ANP granules were membrane-bound larger in diameter (320 nm), and less electron dense, and lacked crystalloid inclusions. Statistical analyses revealed a significant larger diameter and a significant smaller number of hypothalamic ANP granules compared to atrial ones. The significantly greater number of atrial ANP positive granules suggests a greater volume capacity for the atrial ANP positive granules as compared to the hypothalamic ones. This may indicate that ANP is secreted primarily from the right atrium and to a lesser extent from the hypothalamus; and that both atrial and hypothalamic ANP are closely related in chemical nature and immunohistochemical characteristics. This supports the suggestion that ANP may play the dual role of peripheral hormone and a neurotransmitter or neuromediator.     

Chromogranin/secretogranin proteins in murine heart: myocardial production of chromogranin A fragment catestatin (Chga364–384)

In the heart, the secretory granules containing the atrial natriuretic peptides (ANP) and B-type myocardial natriuretic peptide (BNP) provide the basis for the endocrine function of this organ. We sought to determine whether atrial and myocardial secretory granules contain chromogranin/secretogranin proteins including chromogranin A (CHGA/Chga), chromogranin B (CHGB/Chgb) and secretogranin II (SCG2/Scg2). Deconvolution microscopy on immunolabeled proteins revealed the presence of Chga, Chgb, and Scg2 in murine cardiac secretory granules. The presence of low plasma catestatin (CST: mChga_364–384) in older mice indicates diminished processing of Chga to CST with advancement of age, which is comparable to that found in humans. We have previously shown that CST (hCHGA_352–372) exerts potent cardio-suppressive effects on frog and rat heart, but the source of CST for such action has remained elusive. In the present study, we found CST-related peptides in cardiomyocytes and in heart, which establishes an autocrine/paracrine function of CST in cardiac tissue. We conclude that cardiac secretory granules contain Chga, Chgb and Scg2 and that Chga is processed to CST in murine heart.     

Heart and plasma atrial natriuretic peptide (ANP) in response to long-term endurance training in rats.

Long-term endurance training effects on heart and plasma ANP were investigated in male Wistar rats. Maximal O2 uptake (VO2max) was significantly higher in trained groups, when they are used as their own control. After 3, 4, and 5 weeks of endurance training, VO2max was respectively increased by 7.7% (p less than 0.05), 13.7% (p less than 0.01), and 18.4% (p less than 0.001). Plasma ANP and glomerular ANP receptor density showed no clear variations in trained rats. However, cardiac ANP content decreased significantly in left and right atrial tissues by 35-36% (p less than 0.05) after 5 weeks of training. ANP immunoreactivity was investigated to show the distribution of ANP within the atria. ANP was found in diffuse and granular forms. The diffuse pattern (immature ANP) disappeared in cardiocytes of trained rats, while the granular form persisted, especially in the left atrial tissue. These data suggest that chronic endurance training might cause a decrease in ANP synthesis with no change in ANP storage. Such results are in agreement with the hypothesis that the left atrium could be especially involved in long-term fluid volume control.     

Presence of atrial natriuretic polypeptide in the pulmonary vein and vena cava

Striated muscle cells and storage granules observed in the atria were found in main branches of the pulmonary veins and superior and inferior venae cavae of the rat, pig, and ox. The presence of atrial natriuretic polypeptide (ANP) in these veins was examined by reverse-phase high-performance liquid chromatography coupled with a radioimmunoassay for ANP. The veins contained 0.6 to 8.0 ng ANP/mg wet tissue with the major molecular form being gamma-ANP. ANP was detected in the peripheral lung tissue in a small quantity, but was not detected in the pulmonary artery. The identification of gamma-ANP and storage granules stained with an anti-ANP antiserum in the pulmonary vein and vena cava suggest that the veins may participate in regulating volume status, blood pressure, and cardiovascular homeostasis through the release of ANP.     

Electronmicroscopical, immunohistochemical, immunocytochemical and biological evidence for the occurrence of cardiac hormones (ANP/CDD) in chondrichthyes

As representatives of the vertebrate class of chondrichthyes the plagostomian species Squalus acanthias, Scyliorhinus canicula and Raja clavata as well as the holocephalan species Chimaera monstrosa were investigated for the presence of cardiac hormones of the atrial natriuretic polypeptide/cardiodilatin- (ANP/CDD-) family. ANP/CDD-immunoreactive cells were detected in the atria and the ventricles of all species studied. While these cells failed to react with antisera raised against the N-terminus of CDD-126 (= gamma-ANP) they reacted with all antisera directed against sequences of the C-terminus of CDD-126 (CDD 99-126) which is identical to alpha-ANP. The ANP/CDD-immunoreactive cells were found in high numbers in all regions of the atria and in moderate density also in the ventricles. In correspondence, in the electron microscope, myoendocrine cells which were characterized by dense-cored secretory granules were identified in the atrial and ventricular myocardium. With the use of the protein A-gold technique, ANP/CDD-immunoreactivity was determined within the secretory granules. Furthermore, in the bioassay, prepurified extracts of the atria and the ventricles of Scyliorhinus and Chimaera exerted dose-dependent relaxations of the pre-contracted mammalian (rabbit) aorta. In both cases the atrial extracts proved to be more potent than the ventricular extracts. The present findings indicate that myoendocrine cells occur in the atria and ventricles of chondrichthyes and that these cells contain homologous cardiac hormones of the ANP/CDD-family in their secretory granules. The results are compared with those obtained earlier for the other vertebrate classes and their phylogenetic and functional significance is discussed.