Immunohistochemical study of atrial natriuretic polypeptides in the embryonic,fetal and neonatal rat heart

Summary An immunohistochemical study of atrial natriuretic polypeptides was carried out on embryonic, fetal and neonatal rat hearts, using an antiserum raised against -human atrial natriuretic polypeptide (-hANP). Weakly immunoreactive cells were seen in both atrial and ventricular walls at 11 days post coitum (pc). After this stage, the immunoreactive cells became more intensely stained in both atrial and ventricular walls. The immunoreactivity during the prenatal period was stronger in the superficial cell layer beneath the endocardium, than in the deep cell layer of the atrial wall. The cells in the trabecular meshwork also had an apparent, but weak, immunoreactivity, which showed a greater intensity in the left ventricle than in the right one. It is suggested that these immunoreactive cells in the ventricle may differentiate, in situ, into the cells of the impulse-conducting system during the further development of the heart.This research was supported in part by Grants-in-Aid for Scientific research to C. ura from the Ministry of Education of Japan (Nos. 5957009, 59570010)     

Quantitative analyses of atrial myoendocrine cells and plasma atrial natriuretic peptides (ANP) of the rat with special reference to the twenty-four-hour variations in secretory granules and plasma ANP concentrations

Summary Subcellular structures of atrial myoendocrine cells in the rat heart and plasma concentrations of atrial natriuretic peptides (ANP) were examined at six evenlyspaced time points over 24 h, using morphometric techniques and radioimmunoassay.Myofibrils and mitochondria of the cells occupied 73.3% of the cytoplasm; 2% of the cytoplasm was occupied by secretory granules, rough endoplasmic reticulum and Golgi complexes, structures characteristic of endocrine cells. Plasma ANP concentration was maximal at 08.00 h, when the individual volume of secretory granules was minimal. The numerical density of secretory granules was increased at 12.00 h. The plasma ANP concentration was minimal at 20.00 h, when the numerical density was minimal and the individual volume was maximal. The fluctuation in plasma ANP concentrations over 24 h was thus parallel to that in the numerical densities of secretory granules and inverse to that in individual volumes.These results suggest that in rats the secretory activity of atrial myoendocrine cells increases at the beginning of the resting period, whereas it decreases at the beginning of the active phase.     

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.     

Effects of expansion of blood volume and bilateral vagotomy on specific heart granules and release of atrial natriuretic peptide in the rat

Summary There was no statistically significant difference in basal concentrations of immunoreactive atrial natriuretic peptide (ANP), as assessed by radioimmunoassay, between right and left atrial muscle of control rats; similarly, stereological analysis showed no statistically significant difference in the fractional volume of myocytes occupied by specific heart granules, or in numerical density of granules, between right and left atria. Nevertheless, correlated radioimmunoassay and ultrastructural investigations showed that the major source of elevated plasma levels of ANP after expansion of blood volume was the right atrium. Substantial expansion of blood volume caused an increase in the proportion of peripherally located granules in myocytes of both atria, but reduction in the number of granules and in the concentration and total content of ANP occurred in the right atrium only. Bilateral cervical vagotomy also caused a statistically significant elevation of plasma ANP concentration, accompanied by a statistically significant reciprocal reduction in right atrial ANP content; no statistically significant change occurred in left atrial ANP. When blood volume was expanded after bilateral vagotomy, there was a further statistically significant increase in plasma ANP concentration; this was accompanied by further reduction in right atrial ANP and, moreover, the combined manoeuvre also elicited a statistically significant reduction of ANP in the left atrium. Ultrastructural studies confirmed that, under these conditions, myocytes in both atria showed a marked depletion of specific heart 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.     

Atrial natriuretic peptide (ANP): a study of ANP and its mRNA in cardiocytes,and of plasma ANP levels in non-obese diabetic mice

Summary Atrial natriuretic peptide (ANP) levels in cardiocytes and plasma were examined by using immunohistochemistry, electron microscopy, and radioimmunoassay in non-obese diabetic mice (NOD). Cardiocyte ANP mRNA expression was measured by the polymerase chain reaction method. ANP immunoreactivity in the auricular cardiocytes was more prominent in hyperglycemic mice (NOD-h) than in normoglycemic mice (NOD-n). Ultrastructural examination showed that auricular cardiocytes of the NOD-h group contained more cytoplasmic granules than cells of the NOD-n group. Ultrastructural morphometry indicated that the number of granules per auricular cardiocyte was significantly larger in the NOD-h group than in the NOD-n group. (P<0.01), whereas the granule diameter was significantly smaller in the NOD-h group (P<0.01). Radioimmunoassay showed that ANP levels in the NOD-h auricular cardiocytes were significantly higher than those in the NOD-n cardiocytes (P<0.01); the opposite was true in plasma. Cardiocyte ANP mRNA expression was lower in the NOD-h group than in the NOD-n group.     

Islet amyloid polypeptide (IAPP) in the gastrointestinal tract and pancreas of man and rat

Summary An immunohistochemical study for islet amyloid polypeptide (IAPP) was made on the gastrointestinal (GI) tract and pancreas of man and rat, using antisera raised against a synthetic peptide of C-terminal human IAPP (24–37) and a synthetic peptide of rat IAPP (18–37). A large number of IAPP-immunoreactive cells were found in the pyloric antrum, and a small number in the body of the stomach in both man and rat. Cytoplasmic processes extended out from the bipolar peripheral region of the immunoreactive cells, rather like neuronal processes, and some appeared to make contact with other immunoreactive cells. In addition, small numbers of immunoreactive cells were also seen in the duodenum and rectum, whereas they were absent from the jejunum, ileum and large intestine. An examination was made for evidence of colocalization of IAPP-immunoreactive material with material immunoreactive for gastrin, somatostatin, vasoactive intestinal polypeptide, pancreatic polypeptide, insulin, and glucagon, but none was found. IAPP-immunoreactive cells were also found in the pancreas of non-diabetic and non-insulin-dependent diabetic patients, but they were completely absent from a patient with insulin-dependent diabetes mellitus despite the presence of IAPP in the plasma. The results of these studies suggest that the peptide may have a biological role in situ in the GI tract and, in addition to the pancreas, may be a possible source of plasma IAPP.     

Ontogeny of rat pancreatic polypeptide (PP) cells

Summary Cells storing pancreatic polypeptide (PP) appear in rat pancreas at the time of parturition, much later than insulin and glucagon cells. At this stage, the pancreatic polypeptide (PP) cells occur scattered in the exocrine parenchyma and in the islets. Subsequently, 5–7 days postnatally, an abrupt increase in the number of PP cells occurs. At this stage, they are fairly numerous in the islets and comparatively rare in the exocrine parenchyma. Not until 8–10 days after birth is the number of PP cells similar to that in the adult pancreas. A few PP cells were seen in the antral mucosa during the first 10 days after birth. They were not seen elsewhere in the gut.     

Morphometric analysis of atrial natriuretic peptide-containing granules in atriocytes of rats with experimental congestive heart failure

The morphometric characteristics of atrial natriuretic peptide-containing granules were studied in atrial myoendocrine cells of rats with aorto-caval fistula, an experimental model of congestive heart failure. A total of 6680 granules of control and aorto-caval rats were analyzed by a computerized image analysis system that evaluated the number and sectioned surface area of granules and their subcellular location. Compared with control animals, rats with congestive heart failure displayed a slight increase in the number of peripheral granules, adjacent to the sarcolemma, but not centrally located in the Golgi areas. The mean sectioned surface area of granules in rats with congestive heart failure was about 50% of that in controls, both in the right and left atria. Rats with aortocaval fistula had a higher percent of small granules and lower percent of large granules compared with controls. The data demonstrate different morphometric characteristics in atrial natriuretic peptide-containing granules in atriocytes in rats with experimental congestive heart failure; this may reflect the enhanced synthesis and release of atrial natriuretic peptide in heart failure.     

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.     

Chicken atrial natriuretic peptide (chANP) and its secretion

Summary An immunohistochemical study using antiserum raised against synthetic chicken natriuretic polypeptide was used to investigate the distribution of this peptide in the chicken heart. Immunoreactive cells, both in the atrial and ventricular walls, were identified by electron microscopy, and electron-dense granules in the atrial and ventricular cardiocytes were revealed to be storage sites of the peptide. The electron-dense material, thought to be the peptide, was found in the sarcoplasmic reticulum, and it is suggested that a secretory pathway of the peptide through the latter to extracellular space, may be present, in addition to an exocytotic one.     

Distribution of the pulmonary artery and vein in the lung of the crab-eating monkey (Macaca fascicularis)

In the lung of the crab-eating monkey (Macaca fascicularis), the right pulmonary artery runs across the ventral side of the right upper lobe bronchiole and the dorsal side of the right middle lobe bronchiole. Thereafter, it courses along the dorso-lateral side of the right bronchus, between the dorsal and lateral bronchiole systems. During this course, the right pulmonary artery gives off arterial branches running mainly along the dorsal or lateral side of each bronchiole. The left pulmonary artery runs across the dorsal side of the left middle lobe bronchiole, and is then distributed as in the right lower lobe. The pulmonary veins run mainly along the ventral or medial side of the bronchiole in the upper and middle lobes whereas, in the lower lobe, they run ventrally, and between the bronchioles. Finally they enter the left atrium as four large veins.     

Distribution of the pulmonary artery and vein in the chimpanzee lung

Lungs of two chimpanzees (Pan troglodytes) were examined. The right pulmonary artery runs across the ventral side of the right upper lobe bronchiole and, then across the dorsal side of the right middle lobe bronchiole. Thereafter, it runs between the dorsal bronchiole system and the lateral bronchiole system, along the right bronchus. During its course, it gives off arterial branches which run along each bronchiole. The left pulmonary artery runs across the dorsal side of the left middle lobe bronchiole and then between the dorsal bronchiole system and the lateral bronchiole system. The branches of the pulmonary artery run mainly along the dorsal or lateral side of the bronchiole. The pulmonary veins run mainly along the ventral or medial side of the bronchioles, and between them. Finally, they enter the left atrium with four large veins, i.e. the common trunk of the right upper lobe vein and the right middle lobe vein, right lower lobe pulmonary venous trunk, left middle lobe vein, and left lower lobe pulmonary venous trunk.     

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.     

Distribution of the pulmonary artery and vein of the orangutan lung

The distribution of the pulmonary artery and vein of the orangutan lung was examined. The right pulmonary artery runs obliquely across the ventral side of the right bronchus at the caudally to the right upper lobe bronchiole. It then runs across the dorsal side of the right middle lobe bronchiole. Thereafter it runs obliquely across the dorsal side of the right bronchus, and then along the dorso-medial side of the right bronchus. This course is different from that in other mammals. During its course, it gives off branches which run mainly along the dorsal or lateral side of each bronchiole. The left pulmonary artery runs across the dorsal side of the left middle lobe bronchiole, then along the dorso-lateral side of the left bronchus, giving off branches which run along each bronchiole. The pulmonary veins run mainly the ventral or medial side of, along or between the bronchioles. In the left lung, the left middle lobe vein has two trunks; one enters the left atrium, and the other enters the left lower lobe pulmonary venous trunk. This is also different from that found in most mammals. Finally, the pulmonary veins enter the left atrium with four large veins.     

Locally different role of atrial natriuretic peptide (ANP) in the pericardial fluid

Pericardial fluid (PF) contains several vasoactive agents in higher concentrations than venous plasma (VP). However, with human atrial natriuretic peptide (ANP) controversial data have been reported in earlier studies performed on a limited number of patients (less than 20). The present study was designed to characterize the ANP levels in human PF and cardiac tissues, and to ascertain whether myocardial ischemic state is a major factor in determining ANP production of the human heart. In a total of 316 consecutive patients undergoing open heart surgery ANP levels in VP, PF, atrial and ventricular tissues were measured by radioimmunoassay and analyzed by high-performance liquid chromatography (HPLC). The data are presented as median and 25th-75th percentiles. Our results showed ANP concentration [ANP] of PF significantly exceeded that of VP and [ANP] in the atrial tissue was significantly higher than in the ventricular tissue (p < 0.001). In patients without myocardial ischemia (valvular heart disease) [ANP] in the PF was 258.3 (189.9-342.5) pg/ml, in the VP 28.4 (11.7-57.6) pg/ml and 151.7 (78.4-447.6) ng/mg in the atrial, 0.4 (0.2-1.6) ng/mg in the ventricular tissue. The corresponding values for patients with coronary artery disease were 208.1 (153.8-318.9) pg/ml in the PF, 19.8 (9.4-27.9) pg/ml in the VP, 129.6 (66.5-455.0) ng/mg in the atrial and 1.0 (0.1-1.8) ng/mg in the ventricular tissue. The ventricular tissue levels correlated to the atrial tissue levels (r = 0.317; p < 0.05). Great difference (p < 0.001) was found in the atrial tissue levels between females [414.6 (119.7-734.4) ng/mg] and males [105.4 (65.3-204.2) ng/mg]. In HPLC analysis the majority of the pericardial fluid and tissue ir-ANP coeluted with human ANP [99-126]. In conclusion, [ANP] in PF of cardiosurgical patients is higher by an order of magnitude than in VP. Intrapericardial ANP may reflect the peptide concentration in the myocardial interstitium and may represent a paracrine regulatory mechanism, which seems independent of ANP-induced putative antiischemic influences.     

Immunohistochemical localisation of natriuretic peptides in the heart and brain of the gulf toadfish Opsanus beta

Summary The distribution of natriuretic peptide immunoreactivity was determined in the heart and brain of the gulf toadfish Opsanus beta using the avidin-biotin peroxidase technique. Four antisera were used: the first raised against porcine brain natriuretic peptide which cross-reacts with atrial natriuretic and C-type natriuretic peptides (termed natriuretic peptide-like immunoreactivity); the second raised against porcine brain natriuretic peptide which cross-reacts with C-type natriuretic peptide but not with atrial natriuretic peptide (termed porcine brain natriuretic peptide-like immunoreactivity); the third raised against rat atrial natriuretic peptide; and the fourth raised against eel atrial natriuretic peptide. Natriuretic peptide- and porcine brain natriuretic peptide-like immunoreactivity was observed in all cardiac muscle cells of the atrium. In the ventricle, natriuretic peptide-like immunoreactivity was found in all cardiac muscle cells, however porcine brain natriuretic peptidelike immunoreactivity was confined to muscle cells adjacent to the epicardium. There was no discernible difference in the distribution of natriuretic peptide-like immunoreactivity and porcine brain natriuretic peptide-like immunoreactivity in the brain. Immunoreactive perikarya were observed only in the preoptic region of the diencephalon, and many immunoreactive fibres were found in the telencephalon, preoptic area, and rostral hypothalamus, lateral to the thalamic region. There was no immunoreactivity in any region of the hypophysis. A pair of distinct immunoreactive fibre tracts ran caudally from the preoptic area to the thalamic region, from which fibres extended to the posterior commissure, area praetectalis, dorsolateral regions of the midbrain tegmentum, and tectum. Many immunoreactive fibres were present in the rostral regions of the inferior lobes of the hypothalamus and in the dorsolateral and ventrolateral aspects of the rhombencephalon. No immunoreactivity was observed in the heart and brain using rat atrial natriuretic and eel natriuretic peptide antisera. Although the chemical structure of natriuretic peptides in the heart and brain of toadfish is unknown, these observations show that a component of the natriuretic peptide complement is similar to porcine brain natriuretic and/or porcine C-type natriuretic peptides. The presence of natriuretic peptides in the brain suggests that they could be important neuromodulators and/or neurotransmitters.     

Immunoreactive atrial natriuretic peptide in the fish heart and blood plasma examined by electron microscopy,immunohistochemistry and radioimmunoassay

Summary The immunoreactivity of atrial natriuretic peptide and ultrastructure of cardiocytes were examined in 5 species each of freshwater and seawater teleosts, as well as in 2 species each of elasmobranchs and cyclostomes. Immunoreactivity was strong in the atria of Cyprinus carpio, Anguilla japonica and Conger myriaster, rather weak in atria of Channa maculata, Lepomis macrochirus, Salmo gairdneri, Oplegnathus fasciatus and Eptatretus burgeri, very weak in atria of Pagrus major, Trachurus japonicus and Triakis scyllia, and not detectable in atria of Hexagrammos otakii, Narke japonica and Lampetra japonica. The immunoreactivity of the atrial cardiocytes was generally stronger in freshwater than seawater fish. Ventricular immunoreactivity was detected only in 7 species, always being weaker than that observed in the atrium. Ultrastructurally, however, secretory granules were found in atria and ventricles of all species examined, being more frequent in the former than the latter. By radioimmunoassay, immunoreactive ANP was detected in the extracts of blood plasma and both atrial and ventricular tissues of all species examined. There were no statistically significant differences in the values between freshwater and seawater species.     

Association of the left common ostium with clinical outcome after pulmonary vein isolation in atrial fibrillation

IntroductionElectrical pulmonary vein isolation (PVI) is used for the invasive treatment of atrial fibrillation (AF). However, despite the procedure’s technical evolution, the rate of AF recurrence due to electrical reconnection of the PVs is high. The aims of this study was to assess the influence of left common pulmonary venous ostium (LCO) on clinical outcomes following PVI.MethodsRetrospective cohort of 254 patients who underwent the first procedure of PVI from the years 2013–2018 was assessed. Patients with persistent AF of long duration and extra-pulmonary focus associated with triggers for arrhythmia were excluded. Patients were stratified into two groups according to the presence of a LCO and received follow up for atrial tachyarrhythmia-free survival. The mean follow-up period was 28 ± 1.73 months.ResultsThe majority were men (68.5%), with a mean age of 54 ± 12 years. With respect to the atrial anatomy, LCO occurred in 23.6% of cases after pulmonary venous angiotomography. The arrhythmia-free survival rate was 79.5% in the follow-up period. The Cox regression model was utilized and the adjusted hazard ratio for LCO was 0.36 (95% CI 0.15–0.87; p = 0.02) in terms of age, body mass index, left atrium diameter, bi-directional blocking of the cavotricuspid isthmus, persistent AF, left ventricular ejection fraction adjusted model.ConclusionAnatomic abnormality with the presence of the LCO is present in a quarter of patients undergoing AF ablation, which is associated with a lower rate of arrhythmia recurrence in our population.