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.     

Qualitative and quantitative analysis of granules in atrial appendage cardiocytes in different physiological states

Summary Atrial appendage cardiocytes of mammals, including man, contain multiple cytoplasmic granules that vary in number in different physiological states. Using morphologic and comprehensive morphometric techniques, these granules were assessed in Sprague-Dawley rats following dehydration for 5 days, volume-loading by substituting 1% NaCl as drinking water for 7 days, unilateral nephrectomy plus volume-loading for 7 days, and in late term pregnant animals (18–20 days; term 21 days). Although principally located in the paranuclear region, granules were observed throughout the sarcoplasm. Cytological features indicative of synthetic activity and granule formation were readily apparent in all groups with the exception of pregnant rats where they were infrequently observed. Granule contents were released by exocytosis and observed in the right appendage of control, dehydrated and nephrectomy/volume-loaded groups and left appendage of volumeloaded animals. Exocytosis was not observed in pregnant animals. By point counting, the proportional volume of cardiocytes occupied by granules (V _v ) in controls was significantly greater for right than for left appendage (2.12±0.22% vs 1.29±0.16%; mean±SEM;p<0.05). A significantly similar difference was found for nephrectomy/volume-loaded animals. There was no significant difference inV _v for right appendage between the control and experimental groups; for left appendage there was a significant increase inV _v to 2.42±0.09% (p<0.05) for volume-loaded animals only. Estimation of the maximum diameter of granule profiles in control animals was 238±9 nm and 230±6 nm for right and left appendages, respectively. The profile diameters in the left appendages of dehydrated (202±9 nm) and pregnant (200±7 nm) animals were significantly (p<0.05) less than those of the control animals. The morphometric findings did not correlate with predictions based upon published biochemical data. In the course of this study, a previously unreported bimembranous, circular to ovoid structure was observed in the cardiocyte sarcoplasm of all animals; the nature and function of this structure is unknown.     

Distribution of atrial natriuretic polypeptide (ANP)-containing cells in the rat heart and pulmonary vein Immunohistochemical study and radioimmunoassay

Summary The distribution of atrial natriuretic polypeptide (ANP) was immunohistochemically surveyed in the rat heart and lung using an antiserum raised against -human ANP. The ANP-immunoreactive cells were seen to be distributed in the atrial walls and proximal portions of the pulmonary vein and venae cavae, but were absent from the aorta, pulmonary arteries, trachea, bronchus, and alveolar cells. The immunoreactive cells were present in a narrow region just beneath the endothelium of the pulmonary vein and vena cavae, and, ultrastructurally and immunocytochemically, were seen to be striated muscle cells with ANP-containing specific granules similar to those seen in atrial cardiocytes. A radioimmunoassay for ANP revealed a content of 604±51 pg/mg wet weight in the pulmonary vein, and 3343±1620 pg/mg wet weight in the venae cavae. In addition to the atrial wall, the proximal portion of both the pulmonary vein and venae cavae are suggested to be constituents of an ANP-producing organ.     

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.     

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.     

胰岛素诱导低血糖大鼠心房肌细胞特殊颗粒的形态计量和心房利钠肽免疫组织化学研究

通过形态计量学和免疫组织化学方法发现胰岛素诱导低血糖大鼠心房肌细胞核周区特殊颗粒（ＡＳＧ）的体密度、面数密度和数密度及平均直径均高于对照组（Ｐ＜０．０５）,但高尔基复合体各参数与对照组比较没有差别（Ｐ＞０．０５）。实验组的心房利钠肽（ＡＮＰ）的免疫反应强度比对照组强（Ｐ＜０．００１）。提示胰岛素诱导低血糖对心房利钠肽的释放具有抑制作用,表明ＡＮＰ作为生理和病理调节递质与代谢刺激相拮抗。     

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.     

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.     

Localization of wheat-germ agglutinin-binding sites in the Golgi complex of cultured rat atrial myocytes

Summary In the Golgi region of cultured rat atrial myocytes, condensed secretory protein was seen in Golgi-associated tubules or cisternae which lay beyond, and often separated from, the remainder of the Golgi stacks. These structures appeared to be involved in packaging of condensed secretory protein into atrial granules. Binding sites of HRP-conjugated wheat-germ agglutinin (WGA) in saponin-treated cultured atrial myocytes were examined by electron microscopy with special reference to atrial granules and the tubular structures associated with the Golgi stacks. HRP reaction products were observed in both trans-cisternae of the Golgi stacks and the associated tubular structures. While the majority of atrial granules were devoid of reaction products, some granules, which were connected to the WGA-positive tubular structures in the vicinity of the Golgi trans-cisternae, showed HRP reaction products at their connected necks. Similar results were obtained when sections of the cells embedded in Lowicryl K4M were labeled with WGA coupled to colloidal gold (G-WGA); the Golgi complex was G-WGA positive, whereas no specific binding of G-WGA to atrial granules was observed. These results suggest that glycoproteins and/or glycolipids with oligosaccharides recognized by WGA in the Golgi transcisternae, may be separated from atrial natriuretic peptides which are packaged into atrial granules.Abbreviations ANP atrial natriuretic peptide - HRP horseradish peroxidase - M199 medium 199 - TGN trans-Golgi network - WGA wheat-germ agglutinin - G-WGA WGA coupled to colloidal gold     

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.     

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     

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。     

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.     

不同生理状态对大鼠右心耳肌细胞特殊颗粒的影响──形态学和计量学研究

本文通过电镜观察和立体计量学方法,研究迷走神经和肾上腺素对ＳＤ大鼠右心耳肌细胞特殊颗粒（ＳＧ）的影响。结果是迷走神经切除组ＳＧ的体密度（ＶＶ）、颗粒平均直径（Ｄ）明显小于对照组（Ｐ＜０．００１）,肾上腺素组ＳＧ的ＶＶ、Ｄ、数密度（Ｎｖ）也明显小于相应对照组（Ｐ＜０．００１）。结果提示迷走神经对心钠素的释放是抑制作用,肾上腺素对心钠素的释放是促进作用。本研究还为ＳＧ的产生与分型,心钠素的多种释放方式提供了形态学资料。     

Immunohistochemical localization of 14 kDa β-galactoside-binding lectin in various organs of rat

Summary Immunohistochemical localization of 14 kDa -galactoside-binding lectin in various organs of adult rat was achieved using a monospecific antibody raised against lectin purified from rat lung. The antibody-stained cells were formed into small aggregates, thin fascicles, or thick bundles in the walls of blood vessels, gastrointestinal tracts and urogenital organs. From the patterns of distribution, as well as their organization, these immunoreactive cells were regarded as smooth muscle cells. This was confirmed by a double immunofluorescence study using a mixture of anti 14 kDa lectin and anti -smooth muscle-specific actin antibodies. Strong 14 kDa lectin immunoreactivity was seen in the pericellular matrix of smooth muscle cells in intact organs as well as in detergent-treated organs from which all cellular components were extracted. From these findings, it is suggested that the 14 kDa lectin may be externalized by smooth muscle cells into their pericellular matrix and participate in the crosslinking of the complementary glycoconjugate(s) localized at that site. The macromolecular complex of glycoconjugates thus formed around smooth muscle cells may play a role in anchoring smooth muscle cells to the pericellular connective tissue thereby permitting the force of muscle contraction to be efficiently transmitted to the surrounding connective tissue proper.     

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.     

Rapid refilling of neurosecretory terminals with secretory granules after an acute stimulus to the isolated neural lobe of the rat

Summary Quantitative ultrastructural techniques were used to study changes in the distribution of intracellular organelles in neurosecretory terminals of the rat neurohypophysis during recovery from a depolarising stimulus in vitro. The volumetric density of neurosecretory granules which, in profiles of nerve terminals sectioned through the area of contact with the basal lamina, was decreased as a result of stimulation, returned to control values within 2 h after cessation of stimulation. We conclude that, even in the absence of the cell body and action potentials propagated from it, granules can migrate quickly within the terminal region of the neurosecretory axons to refill a depleted compartment.     

Expression of fascin-1, an actin-bundling protein,in migrating hepatoblasts during rat liver development

Fascin-1 is an actin-bundling protein localized at the core actin bundles within microvillar projections and filopodial extensions in migrating cells. It is expressed at a low level in normal epithelial cells, but at a high level in tumor cells, indicating its importance in the invasion and motility of tumor cells. In addition, fascin-1 is expressed in human and murine embryos, occurring at high levels especially in developing nervous tissues. In this study, we have investigated the expression patterns of fascin-1 immunohistochemically during the early stages of rat hepatogenesis. A high expression of fascin-1 was detected in the liver bud and hepatoblasts at embryonic day (ED) 10.5, ED11.5, and ED12.5. Expression fell by ED13.5 and was not detectable at ED14.5. These observations demonstrate that the expression of fascin-1 is correlated with the migration activity of hepatoblasts during the early stages of liver development in rats. This study was supported in part by grant-in-aid for scientific research from the Japan Society for the Promotion of Science and by a grant from the Smoking Research Foundation, Japan.     

α-Gustducin immunoreactivity in the airways

The G-protein subunit -gustducin is a marker of chemoreceptive cells. In the present study, we examined the immunohistochemical localization of -gustducin in rat airway epithelium both by light and electron microscopy. -Gustducin immunoreactivity was found in solitary cells that presented ultrastructural features of chemoreceptor cells, i.e. flask-shaped or pear-shaped, with an apical process with thin microvilli protruding into the lumen. The immunostaining was mainly concentrated in the apical process and along the basolateral cell surface. To investigate whether -gustducin-immunoreactive cells represented a distinct cell subset in rat airways, we performed double-label immunocytochemistry with antibodies to protein gene groduct (PGP) 9.5, a marker of neuroendocrine cells, and to phospholipase C beta2 (PLC2), a component of the bitter signalling pathway. -Gustducin-immunoreactive cells were present in a subset of PGP-9.5-immunoreactive elements, although not all -gustducin-positive cells expressed PGP 9.5 labelling. In addition, a subset of -gustducin-expressing cells colocalized PLC2. This work thus demonstrates that solitary -gustducin-immunoreactive cells exist throughout the airways and represent a specialized cell type with morphological and immunohistochemical characteristics of chemoreceptor cells.