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。     

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     

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。     

糖尿病大鼠心房肌细胞心房特殊颗粒和心房肽免疫反应强度的观察

本研究运用透射电镜及立体计量学方法结合免疫组化技术对糖尿病大鼠右心耳肌细胞心房特殊颗粒(ASG)和心房肽(ANP)免疫反应强度进行了观察和定量研究.实验动物为体重150～250克之间的SD大鼠,糖尿病的诱导采用四氧嘧啶(150mg/kg)右腹股沟区皮下注射.电镜超微结构发现糖尿病未治疗组均出现线粒体肿胀、肌原纤维缩短等病理性改变.ASG膜溶解、排空等现象多见,ASG体密度明显减少.免疫组化分析表明糖尿病未治疗组心肌细胞ANP免疫反应强度减弱.胰岛素治疗组各指标与对照组相比均无显著差别.     

ULTRASTRUCTURE OF UNICELLULAR N2 FIXING CYANOBACTERIA FROM THE TROPICAL NORTH ATLANTIC AND SUBTROPICAL NORTH PACIFIC OCEANS1

Nitrogen fixing unicellular marine cyanobacteria may have a major role in the global biogeochemistry of N; nevertheless, little is known about their phylogeny and morphology. We isolated N₂ fixing unicellular cyanobacteria from the tropical North Atlantic and subtropical North Pacific Oceans and examined ultrastructural dynamics during dark:light cycles when grown in incubators. The isolate from the subtropical North Pacific was larger and showed a size variation from 3 to 7 μm but had similar morphology and cell division‐plane characteristics as the isolate from the North Atlantic (2.5 μm). Nitrogen fixation only occurred during the dark phase, and ultrastructural analysis demonstrated changes in the appearance and quantity of large carbohydrate‐like granules present in the cells. To verify the composition of these carbohydrate‐like granules, staining with periodic acid, thioacetic acid, and silver was carried out, and a positive reaction was obvious in all cells. The cells from the Atlantic seemed to empty their polysaccharide granules during the night, whereas those from the Pacific showed a decrease in the number of their granules. Our work suggests that phylogenetically related strains of unicellular N₂ fixing cyanobacteria from different oceans showed similar carbohydrate‐like granules that could be used to fuel N₂ fixation during darkness.     

The physical characteristics of anaerobic granular sludges in relation to their internal architecture

A range of granular sludges was taken from industrial anaerobic sludge blanket reactors treating a wide variety of wastewaters and a comparison was made between the polymers which were extractable from the granules and their internal structures. The study of the internal structure, using sequential staining of ultra-thin sections, showed the complexity of granular sludges. Much of the area was occupied by Gram-negative cells and the area which stained positive for protein was found to increase nearer the centre of the granules. This was accompanied by a decrease in the carbohydrate positive areas. Positive areas for lipid were widespread throughout the granules. Changes in the internal structure were observed when the type of wastewater treated by the granules was changed and a comparison between sludges treating the same type of wastewater showed that factors other than the nature of the substrate must be considered as parameters which will affect the structure of the granules. Although an appreciable variation in the granule strengths was noted, it was not possible to relate these differences, on an overall basis, to either the internal structure or the chemical composition of the extracted polymers. However, an examination of data for granules produced during the treatment of nominally similar wastes did suggest that there would be a relationship between polymer composition and granule strength in these cases.     

Comparative ultrahistochemistry of the adepidermal granules of Salmo irideus, Lebistis reticulatus and Hynobiuo tokyoensis. Enzyme digestive experiment for the epoxy-embedded sections;.

A comparative ultrahistochemical investigation of the adepidermal granules of Salmo irideus, Lebistis reticulatus and Hynobius tokyoensis was carried out using enzyme digestion methods on epoxy-embedded sections. The granules of S. irideus larvae were decomposed by periodic acid, and digested by lipase without periodic acid pretreatmenetection of the granules. The secondary postosmificated granules were digested by lipase as in S; irideus, but complete decomposition by periodic acid was not observed in this experiment; Both periodic acid and lipase changed the shape of the adepidermal granules of H. tokyoensis to suggest partial digestion, but it appeared that these granules show rather stronger resistance to periodic acid and lipase than those of S. irideus. The granules of H. tokyoensis were completely digested when the sections were treated sequentially with phospholipase C, neuraminidase and lipase.     

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 specific granules of the rat heart: light microscopic staining and histochemical reactions.

An investigation was carried out to determine general staining and histochemical properties of rat atrial specific granules. It was found that these granules may be demonstrated using aldehyde fuchsin after pretreatments which involve oxidation or thiosulfation. This new way of demonstrating atrial granules is compared to other staining methods in terms of sensitivity and selectivity as well as to the nature of reactive groups that may be involved in the staining reactions. No lipid or carbohydrate were detected histochemically. Overall assessment of reactions suggests that atrial granules are a site of storage for a protein or polypeptide. Some of the tests indicate that these may contain tryptophan and sulfur-containing amino acids.     

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。     

Effect of colchicine on the intracellular transport of secretory proteins in rat liver parenchymal cells. Immunocytochemical observations

We studied the effects of colchicine on the intracellular transport of secretory proteins in rat liver parenchymal cells using the direct immunoenzyme technique. Livers were perfusion-fixed 0.5, 1, and 2 h after injection of colchicine. Vibratome sections of the fixed liver were stained using peroxidase-conjugated Fab' of anti-albumin or anti-fibrinogen. By light microscopy, reaction deposits showing albumin and fibrinogen were observed in the cytoplasmic granules of hepatocytes. Such stained granules decreased 30 min after injection, but later increased gradually and crowded in the cytoplasm. The Golgi complex stained for the proteins decreased after 30 min but increased in the juxtanuclear region after 60 min. The analysis of serial sections showed that colchicine severely disturbed the spatial relationship between the Golgi apparatus and the bile canaliculus. We obtained similar results by electron microscopy; a positive reaction for albumin and fibrinogen was observed in a small number of the cytoplasmic granules after 30 min. After 1 h of treatment, most of the Golgi complexes were fragmented and lost their stacked cisternae. However, they reappeared accompanied with vacuolated cisternae and secretory granules, which were partially stained for albumin and fibrinogen. After 2 h, the secretory granules positive for both proteins accumulated further. Some of them lined a long the plasma membrane, and others made a cluster in the cytoplasm. The profiles showing exocytosis were very rarely seen. These results showed that in the first 30 min, colchicine primarily disturbs partially the Golgi assembly but does not affect the post Golgi secretory pathway much. Later, the drug affects both the post Golgi pathway and the Golgi assembly, and it causes a marked accumulation of secretory granules.     

Vacuolar granules in Chlamydomonas reinhardtii: polyphosphate and a 70-kDa polypeptide as major components

The alga Chlamydomonas reinhardtii contains cytoplasmic vacuoles that are often filled with a dense granule that is released from the cell by exocytosis. Purified granules contained polyphosphate, complexed with calcium and magnesium, as the predominant inorganic components. Antiserum was raised against the major 70-kDa protein in granules purified from wall-deficient (cw15) mutants, which reacted on immunoblots with larger glycoprotein complexes in purified cell wall fractions from wild-type cells. Confocal fluorescence microscopy detected binding of these antibodies predominantly at the periphery of wall-containing C. reinhardtiiy1 cells but primarily to loci in the interior of cells of the cw15 strain. Immunoelectron microscopy demonstrated that the 70-kDa protein was localized in vacuolar granules and the trans-Golgi network in sections of cw15 cells but not in the cytosol or chloroplast. Treatment of cells with a dye, fluorescent in its protonated form, indicated that the pH within vacuoles was lower than that in the cytosol, which suggested that the vacuoles are similar to lysosomes. Thus, the vacuoles may serve a dual function to provide an environment for degradation within the cell and also serve as a vehicle for secretion of specific proteins. Received: 29 September 1999 / Accepted: 20 November 1999     

Different glycosphingolipid composition in human neutrophil subcellular compartments

The binding of a number of carbohydrate-recognizing ligands to glycosphingolipids and polyglycosylceramides of human neutrophil subcellular fractions (plasma membranes/secretory vesicles of resting and ionomycin-stimulated cells, specific and azurophil granules) was examined using the chromatogram binding assay. Several organelle-related differences in glycosphingolipid content were observed. The most prominent difference was a decreased content of the GM3 ganglioside in plasma membranes of activated neutrophils. Gangliosides recognized by anti-VIM-2 antibodies were detected mainly in the acid fractions of azurophil and specific granules. Slow-migrating gangliosides and polyglycosylceramides with Helicobacter pylori-binding activity were found in all acid fractions. A non-acid triglycosylceramide, recognized by Gal4Gal-binding Escherichia coli, was detected in the plasma membrane/secretory vesicles but not in the azurophil and specific granules.Although no defined roles of glycosphingolipids have yet been conclusively established with respect to neutrophil function, the fact that many of the identified glycosphingolipids are stored in granules, is in agreement with their role as receptor structures that are exposed on the neutrophil cell surface upon fusion of granules with the plasma membrane. Accordingly, we show that neutrophil granules store specific carbohydrate epitopes that are upregulated to the plasma membrane upon cell activation.     

Immunocytochemical localization of atrial natriuretic peptide in the venae cavae and the pulmonary veins of the rat

Summary In the present investigation atrial natriuretic peptide (ANP) was localized in striated myocytes of the venae cavae and the pulmonary veins in the rat by the use of immunohistochemical and immunocytochemical staining techniques. ANP was stored in granules which appeared to be morphologically similar to the atrial specific granules (ASG) of the atria. In general, the amount of ASG in the great thoracic veins was less than observed in the atria, and the specific granules appeared to be more evenly distributed throughout the sarcoplasm. However, the presence of ANP-containing specific granules in the venae cavae and the pulmonary veins may suggest participation of these veins in the production and secretion of the hormone.     

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.     

High-affinity binding sites for bombesin on mouse colonic mucosal membranes

Summary We examined the possibility that rat atrial granules may contain a pro-ANF processing protease. Isolated atrial granules were lysed either by detergent, osmotic shock or sonication and incubated at 37° C. Pro-ANF processing and/or degradation were followed by radioimmunoassays and Western blotting using three antibodies which are specific either to the N-terminus, the C-terminus or the processing site (98–99) of pro-ANF. Whatever the method used for the lysis of the granules, we failed to detect any production of ANF (99–I26) and ANF (1–98). However, slight degradation of pro-ANF was recorded probably due to contamination by lysosomal proteases. The in vitro system was validated by addition of thrombin to lysed granules which resulted in a rapid disappearance of the immunoreactivity related to the processing site. These results suggest that the rat atrial granules do not contain any active processing enzyme unless adequate incubation conditions were not met to express its enzymatic activity. The atrial granules may not be directly involved in the maturation of pro-ANF.     

Chromogranin A: Immunocytochemical localization in secretory granules of frog atrial cardiomyocytes

Chromogranin A (CgA) belongs to the granin family of acidic proteins that are present in the secretory granules of many endocrine, neuroendocrine, and nerve cells. CgA has been shown to be stored in cardiomyocyte secretory granules of the rat heart atrium together with atrial natriuretic peptide (ANP). CgA-derived peptides (vasostatins) are known to produce a cardiosuppressive effect on isolated and working in vitro frog and rat hearts. Recently, CgA-derived vasostatin-containing peptides have been identified in rat hearts, whereas no data are available so far about the presence of CgA in the frog heart. In our work, we have studied the subcellular CgA localization in atrial myocytes of the adult frog R. temporaria heart by using an ultraimmunocytochemical method. Immunocytochemical staining of the frog atrial tissue for CgA and ANP showed the presence of the CgA-immunoreactive material in two types (A and B) of large specific atrial secretory granules, whereas no gold particles were revealed over the small granules (D) with a high electron density core. Similar results were obtained during the immunocytochemical staining by an antibody to ANP of the drog atrial cardiomyocytes. The data of the present work allow for the suggestion that CgA revealed in frog atrial cardiomyocytes, like CgA in rat cardiomyocytes, can be considered to be a precursor of intracardial vasostatins that, together with ANP, can play an important cardioprotector role under conditions of stress.     

Ultrastructural cytochemistry of atrial muscle cells

Summary Sections of atrial cardiocytes from young rats were subjected to radioautography after a single intravenous injection of L-leucine-4,5 ³H to identify the sites of synthesis and to follow the migration of newly-formed proteins. As early as 2 min after injection of L-leucine ³H, the label was highest in the rough endoplasmic reticulum (RER), suggesting that cisternal ribosomes are sites of protein synthesis. By 5 min, most of the label had migrated from the RER to the Golgi complex. Some label was already present over specific granules by 2 min but the peak was reached at 1 h. By 4 h, the label over the specific granules had diminished, possibly indicating a release of newly-synthetized secretory material outside the cell. The label over myofilaments and Z-bands was relatively high at most time intervals, suggesting an early and important incorporation of leucine into the contractile and structural proteins of these organelles. The label over the cytosol was initially high and increased even further at 5 and 20 min but decreased to a very low level at 4 h. In contrast, the label over the cell surface rose continuously and peaked at 4 h. The pattern of increment of the label over the cell surface suggests that the newly-formed proteins of these sites are also synthetized in the RER, pass through the Golgi complex and are transported in the cytosol before reaching their destination.     

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

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

Differential Staining of Mucin Granules from Epoxy Resin Sections by a Phosphotungstic Acid-Methyl Green Procedure

After treatment of epoxy resin semithin sections from glutaraldehyde fixed rat large intestine with 5% aqueous phosphotungstic acid (PTA), staining with unpurified 0.2% solutions of methyl green at 60 C for 5 min produces a color differentiation between mucin granules of goblet cells. Some mucin granules and the glycocalyx appear deep green while the remaining granules, luminal mucin and collagen fibers are pink. The known contamination of unpurified methyl green with crystal violet seems to be responsible for the pink staining reaction of the latter structures, which also present an orange-red fluorescence under green exciting light. Electron microscopic observations show selective contrast of mucin granules which appear with a different amount of PTA deposits. This procedure is useful to reveal the heterogeneity of mucin granules in light and electron microscopy.