Cytochemical analysis of intracellular calcium distribution in the anterior pituitary of the rat

Summary In an attempt to assign morphologic identities to previously distinguished functional calcium compartments in the anterior pituitary of the rat, we employed the potassium pyroantimonate technique for cation localization. Tissues were incubated for In at 37°C in control medium; with 10mM theophylline; or with depolarizing amounts of potassium. Precipitate was quantified on photomicrographs of tissue prepared for electron microscopy with a Talos Systems Digitizer. The nature of the electron dense precipitate was dependent on the experimental state of the tissue. Treatment with 5 mM EGTA abolished the dense precipitate. Electron microprobe analysis also confirmed that calcium was the predominant cation in the observed precipitate. The most significant changes in precipitate deposition occurred along the plasma membrane, the limiting membrane of secretory granules and within mitochondria. Dense precipitate was present along the plasma membrane only in cells treated with potassium. Control tissue exhibited higher levels of precipitate associated with the limiting membrane of secretory granules than either theophylline-treated or potassium-treated tissue. Mitochondria contained more precipitate in potassium-treated tissue than in controls; the mitochondria of theophylline-treated tissue contained intermediate levels of precipitate. Addition of either theophylline or depolarizing amounts of potassium has been associated with hormone secretion in anterior pituitary tissue of normal rats. Kinetic studies in our laboratory indicate that intracellular calcium shifts occur. The pyroantimonate technique is useful in verifying morphologically the calcium compartments involved in shifts in intracellular calcium.     

A microprobe analysis of Gomori-positive glial cells in the rat arcuate nucleus.

Astrocytes and microglial cells in the arcuate nucleus of the rat hypothalamus contain lipofuscin-like granules which react with chrome alum gallocyanin and exhibit endogenous peroxidase activity. These granules were assessed with energy dispersive X-ray microanalysis and compared to neuronal dense bodies and glial cytoplasm. The granules are distinguished by a consistent content of sulphur and a frequent presence of calcium. The localization of other elements such as iron, copper, potassium and chlorine is impaired by methodical difficulties. The sulphur content as well as the endogenous peroxidase activity is interpreted as indicating a special variant of lipofuscin. The presence of calcium is discussed with respect to recent concepts of glia as a regulator of the ionic environment of the CNS.     

A microprobe analysis of Gomori-positive glial cells in the rat arcuate nucleus

Summary Astrocytes and microglial cells in the arcuate nucleus of the rat hypothalamus contain lipofuscin-like granules which react with chrome alum gallocyanin and exhibit endogenous peroxidase activity. These granules were assessed with energy dispersive X-ray microanalysis and compared to neuronal dense bodies and glial cytoplasm. The granules are distinguished by a consistent content of sulphur and a frequent presence of calcium. The localization of other elements such as iron, copper, potassium and chlorine is impaired by methodical difficulties. The sulphur content as well as the endogenous peroxidase activity is interpreted as indicating a special variant of lipofuscin. The presence of calcium is discussed with respect to recent concepts of glia as a regulator of the ionic environment of the CNS.     

Identification of organelles in bacteria similar to acidocalcisomes of unicellular eukaryotes

Acidocalcisomes are acidic calcium storage compartments described in several unicellular eukaryotes, including trypanosomatid and apicomplexan parasites, algae, and slime molds. In this work, we report that the volutin granules of Agrobacterium tumefaciens possess properties similar to the acidocalcisomes. Transmission electron microscopy revealed that each intracellular granule was surrounded by a membrane. X-ray microanalysis of the volutin granules showed large amounts of phosphorus, magnesium, potassium, and calcium. Calcium in the volutin granules increased when the bacteria were incubated at high extracellular calcium concentration. Immunofluorescence and immunoelectron microscopy, using antisera raised against peptide sequences conserved in the A. tumefaciens proton pyrophosphatase, indicated localization in intracellular vacuoles. Purification of the volutin granules using iodixanol density gradients indicated a preferential localization of the pyrophosphatase activity in addition to high concentrations of phosphate, pyrophosphate, short- and long-chain polyphosphate, but lack of markers of the plasma membrane. The pyrophosphatase activity was potassium-insensitive and inhibited by the pyrophosphate analogs, amynomethylenediphosphonate and imidodiphosphate, by dicyclohexylcarbodiimide, and by the thiol reagent N-ethylmaleimide. Polyphosphate was also localized to the volutin granules by 4',6'-diamino-2-phenylindole staining. The organelles were acidic, as demonstrated by staining with LysoSensor blue DND-167, a dye especially used to detect very acidic compartments in cells, and cycloprodigiosin, a compound isolated from a marine bacterium that has been shown to uncouple proton pyrophosphatase activity acting as a chloride/proton symport. The results suggest that acidocalcisomes arose before the prokaryotic and eukaryotic lineages diverged.     

Histochemical, ultrastructural and X-ray microprobe analytical studies of localization of calcium in the mucous lining of the rat duodenum

Localization of calcium in a rapid frozen and freeze substituted duodenum of normal, starved or calcium-repleted rat was examined using either of the glyoxal bis(2-hydroxyanil) (GBHA) staining method, a sensitive histochemical calcium stain or electron microscopy. In normally-fed rats, a majority of absorptive cells of the duodenum showed numerous discrete red granular GBHA reactions, approximately 1 micron or less in diameter, located primarily along their lateral plasma membranes and within intercellular spaces. Electron microscopy also revealed electron-dense granules, 30-100 nm in diameter, showing a similar distribution as the GBHA granules in the respective absorptive cells, and confirmed their absence in mitochondria and other intracellular compartments. Some of the absorptive cells located exclusively at the tip of each villus contained highly GBHA-reactive tubulo-vesicular structures extending throughout the cytoplasm. However, they displayed virtually no granular GBHA reaction. In these cells, electron microscopy revealed numerous electron-dense granules in the nucleus, mitochondria and in other unidentified organelles. X-ray microprobe analyses of ultrathin sections confirmed the presence of calcium within electron-dense granules associated with both types of absorptive cells. The number and intensity of all GBHA reactions fluctuated according to luminal calcium concentration. In calcium-repleted rats, strong GBHA reactions appeared in a narrow zone of lamina propria at the tip of the villus, overlaid, predominantly, with absorptive cells showing tubulo-vesicular GBHA reactions. These results suggest the existence of distinct types of absorptive epithelial cells in the rat duodenum, with respect to patterns of calcium localization which they display.     

Histochemical,ultrastructural and X-ray microprobe analytical studies of localization of calcium in the mucous lining of the rat duodenum

Summary Localization of calcium in a rapid frozen and freeze substituted duodenum of normal, starved or calciumrepleted rat was examined using either of the glyoxal bis(2-hydroxyanil) (GBHA) staining method, a sensitive histochemical calcium stain or electron microscopy. In normallyfed rats, a majority of absorptive cells of the duodenum showed numerous discrete red granular GBHA reactions, approximately 1 m or less in diameter, located primarily along their lateral plasma membranes and within intercellular spaces. Electron microscopy also revealed electron-dense granules, 30–100 nm in diameter, showing a similar distribution as the GBHA granules in the respective absorptive cells, and confirmed their absence in mitochondria and other intracellular compartments. Some of the absorptive cells located exclusively at the tip of each villus contained highly GBHA-reactive tubulo-vesicular structures extending throughout the cytoplasm. However, they displayed virtually no granular GBHA reaction. In these cells, electron microscopy revealed numerous electron-dense granules in the nucleus, mitochondria and in other unidentified organelles. X-ray microprobe analyses of ultrathin sections confirmed the presence of calcium within electron-dense granules associated with both types of absorptive cells. The number and intensity of all GBHA reactions fluctuated according to luminal calcium concentration. In calcium-repleted rats, strong GBHA reactions appeared in a narrow zone of lamina propria at the tip of the villus, overlaid, predominantly, with absorptive cells showing tubulo-vesicular GBHA reactions. these results suggest the existence of distinct types of absorptive epithelial cells in the rat duodenum, with respect to patterns of calcium localization which they display.     

Ultrastructural localization of calcium around the membrane of the surface connected system in the human platelet.

The localization of calcium in the membrane system of human platelets was determined by ultrahistochemical methods equipped with an electron probe x-ray microanalyzer. After potassium oxalate-glutaraldehyde treatment large amounts of electron opaque precipitates were observed around the membrane of the surface connected system. Electron probe x-ray microanalysis clearly defined that the precipitates were composed of calcium oxalate. The localization of calcium on the membrane of the surface connected system was also confirmed even after treatment of the platelets with potassium antimonate-OsO4. These results support a model which depicts the surface connected membrane system taking part in the store and the transport of calcium.     

Ultrastructural localization of calcium around the membrane of the surface connected system in the human platelet

Summary The localization of calcium in the membrane system of human platelets was determined by ultrahistochemical methods equipped with an electron probe x-ray microanalyzer. After potassium oxalate-glutaraldehyde treatment large amounts of electron opaque precipitates were observed around the membrane of the surface connected system. Electron probe x-ray microanalysis clearly defined that the precipitates were composed of calcium oxalate. The localization of calcium on the membrane of the surface connected system was also confirmed even after treatment of the platelets with potassium antimonate-OsO₄. These results support a model which depicts the surface connected membrane system taking part in the store and the transport of calcium.This investigation was supported in part by the Mitsubishi-Foundation, 1976 to Professor V. Mizuhira     

Intracellular localization of serotonin in mast cells of the colon in normal and colitis rats

Intracellular localization of serotonin (5-HT) in the mast cells of two phenotypes in normal rat colon and dextran sodium sulphate-induced colitis was studied by immunoelectron microscopy with a quantitative analysis of the distribution of immunogold labelling. Mucosal mast cells in normal rats contained round shape secretory granules with varying electron density. Immunogold labelling for 5-HT was concentrated over the secretory granules. In mucosal mast cells from colitis rats, vacuolated granules without 5-HT labelling were frequently observed and immunogold labelling over the secretory granules was significantly increased compared to controls. On the other hand, connective tissue mast cells in normal rats contained oval shape secretory granules with homogeneous electron density. Their immunogold labelling was diffusely scattered over the secretory granules as well as over the cytoplasm. In connective tissue mast cells from colitis rats, secretory granules with high electron density were increased and the immunogold labelling over the secretory granules was much higher than that in controls. The present results suggest that intracellular localization of 5-HT is different in two phenotypes of mast cells and they may release 5-HT in a different manner. Mucosal mast cells may release 5-HT by a degranulation or exocytosis, while connective tissue mast cells may release 5-HT by a diacrine manner of secretion.     

Cytochemical studies on the localization and functional properties of calcium in anterior pituitary cells

The localization of calcium and its functional properties in anterior pituitary cells were studied using a potassium pyroantimonate technique. In all kinds of secretory cells, the precipitates of the calcium-pyroantimonate complex were distributed on the limiting membrane of the secretory granule. They were present also in the cytoplasmic matrix, the mitochondrial matrix, small smooth vesicles, coated vesicles, and in the nuclear euchromatin area. The precipitates were usually seen at the contact region between the limiting membranes of two adjacent secretory granules, or between the granule limiting membrane and the plasma membrane. When the tissues were incubated in the medium containing A23187 (10 microM) for 5 min, the deposits on the granule limiting membrane were increased in number and those on the mitochondrial matrix were decreased; the reaction products almost disappeared on the limiting membranes of the secretory granules after membrane fusion following single or multigranular exocytosis induced by A23187-treatment. In addition, small vesicles in the capillary endothelium contained reaction precipitates. Based on these results we propose a hypothetical model for the relationship between the localization of calcium and secretory activity.     

Distribution of calcium ions in the muscle fibers of the rat diaphragm depending upon their state and the method of histochemical treatment

Distribution of calcium ions in the rat diaphragm muscle fibers has been studied electron histochemically using various fixation techniques and chemical treatment of the tissue. When potassium pyroantimonate in water solution is used after a short perfusate fixation with aldehydes, the reaction product granules are revealed in mitochondria, in the disk I, in the center of the disk A, more seldom the precipitate is found in the sarcoplasmic reticulum (SR) and in the T-system. The presence of calcium ions in the precipitate is proved by means of treatment the preparations with ethylenglycol- and ethylen-diamine-tetra-acetic acids. When contracture is resulted from potassium rhodanide administration, in mitochondria the reaction product granules decrease in their number, the precipitate disappears from the central part of the disk A, while the number of the granules increases in the SR terminal cisterns. The data obtained are compared with calcium ions distribution observed at the freezing-substitution method without an additional chemical fixation, as well as the histochemical fixations after Oschman method and at a usual fixation with OsO4. Certain similarity is revealed in distribution of the calcium pyroantimonate granules at aldehyde fixation and when the freezing-substitution method is used.     

Schistosoma mansoni: localization of calcium-detecting reagents in electron-lucent areas of specific preacetabular gland granules

In an attempt to establish the exact location of calcium within the preacetabular glands of cercariae of Schistosoma mansoni, these larvae were exposed to reagents (potassium oxalate, potassium pyroantimonate, chloranilic acid, and silver nitrate) useful in the detection of calcium, and were subsequently observed with the aid of light and electron microscopes. Cercariae incubated in potassium oxalate and viewed in polarized light showed birefringence only in the preacetabular gland funduses. At the ultrastructural level, the preacetabular glands of potassium oxalate-treated cercariae had no electron-dense precipitate, but instead had translucent, irregularly shaped inclusions, similar to spaces left by volatilized calcium oxalate as described by others. Pyroantimonate treatment, on the other hand, localized the reaction in the electron-lucent areas of the light-spotted granules. The von Kossa silver nitrate procedure destroyed the secretory granules; therefore, an electron-dense precipitate was distributed throughout the gland. However, pretreatment with chloranilic acid before fixation preserved the granules, and subsequent exposure to the von Kossa silver nitrate gave a reaction identical to that obtained with the pyroantimonate alone. When viewed in polarized light, chloranilic acid-incubated cercariae showed birefringence in the fundus and duct areas.     

Ultrastructural localization of potassium and calcium in an insect ommatidium as demonstrated by X-ray microanalysis

Summary Ultrastructural localization of potassium and calcium in the ommatidium of the house-cricketGryllus domeslicus L. was studied by X-ray microprobe analysis using samples prepared as thin sections (2 or 5 m) of freeze-dried and embedded tissue. Real resolution was limited by the size of ice crystals (Fig. 2) and estimated as about 1 m.Average values for potassium, calcium, sodium and phosphorus in different cells of the compound eye are given in Table 1.Striking non-uniformity in distribution of these elements over the cells and their compartments was found by probe scanning (Figs. 3, 4, 5). The highest potassium and calcium concentrations were measured in the pigmented zones of photoreceptors and pigment cells. The pigment granules are thought to be the ionic depots of the eye.Potassium and sodium are fully accessible to water in sections of embedded tissue, whereas all the calcium and half of the phosphorus are not.The functional significance of the non-uniformity discovered is briefly discussed.     

Histochemical and electron probe analysis of secretory ameloblasts of developing rat molar teeth

Calcium was not found in secretory ameloblasts and stratum intermedium cells when treated with OsO4-pyroantimonate or when surfaces prepared by fracturing fresh, rapidly frozen, developing molar tooth germs were subject to electron probe X-ray analysis. Pyroantimonate reaction product, considered to be calcium, was found in mitochondria of enamel organ cells which were first placed in a bath containing calcium and potassium. The plasma membrane was disrupted in cells ehich showed mitochondrial localization of reaction product. The results provide no data which indicates that enamel organ cells have a direct, active role in the movement of calcium into the enamel. Rather, it is suggested that the secretory enamel organ might serve as a selective barrier in regulating the initial mineralization of enamel.     

Cytochemical studies on the localization and functional properties of calcium in anterior pituitary cells

Summary The localization of calcium and its functional properties in anterior pituitary cells were studied using a potassium pyroantimonate technique. In all kinds of secretory cells, the precipitates of the calcium-pyroantimonate complex were distributed on the limiting membrane of the secretory granule. They were present also in the cytoplasmic matrix, the mitochondrial matrix, small smooth vesicles, coated vesicles, and in the nuclear euchromatin area. The precipitates were usually seen at the contact region between the limiting membranes of two adjacent secretory granules, or between the granule limiting membrane and the plasma membrane. When the tissues were incubated in the medium containing A23187 (10 M) for 5 min, the deposits on the granule limiting membrane were increased in number and those on the mitochondrial matrix were decreased; the reaction products almost disappeared on the limiting membranes of the secretory granules after membrane fusion following single or multigranular exocytosis induced by A23187-treatment. In addition, small vesicles in the capillary endothelium contained reaction precipitates. Based on these results we propose a hypothetical model for the relationship between the localization of calcium and secretory activity.This study was supported by grants from the Japan Ministry of Education     

Histochemical and electron probe analysis of secretory ameloblasts of developing rat molar teeth

Summary Calcium was not found in secretory ameloblasts and stratum intermedium cells when treated with OsO₄-pyroantimonate or when surfaces prepared by fracturing fresh, rapidly frozen, developing molar tooth germs were subject to electron probe X-ray analysis.Pyroantimonate reaction product, considered to be calcium, was found in mitochondria of enamel organ cells which were first placed in a bath containing calcium and potassium. The plasma membrane was disrupted in cells which showed mitochondrial localization of reaction product.The results provide no data which indicates that enamel organ cells have a direct, active role in the movement of calcium into the enamel. Rather, it is suggested that the secretory enamel organ might serve as a selective barrier in regulating the initial mineralization of enamel.     

应急大鼠肾上腺髓质嗜铬细胞颗粒数目与其钙含量的关系

采用电镜细胞立体形态计量法及电镜Ｘ射线显微定量分析术,对制动应急大鼠的肾上腺髓质细胞内嗜铬颗粒数密度和颗粒内Ｃａ浓度变化进行测量。结果显示,两者在制动过程中均呈进行性下降,但颗粒内钙浓度的下降快于颗粒数目的减少（Ｐ〈０．０１）。该结果支持颗粒内钙释放入胞质,参与胞质内游离钙浓度的升高,进而激发颗粒胞吐的假设,为主宰嗜铬颗粒是一种细胞内钙库并参与细胞分泌提供了实验依据。     

Intranuclear silicon detection in a subcutaneous connective tissue cell by energy-dispersive x-ray miscroanalysis using fresh air-dried spread.

Silicon was detected by energy-dispersive x-ray microanalysis in the nucleus of a subcutaneous connective tissue cell of mice fed normally. To eliminate contamination, pieces of connective tissue were spread on copper grids and examined without any treatment by an energy-dispersive spectrometer with a scanning transmission apparatus attached to an electron microscope. Scanning transmission electron microscopy of the spread has demonstrated a well-preserved ultrastructure. Fibrous structures, nuclei and nucleoli of cells and mitochondrial granules were recognized. Electron probe analysis showed peaks for silicon at three spots on the nucleus of a cell in addition to those for phosphorus, sulfur, chlorine, potassium and calcium, whereas no peak of silicon could be detected at the spots on nuclei of other cells, mitochondrial granules and electron-lucent area on the same grid as the above. Silicon appears to play a significant role in the nucleus. Applicability of the technique to know the distribution of easily contaminating elements and diffusible substances is shown.     

Immunocytochemical localization of cathepsin B in rat anterior pituitary endocrine cells, with special reference to its co-localization with renin and prorenin in gonadotrophs

We examined by immunocytochemistry the localization of cathepsin B in endocrine cells of rat anterior pituitary lobe, using a monospecific antibody to cathepsin B. By light microscopy, granular immunodeposits for cathepsin B were detected in most endocrine cells of anterior pituitary lobe. Cells immunoreactive for luteinizing hormone (LH) were diffusely immunostained by anti-cathepsin B. By electron microscopy, immunogold particles for cathepsin B were localized in lysosomes of thyrotrophs, somatotrophs, and mammotrophs. In mammotrophs, immunogold particles for cathepsin B were also detected in crinophagic bodies. Double immunostaining co-localized immunogold particles for LH and cathepsin B in secretory granules of gonadotrophs. Immunocytochemistry was also applied to demonstrate localization of renin and prorenin in LH-producing gonadotrophs; immunogold particles for renin were co-localized with those for LH, cathepsin B, or prorenin in their secretory granules. Immunogold particles for prorenin were also co-localized with those for LH or cathepsin B in secretory granules, but prorenin-positive granules appeared less frequently than renin-positive granules. These results suggest that cathepsin B not only plays a role in the protein degradation in lysosomes of anterior pituitary endocrine cells but also participates in the activation of renin in gonadotrophs, as has been demonstrated in secretory granules of juxtaglomerular cells.     

Localization of calcium in the cyanobiont and gonidial zone ofCycas revoluta Thunb. by microelectrodes,chlorotetracycline, electron spectroscopic imaging and electron energy loss spectroscopy

Summary Ionic calcium concentration was measured in the gonidial zone of fresh coralloid roots by means of calcium microelectrodes. It was 10⁻⁶ M in the apical segments of coralloid roots and increased to 10⁻⁵ M in the gonidial zones of median and basal segments. Loosely membrane-bound calcium was evidenced by using chlorotetracycline (CTC) or ethylene glycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) and CTC, in cell walls of columnar cells ofCycas and in the cytoplasm of cyanobiont. Sub-cellular localization of calcium was obtained by electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS) analyses applied at transmission electron microscopy on thin, unstained sections of gonidial zone of coralloid roots. By means of these techniques, bound-calcium was detected inside the mucilage of apical and median segments whereas, in the basal segments, it was completely absent. In the heterocysts of apical segments of coralloid, calcium was localized on the envelope, cell walls, thylakoids and cyanophycin granules. In the gonidial zone of the basal segments, dead or degenerating heterocysts completely lacked calcium. Therefore, the high ionic calcium amounts detected in the gonidial zone of median and basal segments could represent a minor calcium uptake by the cells or release by lysed ones. The decreases in nitrogenase activity recorded in the median and basal segments of the coralloid roots paralleled the decrease in calcium amount in heterocyst envelope.