Distribution of elements in the pancreatic exocrine cells determined by electron probe X-ray microanalysis

We measured the intracellular electrolytes of acinar cells by making electron probe X-ray microanalysis of hydrated and dehydrated sections of freshly frozen dog pancreas.The concentrations of electrolytes in the cytoplasm were: Na 4.8±2.1, K 132±15, Cl 14±4.7, P 165±36, S 19±2.8 and in zymogen granules: Na 6±5, K 60±16, Cl 31±20, P 36±8, S 172±25, Ca 7±5 (mean±S.D. mmol/kg wet weight).The cytoplasm, which is rich in rough endoplasmic reticulum, had low Na and high K concentrations, as compared with levels in the acinar cells of other exocrine glands such as the submandibular gland, the bulk of which is occupied by secretory granules. Though the representative feature of secretory granules was a high S content, occasionally low S peaks of spectra from secretory granules were obtained. These findings may reflect the content of mature zymogen granules and immature condensing vacuoles.Pilocarpine stimulation increased cytoplasmic Na, Cl and Ca and decreased K levels in the pancreatic acinar cells. This indicates that secretory stimulation increases the permeability of the cell membrane to Na, Cl and K ions and that there is a simultaneous Ca release from the intracellular Ca stores such as zymogen granules and endoplasmic reticulum, and/or Ca influx from the extracellular space.     

Electrolyte redistribution in cystic fibrosis fibroblasts studied by electron probe X-ray microanalysis

The elemental composition of fibroblasts from cystic fibrosis (CF) patients and from healthy controls was compared by means of electron probe X-ray micro-analysis. Significantly lower sodium levels (p < .01) and higher calcium levels (p < .02) were found in CF fibroblasts, indicating a disturbance of ion regulation in these cells. Treatment with Staphylococcus aureus protein A and gammaglobulin (proteins that may influence the ciliotoxic CF factor) did not change the elemental composition of the fibroblasts.     

Effects of chronic furosemide treatment on rat exocrine glands

It has been suggested that a defective chloride transport is the primary cellular basis for the disease cystic fibrosis (CF). Therefore, the effects of chronic furosemide treatment on the structure and function of rat exocrine glands were investigated. X-ray microanalysis of the submandibular gland showed an increase in the cellular Ca and Mg concentrations, and a decrease in the cellular Cl concentration. Transmission electron microscopy showed intracellular accumulation of mucus and the presence of mucus in acinar and ductal lumina. The volume of saliva secreted by the submandibular gland after pilocarpine stimulation was markedly reduced in furosemide-treated animals; the salivary concentrations of Na and Ca were higher, and that of K was lower, than in control animals. The protein concentration in submandibular saliva was not significantly affected. The response of the submandibular gland to isoproterenol stimulation was reduced in furosemide-treated animals. In the parotid gland, chronic furosemide treatment caused an accumulation of immature zymogen granules in the acinar cells and a decrease in the cellular Cl concentration. In the pancreas, the acinar lumen was dilated and completely filled with secretory material, and the acinar cells contained less Na and somewhat less Cl than in control animals. The chronically furosemide-treated rat shows a number of parallels with other animal models for CF, in particular the chronically reserpinized rat. There is also agreement with the human disease itself.     

Subcellular elemental composition in isolated cardiac myocytes from rabbit following chronic potassium depletion and acute repletion, studied by X-ray microanalysis

X-ray microanalysis was used to estimate K, Na, Cl, Mg, P and S within the mitochondria and the myofibrillar regions of cardiac myocytes from rabbits, following dietary potassium depletion. K depletion caused no changes in mitochondrial K, but myofibrillar K was reduced. There were no changes in Na, Mg or Cl. Both areas showed increases in P but decreases in S. Acute K repletion resulted in a significant overshoot of K in both areas, coupled with a profound decrease in Cl. P was reduced in the mitochondria alone. These results may be partly explained by adaptive changes in Na+ pump activity.     

Effects of pituitary adenylate cyclase activating polypeptide-27 (PACAP) and vasoactive intestinal polypeptide (VIP) on chloride in HT29 cells studied by X-ray microanalysis

The colon cancer cell line HT29 is a useful model to study intestinal chloride secretion. These cells have both cAMP-activated and calcium-activated chloride channels. Changes in elemental content of the cells after stimulation with agonists were determined by X-ray microanalysis in the scanning or scanning transmission electron microscope. Exposure of HT29 cells to pituitary adenylate cyclase activating polypeptide-27 (PACAP) caused a transient decrease in the cellular Cl and K concentrations, indicating (net) efflux of chloride. The effect of PACAP is inhibited by somatostatin, which is known to inhibit cAMP-activated as well as calcium-activated chloride secretion and by U-73122, an inhibitor of phospholipase C. Alloxan, an inhibitor of adenylate cyclase, did not significantly affect the PACAP-induced loss of chloride. The calcium-chelating agent EGTA inhibited the PACAP-induced loss of chloride, indicating the need for extracellular calcium ions. Also vasointestinal polypeptide (VIP) caused a decrease of the cellular chloride concentration in HT29 cells. VIP-induced loss of chloride could be inhibited by pre-treating the cells with somatostatin or UK14,304, an alpha-2 adrenergic agonist that has been shown previously to inhibit purinergically activated chloride efflux. Our results indicate that there is cross-talk between the cAMP- and the calcium-activated pathways for chloride secretion in HT29 cells.     

Growth of human basophil lines derived from chronic myelocytic leukaemia cells in vitro: ultrastructure and X-ray microanalysis studies.

Mononuclear cells from the peripheral blood of patients with chronic myelocytic leukaemia were grown and expanded in liquid culture in the presence of Con A-conditioned medium. An accelerated development of cells of the basophilic lineage was observed and resulted in the appearance of 85% mature basophils after 14 days of incubation. Transmission electron microscopy of developing basophils showed changes in the nucleus and active granule formation in the cytoplasm. By scanning electron microscopy, the immature cells were relatively smooth in comparison with the mature basophils which showed membranous microvilli. The chemical content of the cells at different days of culture was detected by X-ray microanalysis. Immature cells were characterized by a high level of phosphorus with a low level of sulphur. As maturation progressed, the amount of phosphorus decreased, while the level of the sulphur increased, reaching its highest peak in the mature basophils. The different amount of sulphur found in the cells during the maturational process most probably represents the amount of heparin located in the cell granules. This finding may be useful for studying the influence of growth factors on the development and differentiation of human basophils.     

Quantitative X-ray microanalysis of alveolar macrophages after long-term treatment with amiodarone

Treatment with the iodine-containing antiarrhythmic drug, amiodarone, can cause pulmonary toxicity. Alveolar macrophages are particularly susceptible to formation of lipidrich lamellar bodies in amiodarone-treated animals. Amiodarone and several of its metabolites accumulate in the cell. Previously, we have reported that the technique of X-ray microanalysis is useful in monitoring the distribution of iodine in freeze-dried cryosections of alveolar macrophages from Fischer 344 rats 24 hr after a single dose of amiodarone. In the present study, we examine the effects of longer term amiodarone treatment of 1 or 9 weeks. Substantial changes in iodine distribution occur in the cells with increasing length of drug treatment. High concentrations of iodine are found early in the lamellar bodies. The iodine levels in the nuclei slowly increase with the length of treatment, and after 9 weeks of treatment, approach those found in the lamellar bodies. It is possible that this accumulation of iodine in the nuclei is due to the presence of polar metabolites. In addition, the potassium concentration in the cell decreases and the sodium increases with treatment duration. These changes in cations are most likely due to altered ion transport in the macrophages by the inhibition of membrane Na-K-ATPase by the drug and its principal metabolite, desethylamiodarone.     

Magnesium transport through the basal plasma membrane of larval malpighian tubules of Drosophila hydei studied by electron probe X-ray microanalysis.

Magnesium besides calcium is the most important excretion product. In the anterior Malpighian tubules of Drosophila, excretion of magnesium takes place via the hindgut by proteoglycan containing concretions. This study reports on magnesium transport through the basal plasma membrane of the principal cells of the proximal segment of the anterior Malpighian tubules. Measurements by electron probe X-ray microanalysis indicate the existence of two antiporters which transfer magnesium in still unknown stoichiometry from the hemolymph space into the cell: Mg/H and Mg/Na.     

Demonstration of iron and thorium in autopsy tissues by X-ray microanalysis

We performed x-ray microanalysis of autopsy specimens using a scanning-transmission electron microscopy mode. Tissues were obtained at necropsy from a patient with history of angiography using thorium dioxide and from a patient with hemochromatosis. X-ray microanalysis confirmed the presence of thorium and iron in their respective tissues. Effects of staining reagents were examined.     

X-ray microanalysis of rat mast cells stimulated with compound 48/80 in combination with quick-freezing method

X-ray microanalysis was performed on rat mast cells prepared by quick-freezing, cryosectioning and freeze-drying (QF-FD) method, or quick-freezing and freeze-substitution (QF-FS) method. Peritoneal cells including mast cells were stimulated with compound 48/80 for 0, 10 or 30 s at 17° C, and the mast cells stimulated for 30 s started exocytosis. In X-ray spectra of the QF-FD specimen, mast cells stimulated for 10 s increased their levels of phosphorus, sodium and chlorine in the intergranular cytoplasm prior to exocytosis, and kept this increase until 30 s after stimulation. In the QF-FS specimen, where soluble elements were removed, peaks of phosphorus, sulphur and potassium could be detected as elements in X-ray spectra. Phosphorus increased and potassium decreased in intergranular cytoplasm of mast cells stimulated for 10 s, and these changes became more obvious after 30 s. However, supplemental increase of other cations such as sodium could not be detected in the QF-FS specimens.     

Ultrastructural analysis of salivary calculus in combination with X-ray microanalysis

Ultrastructural studies of salivary calculi were performed. Scanning electron microscopic examination of the calculi revealed lamellar and concentric structures. Granular or globular structures and pyramid structures were found on the surface of the calculi, and in some cases a scaly structure corresponding to fiber and bacteria was recognized. X-ray microanalysis showed the main constitutes of the calculi to be Ca and P. Transmission electron microscopic examination revealed a fine fibrous structure near the degenerated organelles, and analyses of the structure by electron diffraction revealed hydroxyapatite. Calcification was found around the degenerative organelles in the form of lipid-like structures, mitochondria, lysosomes, and microbial structures. Judging from our results, as one of the processes leading to calculi formation, it is speculated that degenerative substances are emitted by saliva, by some phenomenon, and calcification around these substances then occurs, contributing to the formation of calculi.     

Rbp-j regulates expansion of pancreatic epithelial cells and their differentiation into exocrine cells during mouse development.

Notch signaling regulates cell fate determination in various tissues. We have reported the generation of mice with a pancreas-specific knockout of Rbp-j using Pdx.cre mice. Those mice exhibited premature endocrine and ductal differentiation. We now generated mice in which the Rbp-j gene was inactivated in Ptf1a-expressing cells using Ptf1a.cre mice. The timing of the Cre-mediated deletion in Rbp-j(f/f) Ptf1a.cre mice is 1 day later than that in Rbp-j(f/f) Pdx.cre mice. In Rbp-j(f/f) Ptf1a.cre mouse pancreases, at E13.5, the reduced Hes1 expression was accompanied by reduced epithelial growth, but premature endocrine cell differentiation was minimal. At E15.5, Pdx1 expression was repressed and acinar cell differentiation was reduced, but an increase in acinar cell proliferation was observed during the perinatal period. Our study indicates that, in addition to its role in preventing premature differentiation of early endocrine cells, Rbp-j regulates epithelial growth, Pdx1 expression, and acinar cell differentiation during mid-pancreatic development.     

Ca2+-induced excess capacitance fluctuation studied by phase-sensitive detection method in exocrine pancreatic acinar cells

A phase-sensitive detection method in combination with whole-cell voltage-clamp was applied to exocrine pancreatic acinar cells from rat. The results have shown that cell-aialysis with a solution containing 5×10^–7 M Ca²⁺ induces stp-wise changes in cell capacitance. The size distribution of increasing and decreasing capacitance steps is compatible with the idea that these capacitance steps are the results of individual granular fusion and retrieval events. This method may enable experiments studying the exo-and endo-cytotic mechanisms in morphologically polarized secretory cells.     

Mineral-associated hepatic injury: a report of seven cases with X-ray microanalysis.

The patterns of hepatic injury associated with various minerals were studied in seven patients. The subjects included one patient who was a sandblaster (silica by inhalation), one patient who was a dental laboratory technician (silica and chromium-cobalt alloy by inhalation), one patient with inhalational talcum powder abuse, and four chronic intravenous (IV) drug abusers (talc by IV injection). In all cases, the liver was examined by light and polarizing microscopy, and by scanning electron microscopy with energy-dispersive x-ray microanalysis. In the two patients with silica exposure, silica-containing sclerohyaline nodules were diffusely present in portal tracts and lobules. Both chromium-cobalt alloy and silica were present in the dental technician. In contrast, in all cases of talc exposure, aggregates of talc-laden macrophages were present in portal and centrilobular areas. Three IV drug abusers and the talcum powder abuser had histologic evidence of chronic hepatitis, most probably of viral etiology. We conclude that mineral type plays an important role in the pathogenesis and fibrogenesis of hepatic lesions. Compared with silica, talc primarily elicits a macrophage response without granuloma formation or fibrosis. Hepatic silicosis is a rare complication in dental laboratory technicians, and chromium-cobalt alloy may contribute to hepatic injury and fibrosis in this setting.     

Acute renal injury by tetraethyl orthosilicate in mice: ultrastructure, histochemistry and X-ray microanalysis.

Tetraethyl orthosilicate (Si(OC2H5)4, or TEOS) is a silicon-containing compound which has widespread industrial applications and which has been documented as biohazardous. The histopathological features and mechanism of TEOS toxicity in the kidney of ICR mice were investigated in a light and electron microscopy study, which included energy dispersive X-ray microanalysis. TEOS was given to mice as intraperitoneal injection of approximately 1,670 mg/kg body weight in experiments based on a 24 h time-scale. Tissues were examined after sampling either immediately on death if this occurred within 24 h or, in the case of surviving animals, after sacrifice at 24 h. Renal injury was considered to be the most probable cause of death, on the basis of the following main findings: 1) acute tubular necrosis (glomerular lesions were absent); 2) a dense deposit of silicon over the microvilli of dead tubular epithelial cells; 3) an abundant aggregation of hydroxyapatite crystals containing calcium in the cytoplasm and mitochondria of the dead tubular epithelial cells; and 4) abundant myelinosomes and some hydroxyapatite crystals in the cytoplasm of viable proximal convoluted tubule epithelial cells. It was speculated that silicon compounds may bind to the plasma membranes of the proximal convoluted tubule epithelial cell microvilli and damage or interfere with membrane calcium channels. The resulting calcium ion imbalance may play a role in the subsequent progression of acute tubular necrosis by TEOS.     

X-ray microanalysis of the secretory granules in the intestinal goblet cells of aging mice

The elemental changes in the goblet cell secretory granules of the mouse jejunum and ascending colon during postnatal development and aging were studied by a quantitative electron probe X-ray microanalysis on quick frozen and freeze-dried cryosections. In the jejunum, sulfur showed the highest peaks, followed by those for K, Cl, Ca, P and Na, respectively. In the colon, on the other hand, potassium showed the highest peaks, followed by those for S, Cl, Ca, P, Na respectively. Sulfur in the jejunum was at its lowest on postnatal day 14, as its highest at 1 month, and then gradually decreased with aging.     

Ultrastructural localization of calcium in the chick yolk sac membrane endodermal cells as revealed by cytochemistry and X-ray microanalysis

The yolk sac membrane (YSM) of the chick embryo transports calcium from the yolk into the embryonic circulation during the first half of development, but the intracellular pathway of calcium transport is poorly understood. In the present study, the ultrastructural localization of calcium was investigated in cells of the YSM of 9-day chick embryos. X-ray microanalysis as well as cytochemical techniques performed on yolk sac membrane cells treated with potassium oxalate, potassium ferricyanide and potassium antimonate demonstrated accumulation of calcium in yolk granules, digested yolk products, electron-dense bodies (EDBs; 100–400 nm diameter) and electron-dense granules (EDGs; 30–50 nm diameter). When strontium ions were injected into the yolk, they were incorporated into the endodermal cells and sequestered specifically in EDGs. From these results, we propose that calcium enters the endodermal cells by endocytosis of calcium-containing yolk granules, as well as through calcium channels in the apical cell membrane. In the cytoplasm, digested yolk products, EDBs, and EDGs act as sites of sequestration and accumulation of calcium. Extrusion of intracellular calcium into the extracellular space and embryonic circulation is accomplished by exocytosis of calcium-containing material and via an ion pump in the basal cell membrane.     

The surface of dendritic cells in the mouse as studied with monoclonal antibodies

A family of dendritic cells has been identified in situ and in vitro by microscopy and immunolabeling. The members of this family include the dendritic cells isolated from lymphoid organs, Langerhans cells [LC] of the epidermis, veiled cells in afferent lymph, and interdigitating cells [IDC] in the T-cell areas. Some common features to all members of the family are high levels of MHC class II antigens, a lack of most B and T cell markers, and an absence or low levels of macrophage/granulocyte antigens. This review summarizes the markers of mouse dendritic cells as assessed by a panel of monoclonal antibodies, and stresses a few recent findings. 1) In spleen, there are two populations of dendritic cells. More than 75% of isolated cells are 33D1+, NLDC145-, and J11d-, while the remainder have the reciprocal phenotype and thus share the NLDC145 antigen of IDC. Thymic dendritic cells, released by collagenase digestion, and epidermal LC also are 33D1-, NLDC145+, J11d+. 2) When epidermal LC are placed in culture, there are changes in cell function and phenotype. There is a decrease in Fc gamma receptors and the F4/80 macrophage antigen, an increase in class I and II MHC products and p55 IL-2 receptors, and persistence of the NLDC145 IDC antigen. The cultured LC thereby resembles the IDC. 3) A new antibody N418 shows that dendritic cells express the p150/90 member of the leukocyte beta 2 integrin family. Immunolabeling of tissue sections of spleen indicates that N418+ dendritic cells not only are present in the periarterial sheaths, the location of IDC, but also in "nests" at the periphery of the T area where 33D1 has been found. The peripheral collections interrupt the marginal zone of macrophages that separates white and red pulp, and places the dendritic cells in the path of T cells as they move through the white pulp. Therefore the members of the dendritic cell family have important markers in common, as well as differences that are associated with state of immunologic function and location.