Mucosubstances of rabbit granulocytes studied by means of electron-microscopic radioautography and X-ray microanalysis

Summary Rabbit bone marrow cells have been studied by means of light-and electron-microscopic radioautography and X-ray microanalysis. Carrier free sulfuric acid was used as the radioactive precursor in this experiment. Immature granulocytes showed more active incorporation than mature ones. Silver grains were observed in the Golgi apparatus and granules in three kinds of granulocytes. Electron-microscopically, immature granules showed the incorporation of inorganic sulfate, while mature ones did not. Sulfur was detected in all kinds of granules of the three granulocyte types by X-ray microanalysis. It is concluded that incorporated inorganic sulfur may be utilized for the synthesis of acid glycosaminoglycans and the sulfur detected by X-ray microanalysis may be that contained in the acid glycosaminoglycan. The sulfur detected in the specific granules of the heterophil probably derives from proteins or polypeptides incorporating sulfur containing amino acids.     

Ion transport in colon cancer cell cultures studied by X-ray microanalysis.

Three colon cancer cell lines (Colo 205, HT29 and T84) were investigated by X-ray microanalysis with respect to elemental composition and the effect of cAMP on the cellular concentrations of Na, K, and Cl. The cultures were not homogeneous with respect to their elemental composition, but appeared to consist of two sub-groups, low-K cells and high-K cells. In all three cell lines, the low-K cells had, in addition, higher Ca, markedly lower Cl, and somewhat lower P and S concentrations. Differences in Na and Mg concentrations were absent or not consistent. Exposure of cells to cAMP caused a decrease of the cellular Cl and K content in high-K (high-Cl) cells. Changes in Na were not significant. No difference between the three cell lines could be noted. Incubation of the cells with phorbol myristate acetate (PMA), which has been shown to down-regulate the expression of the cystic fibrosis (CF) transmembrane conductance regulator gene and thus confer CF-like characteristics on the cells, significantly decreased the response in the cellular Cl concentration to cAMP stimulation. It is concluded that cAMP initially activates predominantly the apical Cl- channel and the basolateral K+ channel.     

Post-mortem elemental redistribution in rat studied by X-ray microanalysis and electron microscopy

Summary Post-mortem elemental redistribution in various tissues from rat was studied by means of electron probe X-ray microanalysis, and correlated with morphological changes in these tissues. Pancreas, liver and cardiac muscle were removed from the animal either immediately, or after some hours after death. Elemental distribution at the cellular level was studied by X-ray microanalysis of thick cryosections. Calcium redistribution at the subcellular level was studied using tissue fixed with glutaraldehyde/oxalate. In all tissues, post-mortem redistribution of electrolytes had taken place within 2 h. The cellular concentrations of Na, Cl and Ca increased markedly, those of Mg and K decreased; no significant changes were found in the concentrations of P and S. The number of oxalate precipitates (indicating the presence of calcium) increased both in the mitochondria and in the cytoplasm and endoplasmic reticulum, reaching a maximum at 2 h. Morphological changes included mitochondrial swelling and vesiculation of the endoplasmic reticulum. Since the post-mortem ion shifts are similar to those encountered in some diseases and types of cell injury, great care has to be taken in the interpretation of X-ray microanalytical results from autopsy material.     

Subcellular distribution of zinc in rat prostate studied by X-ray microanalysis. I. Normal prostate

Synopsis Zinc is distributed subcellularly throughout the lateral prostate of the rat in both the stromal and epithelial elements. The connective tissue appears to be a major store of zinc. Within the epithelium, the highest concentrations of the element are found in the lysosomes, nucleoli, nuclear chromatin, secretory granules and luminal secretion. Histochemical studies indicate that the metal is bound relatively tightly within the nucleoli (associated with RNA) and in the secretory products of the cytoplasm. Changes in tissue zinc concentration, observed by other workers, following changes in various external stimuli, may not necessarily be reflected by proportionate changes in epithelial concentrations. The role of zinc in the epithelium is considered to be at least two-fold: firstly, for incorporation into vital cellular mechanisms necessary for cell maintenance and, secondly, for involvement in secretory products. It is also possible that the metal participates in the physiology of the sub-epithelial stroma.     

Changes in elemental content during apoptotic cell death studied by electron probe X-ray microanalysis

Recent data suggest that changes in ionic content, primarily potassium, play a pivotal role in the progression of apoptosis. However, the changes in total element content, i.e., sodium (Na), magnesium (Mg), phosphorous (P), chlorine (Cl), potassium (K), and calcium (Ca), during apoptosis have not been evaluated. Electron probe X-ray microanalysis (EPXMA) was used to measure total element content in U937 cells before and after the induction of apoptosis. As an experimental model we used U937 cells irradiated with ultraviolet (UV) light. Apoptosis was evaluated with phase-contrast microscopy, with scanning and transmission electron microscopy, and with the fluorescent dye bisbenzimide (Hoechst 33342). Plasma membrane permeability as a measure of cell death was determined by trypan blue dye exclusion. To investigate element content with EPXMA, cells were cryoprepared, i.e., cryofixed and freeze-dried, and analyzed as whole cells using a scanning electron microscope. We found that the UV irradiation induced rapid (within 2 h) morphological changes associated with apoptosis, such as plasma membrane blebbing, condensation of the chromatin, and the formation of membrane-bound apoptotic bodies. At this time, 95% of the apoptotic cells excluded trypan blue dye. EPXMA results demonstrated that UV light-irradiated apoptotic cells (cells with membrane-bound apoptotic bodies) had a lower Cl content (P < 0.001) and K content (P < 0.001) and a higher Na content (P < 0.001) in comparison with nonirradiated control cells. Also, P and Ca content was higher in apoptotic cells than in control cells, but this difference did not reach statistical significance. No differences were found in Mg. These data indicated that morphological changes characteristic of apoptotic cell death are related with significant changes in sodium, chlorine, and potassium content. In addition, we demonstrated that these changes in elemental composition were not associated with loss of cell membrane integrity.     

Chloride secretion in the submandibular gland of adult and early postnatal rats studied by X-ray microanalysis

Submandibular acinar cells of 1-day-old, 7-day-old, and adult rats were analyzed with X-ray microanalysis after stimulation with carbachol for different time periods (2–7 min). In unstimulated animals, marked differences in elemental content between compartments could be observed: secretory granules had a higher Ca and lower P and K content than other cell compartments. Comparison between different age groups showed significant differences for Ca, which increased with age in all compartments; Mg increased with age in the secretory granules and the apical cytoplasm. Only the glands from adult animals showed a significant effect of cholinergic stimulation: a transient decrease in Cl and K. The Cl concentration in the secretory granules decreased to 60% of the control value, which suggests that the granules release Cl upon stimulation. In young animals, no or little change in elemental distribution was observed after stimulation. This may indicate that Cl-secretion mechanisms are much less prominent in young animals. The ultrastructure of submandibular secretory granules depends on the preparation method: condensed and electrondense in freeze-substituted unfixed tissue, decondensed and more translucent in aldehyde-fixed tissue. This may indicate that the granules can transport water, and swell during the process of aldehyde fixation.     

The formation of type-I concretions in Drosophila Malpighian tubules studied by electron microscopy and X-ray microanalysis

There are two types of concretions in Drosophila Malpighian tubules: Type-I concretions originate in the distal segments of the anterior tubules, type-II concretions in the adjacent transitional segment between the apical microvilli. Type-I concretions are formed with the aid of carbonic anhydrase within intracellular vesicles, which migrate to the apical cell membrane where they are discharged into the lumen by exocytosis. The carbonic anhydrase inhibitors acetazolamide or hydrochlorothiazide prevent the formation of concretions by interruption of bicarbonate supply. In addition, the formation of concretions can be reduced by feeding with sodium cellulose phosphate.     

Mast cell origin studied by H3-thymidine radioautography

Reproduction of mast cells has been investigated by means of H3-thymidine radioautography in 26 mature mice of CC-57 White line and in 40 new-born nonlinear albino rats under normal conditions and experimental cold effect. It is established that differentiated mast cells incorporate H3-thymidine and can enter the phase of DNA synthesis of a proliferative cycle.     

Demonstration of endocytosis vesicles by means of electron-microscopic histochemistry

Summary A phosphatase activity similar to that of the plasma membrane is demonstrated at the inside of the membrane, which delimits the lipid-containing lysosomes of chicken liver parenchymal cells. This is taken as a definite confirmation of their endocytotic origin.It should be possible to demonstrate endocytosis vesicles in other cells in this way. It is, however, pointed out, that cells with a weak plasma membrane activity may require particular attention to certain steps in the technical procedure.This study was supported in part by a grant from the Swedish Medical Research Council.     

Protein synthesis in the livers of aging mice studied by electron microscopic radioautography.

The application of 3H-leucine results in labeling of the liver cells of mice in which protein is synthesized at various ages of the animals. Quantitative changes of protein synthesis in the hepatocytes of aging mice were studied by electron microscopic radioautography. The silver grains in the hepatocytes were mainly located over the rough surfaced endoplasmic reticulum, mitochondria, Golgi apparatus, cytoplasmic matrix, and a few over the nuclei. The number of silver grains in the cytoplasm and nuclei of the hepatocytes gradually increased after birth, reached the maximum at 1 month after birth, thereafter it continued to decrease with aging until the 24th month. The number of silver grains in the hepatocyte cytoplasm was more than that in nuclei at various ages. The number of silver grains in the rough surfaced endoplasmic reticulum and mitochondria gradually increased from embryo to 1 month after birth, thereafter it continued to decrease with aging until the 24th month. The number of silver grains in the Golgi apparatus showed almost no change from fetal stage to 6 months after birth, thereafter it continued to decrease with aging until the 24th month. The number of silver grains in the cytoplasmic matrix gradually increased from fetal stage to 2 months after birth, then decreased with aging until the 24th month. These changes reflect the quantity of protein synthesized in each cell organelle at various ages of animals.     

Proteoglycan-degrading enzymes of rabbit fibroblasts and granulocytes.

A neutral proteinase secreted by rabbit synovial fibroblasts in parallel with specific collagenase was partially purified by ion-exchange chromatography. At pH 7.6 this proteinase degraded 35S-labelled bovine nasal proteoglycan and azo-casein. The enzymic activity was inhibited by EDTA, 1,10-phenanthroline and serum, whereas di-isopropyl phosphorofluoridate and soya-bean trypsin inhibitor had little effect. By gel filtration the apparent mol.wt. of the enzyme was 25000. The fibroblast neutral proteinase was compared with the proteoglycan-degrading neutral proteinases of rabbit polymorphonuclear-leucocyte granules. Two distinct activities were found in the granules: one was inhibited by soya-bean trypsin inhibitor and the other by EDTA. The proteoglycan-degrading proteinases of rabbit fibroblasts and polymorphonuclear leucocytes at acid pH also were examined. Both cathepsin D and a thiol-dependent proteinase contributed to the degradation of proteoglycan at pH 4.5.     

Ion transport in primary cultures from human sweat gland coils studied with X-ray microanalysis

Chloride secretion in primary cultures of cells originating from the secretory coil of human sweat glands was investigated by electron probe X-ray microanalysis. The total intracellular Cl concentration was lowered by muscarinic agonists (carbachol and acetylcholine), as well as by the calcium ionophore A23187. The muscarinic agonists also lowered the cellular K concentration. Cl^- secretion induced by these agonists could be inhibited by the chloride channel blocker NPPB. After cAMP stimulation, the frequency distribution of the Cl concentration changed from Gaussian to bimodal, indicating that cAMP induces Cl^- secretion only from a subpopulation of the cells. Also ATP stimulated Cl^- secretion, indicating the presence of purinergic receptors. The results suggest that some of the cells in addition to Ca²⁺ -regulated Cl^- channels also possess cAMP-activated Cl^- channels. Hence, the primary cultures still possess the Cl^- transport mechanisms known to be present in intact glands. It can, however, not be excluded that some coil cells have acquired ductal characteristics during culture.     

Electron microscopic dry-mounting radioautography for diffusible compounds by means of ultracryotomy

Summary Cultured HeLa cells or mouse liver and pancreas tissues were labeled with ³H-thymidine, -uridine or -glycine for varying periods in vitro, frozen in liquid nitrogen and cut on an LKB ultrotome equipped with LKB Cryokit. Dry ultrathin sections were mounted on grid meshes and were either air-dried, freeze-substituted or freeze-dried, and were covered with dry films of radioautographic emulsions, exposed, developed, stained and were observed in electron microscopes.After a number of trials, it was possible to obtain fairly good preservation of both cell structure and radioisotopes by means of freeze-dried and drymounted ultrathin frozen sections. However, the results are not completely satisfactory at the present time.The outline of this paper was presented at the Symposium on Radioautography at the 5th International Congress of Histochemistry and Cytochemistry held in Bucharest, Romania, 30 August–2 September 1976     

The mechanism of fluid secretion in the rabbit pancreas studied by means of various inhibitors

In order to increase our understanding of the mechanism of pancreatic fluid secretion we have studied the effects of various transport inhibitors on this process in the isolated rabbit pancreas. In this preparation, a high rate of unstimulated fluid secretion occurs, which probably originates from the ductular cells. Inhibitory are ouabain, furosemide, bumetanide, piretanide, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and acetazolamide, with their half-inhibitory concentrations: 2 X 10(-6) M (ouabain), 1.3 X 10(-3) M (furosemide), 2.2 X 10(-3) M (bumetanide and piretanide) and 1.4 X 10(-4) M (SITS). With acetazolamide a maximal inhibition of only 20% is found at 10(-3) M. Amiloride (10(-3) M) has no effect on pancreatic fluid secretion. The inhibitory effects on HCO-3 output are always larger and those on Cl- output lower than those on fluid secretion. The results suggest that the ouabain-sensitive (Na+ + K+)-ATPase system provides the energy for a Na+-gradient-driven Cl--HCO-3-exchange transport system, sensitive to the loop diuretics furosemide, bumetanide and piretanide and to SITS. This system would drive the transcellular transport of HCO-3 and secondarily that of cations, Cl- and water.     

Innervation of the rabbit cornea. A histochemical and electron-microscopic study

The innervation of the rabbit cornea was investigated histochemically and electron-microscopically with special reference to the autonomic nerves. Both formaldehyde- and glyoxylic-acid-induced fluorescence methods revealed adrenergic nerves in the stroma; a few fibres were also observed between the basal epithelial cells near the limbus. Acetylcholinesterase- (AChE-) positive nerves were found both in the stroma and in the epithelium, whereas nonspecific cholinesterase (NsChE) activity appeared only in the stromal nerves. Under the electron microscope, both AChE and NsChE activities were observed to be located in the axon membranes. A weak NsChE reaction also appeared in the Schwann cells. When the specimens fixed with KMnO4 were examined under the electron microscope, most nerve fibres did not contain any special axoplasmic structures, although several axons contained mitochondria. Moreover, two vesicle-containing axon types were found in the stromal nerves; axons with small granular vesicles and axons containing small agranular vesicles. In the epithelium, two types of fibres were observed; one type containing only mitochondria while the other showed both agranular vesicles and mitochondria.     

X-ray microanalysis of black piedra

The elements present in the fungal structures produced by Piedraia hortae in vivo and in vitro have been investigated using electron microscopy X-ray microanalysis. Phosphorus, sulphur and calcium were detected in the nodules which developed on hair and on colonies on culture. These elements belong to the extracellular material that compacts the pseudoparenchymatous organization of the fungus. They may be present due to the capacity of melanin-like pigments to sequester ions and/or they may form part of the sulphates and phosphates of the polyanionic mucopolysaccharides that constitute the extracellular material. Environmental contaminants such as aluminium, silicon and iron were detected exclusively on the surface of the nodule. They were deposited or linked to the residual molecules produced during the breakdown of the cuticular keratin. The advantages of these techniques for elucidating the chemical nature of fungal structures are discussed.