Calcium and sulphur in neurosecretory granules and calcium in mitochondria as determined by electron microscope X-ray microanalysis

Summary Sections of neurosecretory cells fixed in glutaraldehyde and osmium tetroxide were studied by means of an EMMA-4 analytical microscope. Secretory granules in neurosecretory cells of the corpus cardiacum and of the brain, both in the desert locust Schistocerca and in the blowfly Calliphora, as well as neurosecretory granules in posterior pituitaries of the frog Rana and of the albino rat all contain a high concentration of calcium. A distinct sulphur peak was also a constant feature.In neurosecretory cells of the corpus cardiacum of Schistocerca the chromatin contained a high concentration of calcium. The mitochondria also contained much calcium, but part of this disappeared during preparation except when fixative and wash contained calcium chloride. By block staining with uranyl acetate most calcium is displaced from the mitochondria, whereas most of the calcium remains in the neurosecretory granules. Since the calcium peaks in spectra from neurosecretory granules appear of similar size, regardless of variations in the preparative procedure, this calcium must be firmly bound. The possible role of the calcium bound to the neurosecretory substance is discussed.The presence of sulphur in insect neurosecretory granules indicates the presence of a protein besides the hormone, i.e., an insect neurophysin.We wish to thank Dennis Greer for the operation of the analytical microscope. This work was supported by a Wellcome-Carlsberg Travelling Research Fellowship awarded to T.N. The EMMA-4 facility is supported by a grant from the British Science Research Council     

Energy dispersive X-ray microanalysis of sulfated glycosaminoglycans in cartilage matrix stained with alcian blue 8GX

X-ray spectra were recorded from 400-700 nm matrix areas of 0.5 micron sections prepared from the articular cartilages of 15- and 23-year-old human cadavers. The X-ray microanalysis was carried out (i) on untreated material; (ii) after removing sulfate group by a methylation procedure; (iii) after staining with a copper containing cationic phatolcyanin dye, alcian blue 8GX, preceded by carboxymethylation. K alpha peaks of sulphur could be detected in methylated (i.e. desulfated) samples. These peaks probably indicated the presence of sulphur-containing amino acids in different matrix proteins. Consequently, the measurements of sulphur despite its general use cannot be recommended for the X-ray microanalysis of sulfated glycosaminoglycans of cartilage matrix. K alpha peaks of copper could be identified after carboxymethylation and staining with alcian blue. After carboxymethylation, alcian blue can only be bound to the dissociated sulfate groups of glycosaminoglycans in the cartilage matrix. According to our spectrophotometric studies, approximately one molecule of alcian blue combined with one sulfate group. These data suggested that this technique could be used for semiquantitative estimation of sulfated glycosaminoglycans in small areas of the cartilage matrix. Using this method, we found a higher occurrence of sulfated glycosaminoglycans in the territorial matrix than in the interterritorial matrix of the intermediate and deep zones of the human articular cartilage.     

Demonstration of sulphur in secretory granules of anterior and posterior pituitary by X-ray microanalysis (author's transl)]

The sulphur content of secretory granules and other subcellular structures in the cells of the anterior pituitary and of neurosecrotory granules in the nerve fibers of the neural lobe have been studied by energy dispersive microanalysis. The highest level has been found in the secretory granules of the neural lobe.     

Energy dispersive X-ray analysis of tissue gold after silver amplification by physical development

Rats were treated intraperitoneally with the gold-containing compounds sodium aurothiomalate (Myocrisin), sodium aurothiosulfate (Sanocrysin), and aurothioglucose. Using stem energy dispersive X-ray analysis, gold and silver were shown to be located at the same point in lysosomes of proximal tubular cells of the kidney, in hepatocytes and in macrophages of lymph glands, spleen and liver. This result indicates that, after exposure to ultraviolet radiation, chemically bound tissue gold is transformed to metallic gold that subsequently can catalyze the reduction of silver ions to silver when subjected to physical development, i.e. exposed to a photographic developer containing silver ions in addition to the reducing molecules.     

Energy dispersive X-ray analysis of tissue gold after silver amplification by physical development

Summary Rats were treated intraperitoneally with the gold-containing compounds sodium aurothiomalate (Myocrisin), sodium aurothiosulfate (Sanocrysin), and aurothioglucose. Using stem energy dispersive X-ray analysis, gold and silver were shown to be located at the same point in lysosomes of proximal tubular cells of the kidney, in hepatocytes and in macrophages of lymph glands, spleen and liver. This result indicates that, after exposure to ultraviolet radiation, chemically bound tissue gold is transformed to metallic gold that subsequently can catalyze the reduction of silver ions to silver when subjected to physical development, i.e. exposed to a photographic developer containing silver ions in addition to the reducing molecules.     

Isolation and characterization of neurosecretory granules of the rat posterior pituitary gland

Summary Neurosecretory granules (NSG) of rat posterior pituitary glands were prepared by differential centrifugation techniques mainly according to the procedure as described by Barer, Heller and Lederis (1963). As revealed by electron microscopy, the recovery of neurophysin and the contents of enzymes, purified NSG were obtained in a pellet at 30 000 g/60 min (0.44 M sucrose). Eighteen h after injection of (³⁵S) cysteine into the supraoptic nucleus 60% of the recovered radioactivity in the neural lobe was found in the NSG, whereas 20% was found in the final supernatant (100 000 g/120 min). Sixteen days after injection the NSG and the final supernatant fraction contained fairly equal amount of (³⁵S) cysteine (approximately 40%). It is suggested that after a period of intragranular maturation neurophysin is extruded into an extragranular pool of neurosecretory material.With the use of conventional polyacrylamide-gel electrophoresis it was shown that the predominating proportion of radioactivity in the NSG after a hypothalamic injection of (³⁵S) cysteine was located within the neurophysin fraction A and in fraction B. Fraction B is suggested to be partly bound to the NSG membranes. When the NSG soluble and NSG insoluble proteins, obtained after lysis of NSG, were separated on polyacrylamide gels in the presence of sodium dodecylsulphate, the highly radioactive soluble protein was shown to consist of two components with average molecular weights of 12 300 and 14 600. Most of the proteins in the lysate were found in the NSG membranes, though less radioactive. A component with a mol.wt. of 37 000 was enriched in the membrane fraction. At longer times after isotope injection the high mol.wt. proteins, particularly those of the NSG membranes, contained increased amounts of radioactivity.Abbreviations NSG neurosecretory granule - NSM neurosecretory material - SON supraoptic nucleus The present investigation was supported by grants from Svenska Livförsäkringsbolags nämnd för medicinsk forskning from Svenska Sällskapet för Medicinsk forskning, from the Medical Faculty, University of Göteborg, and from the Swedish Medical Research Council (B 72-12X-2543-04A).We are indebted to Mrs. Marie-Louise Eskilsson, Mrs. Wally Holmberg and Mrs. Ulla Svedin for technical assistance, and to Miss Gull Grönstedt for careful secreterial work.     

X-ray microanalysis of cardiocytes in the Snell dwarf mouse

X-ray microanalysis has been used to determine elemental content in the nucleus, myofibrillar cytoplasm and mitochondrially enriched cytoplasm of cardiocytes in Snell dwarf mice in comparison with phenotypically normal mice from the same strain. It was found that there was significantly lower chlorine concentration in all three subcellular locations and significantly lower sodium concentration in the nucleus of dwarf mouse cardiocytes. In both normal and dwarf mice, statistically significant subcellular compartmentalization was found for phosphorus, sulfur, and potassium.     

X-ray microanalysis of airway surface liquid in the mouse

The ionic composition of airway surface liquid (ASL) has been debated, and, in particular for the mouse, a wide range of values has been published. Two techniques were developed to measure the elemental composition of the ASL. X-ray microanalysis of ASL was carried out at low temperature on trachea removed from isoflurane-anesthetized animals and shock-frozen. In the second technique, dextran beads were placed on top of the epithelium of the trachea removed from pentobarbital-anesthetized animals, left to equilibrate with the ASL, dried, and subjected to X-ray microanalysis. Both techniques showed that mouse tracheal ASL has significantly lower concentrations of Na and Cl (approximately 60-80 mM) than serum. Differences between the two techniques were due to different sampling of mucus. CFTR(-/-) mice had significantly higher concentrations of Na and Cl in their ASL than age-matched controls. Pilocarpine or isoproterenol stimulation significantly reduced the ion concentrations in tracheal ASL. ASL was also collected with the dextran bead method from the nasal cavity in situ in pentobarbital-anesthetized animals. In control animals, the elemental composition of nasal fluid was similar to that of tracheal ASL. Pilocarpine stimulation caused a significant increase in Na, Cl, and K; stimulation with isoproterenol or phenylephrine caused a significant increase only in K. It is concluded that mouse ASL under unstimulated conditions is hypotonic, which may be related to the relative paucity of submucosal glands in the mouse trachea.     

Electron microscopy of human melanosomes in unstained,fresh air-dried hair bulbs and their examination by electron probe microanalysis

Summary Electron microscopy of unstained, fresh air-dried spreads of plucked human hair bulbs revealed relatively well preserved melanocytes and dense storage granules of melanin. Energy dispersive X-ray microanalysis of melanosomes and premelanosomes from black facial hair bulbs disclosed high peaks for potassium and sulfur, intermediate peaks for magnesium and calcium, and low but distinct ones for phosphorus and chlorine. No peaks for magnesium were found in the central portion of mature melanosomes, but at their edges and in premelanosomes conspicuous peaks for this element were obtained.Supported by a grant from the Ministry of Education of JapanThe author thanks Dr. H. Fujita for his advice and Messrs K. Yonehara, S. Okamura, T. Fukuoka, and T. Asai, Naka Works, Hitachi Ltd., Katsuta, Ibaraki, for use of their X-ray microanalyzer and for their assistance in electron probe analysis     

Quantitative energy dispersive X-ray microanalysis of eight elements in pancreatic endocrine and exocrine cells after cryo-fixation

Quantitative X-ray microanalysis of 8 elements was performed on ultrathin, freeze-dried sections of islets and pancreas pieces from non-inbredob/ob-mice. Diffusion of elements was reduced to a minimum by rapidly freezing the tissue samples between nitrogen-cooled polished copper surfaces and avoiding the use of chemical fixatives and stains. The ultrastructural morphology was adequately maintained to allow measurements on secretory granules, mitochondria, cell nuclei, and cytoplasm free of these organelles. The distribution of the various elements between cellular compartments was similar in islet -cells and exocrine pancreas cells. However, the insulin secretory granules were outstanding in exhibiting the highest concentrations of zinc and calcium. In comparison with cytoplasm in the -cells, the insulin granules accumulated calcium 2-fold and zinc as much as 40-fold. As no correlation could be made for endoplasmic reticulum in the cytoplasmic measurements areas, the true accumulations above cytosol are likely to be even higher.     

Quantitative X-ray microanalysis of calcium with the Camebax-TEM system in frozen, freeze-substituted and resin-embedded tissue sections. Application to molluscan glio-interstitial granules

The relevance of the continuum method for a quantitative X-ray microanalysis of epon embedded tissue sections in the particular conditions offered by the Camebax-TEM system was tested and an improved model of specimen holder is proposed. The absolute calcium concentration [Ca] of membrane-bound intracellular glio-interstitial granules was determined by X-ray microanalysis in transmission electron microscopy of Mytilus retractor muscle. The Ca peak and background values were measured by the wavelength-dispersive spectrometer of the Camebax; the mass thickness of the section was recorded simultaneously with an added energy-dispersive detector. The tissue was frozen at approximately equal to 77 K in a mixture of liquid propane and butane, freeze-substituted in the presence of oxalic acid and embedded in epoxy resin. The calcium concentration of glio-interstitial granules can be as high as 180 mmol.kg-1 of epoxy-embedded tissue, with an average of 40 mmol.kg-1. The sampling of the data through repeated experiments is discussed and it is proposed that the cell would be the main level of variation. The Ca content of glio-interstitial granules is significantly lower in the tissues of animals submitted to high-potassium artificial sea-water for 10 min. This finding was predicted by the hypothesis that glio-interstitial tissue is a regulator of calcium concentration in extracellular spaces.     

Energy dispersive X-ray analysis of protein bodies in Protea compacta cotyledons

Summary The elemental composition of globoids in the protein bodies of Protea compacta cotyledons was studied by means of energy dispersive X-ray analysis. The globoid crystal was rich in phosphorus and calcium with lesser amounts of magnesium and potassium suggesting the presence of phytin in these structures.Abbreviation EDX energy dispersive X-ray analysis     

Presence of calcium in polychaete cortical granules demonstrated by X-ray microanalysis on ultrathin cryo-sections of oocytes and eggs

Summary Cortical granules from fertilized eggs, oocytes and nurse cells of Ophryotrocha labronica have been analyzed for the presence of calcium using cryo-ultramicrotomy and X-ray microprobe analysis. All cortical granules showed a significant peak for calcium, but yolk granules were without calcium. These results support the hypothesis that the discharge of cortical granules shortly after fertilization is a self-propagating phenomenon involving the diffusion of Ca²⁺ from bursting granules.     

The isolation of neurophysin-I and -II from bovine pituitary neurosecretory granules separated on a large scale from other subcellular organelles. Demonstration of slow equilibration of neurosecretory granules during centrifugation in a sucrose density gradient

1. An improved procedure for the isolation of neurosecretory granules from the posterior lobe of the bovine pituitary gland is described. 2. Of the total oxytocic and pressor activities present in the original tissue 80% was sedimentable. 3. The granules were separated from mitochondria by prolonged centrifugation in a sucrose density gradient. During a sedimentation period of 5hr. the granules moved progressively into denser regions of the gradient and the mitochondria remained at the top. 4. The biological activities of the granules were measured: the oxytocic activity was 11·56±1·63 and the pressor activity was 15·60±3·91 units/mg. of protein. 5. A protein was isolated from a lysate of granules prepared from 40 pituitary glands. Amino acid analysis showed that it consisted of a mixture of neurophysin-I and neurophysin-II in equal proportions. It accounted for 60% of the soluble granule protein and for 50% of the total granule protein. 6. The neurophysins present in the granules are associated with 19·1 units of oxytocic and 21·1 units of pressor activity/mg. of protein. 7. Starch-gel electrophoresis revealed the presence of both neurophysins in extracts of 15 pituitary glands studied individually. 8. We conclude that the polypeptide hormones, oxytocin and [8-arginine]-vasopressin, are normally closely associated with the two neurophysins within neurosecretory granules of the pituitary gland.