The localization of calcium by X-ray microanalysis in myopathic muscle fibers

Summary An electron histochemical study was undertaken to localize calcium with ammonium oxalate precipitation technique in soleus muscle of rat in normal cases and in myopathy induced experimentally by a prolonged treatment of 2,4-dichlorophenoxyacetate (2,4-D). The calcium content of precipitates was detected by energy-dispersive X-ray microanalysis. In normal cases, the electron dense precipitates containing calcium were mainly found in the vesicles of sarcoplasmic reticulum, whereas in 2,4-D induced myopathy the deposits were shifted near the Z line into the myofibrils. Calcium, because the uptake into sarcoplasmic vesicles was inhibited by 2,4-D, could attach to other binding sites, such as to the troponin-C.A long-lasting binding of calcium might lead to a prolonged activation of the actin-myosin system.Fellow of the Max-Planck Society at the Abteilung Neurobiologie, Neuroanatomie, Max-Planck-Institut für biophysikalische Chemie, Göttingen. Federal Republic of Germany     

Evaluation of the pyroantimonate method for detecting intracellular calcium localization in smooth muscle fibers by the X-ray microanalysis of cryosections

Summary The validity of the pyroantimonate method, which has been used for detecting intracellular Ca localization and translocation in smooth muscles, was examined by making cryosections of the relaxed anterior byssal retractor muscle (ABRM) of Mytilus edulis at various stages of procedures for preparing ordinary Epon-embedded sections and determining the elemental concentration ratios of the pyroantimonate precipitate, localized along the inner surface of the plasma membrane, with an energy dispersive X-ray microanalyzer. The concentration of Ca (relative to that of Sb) in the precipitate stayed constant after the procedures of fixation, dehydration and Epon-embedding, while the concentrations of K, Mg, Na and Os showed their respective characteristic changes after the above procedures, being lower than that of Ca in the Epon-embedded sections. The presence of Ca in the precipitate was also demonstrated with an electron energy-loss spectrometer. The localization of Ca underneath the plasma membrane was also observed in the cryosections of the ABRM fibers prepared after mild fixation with acrolein vapor without using pyroantimonate. These results indicate that the pyroantimonate precipitate serves as a valid measure of intracellular Ca localization.     

Evaluation of the pyroantimonate method for detecting intracellular calcium localization in smooth muscle fibers by the X-ray microanalysis of cryosections

The validity of the pyroantimonate method, which has been used for detecting intracellular Ca localization and translocation in smooth muscles, was examined by making cryosections of the relaxed anterior byssal retractor muscle (ABRM) of Mytilus edulis at various stages of procedures for preparing ordinary Epon-embedded sections and determining the elemental concentration ratios of the pyroantimonate precipitate, localized along the inner surface of the plasma membrane, with an energy dispersive X-ray microanalyzer. The concentration of Ca (relative to that of Sb) in the precipitate stayed constant after the procedures of fixation, dehydration and Epon-embedding, while the concentrations of K, Mg, Na and Os showed their respective characteristic changes after the above procedures, being lower than that of Ca in the Epon-embedded sections. The presence of Ca in the precipitate was also demonstrated with an electron energy-loss spectrometer. The localization of Ca underneath the plasma membrane was also observed in the cryosections of the ABRM fibers prepared after mild fixation with acrolein vapor without using pyroantimonate. These results indicate that the pyroantimonate precipitate serves as a valid measure of intracellular Ca localization.     

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.     

Ultrastructural localization of calcium, by electron-probe X-ray microanalysis, in the small intestine of suckling rats

The subcellular distribution of calcium has been investigated in samples, from the intestinal mucosa of 10-day rats, prepared for X-ray microanalysis by various techniques designed to minimize the loss of this element. Calcium retention and its threshold of detection was most satisfactory in freeze-dried frozen thin sections. In resin-embedded samples the best retention of calcium was found in specimens fixed in absolute ethanol, embedded without osmication, and sectioned onto glycerol. The results of this investigation indicate the presence of calcium in the supranuclear vacuole of enterocytes in the distal intestine of the neonatal rat. This calcium is probably taken up during the endocytosis of material from the intestinal lumen. The same mechanism may also be important in the uptake of other metals by suckling animals.     

Ultrastructural localization of intracellular calcium stores in Xenopus ovarian follicles as revealed by cytochemistry and X-ray microanalysis

The ultrastructural localization of calcium in full-grown ovarian follicles of Xenopus laevis was demonstrated after fixation in the presence of fluoride ions and by means of energy dispersive X-ray microanalysis. In hormonally untreated follicles (prophase I-arrested oocytes), two calcium sites were detected: follicle cells and oocyte pigment granules. In follicle cells, calcium containing deposits were preferentially associated with macrovilli, which ended by gap junctions. In human chorionic gonadotropin treated follicles (meiotically reinitiated oocytes), deposits were only seen in follicle cells. This is the first report of the cytochemical detection of intracellular Ca²⁺ in follicle cells of amphibians. The possible involvements of these Ca²⁺ stores in mediating the hormonal control of meiotic maturation are discussed.     

X-ray microanalysis of calcium binding sites in Paramecium

Summary In Paramecium cells Ca⁺⁺-stimulated triggering of the exocytosis of secretory vesicles (trichocysts) was achieved by ionophores X-537 A or A 23187. Under triggering conditions electron dense deposits were present in some resting trichocysts and regularly in discharging trichocysts; upon subsequent fixation deposits occurred on the trichocyst membrane (on the inner side or within the membrane) and on the inner lamellar sheath from where deposits seemed to radiate into the secretory materials. Similar results were obtained with glutardialdehyde fixation alone which also triggers exocytosis but only at low concentrations. Element analysis by energy dispersive x-ray microanalysis ascertained the presence of Ca and P in deposits occurring in trichocysts. Those resting trichocysts which were devoid of deposits did not contain Ca or P enriched. Hence, an abrupt Ca⁺⁺-influx into individual trichocysts just before exocytosis seems to be involved in the triggering mechanism, possibly in combination with the sudden activation of an ATPase systemlocalized at those sites of the trichocysts which primarily contain the deposits. When paramecia were treated only with Ca⁺⁺ and then fixed with OsO₄ plus oxalate or merely with glutardialdehyde, electron scattering deposits were formed also on the inner side of the cell membrane and within the ciliary shaft (but rarely in trichocysts). Deposits obtained on cilia (including ciliary granule plaques) also contained Ca, P and S. Cells contain osmiophilic calcium-storing vacuoles which were selectively rich in Ca and S but devoid of P.     

Electron probe X-ray microanalysis of human muscle biopsies

Summary The elemental composition of human muscle fibres have been determined by electron probe microanalysis. In order to distinguish between different types of fibres, two approaches were used. In one approach individual fibres were isolated, portions of them used for a typing by histochemical methods and the main part used for X-ray microanalysis. In the other approach the muscle biopsy was serial-sectioned, some sections used for a histochemical typing and the others (16 m thick cryosections) used for X-ray microanalysis in the electron microscope.The comparison of the ratios between P, S and K in Study No. 1 and 2 indicates different concentrations of sulphur in the subsarcolemmal zone and in the interior of the fibre. Both routes give information on all elements (except the ten lightest ones) contained in the fibres or in sections of them, provided the concentration is high enough. In order to obtain quantitative data, expressed as mmol/kg_dw, the spectra of the specimens were compared to those of standards of known composition and the data subjected to a so called ZAF-correction (corrections for the atomic number effect, absorption of X-rays in the specimen and secondary fluorescence). Quantitative data concerning phosphorus, sulphur, chlorine and potassium were obtained in Study No. 2. A significantly higher sulphur concentration was found in type IIA muscle fibres as compared to those of type I.     

Ca++ localization in boar spermatozoa by the pyroantimonate technique and X-ray microanalysis

Intracellular, loosely bound Ca++ has been localized electron microscopically in freshly ejaculated boar spermatozoa by in situ precipitation with potassium antimonate. Ca++ was identified as the cation precipitated by testing the EGTA-sensitivity of the precipitates and by X-ray microprobe analysis. The data obtained revealed that the outer acrosomal membrane is the preferential site for Ca++ precipitation in the sperm head.     

Calcium localization in oat aleurone cells using chlorotetracycline and X-ray microanalysis

Calcium distribution was studied in oat caryopses. Using the chlorotetracycline method it was found that membrane-associated Ca²⁺ was present in the aleurone layer. X-ray microanalysis confirmed the presence of calcium in aleurone cells; it also demonstrated the presence of considerable amounts of calcium in the cell wall surrounding these cells.Abbreviation CTC chlorotetracycline     

The role of free calcium ion in calcium release in skinned muscle fibers

Major questions in excitation--contraction coupling of fast skeletal muscle concern the mechanism of signal transmission between sarcolemma and sarcoplasmic reticulum (SR), the mechanism of SR Ca release, and operation of the SR active transport system during excitation. Intracellular Ca movement can be studied in skinned muscle fibers with more direct control, analysis of 45Ca flux, and simultaneous isometric force measurements. Ca release can be stimulated by bath Ca2+ itself, ionic "depolarization," Mg2+ reduction, or caffeine. The effectiveness of bath Ca2+ has suggested a possible role for Ca2+ in physiological release, but this response is difficult to analyze and evaluate. Related evidence emerged from analysis of other responses: with all agents studied, stimulation of 45Ca efflux is highly Ca2+-dependent. The presence of a Ca chelator prevents detectable stimulation by ionic "depolarization" or Mg2+ reduction and inhibits the potent caffeine stimulus; inhibition is graded with chelator concentration and caffeine concentration, and is synergistic with inhibition by increased Mg2+. The results indicate that a Ca2+-dependent pathway mediates most or all of stimulated 45Ca efflux in skinned fibers, and has properties compatible with a function in physiological Ca release.     

X-ray microanalysis of cellular localization of ferritin in mammalian spleen and liver

X-ray microanalysis of mineral core of cellular localizations of ferritin in horse, sheep and rat spleen macrophages and in parenchymal cells of normal and pathological human liver was performed to obtain the net intensities of iron and phosphorus in the irradiated areas and to calculate the P:Fe ratios.For comparison the same analysis was performed on commercially produced horse spleen ferritin in two processings: unembedded and after treatment similar to tissue and embedded in Epon. Our analytical results of unembedded commercially produced horse spleen ferritin particles (1:15) confirmed the weight ratio suggested by Granick and Hahn (J. biol. Chem., 155: 661–669, 1944) for isolated crystallizable horse spleen ferritin in their chemical studies (1:16 or 1:14). After application of EM-tissue processing procedures to commercially produced horse spleen ferritin the ratio changed into 1:22, presumably by the loss of phosphorus. In spleen of three species the X-ray analytical results of ferritin particles in situ showed that in both localizations (clusters and lysosomes) the P:Fe ratios varied widely and the mean P:Fe ratios were generally higher than in embedded commercially produced horse spleen ferritin. Within these three species the mean P:Fe ratios of ferritin particles in two localizations of sheep and rat spleen were higher than in horse spleen. Moreover in sheep and rat spleen one third of the analysed clusters and lysosomes contained ferritin particles with zero phosphorus although sufficient iron was detected. Within all three species we found no statistically significant difference in mean P:Fe ratios between clusters and lysosomes.The X-ray analytical results in normal human liver parenchymal cells showed that as a result of very variable P:Fe ratios in ferritin-containing lysosomes, the mean P:Fe ratio was higher than in embedded commercially produced horse spleen ferritin and was nearly the same as in ferritin within clusters and lysosomes of horse spleen. In human liver with haemochromatosis, there were no significant variations in P:Fe ratios. The mean P:Fe ratio for ferritin particles in lysosomes was 1:13, much lower than in normal liver (1:39) and nearly the same as in unembedded commercially produced horse spleen ferritin (1:15). Our findings led us to conclude that in spleen macrophages and in parenchymal cells of normal liver among the populations of ferritin particles the iron-poor ferritin particles are more extensively present (especially in sheep and rat spleen) than in isolated crystallized horse spleen ferritin or ferritin-containing lysosomes of pathological human liver. In these iron-poor ferritin molecules the P:Fe ratio is variable from molecule to molecule and different from that suggested in the literature. The hypothesis of a constant ratio P:Fe for ferritin with different iron content is rejected. The formula for the composition of the mineral core of ferritin, as proposed by Granick and Hahn (1944) can only be considered correct for ferritin as iron-rich as isolated from horse spleen.     

Ultrastructural localization of calcium in post-mortem bovine muscle: A cytochemical and X-ray microanalytical study

Summary Calcium localization was demonstrated in bovine longissimus muscle using the antimonate precipitation technique in combination with electron probe X-ray microanalysis. Samples were taken each hour during the first 24 h post-mortem, and then after a storage period of 8 and 15 days. For all sampling times analysed, heavy precipitates were seen in dense parts of nuclei and on N-lines of myofibrils. Up to 18–20 h post-mortem, deposits were observed in sarcoplasmic reticulum at the level of triads. In comparison with the earlier post-mortem samples, myoplasmic precipitates were strongly increased at 4 h post-mortem, and just before rigor onset, at 19 h where intermyofibrillar spaces were completely blackened and triads were no more visible. These localizations of precipitates were still observed up to 15 days post-mortem. At these storage times, myofibril disruptions were seen at the level of N-lines. Wavelength-dispersive and energy-dispersive spectrometric analyses indicated that significant amounts of calcium occurred in the dense precipitates observed.     

Regulation by magnesium of intracellular calcium movement in skinned muscle fibers

The effect of Mg on Ca movement between the sarcoplasmic reticulum (SR) and myofilament space (MFS) was studied in skinned muscle fibers by using isometric force as an indicator of MFS Ca. In Ca-loaded fibers at 20 degrees C, the large force spike induced by Ca in 1 mM Mg (5 mM ATP) was strongly inhibited in 3 mM Mg, and force development was extremely slow. After a brief Ca stimulus in 1 mM Mg, relaxation in Ca-free solution was significantly faster in 3 mM Mg. These changes were due to altered Ca movements, since the effect of 3 mM Mg on steady force in CaEGTA solutions was small. Changes in Mg alone induced force transients apparently due to altered Ca movement. In relaxed fibers, decreasing the Mg to 0.25 mM caused phasic force development. In contracting fibers in Ca solutions, increasing the Mg caused a large transient relaxation. The effects of increased Mg were antagonized by 0.5 mM Cd, an inhibitor of the SR Ca transport system. The results indicate that active Ca uptake by the SR in situ is stimulated by Mg, and that it can affect local MFS [Ca++] in the presence of a substantial Ca source. These results provide evidence that an increased rate of Ca uptake in 3 mM Mg could account for inhibition of the large force spike associated with Ca-induced Ca release in skinned fibers.     

The role of calcium in excitation--contraction coupling in crustacean muscle fibers

The evidence that calcium (Ca) plays an important role in electrical activity and an essential role in excitation--contraction (E--C) coupling in crustacean muscles is reviewed. These muscles produce graded electrical and mechanical responses to applied depolarizations. Removal of Ca from the bath solution eliminates both responses. Addition of Ba2+ or Sr2+ to Ca-free saline restores membrane electrogenesis, and all-or-none action potentials can be induced. With Sr2+ vigorous contractions are produced, whereas Ba action potentials evoke minimal or no tension, showing that rapid depolarization of the membrane potential is not sufficient per se for E--C coupling in crab and barnacle muscle. Several inorganic (e.g., multivalent cations) and organic (e.g., aminoglycoside antibiotics) which block membrane Ca channels block electrogenesis and contraction. However, the "Ca antagonists" verapamil and D600 also block Ca uptake at intracellular storage sites, resulting in spontaneous contractions and the delayed relaxation of small contractions associated with residual Ca currents. The evidence that the Ca which enters the fibres needs to release Ca from intracellular storage sites to produce contractions is detailed and discussed. Finally, a model for E--C coupling is discussed. This model includes the sites and mechanisms of action for several chemicals which modify E--C coupling in crustacean muscle fibres.     

Activation of skinned muscle fibers by calcium and strontium ions

Intact and mechanically skinned skeletal muscle fibers of the crab Carcinus maenas have been used. The aim of the experiments was to determine the origin of the mechanical activity recorded in intact crab muscle fibers exhibiting an inward strontium current in strontium solution without calcium. To do so, the effect of strontium ions in inducing activation of contractile proteins and calcium release from the sarcoplasmic reticulum has been studied. The properties of the sarcoplasmic reticulum membrane towards strontium ions, i.e., the efficiency of the calcium ATPase towards strontium ions and the capability to release strontium ions have been investigated. Results show that the contractile proteins have a lower affinity for strontium than for calcium ions. However, the maximum bound strontium is identical to the maximum bound calcium. As for the sarcoplasmic reticulum, strontium ions can induce a calcium release and also can be taken up by the calcium ATPase and be released. We concluded that the mechanical activity in intact fibers bathed in a strontium medium has two origins: first, a direct and partial activation of the contractile proteins by strontium ions flowing through the calcium channel; second, a contractile proteins activation of calcium ions released by the sarcoplasmic reticulum by a "strontium-induced calcium release" mechanism.     

Electron probe X-ray microanalysis of human skeletal muscle involved in rheumatoid arthritis

Summary Electron probe X-ray microanalysis in the scanning microscope was used to determine the elemental composition of muscle fibes from patients with rheumatoid arthritis (RA). Quantitative data concerning phosphorus, sulphur, chlorine and potassium were correlated to the fibre type by a routine method based on serial cryosectioning and histochemical staining of adjacent sections. Significantly lowered sulphur values were found in type II A and II B muscle fibres of RA patients as compared to those of healthy controls. Traces of gold were detected in muscles of two patients treated with gold salts. The basis and mechanism for the decreased sulphur content in RA muscles are so far unknown, but may depend on the decreased amount of sulphur-rich protein(s).     

X-ray microanalysis and chlorotetracycline staining of calcium vesicles in the green alga Mougeotia

Calcium ions have been proposed to play a key role in the sensory transduction of phytochrome-governed chloroplast movement in the green alga Mougeotia. To test this hypothesis, the intracellular pattern of calcium distribution was studied in this alga by two independent techniques, namely, X-ray microanalysis of fixed and of unfixed frozen-hydrated cells, as well as in vivo fluorescence by chlorotetracycline. Both methods of detection reveal a significant compartmentation of calcium in vesicles close to the chloroplast edge and, less frequently, in the cortical cytoplasm. Microfilaments, presumably actin, which could function in driving chloroplast movement, have been observed running between the chloroplast edge and the cortical cytoplasm (Wagner, G., Klein, K. (1978) Photochem. Photobiol. 27, 137). The vesicular calcium concentration is stable and decays only slowly in the absence of extracellular calcium much in the same way as the ability of the chloroplast to perform movements decreases. A functional relationship between vesicular calcium compartmentation and phytochrome-governed chloroplast movement in the green alga Mougeotia seems indicated.Abbreviation CTC chlorotetracycline     

Quantitative X-ray microanalysis of calcium in sea urchin eggs after quick-freezing and freeze-substitution

Summary Freeze-substitution was used to study the distribution of calcium in sea urchin eggs, and the validity of the technique was assessed. We followed the fate of both total and exchangeable calcium of sea urchin eggs in two species (Paracentrotus lividus and Arbacia lixula) after the various treatments needed for freeze-substitution and embedding. We compared the calcium content either by X-ray microanalysis of Epon-embedded sections of freeze-substituted eggs (6.2±0.71 mmoles/kg of Epon-embedded tissue) or by flame spectrometry analysis of living eggs (32.3±1.30 nmoles/mg protein). After standardization of units, both values lead to similar total calcium content. We also measured the movements of ⁴⁵Ca from prelabelled eggs. Exchangeable ⁴⁵Ca as well as total calcium appeared unaffected by the preparative treatment for X-ray microanalysis. In conclusion, our preparative technique for X-ray microanalysis can be considered appropriate for our material and allows us to undertake a subcellular quantification of calcium in various organelles.