Selective subcellular localization of cations with variants of the potassium (pyro)antimonate technique.

Fixation of rat parotid with an unbuffered osmium tetroxide solution containing nearly saturated potassium (pyro)antimonate resulted in abundant deposition of cation-antimonate precipatates in acinar cells. Altering the antimonate concentration, including buffers or chelators in the solution or changing the primary fixative resulted in an altered intensity and distribution of the precipitates formed in the tissue, apparently reflecting a degree of selectivity in ion localization. Decreasing the concentration of pyroantimonate to about half-saturation preserved predominantly the less soluble antimonate salts (e.g., Na+, Ca++) and resulted in preferential retention of deposits along the plasmalemma and in mitochondrial "dense bodies," with loss of most cytoplasmic and nuclear precipitates. A similar pattern was seen if fixation with the high concentration antimonate-osmium procedure was followed by a prolonged rinse. Adding phosphate or collidine buffers markedly decreased precipitates in the nuclei and on granular reticulum as well. Phosphate buffer or ehtyleneglycoltetraacetate inhibited in vitro precipitation of calcium and sodium and decreased or abolished plasmalemmal deposits. Glutaraldehyde fixation, either in the presence of antimonate or prior to antimonate-containing osmium tetroxide, abolished heterochromatin deposits. Mitochondrial dense bodies were of two types, one containing precipitate and the other inherently osmiophilic. The latter were also observed in pyrophosphate-osmium controls. Results from in vitro titrations of cations with the various antimonate methods and from neutron activation analyses of fixed tissues supported conclusions drawn from fine structural distribution patterns and were interpreted as follows. In rat parotid acinar cells, deposits in heterochromatin and on granular reticulum probably arose from precipitation in sites of high K+ and H+ as well as--NH3+-rich histones. Plasmalemmal antimonate deposits demonstrated sites of sodium and/or calcium accumulation. Some mitochondrial dense bodies contained Ca++ whereas others were inherently osmiophilic. Large, extracellular deposits were probably predominantly sodium precipitates.     

Cytochemical study of intracellular calcium in hamster spermatozoa during the acrosome reaction

Calcium was localized by a pyroantimonate technique in hamster spermatozoa during the acrosome reaction and pyroantimonate precipitates were observed in the anterior region of the acrosome. The calcium was also localized in the postacrosomal lamina of spermatozoa undergoing the acrosome reaction. Spermatozoa, incubated in capacitating medium containing verapamil, showed denser precipitates with an increase in concentration of this drug. Ionophore A23187 enhanced binding of calcium to the acrosomal region. The sodium channel inhibitor amiloride inhibited the acrosome reaction and the pyroantimonate precipitates were absent in these spermatozoa, whereas ionophore monensin enhanced the acrosome reaction. This suggests that the Na⁺/Ca⁺⁺ antiporter may be responsible for intracellular Ca⁺⁺ regulation during the acrosome reaction in hamster spermatozoa.     

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.     

Intracellular calcium in hamster spermatozoa in testis,epididymis and during the acrosome reaction

Hamster spermatozoa from the testis and cauda epididymis were examined for intracellular localization of calcium using a pyroantimonate technique. Calcium precipitates were found in spermatozoa from the testis but not in those from cauda epididymis. Spermatozoa from the epididymis which had been induced to undergo capacitation and the acrosome reaction in vitro, were also investigated. In these, the anterior region of the acrosome showed precipitates indicating calcium binding at specific sites, possibly prior to transport across the membranes. The additional presence of calcium precipitates in the postacrosomal region suggests that the appearance of free calcium in that region is an important step in the acrosome reaction.     

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     

A variant of the pyroantimonate technique suitable for localization of calcium in ovarian tissue.

Osmium-pyroantimonate solutions for the precipitation of cations are unsuitable for use with delicate mammalian oocytes. A variant of the pyroantimonate technique employing a mixture of pyroantimonate and glutaraldehyde has been found to give successful and repeatable results if a fixation time of 4 hr is used. Calcium-containing antimonate precipitates were localized principally in nuclei, smooth endoplasmic reticulum, Golgi apparatus, mitochondria, and cytoplasmic processes of both oocytes and follicle cells, and along the plasma membrane in small oocytes. Deposits were also concentrated around the periphery of lipid droplets in the follicle cells. The presence of calcium in the precipitates was confirmed by x-ray microprobe analysis.     

Abnormal calcium distribution in human parathyroid adenomas as possible cause of primary hyperparathyroidism

Current knowledge suggests that normal parathyroid glands and parathyroid adenomas have different sensitivities to environmental calcium. In search for morphological equivalents, 5 normal human and 10 porcine parathyroid glands, as well as 10 human parathyroid adenomas were investigated with regard to intracellular and extracellular calcium distribution. The glands were incubated for 2, 4, 6 and 20 h in tissue cultures using HAM's F10 medium with various calcium concentrations. For visualization of the calcium distribution in the tissue the method of pyroantimonate precipitation was applied. Specificity of the reaction was controlled by X-ray microanalysis. Shifts of the calcium pyroantimonate precipitates were quantitated by morphometry using an area-counting system. The results demonstrate that in normal parathyroid glands calcium precipitates are distributed randomly. Incubation of normal glands in medium with low calcium concentration (0.6 mM) provoked reduced amounts of intracellular and extracellular calcium complexes. When the incubations were performed in medium with high calcium content (2.6 mM), calcium accumulated inside parathyroid and stroma cells. In contrast to normal parathyroid glands, parathyroid adenomas fixed immediately after surgery showed an atypical calcium distribution with low amounts of intracellular and high amounts of extracellular calcium grains. The data suggest that in normal parathyroid glands the intracellular calcium concentration follows the extracellular environmental calcium concentration. Thus, calcium modulates parathyroid hormone (PTH) secretion via intracellular regulatory mechanisms. In parathyroid adenomas the calcium transport via the tumor cell membrane appears to be disturbed resulting in lowered intracellular calcium levels. This is remarkable since the environmental calcium concentration is elevated due to the hypercalcemia of primary hyperparathyroidism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium sensitivity of hybrid complexes of muscle myosin and Physarum proteins.

1. A myosin-actin hybrid complex was used to study actin-associated calcium sensitivity of a "cytoplasmic" actomyosin. The approach should be generally applicable. 2. Low salt extracts of Physarum polycephalum contain actin which remains in solution after centrifugation at 46 000 times g or at 100 000 times g for 1 h. The actin was precipitated by the addition of muscle myosin to the supernatants and detected in the hybrid complex by electron microscopy, sodium dodecyl sulfate gel analysis, super-precipitation and activation of the myosin ATPase activity. Actin was also precipitable from high speed supernatants of brain tissue or platelets. 3. The hybrid complexes from Physarum possessed 1.5-5-fold calcium dependency which could be removed by washing. Reincubation of the washed complex with concentrated wash solution resulted in high calcium sensitivity. On sodium dodecyl sulfate gels, unwashed complexes from Physarum contained high molecular weight material in addition to bands of molecular weights less than actin. The bands in the size range of 39 000 to 18 000 were primarily lost from the Physarum complex concomitantly with loss of calcium dependence. 4. When the Physarum supernatants were made 40 mM in MgCl2, precipitates were formed containing actin which possessed calcium sensitivity which was also lost on washing with low ionic strength solutions. This calcium dependency was partially reversed by the addition of desensitized rabbit actin to the precipitate before assay. 5. Conclusion: calcium regulation of actomyosin in Physarum is mediated primarily by factors that are bound to the actin component. The regulatory factors are soluble in low salt buffers. The molecular weights of the polypeptide chains of several of these factors are similar to those of the troponin polypeptides of striated muscle. In Physarum but not in platelet or brain a prominent polypeptide chain of approx. 55 000 molecular weight also occurs which coprecipitates with the hybrid complex and which is not easily removed.     

华北落叶松花粉发育过程中的钙动态分布

运用焦锑酸钾沉淀法研究了华北落叶松(Larix principis-rupprechtii Mayr)小孢子发育过程中不同阶段Ca2 的分布情况.减数分裂时期,小孢子囊壁表皮和中层细胞的细胞壁及细胞间隙Ca2 分布较多,绒毡层只有外切向面的细胞膜有Ca2 分布,小孢子母细胞的各部位则很少有Ca2 ;四分体时期,包围四分小孢子的胼胝质壁上有大量的Ca2 分布,在四分孢子壁上也有较多沉淀;游离小孢子时期,钙离子在小孢子壁的分布较四分体时期有所减少,而到花粉成熟时又逐渐增多;从四分体到花粉成熟,乌氏体周围的Ca2 有增多的趋势.对四分体外壁Ca2 的大量分布与花粉壁的形成及信号物质在花粉表面贮存的关系,以及小孢子囊的外壁、绒毡层和乌氏体在Ca2 向花粉运输中所起的作用进行了讨论.     

THE INTRACELLULAR LOCALIZATION OF INORGANIC CATIONS WITH POTASSIUM PYROANTIMONATE : Electron Microscope and Microprobe Analysis

Potassium pyroantimonate, when used as fixative (saturated or half-saturated, without addition of any conventional fixative) has been demonstrated to produce intracellular precipitates of the insoluble salts of calcium, magnesium, and sodium and to preserve the general cell morphology. In both animal and plant tissues, the electron-opaque antimonate precipitates were found deposited in the nucleus—as well as within the nucleolus—and in the cytoplasm, largely at the site of the ribonucleoprotein particles; the condensed chromatin appeared relatively free of precipitates. The inorganic cations are probably in a loosely bound state since they are not retained by conventional fixatives. The implications of this inorganic cation distribution in the intact cell are discussed in connection with their anionic counterparts, i.e., complexing of cations by fixed anionic charges and the coexistence of a large pool of inorganic orthophosphate anions in the nucleus and nucleolus.     

Localization of a thrombin-sensitive calcium pool in platelets.

Electron microscopic x-ray microprobe analysis of pyroantimonate precipitates in platelets fixed in osmium tetroxide-pyroantimonate revealed calcium localization in the nucleoids of alpha-granules. This pool of calcium had largely disappeared within 10 sec after stimulation of platelets by thrombin. Such a rapid change suggests that this calcium pool may have a regulatory role in stimulus-response coupling.     

Physiological and cytochemical studies on activator calcium in contraction by smooth muscle of a sea cucumber,Isostichopus badionotus

Summary The physiological properties of mechanical responses and the intracellular localization and translocation of calcium as a pyroantimonate precipitate were studied in the longitudinal retractor muscle (LRM) of a Bermuda sea cucumber. Acetylcholine (ACh)-induced contraction was reduced by lowering the external Ca concentration, and suppressed completely by prolonged soaking in Ca-free solution. The magnitude of ACh-induced contraction was decreased by Mn and La ions. Furthermore, procaine reduced the ACh-induced contraction. The complete removal of Ca and Mg ions from the external medium induced a socalled Ca · Mg-removal contraction. Electron microscopically, numerous subsarcolemmal vesicles were observed in the LRM fibers. In the resting fibers, pyroantimonate precipitates were localized in the subsarcolemmal vesicles and along the inner surface of plasma membrane. While, in the fiber fixed during mechanical activity, the pyroantimonate precipitates were decreased remarkably in the subsarcolemmal vesicles and at the plasma membrane, and diffusely distributed in the myoplasm. Electronprobe X-ray microanalysis showed that the precipitate contains Ca in a significant amount. These results indicate that the contraction of the LRM fibers is caused not only by Ca-influx but also by Ca-release from the intracellular storage sites, such as the subsarcolemmal vesicles and the inner surface of plasma membrane.     

华北落叶松花粉发育过程中的钙动态分布

运用焦锑酸钾沉淀法研究了华北落叶松(Larixprincipis-rupprechtiiMayr)小孢子发育过程中不同阶段Ca2 的分布情况。减数分裂时期,小孢子囊壁表皮和中层细胞的细胞壁及细胞间隙Ca2 分布较多,绒毡层只有外切向面的细胞膜有Ca2 分布,小孢子母细胞的各部位则很少有Ca2 ;四分体时期,包围四分小孢子的胼胝质壁上有大量的Ca2 分布,在四分孢子壁上也有较多沉淀;游离小孢子时期,钙离子在小孢子壁的分布较四分体时期有所减少,而到花粉成熟时又逐渐增多;从四分体到花粉成熟,乌氏体周围的Ca2 有增多的趋势。对四分体外壁Ca2 的大量分布与花粉壁的形成及信号物质在花粉表面贮存的关系,以及小孢子囊的外壁、绒毡层和乌氏体在Ca2 向花粉运输中所起的作用进行了讨论。     

Programmed cell death. Cytochemical evidence for accumulation of calcium in mitochondria and its translocation into lysosomes: X-ray microanalysis in metamorphosing insect muscles

Summary The intersegmental muscles in the metamorphosing silkmothAntheraea polyphemus were examined by two electron cytochemical procedures for demonstration of calcium compartmentation during the two-day period of degeneration after emergence. Muscle fibres were treated with either oxalate—pyroantimonate, or phosphate—pyroantimonate procedures. The elemental composition of the reaction product arising from the oxalate procedure was determined with electron probe X-ray microanalysis of unstained thin sections by energy dispersive spectrometry and wavelength dispersive spectrometry. The wavelength dispersive data revealed high peaks of calcium and antimony in the electron-dense precipitates. No reaction was obtained in muscles after treatment with the phosphate—pyroantimonate method.Shortly after the emergence of the moth, very few calcium deposits were found in the mitochondria, which also contained amorphous matrix densities. During the rapid lytic phase (17 and 30 h after ecdysis), the mitochondria, autophagic vacuoles sequestering mitochondria, and lysosomal dense bodies issuing from the latter were highly reactive in each muscle fibre.These results demonstrate that the collapse of tracheae (hypoxic conditions) is correlated with the calcium overload of mitochondria when the cell calcium homeostasis is apparently lost. Such calcium overload of the mitochondria appears to cause irreversible damage to these organelles which are then sequestered in autophagic vacuoles. This mitochondrial autophagic process leads to calcium translocation into a lysosomal compartment. We suggest that the calcium lysosomal stores may have a transient function of cell detoxification and stimulation of calcium-dependent degradative processes prior to the final muscle collapse.     

Ultrastructural localization of calcium ions in ram spermatozoa before and after cold shock as demonstrated by a pyroantimonate technique

Ram spermatozoa were subjected to cold shock before fixation in pyroantimonate-osmium. Ultrathin sections revealed an electron-dense particulate precipitate in association with the cells. The precipitate was shown to be related to the presence of calcium by exposure of the material to EGTA which reduced or completely eliminated the deposits. In the acrosome region, very little precipitate was evident when the plasma membrane was intact. Cold shock resulted in the disruption of the plasma membrane. When the acrosome remained intact, precipitate was concentrated just anterior to the equatorial segment, but many cells also had acrosomal disruption and then a more even distribution of precipitate was seen on the outer acrosomal membrane. Precipitate was rarely visible within or beneath the acrosome. Post-acrosomally, calcium pyroantimonate deposits were frequently present in the dense lamina beneath the plasma membrane and these became more intense after cold shock. Midpiece sections revealed a few large granules beneath the plasma membrane and a fine particulate precipitate within mitochondria. Similarly, the fine precipitate was also associated with the outer dense fibres in midpieces and tails. Cold shock did not apparently increase the extent or intensity of precipitates in these sites.     

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

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.     

Excitation-contraction coupling in heart. XIX. Effect of hypoxia on calcium transport by subcellular particles in the isolated perfused rat heart.

To examine the role of changes in calcium transport by subcellular particles in the pathogenesis of contractile failure due to oxygen lack, both mitochondrial and microsomal fractions were obtained from the isolated hypoxic rat hearts and their calcium binding and uptake abilities were determined by the Millipore filtration technique. The contractile force decreased by about 40, 60 and 70% of the control within 5, 10 and 30 min respectively, of perfusing the heart with hypoxic medium containing glucose. In hearts perfused for 10 min with hypoxic medium containing glucose, calcium binding and uptake by the microsomal fraction decreased significantly. However, mitochondrial calcium binding, but not uptake, decreased significantly on perfusing the hearts with hypoxic medium containing glucose for 20 to 30 min when the microsomal calcium transport was markedly depressed. Reduction in contractile force, calcium binding and uptake by the microsomal fraction as well as calcium binding by mitochondria of hearts made hypoxic for 30 min recovered towards normal upon reperfusion with control medium for 15 min. On the other hand, omitting glucose from the hypoxic medium significantly decreased calcium binding by mitochondrial and microsomal fractions within 10 min of perfusion in comparison to the control and accelerated the effects of hypoxia upon contractile force and microsomal calcium uptake. In contrast to the hypoxic hearts, the mitochondrial calcium uptake decreased significantly and the magnitude of depression in the microsomal calcium binding was appreciably greater in hearts made to fail to a comparable degree upon perfusion with substrate-free medium. The observed defects in calcium transporting properties of microsomal and mitochondrial membranes appear secondary to the contactile failure in hypoxic hearts.     

Experimental study of follicle formation in suppressed parathyroid glands of Mongolian gerbils.

Parathyroid follicle formation was studied in Mongolian gerbils subjected to different concentrations of calcium in vivo and in vitro, using light and electron microscopic methods, including the potassium pyroantimonate technique and x-ray microanalysis for identification of cations. Follicles were frequent at high calcium concentration, but sparse at intermediate and low levels of calcium. Two main types of follicle were differentiated: "degenerative follicles" containing cellular debris and lined by smooth-surfaced epithelium which occasionally showed degenerative changes; and "secretory follicles" characterized by amorphous and granular contents, and an epithelium possessing microvilli and cytoplasmic projections. Amorphous masses were also seen in dilated intercellular spaces and in dilated cisterns of rough endoplasmic reticulum in the follicle epithelium. Calcium-containing precipitates were found in degenerating chief cells, and between degenerating cells and follicles. Parathyroid follicles are believed to be formed by degeneration of suppressed chief cells (degenerative follicles), and by secretion of hormonal and/or other substances into dilated intercellular spaces which progressively increase in size to form follicular cavities (secretory follicles), thereby possibly reducing the level of metabolically active parathyroid hormone. Functional suppression is believed to underlie the development of parathyroid follicles.     

Regulation of mitochondrial dehydrogenases by calcium ions

Studies in Bristol in the 1960s and 1970s, led to the recognition that four mitochondrial dehydrogenases are activated by calcium ions. These are FAD-glycerol phosphate dehydrogenase, pyruvate dehydrogenase, NAD-isocitrate dehydrogenase and oxoglutarate dehydrogenase. FAD-glycerol phosphate dehydrogenase is located on the outer surface of the inner mitochondrial membrane and is influenced by changes in cytoplasmic calcium ion concentration. The other three enzymes are located within mitochondria and are regulated by changes in mitochondrial matrix calcium ion concentration. These and subsequent studies on purified enzymes, mitochondria and intact cell preparations have led to the widely accepted view that the activation of these enzymes is important in the stimulation of the respiratory chain and hence ATP supply under conditions of increased ATP demand in many stimulated mammalian cells. The effects of calcium ions on FAD-isocitrate dehydrogenase involve binding to an EF-hand binding motif within this enzyme but the binding sites involved in the effects of calcium ions on the three intramitochondrial dehydrogenases remain to be fully established. It is also emphasised in this article that these three dehydrogenases appear only to be regulated by calcium ions in vertebrates and that this raises some interesting and potentially important developmental issues.     

THE SUBCELLULAR LOCALIZATION OF CALCIUM ION IN MAMMALIAN MYOCARDIUM

This study was designed to investigate the proposition that subcellular calcium is sequestered in specific sites in mammalian myocardium. 29 functioning dog papillary muscles were fixed through the intact vascular supply by means of osmium tetroxide containing a 2% concentration of potassium pyroantimonate (K₂H₂Sb₂O₇·₄H₂O). Tissue examined in the electron microscope showed a consistent and reproducible localization of the electron-opaque pyroantimonate salts of sodium and calcium to distinct sites in the tissue. Sodium pyroantimonate was found exclusively in the extracellular space and clustered at the sarcolemmal membrane. Calcium pyroantimonate, on the other hand, identified primarily by its susceptibility to removal by chelation with EGTA and EDTA, was consistently found densely concentrated in the lateral sacs of the sarcoplasmic reticulum and over the sarcomeric I bands. M zones were virtually free of precipitate. The implications of these findings with respect to various parameters of muscle function are discussed.