Distribution of calcium ions in the muscle fibers of the rat diaphragm depending upon their state and the method of histochemical treatment

Distribution of calcium ions in the rat diaphragm muscle fibers has been studied electron histochemically using various fixation techniques and chemical treatment of the tissue. When potassium pyroantimonate in water solution is used after a short perfusate fixation with aldehydes, the reaction product granules are revealed in mitochondria, in the disk I, in the center of the disk A, more seldom the precipitate is found in the sarcoplasmic reticulum (SR) and in the T-system. The presence of calcium ions in the precipitate is proved by means of treatment the preparations with ethylenglycol- and ethylen-diamine-tetra-acetic acids. When contracture is resulted from potassium rhodanide administration, in mitochondria the reaction product granules decrease in their number, the precipitate disappears from the central part of the disk A, while the number of the granules increases in the SR terminal cisterns. The data obtained are compared with calcium ions distribution observed at the freezing-substitution method without an additional chemical fixation, as well as the histochemical fixations after Oschman method and at a usual fixation with OsO4. Certain similarity is revealed in distribution of the calcium pyroantimonate granules at aldehyde fixation and when the freezing-substitution method is used.     

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.     

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.     

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.     

THE CELL JUNCTION IN A LAMELLIBRANCH GILL CILIATED EPITHELIUM : Localization of Pyroantimonate Precipitate

The junctional complex in the gill epithelium of the freshwater mussel (Elliptio complanatus) consists of an intermediary junction followed by a 2–3 µ long septate junction. Homologous and heterologous cell pairs are connected by this junction. After fixation with 1% OsO₄ containing 1% potassium pyroantimonate, electron microscopy of the gill reveals deposits of electron-opaque precipitate, specifically and consistently localized along cellular membranes. In both junctional and nonjunctional membrane regions, the precipitate usefully outlines the convolutions without obliterating the 150 A intercellular space, which suggests the rarity or absence of either vertebrate-type gap or tight junctions along the entire cell border. The precipitate appears on the cytoplasmic side of the limiting unit membranes of frontal (F), laterofrontal (LF), intermediate (I), lateral (L), and postlateral (PL) cells. The membrane surfaces of certain vesicles of the smooth endoplasmic reticulum, of multivesicular bodies, and of mitochondrial cristae contain precipitate, as does the nucleolus. In other portions of the cell, precipitate is largely absent. The amount of over-all deposition is variable and depends on the treatment of the tissue prior to fixation. Deposition is usually enhanced by pretreatment with 40 mM NaCl as opposed to 40 mM KCl, which suggests that the precipitate is in part sodium pyroantimonate. Treatment with 0.2 mM ouabain does not enhance deposition. Regional differentiation of cell membranes with respect to their ability to precipitate pyroantimonate is found in at least three instances: (a) between the ciliary membranes and other portions of the cell membrane: the precipitate terminates abruptly at the ciliary base, (b) between the LF and I cell borders: the precipitate is asymmetric, favoring the LF side of the junction, and (c) between the septate junctional membrane and adjacent membrane: the precipitate occurs periodically throughout the septate junction region with the periodicity corresponding to the spacing of the septa. This suggests that different regions of the cell membrane may have differing ion permeability properties and, in particular, that the septa may be the regions of high ion permeability in the septate junction.     

THE DISTRIBUTION OF INORGANIC CATIONS IN MOUSE TESTIS : Electron Microscope and Microprobe Analysis

For localization of pyroantimonate-precipitable cations, mouse testes were fixed with a saturated aqueous solution of potassium pyroantimonate (pH about 9.2, without addition of any conventional fixative), hardened with formaldehyde, and postosmicated. A good preservation of the cell membranes and over-all cell morphology is obtained as well as a consistent and reproducible localization of the insoluble antimonate salts of magnesium, calcium, and sodium. Four sites of prominent antimonate deposits are revealed, besides a more or less uniform distribution of the precipitates. These sites are: (a) In the walls of the seminiferous tubules, localized in two concentric layers corresponding to the inner and outer layers of the tubular wall; (b) Around the blood vessels and adjacent connective tissue; (c) At the area of contact between the Sertoli cell and spermatids, where a double line of precipitate surrounds the head of the mature spermatids; and (d) In the cell nuclei, disposed between regions of the condensed chromatin. The nucleus of mature spermatids did not show any sign of antimonate precipitation. The implications of this inorganic cation distribution are discussed with relation to their anionic counterparts, their localization in other animal and plant tissues, and the possibility that those sites may represent barriers to the free passage of ions.     

Cytochemical analysis of intracellular calcium distribution in the anterior pituitary of the rat

Summary In an attempt to assign morphologic identities to previously distinguished functional calcium compartments in the anterior pituitary of the rat, we employed the potassium pyroantimonate technique for cation localization. Tissues were incubated for In at 37°C in control medium; with 10mM theophylline; or with depolarizing amounts of potassium. Precipitate was quantified on photomicrographs of tissue prepared for electron microscopy with a Talos Systems Digitizer. The nature of the electron dense precipitate was dependent on the experimental state of the tissue. Treatment with 5 mM EGTA abolished the dense precipitate. Electron microprobe analysis also confirmed that calcium was the predominant cation in the observed precipitate. The most significant changes in precipitate deposition occurred along the plasma membrane, the limiting membrane of secretory granules and within mitochondria. Dense precipitate was present along the plasma membrane only in cells treated with potassium. Control tissue exhibited higher levels of precipitate associated with the limiting membrane of secretory granules than either theophylline-treated or potassium-treated tissue. Mitochondria contained more precipitate in potassium-treated tissue than in controls; the mitochondria of theophylline-treated tissue contained intermediate levels of precipitate. Addition of either theophylline or depolarizing amounts of potassium has been associated with hormone secretion in anterior pituitary tissue of normal rats. Kinetic studies in our laboratory indicate that intracellular calcium shifts occur. The pyroantimonate technique is useful in verifying morphologically the calcium compartments involved in shifts in intracellular calcium.     

Subcellular distribution of calcium in zona fasciculata cells of the rat adrenal cortex

Zona fasciculata cells from the adrenal cortex of female Sprague-Dawley rats were fixed by immersion in potassium pyroantimonate-osmium tetroxide and potassium pyroantimonate-glutaraldehyde to study the distribution of calcium. Potassium pyroantimonate-osmium tetroxide treatment gave reproducible patterns of electron-opaque precipitate, whereas inconsistent deposits of reaction product were seen after potassium pyroantimonate-glutaraldehyde fixation. Nuclei showed sparse precipitate over heterochromatin and dense aggregates over areas of nucleoli surrounded by portions of the nucleolar-dense component. Two major cytoplasmic sites of precipitate were identified: mitochondria and vesicles of smooth endoplasmic reticulum. Most of the intramitochondrial precipitate was localized to the intracristal space. Precipitate was also seen in vesicles of Golgi apparatus. The extracellular space was filled with closely packed electron-opaque particles. Observation of tissues treated with control fixative saturated with EGTA showed little if any reaction, confirming that calcium was the primary cation precipitated by potassium pyroantimonate. Our results provide a method suitable for accurate localization of calcium in adrenocortical cells.     

INORGANIC CATIONS IN RAT KIDNEY : Localization with Potassium Pyroantimonate-Perfusion Fixation

For localization of pyroantimonate-precipitable cations, rat kidney was fixed by perfusion with a saturated aqueous solution of potassium pyroantimonate (pH about 9.2, without addition of any conventional fixative). A remarkably good preservation of the tissue and cell morphology was obtained as well as a consistent and reproducible localization of the insoluble antimonate salts of magnesium, calcium, and sodium. All proximal and distal tubules and glomeruli were delimited by massive electron-opaque precipitates localized in the basement membrane and, to a lesser extent, in adjacent connective tissue. In the intraglomerular capillaries the antimonate precipitate was encountered in the basement membranes and also between the foot processes. In addition to a more or less uniform distribution in the cytoplasm and between the microvilli of the brush border, antimonate precipitates were found in all cell nuclei, mainly between the masses of condensed chromatin. The mitochondria usually contained a few large antimonate deposits which probably correspond to the so-called "dense granules" observed after conventional fixations.     

The penetration of sodium into the brain following a cisternal injection of sodium chloride with particular emphasis on the area postrema

Summary Following an intracisternal injection of sodium chloride, sodium has been localized in paraventricular and subpial tissues of the posterior fossa by means of the pyroantimonate histochemical technique, with the use of a buffered pyroantimonate medium. The electron dense deposit is present in these tissues within 4 minutes after injection and is found only extracellularly except in the area postrema. This finding supports the contention that sodium is chiefly an extracellular ion and that the cerebrospinal fluid and the extracellular fluid are in equilibrium at these sites. In the area postrema, an intracellular precipitate is noted in the vesicular structures of the atypical astrocytes of this structure and in pinocytic vesicles of the large blood vessels. The intraglial localization of sodium in the area postrema is discussed in relation to a possible function of this structure as a regulator of cerebrospinal fluid ionic content.This work was supported by grant number NB-08549-02 from the National Institute of Neurological Disease and Stroke, Bethesda, Maryland.     

Localization of cations by pyroantimonate. I. Influence of fixation on distribution of calcium and sodium. An approach by analytical ion microscopy

A modification of the potassium pyroantimonate (PA) method for localization of calcium and sodium was tested using skeletal muscle of mouse. Massive diffusion of these cations, depending on the method of fixation, was demonstrated by analytical ion microscopy (AIM) images on the optical microscopy level. Rapid penetration of the fixative appeared to be the principal condition that reduced diffusion of Ca2+ and Na+. Paraformaldehyde (2%) appeared more efficient than glutaraldehyde (1%) for preserving metal composition. Addition of 1% phenol strikingly improved the quality of the AIM images. Supersaturated PA (4%) appeared to retain about 10 times more sodium in the tissue than insaturated PA (2%). The role of different buffers is also discussed, particularly collidine, which permitted better preservation of sodium. Fixation with this buffer should be very useful for study by AIM of large-scale distribution of sodium. These results are analyzed at the ultrastructural level in the accompanying report.     

Ultrastructural and physiological studies on the longitudinal body wall muscle of Dolabella auricularia. II. Localization of intracellular calcium and its translocation during mechanical activity

The localization of Ca-accumulating structures in the longitudinal body wall muscle (LBWM) of the opisthobranch mollusc Dolabella auricularia and their role in the contraction-relaxation cycle were studied by fixing the LBWM fibers at rest and during mechanical response to 400 mM K or to 10(-4)--10(-3) M acetylcholine in a 1% OsO4 solution containing 2% K pyroantimonate. In the resting fibers, electron-opaque pyroantimonate precipitate was mostly localized at the peripheral structures, i.e., along the inner surface of the plasma membrane, at the membrane of the surface tubules, and at the sarcoplasmic reticulum. In the fibers fixed during mechanical activity, the precipitate was diffusely distributed in the myoplasm in the form of numerous particles with corresponding decrease in the amount of the precipitate at the peripheral structures. Electron-probe X-ray microanalysis showed the presence of Ca in the precipitate, indicating that the precipitate may serve as a measure of Ca localization. These results are in accord with the view that, in the LBWM, the Ca stored in the peripheral structures is released into the myoplasm to activate the contractile mechanism.     

Modifications of S100-protein immunoreactivity in rat brain induced by tissue preparation

Immunocytochemistry using antibodies against various molecular forms of the Ca⁺⁺ and Zn⁺⁺-binding S100 proteins predominantly labelled astrocytes. However, especially in the neocortex the staining pattern is variable. Methods of tissue preparation have been evaluated with the aim to preserve as much S 100 immunoreactivity as possible. Optimal results were obtained after perfusion fixation with 4–5% aldehydes, 0.1M sodium cacodylate, 0.1% CaCl₂, pH 7.3. In such preparations, astrocytes were completely labelled including their lamellar compartments in large parts of the central nervous system. Ca⁺⁺-withdrawal had adverse affects on S100 immunoreactivity. Cryostat sections treated with EDTA-containing solutions before fixation showed that Ca⁺⁺-free S100 can apparently not be fixed to the tissue. Perfusion fixatives containing EDTA resulted in inhomogeneous loss of S100 staining, indicating a differential susceptibility of astrocytic subpopulations. A different type of reduction in S100 immunoreactivity occurred around large neocortical blood vessels. Perivascular defects in immunostaining occasionally appeared even after optimal fixation, but could be regularly provoked by mildly acidic fixation (pH 6.6) or prolonged barbiturate anaesthesia. These defects might be based on S100 release into the cerebrospinal fluid. Presumably under none of the conditions studied can the immunoreactivity of all S100-forms and-fractions be completely preserved in the tissue. However, recommendations are presented for optimizing tissue preparation, to the extent that premortal modifications affecting the stainability of astrocytes may be detected by S100 immunohistochemistry in fixed brain tissue.     

陆地棉雌蕊的花粉管生长途径中钙分布的超微细胞化学定位

用焦锑酸盐沉淀法对陆地棉(Gossypium hirsutum L.)授粉前后的柱头、花柱、珠孔与珠心组织中的钙进行了超微细胞化学定位。X射线波谱与能谱分析证明所定位的沉淀确系焦锑酸钙。观察结果表明:在整个花粉管生长途径中的雌蕊组织,钙分布均较其它相邻组织密集;钙主要分布在细胞壁与胞间基质等质外体系统中。在雌蕊中生长的花粉管,其尖端细胞器区也有丰富的钙。     

Calcium binding to intestinal membranes

Flame photometry reveals that glutaraldehyde and buffer solutions in routine use for electron microscopy contain varying amounts of calcium. The presence of electron-opaque deposits adjacent to membranes in a variety of tissues can be correlated with the presence of calcium in the fixative. In insect intestine (midgut), deposits occur adjacent to apical and lateral plasma membranes. The deposits are particularly evident in tissues fixed in glutaraldehyde without postosmication. They are also observed in osmicated tissue if calcium is added to wash and osmium solutions. Deposits are absent when calcium-free fixatives are used, but are present when traces of CaCl₂ (as low as 5 x 10^-5 M) are added. The deposits occur at regular intervals along junctional membranes, providing images strikingly similar to those obtained by other workers who have used pyroantimonate in an effort to localize sodium. Other divalent cations (Mg⁺⁺, Sr⁺⁺, Ba⁺⁺, Mn⁺⁺, Fe⁺⁺) appear to substitute for calcium, while sodium, potassium, lanthanum, and mercury do not. After postfixing with osmium with calcium added, the deposits can be resolved as patches along the inner leaflet of apical and lateral plasma membranes. The dense regions may thus localize membrane constituents that bind calcium. The results are discussed in relation to the role of calcium in control of cell-to-cell communication, intestinal calcium uptake, and the pyroantimonate technique for ion localization.     

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.     

Evidence for extracellular localization of activator calcium in dog coronary artery smooth muscle as studied by the pyroantimonate method

Summary Correlated physiological and electron-microscopic studies were made on the source of calcium activating the contractile system (activator calcium) in dog coronary artery smooth muscle fibers. The magnitude of contracture tension induced by 100 mM K⁺ was dependent on external Ca²⁺ concentration and reduced or eliminated by factors known to reduce the Ca²⁺ spike or ca²⁺ influx. Little or no mechanical response was elicited by treatments known to cause release of intracellularly stored calcium. These results indicated that the contractile system is mainly activated by the inward movement of extracellular calcium. In accordance with the physiological experiments, electron-opaque pyroantimonate precipitate containing calcium was found in the lumina of caveolae, but not in any intracellular structures close to the plasma membrane, when the relaxed fibers were fixed in a 1% osmium tetroxide solution containing 2% potassium pyroantimonate. If the contracted fibers were fixed in the same solution, the pyroantimonate precipitate was diffusely distributed in the myoplasm in the form of numerous particles, while the precipitate in the caveolar lumina was scarcely seen. These findings are discussed in connection with the regulation of intracellular Ca²⁺ concentration in dog coronary artery smooth muscle.     

Localization artefacts in ultracytochemical ion precipitation reactions

Summary The precipitation patterns of the following ultracytochemical methods in rat muscle cells were compared and examined critically: the potassium pyroantimonate method for calcium demonstration; the calcium phosphate technique for the Ca²⁺ — ATPase reaction; the formazan reaction for the demonstration of creatine kinase activity (all performed on heart muscle); and the lead phosphate technique for the Mg²⁺ — ATPase reaction in skeletal muscle. Using X-ray microanalysis, it was found that the antimonate precipitate contains only calcium as the precipitated ion in the vast majority of cases. Most probably it consists of pure calcium pyroantimonate. However, in myocytes showing the well-established precipitation pattern, the concentration of calcium was estimated to be about two orders of magnitude higher than the native concentration of total intracellular calcium. It is concluded that calcium ions diffuse freely from the extracellular space and from adjacent cells into cells containing antimonate and are precipitated mostly at sites where heterogeneous nucleation is facilitated by intracellular catalysts (biopolymers).As shown by the similar precipitation patterns for the four reactions compared, these catalysts are not specific to any of these reactions and are most probably neither calcium-binding sites nor sites of any one of the enzymes examined in the native cell.     

AN ELECTRON MICROSCOPIC CHARACTERIZATION OF CLASSES OF SYNAPTIC VESICLES BY MEANS OF CONTROLLED ALDEHYDE FIXATION

Examination of variables of aldehyde fixation that may affect the shape of agranular synaptic vesicles has revealed that even brief storage of aldehyde-perfused nervous tissue pieces in cacodylate buffer, prior to hardening in osmium tetroxide, has an unusually severe flattening effect on agranular vesicles of a particular type. These are the vesicles of peripheral cholinergic axon endings, and of certain central synaptic bulbs. Types of synaptic bulbs can now be further defined on the basis of shape of agranular synaptic vesicles under controlled conditions of aldehyde fixation. Previously described "S" bulbs in the spinal cord contain uniformly spheroid vesicles, which are wholly resistant to flattening. Previously described "F" bulbs contain somewhat smaller agranular vesicles that are flattened after aldehyde fixation, even when this is followed by prompt hardening in osmium tetroxide solution. A third type, previously characterized as having irregularly round agranular vesicles after the above treatment, contains only severely flattened vesicles when the osmium tetroxide hardening is preceded by even a brief wash with sodium cacodylate buffer containing sucrose. Moreover, the "third" type is characteristic of all cholinergic peripheral axon endings examined, as well as the large axosomatic ("L") synaptic bulbs of the spinal cord.