Distribution of calcium differs in relaxed and contracted myocardial cells of the rat

Summary Myocardial cells from left ventricles of beating hearts of rats were fixed by immersion in an osmium tetroxide solution containing potassium pyroantimonate to study the electron-microscopic distribution of calcium, the cation being precipitated as an electron-opaque salt (calcium antimonate) by this cytochemical technique. The observed myocytes could be divided into two groups according to their contractile state, evaluated by sarcomere length measurements. In contracted cells (mean sarcomere length 1.43 m) the intramyoflbrillar precipitate was confined to areas of I-bands bordering the A-bands, the intermyofibrillar space showing scarce content in reaction product. Relaxed cells (mean sarcomere length 1.69 m) presented a heavy deposition of reaction product over the sarcomeres, the electron-opaque dots being absent on the H and Z bands. The sarcotubular system and mitochondria were also clearly marked by the reaction product. This second pattern of calcium distribution has not been previously described in heart muscle cells and is interpreted as corresponding to the phase of rise of intracellular calcium which is mediated by membrane depolarization. Our results suggest that different bands of heart sarcomeres show different abilities to bind calcium. The I bands retain the cation even in cells under sustained contraction, probably due to their content in calmodulin; Z and M bands are apparently not involved in calcium sequestration, whereas the content in calcium of the A bands seems to be dependent on the contraction-relaxation cycle of heart myocytes.Supported by Grant HL 06975 from the National Heart, Lung and Blood Institute and AM 18141 from the National Institute of Arthritis, Metabolism and Digestive DiseasesThe authors are grateful to Maria Kapuscinski, Luther B. Joseph, Elisabeth Lawson, Robert Linsmair and Miriam Alojipan for skilled technical assistance     

Localization of calcium in roots and microsomal membranes of corn by direct pyroantimonate precipitation

Many cell membrane systems, including microsomal vesicles of corn, are able to regulate calcium levels both in vivo and in vitro, often in an ATP-dependent, calmodulin-stimulated fashion. The purpose of this study was to determine calcium distribution in meristematic cells of intact tissue and microsomal vesicles from corn roots using direct pyroantimonate-osmium fixation. In root cells, precipitates were localized in mitochondria, plastids, the nucleus, endoplasmic reticulum, Golgi apparatus, and along the plasma membrane. Plasma membrane-enriched microsomal vesicles isolated from corn roots incubated in media to permit calcium transport before pyroantimonate-osmium fixation show internal precipitates associated with the membrane and in the lumen of the vesicles. De-staining of the sections with 1 mM EDTA or EGTA removed precipitate from the sections, confirming the presence of calcium in the antimonate precipitates. These data support biochemical data that this same membrane preparation exhibited ATP-dependent calcium sequestration that was stimulated by calmodulin, as measured by retention of 45Ca. This provides evidence that these membranes are responsible for ATP-requiring, calmodulin-stimulated calcium transport in the intact cell.     

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.     

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.     

An electron microscopic study on the effects of reserpine on the subclavian glomera of the rabbit.

Young male and female New Zealand white rabbits were given a daily subcutaneous injection of reserpine (Serpasil, Ciba; 3 mg/kg) for two days and were sacrificed 24 hours after the last injection. The subclavian glomera (aortic bodies) were processed for electron microscopy to determine the effects of this biogenic amine depleting agent on the electron-opaque cytoplasmic granules of the parenchymal type I cells. Observations of glutaraldehyde-osmium tetroxide fixed glomera from reserpinized animals showed a slight decrease in granule density of the type I cells. Glomera fixed in glutaraldehyde and incubated in potassium dichromate (pH 4.1) demonstrated a reduction in granule opacity following reserpine treatment. Control glomera incubated in potassium dichromate displayed electron-opaque granules. These results indicate that reserpine does deplete the amines without granule disappearance or changes in granule population. The positive reaction of the control tissue granules to potassium dichromate incubation suggests that the predominant biogenic amines in the electron-opaque granules are unsubstituted monoamines. Persistence of the opaque granules following reserpinization and glutaraldehyde-osmium tetroxide double fixation, may be due to amine-binding protein within the granules. The mode of granule depletion could not be ascertained with certainty.     

Fine structure of dense-cored vesicles in glomus cells of rat carotid body after fixation with permanganate or glutaraldehyde.

Glomus (Type I) cells of the carotid body of adult rats were studied electron microscopically after fixation with potassium permanganate or with glutaraldehyde and osmium tetroxide. Two permanganate fixation methods (using Krebs-Ringer-glucose, pH 7.0, or acetate buffer, pH 5.0) were compared. Numerous dense-cored vesicles were observed only in about one tenth of the glomus cells when neutral permanganate was used for fixation, although all glomus cells showed such vesicles after fixation with glutaraldehyde and osmium tetroxide. Numerous vesicles with a dense core were observed in about one third of the cells after fixation with acid potassium permanganate. With this fixation, small dense-cored vesicles similar to those in adrenergic nerve terminals were occasionally seen in the cytoplasm of glomus cells. It is tentatively concluded that the amine-storing vesicles of the carotid body are different from those in the small intensely fluorescent (SIF) cells and those in adrenergic nerve terminals.     

Fine structure of dense-cored vesicles in glomus cells of rat carotid body after fixation with permanganate or glutaraldehyde

Summary Glomus (Type I) cells of the carotid body of adult rats were studied electron microscopically after fixation with potassium permanganate or with glutaraldehyde and osmium tetroxide. Two permanganate fixation methods (using Krebs-Ringer-glucose, pH 7.0, or acetate buffer, pH 5.0) were compared. Numerous dense-cored vesicles were observed only in about one tenth of the glomus cells when neutral permanganate was used for fixation, although all glomus cells showed such vesicles after fixation with glutaraldehyde and osmium tetroxide. Numerous vesicles with a dense core were observed in about one third of the cells after fixation with acid potassium permanganate. With this fixation, small dense-cored vesicles similar to those in adrenergic nerve terminals were occasionally seen in the cytoplasm of glomus cells. It is tentatively concluded that the amine-storing vesicles of the carotid body are different from those in the small intensely fluorescent (SIF) cells and those in adrenergic nerve terminals.     

Localization of intracellular calcium during the acrosome reaction in ram spermatozoa

Calcium was identified by a pyroantimonate-osmium fixation technique in ram spermatozoa undergoing a spontaneous acrosome reaction induced by incubation of diluted semen at 39°C. Intracellular calcium was only detected in diluted spermatozoa and increased in amount and distribution over 4 hr At 4 hr, the majority of the spermatozoa displayed ultrastructural evidence of an acrosome reaction. Calcium was initially evident on the outer acrosomal membrane in multiparticulate clusters, which were seen to be located on scalloped crests of acrosomal membrane as fusion developed; it was also located in the region of the acrosomal ridge beneath the outer acrosomal membrane. Vesiculation commenced just anterior to the equatorial segment and proceeded anteriorly. As vesiculation advanced, calcium particles became associated with the periphery of the vesicles attached in the region of the fusion between the two membranes, but were never seen inside the vesicles. The equatorial segment was not labelled until much later in the reaction, at which time calcium particles were also evident on the nuclear membrane; vesiculation of the equatorial segment was also noted at this time. Dense labelling of the postacrosomal dense lamina was seen in all incubated spermatozoa. At the anterior margin of this structure the labelling was seen to be in a “sawtooth” arrangement. The disposition of the calcium both temporally and spatially is discussed in relation to its possible mechanisms in bringing about membrane fusion. © 1995 Wiley-Liss, Inc.     

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.     

Relationship between calcium binding sites and membrane fusion during the acrosome reaction induced by ionophore in ram spermatozoa

Ram spermatozoa treated with the ionophore A23187, to induce the acrosome reaction, were fixed in pyroantimonate-osmium in order to demonstrate calcium at an ultrastructural level. Prior to vesiculation, granules of precipitate occurred at points of contact between plasma and outer acrosomal membranes, suggesting a discrete role for calcium during membrane fusion. The earliest stages of vesiculation always occurred in the region just anterior to the equatorial segment and it was this same site where a marked concentration of precipitate, indicating calcium binding sites, could be demonstrated.     

Electron-dense precipitates in glomus cells of rat carotid body after fixation in glutaraldehyde and pyroantimonate-osmium tetroxide mixture as possible indicators of calcium localization

Summary An attempt was made to study the subcellular localization of calcium in carotid body glomus cells of adult rats using fixation with glutaraldehyde followed by treatment with a mixture of pyroantimonate and osmium tetroxide. Precipitates were seen as electron-dense particles (EDP) in the glomus cells, mostly within membrane-bound organelles, such as dense-cored vesicles, mitochondria, small clear vesicles, multivesicular bodies, and especially in lysosomes. However, EDP were also seen in the nuclei and in the free cytoplasm of the glomus cells and even outside them.Preincubation of carotid bodies in media containing calcium and either high potassium or calcium-ionophore A 23187 resulted in a marked increase in the general precipitation pattern, there being an increased amount of EDP both in the glomus cell nuclei and in the cytoplasm. Dense-cored vesicles more often showed precipitates than those in the controls. Some dense-cored vesicles contained multiple precipitates, typically located in the electron-lucent area between core and vesicle membrane.Extensive diffusion of ions probably occurred during fixation before precipitation, making the localization of calcium and other precipitating cations unreliable. However, it is possible that precipitates, which were regularly seen in the dense-cored vesicles, may reflect the content of bound calcium. The possible significance of calcium in glomus cell function is discussed, and the need for more adequate methods is emphasized.The present study has been supported by grants from the Finska Läkaresällskapet and the Sigrid Jusélius Foundation, Helsinki, FinlandWe wish to express our gratitude to Dr. Robert Hamill of Eli Lilly Co. for kindly providing us with the ionophore A 23187. Technical assistance by Mrs. S. Huhtaniitty and Mrs. T. Stjernvall is gratefully acknowledged     

Structure of amyloplasts and endoplasmic reticulum in the root caps of Lepidium sativum and Zea mays observed after selective membrane staining and by high-voltage electron microscopy

The structure of plastids in the root cap of cress and maize was studied by low- and high-voltage electron microscopy after staining their membranes with a mixture of zinc iodide and osmium tetroxide. In plastids of both species electron-opaque membranes were found in the plastid interior while membranes of lesser electron-opacity comprised the outer envelope and vesicles and cisternae underlying it. Electron-opaque tubules, often in groups attached to the inner membrane of the amyloplast envelope, were found in cress but not in maize. The internal, less-opaque membranes were often found associated with the starch grains. No specific association could be seen between amyloplasts and endoplasmic reticulum (ER); their surfaces showed no regular contact or connexion, though the amyloplasts clearly indented the underlying ER. The ER in statocytes was predominantly tubular in cress but predominantly cisternal in maize.Abbreviations ER endoplasmic reticulum - ZIO zinc iodideosmium tetroxide     

Ultrastructural localization of calcium in matrix vesicles and preodontoblasts of developing rat molar tooth germs during initial dentinogenesis

We investigated the ultrastructural localization of calcium in progenitor predentine and preodontoblasts of developing rat molar tooth germs using the potassium pyroantimonate technique. At the precalcification stage, antimonate reaction product was sparsely, randomly distributed in the preodontoblasts and in the progenitor predentine but no significant reaction could be noticed associated with matrix vesicles. At the matrix vesicle calcification stage, large amounts of antimonate reaction product tended to be localized in the region adjacent to the distal, outer surface membrane of preodontoblasts in which moderate antimonate reaction activity could be observed in mitochondria. Strong antimonate reaction was detected preferentially on the outer surface membrane of some matrix vesicles at this stage. At the subsequent collagen calcification stage, definite antimonate reaction was no longer seen within mitochondria of the late preodontoblasts, instead precipitate was mainly distributed in Golgi area, secretory granules and lateral intercellular spaces. It is suggested that although matrix vesicles contain few calcium capable of reacting to antimonate immediately after their biogenesis, subsequently, large amounts of calcium are accumulated associated with the outer surface membrane of matrix vesicles in the extracellular matrix.     

The structure of the endoplasmic reticulum revealed by osmium tetroxide-potassium ferricyanide staining

Postfixation of plant tissues with a mixture of osmium tetroxide and potassium ferricyanide (OsFeCN) yields a selective staining of the endoplasmic reticulum (ER) and nuclear envelope (NE). The other cytoplasmic organelles and inclusions are evident, but by comparison with the NE-ER they are weakly contrasted. Demarcation of the NE-ER results from the enhanced deposition of an electron-opaque reaction product on the inner leaflet of the membrane that extends into the cisternal space. The procedure thus renders the NE-ER readily apparent even when the elements are sectioned parallel to their surface and makes it possible to easily visualize their cellular pattern. Ultrastructural studies reveal with clarity tubular reticula and fenestrated lamellae that are extensively interconnected into one continuous membrane system. Problems with the OsFeCN procedure include the inability of the reagent to stain the NE-ER in all cells of a tissue, the occasional staining of non-ER such as dictyosomal cisternae and plastids, and the failure to selectively stain the NE-ER in protoplasts or single wall-less cells. Results obtained with OsFeCN are compared with other ER fixatives and stains including potassium permanganate and zinc iodide-osmium tetroxide. Despite its problems, under optimal circumstances OsFeCN is judged to be superior to other stain-fixatives for selectively contrasting the NE-ER compartment and is recommended generally for ultrastructural investigations.     

The use of the potassium pyroantimonate-osmium method as a means of identifying and localizing calcium at the ultrastructural level in the cells of calcifying systems.

Potassium pyroantimonate-osmium has been used to localize calcium as an electron dense precipitate in the odontoblast, ameloblast and early hypertrophic chondrocytes of the mandibular condylar growth cartilage. The precipitate exhibited a specific association with various cell organelles. The presence of calcium in the precipitates was confirmed using energy dispersive analysis by x-rays. It is suggested that this K-pyroantimonate method provides an accurate technique for the investigation of the subcellular localization of calcium in calcifying systems.     

THE EFFECTS OF RESERPINE AND HYPOXIA ON THE AMINE-STORING GRANULES OF THE HAMSTER CAROTID BODY

The carotid bodies from control, reserpine-treated, and hypoxia-treated hamsters were fixed with phosphate-buffered glutaraldehyde and osmium tetroxide, s-Collidine-buffered osmium tetroxide, or phosphate-buffered glutaraldehyde followed by potassium dichromate incubation. Following glutaraldehyde-osmium tetroxide fixation no differences in density or population of the electron-opaque granules in the glomus cells of either control or experimental animals were observed. With s-Collidine-buffered osmium tetroxide and the glutaraldehyde-dichromate technique a marked decrease in density without an appreciable reduction in number of granules was noted after reserpine treatment, while in hypoxia-treated hamsters the density and population of the granules were not different from those of the controls. The results indicate that reserpine depletes the amines without granule disappearance and that hypoxia does not affect the amine content of the granules. It is suggested that following glutaraldehyde-osmium tetroxide double fixation, persistence of the density of the granules in reserpine-treated animals is due primarily to the nonamine content, and that the amines in the glomus cells are probably not directly involved in the respiratory reflex.     

Calcium binding sites in plasmodia of Physarum polycephalum as revealed by the pyroantimonate technique

Plasmodia of the acellular slime mold, Physarum polycephalum, were treated with an osmium tetroxide fixative containing potassium pyroantimonate to precipitate calcium and thereby localize calcium binding sites and sites of increased calcium concentration. Dense calcium pyroantimonate precipitates were detected within the nucleoli. The distribution of these precipitates during interphase and mitosis coincides with the distribution of the unique minichromosomes in Physarum, i.e., the numerous short pieces of extrachromosomal nucleolar chromatin containing segments of amplified DNA coding for ribosomal RNA. Calcium pyroantimonate precipitates were present as frequent dense granules in the mitochondrial matrix and as fine precipitates in the mitochondrial nucleoid. Large calcium-containing precipitates were seen within cytoplasmic vacuoles, confirming reports by others. In addition, we have identified calcium binding sites along the cytoplasmic surface of the plasma membrane. The distribution of calcium within the plasmodium is discussed in relation to the assembly of the mitotic spindle and the regulation of cell motility.     

Ultrastructural localization of intracellular calcium stores by a new cytochemical method

We describe a new cytochemical method for ultrastructural localization of intracellular calcium stores. This method uses fluoride ions for in situ precipitation of intracellular calcium during fixation. Comparisons made using oxalate, antimonate, or fluoride showed that fluoride was clearly superior for intracellular calcium localization in eggs of the sea urchin Strongylocentrotus purpuratus. Whereas oxalate generally gave no intracellular precipitate and antimonate gave copious but random precipitate, three prominent calcium stores were detected using fluoride: the tubular endoplasmic reticulum, the cortical granules, and large, clear, acidic vesicles of unknown function. The mitochondria of these eggs generally showed no detectable calcium deposits. X-ray spectra confirmed the presence of calcium in the fluoride precipitates, although in some cases magnesium was also detected. Rat skeletal muscle and sea urchin sperm were used to test the reliability of the fluoride method for calcium localization. In rat skeletal muscle, most fluoride precipitate was confined to the sarcoplasmic reticulum. Using sea urchin sperm, which transport calcium into the mitochondria after exposure to egg jelly to induce the acrosome reaction, the expected result was also obtained. Before the acrosome reaction, sperm mitochondria contain no detectable calcium-containing precipitate. Within 4 min after induction of the acrosome reaction, the expected result was also obtained. Before the acrosome reaction, sperm mitochondria displayed many foci of calcium-containing precipitate. The use of fluoride for intracellular calcium localization therefore appears to be a substantial improvement over previous cytochemical methods.     

THE FINE STRUCTURE OF THE ELECTRIC ORGAN OF TORPEDO MARMORATA

The fine structure of the electric organ of the fish Torpedo marmorata has been examined after osmium tetroxide or potassium permanganate fixation, acetone dehydration, and Araldite embedment. This organ consists of stacks of electroplaques which possess a dorsal noninnervated and a ventral richly innervated surface. Both surfaces are covered with a thin basement membrane. A tubular membranous network whose lumen is continuous with the extracellular space occupies the dorsal third of the electroplaque. Nerve endings, separated from the ventral surface of the electroplaque by a thin basement membrane, contain synaptic vesicles (diameter 300 to 1200 A), mitochondria, and electron-opaque granules (diameter 300 A). Projections from the nerve endings occupy the lumina of the finger-like invaginations of the ventral surface. The cytoplasm of the electroplaques contains the usual organelles. A "cellular cuff" surrounds most of the nerve fibers in the intercellular space, and is separated from the nerve fibre and its Schwann cell by a space containing connective tissue fibrils. The connective tissue fibrils and fibroblasts in the intercellular space are primarily associated with the dorsal surface of the electroplaque.     

Cytochemical characterization of the endomembranous system during oocyte secondary growth in Serrasalmus spilopleura (Teleostei, Characiformes, Characidae)

The endomembranous system of Serrasalmus spilopleura oocyte secondary growth was analysed using structural and ultrastructural cytochemical techniques. In vitellogenic oocytes, the endoplasmic reticulum components, the nuclear envelope intermembranous space, some Golgi dictiossomes, lysosomes, yolk granules, regions of the egg envelope and sites of the follicle cells react to acid phosphatase detection (AcPase). The cortical alveoli, some heterogeneous cytoplasmic structures, regions of the egg envelope, and sites of the follicle cells are strongly contrasted by osmium tetroxide and zinc iodide impregnation (ZIO). The endoplasmic reticulum components, some vesicles, and sites of the follicle cells also react to osmium tetroxide and potassium iodide impregnation (KI). The biosynthetic pathway of lysosomal proteins, such as acid phosphatase, required for vitellogenesis, involves the endoplasmic reticulum, Golgi complex, vesicles with inactive hydrolytic enzymes, and, finally, lysosomes. In S. spilopleura oocytes at secondary growth, the endomembranous system takes part in the production of the enzymes needed for vitellogenesis, and in the metabolism of yolk exogenous components (AcPase detection). The endomembranous system compartments also show reduction capacity (KI reaction) and are involved in the metabolism of proteins rich in SH‐groups (ZIO reaction).