Glycosaminoglycans (GAGs)are essential components of the extracellular matrix of various tissues.They have been known to regulate cellular and extracellular environments for cell survival and differentiation in various physiologic and pathologic conditions.The present study was conducted to identify the distribution of GAGs in subepithelial cortical fibers of normal and traumatic cataractous lens of rabbits. Traumatic cataract was made by piercing the anterior lens capsule with 25-gauge sharp needle and the rabbits were killed at different time intervals (1, 2, 3 and 4 weeks).From the enucleated eyes subepithelial lens cortical fibers were obtained.A normal rabbit lens was used for the control. The specimens were stained with 0.05%ruthenium red (RR)and processed for histochemical electron microscopy. RR reactive materials were identified as fine granular or filamentous structures.In normal rabbit lens they were present mainly along the surface of lens epithelial cells, the surface of the subepithelial cortical fibers. In cataractous rabbits lens, strong RR positive reactions were observed along the surface of the lens epithelial cells and subepthelial cortical fibers as well as at the intercortical fiber spaces and even within its micro-organelles. This investigation resulted in an illustration of the ultrastructural distribution of GAGs in normal and traumatic cataractous lens of rabbit.this may suggest altered GAG distribution may closely related to the formation of lens opacity.
Cataract* ; Cell Survival ; Epithelial Cells ; Extracellular Matrix ; Glycosaminoglycans ; Microscopy, Electron ; Needles ; Rabbits* ; Ruthenium Red* ; Ruthenium*

N-acetyl-L-cysteine (NAC) and cysteine have been implicated in a number of human neutrophils' functional responses. However, though Ca²⁺ signaling is one of the key signalings contributing to the functional responses of human neutrophils, effects of NAC and cysteine on intracellular calcium concentration ([Ca²⁺]ᵢ) in human neutrophils have not been investigated yet. Thus, this study was carried out with an objective to investigate the effects of NAC and cysteine on [Ca²⁺]ᵢ in human neutrophils. We observed that NAC (1 µM ~ 1 mM) and cysteine (10 µM ~ 1 mM) increased [Ca²⁺]ᵢ in human neutrophils in a concentration-dependent manner. In NAC pre-supplmented buffer, an additive effect on N-formyl-methionine-leucine-phenylalanine (fMLP)-induced increase in [Ca²⁺]ᵢ in human neutrophils was observed. In Ca²⁺-free buffer, NAC- and cysteine-induced [Ca²⁺]ᵢ increase in human neutrophils completely disappeared, suggesting that NAC- and cysteine-mediated increase in [Ca²⁺]ᵢ in human neutrophils occur through Ca²⁺ influx. NAC- and cysteine-induced [Ca²⁺]ᵢ increase was effectively inhibited by calcium channel inhibitors SKF96365 (10 µM) and ruthenium red (20 µM). In Na⁺-free HEPES, both NAC and cysteine induced a marked increase in [Ca²⁺]ᵢ in human neutrophils, arguing against the possibility that Na⁺-dependent intracellular uptake of NAC and cysteine is necessary for their [Ca²⁺]ᵢ increasing activity. Our results show that NAC and cysteine induce [Ca²⁺]ᵢ increase through Ca²⁺ influx in human neutrophils via SKF96365- and ruthenium red-dependent way.
Acetylcysteine* ; Calcium Channels ; Calcium* ; Cysteine* ; HEPES ; Humans* ; Neutrophils* ; Ruthenium ; Ruthenium Red

This study was performed to evaluate the histochemical changes of the conjunctival epithelial cell in eyes with dry eye syndrome. The authors selected 10 eyes clinically diagnosed as dry eye syndrome and 2 eyes which were enucleated due to choroidal melanoma as control group. The conjunctiva was stained with ruthenium red to enhance the detection of glycoprotein in conjunctival epithelium and observed by electron microscopy. The conjunctiva with dry eye syndrome showed the epithelial stratification, elon gation of the epithelial cells, reduction of microplicae in number, and irregular distribution of mucin. These results suggest that the mucin in the conjunctiva with dry eye syndrome may be abnormal in function and may lead to clumping.
Choroid ; Conjunctiva ; Dry Eye Syndromes* ; Epithelial Cells ; Epithelium ; Glycoproteins ; Melanoma ; Microscopy, Electron* ; Mucins ; Ruthenium Red

The distribution of glycosaminoglycans in Bruch`s membrane and sensory retina of human eye was evaluated with histochemical electron microscope using 0.05%ruthenium red (RR). Seven donated or enucleated eyeballs without chorioretinal disease were used.Electron-dense RR reactive materials were identified as fine discrete granules along the basement membrane of the retinal pigment epithelium, in the inner and outer collagen layer and central elastic layer of Bruch 's membrane, and in the interphotorecep- tor matrix. In addition, they were observed at the cytoplasmic wall of photoreceptor cells, their intercellular spaces, and the internal limiting membrane. These results demonstrated that GAGs are widely distributed within the interphotoreceptor matrix, Bruch`s membrane, and intercellular spaces of retinal neurons. They are considered to function as a mechanical supporter as well as a selective filtration barrier through which the metabolitespass.
Basement Membrane ; Collagen ; Cytoplasm ; Extracellular Space ; Filtration ; Glycosaminoglycans* ; Humans ; Membranes* ; Photoreceptor Cells ; Retina* ; Retinal Neurons ; Retinal Pigment Epithelium ; Ruthenium Red* ; Ruthenium*

The secretion of insulin from pancreatic beta-cells is triggered by the influx of Ca2+ through voltage-dependent Ca2+ channels. The resulting elevation of intracellular calcium ([Ca2+]i) triggers additional Ca2+ release from internal stores. Less well understood are the mechanisms involved in Ca2+ mobilization from internal stores after activation of Ca2+ influx. The mobilization process is known as calcium-induced calcium release (CICR). In this study, our goal was to investigate the existence of and the role of caffeine-sensitive ryanodine receptors (RyRs) in a rat pancreatic beta-cell line, INS-1 cells. To measure cytosolic and stored Ca2+, respectively, cultured INS-1 cells were loaded with fura-2/AM or furaptra/AM. [Ca2+]i was repetitively increased by caffeine stimulation in normal Ca2+ buffer. However, peak [Ca2+]i was only observed after the first caffeine stimulation in Ca2+ free buffer and this increase was markedly blocked by ruthenium red, a RyR blocker. KCl-induced elevations in [Ca2+]i were reduced by pretreatment with ruthenium red, as well as by depletion of internal Ca2+ stores using cyclopiazonic acid (CPA) or caffeine. Caffeine-induced Ca2+ mobilization ceased after the internal stores were depleted by carbamylcholine (CCh) or CPA. In permeabilized INS-1 cells, Ca2+ release from internal stores was activated by caffeine, Ca2+, or ryanodine. Furthermore, ruthenium red completely blocked the CICR response in permeabilized cells. RyRs were widely distributed throughout the intracellular compartment of INS-1 cells. These results suggest that caffeine-sensitive RyRs exist and modulate the CICR response from internal stores in INS-1 pancreatic beta-cells.
Animals ; Caffeine ; Calcium ; Carbachol ; Cytosol ; Indoles ; Insulin ; Insulinoma ; Rats ; Ruthenium Red ; Ryanodine ; Ryanodine Receptor Calcium Release Channel

Mast cells are primary mediators of allergic inflammation. Beta-1,3-glucan (BG) protects against infection and shock by activating immune cells. Activation of the BG receptor induces an increase in intracellular Ca2+, which may induce exocytosis. However, little is known about the precise mechanisms underlying BG activation of immune cells and the possible role of mitochondria in this process. The present study examined whether BG induced mast cell degranulation, and evaluated the role of calcium transients during mast cell activation. Our investigation focused on the role of the mitochondrial calcium uniporter (MCU) in BG-induced degranulation. Black mouse (C57) bone marrow-derived mast cells were stimulated with 0.5 microg/ml BG, 100 microg/ml peptidoglycan (PGN), or 10 microM A23187 (calcium ionophore), and dynamic changes in cytosolic and mitochondrial calcium and membrane potential were monitored. BG-induced mast cell degranulation occurred in a time-dependent manner, and was significantly reduced under calcium-free conditions. Ruthenium red, a mitochondrial Ca2+ uniporter blocker, significantly reduced mast cell degranulation induced by BG, PGN, and A23187. These results suggest that the mitochondrial Ca2+ uniporter has an important regulatory role in BG-induced mast cell degranulation.
Animals ; Calcimycin ; Calcium* ; Cytosol ; Exocytosis ; Inflammation ; Ion Transport* ; Mast Cells* ; Membrane Potentials ; Mice* ; Mitochondria ; Peptidoglycan ; Ruthenium Red ; Shock

PURPOSE: This study was performed to investigate the distribution of glycosaminiglycans by the activated retinal neuronal cell on the cultured retinal tissue. METHODS: The retinal tissue was obtained from the donor eyeball without the pathological findings of the retina. The dissected retinal tissue was cultured for 2 weeks in the culture media and reacted with ruthenium red dye to observe the glycosaminoglycans reaction with transmission electron microscopy. RESULTS: The histochemical reaction to ruthenium red on the cultured retina tissue was prominent on the internal limiting membrane, intercellular space between the axons and dendrites at the outer and inner plexiform layers and interphotoreceptor matrix. CONCLUSIONS: These findings suggest that GAGs may be released into the extracellular spaces between the axons and dendrites, which modulate the retinal circuits.
Axons ; Culture Media ; Dendrites ; Extracellular Space ; Glycosaminoglycans* ; Humans ; Membranes ; Microscopy, Electron ; Microscopy, Electron, Transmission ; Retina* ; Retinal Neurons ; Retinaldehyde ; Ruthenium Red ; Tissue Donors

Reciprocal exchange of morphogenetic proteins between epithelial and mesenchymal cells in a stem/progenitor cell niche results in formation of a nephron. To maintain diffusion of morphogenetic proteins, it is assumed that a close contact exists between involved cells. However, recent publications underline that both types of stem/progenitor cells are separated by a striking interface. To explore this microarchitecture in detail, neonatal rabbit kidneys were fixed in traditional glutaraldehyde (GA) solution for transmission electron microscopy. For contrast enhancing specimens were fixed in GA solution including cupromeronic blue, ruthenium red or tannic acid. To record same perspectives, embedded blocks of parenchyma were cut in exactly orientated vertical and transverse planes to lining collecting ducts. Electron microscopy of specimens fixed by traditional GA solution illustrates a spatial separation of stem/progenitor cells and an unobstrusively looking interface. In contrast, advanced fixation of specimens in GA solution including cupromeronic blue, ruthenium red and tannic acid unmasks earlier not visible extracellular matrix. In addition, projections of mesenchymal cells covered by matrix cross the interface to contact epithelial cells. Surprisingly, the end of a mesenchymal cell projection does not dangle but is enclosed in a fitting sleeve and connected via tunneling nanotubes with the plasma membrane of an epithelial cell. Regarding this complex ensemble the question is to what extent illustrated cell-cell connections and extracellular matrix are involved in communication and transmission of morphogenetic proteins during induction of a nephron.
Cell Membrane ; Diffusion ; Epithelial Cells ; Extracellular Matrix* ; Glutaral ; Kidney ; Microscopy, Electron ; Microscopy, Electron, Transmission ; Nanotubes ; Nephrons ; Ruthenium Red ; Strikes, Employee ; Tannins

To investigate the properties of ryanodine binding sites of the bird skeletal SR vesicles, SDS PAGE, purification of RyR, and (3H)ryanodine binding study were carried out in the SR vesicles prepared from the chicken pectoral muscle. The chicken SR vesicles have two high molecular weight (HMW) protein bands as in eel SR vesicles on SDS PAGE. The HMW bands on SDS PAGE were found in the (3H) ryanodine peak fraction (Fr3-5) obtained from the purification step of the ryanodine receptor protein. Bmax and KD of the chicken (3H)ryanodine binding sites were 12.52 pmol/mg protein and 14.53 nM, respectively. Specific (3H)ryanodine binding was almost maximal at 50~100 micrometer Ca2+, but was not increased by 5 mM AMP and not inhibited by high Ca2+. Binding was significantly inhibited by 20~100 micrometer ruthenium red and 1 mM tetracaine, but slightly inhibited by Mg2+. From the above results, it is suggested that chicken SR vesicles have the ryanodine binding sites to which the binding of ryanodine is almost maximal at 50~10 micrometer Ca2+, is significantly inhibited by ruthenium red and tetracaine, slightly inhibited by Mg2+, but not affected by AMP and not inhibited by high Ca2+.
Binding Sites* ; Birds ; Chickens* ; Eels ; Electrophoresis, Polyacrylamide Gel ; Molecular Weight ; Ruthenium Red ; Ryanodine ; Ryanodine Receptor Calcium Release Channel ; Sarcoplasmic Reticulum ; Tetracaine

Intracellular calcium (Ca²⁺) oscillation is an initial event in digestive enzyme secretion of pancreatic acinar cells. Reactive oxygen species are known to be associated with a variety of oxidative stress-induced cellular disorders including pancreatitis. In this study, we investigated the effect of hydrogen peroxide (H₂O₂) on intracellular Ca²⁺ accumulation in mouse pancreatic acinar cells. Perfusion of H₂O₂ at 300 µM resulted in additional elevation of intracellular Ca²⁺ levels and termination of oscillatory Ca²⁺ signals induced by carbamylcholine (CCh) in the presence of normal extracellular Ca²⁺. Antioxidants, catalase or DTT, completely prevented H₂O₂-induced additional Ca²⁺ increase and termination of Ca²⁺ oscillation. In Ca²⁺-free medium, H₂O₂ still enhanced CCh-induced intracellular Ca²⁺ levels and thapsigargin (TG) mimicked H₂O₂-induced cytosolic Ca²⁺ increase. Furthermore, H₂O₂-induced elevation of intracellular Ca²⁺ levels was abolished under sarco/endoplasmic reticulum Ca²⁺ ATPase-inactivated condition by TG pretreatment with CCh. H₂O₂ at 300 µM failed to affect store-operated Ca²⁺ entry or Ca²⁺ extrusion through plasma membrane. Additionally, ruthenium red, a mitochondrial Ca²⁺ uniporter blocker, failed to attenuate H₂O₂-induced intracellular Ca²⁺ elevation. These results provide evidence that excessive generation of H₂O₂ in pathological conditions could accumulate intracellular Ca²⁺ by attenuating refilling of internal Ca²⁺ stores rather than by inhibiting Ca²⁺ extrusion to extracellular fluid or enhancing Ca²⁺ mobilization from extracellular medium in mouse pancreatic acinar cells.
Acinar Cells* ; Animals ; Antioxidants ; Calcium* ; Carbachol ; Catalase ; Cell Membrane ; Cytosol ; Extracellular Fluid ; Hydrogen Peroxide* ; Hydrogen* ; Ion Transport ; Mice* ; Pancreatitis ; Perfusion ; Reactive Oxygen Species ; Reticulum ; Ruthenium Red ; Thapsigargin