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

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

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

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

Hair cells at the base of the cochlea appear to be more susceptible to damage by the aminoglycoside gentamicin than those at the apex. However, the mechanism of base-to-apex gradient ototoxicity by gentamicin remains to be elucidated. We report here that gentamicin caused rodent cochlear hair cell damages in a time- and dose-dependent manner. Hair cells at the basal turn were more vulnerable to gentamicin than those at the apical turn. Gentamicin-conjugated Texas Red (GTTR) uptake was predominant in basal turn hair cells in neonatal rats. Transient receptor potential vanilloid 1 (TRPV1) and 4 (TRPV4) expression was confirmed in the cuticular plate, stereocilia and hair cell body of inner hair cells and outer hair cells. The involvement of TRPV1 and TRPV4 in gentamicin trafficking of hair cells was confirmed by exogenous calcium treatment and TRPV inhibitors, including gadolinium and ruthenium red, which resulted in markedly inhibited GTTR uptake and gentamicin-induced hair cell damage in rodent and zebrafish ototoxic model systems. These results indicate that the cytotoxic vulnerability of cochlear hair cells in the basal turn to gentamicin may depend on effective uptake of the drug, which was, in part, mediated by the TRPV1 and TRPV4 proteins.
Animals ; Cell Death/drug effects ; Cell Polarity/drug effects ; Cell Survival/drug effects ; Dose-Response Relationship, Drug ; Gadolinium/metabolism ; Gentamicins/*metabolism/pharmacology ; Hair Cells, Auditory/drug effects/*metabolism ; Hair Cells, Auditory, Inner/drug effects/metabolism ; Rats ; Rats, Sprague-Dawley ; Ruthenium Red/metabolism ; TRPV Cation Channels/*metabolism ; Time Factors ; Xanthenes/metabolism ; Zebrafish

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

Recent studies have implicated reactive oxygen species (ROS) as determinants of the pathological pain caused by the activation of peripheral neurons. It has not been elucidated, however, how ROS activate the primary sensory neurons in the pain pathway. In this study, calcium imaging was performed to investigate the effects of NaOCl, a ROS donor, on the intracellular calcium concentration ([Ca2+]i) in acutely dissociated dorsal root ganglion (DRG) neurons. DRG was sequentially treated with 0.2 mg/ml of both protease and thermolysin, and single neurons were then obtained by mechanical dissociation. The administration of NaOCl then caused a reversible increase in the [Ca2+]i, which was inhibited by pretreatment with phenyl-N-tert-buthylnitrone (PBN) and isoascorbate, both ROS scavengers. The NaOCl-induced [Ca2+]i increase was suppressed both in a calcium free solution and after depletion of the intracellular Ca2+ pool by thapsigargin. Additionally, this increase was predominantly blocked by pretreatment with the transient receptor potential (TRP) antagonists, ruthenium red (50 microM) and capsazepine (10 microM). Collectively, these results suggest that an increase in the intracellular calcium concentration is produced from both extracellular fluid and the intracellular calcium store, and that TRP might be involved in the sensation of pain induced by ROS.
Animals ; Calcium ; Capsaicin ; Diagnosis-Related Groups ; Dissociative Disorders ; Extracellular Fluid ; Ganglia, Spinal ; Humans ; Neurons ; Rats ; Reactive Oxygen Species ; Ruthenium Red ; Sensation ; Sensory Receptor Cells ; Spinal Nerve Roots ; Thapsigargin ; Thermolysin ; Tissue Donors

OBJECTIVETo observe the effect of ruthenium red (RR) on the body temperature of rats with lipopolysaccharide (LPS)-induced fever and investigate the possible mechanism.

METHODSRat models of fever were established with lipopolysaccharide and the effects of RR at different doses were observed on the body temperature of the rats and the content of TRPV4 in the hypothalamus.

RESULTSCompared with those in LPS group, the rats with LPS-induced fever receiving RR treatment showed a dose-dependent lowering of the body temperature. The rats with RR treatment had lower body temperature than those with saline injection. The content of TRPV4 in the saline group was significantly higher than that in RR+LPS and RR group.

CONCLUSIONSRR inhibits LPS-induced fever in rats and regulates the hypothalamal expression of TRPV4 channels, which may participate in the maintenance of normal body temperature.

Animals ; Blotting, Western ; Body Temperature ; drug effects ; Dose-Response Relationship, Drug ; Fever ; chemically induced ; metabolism ; physiopathology ; Hypothalamus ; drug effects ; metabolism ; Lipopolysaccharides ; Male ; Rats ; Rats, Sprague-Dawley ; Ruthenium Red ; pharmacology ; TRPV Cation Channels ; biosynthesis

Fatigue occurs when the interval of intermittent tetanic contraction of skeletal muscle is shortened to a certain degree and the contractile tension declines. After fatigue, prolongation of the contraction interval can make the contractile tension recover. In atrophic soleus, the recovery rate is slower. It has been shown that a decrease in the contractile tension is caused by the inhibition of the myofibrils and sarcoplasmic reticulum Ca(2+) release channels during fatigue. So the mechanism of the recovery of contractile tension is the recovery of the inhibited myofibrils and sarcoplasmic reticulum Ca(2+) release channels. But how the inhibition affects the recovery course is still unclear. To specify the factors modulating the recovery rate after intermittent tetanic fatigue in soleus, and to seek the reasons for the decrease in recovery rate in atrophic soleus, we observed the recovery time course of different types of fatigue in isolated soleus muscle strips. The 10% or 50% decrease in the maximal tetanic contractile tention (P(0)) was defined respectively as slight or moderate fatigue. After short-term (S10P, 10 s) and long-term (L10P, 300 s) slight fatigue, the tetanic contractile tension recovered to nearly 100% P(0) at the 20th minute. In both slight fatigue groups, perfusion with 10 mumol/L of ruthenium red (an inhibitor of Ca(2+) release channels in sarcoplasmic reticulum) slowed down the recovery rate. It was suggested that slight fatigue only induced inhibition of myofibrils. After short-term (S50P, 60 s) or long-term (L50P, 300 s) moderate fatigue, the tetanic contractile tension at the 20th minute recovered to about 95% P(0) in S50P group and 90% P(0) in L50P group, respectively. The recovery rate in L50P group was significantly lower than that in S50P group. So the recovery rate after moderate fatigue was related to the tetanic contraction duration. In both moderate fatigue groups, perfusion with 5 mmol/L of caffeine (an opener of Ca(2+) release channels in sarcoplasmic reticulum) resulted in nearly 100% recovery at the 5th minute. It was suggested that moderate fatigue induced inhibition of myofibrils and sarcoplasmic reticulum Ca(2+) release channels. In 1-week tail-suspended rats, soleus muscles showed a 40% of atrophy. After slight fatigue, the tetanic contractile tension in unloaded soleus recovered to 94% P(0) in S10P group and 95% P(0) in L10P. After moderate fatigue, the tetanic contractile tension in unloaded soleus recovered to 92% P(0) in S50P and 84% P(0) in L50P at the 20th minute. There were significant decreases in all of the fatigue groups as compared with the control groups. These results suggest that both slight and moderate fatigue inhibit the myofibrils and sarcoplasmic reticulum Ca(2+) release channels in 1-week unloaded soleus, so the recovery rate after tetanic fatigue is slower than that in the control group.
Animals ; Caffeine ; pharmacology ; Calcium ; metabolism ; Hindlimb Suspension ; Male ; Muscle Fatigue ; physiology ; Muscle, Skeletal ; pathology ; physiopathology ; Muscular Atrophy ; physiopathology ; Rats ; Rats, Sprague-Dawley ; Ruthenium Red ; pharmacology ; Ryanodine Receptor Calcium Release Channel ; physiology

BACKGROUND: Capsaicin has been shown to have different biologic and toxic effects, depending on non-neuronal cells and several transformed cells, however no study has been reported from cultured human skin fibroblast. OBJECTIVE: Present study was aimed to evaluate the cytotoxicity and its mechanism of capsacin on the cultured human skin fibroblast. MATERIAL AND METHOD: Normal neonatal human fibroblasts were used, and changes of cell survival were measured by MTT assay after the cells were pre-treated with growth factors, receptor antagonist, antioxidants, calcium modulators were pre-treated or co-treated with capsaicin. RESULTS: Suvival of fibroblast was significantly increased by treatment with EGF (10ng/ml), bFGF (10ng/ml), capsazepine (10M) but inhibited by cycloheximide (1g/ml). When 200 M capsaicin was added to fibroblasts, chromatin condensations were observed at 12 hours and cell survival rate was reduced to 25-50% at 24 hours. Vanilloid receptor antagonists, capsazepine and ruthenium red, did not prevent the toxic effect of capsaicin, and 10M capsazepine paradoxically rather enhanced the cytotoxicity. In contrast to bFGF (10ng/ml), EGF (10, 100ng/ml) enhanced the cytotoxicity of capsaicin. Neuropeptides, substance P (1, 10nM) and CGRP (1, 10nM), and a structural analogue to capsaicin, tyrosine (0.3-1.2mM) did not affect the cytotoxicity. However, antioxidants such as trolox (100M) and ascorbic acid (0.1, 0.3 mM) reduced the capsaicin cytotoxicity. Of calcium modulating agents, nifedifine, a Ca2+ channel blocker (10, 20M) and cyclopiazonic acid, a Ca2+-ATPase inhibitor in ER (10M) did not influence the cytotoxicity, however BAPTA/AM (10M) as a chelater for cytoplasmic free calcium ion (10M) significantly decreased capsaicin cytotoxicity. Unlike cycloheximide, z-VAD-FMK, a protein synthesis inhibitor and a non-specific caspase inhibitor, prevented the capsaicin cytotoxicity. The DNA ladder and TUNEL positive cells were observed among the capsaicin treated fibroblasts and Western blot revealed caspase-3 activity. CONCLUSION: The capsaicin-induced cytotoxicity on human skin fibroblasts is likely to suggest the mechanism of an apoptotic pathway, which can possibly be prevented by antioxidants.
Antioxidants ; Ascorbic Acid ; Blotting, Western ; Calcium ; Capsaicin* ; Caspase 3 ; Cell Survival ; Chromatin ; Cycloheximide ; Cytoplasm ; DNA ; Epidermal Growth Factor ; Fibroblasts* ; Humans* ; In Situ Nick-End Labeling ; Intercellular Signaling Peptides and Proteins ; Neuropeptides ; Ruthenium Red ; Skin* ; Substance P ; Tyrosine