Fluorescence Intensity Of Fluo-3 Research Articles

Myricetin-induced suicidal erythrocyte death.

Myricetin, a type of flavonol commonly found in fruits and herbs, has demonstrated anticancer properties by triggering the process of apoptosis or programmed cell death in tumor cells. Despite the absence of mitochondria and nuclei, erythrocytes can undergo programmed cell death, also known as eryptosis.This process is characterized by cell shrinkage, externalization of phosphatidylserine (PS) on the cell membrane, and the formation of membrane blebs. The signaling of eryptosis involves Ca2+ influx, the formation of reactive oxygen species (ROS), and the accumulation of cell surface ceramide. The present study explored the effects of myricetin on eryptosis. Human erythrocytes were exposed to various concentrations of myricetin (2-8 µM) for 24h. Flow cytometry was used to assess the markers of eryptosis, including PS exposure, cellular volume, cytosolic Ca2+ concentration, and ceramide accumulation. In addition, the levels of intracellular ROS were measured using the 2',7'-dichlorofluorescin diacetate (DCFDA) assay. The myricetin-treated (8 µM) erythrocytes significantly increased Annexin-positive cells, Fluo-3 fluorescence intensity, DCF fluorescence intensity, and the accumulation of ceramide. The impact of myricetin on the binding of annexin-V was significantly reduced, but not completely eliminated, by the nominal removal of extracellular Ca2+. Myricetin triggers eryptosis, which is accompanied and, at least in part, caused by Ca2+ influx, oxidative stress and increase of ceramide abundance.

Mitochondrial dysfunction in cumulus cells is related to decreased reproductive capacity in advanced-age women

To reveal the relationship between mitochondrial function in cumulus cells (CCs) and the aging-related decline in ovarian function and reproductive capacity. Retrospective and transcriptome analysis of human tissue. University hospital. A total of 186 infertile women with normal weight and no other diseases, including 86 young women (aged <38 years) with normal ovarian reserve and 100 advanced-age women (aged ≥38 years) with diminished ovarian reserve. None. Embryo development data were analyzed. The mitochondrial ultrastructure of CCs was observed by transmission electron microscopy. Mitochondrial activity was detected by immunofluorescence. The per-CC mitochondrial deoxyribonucleic acid copy numbers and cellular adenosine triphosphate levels were quantified. Unbiased comprehensive genome-wide transcriptomics was performed. The functions of all annotated genes and biologic pathways were analyzed by the Kyoto Encyclopedia of Genes and Genomes. Advanced-age women with diminished ovarian function had significantly fewer retrieved oocytes and lower embryo quality. The normal mitochondrial rate in CCs was significantly lower. In addition to mitochondrial morphology and structural changes, the fluorescence intensity of Fluo-3/AM was significantly higher, and that of MitoTracker was lower than that of young women with normal ovarian reserve, suggesting that age negatively affects the mitochondrial activity of CCs. The per-CC mitochondrial deoxyribonucleic acid copy number and adenosine triphosphate levels significantly decreased in the advanced-age group. The Kyoto Encyclopedia of Genes and Genomes analysis showed that differentially expressed genes were significantly enriched in the oxidative phosphorylation pathway. Additionally, most mitochondrially encoded genes related to oxidative phosphorylation were significantly down-regulated in the advanced-age group. We present current evidence that mitochondrial dysfunction in CCs may play a key role in the age-related decline in ovarian function and reproductive capacity.

Thiopental Elevates Steady-State Levels of Intracellular Ca2+ and Zn2+ in Rat Thymic Lymphocytes

Thiopental is an ultra-short-acting barbiturate and has been used commonly in the induction phase of general anesthesia. However, the toxic effect of thiopental is not completely clear. The effect of thiopental on intracellular Ca2+ ([Ca2+]i) levels was investigated in non-excitable cells. Experiments were carried out using a flow-cytometric technique, rat thymic lymphocytes (as non-excitable cells), and appropriate fluorescent probes. Treatment of cells with 300 µM thiopental increased Fluo-3 fluorescence intensity, indicating elevation of [Ca2+]i. This increase was partially attenuated by a chelator of intracellular Zn2+. Thus, thiopental elevated both [Ca2+]i and intracellular Zn2+ ([Zn2+]i) levels. Under intracellular Zn2+-free conditions, 100–300 µM thiopental was still able to induce a statistically significant increase in [Ca2+]i, whereas removal of extracellular Ca2+ greatly reduced the increase in [Ca2+]i induced by this dose of thiopental. Therefore, the thiopental-induced increase in [Ca2+]i was mainly due to an increased influx of Ca2+. Treatment of cells with 300 µM thiopental increased FluoZin-3 fluorescence intensity, indicating the presence of [Zn2+]i, both in the presence and absence of extracellular Zn2+. The thiopental-induced elevation of [Zn2+]i was due to an increase in both influx of Zn2+ and intracellular Zn2+ release. Concanavalin A (10 µg/mL) augmented Fluo-3 fluorescence in the presence of an intracellular Zn2+ chelator. The combination of concanavalin A and 100–300 µM thiopental synergistically increased [Ca2+]i. Results suggest that thiopental increases [Ca2+]i in both quiescent and activated lymphocytes, possibly resulting in modulation of immune system function.&#x0D;

Abstract PR031

Background & Objectives: The physiological mechanisms that regulate tissue blood flow sufficiently are involved in the control of endothelium-dependent vasodilation. Nitric oxide, which is produced by endothelial nitric oxide synthase (eNOS), acts as an endothelium-dependent vasodilator. eNOS activity is regulated by several mechanisms including intracellular calcium concentration ([Ca2+]i) and phosphorylation of eNOS. Phosphorylation of Ser1177 increases Ca2+ sensitivity of the enzyme (1). Endothelium-dependent vasodilation is impaired by sevoflurane (2). The interaction of desflurane locally with the action of endothelial cells remains unknown. This study was carried out to elucidate the effects of desflurane and sevoflurane on endothelium-dependent vasodilation and the mechanisms of regulation of eNOS activity. Materials & Methods: With institutional approval, a rat descending aorta with the endothelium was suspended in a temperature-controlled organ bath for measurement of isometric tension. Changes in tension in response to phenylephrine (3×10–7 M) followed by acetylcholine (10–6 M) were recorded in the presence or absence of desflurane (5.7%, 11.4%; 1, 2 MAC for normal rats) or sevoflurane (2.4%, 4.8%; 1, 2 MAC for normal rats) to test endothelium-dependent vasodilation. Cultured bovine aortic endothelial cells were treated with bradykinin (10–6 M) in the presence or absence of desflurane or sevoflurane (11.4% and 4.8%, respectively). After treatments, Western blotting was used to determine eNOS Ser1177 phosphorylation, and measurement of the fluorescence intensity of Fluo-3/AM, as a [Ca2+]i indicator, was used to study the changes of [Ca2+]i. Statistical analysis was performed using Kruskal-Wallis’ H-test followed by Newman-Keuls test for multiple comparisons. A P value < 0.05 was considered statistically significant. Results: Endothelium-dependent vasodilation of the rat aorta and production of NO was inhibited by both anesthetics. The inhibitory effect of desflurane was greater than that of sevoflurane (Figure 1A, 1B). Western blotting revealed that phosphorylation of eNOS Ser1177 was inhibited by sevoflurane but not by desflurane (Figure 1C). Live cell calcium imaging revealed that bradykinin-induced increase in [Ca2+]i was suppressed by desflurane but not by sevoflurane (Figure 1D).Conclusion: The findings suggest that sevoflurane inhibits endothelium-dependent vasodilation caused by inhibition of eNOS Ser1177 phosphorylation, whereas desflurane inhibits vasodilation by inhibition of calcium signal transduction of eNOS activity. This difference in inhibitory mechanisms might reflect the different degrees of inhibitory effects of sevoflurane and desflurane on endothelium-dependent vasodilation.

Temperature-induced labelling of Fluo-3 AM selectively yields brighter nucleus in adherent cells

Fluo-3 is widely used to study cell calcium. Two traditional approaches: (1) direct injection and (2) Fluo-3 acetoxymethyl ester (AM) loading, often bring conflicting results in cytoplasmic calcium ([Ca2+]c) and nuclear calcium ([Ca2+]n) imaging. AM loading usually yields a darker nucleus than in cytoplasm, while direct injection always induces a brighter nucleus which is more responsive to [Ca2+]n detection. In this work, we detailedly investigated the effects of loading and de-esterification temperatures on the fluorescence intensity of Fluo-3 in response to [Ca2+]n and [Ca2+]c in adherent cells, including osteoblast, HeLa and BV2 cells. Interestingly, it showed that fluorescence intensity of nucleus in osteoblast cells was about two times larger than that of cytoplasm when cells were loaded with Fluo-3 AM at 4°C and allowed a subsequent step for de-esterification at 20°C. Brighter nuclei were also acquired in HeLa and BV2 cells using the same experimental condition. Furthermore, loading time and adhesion quality of cells had effect on fluorescence intensity. Taken together, cold loading and room temperature de-esterification treatment of Fluo-3 AM selectively yielded brighter nucleus in adherent cells.

Role of store-operated Ca2+ channels in ethanol-induced intracellular Ca2+ increase in HepG2 cells

To investigate the mechanism of ethanol-induced calcium overload in hepatocytes and the related role of store-operated calcium channels (SOCs). HepG2 cells were treated an ethanol concentration gradient with or without intervention treatment with the extracellular calcium chelator EGTA or the SOCs inhibitor 2-aminoethoxydiphenyl borate (2-APB). Effects on cell viability were assessed by the CCK8 assay. Effects on leakage of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined by automatic biochemical analyzer measurements of the culture supernatants. Effects on cytoplasmic free Ca2+ concentration ([Ca2+]i) were accessed by detecting fluorescence intensity of the calcium indicator Fluo-3/AM with a flow cytometer. Effects on mRNA and protein expression levels of SOCs, stromal interacting factor 1 (STIM1), and calcium release-activated calcium channel protein 1 (Orai1) were evaluated by qPCR and western blotting. The ethanol treatment produced dose-dependent reduction in cell viability (r = -0.985, P less than 0.01) and increases in leakage of ALT (F = 15.286, P less than 0.01) and AST (F = 39.674, P less than 0.01). Compared to untreated controls, the ethanol treatments of 25, 50, 100, 200 and 400 mM induced significant increases in [Ca2+]i level (1.25+/-0.36, 1.31+/-0.15, 1.41+/-0.18, 2.29+/-0.25, 2.58+/-0.19; F = 15.286, P less than 0.01). Both intervention treatments, EGTA and 2-APB, significantly reduced the 200 mM ethanol treatment-induced [Ca2+]i increase (2.32+/-0.08 reduced to 1.79+/-0.15 (t = 7.201, P less than 0.01) and 1.86+/-0.09 (t = 8.183, P less than 0.01) respectively). EGTA and 2-APB also increased the ethanol-treated cells' viability and reduced the ALT and AST leakage. The 200 mM ethanol treatment stimulated both gene and protein expression of STIM1 and Orai1, and the up-regulation effect lasted at least 72 h after treatment. Ethanol-induced dysregulation of SOCs may be an important molecular mechanism of ethanol-induced [Ca2+]i rise in hepatocytes and the related liver cell injury.

Fluoxetine Induced Suicidal Erythrocyte Death

The antidepressant fluoxetine inhibits ceramide producing acid sphingomyelinase. Ceramide is in turn known to trigger eryptosis the suicidal death of erythrocytes characterized by cell shrinkage and exposure of phosphatidylserine at the erythrocyte surface. Ceramide is effective through sensitizing the erythrocytes to the pro-eryptotic effect of increased cytosolic Ca2+ activity ([Ca2+]i). In nucleated cells, fluoxetine could either inhibit or stimulate suicidal death or apoptosis. The present study tested whether fluoxetine influences eryptosis. To this end cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin V binding, hemolysis from hemoglobin release and [Ca2+]i from Fluo-3 fluorescence intensity. As a result, a 48 h exposure of erythrocytes to fluoxetine (≥25 µM) significantly decreased forward scatter, increased annexin V binding and enhanced [Ca2+]i. The effect on annexin V binding was significantly blunted, but not abolished, in the absence of extracellular Ca2+. In conclusion, fluoxetine stimulates eryptosis, an effect at least in part due to increase of cytosolic Ca2+ activity.

The relationship between calcification and photosynthesis in the coccolithophorid Pleurochrysis carterae

Coccolithophorids are one of the dominant groups of marine phytoplankton. They are found in large numbers throughout the surface euphotic zone of the ocean, and are able to form large-scale blooms that persist for long periods of time. Coccolithophorid cells are covered by species-specific calcium carbonate crystals of various structures. In the process of calcification in coccolithophorids, Ca 2+ is absorbed into cells from the culture medium, and a coccolith unit is formed inside the cell. Then, the coccolith unit extrudes to the cell surface where it is constructed into crystal layers. The formation of these crystals is regulated by cellular metabolism under different environmental conditions. The carbon biogeochemical cycle in the coccolithophorids involves both photosynthetic and calcification processes, which not only play an important role in population dynamics, but also in the global carbon cycle and climate change. However, one important question remains, namely, whether the relationship between photosynthesis and calcification is species-dependent. Previous studies have yielded controversial results, even in the same species. In this paper, we selected Pleurochrysis carterae, a coccolithophore species that frequently blooms in coastal areas, to study the relationship between calcification and photosynthesis. First, we studied population growth in a batch culture over several days. For batch cultures, P. carterae was inoculated into a 10 L bioreactor at an initial cell density of approximately 5 × 10 4 cells mL −1. The culture conditions were optimal for cell growth. Dissolved oxygen (DO) was detected during all the culture period, and the rate of photosynthetic oxygen evolution was calculated according the DO changes during the 12-h illumination period. Algal samples (10 mL) were collected during the population growth phases. The calcium carbonate content on the cell surface was determined each day by chemical titration. Next, we studied the relationship between photosynthesis and calcification at the cellular level by observing patterns of recalcification during a 12-h period. In this study, non-calcified cells were obtained by decalcifying calcified cells collected during the exponential growth period in MES-NaOH buffer solution (pH 5.5). The non-calcified cells were inoculated into culture media containing different concentrations of Ca 2+ (0, 5, 20, 40, 50, or 100 mg L −1). The rate of recalcification was determined by microscopic analyses in which the number of recalcified cells per 100 cells was counted at 0, 3, 6, 9, and 12 h of culture. Ca 2+ absorbed into the cell was detected by measuring the fluorescence intensity of Fluo-3/AM labeled Ca 2+. The rate of photosynthetic oxygen evolution in the non-calcified cell cultures was detected by measuring the changes in dissolved oxygen during the 12-h illumination period. The results showed that during the population growth period, the rate of photosynthetic oxygen evolution was inversely related to the calcium carbonate content per cell. When the amount of calcium carbonate on the cell surface increased, the relative photosynthetic ability (the rate of photosynthetic oxygen evolution) decreased, and vice versa. Both recalcification rates and photosynthetic oxygen evolution were affected by the extracellular calcium concentration. Non-calcified cells showed different recalcification abilities at different extracellular Ca 2+ concentrations. The recalcification rate of non-calcified cells was positively correlated with the extracellular calcium concentration when [Ca 2+] in the medium ranged from 0 to 100 mg L −1. However, photosynthetic oxygen evolution was suppressed at higher cell calcification rates, especially when extracellular [Ca 2+] was 50–100 mg L −1. Our analyses of the population growth process and the cell recalcification process confirmed that photosynthesis is inversely related to calcification in P. carterae.

Effects of hypothermic storage on intracellular calcium, reactive oxygen species formation, mitochondrial function, motility, and plasma membrane integrity in striped bass (Morone saxatilis) sperm

Experiments were conducted to determine the effect of hypothermic 24 h storage on striped bass sperm cell plasma membrane integrity, free intracellular Ca2+ ([Ca2+]i), mitochondrial membrane potential (ΔΨm), and reactive oxygen species (ROS) formation (oxidation of hydroethidine to ethidium) as determined by flow cytometry; motion activation and ATP concentration as determined by Luciferin-Luciferase bioluminescence assay. Semen was stored for 1 or 24 h at 4 °C in an O2 atmosphere undiluted or diluted (one volume semen with 3 volumes diluent) with T350 (20 mM TRIS base-NaCl, 350 mOsm/mL, pH 8) or with seminal plasma in the presence of various treatments. Viability (% cells excluding propidium iodide) approached 100% after 1 h storage in undiluted or diluted semen. After 1 h of storage the [Ca2+]i marker, Fluo-3, was detected in only 3% of sperm cells in undiluted or diluted semen. In contrast to storage for 1 h, after 24 h the incidence Fluo-3 fluorescence intensity was increased (P < 0.05) in > 50% of the viable cells in undiluted and diluted semen along with increased cell death; the presence of 1 mM ethylene glycol tetraacetic acid (EGTA) blocked CaCl2-induced Fluo-3 fluorescence and cell death. Activation of sperm motility was 82% after 1 h in T350 and decreased (P < 0.05) to 30% after 24 h. However, motility activation failed in the presence of EGTA at 1 or 24 h. During storage ΔΨm was not affected by storage time or treatment. In contrast, sperm ATP was greater (P < 0.05) at 1 h than at 24 h and was greater in sperm stored in diluted than undiluted form. While ROS formation was induced by menadione treatment, there was no evidence of storage-induced ROS formation in the absence of menadione. The increased [Ca2+]i found after 24 h indicates a storage induced defect in the maintenance of cellular calcium homeostasis which may be detrimental to sperm activation.

Elevation of Intracellular Ca2+ Level by Triclosan in Rat Thymic Lymphocytes: Increase in Membrane Ca2+ Permeability and Induction of Intracellular Ca2+ Release

Triclosan is an antibacterial agent used in household items and personal care products. Because wild animals and humans can harbor this compound in their systems, the toxic effects of triclosan are a possibility and are suspected. Therefore, we examined the effects of triclosan on intracellular Ca2+ concentration in rat thymocytes by cytometric techniques using fluorescent probes. Triclosan doses of 1-10 μM significantly increased the intensity of Ca2+-detecting Fluo-3 fluorescence, indicating an increase in intra-cellular Ca2+ concentrations. The augmentation of Fluo-3 fluorescence became more profound in a dose-dependent manner after the addition of an external source of Ca2+. Conversely, the removal of external Ca2+ greatly attenuated the triclosan-induced augmentation of Fluo-3 fluorescence. These results suggest that triclosan treatment allows external Ca2+ to pass through cell membranes. This phenomenon was not specific for Ca2+ because external Mn2+ quenched the triclosan-induced augmentation of Fluo-3 fluo-rescence, indicating that triclosan can also mediate Mn2+ permeation across membranes. Therefore, these results suggest that triclosan increases membrane permeability to divalent metal cations. Furthermore, triclosan induces Ca2+ release from intracellular stores because the Fluo-3 fluorescence intensitystill increased slightly after triclosan treatment, even under conditions free from external Ca2+. Additionally, triclosan did not increase the intensity of Fluo-3 fluorescence when Ca2+ was depleted from intracellular Ca2+ stores by A23187 under the external Ca2+-free condition. Taken together, these data suggest that micromolar concentrations of triclosan affect intracellular Ca2+ homeostasis in thymocytes, possibly resulting in cellular malfunction.

Micromolar Zn 2+ potentiates the cytotoxic action of submicromolar econazole in rat thymocytes: Possible disturbance of intracellular Ca 2+ and Zn 2+ homeostasis

Econazole, one of imidazole antifungals, has been reported to exhibit an inhibitory action on Mycobacterium tuberculosis and its multidrug-resistant strains under in vitro and ex vivo conditions. There is a chemotherapeutic potential of econazole against tuberculosis. We have revealed that Zn 2+ at micromolar concentrations potentiates the cytotoxicity of imidazole antifungals by increasing membrane Zn 2+ permeability. It is reminiscent of a possibility that econazole exhibits harmful action on human in the presence of Zn 2+ at a physiological range when the agent is systemically administered. Because it is necessary to characterize the cytotoxic action of econazole in the presence of Zn 2+, we have cytometrically examined the effects of econazole, ZnCl 2, and their combination on rat thymocytes. ZnCl 2 at concentrations ranging from 1 μM to 30 μM significantly increased the lethality induced by 10 μM econazole in a concentration-dependent manner. Econazole at a sublethal concentration of 1 μM significantly augmented the intensity of side scatter in the presence of micromolar ZnCl 2, suggesting the change in an intracellular circumstance by the combination of econazole and ZnCl 2. Econazole at 0.3 μM or more in the presence of ZnCl 2 increased the intensity of Fluo-3 fluorescence, an indicator for intracellular Ca 2+. Furthermore, the intensity of FluoZin-3 fluorescence, an indicator for intracellular Zn 2+, was also augmented by econazole at 0.1 μM or more in the presence of ZnCl 2. Results suggest that the combination of submicromolar econazole with micromolar ZnCl 2 may increase the intracellular concentration of Ca 2+ and Zn 2+, leading to disturbance of intracellular Ca 2+ and Zn 2+ homeostasis that triggers cytotoxic action.

Zn2+, derived from cell preparation, partly attenuates Ca2+-dependent cell death induced by A23187, calcium ionophore, in rat thymocytes

A23187, a calcium ionophore, is used to induce Ca(2+)-dependent cell death by increasing intracellular Ca(2+) concentration ([Ca(2+)](i)) under in vitro condition. Since this ionophore also increases membrane permeability of metal divalent cations such as Zn(2+) and Fe(2+) rather than Ca(2+), trace metal cations in cell suspension may affect Ca(2+)-dependent cell death induced by A23187. Therefore, the effects of chelators for divalent metal cations, EDTA and TPEN, on the A23187-induced cytotoxicity were cytometrically examined in rat thymocytes. The cytotoxicity of A23187 was attenuated by 1mM EDTA while it was augmented by 50 microM EDTA and 10 microM TPEN. These changes were statistically significant. The A23187-induced increase in Fluo-3 fluorescence intensity, a parameter for [Ca(2+)](i), was significantly reduced by 1mM EDTA while it was not the case for 50 microM EDTA and 10 microM TPEN. The intensity of FluoZin-3 fluorescence, a parameter for [Zn(2+)](i), increased by A23187 was respectively reduced by 50 microM EDTA and 10 microM TPEN. It is suggested that the attenuation of A23187-induced cytotoxicity by 1mM EDTA is due to the chelation of extracellular Ca(2+) and Zn(2+) while the augmentation by 50 microM ETDA or 10 microM TPEN is due to the chelation of extracellular Zn(2+). The Tyrode's solution without thymocytes contained 32.4 nM of zinc while it was 216.9 nM in the cell suspension. In conclusion, trace Zn(2+), derived from cell preparation, partly attenuates the Ca(2+)-dependent cell death induced by A23187.

Clotrimazole, an antifungal drug possessing diverse actions, increases membrane permeation of cadmium in rat thymocytes

In previous study, clotrimazole, an antifungal drug, exerted potent cytotoxic action on rat thymocytes in presence of metal divalent cations such as Cd 2+ and Pb 2+. To reveal one of toxicological characteristics of clotrimazole, we examined the effect of clotrimazole on intracellular concentration of metal divalent cations by flow cytometer with fluo-3, a fluorescent. Simultaneous application of clotrimazole and CdCl 2 significantly decreased the cell viability although their concentrations were not cytotoxic, respectively. Clotrimazole alone increased the intensity of fluo-3 fluorescence, suggesting an increased concentration of intracellular Ca 2+. The intensity of fluo-3 fluorescence augmented by the combination of clotrimazole and CdCl 2 was much higher than that by respective agents. Removal of external Ca 2+ further increased the intensity of fluorescence augmented by the combination. Furthermore, the application of MnCl 2 did not attenuate the intensity in the presence of CdCl 2. Therefore, it is suggested that the augmentation of fluo-3 fluorescence in the simultaneous presence of clotrimazole and CdCl 2 is Cd 2+-dependent. Clotrimazole may increase membrane permeation of Cd 2+.

Integrin Ligands Mobilize Ca2+ from Ryanodine Receptor-gated Stores and Lysosome-related Acidic Organelles in Pulmonary Arterial Smooth Muscle Cells

Extracellular matrix (ECM) protein receptors, or integrins, participate in vascular remodeling and the systemic myogenic response. Synthetic ligands and ECM fragments regulate the vascular smooth muscle cell contractile state by altering intracellular Ca2+ levels ([Ca2+]i). Information on the Ca2+ effect of integrins in vascular smooth muscle cells is limited, but nonexistent in pulmonary arterial smooth muscle cells (PASMCs). We therefore characterized integrin expression in endothelium-denuded pulmonary arteries, and explored [Ca2+]i mobilization pathways induced by soluble ligands in rat PASMCs. Reverse transcriptase-PCR showed mRNA expression of integrins alpha1, alpha2, alpha3, alpha4, alpha5, alpha7, alpha8, alpha(v), beta1, beta3, and beta4, and immunoblots of alpha5, alpha(v), beta1, and beta3 confirmed protein expression. Exposure of PASMCs to integrin-binding peptides (0.5 mM) containing the arginine-glycine-aspartate (RGD) motif elicited [Ca2+]i responses with an order of potency of GRGDNP > GRGDSP > GRGDTP = cyclo-RGD. Pharmacological analysis revealed that the GRGDSP-induced Ca2+ response was unrelated to Ca2+ influx and the inositol triphosphate receptor-gated Ca2+ store, but partially blocked by ryanodine or inhibition of lysosome-related acidic organelles with bafilomycin A1. Simultaneous inhibition of both pathways was necessary to abolish the response. GRGDSP treatment increased cyclic ADP-ribose, the endogenous activator of ryanodine receptors, by 70%. GRGDSP also rapidly reduced Lysotracker Red accumulation, confirming direct modulation of acidic organelles. These data are the first demonstration of integrin-mediated Ca2+ regulation in PASMCs. The presence of an array of integrins, and activation of ryanodine-sensitive Ca2+ stores and lysosome-like organelles by GRGDSP suggest important roles for integrin-dependent Ca2+ signaling in regulating PASMC function.

Ca 2+ stimulates COX-2 expression through calcium-sensing receptor in fibroblasts

Fibroblasts isolated from jaw cysts expressed calcium-sensing receptor (CasR). In the fibroblasts elevated extracellular Ca 2+ ([Ca 2+] o) increased fluo-3 fluorescence intensity, and the production of inositol(1,4,5)trisphosphate and active protein kinase C. Phospholipase C inhibitor U-73122 attenuated the Ca 2+-induced increase in fluo-3 fluorescence intensity. Elevated [Ca 2+] o enhanced the expression of cyclooxygenase-2 (COX-2) mRNA and protein, and the secretion of prostaglandin E 2 in the fibroblasts. CasR activator neomycin also increased the expression of COX-2 mRNA, and U-73122 attenuated the Ca 2+-induced expression of COX-2 mRNA. Elevated [Ca 2+] o-induced phosphorylation of extracellular signal-regulated protein kinase-1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK), and U-73122 inhibited the Ca 2+-induced phosphorylation. The inhibitors for each kinase, PD98059, SB203580, and SP600125, attenuated the Ca 2+-induced expression of COX-2 mRNA. These results suggest that in jaw cyst fibroblasts elevated extracellular Ca 2+ may enhance COX-2 expression via the activation of ERK1/2, p38 MAPK, and JNK through CasR.

Effect of manganese on cytosolic free calcium concentration in cortical neurons

To investigate the change of free Ca(2+) in cytoplasma in the neurotoxicity of the manganese (Mn). The cortical neurons were separated from the neonatal Wistar rats and cultured in vitro. The neurons were grouped as the Mn-treated groups and the untreated group. The neurons in the Mn-added groups were incubated in the culture media containing lower, medium and high dosage manganese chloride (MnCl(2 x 4) H2O) with the concentration at 0.2, 0.6, 1.0 mmol/L respectively. Meanwhile, neurons in control were cultured in the normal culture media. All treatments stopped 24 h later. Neurons were labeled Ca(2+) sensitive prober, Fluo-3/AM. The fluorescence intensity of Fluo-3 combined with Ca(2+) was examined by LSCM (Laser scanning confocal microscope) and was treated by the picture analysis technique. The intensity was equal to the free Ca(2+) concentrations in cytoplasma of neurons. MnCl(2) can induce free Ca(2+) overloaded in cytoplasma of neurons, but the increasing degree varied in MnCl(2) dosage. Cytoplasma Ca(2+) concentration in the moderate dosage The moderate dosage MnCl(2) group and the high dosage MnCl(2) group were significantly higher than that in the lower dosage MnCl(2) group and the control group (P < 0.05). The Ca(2+) overload is involved in the neurotoxicity of manganese, and a dosage response relationship is found between the manganese chloride dose and Ca(2+) overload in cortical neurons.

Increase in intracellular Cd 2+ concentration of rat cerebellar granule neurons incubated with cadmium chloride: Cadmium cytotoxicity under external Ca 2+-free condition

In order to examine the cadmium cytotoxicity unrelated to external Ca 2+, the effects of micromolar CdCl 2 on intracellular Cd 2+ concentration, cellular content of glutathione, and cell viability of rat cerebellar granule neurons were examined under normal Ca 2+ and external Ca 2+-free conditions, using a laser confocal microscope with fluorescent probes, fluo-3-AM, 5-chloromethylfluorescein (CMF) diacetate, and propidium iodide. CdCl 2 (10–300 μM) dose-dependently increased the intensity of fluo-3 fluorescence. Exposure to CdCl 2 equally enhanced the fluo-3 fluorescence under both Ca 2+ conditions and MnCl 2 did not quench the CdCl 2-enhanced fluorescence. The results indicate that the enhancement of fluo-3 fluorescence is due to the increase in intracellular Cd 2+ concentration. CdCl 2 at 100–300 μM decreased the intensity of CMF fluorescence, indicating the decrease in cellular content of glutathione. The population of cells stained with propidium (dead cells) was increased by 100–300 μM CdCl 2. Similar results described above were also observed under external Ca 2+-free condition. It is suggested that some of cytotoxic actions of CdCl 2 on neurons are unrelated to external Ca 2+, one of main sources for increasing intracellular Ca 2+ concentration.

Prostaglandin E2 Stimulates Fibronectin Expression through EP1 Receptor, Phospholipase C, Protein Kinase Cα, and c-Src Pathway in Primary Cultured Rat Osteoblasts

Fibronectin (Fn) is involved in the early stages of bone formation, and prostaglandin E (PGE) is an important factor regulating osteogenesis. Here we found that PGE(2) enhanced extracellular Fn assembly in rat primary osteoblasts, as shown by immunofluorescence staining and enzyme-linked immunosorbent assay. PGE(2) also increased the protein levels of Fn by using Western blotting analysis. By using pharmacological inhibitors or activators or genetic inhibition by the EP receptor, antisense oligonucleotides revealed that the EP(1) receptor but not other PGE receptors is involved in PGE(2)-mediated up-regulation of Fn. At the mechanistic level, Ca(2+) chelator (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester)), phosphatidylinositol-phospholipase C inhibitor (U73122), or Src inhibitor (PP2) attenuated the PGE(2)-induced Fn expression. Protein kinase C (PKC) inhibitor (GF109203X) also inhibited the potentiating action of PGE(2). Furthermore, treatment with antisense oligonucleotides of various PKC isoforms, including alpha, beta, epsilon, and delta, demonstrated that alpha isozyme plays an important role in the enhancement action of PGE(2) on Fn assembly. Flow cytometry and reverse transcription-PCR showed that PGE(2) and 17-phenyl trinor PGE(2) (EP(1)/EP(3) agonist) increased the surface expression and mRNA level of alpha5 or beta1 integrins. Fn promoter activity was enhanced by PGE(2) and 17-phenyl trinor PGE(2) in cells transfected with pGL2F1900-Luc. Cotransfection with dominant negative mutants of PKCalpha or c-Src inhibited the potentiating action of PGE(2) on Fn promoter activity. Local administration of PGE(2) or 17-phenyl trinor PGE(2) into the metaphysis of the tibia via the implantation of a needle cannula significantly increased the Fn and alpha5beta1 integrin immunostaining and bone volume of secondary spongiosa in tibia. Taken together, our results provided evidence that PGE(2) increased Fn and promoted bone formation in rat osteoblasts via the EP(1)/phospholipase C/PKCalpha/c-Src signaling pathway.

In situ Ca 2+ dynamics of Purkinje fibers and its interconnection with subjacent ventricular myocytes

Purkinje fibers play essential roles in impulse propagation to the ventricles, and their functional impairment can become arrhythmogenic. However, little is known about precise spatiotemporal pattern(s) of interconnection between Purkinje-fiber network and the underlying ventricular myocardium within the heart. To address this issue, we simultaneously visualized intracellular Ca 2+ dynamics at Purkinje fibers and subjacent ventricular myocytes in Langendorff-perfused rat hearts using multi-pinhole type, rapid-scanning confocal microscopy. Under recording of electrocardiogram at room temperature spatiotemporal changes in fluo3-fluorescence intensity were visualized on the subendocardial region of the right-ventricular septum. Staining of the heart with either fluo3, acetylthiocholine iodide (ATCHI), or di-4-ANEPPS revealed characteristic structures of Purkinje fibers. During sinus rhythm (about 60 bpm) or atrial pacing (up to 3 Hz) each Purkinje-fiber exhibited spatiotemporally synchronous Ca 2+ transients nearly simultaneously to ventricular excitation. Ca 2+ transients in individual fibers were still synchronized within the Purkinje-fiber network not only under high-K + (8 mM) perfusion-induced Purkinje-to-ventricular (P–V) conduction delay, but also under unidirectional, orthodromic P–V block produced by 10-mM K + perfusion. While spontaneous, asynchronous intracellular Ca 2+ waves were identified in injured fibers of Purkinje network locally, surrounding fibers still exhibited Ca 2+ transients synchronously to ventricular excitation. In summary, these results are the first demonstration of intracellular Ca 2+ dynamics in the Purkinje-fiber network in situ. The synchronous Ca 2+ transients, preserved even under P–V conduction disturbances or under emergence of Ca 2+ waves, imply a syncytial role of Purkinje fibers as a specialized conduction system, whereas unidirectional block at P–V junctions indicates a substrate for reentrant arrhythmias.

Flow-cytometric analysis on adverse effects of polysorbate 80 in rat thymocytes.

The effects of polysorbate 80, a non-ionic surfactant widely used in pharmaceutical products, on rat thymocytes were examined to reveal its toxic property at the cellular level. Polysorbate 80 at concentrations of 1-100 microg/ml did not significantly affect the cell viability. This surfactant at 30 microg/ml or more augmented the intensity of fluo-3 fluorescence, indicating the increase in intracellular Ca(2+) concentration. Such an augmentation of fluo-3 fluorescence by polysorbate 80 was not seen under the Ca(2+)-free condition, suggesting that polysorbate 80 increased membrane Ca(2+) permeability. The concentration-dependent polysorbate 80 at 10 microg/ml or more attenuated the intensity of 5-chloromethylfluorescein, indicating a decrease in cellular content of glutathione by polysorbate 80. Furthermore, the agent at 1 microg/ml or more attenuated the intensity of bis-(1,3-dibutylbarbituric acid) trimethine oxonol fluorescence, being independent from the changes in membrane potential. This phenomenon indicates that polysorbate 80 at 1 microg/ml or more may attenuate the incorporation of anionic compounds into the membranes. It can be suggested that polysorbate 80 modifies some of membranes and intracellular physiological parameters without affecting the cell viability.