Reduction and uptake of methylene blue by human erythrocytes

2004 ◽  
Vol 286 (6) ◽  
pp. C1390-C1398 ◽  
Author(s):  
James M. May ◽  
Zhi-chao Qu ◽  
Charles E. Cobb
Keyword(s):  
Methylene Blue ◽  
Reductase Activity ◽  
Oxidant Stress ◽  
Pentose Phosphate ◽  
Human Erythrocytes ◽  
Dependent Manner ◽  
Phenylarsine Oxide ◽  
Concentration Dependent Manner ◽  
Ferricyanide Reduction ◽  
Thiazine Dye

A thiazine dye reductase has been described in endothelial cells that reduces methylene blue (MB), allowing its uptake into cells. Because a different mechanism of MB uptake in human erythrocytes has been proposed, we measured MB uptake and reduction in this cell type. Oxidized MB (MB+) stimulated reduction of extracellular ferricyanide in a time- and concentration-dependent manner, reflecting extracellular reduction of the dye. Reduced MB was then taken up by the cells and partially oxidized to MB+. Both forms were retained against a concentration gradient, and their redox cycling induced an oxidant stress in the cells. Whereas concentrations of MB+ <5 μM selectively oxidized NAD(P)H, higher concentrations also oxidized both glutathione (GSH) and ascorbate, especially in the absence of d-glucose. MB+-stimulated ferricyanide reduction was inhibited by thiol reagents with different mechanisms of action. Phenylarsine oxide, which is selective for vicinal dithiols in proteins, inhibited MB+-dependent ferricyanide reduction more strongly than it decreased cell GSH and pentose phosphate cycle activity, and it did not affect cellular NADPH. Open erythrocyte ghost membranes facilitated saturable NAD(P)H oxidation by MB+, which was abolished by pretreating ghosts with low concentrations of trypsin and phenylarsine oxide. These results show that erythrocytes sequentially reduce and take up MB+, that both reduced and oxidized forms of the dye are concentrated in cells, and that the thiazine dye reductase activity initially responsible for MB+ reduction may correspond to MB+-dependent NAD(P)H reductase activity in erythrocyte ghosts.

scholarly journals Involvement of GR and p300 in the Induction of H6PD by Cortisol in Human Amnion Fibroblasts

Endocrinology ◽  
2012 ◽  
Vol 153 (12) ◽  
pp. 5993-6002 ◽  
Author(s):  
Weihua Wang ◽  
Chunming Guo ◽  
Wenjiao Li ◽  
Jianneng Li ◽  
Wangsheng Wang ◽  
...  
Keyword(s):  
Fetal Development ◽  
Reductase Activity ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Reduced Form ◽  
Fetal Membranes ◽  
Dependent Manner ◽  
Forward Induction ◽  
Concentration Dependent Manner ◽  
Human Amnion ◽  
Feed Forward

Abstract Human fetal membranes express 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which reduces biologically inert cortisone to active cortisol and may provide an extraadrenal source of cortisol mediating fetal development and parturition. The reductase activity of 11β-HSD1 depends on the availability of the cofactor reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) derived from the enzymatic activity of hexose-6-phosphodehydrogenase (H6PD). Based on the feed-forward induction of 11β-HSD1 by glucocorticoids in human fetal membranes, we hypothesize that glucocorticoids simultaneously induce H6PD in the fetal membranes. We found a parallel distribution of H6PD and 11β-HSD1 in the amnion, chorion, and decidua. In cultured human amnion fibroblasts, small interfering RNA-mediated knockdown of H6PD expression significantly attenuated the conversion of cortisone to cortisol. Cortisol (0.01–1 μm) induced H6PD expression in a concentration-dependent manner, which was attenuated by glucocorticoid receptor (GR) antagonist RU486. Cortisol induced the expression of p300, a histone acetyltransferase, whereas C646, an inhibitor of p300, attenuated the induction of H6PD by cortisol. Coimmunoprecipitation revealed GR and p300 in the same nuclear protein complex upon cortisol stimulation. Chromatin immunoprecipitation showed that cortisol increased the binding of p300 and GR to H6PD promoter and the acetylation of histone 3 lysine 9 on the promoters. In conclusion, the induction of H6PD by cortisol requires the participation of GR and p300 as well as the acetylation of H3K9 by p300. This may be a prerequisite for the parallel induction of reductase activity of 11β-HSD1 in human amnion fibroblasts in a feed-forward loop that may influence fetal development and the onset of parturition.

The cytoprotective effects of bilirubin and biliverdin on rat hepatocytes and human erythrocytes and the impact of albumin

1991 ◽  
Vol 69 (12) ◽  
pp. 828-834 ◽  
Author(s):  
Tai-Wing Wu ◽  
Doug Carey ◽  
Jun Wu ◽  
Hiroshi Sugiyama
Keyword(s):  
Rat Hepatocytes ◽  
Human Erythrocytes ◽  
Red Cells ◽  
Peroxyl Radicals ◽  
Blood Levels ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Hepatocyte Necrosis ◽  
The Impact ◽  
First Time

The hypothesis that unconjugated bilirubin and biliverdin are cytoprotective antioxidants has been examined for the first time in systems containing cells. In primary rat hepatocytes exposed to xanthine oxidase and hypoxanthine, bilirubin (0–60 μM) failed to prolong cell survival. In contrast, biliverdin (20–100 μM) markedly delayed hepatocyte necrosis in a concentration-dependent manner. When 0.3 mM of albumin was present, bilirubin (0–50 μM) became protective of hepatocytes, while biliverdin was less dramatically enhanced in its cytoprotective effect. In human erythrocytes exposed to peroxyl radicals, bilirubin and biliverdin inhibited 50% cell lysis at lower concentrations than Trolox and ascorbate, respectively. Albumin alone appeared less cytoprotective in red cells than in hepatocytes, but its presence enhanced the effects of both pigments on erythrocytes. Of probable physiologic relevance, bilirubin with albumin present or biliverdin alone protected hepatocytes substantially (and to a lesser extent red cells) at the normal blood levels of bilirubin (3.4–26 μM). Moreover, the fact that the pigments are cytoprotective at higher bilirubin levels (e.g., 50–100 μM) tempts the speculation that they may be circulating cytoprotectors of overlooked importance in jaundice.Key words: cytoprotection, biliverdin, bilirubin, albumin.

scholarly journals Toxicity Effects of Methylene Blue on Rat Intervertebral Disc Annulus Fibrosus Cells

2019 ◽  
Vol 2 (22.2) ◽  
pp. 155-164
Author(s):  
Liang Zhang
Keyword(s):  
Methylene Blue ◽  
Cell Apoptosis ◽  
Annulus Fibrosus ◽  
Caspase 3 ◽  
In Vitro Study ◽  
Collagen Type I ◽  
Type I ◽  
Dependent Manner ◽  
Concentration Dependent Manner

Background: There is an increasing local application of methylene blue (MB) in the treatment of discogenic low back pain (LBP) and percutaneous transforaminal endoscopic discectomy (PTED) procedures. MB could generate DNA damage and induce apoptosis in different cell types; however, the effects of MB on intervertebral disc (IVD) annulus fibrosus (AF) cells are not clearly understood. Objective: The objective of this study was to investigate the effects of different concentrations of MB on rat AF cells in vitro. Study Design: This study used an experimental design. Setting: This research was conducted at the Orthopaedic Institute of the Clinical Medical College of Yangzhou University. Methods: AF cells were isolated and cultured with different concentrations of MB (0, 2, 20, and 200 μg/mL) and assessed to determine the possible cytotoxic effects of MB. The cell proliferation was detected by Cell Counting Kit-8 (CCK-8) assay. The inverted phase-contrast microscopy was used to perform morphological observation of apoptotic cells, and flow cytometry was used to measure the incidence of cell apoptosis. The mRNA and protein expression levels of apoptosis-associated genes (caspase-3, Bcl-2, and Bax) and other related genes (collagen type I, transforming growth factor β1 [TGF-β1], fibroblast growth factor [bFGF], and tissue inhibitor of metalloproteinase-1 [TIMP-1]) were analyzed by quantitative real-time PCR (RT-PCR) and Western blotting. Results: Our results indicated that MB reduced cell viability in a concentration- and timedependent manner. MB also induced marked AF cell apoptosis in a concentration-dependent manner observed by inverted phase-contrast microscopy, flow cytometry, and indicated by the increased expression of caspase-3. Both RT-PCR and Western blotting revealed significant upregulation of Bax and caspase-3 expression levels accompanied by decreased expression of Bcl2 in a concentration-dependent manner. Moreover, collagen type I, TGF-β1, bFGF, and TIMP-1 mRNA and protein levels were also found to be decreased by MB in a concentration-dependent manner. Limitations: Limitations of this study were the in vitro study design and lack of in vivo validation of the observed effects of MB on human IVD cells. Conclusions: Our results indicate that a high concentration of MB can not only inhibit proliferation and paracrine function of AF cells, but can also induce cell apoptosis in a concentration-dependent manner, suggesting that it is necessary to choose low concentrations of MB in practical application and limit the use of MB in the treatment of discogenic LBP to research protocols. Key words: Methylene blue, annulus fibrosus cell, proliferation, apoptosis, paracrine

Urea activation of K-Cl transport in human erythrocytes

1995 ◽  
Vol 268 (4) ◽  
pp. C1018-C1025 ◽  
Author(s):  
D. M. Kaji ◽  
C. Gasson
Keyword(s):  
Time Lag ◽  
Renal Medulla ◽  
Normal Subjects ◽  
Lag Time ◽  
Human Erythrocytes ◽  
Cell Swelling ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Activated State ◽  
High Concentrations

This report prompted us to examine the effect of urea on K-Cl cotransport in human erythrocytes. In human erythrocytes, urea activated K-Cl cotransport reversibly and in a concentration-dependent manner. Pretreatment with okadaic acid abolished the urea activation of transport, suggesting that exposure to urea resulted in net dephosphorylation of the transporter or a key regulator and that the action of urea was exerted proximal to the phosphorylation-dephosphorylation step. At a concentration of 200 mM, urea activated K-Cl cotransport without any delay, even in the absence of cell swelling. However, with increasing urea concentrations, an appreciable increase in lag time was observed before the final steady-state flux was reached, suggesting that urea inhibits a regulatory kinase. The latter conclusion was also supported by the finding that, at any given urea concentration, the lag time for activation was greater than the lag time for deactivation. Mg depletion activated cotransport, and urea had no additional stimulatory effect in Mg-depleted cells. In urea-pretreated cells, swelling further activated cotransport, but without any measurable delay, in contrast to a time lag of 8 min when control cells (not exposed to urea) were swollen. The latter finding suggests that urea promotes the conversion of transporters from the resting to the partially activated state. These findings raise the possibility that high concentrations of urea in the renal medulla may play a role in the decrease in cell volume that occurs during the maturation of reticulocytes and young erythrocytes, both in normal subjects and in subjects with hemoglobinopathies.

Bisphenol A decreases the spontaneous contractions of rat uterus in vitro through a nitrergic mechanism

2018 ◽  
Vol 29 (6) ◽  
pp. 593-598 ◽  
Author(s):  
Hemlata Gupta ◽  
Shripad B. Deshpande
Keyword(s):  
Methylene Blue ◽  
Bisphenol A ◽  
Estrogenic Activity ◽  
Dependent Manner ◽  
Uterine Contractility ◽  
Adult Rats ◽  
Concentration Dependent Manner ◽  
Uterine Contractions ◽  
Synthase Inhibitor

Abstract Background: Bisphenol A (BPA), a chemical used in the manufacture of plastics, has toxic effects on various systems of the human body including the reproductive system. BPA possesses estrogenic activity and is implicated in altering oogenesis, ovulation, and fertility. In addition to ovulatory changes, uterine contractility is an important factor for fertility. However, the effects of BPA on myometrial contractions are not known. Therefore, we examined the effect of BPA on rat uterine contractions. Methods: The uterus was isolated from adult rats showing estrous phase, and spontaneous in vitro contractions were recorded (35±1 °C). The effect of cumulative concentrations of BPA was determined. Further, the involvement of nitric oxide (NO) and guanylyl cyclase (GC) for the BPA-induced changes on uterine contractility was evaluated using the NO synthase inhibitor (L-NAME) or GC inhibitor (methylene blue). Results: BPA decreased the amplitude and frequency of spontaneous uterine contractions in a concentration-dependent manner. A decrease of 50% occurred at 1 and 3 μM for amplitude and frequency, respectively. L-NAME (N-ω-nitro-l-arginine methyl ester) blocked the BPA-induced decrease in amplitude at all concentrations but antagonized the frequency only at the maximum concentration (10 μM). Methylene blue (a GC inhibitor) did not block the BPA-induced responses but for the frequency at 10 μM of BPA. Conclusions: The results indicate that BPA decreased the amplitude and frequency of spontaneous uterine contractions by involving the nitrergic mechanism; however, the GC mechanism is not involved in the depression.

Antrodia Camphorata in Submerged Culture Protects Low Density Lipoproteins Against Oxidative Modification

2006 ◽  
Vol 34 (02) ◽  
pp. 217-231 ◽  
Author(s):  
Hsin-Ling Yang ◽  
You-Cheng Hseu ◽  
Jing-Yi Chen ◽  
Yi-Jen Yech ◽  
Fung-Jou Lu ◽  
...  
Keyword(s):  
Oxidized Ldl ◽  
Oxidant Stress ◽  
Antioxidant Properties ◽  
Low Density Lipoproteins ◽  
Oxidative Modification ◽  
Low Density ◽  
Dependent Manner ◽  
Antrodia Camphorata ◽  
Concentration Dependent Manner ◽  
Apo B

Antrodia camphorata is well known in Taiwan as a traditional Chinese medicine. In this study, we have investigated the antioxidant properties of a fermented culture broth of Antrodia camphorata (FCBA) and the aqueous extracts of mycelia from Antrodia camphorata (AEMA) on the oxidative modification of human low-density lipoproteins (LDL), as induced by either copper sulfate ( CuSO 4) or 2,2′-azo-bis(2-amidinopropane) hydrochloride (AAPH). Under such oxidant stress, FCBA and AEMA appear to possess antioxidant properties with respect to oxidation of LDL in a time-and concentration-dependent manner, as assessed by inhibition of thiobarbituric acid-reactive substances (TBARS) formation, conjugated diene production, and cholesterol degradation of oxidized LDL. In addition, both FCBA and AEMA exhibited a remarkable ability to rescue the relative electrophoretic mobility and fragmentation of the Apo B moiety of the oxidized LDL. Furthermore, FCBA and AEMA effectively protected the endothelial cells from the damaging effects of the CuSO 4-oxidized LDL. Our findings suggest that the antioxidant properties of Antrodia camphorata may also provide effective protection from atherosclerosis.

Oxidative stress inhibits bradykinin-stimulated 45Ca2+ flux in pulmonary vascular endothelial cells

1991 ◽  
Vol 260 (2) ◽  
pp. H549-H556
Author(s):  
S. J. Elliott ◽  
W. P. Schilling
Keyword(s):  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Reductase Activity ◽  
Oxidant Stress ◽  
K Channel ◽  
Dependent Manner ◽  
Vascular Endothelial ◽  
Prolonged Incubation ◽  
Glutathione Reductase Activity ◽  
Tetrabutylammonium Chloride

The effects of oxidant stress and altered glutathione reductase activity on agonist-induced flux of Ca2+ were studied in cultured calf pulmonary artery endothelial cells using radioisotopic 45Ca2+. Bradykinin-stimulated uptake of 45Ca2+ was determined after cells were incubated with the membrane-permeant oxidant t-butylhydroperoxide (0.4 mM) for various durations. t-Butylhydroperoxide increased uptake of 45Ca2+ under basal conditions and significantly decreased bradykinin-stimulated uptake in a time-dependent manner through incubation periods of 2 h. Preincubation of cells with 1,3-bis(chloroethyl)-1-nitrosourea markedly reduced bradykinin-stimulated uptake in cells subsequently treated with t-butylhydroperoxide. Bradykinin-stimulated efflux of 45Ca2+ and 86Rb+ was examined in control and oxidant-stressed endothelial cells. t-Butylhydroperoxide initially decreased bradykinin-stimulated efflux of 45Ca2+ but had no effect on 86Rb+ efflux. After more prolonged incubation with the oxidant, stimulated 45Ca2+ efflux was further inhibited, and basal efflux of 86Rb+ was increased to a rate similar to that observed with bradykinin stimulation. Elevated basal 86Rb+ efflux was blocked by tetrabutylammonium chloride, a selective inhibitor of Ca2(+)-dependent K+ channels in endothelial cells. These findings, together with our previously described results using fura-2, suggest that oxidant stress initially inhibits bradykinin-stimulated Ca2+ influx and later inhibits stimulated Ca2+ efflux. Finally, cytosolic free Ca2+ concentration becomes persistently elevated and is associated with elevated basal efflux of K+ via the Ca2(+)-dependent K+ channel.

Nucleoside Transport. II. Inhibition by p-Nitrobenzylthioguanosine and Related Compounds

1971 ◽  
Vol 49 (2) ◽  
pp. 271-274 ◽  
Author(s):  
A. R. P. Paterson ◽  
J. M. Oliver
Keyword(s):  
Potent Inhibitor ◽  
Facilitated Transport ◽  
Human Erythrocytes ◽  
Nucleoside Transport ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Uridine Uptake ◽  
Related Compounds

p-Nitrobenzylthioguanosine (NBTGR) was found to be a potent inhibitor of nucleoside transport by human erythrocytes; initial rates of uridine uptake were reduced to zero upon exposure of cells to 10−6 M NBTGR. The inhibitor was firmly bound because repeated washing did not restore uridine transport capability to NBTGR-treated cells. NBTGR inhibited the influx of uridine, inosine, and cytidine, without inhibiting the uptake of the corresponding bases, or that of D-glucose or L-leucine. Uridine antagonized the NBTGR inhibition of uridine transport in a concentration-dependent manner. NBTGR and related compounds appear to interact with the mechanism for the facilitated transport of nucleosides.

scholarly journals Methylene Blue Inhibits the SARS-CoV-2 Spike–ACE2 Protein-Protein Interaction–a Mechanism that can Contribute to its Antiviral Activity Against COVID-19

2021 ◽  
Vol 11 ◽  
Author(s):  
Damir Bojadzic ◽  
Oscar Alcazar ◽  
Peter Buchwald
Keyword(s):  
Methylene Blue ◽  
Antiviral Activity ◽  
Protein Interactions ◽  
Inhibitory Activity ◽  
Organic Dyes ◽  
Chemical Space ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Viral Attachment ◽  
Protein Protein Interaction

Due to our interest in the chemical space of organic dyes to identify potential small-molecule inhibitors (SMIs) for protein-protein interactions (PPIs), we initiated a screen of such compounds to assess their inhibitory activity against the interaction between SARS-CoV-2 spike protein and its cognate receptor ACE2, which is the first critical step initiating the viral attachment and entry of this coronavirus responsible for the ongoing COVID-19 pandemic. As part of this, we found that methylene blue, a tricyclic phenothiazine compound approved by the FDA for the treatment of methemoglobinemia and used for other medical applications (including the inactivation of viruses in blood products prior to transfusion when activated by light), inhibits this interaction. We confirmed that it does so in a concentration-dependent manner with a low micromolar half-maximal inhibitory concentration (IC50 = 3 μM) in our protein-based ELISA-type setup, while chloroquine, siramesine, and suramin showed no inhibitory activity in this assay. Erythrosine B, which we have shown before to be a promiscuous SMI of PPIs, also inhibited this interaction. Methylene blue inhibited the entry of a SARS-CoV-2 spike bearing pseudovirus into ACE2-expressing cells with similar IC50 (3.5 μM). Hence, this PPI inhibitory activity could contribute to its antiviral activity against SARS-CoV-2 even in the absence of light by blocking its attachment to ACE2-expressing cells and making this inexpensive and widely available drug potentially useful in the prevention and treatment of COVID-19 as an oral or inhaled medication.

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