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    Arsenate V induced glutathione efflux from human erythrocytes
    (Elsevier Gmbh, 2012) Yildiz, Deniz; Cakir, Yeliz
    Objective: The objective of the present study was to investigate if arsenate V exposure results in glutathione efflux from human erythrocytes. Procedure: The changes in intracellular and extracellular nonprotein sulfhydryl and glutathione levels were determined in arsenate (V) exposed erythrocytes. Presence of any cellular membrane damage was assessed by lactate dehydrogenase activity measurement in the supernatant. Results: When erythrocytes were exposed to 10 mM of arsenate (V) for 4h, the intracellular NPSH level decreased to 0.28 +/- 0025 mu mol/ml erythrocyte. In contrast, extracellular nonprotein thiol level was increased to 0.180 +/- 0.010 mu mol/ml erythrocyte in 4h. Extracellular glutathione levels reached to 0.028 +/- 0.001, 0.052 +/- 0.002, and 0.054 +/- 0.004 mu mol/ml erythrocyte with 1, 5, and 10 mM of arsenate (V), respectively. Utilization of MK571 a multi drug resistance-associated protein 1 inhibitor decreased the rate of glutathione efflux from erythrocytes suggesting a role for this membrane transporter in the process. Conclusion: The results of the present study indicate that erythrocytes efflux glutathione when exposed to arsenate (V). (C) 2011 Elsevier GmbH. All rights reserved.
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    Efflux of Glutathione and Glutathione Complexes from Human Erythrocytes in Response to Inorganic Arsenic Exposure
    (Humana Press Inc, 2012) Yildiz, Deniz; Cakir, Yeliz
    The objective of the present study was to investigate if arsenic exposure results in glutathione efflux from human erythrocytes. Arsenite significantly depleted intracellular nonprotein thiol level in a time- and concentration-dependent manner. The intracellular nonprotein thiol level was decreased to 0.767 +/- 0.0017 mu mol/ml erythrocyte following exposure to 10 mM of arsenite for 4 h. Extracellular nonprotein thiol level was increased concomitantly with the intracellular decrease and reached to 0.481 +/- 0.0005 mu mol/ml erythrocyte in 4 h. In parallel with the change in extracellular nonprotein thiol levels, significant increases in extracellular glutathione levels were detected. Extracellular glutathione levels reached to 0.122 +/- 0.0013, 0.226 +/- 0.003, and 0.274 +/- 0.004 mu mol/ml erythrocyte with 1, 5, and 10 mM of arsenite, respectively. Dimercaptosuccinic acid treatment of supernatants significantly increased the glutathione levels measured in the extracellular media. Utilization of MK571 and verapamil, multidrug resistance-associated protein 1 and Pgp inhibitors, decreased the rate of glutathione efflux from erythrocytes suggesting a role for these membrane transporters in the process. The results of the present study indicate that human erythrocytes efflux glutathione in reduced free form and in conjugated form or forms that can be recovered with dimercaptosuccinic acid when exposed to arsenite.
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    Efflux of glutathione and glutathione complexes from human erythrocytes in response to vanadate
    (Academic Press Inc Elsevier Science, 2013) Cakir, Yeliz; Yildiz, Deniz
    The main objective of the present study was to investigate if vanadate is extruded from the cells in a glutathione dependent manner resulting in the appearance of extracellular glutathione and complexes of glutathione with vanadium. Vanadate significantly depleted intracellular non-protein sulfhydryl (NPSH) levels in a time- and concentration-dependent manner. The intracellular NPSH level was decreased to 0.0 +/- 0.0 mu mol/ml erythrocyte when exposed to 10 mM of vanadate for 4 h. Extracellular NPSH level was increased concomitantly with the intracellular decrease and reached to 0.1410 +/- 0.005 mu mol/ml erythrocyte in 4 h. Intracellular decrease and extracellular increase in NPSH levels were significantly inhibited in the presence of DIDS, a chloride-bicarbonate exchanger which also mediates phosphate and arsenate transport in erythrocytes. In parallel with the increase in extracellular NPSH levels, significant increases in extracellular glutathione levels were detected following exposure to vanadate. Extracellular glutathione levels reached to 0.0150 +/- 0.0.001, 0.0330 +/- 0.001, and 0.0576 +/- 0.002 mu mol/ml erythrocyte with 1, 5, and 10 mM of vanadate respectively. Dimercaptosuccinic acid treatment of supematants significantly increased the glutathione levels measured in the extracellular media. Utilization of MK571 an MRP inhibitor decreased the rate of glutathione efflux from erythrocytes suggesting a role for this membrane transporter in the process. A known methylation inhibitor periodate oxidized adenosine decreased the rate of glutathione efflux from erythrocytes. This observed decrease in extracellular GSH levels suggests that GSH release partly requires a proper cellular methylation process and that part of GSH detected in the extracellular media may arise from GSH-vandium complexes. The results of the present study indicate that human erythrocyte efflux glutathione in reduced free form and in conjugated form/s that can be recovered with dimercaptosuccinic acid when exposed to vanadate. (C) 2012 Elsevier Inc. All rights reserved.
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    Homocysteine influx and efflux: Participation of erythrocytes in homocysteine homeostasis of the plasma
    (Gazi University Eti Mahallesi, 2010) Yildiz, Deniz; Civi, Zuleyha; Cakir, Yeliz
    One of the main objective of the present study was to determine if erythrocytes play a role in regulation of plasma homocysteine concentration. Another objective was to investigate if erythrocytes convert homocystine into homocysteine. In the present study, we exposed erytrocytes to different concentrations of homocysteine and then measured the nonprotein sulfhydryl (NPSH) concentrations. Erythrocytes treated in the same manner were later utilized for the homocysteine efflux studies. The effect of temperature on the influx and the efflux processes were also evaluated. We also determined the rate of homocysteine influx in the presence of different amino acids. The homocysteine influx studies demonstrated that erythrocytes can respond to increases in homocysteine concentration in the extracellular media and influx homocysteine in a concentration-dependent manner. NPSH concentrations in erythrocytes treated with 1 mM homocysteine reached to 1.47 ± 0.01 imol/ml erythrocyte in 1 h whereas this concentration reached to 2.01 ± 0.1 imol/ml erythrocyte in 3 mM homocysteine treated erytrocytes. The homocysteine efflux is also determined to be time-and concentration -dependent. Extracellular concentration of NPSH in 1 mM homocysteine pre-treated erythrocytes reached to 0.266 ± 0.02 imol/ml erythrocyte in 1 h whereas this concentration reached to 0.64 ± 0.01 imol/ml erythrocyte with 3 mM homocysteine pre-treated erythrocytes. Our results also indicate that erythrocytes convert extracellulary applied homocystine into homocysteine. Depending on our results, it could be concluded that erythrocytes may play a significant role in the regulation of plasma homocysteine homeostasis.
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    Homocysteine Influx and Efflux: Participation of Erythrocytes in Homocysteine Homeostasis of the Plasma
    (Gazi Univ, 2010) Yildiz, Deniz; Civi, Zuleyha; Cakir, Yeliz
    One of the main objective of the present study was to determine if erythrocytes play a role in regulation of plasma homocysteine concentration. Another objective was to investigate if erythrocytes convert homocystine into homocysteine. In the present study, we exposed erytrocytes to different concentrations of homocysteine and then measured the nonprotein sulfhydryl (NPSH) concentrations. Erythrocytes treated in the same manner were later utilized for the homocysteine efflux studies. The effect of temperature on the influx and the efflux processes were also evaluated. We also determined the rate of homocysteine influx in the presence of different amino acids. The homocysteine influx studies demonstrated that erythrocytes can respond to increases in homocysteine concentration in the extracellular media and influx homocysteine in a concentration-dependent manner. NPSH concentrations in erythrocytes treated with 1 mM homocysteine reached to 1.47 +/- 0.01 mu mol/ml erythrocyte in 1 h whereas this concentration reached to 2.01 +/- 0.1 mu mol/ml erythrocyte in 3 mM homocysteine treated erytrocytes. The homocysteine efflux is also determined to be time-and concentration -dependent. Extracellular concentration of NPSH in 1 mM homocysteine pre-treated erythrocytes reached to 0.266 +/- 0.02 mu mol/ml erythrocyte in 1 h whereas this concentration reached to 0.64 +/- 0.01 mu mol/ml erythrocyte with 3 mM homocysteine pre-treated erythrocytes. Our results also indicate that erythrocytes convert extracellulary applied homocystine into homocysteine. Depending on our results, it could be concluded that eryhtrocytes may play a significant role in the regulation of plasma homocysteine homeostasis.
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    Hyperosmolarity induced cystine transport and cystine-cysteine cycle between erythrocytes and the plasma
    (Pharmaceutical Soc Japan, 2008) Yildiz, Deniz; Cakir, Yeliz
    The objective of the present study was to determine if a cystine-cysteine cycle operates between the erythrocytes and the plasma. In the present study we incubated the erythrocytes in krebs ringer phosphate buffer with different osmolarity containing different amounts of cystine. Our results show that erythrocytes do not uptake cystine from the environment when the osmolarity of the buffer is 310mOsmol/l.Erythrocytes also do not operate a cystine-cysteine cycle in this isoosmolar buffer. However, when exposed to hyperosmolar buffer in the ranges that occur in the kidney medulla which is in between 1200-1400 mOsmol/l erythrocytes start to uptake cystine from the environment and induce a cystine-cysteine cycle. The cystine uptake and cystine-cysteine cycle were characterized by measurement of changes in the free -SH concentrations in erythrocytes and in the buffer. Following incubation of erythrocytes in 1 mM cystine containing 1250 and 1300 mOsmol/l buffer, the free -SH concentrations in the buffer reached to 0. 102 +/- 0.002 and 0.241 +/- 0.013 mu mol/ml erythrocyte respectively. Our results demonstrate that erythrocytes display a cystine-cysteine cycle in hyperosmolar environment which is prevailed mainly in the kidney medulla. Our results also display that this process is biologically active and energy dependent. The observed cystine-cysteine cycle is inhibited when the erythrocytes are incubated at lower temperatures and in the absence of glucose. Our results suggest that erythrocytes uptake cystine, intracellulary reduce it to cysteine and release it back to the environment when exposed to hyperosmolar conditions. Erythrocytes may have a role in the regulation of plasma cystine and cysteine concentrations and may contribute to the regulation of plasma redox status.