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      Protective Effect of Pyruvate upon Cultured Mesothelial Cells Exposed to 2 m M Hydrogen Peroxide

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          Abstract

          Rat peritoneal mesothelial cells in culture have the capability of generating hydrogen peroxide. Exposure of these cells to glucose-enriched, lactated-buffered fluids for peritoneal dialysis significantly increases the production of H<sub>2</sub>O<sub>2</sub>. Increased liberation of oxygen radicals also involves the risk of damaging the peritoneal membrane. Pyruvate being a natural oxidant scavenger abundantly present in mammalian cells, we hypothesized that its protective effects facing H<sub>2</sub>O<sub>2</sub> can eventually be of relevance for the mesothelial monolayer of patients on long-term peritoneal dialysis. So far, we designed an experimental study in which rat peritoneal mesothelial cells in culture were exposed to 2 m M H<sub>2</sub>O<sub>2</sub>. Cell damage was estimated in terms of decreased capability of the mitochondrial dehydrogenases to reduce MTT. Addition of 2 m M sodium pyruvate to the medium prevented the negative effect of hydrogen peroxide. The MTT/protein values for the control group were 0.00357 ± 0.00075. The ratio after exposure to 2 m M H<sub>2</sub>O<sub>2</sub> was 0.00217 ± 0.00028, whereas that detected in cells incubated in H<sub>2</sub>O<sub>2</sub> plus pyruvate was 0.00325 ± 0.0082 (p < 0.05). These results indicate that pyruvate protected rat peritoneal mesothelial cells in culture against oxidant injury. These data are one more piece of evidence pointing at pyruvate as a potentially useful buffer for peritoneal dialysis solutions.

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          Most cited references 8

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          Blebbing, free Ca2+ and mitochondrial membrane potential preceding cell death in hepatocytes.

          Cell surface 'blebbing' is an early consequence of hypoxic and toxic injury to cells. A rise in cytosolic free Ca2+ has been suggested as the stimulus for bleb formation and the final common pathway to irreversible cell injury. Here, using digitized low-light video microscopy, we examine blebbing, cytosolic free Ca2+, mitochondrial membrane potential and loss of cell viability in individual cultured hepatocytes. Unexpectedly, we found that after 'chemical hypoxia' with cyanide and iodoacetate, cytosolic free Ca2+ does not change during bleb formation or before loss of cellular viability. Cell death was precipitated by a sudden breakdown of the plasma membrane permeability barrier, possibly caused by rupture of a cell surface bleb.
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            Hydrogen peroxide-induced increase in lung endothelial and epithelial permeability--effect of adenylate cyclase stimulation and phosphodiesterase inhibition.

            Neutrophil-derived hydrogen peroxide (H2O2) is believed to play an important role in inflammatory lung injury. We investigated the influence of pharmacological agents that increase intracellular c-AMP levels on endothelial and epithelial leakage in response to intravascular H2O2 challenge in buffer-perfused rabbit lungs. Endothelial permeability was assessed by determination of the capillary filtration coefficient (Kfc) and lung weight gain. Measurement of the clearance rate of inhaled aerosolized technetium-99m-labeled diethylenetriamine pentaacetic acid ([99mTc]DTPA) from the lungs into the perfusion fluid was used as an index of alveolar epithelial permeability. Experiments were performed in the presence of acetylsalicylic acid to suppress H2O2-induced lung prostanoid generation and concomitant vasoconstriction. Under these conditions, H2O2 admixture to the perfusate (250 microM) caused a greater than eight-fold increase in Kfc values, resulting in > 30 g lung weight gain within 30 min in the absence of any significant vasopressor response. Pretreatment with the adenylate cyclase activators prostaglandin E1 (0.1 microM) and forskolin (0.1 microM), the dual phosphodiesterase type III/IV inhibitor zardaverine (10 microM) as well as combinations of these drugs all caused a nearly complete suppression of this early Kfc increase; and severe edema formation (> 30 g) was retarded to approximately 50-55 min. In addition to the microvascular leakage response, H2O2 caused a four- to five-fold increase in the [99mTc]DTPA clearance rate, starting within 15 min and culminating after approximately 35 min. Adenylate cyclase activation reduced this epithelial leakage response by approximately 30%, whereas zardaverine exerted no significant effect. We conclude that both microvascular endothelial and alveolar epithelial barrier function are severely compromised by intravascular H2O2 challenge in intact lungs. Pharmacological approaches to increase c-AMP levels, including both adenylate cyclase activation and phosphodiesterase inhibition, partially block the endothelial response and, to a lesser extent, the epithelial response.
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              A colorimetric microtiter assay for the quantitation of cytokine activity on adherent cells in tissue culture.

               Yona Keisari (1992)
              A colorimetric microtiter assay was developed for the quantitation of adherent cells in culture, which is based on the staining of cells with a commercially available staining kit. Adherent L929, A375 cell lines and human monocytes were stained with Hemacolor reagents and the color eluted with SDS 0.5% was determined spectrophotometrically with an ELISA plate reader at 630 nm. The method enabled the detection of cells and was linear up to 3 x 10(4) L929 cells/well. The Hemacolor staining assay was compared to the crystal violet staining assay and the MTT reduction assay, and was found to be sensitive, accurate and reproducible, and has the advantage of enabling microscopic inspection of the stained cells prior to color elution. The assay was found to be suitable for the determination of cytotoxic cytokines, and the enumeration of adherent monocytes. This method might be also used for the quantitation of cytotoxic drugs, and the cytophatic activity of viruses.
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                Author and article information

                Journal
                NEF
                Nephron
                10.1159/issn.1660-8151
                Nephron
                S. Karger AG
                1660-8151
                2235-3186
                2000
                April 2000
                30 March 2000
                : 84
                : 4
                : 362-366
                Affiliations
                Department of Nephrology and Hypertension and Research Center for Experimental Nephrology, ‘Ha’ Emek’ Medical Center, Afula, Israel
                Article
                45612 Nephron 2000;84:362–366
                10.1159/000045612
                10754414
                © 2000 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                Figures: 1, References: 52, Pages: 5
                Product
                Self URI (application/pdf): https://www.karger.com/Article/Pdf/45612
                Categories
                Original Paper

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