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      The Water-Soluble Fraction (<10 kD) of Bee Venom (Apis mellifera) Produces Inhibitory Effect on Apical Transporters in Renal Proximal Tubule Cells

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          Abstract

          Human envenomation caused by bee stings has been reported to cause acute renal failure and the pathogenetic mechanisms of these renal functional changes are still unclear. Bee venom is also a complex mixture of enzymes and proteins. Thus, this study was conducted to examine the effects of bee venom (BV, Apis mellifera) fractions on apical transporters’ activity and its related signal pathways in primary cultured renal proximal tubule cells. Whole BV was extracted into three fractions according to solubility [a water-soluble fraction (BVA), an ethylacetate-soluble fraction (BVE), and a hexane-soluble fraction (BVH)]. BVA fraction was further separated to three portions according to molecular weights: BF1 (>20 kD), BF2 (10–20 kD), and BF3 (<10 kD). Each fraction was treated to the PTCs to the ratio of BV (1 µg/ml). BVA (930 ng/ml) significantly decreased cell viability, but BVH (27 ng/ml) and BVE (43 ng/ml) did not. BF3 (710 ng/ml) among BVA fractions predominantly decreased cell viability and inhibited α-methyl- D-glucopyranoside (α-MG), phosphate (Pi), and Na<sup>+</sup> uptake. In addition, BF3 increased [<sup>3</sup>H] arachidonic acid release, lipid peroxide formation, and Ca<sup>2+</sup> uptake. These effects of BF3 were blocked by mepacrine and AACOCF<sub>3</sub> (phospholipase A<sub>2</sub> inhibitors) or N-acetylcysteine, vitamin C, and vitamin E (antioxidants). In conclusion, BF3 (<10 kD) among BV fractions is the most effective portion in BV-induced inhibition of α-MG, P<sub>i</sub>, and Na<sup>+</sup> uptake and these effects of BF3 are associated with phospholipase A<sub>2</sub>-oxidative stress-Ca<sup>2+</sup> signal cascade in the primary cultured rabbit renal proximal tubule cells.

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          Most cited references 9

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          Characterization of primary rabbit kidney cultures that express proximal tubule functions in a hormonally defined medium

          Primary cultures of rabbit-kidney epithelial cells derived from purified proximal tubules were maintained without fibroblast overgrowth in a hormone-supplemented serum-free medium (Medium RK-1). A hormone- deletion study indicated that the primary cultures derived from purified rabbit proximal tubules required all of the three supplements in Medium RK-1 (insulin, transferrin, and hydrocortisone) for optimal growth but did not grow in response to EGF and T3. In contrast, the epithelial cells in primary cultures derived from an unpurified preparation of rabbit kidney tubules and glomeruli grew in response to EGF and T3, as well as insulin, transferrin, and hydrocortisone. These observations suggest that kidney epithelial cells derived from different segments of the nephron grow differently in response to hormones and growth factors. Differentiated functions of the primary cultures derived from proximal tubules were examined. Multicellular domes were observed, indicative of transepithelial solute transport by the monolayers. The proximal tubule cultures also accumulated alpha- methylglucoside (alpha-MG) against a concentration gradient. However, little or no alpha-MG accumulation was observed in the absence of Na+. Metabolic inhibitor studies also indicated that alpha-MG uptake by the primaries is an energy-dependent process, and depends upon the activity of the Na+/K+ ATPase. Phlorizin at 0.1 mM significantly inhibited 1 mM alpha-MG uptake whereas 0.1 mM phloretin did not have a significant inhibitory effect. Similar observations have been made concerning the Na+-dependent sugar-transport system located on the lumenal side of the proximal tubule, whereas the Na+-independent sugar transporter on the peritubular side is more sensitive to inhibition by phloretin than phlorizin. The cultures also exhibited PTH-sensitive cyclic AMP synthesis and brush-border enzymes typical of proximal cells. However, the activities of the enzymes leucine aminopeptidase, alkaline phosphatase, and gamma-glutamyl-transpeptidase were lower in the cultures than in purified proximal-tubule preparations from which they are derived.
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            Induction of apoptosis by arachidonic acid in human retinoblastoma Y79 cells: involvement of oxidative stress.

            Arachidonic acid administration caused apoptosis in Y79 cells, as shown by typical morphological changes, phosphatidylserine externalization, chromatin condensation, processing and activation of caspase-3 and cleavage of the endogenous caspase substrate poly-(ADP-ribose)-polymerase. Arachidonic acid also caused lamin B cleavage, suggesting caspase-6 activation. Arachidonic acid treatment was accompanied by increased formation of the lipid peroxidation end products malondialdehyde and 4-hydroxy-2-nonenal, lowering in reduced glutathione content and in mitochondrial membrane potential. Inhibiting glutathione synthesis sensitized Y79 cells to apoptosis-inducing stimuli, whilst replenishing reduced glutathione attenuated arachidonic acid toxicity. Similar findings were obtained using hydroperoxyeicosatetranoic acids (oxygenated metabolites of arachidonic acid which deplete the reduced glutathione pool) and nordihydroguaretic acid, a general inhibitor of lipooxygenase pathway. which may also trigger rapid depletion of reduced glutathione. Melittin, which is known to activate phospholipase A2, also potently induced apoptosis. Arachidonic acid toxicity was inversely related to cell density. This could depend on an increased production of molecules with antiapoptotic effect; insulin-like growth factors could most likely be one of these molecules. These results propose a role for oxidative stress in the cytotoxicity induced by arachidonic acid in Y79 cells and suggest that these cells could be protected from such toxicity as long as sufficient levels of reduced glutathione and survival factors are present.
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              Possible mechanisms of action of cobra snake venom cardiotoxins and bee venom melittin.

              Cobra snake venom cardiotoxins and bee venom melittin share a number of pharmacological properties in intact tissues including hemolysis, cytolysis, contractures of muscle, membrane depolarization and activation of tissue phospholipase C and, to a far lesser extent, an arachidonic acid-associated phospholipase A2. The toxins have also been demonstrated to open the Ca2+ release channel (ryanodine receptor) and alter the activity of the Ca(2+)+Mg(2+)-ATPase in isolated sarcoplasmic reticulum preparations derived from cardiac or skeletal muscle. However, a relationship of these actions in isolated organelles to contracture induction has not yet been established. The toxins also bind to and, in some cases, alter the function of a number of other proteins in disrupted tissues. The most difficult tasks in understanding the mechanism of action of these toxins have been dissociating the primary from secondary effects and distinguishing between effects that only occur in disrupted tissues and those that occur in intact tissue. The use of cardiotoxin and melittin fractions contaminated with trace ('undetectable') amounts of venom-derived phospholipases A2 has continued to be common practice, despite the problems associated with the synergism between the toxins and enzymes and the availability of methods to overcome this problem. With adequate precautions taken with regard to methodology and interpretation of results, the cobra venom cardiotoxins and bee venom melittin may prove to be useful probes of a number of cell processes, including lipid metabolism and Ca2+ regulation in skeletal and cardiac muscle.
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                Author and article information

                Journal
                KBR
                Kidney Blood Press Res
                10.1159/issn.1420-4096
                Kidney and Blood Pressure Research
                S. Karger AG
                1420-4096
                1423-0143
                2002
                2002
                26 February 2003
                : 25
                : 6
                : 375-383
                Affiliations
                aDepartment of Veterinary Physiology, College of Veterinary Medicine, Chonnam National University, Kwangju; bCollege of Veterinary Medicine, Chonbuk National University, Jeonju; cCollege of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Suwon, and dCollege of Pharmacy & Natural Products Research Institute, Seoul National University, Seoul, Korea
                Article
                68696 Kidney Blood Press Res 2002;25:375–383
                10.1159/000068696
                12590201
                © 2002 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: 9, Tables: 2, References: 37, Pages: 9
                Product
                Self URI (application/pdf): https://www.karger.com/Article/Pdf/68696
                Categories
                Original Paper

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