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      Stress Response in a Leporine Renal Cell Model

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          Abstract

          It is well established that renal proximal tubule (RPT) cells grown under standard in vitro conditions attenuate many of their in vivo properties and functions. Thus, the study of renal stress response mechanisms requires an appropriate cell culture model. In the present study, we compared the heat stress (10 min, 45°C) response of freshly isolated RPT cells with that of RPT cells grown in vitro for 6 days under two different culture conditions: (1) SHAKE conditions, where oxygen levels and physiological functions are maintained via continuous media motion [Nowak G, Schnellmann RG: Am J Physiol 1996;271:C2072–2080] and (2) STILL conditions, involving standard cell culture which leads to partial hypoxia and a marked reduction in physiological functions. The freshly isolated RPT cells progressively synthesized heat shock proteins (HSPs) and stress glycoproteins (SGs) during a 3-hour culture period in vitro. Under these conditions, heat stress did not further increase HSP and SG synthesis. In RPT cells grown under SHAKE conditions, HSP70 synthesis was detected 1 h after heat stress and decreased below detection by 3 h. In contrast, the uptake of radiolabeled mannose into (glycoprotein) GP62 (M<sub>r</sub> 62,000), GP50, and GP38 was observed in control SHAKE cultures and was not further increased after heat stress. These results are consistent with immunohistochemistry studies, where similar changes in HSP70 and GP50 expression were noted. RPT cells grown under STILL conditions showed both increased synthesis of HSP70 and increased glycosylation of GP62, GP50, and GP38 as early as 1 h after heat stress, but in contrast to SHAKE conditions, this heat-induced stress response further intensified at 3 h after heat stress. By 7 h after heating, HSP synthesis returned to control levels, while glycosylation of GP62 and GP50 remained elevated. Based on our results, we conclude that freshly isolated RPT cells exhibit a stress response that may be caused by acute cell isolation/culture stress. While this stress response unfolds, freshly isolated RPT cells appear unable to respond to additional heat stress. RPT cells grown under SHAKE and STILL conditions exhibit high rates of SG glycosylation, especially that of GP62, possibly reflecting a ‘stress’ condition associated with growth on plastic substrate. Concurrently, RPT cells from STILL cultures show a higher capacity for responding to acute heat stress than SHAKE cultures, evidenced by the transiently increased HSP synthetic rates. The interpretation of the renal stress response capacity, therefore, must be linked to a specific culture condition.

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          Prompt protein glycosylation during acute heat stress.

          Constitutive patterns of protein synthesis and protein glycosylation are severely disrupted by acute heat stress. Stressed cells respond by preferential synthesis of specific proteins, e.g., the well-known family of heat shock proteins. We observed another response that rapidly occurs during heating periods as short as 10 min at 45 degrees C. During that period, CHO cells began to glycosylate specific proteins, designated as "prompt" stress glycoproteins (P-SG), while constitutive protein glycosylation ceased. Labeling of P-SGs showed a dose response with time and with temperature and appeared regardless of the label used (D-[3H]mannose or D-[3H]glucose). On SDS-PAGE, the major P-SG was characterized by M(r) approximately 67 kDa (P-SG67) and pI = 5.1. Other less prominent P-SGs appeared at M(r) 160, 100, 64, 60, and 47 kDa; incorporated label showed little turnover during 24 h at 37 degrees C. Prompt glycosylation was inhibited by tunicamycin, and label incorporated into P-SGs was sensitive to N-glycosidase F, but not to O-glycosidase. Analysis of enzymatically digested P-SG67 indicated that label had been incorporated into both high-mannose (Man9GlcNAc) and complex-type oligosaccharides. Brefeldin A did not eliminate P-SG67 labeling, but caused the further appearance of novel, Brefeldin-associated P-SGs. Labeling of P-SG67 oligosaccharides occurred without significant concomitant protein synthesis, suggesting that addition of labeled oligosaccharides largely occurred on mature, rather than nascent proteins. The functional significance of prompt glycosylation remains to be defined, but we propose that this novel phenomenon is an integral part of the cellular heat stress response.
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              Author and article information

              Journal
              NEF
              Nephron
              10.1159/issn.1660-8151
              Nephron
              S. Karger AG
              1660-8151
              2235-3186
              1998
              January 1998
              19 December 1997
              : 78
              : 1
              : 54-62
              Affiliations
              Departments of a Medicine, Division of Hematology/Oncology, and b Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Ark., c Department of Biological Sciences, SUNY at Albany, N.Y., USA
              Article
              44883 Nephron 1998;78:54–62
              10.1159/000044883
              9453405
              © 1998 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: 5, References: 30, Pages: 9
              Product
              Self URI (application/pdf): https://www.karger.com/Article/Pdf/44883
              Categories
              Original Paper

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