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      Altered Dye Diffusion and Upregulation of Connexin37 in Mouse Aortic Endothelium Deficient in Connexin40

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          Abstract

          Connexin40 (Cx40), connexin37 (Cx37) and connexin43 (Cx43) are subunit proteins of gap junction channels in the vascular wall which are presumably involved in the propagation of vasomotor signals. In this study we have investigated in Cx40-deficient versus wild-type aortic endothelium to which extent loss of Cx40 impairs intercellular communication. We show in Cx40-deficient mice that expression of both Cx37 and Cx43 protein was increased approximately 3- and 2-fold over the level in wild-type endothelium, respectively. Furthermore, Cx37 immunosignals were distributed more homogeneously on contacting plasma membranes in Cx40-deficient versus with wild-type endothelium. Cx43 was not detected in endothelium but only in smooth muscle cells of the vessel wall. Iontophoretic injection of Lucifer Yellow or neurobiotin into aortic endothelium of Cx40-deficient mice showed extensive intercellular transfer of neurobiotin but not of Lucifer Yellow. In contrast, intercellular spreading of Lucifer Yellow was observed in endothelium of wild-type aorta. As shown by electron microscopy, gap junctions in Cx40-deficient endothelium were morphologically different from those of wild-type vessels. These results demonstrate that dye diffusibility of endothelial gap junctions is different in Cx40-deficient and wild-type mice, although Cx40-deficient mice retain the capability of intercellular communication. Apparently, Cx40-deficient endothelial cells upregulate and redistribute Cx37 as a molecular adaptation to the lack of Cx40.

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          Most cited references 3

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          Genetic diseases and gene knockouts reveal diverse connexin functions.

          Intercellular channels present in gap junctions allow cells to share small molecules and thus coordinate a wide range of behaviors. Remarkably, although junctions provide similar functions in all multicellular organisms, vertebrates and invertebrates use unrelated gene families to encode these channels. The recent identification of the invertebrate innexin family opens up powerful genetic systems to studies of intercellular communication. At the same time, new information on the physiological roles of vertebrate connexins has emerged from genetic studies. Mutations in connexin genes underlie a variety of human diseases, including deafness, demyelinating neuropathies, and lens cataracts. In addition, gene targeting of connexins in mice has provided new insights into connexin function and the significance of connexin diversity.
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            Endothelium-specific replacement of the connexin43 coding region by a lacZ reporter gene.

            The murine gap junction protein connexin43 (Cx43) is expressed in blood vessels, with vastly different contribution by endothelial and smooth muscle cells. We have used the Cre recombinase under control of TIE2 transcriptional elements to inactivate a floxed Cx43 gene specifically in endothelial cells. Cre-mediated deletion led to replacement of the Cx43 coding region by a lacZ reporter gene. This allowed us to monitor the extent of deletion and to visualize the endothelial expression pattern of Cx43. We found widespread endothelial expression of the Cx43 gene during embryonic development, which became restricted largely to capillaries and small vessels in all adult organs examined. Mice lacking Cx43 in endothelium did not exhibit altered blood pressure, in contrast to mice deficient in Cx40. Our results show that lacZ activation after deletion of the target gene allows us to determine the extent of cell type-specific deletion after phenotypical investigation of the same animal.
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              Molecular cloning and functional expression of the mouse gap junction gene connexin-57 in human HeLa cells.

              A new mouse connexin gene has been isolated that codes for a connexin protein of 505 amino acid residues. Based on the predicted molecular mass of 57.115 kDa, it has been designated connexin-57. Similar to most other mouse connexin genes, the coding region of connexin-57 is not interrupted by introns and exists in the mouse genome as a single-copy gene. Within the connexin family, this new gene shows highest sequence identity to porcine connexin-60 in the alpha group of connexins. The connexin-57 gene was mapped to a position on mouse chromosome 4, 30 centimorgans proximal to a cluster of previously mapped connexin genes. Low levels of connexin-57 mRNA were detected in skin, heart, kidney, testis, ovary, intestine, and in the mouse embryo after 8 days post coitum, but expression was not detected in brain, sciatic nerve or liver. In order to analyze gene function, the connexin-57 coding region was expressed by transfection in human HeLa cells, where it restored homotypic intercellular transfer of microinjected neurobiotin. Heterotypic transfer was observed between HeLa connexin-57 transfectants and HeLa cells, expressing murine connexin-43, -37, or -30.3. Double whole-cell voltage clamp analyses revealed that HeLa-connexin-57 transfectants expressed about 10 times more channels than parental HeLa cells. Voltage gating by transjunctional and transmembrane voltages as well as unitary conductance ( approximately 27 picosiemens) were different from intrinsic connexin channels in parental HeLa cells.
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                Author and article information

                Journal
                JVR
                J Vasc Res
                10.1159/issn.1018-1172
                Journal of Vascular Research
                S. Karger AG
                1018-1172
                1423-0135
                2002
                April 2002
                10 May 2002
                : 39
                : 2
                : 160-172
                Affiliations
                aInstitut für Genetik, Abteilung Molekulargenetik und bInstitut für Zellbiologie, Universität Bonn, Deutschland; cDépartement de Zoologie et Biologie Animale, Université de Genève, Suisse
                Article
                57764 J Vasc Res 2002;39:160–172
                10.1159/000057764
                12011587
                © 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: 6, Tables: 1, References: 31, Pages: 13
                Categories
                Research Paper

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