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      Functional Nucleotide Receptor Expression and Sarcoplasmic Reticulum Morphology in Dedifferentiated Porcine Coronary Smooth Muscle Cells

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          Abstract

          The phenotypic dedifferentiation of vascular smooth muscle cells (SMCs) is an early event associated with cell culturing and vascular injury. The purpose of this study was to evaluate the SMC phenotype underlying the functional responsiveness of SMCs to nucleotides in organ culture. Porcine coronary arteries were either used fresh, cold stored (5°C) 4 days, or organ cultured (37°C) 4 days. Fura-2 digital imaging of single SMCs was used to measure the myoplasmic calcium (Ca<sub>m</sub>) response to 10 µ M of the following nucleotide receptor agonists: UTP, UDP, ATP, ADP, and 2-MeSATP. In contrast to the nucleotides UDP, ATP, ADP, and 2-MeSATP, the Ca<sub>m</sub> response increased 10-fold and the number of cells that responded to UTP increased 5-fold in SMCs from organ culture compared to SMCs from fresh or cold-stored arteries. Simultaneous imaging of Ca<sub>m</sub>, DNA content, and SR distribution in SMCs from organ culture indicated that the UTP-induced Ca<sub>m</sub> increase occurred exclusively in SMCs that had a dedifferentiated cell phenotype. Three-dimensional image reconstruction of the nucleus and sarcoplasmic reticulum (SR) revealed a novel transnuclear SR distribution that intertwined with the nucleus in fresh SMCs, while in SMCs from organ culture the SR was predominantly perinuclear and cytoplasmic. This study demonstrates that the functional up-regulation of UTP-sensitive receptors and the disappearance of the transnuclear SR distribution are novel features of dedifferentiated coronary SMCs.

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          Most cited references 5

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          Spatially and functionally distinct Ca2+ stores in sarcoplasmic and endoplasmic reticulum.

          The organization of calcium (Ca2+) stores in the sarcoplasmic and endoplasmic reticulum (S-ER) is poorly understood. The dynamics of the storage and release of calcium in the S-ER of intact, cultured astrocytes and arterial myocytes were studied with high-resolution imaging methods. The S-ER appeared to be a continuous tubular network; nevertheless, calcium stores in the S-ER were organized into small, spatially distinct compartments that functioned as discrete units. Cyclopiazonic acid (an inhibitor of the calcium pump in the S-ER membrane) and caffeine or ryanodine unloaded different, spatially separate compartments. Heterogeneity of calcium stores was also revealed in cells activated by physiological agonists. These results suggest that cells can generate spatially and temporally distinct calcium signals to control individual calcium-dependent processes.
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            Cloning and functional expression of a human uridine nucleotide receptor.

            In order to isolate new subtypes of P2 purinoceptors, sets of degenerate oligonucleotide primers were synthesized on the basis of the best conserved segments in the published sequences of the chick brain P2Y/P2Y1 and murine neuroblastoma P2U/P2Y2 receptors. Their use in polymerase chain reaction (PCR) experiments on human genomic DNA amplified, among other things, a 712-base pair sequence, that was used as a probe to screen a human genomic DNA library. Several clones corresponding to a single locus were isolated, and the sequence analysis revealed an intronless 1095-base pair open reading frame. The deduced amino acid sequence is consistent with a G protein-coupled receptor and exhibits 51% identity with the human P2Y2 receptor and 35% with the chick P2Y1 receptor. A close comparison with the human P2Y2 sequence reveals the conservation of histidine 262, arginine 265, lysine 289, and arginine 292, which were reported to be involved in nucleotide binding (Erb, L., Garrad, R., Wang, Y., Quinn, T., Turner, J. T., and Weisman, G. A. (1995) J. Biol. Chem. 270, 4185-4188). Northern blot analysis detected a 1.8-kilobase messenger RNA in human placenta. The coding sequence was inserted in the pcDNA3 vector in order to transfect 1321N1 human astrocytoma cells. In cells stably expressing the receptor, UTP and UDP stimulated the formation of inositol phosphates with equivalent potency and maximal effect, ATP behaved as a partial agonist, and ADP was almost inactive. We have thus cloned a new member of the G protein-coupled P2 purinergic receptor family, which functionally behaves as a pyrimidinergic receptor.
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              Cloning, expression, and chromosomal localization of the human uridine nucleotide receptor gene.

              Extracellular ATP and ADP mediate diverse physiological responses in mammalian cells, in part through the activation of G protein-coupled P2 purinoceptors. The cloning and expression of cDNAs encoding several P2 purinoceptor subtypes have enabled rapid advances in our understanding of the structural and functional properties of these receptors. The current report describes the isolation of a gene from a human genomic library that encodes a protein with the greatest similarity to the human P2U purinoceptor, a subtype that is distinguished by its ability to be activated by uridine nucleotides as well as adenine nucleotides. When expressed in a mammalian cell line, this novel receptor is activated specifically by UTP and UDP but not by ATP and ADP. Activation of this uridine nucleotide receptor resulted in increased inositol phosphate formation and calcium mobilization. Fluorescence in situ hybridization revealed that the gene encoding the uridine nucleotide receptor is located in region q13 of the X chromosome. Dendrogram analysis of the G protein-coupled P2 purinoceptors and the uridine nucleotide receptor indicates that these receptors belong to a family that may be more aptly named nucleotide receptors.
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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
                2001
                October 2001
                17 September 2001
                : 38
                : 5
                : 432-443
                Affiliations
                aDepartments of Physiology and bInternal Medicine, cSchool of Medicine, Dalton Cardiovascular Research Center, and dDiabetes & Cardiovascular Biology Program, University of Missouri, Columbia, Miss., USA
                Article
                51076 J Vasc Res 2001;38:432–443
                10.1159/000051076
                11561145
                © 2001 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: 7, References: 47, Pages: 12
                Categories
                Research Paper

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