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      Endothelin-1 Stimulates Hydrolysis of Phosphatidylcholine by Phospholipases C and D in Intact Rat Mesenteric Arteries


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          A characteristic of endothelin-1 (ET-1)-induced contraction is the prolonged duration of the response. Phosphatidylcholine (PC) hydrolysis has been implicated in sustained agonist-induced effects through the formation of the second messengers phosphatidic acid (PA) and diacylglycerol (DAG). Therefore, we have investigated the activation of PC-phospholipase D (PLD) and PC-phospholipase C (PLC) in intact rat mesenteric small arteries stimulated with ET-1 and determined whether PC-derived DAG is necessary for ET-1-induced contraction. PLD activity, measured as phosphatidylethanol (PEt) production in vessels labelled with [<sup>3</sup>H] or [<sup>14</sup>C]myristate, was increased in a concentration- and time-dependent manner following ET-1 stimulation, as were [<sup>3</sup>H]PA levels, peaking at 10 min (ET-1 100 nM) and remaining above control levels for up to 20 min. Inclusion of 0.5% ethanol during ET-1 stimulation reduced [<sup>3</sup>H]PA levels, but did not alter the time course of formation. In addition, [<sup>14</sup>C]choline release was increased, confirming PLD-mediated PC hydrolysis. In contrast, DAG levels, measured by [<sup>3</sup>H]myristate labelling and mass assay, increased transiently at 5 min of ET-1 stimulation only. Analysis of the subclasses of DAG demonstrated an increase in all types of DAG without any enrichment with arachidonate-containing species, indicating PC, not inositol lipid hydrolysis was the source of DAG. The PC-PLC inhibitor D609, 2.5 µg/ml, completely abolished the ET-1-induced increase in [<sup>14</sup>C]DAG without affecting the increase in [<sup>14</sup>C]PA, PLD activity or the contractile response. In [<sup>14</sup>C]choline-labelled vessels, [<sup>14</sup>C]phosphocholine levels were increased by ET-1 with a similar time course to DAG production. Removal of extracellular Ca<sup>2+</sup> and addition of 0.1 or 2 mM EGTA completely inhibited ET-1-stimulated PLD activity. The tyrosine kinase inhibitor tyrphostin A23 (100 µM) abolished ET-1-induced [<sup>3</sup>H]PA, [<sup>3</sup>H]PEt and [<sup>14</sup>C]DAG increases, whilst the negative analogue A1 was without effect. These data suggest that ET-1 couples via a calcium-dependent tyrosine kinase mechanism to PLD and PC-PLC in vascular smooth muscle. PC-derived DAG did not appear to be necessary for the contractile response, whereas sustained formation of PA, generated by PLD activity, implies that this lipid second messenger could be involved in the prolonged contractile response to this peptide.

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          Most cited references 11

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           E. R. Levin (1995)
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            Identification of an Essential Signaling Cascade for Mitogen-activated Protein Kinase Activation by Angiotensin II in Cultured Rat Vascular Smooth Muscle Cells

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              Unitary anion currents through phospholemman channel molecules.

              Phospholemman (PLM) is a 72-amino-acid peptide with a single transmembrane domain, the expression of which induces chloride currents in Xenopus oocytes. It has remained unknown whether PLM is an ion channel or acts as a channel regulator. Here we show, by measuring unitary anion currents across planar phospholipid bilayers to which immunoaffinity-purified recombinant PLM was added, that it does indeed form ion channels. Excised patches of oocytes expressing PLM had similar currents. Of the ions tested, the sulphonic amino acid taurine was the most permeant, and expression of PLM increased fluxes of radiolabelled taurine in oocytes. Phospholemman is the smallest protein in cell membranes known to form an ion channel and the taurine selectivity suggests that it is involved in cell volume regulation.

                Author and article information

                J Vasc Res
                Journal of Vascular Research
                S. Karger AG
                February 1999
                01 March 1999
                : 36
                : 1
                : 35-46
                Department of Medicine, Manchester Royal Infirmary, Manchester, UK
                25624 J Vasc Res 1999;36:35–46
                © 1999 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, Tables: 3, References: 60, Pages: 12
                Research Paper


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