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      The Duration and Amplitude of the Plateau Phase of ATP- and ADP-Evoked Ca 2+ Signals Are Modulated by Ectonucleotidases in in situ Endothelial Cells of Rat Aorta

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          Abstract

          ATP has a long-lasting vasodilatory effect, possibly due to its capability to induce a prolonged increase in the intracellular Ca<sup>2+</sup> concentration ([Ca<sup>2+</sup>]<sub>i</sub>) in endothelial cells (EC) and activate constitutive nitric oxide synthase. However, contradictory data have been reported regarding the time course of ATP-evoked Ca<sup>2+</sup> signals in in situ EC. In particular, short-duration Ca<sup>2+</sup> signals have been reported, which might be thought to be unable to sustain a prolonged, NO-induced vasodilation. The current experiments were therefore performed in in situ EC of rat aorta in order to more fully define the time course of ATP-evoked Ca<sup>2+</sup> signals. 20 µ M ATP evoked a short-lasting Ca<sup>2+</sup> signal. However, medium stirring, high agonist concentrations, inhibition of ectonucleotidases and application of a poorly hydrolyzable agonist evoked long-lasting Ca<sup>2+</sup> signals (up to 20 min at 37°C). These studies suggest that ATP is able to sustain a prolonged [Ca<sup>2+</sup>]<sub>i</sub> increase, unless ectonucleotidase activity reduces the agonist concentration near the EC surface to subthreshold values, quickly cutting the Ca<sup>2+</sup> signal. Furthermore, the amplitude of the long-lasting phase of the Ca<sup>2+</sup> signal depended on the balance between agonist degradation by ectonucleotidases and agonist transport, by diffusion and convection, from bulk solution to the EC surface.

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          Most cited references 4

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          Endothelial Cell E- and P-Selectin and Vascular Cell Adhesion Molecule-1 Function as Signaling Receptors

          Previous studies have shown that polymorphonuclear leukocyte (PMN) adherence to endothelial cells (EC) induces transient increases in EC cytosolic free calcium concentration ([Ca2+]i) that are required for PMN transit across the EC barrier (Huang, A.J., J.E. Manning, T.M. Bandak, M.C. Ratau, K.R. Hanser, and S.C. Silverstein. 1993. J. Cell Biol. 120:1371–1380). To determine whether stimulation of [Ca2+]i changes in EC by leukocytes was induced by the same molecules that mediate leukocyte adherence to EC, [Ca2+]i was measured in Fura2-loaded human EC monolayers. Expression of adhesion molecules by EC was induced by a pretreatment of the cells with histamine or with Escherichia coli lipopolysaccharide (LPS), and [Ca2+]i was measured in single EC after the addition of mAbs directed against the EC adhesion proteins P-selectin, E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), or platelet/endothelial cell adhesion molecule-1 (PECAM-1). Both anti–P- and anti–E-selectin mAb, as well as anti–VCAM-1 mAb, induced transient increases in EC [Ca2+]i that were comparable to those induced by 200 μM histamine. In contrast, no effect was obtained by mAbs directed against the endothelial ICAM-1 or PECAM-1. PMN adherence directly stimulated increases in [Ca2+]i in histamine- or LPS-treated EC. mAbs directed against leukocyte CD18 or PECAM-1, the leukocyte counter-receptors for endothelial ICAM-1 and PECAM-1, respectively, did not inhibit PMN-induced EC activation. In contrast, mAb directed against sialyl Lewis x (sLex), a PMN ligand for endothelial P- and E-selectin, completely inhibited EC stimulation by adherent PMN. Changes in EC [Ca2+]i were also observed after adherence of peripheral blood monocytes to EC treated with LPS for 5 or 24 h. In these experiments, the combined addition of mAbs to sLex and VLA-4, the leukocyte counter-receptor for endothelial VCAM-1, inhibited [Ca2+]i changes in the 5 h–treated EC, whereas the anti–VLA-4 mAb alone was sufficient to inhibit [Ca2+]i changes in the 24 h-treated EC. Again, no inhibitory effect was observed with an anti-CD18 or anti–PECAM-1 mAb. Of note, the conditions that induced changes in EC [Ca2+]i, i.e., mAbs directed against endothelial selectins or VCAM-1, and PMN or monocyte adhesion to EC via selectins or VCAM-1, but not via ICAM-1 or PECAM-1, also induced a rearrangement of EC cytoskeletal microfilaments from a circumferential ring to stress fibers. We conclude that, in addition to their role as adhesion receptors, endothelial selectins and VCAM-1 mediate endothelial stimulation by adhering leukocytes.
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            P2y1 and P2y2 receptor-operated Ca2+ signals in primary cultures of cardiac microvascular endothelial cells.

            Intracellular Ca2+ signals elicited by nucleotide agonists were investigated in primary cultures of rat cardiac microvascular endothelial cells using the fura-2 technique. UTP increased the intracellular [Ca2+] in 94% of the cells, whereas 2MeSATP was active in 32%. The rank order of potency was ATP = UTP > 2MeSATP and the maximal response to 2MeSATP was lower compared to UTP and ATP. ATP and UTP showed strong homologous and heterologous desensitization. ATP fully inhibited the 2MeSATP response, while UTP abolished 2MeSATP-elicited transients in 25% of cells. 2MeSATP did not desensitize the UTP or ATP response. Adenosine 2',5'-diphosphate inhibited the response to 2MeSATP, while it did not modify the response to ATP and UTP. 2MeSATP was more sensitive to suramin than UTP and ATP. These results indicate that P(2Y1) and P(2Y2) receptors may be coexpressed in CMEC. Nucleotide-induced Ca2+ signals lacked a sustained plateau and were almost independent from extracellular Ca2+. ATP and UTP elicited Ca2+ transients longer than 2MeSATP-evoked transients. The kinetics of Ca2+ responses was not affected by bath solution stirring or ectonucleotidase inhibition. Furthermore, the nonhydrolyzable ATP analogue AMP-PNP induced Ca2+ signals similar to those elicited by ATP and UTP. These results suggest that the distinct kinetics of nucleotide-evoked Ca2+ responses do not depend on the activity of ectonucleotidases or ATP autocrine stimulation. The possibility that Ca2+ signals with different time courses may modulate different cellular responses is discussed. Copyright 2001 Academic Press.
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              Pharmacological analysis of responses to ATP in the isolated and perfused canine coronary artery

              Vascular responses of the isolated and perfused canine coronary artery to adenosine 5'-triphosphate (ATP) were analyzed pharmacologically. At basal perfusion pressure, ATP induced a vasoconstriction followed by a vasodilation dose-dependently. The potency order for vasoconstriction was alpha,beta-methylene ATP > 2-methylthio ATP > UTP > ATP. That for vasodilation was ATP > 2-methylthio ATP > alpha,beta-methylene ATP > UTP in the preparations precontracted by 20 mM KCl. Aminophylline inhibited the vasodilation induced by adenosine, but not that induced by ATP. Alpha,beta-methylene ATP and suramin inhibited the vasoconstriction induced by ATP. Reactive blue 2 inhibited the vasodilation induced by ATP, but not the vasoconstriction. Removal of the endothelium by saponin and L-N(G)-nitroarginine inhibited the vasodilation induced by ATP, but indomethacin did not. The results suggest that ATP induces vasoconstriction via P2X purinoceptors on the smooth muscle and vasodilation via P2Y purinoceptors on the endothelium through mainly the release of nitric oxide in the canine coronary artery, respectively.
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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
                2004
                April 2004
                21 April 2004
                : 41
                : 2
                : 166-173
                Affiliations
                Department of Physiological and Pharmacological Sciences, University of Pavia, Pavia, Italy
                Article
                77146 J Vasc Res 2004;41:166–173
                10.1159/000077146
                15004436
                © 2004 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: 24, Pages: 8
                Categories
                Research Paper

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