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      Phosphorylation of Neuronal and Endothelial Nitric Oxide Synthase in the Kidney with High and Low Salt Diets

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          Abstract

          Background: Renal nitric oxide (NO) synthesis increases with increasing salt intake, however, the mechanisms underlying this are poorly understood. We hypothesized that activating or inhibitory phosphorylation of neuronal and endothelial nitric oxide synthase (nNOS, eNOS) regulates renal NO production in response to altered dietary salt. Methods:Sprague-Dawley rats were fed low, normal or high salt diets for 12 h or 2 weeks, and kidney NOS phosphorylation was analyzed by Western blot using phosphopeptide antibodies against the sites nNOS-Ser<sup>1412</sup>, nNOS-Ser<sup>847</sup>, eNOS-Ser<sup>1176</sup> and eNOS-Thr<sup>494</sup>. Results:At 12 h, total nNOS increased 1.4-fold (p < 0.01) in the high salt group and decreased by 26% (p < 0.05) in the low salt group. Changes in expression of phospho-nNOS at 12 h were accounted for by the changes in total nNOS. No change in total or phospho-eNOS was seen at 12 h. At 2 weeks, in the low salt group expression of total nNOS increased 1.8-fold (p < 0.05) whereas expression of nNOS phosphorylated at the inhibitory site Ser<sup>847</sup> increased 4.3-fold (p < 0.01). Total eNOS was increased 3-fold in the low salt group (p < 0.01), with parallel increases in eNOS phosphorylated at both activating and inhibitory sites (p < 0.05). In the 2-week high salt group no changes in NOS expression or phosphorylation were seen, despite the observed increased excretion of urinary NO metabolites. Conclusion:In summary, changes in phospho-nNOS and phospho-eNOS expression occurred in parallel with changes in total expression, thus, the overall activating and inhibitory effects of nNOS and eNOS phosphorylation at the sites studied were not changed by altered dietary salt.

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          Most cited references 29

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          AMP-activated protein kinase phosphorylation of endothelial NO synthase.

          The AMP-activated protein kinase (AMPK) in rat skeletal and cardiac muscle is activated by vigorous exercise and ischaemic stress. Under these conditions AMPK phosphorylates and inhibits acetyl-coenzyme A carboxylase causing increased oxidation of fatty acids. Here we show that AMPK co-immunoprecipitates with cardiac endothelial NO synthase (eNOS) and phosphorylates Ser-1177 in the presence of Ca2+-calmodulin (CaM) to activate eNOS both in vitro and during ischaemia in rat hearts. In the absence of Ca2+-calmodulin, AMPK also phosphorylates eNOS at Thr-495 in the CaM-binding sequence, resulting in inhibition of eNOS activity but Thr-495 phosphorylation is unchanged during ischaemia. Phosphorylation of eNOS by the AMPK in endothelial cells and myocytes provides a further regulatory link between metabolic stress and cardiovascular function.
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            Coordinated control of endothelial nitric-oxide synthase phosphorylation by protein kinase C and the cAMP-dependent protein kinase.

            Endothelial nitric-oxide synthase (eNOS) is an important regulatory enzyme in the cardiovascular system catalyzing the production of NO from arginine. Multiple protein kinases including Akt/PKB, cAMP-dependent protein kinase (PKA), and the AMP-activated protein kinase (AMPK) activate eNOS by phosphorylating Ser-1177 in response to various stimuli. During VEGF signaling in endothelial cells, there is a transient increase in Ser-1177 phosphorylation coupled with a decrease in Thr-495 phosphorylation that reverses over 10 min. PKC signaling in endothelial cells inhibits eNOS activity by phosphorylating Thr-495 and dephosphorylating Ser-1177 whereas PKA signaling acts in reverse by increasing phosphorylation of Ser-1177 and dephosphorylation of Thr-495 to activate eNOS. Both phosphatases PP1 and PP2A are associated with eNOS. PP1 is responsible for dephosphorylation of Thr-495 based on its specificity for this site in both eNOS and the corresponding synthetic phosphopeptide whereas PP2A is responsible for dephosphorylation of Ser-1177. Treatment of endothelial cells with calyculin selectively blocks PKA-mediated dephosphorylation of Thr-495 whereas okadaic acid selectively blocks PKC-mediated dephosphorylation of Ser-1177. These results show that regulation of eNOS activity involves coordinated signaling through Ser-1177 and Thr-495 by multiple protein kinases and phosphatases.
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              Reciprocal phosphorylation and regulation of endothelial nitric-oxide synthase in response to bradykinin stimulation.

              Endothelial nitric-oxide synthase (eNOS) is phosphorylated at Ser-1179 (bovine sequence) by Akt after growth factor or shear stress stimulation of endothelial cells, resulting in increased eNOS activity. Purified eNOS is also phosphorylated at Thr-497 by purified AMP-activated protein kinase, resulting in decreased eNOS activity. We investigated whether bradykinin (BK) stimulation of bovine aortic endothelial cells (BAECs) regulates eNOS through Akt activation and Ser-1179 or Thr-497 phosphorylation. Akt is transiently activated in BK-stimulated BAECs. Activation is blocked completely by wortmannin and LY294002, inhibitors of phosphatidylinositol 3-kinase, suggesting that Akt activation occurs downstream from phosphatidylinositol 3-kinase. BK stimulates a transient phosphorylation of eNOS at Ser-1179 that is correlated temporally with a transient dephosphorylation of eNOS at Thr-497. Phosphorylation at Ser-1179, but not dephosphorylation at Thr-497, is blocked by wortmannin and LY294002. BK also stimulates a transient nitric oxide (NO) release from BAECs with a time-course similar to Ser-1179 phosphorylation and Thr-497 dephosphorylation. NO release is not altered by wortmannin. BK-stimulated dephosphorylation of Thr-497 and NO release are blocked by the calcineurin inhibitor, cyclosporin A. These data suggest that BK activation of eNOS in BAECs primarily involves deinhibition of the enzyme through calcineurin-mediated dephosphorylation at Thr-497.
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                Author and article information

                Journal
                NEP
                Nephron Physiol
                10.1159/issn.1660-2137
                Nephron Physiology
                S. Karger AG
                1660-2137
                2006
                January 2006
                09 January 2006
                : 102
                : 2
                : p36-p50
                Affiliations
                aThe Austin Research Institute, bDepartment of Nephrology, and cUniversity of Melbourne (Department of Medicine) Austin Hospital, and dSt. Vincent’s Institute of Medical Research, St. Vincent’s Hospital, Heidelberg, Australia, and eCell Physiology, Faculty of Medicine, Kagawa University, Kagawa, Japan
                Article
                89092 Nephron Physiol 2006;102:p36–p50
                10.1159/000089092
                16244499
                © 2006 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: 8, Tables: 3, References: 50, Pages: 1
                Product
                Self URI (application/pdf): https://www.karger.com/Article/Pdf/89092
                Categories
                Original Paper

                Cardiovascular Medicine, Nephrology

                nNOS, Kidney, Nitric oxide, Salt, Phosphorylation, eNOS

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