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      Serine 68 Phospholemman Phosphorylation during Forskolin-Induced Swine Carotid Artery Relaxation


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          Background: Phospholemman (PLM) is an abundant phosphoprotein in the plasma membrane of cardiac, skeletal and smooth muscle. It is a member of the FXYD family of proteins that bind to and regulate the Na,K-ATPase. Protein kinase A (PKA) is known to phosphorylate PLM on serine 68 (S68), although the functional effect of S68 PLM phosphorylation is unclear. We therefore evaluated S68 PLM phosphorylation in swine carotid arteries. Methods: Two anti-PLM antibodies, one to S68 phosphorylated PLM and one to unphosphorylated PLM, were made to PLM peptides in rabbits and tested with purified PLM and PKA-treated PLM. Swine carotid arteries were mounted isometrically, contracted, relaxed with forskolin and then homogenized. Proteins were separated on SDS gels and the intensity of immunoreactivity to the two PLM antibodies determined on immunoblots. Results: The antipeptide antibody ‘C2’ primarily reacted with unphosphorylated PLM, and the antipeptide antibody ‘CP68’ detected S68 PLM phosphorylation. Histamine stimulation of intact swine carotid artery induced a contraction, increased the CP68 PLM antibody signal and reduced the C2 PLM antibody signal. High extracellular [K<sup>+</sup>] depolarization induced a contraction without altering the C2 or CP68 PLM signal. Forskolin-induced relaxation of histamine or extracellular [K<sup>+</sup>] contracted arteries correlated with an increased CP68 signal. Nitroglycerin-induced relaxation was not associated with changes in the C2 or CP68 PLM signal. Conclusions: These data suggest that a contractile agonist increased S68 PLM phosphorylation. Agents that increase [cAMP], but not agents that increase [cGMP], increased S68 PLM phosphorylation. S68 PLM phosphorylation may be involved in cAMP-dependent regulation of smooth muscle force.

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          Most cited references14

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          Phospholemman (FXYD1) associates with Na,K-ATPase and regulates its transport properties.

          A family of small, single-span membrane proteins (the FXYD family) has recently been defined based on their sequence and structural homology. Some members of this family have already been identified as tissue-specific regulators of Na,K-ATPase (NKA). In the present study, we demonstrate that phospholemman (PLM) (FXYD1), so far considered to be a heart- and muscle-specific channel or channel-regulating protein, associates specifically and stably with six different alpha-beta isozymes of NKA after coexpression in Xenopus oocytes, and with alpha1-beta, and less efficiently with alpha2-beta isozymes, in native cardiac and skeletal muscles. Stoichiometric association of PLM with NKA occurs posttranslationally either in the Golgi or the plasma membrane. Interaction of PLM with NKA induces a small decrease in the external K+ affinity of alpha1-beta1 and alpha2-beta1 isozymes and a nearly 2-fold decrease in the internal Na+ affinity. In conclusion, this study demonstrates that PLM is a tissue-specific regulator of NKA that may play an essential role in muscle contractility.
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            Molecular cloning and immunological characterization of the gamma polypeptide, a small protein associated with the Na,K-ATPase

            The gamma subunit of the Na,K-ATPase is a small membrane protein that copurifies with the alpha and beta subunits of the enzyme. Strong evidence that the gamma subunit is a component of the Na,K-ATPase comes from studies indicating that the subunit is involved in forming the site for cardiac glycoside binding. We have isolated and characterized the cDNAs coding the gamma subunit from several species. The gamma subunit is a highly conserved protein consisting of 58 amino acids with a molecular weight of 6500. Hydropathy analysis reveals the presence of a single hydrophobic domain that is sufficient to cross the membrane. There are no sites for N-linked glycosylation. Northern blot analysis revealed that the gamma subunit mRNA is expressed in a tissue-specific fashion and is present in all tissues characterized. gamma-specific antibodies have been used to verify that the sequenced protein is the same protein labeled by [3H]nitroazidobenzoyl-ouabain (NAB-ouabain), and that this protein, the gamma subunit of the Na,K-ATPase, has a distribution pattern along nephron segments that is identical with the alpha subunit. In addition, coimmunoprecipitation of the alpha, beta and gamma subunits demonstrate specific association of the subunits. These results are consistent with the notion that the gamma subunit is specifically associated with and may be an important component of the Na,K-ATPase.
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              Serine 68 phosphorylation of phospholemman: acute isoform-specific activation of cardiac Na/K ATPase.

              The mechanism by which the cardiac Na/K ATPase (NKA) is regulated by phosphorylation is controversial. We have used the perforated-patch technique to limit cell dialysis and maintain conditions as near physiological as possible. NKA pump current (I(p)) was measured in isolated guinea pig ventricular myocytes, and its components (I(alpha 1) and I(alpha 2)) defined by their differing dihydroouabain sensitivities. Treatment with 1 micromol/l forskolin for 4 min at 35 degrees C caused a significant increase in I(alpha1) of 36+/-15% (P<0.05, n=6), but no change in I(alpha2). The presence of the PKA selective inhibitor H89 (50 micromol/l) throughout the protocol blocked the effect of the forskolin on I(alpha1). Treatment with H89 alone did not change I(alpha 1) or I(alpha 2). Isoelectric focusing gels of the NKA alpha1 subunit demonstrated six charge states, which were unaltered following treatment with forskolin. Western blots using an antibody specific for the PKA phosphorylation consensus site on the alpha1 subunit showed no change in the phosphorylation status of this residue following forskolin treatment. The sarcolemmal protein phospholemman (PLM) was found associated with NKA alpha 1 but not alpha 2 subunits by immunoprecipitation and immunofluorescence. PLM was phosphorylated at serine 68, but not 63, following treatment with forskolin. PKA-dependent, alpha 1-specific NKA activation may be mediated through phosphorylation of the accessory protein PLM, rather than direct alpha1 subunit phosphorylation.

                Author and article information

                J Vasc Res
                Journal of Vascular Research
                S. Karger AG
                December 2005
                20 October 2005
                : 42
                : 6
                : 483-491
                aCardiovascular Division, Department of Internal Medicine, bCardiovascular Research Center, cDepartment of Molecular Physiology and Biological Physics, University of Virginia Health System, Charlottesville, Va., dThe Burnham Institute, La Jolla, Calif., and eDepartment of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pa., USA
                88102 PMC1266286 J Vasc Res 2005;42:483–491
                © 2005 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.

                : 05 January 2005
                : 26 June 2005
                Page count
                Figures: 6, References: 43, Pages: 9
                Research Paper

                General medicine,Neurology,Cardiovascular Medicine,Internal medicine,Nephrology
                Phospholemman,Cyclic adenosine monophosphate,Vascular smooth muscle,FXYD protein,Phosphorylation


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