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      Sevoflurane and isoflurane inhibit KCl-induced Class II phosphoinositide 3-kinase α subunit mediated vasoconstriction in rat aorta

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          Abstract

          Background

          Class II phosphoinositide 3-kinase α-isoform (PI3K-C2α) is involved in regulating KCl-induced vascular smooth muscle contraction. The current study was to investigate the effects of sevoflurane (SEVO) and isoflurane (ISO) on KCl-elicited PI3KC2α mediated vasoconstriction in rat aortic smooth muscle.

          Methods

          Isometric force, in the absence or presence of SEVO or ISO (1 ~ 3 minimum alveolar concentration, MAC), PI3K inhibitor LY294002, Rho kinase inhibitor Y27632, and membrane translocation of PI3K-p85, PI3K-C2α, Rho kinase (Rock II), or phosphorylation of MYPT1/Thr853, MYPT1/Thr696, CPI-17/Thr38 and MLC in response to KCl (60 mM) was measured by using isometric force transducer and western blotting analysis, respectively.

          Results

          KCl elicited a rapid and sustained contraction of rat aortic smooth muscle that was inhibited by both SEVO and ISO in a concentration-dependent manner, and also suppressed by LY294002 (1 mM) and Y27632 (1 uM). LY294002 (1 mM) and Y27632 (1 uM) also inhibited KCl-induced MLC phosphorylation. LY294002 (1 mM) inhibited KCl-induced PI3K-p85, PI3K-C2α membrane translocation in response to KCl ( p <0.05, p < 0.01, respectively). Not only Y27632 (1 uM), but also LY294002 (1 mM), inhibited KCl-induced Rock-II membrane translocation ( p < 0.01). SEVO and ISO inhibited KCl-stimulated MLC phosphorylation, PI3K-C2α and Rock-II,not PI3K p85 membrane translocation in a concentration-dependent manner in rat aorta. Both SEVO and ISO suppressed the MYPT1/Thr853, not MYPT1/Thr696 and CPI-17/Thr38, MLC phosphorylation in response to KCl.

          Conclusion

          PI3K-C2α mediates part of SEVO and ISO-mediated vasodilation in rat aorta. The cellular mechanisms underlying the inhibitory effect of volatile anesthetics might be mediated by KCl/PI3K-C2α/Rho kinase/MYPT1/MLC pathway.

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          Most cited references29

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          The role of phosphoinositide 3-kinase lipid products in cell function.

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            Inhibitory phosphorylation site for Rho-associated kinase on smooth muscle myosin phosphatase.

            It is clear from several studies that myosin phosphatase (MP) can be inhibited via a pathway that involves RhoA. However, the mechanism of inhibition is not established. These studies were carried out to test the hypothesis that Rho-kinase (Rho-associated kinase) via phosphorylation of the myosin phosphatase target subunit 1 (MYPT1) inhibited MP activity and to identify relevant sites of phosphorylation. Phosphorylation by Rho-kinase inhibited MP activity and this reflected a decrease in V(max). Activity of MP with different substrates also was inhibited by phosphorylation. Two major sites of phosphorylation on MYPT1 were Thr(695) and Thr(850). Various point mutations were designed for these phosphorylation sites. Following thiophosphorylation by Rho-kinase and assays of phosphatase activity it was determined that Thr(695) was responsible for inhibition. A site- and phosphorylation-specific antibody was developed for the sequence flanking Thr(695) and this recognized only phosphorylated Thr(695) in both native and recombinant MYPT1. Using this antibody it was shown that stimulation of serum-starved Swiss 3T3 cells by lysophosphatidic acid, thought to activate RhoA pathways, induced an increase in Thr(695) phosphorylation on MYPT1 and this effect was blocked by a Rho-kinase inhibitor, Y-27632. In summary, these results offer strong support for a physiological role of Rho-kinase in regulation of MP activity.
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              Regulation of smooth muscle calcium sensitivity: KCl as a calcium-sensitizing stimulus.

              KCl has long been used as a convenient stimulus to bypass G protein-coupled receptors (GPCR) and activate smooth muscle by a highly reproducible and relatively "simple" mechanism involving activation of voltage-operated Ca2+ channels that leads to increases in cytosolic free Ca2+ ([Ca2+]i), Ca2+-calmodulin-dependent myosin light chain (MLC) kinase activation, MLC phosphorylation and contraction. This KCl-induced stimulus-response coupling mechanism is a standard tool-set used in comparative studies to explore more complex mechanisms generated by activation of GPCRs. One area where this approach has been especially productive is in studies designed to understand Ca2+ sensitization, the relationship between [Ca2+]i and force produced by GPCR agonists. Studies done in the late 1980s demonstrated that a unique relationship between stimulus-induced [Ca2+]i and force does not exist: for a given increase in [Ca2+]i, GPCR activation can produce greater force than KCl, and relaxant agents can produce the opposite effect to cause Ca2+ desensitization. Such changes in Ca2+ sensitivity are now known to involve multiple cell signaling strategies, including translocation of proteins from cytosol to plasma membrane, and activation of enzymes, including RhoA kinase and protein kinase C. However, recent studies show that KCl can also cause Ca2+ sensitization involving translocation and activation of RhoA kinase. Rather than complicating the Ca2+ sensitivity story, this surprising finding is already providing novel insights into mechanisms regulating Ca2+ sensitivity of smooth muscle contraction. KCl as a "simple" stimulus promises to remain a standard tool for smooth muscle cell physiologists, whose focus is to understand mechanisms regulating Ca2+ sensitivity.
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                Author and article information

                Contributors
                qifeng66321@sina.com
                Journal
                BMC Anesthesiol
                BMC Anesthesiol
                BMC Anesthesiology
                BioMed Central (London )
                1471-2253
                18 August 2016
                18 August 2016
                2015
                : 16
                : 63
                Affiliations
                Department of Anesthesiology, Qilu Hospital of Shandong University, Jinan, 250012 China
                Article
                227
                10.1186/s12871-016-0227-9
                4991059
                27538808
                60d17a87-6349-4637-a142-8d7be960c33a
                © The Author(s). 2016

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 25 November 2015
                : 9 August 2016
                Funding
                Funded by: the National Natural Science Foundation of China
                Award ID: (No. 81171864)
                Award Recipient :
                Funded by: the Fundamental Research Funds of Shandong University, China
                Award ID: 2014YQ014
                Award Recipient :
                Categories
                Research Article
                Custom metadata
                © The Author(s) 2016

                Anesthesiology & Pain management
                sevoflurane,isoflurane,kcl,phosphoinositide 3 kinase,rho kinase,myosin light chain phosphatase

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