Propofol and Protein Kinase C: Molecular Interactions in Cardiomyocytes

异丙酚和蛋白激酶 C：心肌细胞中的分子相互作用

基本信息

批准号：
7671426
负责人：
DEREK Scott DAMRON
金额：
$ 32.23万
依托单位：
KENT STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-12-01 至 2012-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7671426
关键词：
1,2-diacylglycerol Actomyosin Adenosinetriphosphatase Adenosine Amino Acids Anesthesia procedures Anesthetics Arachidonic Acids Area Binding Binding Sites Biological Assay Biological Models Biological Preservation Cardiac Cardiac Myocytes Cell Survival Cell membrane Cells Complement component C1s Coupled Cultured Cells DAG/PE-Binding Domain Data Detection Development Diazomethane Diglycerides Doctor of Philosophy Down-Regulation Enzyme Activation Enzymes Fluorescence Funding G-Protein-Coupled Receptors GTP-Binding Proteins Goals Heart High Pressure Liquid Chromatography Injury Intravenous Anesthetics Investigation Ischemia Knockout Mice Knowledge Label Laboratories Ligands Lipids Measurement Mediating Microfilaments Molecular Mus Muscarinics Myocardial Myofibrils Neurons Organ Pathway interactions Peptides Phorbol Esters Phosphorylation Play Postoperative Period Propofol Protein Isoforms Protein Kinase C Proteins Recombinant Proteins Recombinants Regulation Reperfusion Injury Research Research Personnel Role Signal Pathway Signal Transduction Signaling Molecule Site System Testing Troponin I Western Blotting Wild Type Mouse Work analog base body system cell growth regulation cell type cellular targeting gel electrophoresis human TYRP1 protein inhibitor/antagonist innovation kit protein loss of function molecular site myosin light chain 2 novel patch clamp preconditioning programs protein kinase C epsilon receptor response tandem mass spectrometry tool

1,2-diacylglycerol Actomyosin Adenosinetriphosphatase Adenosine Amino Acids Anesthesia procedures Anesthetics Arachidonic Acids Area Binding Binding Sites Biological Assay Biological Models Biological Preservation Cardiac Cardiac Myocytes Cell Survival Cell membrane Cells Complement component C1s Coupled Cultured Cells DAG/PE-Binding Domain Data Detection Development Diazomethane Diglycerides Doctor of Philosophy Down-Regulation Enzyme Activation Enzymes Fluorescence Funding G-Protein-Coupled Receptors GTP-Binding Proteins Goals Heart High Pressure Liquid Chromatography Injury Intravenous Anesthetics Investigation Ischemia Knockout Mice Knowledge Label Laboratories Ligands Lipids Measurement Mediating Microfilaments Molecular Mus Muscarinics Myocardial Myofibrils Neurons Organ Pathway interactions Peptides Phorbol Esters Phosphorylation Play Postoperative Period Propofol Protein Isoforms Protein Kinase C Proteins Recombinant Proteins Recombinants Regulation Reperfusion Injury Research Research Personnel Role Signal Pathway Signal Transduction Signaling Molecule Site System Testing Troponin I Western Blotting Wild Type Mouse Work analog base body system cell growth regulation cell type cellular targeting gel electrophoresis human TYRP1 protein inhibitor/antagonist innovation kit protein loss of function molecular site myosin light chain 2 novel patch clamp preconditioning programs protein kinase C epsilon receptor response tandem mass spectrometry tool

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项目摘要

DESCRIPTION (provided by applicant): Anesthetic agents can provide organ protection in the setting of ischemia-reperfusion injury, which is clinically important in both the peri-operative and post-operative setting. The cellular and molecular mechanisms by which anesthetics preserve organ systems and promote cell survival from an ischemic challenge are not clearly established. It is believed that the cellular actions of anesthetics in the CNS are mediated via interactions with G-protein coupled receptors (GPCRs), particularly the GABAA receptors. However, because anesthetics readily pass through cell membranes, they may also directly interact with soluble intracellular proteins resulting in direct regulation and/or an allosteric modulation of molecular interactions with other signaling molecules derived from GPCR stimulation. Activation of protein kinase C epsilon (PKCe) has been shown to play a key role in mediating anesthesia-induced myocardial protection, however the cellular and molecular mechanisms of activation, whether by stimulation of GPCRs, or direct activation of the enzyme, have not been investigated and therefore represent a clinically important area of laboratory based research. Our primary goal is to identify the cellular signaling pathways by which the intravenous anesthetic, propofol, acts as a ligand to activate PKCe, and to delineate the molecular mechanism by which interaction and activation of the enzyme occurs. Our overarching hypothesis is that propofol activates the PKCe isoform indirectly via actions on GPCRs, and directly via a molecular interaction with the enzyme at or near the diacylglycerol/phorbol ester binding domain. To achieve our goal, we will utilize a gain or loss of function approach using isolated cardiomyocytes from wild type and PKCe null mice in combination with recombinant PKCe and synthesized PKCe regulatory sub-domains (C1A and C1B) to investigate cellular and molecular mechanisms of PKCe activation. This innovative approach encompasses the use of PKCs activator and inhibitor peptides, down regulation and re-expression of PKCe in cultured cells, recombinant PKCe and synthesized sub-domains combined with photoactivable diazirine propofol analogs to assess direct propofol-induced activation of PKCe. Endpoint measurements include intracellular Ca2+ concentration, contractility, myofilament Ca2+ sensitivity, protein phosphorylation, PKCe activity, translocation and autophosphorylation, and molecular binding studies. Our experimental approach is comprehensive, ranging from molecular interactions to functional assessments of cellular regulation. Cardiomyocytes and the PKCe isoform were chosen as the model system to investigate because they represent a direct extension of our studies from the previous funding period, and because anesthetics are believed to provide myocardial protection during ischemia-reperfusion injury via activation of PKCe. Specific Aim 1 will determine the role of PKCe in mediating propofol-induced effects on intracellular Ca2+ concentration and myofilament Ca2+ sensitivity, the key regulators of myocardial contractility. Specific Aim 2 will identify the cellular signaling pathways involved in propofol-induced activation of PKCe. Specific Aim 3 will determine the molecular mechanism of interaction between propofol and PKCe. We believe these studies represent a logical extension of our previous work in cardiomyocytes and provide an innovative approach to better understand the cellular and molecular mechanisms of anesthetic action in the heart.

描述（由申请人提供）：麻醉剂可以在缺血 - 重新灌注损伤的情况下提供器官保护，这在围手术期和术后环境中在临床上都很重要。没有明确确定麻醉剂保护器官系统并从缺血性挑战中促进细胞存活的细胞和分子机制。据认为，中枢神经系统中麻醉药的细胞作用是通过与G蛋白偶联受体（GPCR）（尤其是GABAA受体）相互作用介导的。但是，由于麻醉剂很容易通过细胞膜，因此它们也可能直接与可溶性细胞内蛋白相互作用，从而导致直接调节和/或分子相互作用与其他信号分子的分子相互作用的变构调节。蛋白激酶C ePsilon（PKCE）的激活已被证明在介导麻醉诱导的心肌保护方面起着关键作用，但是，无论是通过刺激GPCR还是直接激活酶的细胞和分子激活机制，均未研究并没有研究临床重要的领域。我们的主要目标是确定静脉麻醉（丙泊酚）充当激活PKCE的配体的细胞信号传导途径，并描绘出酶相互作用和激活酶的分子机制。我们的总体假设是，丙泊酚通过在GPCR上的作用并直接通过与二酰基甘油/phorbol酯结合结构域或附近的酶的分子相互作用间接地激活PKCE同工型。为了实现我们的目标，我们将使用来自野生型和PKCE无效小鼠的孤立心肌细胞与重组PKCE以及合成的PKCE调节子域（C1A和C1B）结合使用功能方法的增益或丧失，以研究PKCE激活的细胞和分子机制。这种创新的方法包括PKCS激活剂和抑制剂肽的使用，在培养的细胞中降低和重新表达PKCE，重组PKCE和合成的亚构域，结合了光活性二嗪丙烯类似物，以评估PKCE的直接丙二抗酚诱导的激活。端点测量值包括细胞内Ca2+浓度，收缩力，肌丝Ca2+敏感性，蛋白质磷酸化，PKCE活性，易位和自磷酸化以及分子结合研究。我们的实验方法是全面的，从分子相互作用到细胞调节的功能评估。选择心肌细胞和PKCE同工型作为研究模型系统，因为它们代表了我们的研究直接扩展到以前的融资期，并且由于据信麻醉药可通过激活PKCE激活缺血 - 再灌注损伤期间提供心肌保护。具体的目标1将确定PKCE在介导丙泊酚诱导的对细胞内Ca2+浓度和肌丝Ca2+灵敏度的作用（心肌收缩力的关键调节剂）中的作用。特定的目标2将识别与丙泊酚诱导的PKCE激活有关的细胞信号传导途径。具体的目标3将确定丙泊酚和PKCE之间相互作用的分子机制。我们认为，这些研究代表了我们先前在心肌细胞中的工作的逻辑扩展，并提供了一种创新的方法来更好地了解心脏麻醉作用的细胞和分子机制。