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：
7820938
负责人：
DEREK Scott DAMRON
金额：
$ 6.22万
依托单位：
KENT STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-06-01 至 2010-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7820938
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

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）：麻醉剂可以在缺血再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具有临床重要意义。麻醉剂保护器官系统和促进细胞在缺血挑战中存活的细胞和分子机制尚不清楚。人们相信麻醉剂在中枢神经系统中的细胞作用是通过与 G 蛋白偶联受体 (GPCR)，特别是 GABAA 受体的相互作用介导的。然而，由于麻醉剂很容易穿过细胞膜，它们也可能直接与可溶性细胞内蛋白质相互作用，从而导致与源自 GPCR 刺激的其他信号分子的分子相互作用的直接调节和/或变构调节。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中发挥关键作用，然而激活的细胞和分子机制，无论是通过刺激 GPCR，还是直接激活该酶，尚不清楚。进行了研究，因此代表了实验室研究的临床重要领域。我们的主要目标是确定静脉麻醉剂丙泊酚作为配体激活 PKCe 的细胞信号传导途径，并描述酶发生相互作用和激活的分子机制。我们的总体假设是，异丙酚通过对 GPCR 的作用间接激活 PKCe 同工型，并直接通过与二酰基甘油/佛波酯结合域或其附近的酶的分子相互作用来激活 PKCe 同工型。为了实现我们的目标，我们将利用功能获得或丧失的方法，使用来自野生型和 PKCe 缺失小鼠的分离心肌细胞，结合重组 PKCe 和合成的 PKCe 调节子结构域（C1A 和 C1B）来研究 PKCe 的细胞和分子机制激活。这种创新方法包括使用 PKC 激活剂和抑制剂肽、培养细胞中 PKCe 的下调和重新表达、重组 PKCe 和合成的子结构域与可光激活的二嗪丙泊酚类似物相结合，以评估丙泊酚直接诱导的 PKCe 激活。终点测量包括细胞内 Ca2+ 浓度、收缩性、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 亚型作为研究的模型系统，因为它们代表了我们之前资助期研究的直接延伸，并且因为麻醉剂被认为可以通过激活 PKCe 在缺血再灌注损伤期间提供心肌保护。具体目标 1 将确定 PKCe 在介导丙泊酚诱导的细胞内 Ca2+ 浓度和肌丝 Ca2+ 敏感性（心肌收缩力的关键调节因子）的影响中的作用。具体目标 2 将确定参与异丙酚诱导的 PKCe 激活的细胞信号传导途径。具体目标 3 将确定丙泊酚和 PKCe 之间相互作用的分子机制。我们相信这些研究代表了我们之前在心肌细胞方面的工作的逻辑延伸，并提供了一种创新方法来更好地理解心脏麻醉作用的细胞和分子机制。