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

• 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
• 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

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.

说明(申请人提供)：麻醉剂可以在缺血再灌注损伤的环境中提供器官保护，这在围手术期和手术后的环境中都具有重要的临床意义。麻醉剂保护器官系统和促进细胞存活免受缺血挑战的细胞和分子机制尚不清楚。据认为，麻醉药在中枢神经系统的细胞作用是通过与G蛋白偶联受体，特别是GABAA受体的相互作用而实现的。然而，由于麻醉剂很容易穿过细胞膜，它们也可能直接与细胞内的可溶性蛋白相互作用，从而直接调节和/或变构调节与来自GPCR刺激的其他信号分子的分子相互作用。蛋白激酶C epsilon(PKCE)的激活在介导麻醉诱导的心肌保护中起着关键作用，然而，激活的细胞和分子机制，无论是通过刺激GPCRs，还是直接激活该酶，都尚未被研究，因此是一个重要的临床实验室研究领域。我们的主要目标是确定静脉麻醉剂异丙酚作为配体激活PKCE的细胞信号通路，并描述发生相互作用和酶激活的分子机制。我们的主要假设是，异丙酚通过对GPCRs的作用间接激活PKCE亚型，并直接通过与二酰甘油/佛波酯结合区或附近的酶的分子相互作用来激活PKCE。为了实现我们的目标，我们将利用从野生型和PKCE缺失小鼠分离的心肌细胞，结合重组的PKCE和合成的PKCE调节亚域(C1A和C1B)来研究PKCE激活的细胞和分子机制。这一创新的方法包括使用PKCs激活剂和抑制肽、在培养细胞中下调和重新表达PKCE、重组PKCE和合成的亚域与可光激活的二氮丙酚类似物相结合来评估异丙酚对PKCE的直接激活。终点测量包括细胞内钙离子浓度、收缩能力、肌丝钙敏感性、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亚型作为模型系统进行研究，是因为它们代表了我们在前一个资助时期的研究的直接延伸，而且因为麻醉药被认为通过激活PKCE来在缺血再灌注损伤中提供心肌保护。具体目的1将确定PKCE在丙泊酚诱导的细胞内钙离子浓度和肌丝钙敏感性中的作用，这是心肌收缩的关键调节因素。特异性目标2将确定参与异丙酚诱导的PKCE激活的细胞信号通路。特异靶3将确定异丙酚与PKCE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中工作的合理扩展，并提供了一种创新的方法，以更好地了解心脏中麻醉作用的细胞和分子机制。