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:7316888
- 负责人:
- 金额:$ 32.09万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2000
- 资助国家:美国
- 起止时间:2000-07-01 至 2012-08-31
- 项目状态:已结题
- 来源:
- 关键词:1,2-diacylglycerolActomyosin AdenosinetriphosphataseAdenosineAmino AcidsAnesthesia proceduresAnestheticsArachidonic AcidsAreaBindingBinding SitesBiological AssayBiological ModelsBiological PreservationCardiacCardiac MyocytesCell SurvivalCell membraneCellsComplement component C1sCoupledCultured CellsDAG/PE-Binding DomainDataDetectionDevelopmentDiazomethaneDiglyceridesDoctor of PhilosophyDown-RegulationEnd PointEnzyme ActivationEnzymesFluorescenceFundingG-Protein-Coupled ReceptorsGTP-Binding ProteinsGoalsHeartHigh Pressure Liquid ChromatographyInjuryIntravenous AnestheticsInvestigationIschemiaKnockout MiceKnowledgeLabelLaboratoriesLigandsLipidsMeasurementMediatingMicrofilamentsMolecularMusMuscarinicsMyocardialMyofibrilsNeuronsOrganPathway interactionsPeptidesPhorbol EstersPhosphorylationPlayPostoperative PeriodPropofolProtein IsoformsProtein Kinase CProteinsPurinergic P1 ReceptorsRangeRecombinant ProteinsRecombinantsRegulationReperfusion InjuryResearchResearch PersonnelRoleSignal PathwaySignal TransductionSignaling MoleculeSiteSystemTestingTroponin IWestern BlottingWild Type MouseWorkanalogbasebody systemcell growth regulationcell typecellular targetinggel electrophoresishuman TYRP1 proteininhibitor/antagonistinnovationkit proteinloss of functionmolecular sitemyosin light chain 2novelpatch clamppreconditioningprogramsprotein kinase C epsilonreceptorresponsetandem mass spectrometrytool
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都具有重要的临床意义。麻醉剂保护器官系统和促进细胞从缺血性挑战中存活的细胞和分子机制尚未明确建立。据信,CNS中麻醉剂的细胞作用通过与G蛋白偶联受体(GPCR),特别是GABAA受体的相互作用介导。然而,因为麻醉剂容易穿过细胞膜,所以它们也可以直接与可溶性细胞内蛋白相互作用,导致与衍生自GPCR刺激的其它信号传导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 β(PKCe)的激活已被证明在介导麻醉诱导的心肌保护中起关键作用,然而,激活的细胞和分子机制,无论是通过刺激GPCR,还是直接激活酶,都没有被研究,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激活PKCe的配体的细胞信号传导途径,并描述酶的相互作用和激活发生的分子机制。我们的总体假设是丙泊酚通过对GPCR的作用间接激活PKCe亚型,并直接通过与二酰基甘油/佛波酯结合结构域处或附近的酶的分子相互作用激活PKCe亚型。为了实现我们的目标,我们将利用从野生型和PKCe裸小鼠中分离的心肌细胞与重组PKCe和合成的PKCe调节亚结构域(C1 A和C1 B)组合的功能获得或丧失方法来研究PKCe激活的细胞和分子机制。这种创新的方法包括使用PKC激活剂和抑制剂肽,在培养的细胞中下调和重新表达PKCe,重组PKCe和合成的亚结构域与可光活化的二氮丙啶丙泊酚类似物组合,以评估直接丙泊酚诱导的PKCe激活。终点测量包括细胞内Ca 2+浓度、收缩力、肌丝Ca 2+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同种型作为模型系统进行研究,因为它们代表了我们先前资助期研究的直接延伸,并且因为认为麻醉剂通过激活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对细胞内Ca 2+浓度和肌丝Ca 2+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将确定涉及丙泊酚诱导的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以前在心肌细胞中工作的逻辑延伸,并提供了一种创新的方法来更好地理解心脏麻醉作用的细胞和分子机制。
项目成果
期刊论文数量(0)
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DEREK Scott DAMRON其他文献
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{{ truncateString('DEREK Scott DAMRON', 18)}}的其他基金
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 - 财政年份:2009
- 资助金额:
$ 32.09万 - 项目类别:
Plasma Volume, Adrenergic Tone, and Hemodynamics in Nul
Nul 中的血浆容量、肾上腺素张力和血流动力学
- 批准号:
7041548 - 财政年份:2004
- 资助金额:
$ 32.09万 - 项目类别:
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:
7671426 - 财政年份:2001
- 资助金额:
$ 32.09万 - 项目类别:
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:
7487875 - 财政年份:2001
- 资助金额:
$ 32.09万 - 项目类别:
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:
7910441 - 财政年份:2001
- 资助金额:
$ 32.09万 - 项目类别:
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:
8127614 - 财政年份:2001
- 资助金额:
$ 32.09万 - 项目类别: