Hypoxic modulation of protein kinase G function in fetal

胎儿蛋白激酶 G 功能的缺氧调节

• 批准号: 6875423
• 负责人: William J. Pearce
• 金额: $ 16.2万
• 依托单位: LOMA LINDA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-01-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6875423
• 关键词: age difference; altitude; autoradiography; cGMP dependent protein kinase; calcium flux; cerebral artery; embryo /fetus; embryo /fetus hypoxia; environmental adaptation; enzyme activity; hypoxia; immunocytochemistry; isozymes; microfilaments; myosin light chain kinase; phosphorylation; receptor expression; second messengers; sheep; vasomotion; western blottings

The transition from fetal to adult life involves dramatic changes in vascular reactivity, particularly in cerebral arteries where both the capacity to contract and the ability to relax improve simultaneously during postnatal life. The rates and patterns of these maturational changes, in turn, are highly susceptible to environmental stresses such as chronic hypoxia, which can potently depress pharmacomechanical coupling through multiple simultaneous effects. Whereas it is clear that chronic hypoxia alters agonist-induced calcium mobilization and myofilament calcium sensitivity, the mechanisms that mediate hypoxia' s effects on vascular reactivity remain unclear. Hypoxia-induced changes in endothelial production and release of NO contribute to the overall cerebrovascular effects of hypoxia, but major changes in reactivity to NO are also involved. Similarly, changes in cGMP metabolism contribute to the cerebrovascular effects of chronic hypoxia, but again, major changes in the mechanisms coupling cGMP to vasorelaxation are an essential component of the cerebrovascular adaptation to chronic hypoxia. The single most important effector of vasorelaxation downstream of cGMP is Protein Kinase G (PKG), which in cerebral arteries mediates almost all vasodilator effects of cGMP. Despite the central importance of PKG, its role in cerebrovascular function has been largely ignored, particularly in immature cerebral arteries. Virtually nothing is known of the effects of chronic hypoxia on cerebrovascular PKG function in any artery type or age. In light of these deficits, and the strong potential for PKG to play a key role in cerebrovascular adaptation to chronic hypoxia, the proposed studies focus on the general mechanisms involved in PKG-mediated vasorelaxation, and how these are modulated by maturation and chronic hypoxia. The general hypothesis addressed by these studies is that chronic hypoxia selectively enhances the ability of Protein Kinase G to elicit cerebral vasodilatation in an age-dependent and artery-specific manner. This main hypothesis, in turn, has four main corollaries, each of which proposes that hypoxia influences a mechanism whereby Protein Kinase G modulates pharmacomechanical coupling: 1) PKG modulates coupling between activation of cell surface receptors and synthesis of the second messenger IP3; 2) PKG alters the ability of second messengers such as IP3 to elicit calcium entry and/or release; 3) PKG influences thick filament reactivity, as indicated by the relation between cytosolic calcium and the extent of myosin light chain phosphorylation; and 4) PKG differentially enhances thin filament reactivity, as indicated by the relation between myosin fight chain phosphorylation and the production of contractile force. To evaluate the main hypothesis and its corollaries, we will conduct experiments designed to: 1. Quantify the distribution, abundance, and activity of cerebrovascular PKG isoforms 1alpha and 1beta using immunohistochemical, immunoblotting, and PKG activity measurements; 2. Quantify the effect of PKG activation on the coupling efficiency between receptor activation and IP3 production using functional measurements of agonist affinity and simultaneous IP3 accumulation; 3. Determine the effect of PKG activation on calcium entry and release using fluorometric measurements of cytosolic and organellar calcium together with microautoradiographic measurements of IP3 receptor density and binding affinity; 4. Determine the effect of PKG activation on thick filament reactivity as indicated by the relation between cytosolic calcium and myosin light chain phosphorylation, measured using fluorometric measurements of cytosolic calcium together with immunoblotting of phospho- and dephospho-myosin light chain; and 5. Determine the effect of PKG activation on thin filament reactivity as indicated by the relation between the extent of myosin fight chain phosphorylation, measured using immunoblotting of phospho- and dephospho-myosin light chain, and force development measured in arterial rings. To address the effects of perinatal maturation on the function of Protein Kinase G, we will conduct these experiments in both term fetuses and non-pregnant adults. To define the importance of arterial size and type, all experiments will be conducted in a series of arteries including the common carotid, basilar, posterior communicating, and middle cerebral arteries. Finally, to enable assessment of the role of changes in Protein Kinase G function associated with hypoxic acclimatization, parallel studies will be carried out in normoxic animals and in animals acclimatized to high altitude hypoxia. Together, the results of these experiments will enable an unprecedented assessment of the mechanisms whereby maturation and hypoxic acclimatization modulate the cerebrovascular role of Protein Kinase G.

从胎儿到成人生活的过渡涉及血管反应性的巨大变化，尤其是在脑动脉中，收缩能力和放松的能力在产后生活期间同时改善了血管反应性。这些成熟变化的速率和模式反过来又易受环境应力（例如慢性缺氧）的影响，慢性缺氧可以通过多种同时效应来有效地降低药物力学耦合。显然，慢性缺氧会改变激动剂诱导的钙动员和肌丝钙敏感性，但介导缺氧对血管反应性的影响的机制尚不清楚。缺氧引起的内皮产生和NO释放的变化有助于缺氧的整体脑血管作用，但反应性对NO的反应性的重大变化也是 涉及。同样，CGMP代谢的变化有助于慢性缺氧的脑血管作用，但同样，将CGMP与血管结构偶联的机制的重大变化是对慢性缺氧的脑血管适应的重要组成部分。 CGMP下游的血管瘤化最重要的效应因子是蛋白激酶G（PKG），在脑动脉中，该蛋白激酶G（PKG）几乎介导了CGMP的所有血管扩张效应。尽管PKG具有至关重要的重要性，但其在脑血管功能中的作用在很大程度上被忽略了，尤其是在未成熟的脑动脉中。实际上，慢性缺氧对任何动脉类型或年龄中的脑血管PKG功能的影响几乎没有任何了解。鉴于这些缺陷，以及 PKG在对慢性缺氧的脑血管适应中发挥关键作用的强大潜力，提出的研究集中于涉及PKG介导的血管结构的一般机制，以及如何通过成熟和慢性缺氧来调节它们。 这些研究解决的一般假设是，慢性缺氧有选择地增强了蛋白激酶G在年龄依赖性和 动脉特异性方式。反过来，该主要假设具有四个主要的推论，每个定义都提出缺氧会影响一种机制，在该机制中，蛋白激酶G调节药物力学耦合：1）PKG调节细胞表面受体的激活与第二梅斯格IP3的合成之间的激活。 2）PKG改变了诸如IP3之类的第二使者引起钙进入和/或释放的能力； 3）PKG影响厚细丝反应性，如胞质钙与肌球蛋白轻链磷酸化程度之间的关系所表明。 4）PKG差异地增强了细丝反应性，如肌球蛋白战链磷酸化与收缩力的产生之间的关系所表明。 为了评估主要假设及其推论，我们将进行旨在： 1。使用免疫组织化学，免疫印迹和PKG活性测量，量化脑血管PKG同工型和1Beta的分布，丰度和活性； 2。使用激动剂亲和力和同时IP3积累的功能测量，量化PKG激活对受体激活和IP3产生之间耦合效率的影响； 3。确定PKG激活对钙进入钙进入和释放的影响，并使用胞质和细胞器钙的荧光测量以及IP3受体密度和结合亲和力的显微运动摄影测量结果； 4。确定PKG激活对厚细丝反应性的影响，如胞质钙和肌球蛋白轻链磷酸化之间的关系所示，该关系使用胞质钙的荧光测量以及对磷酸化和去磷蛋白甲基摩西蛋白蛋白轻度链的免疫印迹进行测量；和 5。确定PKG活化对肌球蛋白战链磷酸化程度之间的关系，该关系是使用磷酸化和去磷膜肌球蛋白轻链的免疫印迹测量的，并在动脉环中测得的力发育。 为了解决围产期成熟对蛋白激酶G功能的影响，我们将在胎儿和非怀孕成年人中进行这些实验。为了定义动脉大小和类型的重要性，所有实验都将在一系列动脉中进行，包括常见的颈动脉，基底，后验交流和大脑中动脉。最后，为了评估与低氧适应性相关的蛋白激酶G功能变化的作用，将在常规动物和适应高海拔低氧的动物中进行平行研究。这些实验的结果在一起将 对机制进行前所未有的评估，从而使成熟和低氧适应性调节蛋白激酶G的脑血管作用。