Hypoxic modulation of protein kinase G function in fetal/adult cerebral arteries

胎儿/成人脑动脉中蛋白激酶 G 功能的缺氧调节

• 批准号: 7541403
• 负责人: William J. Pearce
• 金额: $ 19.31万
• 依托单位: LOMA LINDA UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7541403
• 关键词: Acclimatization; Address; Adult; Affinity; Age; Agonist; Altitude; Animals; Arteries; Binding; Blood Vessels; Calcium; Cell Surface Receptors; Cerebrum; Chronic; Contracts; Coupling; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Depressed mood; Development; Fetus; GTP-Binding Proteins; Hypoxia; Immunoblotting; Life; Light; Measurement; Measures; Mediating; Metabolism; Microfilaments; Myosin ATPase; Myosin Light Chains; Numbers; Patient currently pregnant; Pattern; Perinatal; Phosphorylation; Play; Production; Protein Isoforms; Rate; Receptor Activation; Role; Second Messenger Systems; Series; Stress; Thick Filament; Thin Filament; Vasodilation; Vasodilation disorder; Vasodilator Agents; age related; cerebral artery; cerebrovascular; design; fetal; fighting; improved; inositol-1,4,5-triphosphate receptor; middle cerebral artery; postnatal; receptor density; release of sequestered calcium ion into cytoplasm; research study; second messenger; size

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.

从胎儿到成人的过渡涉及血管反应性的巨大变化，特别是在脑动脉中，收缩能力和舒张能力在出生后同时提高。反过来，这些成熟变化的速率和模式对环境应激（如慢性缺氧）高度敏感，这可能通过多种同时作用有效抑制药物力学偶联。尽管慢性缺氧明显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影响的机制仍不清楚。缺氧诱导的内皮细胞产生和释放NO的变化有助于缺氧的总体脑血管效应，但对NO反应性的主要变化也是缺氧引起的。 涉案类似地，cGMP代谢的变化有助于慢性缺氧的脑血管效应，但同样，将cGMP与血管舒张耦合的机制的重大变化是脑血管适应慢性缺氧的重要组成部分。环鸟苷酸下游血管舒张的最重要效应物是蛋白激酶G（PKG），它在脑动脉中介导环鸟苷酸的几乎所有血管扩张作用。尽管PKG的核心重要性，其在脑血管功能中的作用在很大程度上被忽视，特别是在未成熟的脑动脉。事实上，慢性缺氧对任何动脉类型或年龄的脑血管PKG功能的影响尚不清楚。鉴于这些缺陷， PKG在脑血管对慢性缺氧的适应中发挥关键作用的强大潜力，建议的研究集中在PKG介导的血管舒张的一般机制，以及这些机制是如何通过成熟和慢性缺氧调节的。 这些研究提出的一般假设是，慢性缺氧选择性地增强蛋白激酶G的能力，以引起脑血管舒张，在一个年龄依赖性， 动脉特异性方式。这一主要假说又有四个主要推论，每个推论都提出缺氧影响蛋白激酶G调节药物力学偶联的机制：1）PKG调节细胞表面受体活化和第二信使IP 3合成之间的偶联; 2）PKG改变第二信使如IP 3引起钙进入和/或释放的能力; 3）PKG影响粗丝反应性，如胞质钙与肌球蛋白轻链磷酸化程度之间的关系所示;以及4）PKG差异性地增强细丝反应性，如肌球蛋白轻链磷酸化与收缩力产生之间的关系所示。 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活性测量定量脑血管PKG亚型1 α和1 β的分布、丰度和活性; 2.使用激动剂亲和力和同时IP 3积累的功能测量来量化PKG活化对受体活化和IP 3产生之间的偶联效率的影响; 3.使用细胞溶质和细胞器钙的荧光测量以及IP 3受体密度和结合亲和力的显微放射自显影测量来确定PKG激活对钙进入和释放的影响; 4.确定PKG激活对粗丝反应性的影响，如胞质钙和肌球蛋白轻链磷酸化之间的关系所示，使用胞质钙的荧光测量以及磷酸化和去磷酸化肌球蛋白轻链的免疫印迹进行测量;和 5.通过肌球蛋白轻链磷酸化程度（使用磷酸化和去磷酸化肌球蛋白轻链的免疫印迹法测量）与动脉环中测得的力发展之间的关系，确定PKG激活对细丝反应性的影响。 为了解决围产期成熟对蛋白激酶G功能的影响，我们将在足月胎儿和非妊娠成人中进行这些实验。为了确定动脉大小和类型的重要性，所有实验将在一系列动脉中进行，包括颈总动脉、基底动脉、后交通动脉和大脑中动脉。最后，为了能够评估与低氧适应相关的蛋白激酶G功能变化的作用，将在常氧动物和适应高海拔低氧的动物中进行平行研究。这些实验的结果将 使成熟和低氧适应调节蛋白激酶G的脑血管作用的机制进行了前所未有的评估。