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: 7373601
• 负责人: William J. Pearce
• 金额: $ 18.86万
• 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/7373601
• 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.

从胎儿到成人生活的过渡涉及血管反应性的戏剧性变化，特别是在大脑动脉，在出生后的生活中，收缩能力和放松能力都同时提高。这些成熟变化的速度和模式反过来又非常容易受到环境压力的影响，如慢性缺氧，这些环境压力可以通过多个同时作用有效地抑制药物-机械耦合。虽然慢性低氧明显改变激动剂诱导的钙动员和肌丝钙敏感性，但介导低氧对血管反应性的S影响的机制尚不清楚。低氧诱导的内皮细胞产生和释放NO的变化有助于低氧对整体脑血管的影响，但对NO的反应性的主要变化也是 牵涉其中。同样，cGMP代谢的变化有助于慢性缺氧对脑血管的影响，但同样，连接cGMP与血管松弛机制的主要变化是脑血管对慢性缺氧适应的重要组成部分。CGMP下游最重要的血管松弛效应物是蛋白激酶G(PKG)，在脑动脉中，它几乎介导了cGMP的所有血管扩张作用。尽管PKG很重要，但它在脑血管功能中的作用被忽视了，特别是在未成熟的脑动脉中。在任何动脉类型和年龄中，慢性低氧对脑血管PKG功能的影响几乎一无所知。鉴于这些赤字，以及 鉴于PKG在脑血管对慢性低氧适应中发挥关键作用的巨大潜力，建议的研究重点是PKG介导的血管松弛的一般机制，以及成熟和慢性低氧是如何调节这些机制的。 这些研究提出的一般假设是，慢性低氧选择性地增强蛋白激酶G在年龄依赖和 动脉特有的方式。这一主要假说又有四个主要推论，每一个都提出低氧影响蛋白激酶G调节药物-机械偶联的机制：1)PKG调节细胞表面受体的激活和第二信使IP3的合成之间的偶联；2)PKG改变第二信使如IP3诱导钙进入和/或释放的能力；3)PKG影响粗丝的反应性，如细胞内钙与肌球蛋白轻链磷酸化程度的关系；以及4)PKG差异性地增强细丝的反应性，如肌球蛋白抗链磷酸化和收缩力量的产生的关系所表明的那样。 为了评估主要假设及其推论，我们将进行旨在： 1.通过免疫组织化学、免疫印迹和PKG活性测定，定量检测脑血管中PKG亚型1α和1β的分布、丰度和活性； 2.通过激动剂亲和力和IP3同时积累的功能测量，量化PKG激活对受体激活和IP3产生之间偶联效率的影响； 3.用细胞内和细胞器内钙的荧光测定以及IP3受体密度和结合亲和力的显微放射自显影测定来确定PKG激活对细胞内钙进入和释放的影响； 4.通过胞浆钙的荧光测量和磷酸化和去磷化肌球蛋白轻链的免疫印迹来测量胞浆钙和肌球蛋白轻链磷酸化之间的关系，确定PKG激活对粗丝反应性的影响；以及 5.确定PKG激活对细丝反应性的影响，通过免疫印迹法检测肌球蛋白轻链和磷酸化肌球蛋白轻链的磷酸化程度与动脉环力量发育的关系来确定PKG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未怀孕的成年人中进行这些实验。为了确定动脉大小和类型的重要性，所有实验都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习服相关的蛋白激酶G功能变化的作用，将在常氧动物和适应高原低氧的动物中进行平行研究。总而言之，这些实验的结果将 能够对成熟和低氧习服调节蛋白激酶G的脑血管作用的机制进行前所未有的评估。