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: 7755430
• 负责人: William J. Pearce
• 金额: $ 19.8万
• 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/7755430
• 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; Development; Fetus; GTP-Binding Proteins; Hypoxia; Immunoblotting; Life; Light; Measurement; Measures; Mediating; Metabolism; Microfilaments; Myosin ATPase; Myosin Light Chains; Pattern; Perinatal; Phosphorylation; Play; Production; Protein Isoforms; Receptor Activation; Role; Second Messenger Systems; Series; Stress; Thick Filament; Thin Filament; Vasodilation; Vasodilator Agents; age related; cerebral artery; cerebrovascular; depressed; design; fetal; fighting; improved; middle cerebral artery; postnatal; pregnant; receptor density; release of sequestered calcium ion into cytoplasm; research study; second messenger

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.

从胎儿到成年的过渡涉及血管反应性的巨大变化，特别是脑动脉，其收缩能力和放松能力在出生后同时改善。反过来，这些成熟变化的速率和模式非常容易受到环境压力的影响，例如慢性缺氧，这可以通过多种同时发生的效应有效抑制药物机械耦合。虽然很明显，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影响的机制仍不清楚。缺氧引起的内皮细胞生成和释放 NO 的变化有助于缺氧对脑血管的整体影响，但对 NO 反应性的主要变化也与缺氧有关。 涉及。同样，cGMP 代谢的变化会导致慢性缺氧对脑血管的影响，但 cGMP 与血管舒张耦合机制的重大变化是脑血管适应慢性缺氧的重要组成部分。 cGMP 下游血管舒张的最重要的单一效应物是蛋白激酶 G (PKG)，它在脑动脉中介导 cGMP 的几乎所有血管舒张作用。尽管 PKG 具有核心重要性，但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中。事实上，对于慢性缺氧对任何动脉类型或年龄的脑血管 PKG 功能的影响一无所知。鉴于这些缺陷，并且 鉴于 PKG 在脑血管适应慢性缺氧方面发挥关键作用的巨大潜力，拟议的研究重点是 PKG 介导的血管舒张的一般机制，以及这些机制如何通过成熟和慢性缺氧进行调节。 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 在年龄依赖性和年龄相关性中引起脑血管舒张的能力。 动脉特定方式。这一主要假设又具有四个主要推论，每个推论都提出缺氧会影响蛋白激酶 G 调节药效耦合的机制： 1) PKG 调节细胞表面受体的激活和第二信使 IP3 的合成之间的耦合； 2) PKG 改变第二信使（如 IP3）引发钙进入和/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； 4) PKG 差异性地增强细丝反应性，如肌球蛋白战斗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，我们将进行旨在以下目的的实验： 1. 使用免疫组织化学、免疫印迹和 PKG 活性测量来量化脑血管 PKG 同工型 1alpha 和 1beta 的分布、丰度和活性； 2. 使用激动剂亲和力和同时 IP3 积累的功能测量来量化 PKG 激活对受体激活和 IP3 产生之间耦合效率的影响； 3. 使用细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放射自显影测量，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和去磷酸肌球蛋白轻链的免疫印迹进行测量；和 5. 确定 PKG 激活对细丝反应性的影响，如肌球蛋白战斗链磷酸化程度（使用磷酸肌球蛋白轻链和去磷酸肌球蛋白轻链的免疫印迹测量）与动脉环中测量的力发展之间的关系所示。 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。这些实验的结果将共同 能够对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前所未有的评估。