MATURATION OF CEREBROVASCULAR RELAXANT MECHANISMS

描述(由申请人提供)：心血管动态平衡在所有血管床的收缩和松弛的平滑肌影响之间微妙地平衡。虽然血管收缩的调节在新生儿中已经得到了很好的研究，但出生后成熟对血管松弛机制的影响仍未得到充分的研究和了解。我们实验室最近的工作有力地表明，未成熟脑动脉典型的收缩能力低下涉及cGMP依赖性血管张力的上调，这不能简单地用cGMP浓度升高来解释。因此，我们认为，在未成熟的脑动脉中，蛋白激酶G诱导脑血管扩张的能力上调。为了解决这一核心假设，本文提出了五个具体目标。目的#1将利用蛋白质印迹、免疫组织化学和酶活性的动力学测量来验证成熟调节蛋白激酶G(PKG)的相对丰度、分布和活性的假设。目的#2将利用受体密度、激动剂亲和力和IPS含量的放射自显影测量来验证成熟改变cGMP/PKG影响G蛋白受体介导的IPS动员的能力的想法。目的#3将利用IPS受体密度和结合亲和力、钙库大小和IPS诱导的钙释放的测量来探索成熟改变PKG减弱IPS介导的钙释放的能力的想法。目的#4验证成熟改变依赖于cGMP的粗丝反应性调节的想法，胞浆钙和肌球蛋白轻链磷酸化之间的关系的变化表明。对于目标#4，将通过尿素凝胶在用cGMP和/或肌球蛋白磷酸酶抑制剂处理的完整和通透性动脉样本中测量钾诱导的肌球蛋白轻链磷酸化的时间进程。目的#5验证成熟改变cGMP/PKG调节细丝反应性的能力的假说，肌球蛋白轻链磷酸化程度与收缩力量之间的关系的变化表明。对于Aim#5，肌球蛋白轻链的磷酸化将与收缩力以及潜在的细丝调节蛋白HSP27和HSP20的相对丰度和磷酸化状态一起被测量。这些实验结果的量化整合将使我们能够对出生后成熟对cGMP介导的血管松弛途径的作用部位进行前所未有的评估，并将提供对每一组主要PKG靶标对胎儿、新生儿和成人整体脑血管内稳态的相对重要性的独特评估。这种方法还应该确定哪些cGMP依赖机制可能最适合危重新生儿的治疗性药理操作。

DESCRIPTION (provided by applicant): Cardiovascular homeostasis is delicately balanced between contractile and relaxant smooth muscle influences in all vascular beds. Whereas regulation of vascular contraction has been reasonably well studied in neonates, the effects of postnatal maturation on mechanisms of vasorelaxation remain understudied and poorly understood. Recent work in our laboratory strongly suggests that the hypocontractility typical of immature cerebral arteries involves an upregulation of cGMP-dependent attenuation of vascular tone that cannot be explained simply by elevated cGMP concentrations. Thus, we propose that the ability of Protein Kinase G to elicit cerebral vasodilatation is upregulated in immature cerebral arteries. To address this core hypothesis, five specific aims are proposed. Aim #1 will employ Western blots, immunohistochemistry, and kinetic measurements of enzyme activity to test the hypothesis that maturation modulates the relative abundance, distribution, and activity of Protein Kinase G (PKG). Aim #2 will utilize autoradiographic measurements of receptor density, agonist affinity and assays for IPS content to test the idea that maturation alters the ability of cGMP/PKG to influence G-protein receptor-mediated IPS mobilization. Aim #3 will use measurements of IPS receptor density and binding affinity, calcium store size, and IPS-induced calcium release to explore the idea that maturation alters the ability of PKG to attenuate IPS-mediated calcium release. Aim #4 tests the idea that maturation alters cGMP-dependent modulation of thick filament reactivity, as indicated by shifts in the relations between cytosolic calcium and myosin light chain phosphorylation. For aim #4 the time courses of myosin light chain phosphorylation induced by potassium will be measured via urea gels in samples from both intact and permeabilized arteries treated with cGMP and/or inhibitors of myosin phosphatase. Aim #5 tests the hypothesis that maturation alters the ability of cGMP/PKG to modulate thin filament reactivity, as indicated by shifts in the relations between the extent of myosin light chain phosphorylation and contractile force. For aim #5, myosin light chain phosphorylation will be measured together with contractile force and the relative abundances and phosphorylation states of the potential thin-filament regulatory proteins HSP27 and HSP20. Quantitative integration of the results of these experiments will enable an unprecedented evaluation of the sites of action of postnatal maturation on cGMP- mediated pathways of vasorelaxation, and will provide a unique assessment of the relative importance of each main group of PKG targets for overall cerebrovascular homeostasis in the fetus, neonate and adult. This approach should also identify which cGMP-dependent mechanisms may be most amenable to therapeutic pharmacological manipulation in the critically ill neonate.