MATURATION OF CEREBROVASCULAR RELAXANT MECHANISMS

心血管动态平衡在收缩和松弛的平滑肌之间保持微妙的平衡 对所有血管床的影响。鉴于血管收缩的调节已经被很好地研究了 在新生儿中，出生后成熟对血管松弛机制的影响仍未得到充分研究。 人们对此知之甚少。我们实验室最近的工作有力地表明，典型的收缩功能低下 未成熟的脑动脉涉及cGMP依赖的血管张力减弱的上调 不能简单地用cGMP浓度升高来解释。因此，我们认为蛋白质的能力 激动型GTO诱导的脑血管扩张在未成熟的脑动脉中上调。来解决这个问题 核心假设，提出了五个具体目标。目标1将使用免疫印迹，免疫组织化学， 以及酶活性的动力学测量，以检验成熟调节相对 蛋白激酶G(PKG)的丰度、分布和活性。AIM#2将利用放射自显影 受体密度、激动剂亲和力的测量和IPS含量的测定以检验成熟的想法 改变cGMP/PKG影响G蛋白受体介导的IPS动员的能力。AIM#3将使用 IPS受体密度和结合亲和力、钙库大小和IPS诱导的钙离子的测量 释放以探索成熟改变PKG减弱IPS介导的钙离子的能力的想法 放手。目的#4测试成熟改变依赖于cGMP的粗丝反应性调制的想法， 细胞内钙和肌球蛋白轻链磷酸化关系的变化表明。为 目的#4测定钾诱导的肌球蛋白轻链磷酸化的时程 经cGMP和/或血管紧张素转换酶抑制剂处理的完整和通透性动脉样本中的尿素凝胶 肌球蛋白磷酸酶。目的#5验证成熟改变cGMP/PKG能力的假设 调节细丝的反应性，如肌球蛋白光范围之间关系的变化所表明的 链的磷酸化和收缩力。对于目标5，肌球蛋白轻链磷酸化将是 与收缩力、相对丰度和磷酸化状态一起测量 潜在的细丝调节蛋白HSP27和HSP20。对这些结果的量化整合 实验将使人们能够前所未有地评估出生后成熟对cGMP的作用部位。 介导的血管松弛途径，并将提供一个独特的评估相对重要性的 每个主要的PKG组的目标是胎儿、新生儿和成人的整体脑血管内稳态。 这种方法还应该确定哪些cGMP依赖机制可能最适合于 危重新生儿的治疗性药理手法。

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 Gto elicit cerebral vasodilatation is upregulated in immature cerebral arteries. Toaddress 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.