MATURATION OF CEREBROVASCULAR RELAXANT MECHANISMS

心血管内稳态在收缩和舒张平滑肌之间微妙地平衡 影响所有血管床。然而，血管收缩的调节已经得到了相当好的研究， 在新生儿中，出生后成熟对血管舒张机制的影响仍未得到充分研究， 不太了解。我们实验室最近的工作强烈表明， 未成熟的脑动脉涉及cGMP依赖性血管张力衰减的上调， 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 引起脑血管舒张的激酶G在未成熟的脑动脉中上调。为了解决这个问题 核心假设，提出了五个具体目标。目标#1将采用蛋白质印迹，免疫组织化学， 和酶活性的动力学测量，以检验成熟调节相对生物量的假设。 蛋白激酶G（PKG）的丰度、分布和活性。目标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，以测试成熟 改变cGMP/PKG影响G蛋白受体介导的IPS动员的能力。目标#3将使用 测量IPS受体密度和结合亲和力、钙储存大小和IPS诱导的钙离子浓度。 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。 release.目的#4测试成熟改变粗丝反应性的cGMP依赖性调节的想法， 如胞质钙和肌球蛋白轻链磷酸化之间关系的变化所示。为 目的#4钾诱导的肌球蛋白轻链磷酸化的时程将通过 来自用cGMP和/或cGMP抑制剂处理的完整和透化动脉的样品中的尿素凝胶 肌球蛋白磷酸酶目的#5测试成熟改变cGMP/PKG的能力的假设， 调节细丝的反应性，如肌球蛋白光的程度之间的关系的变化所示。 链磷酸化和收缩力。对于目标#5，肌球蛋白轻链磷酸化将被抑制。 与收缩力和相对丰度和磷酸化状态一起测量， 潜在的细丝调节蛋白HSP 27和HSP 20。这些结果的定量整合 实验将能够对出生后成熟对cGMP的作用位点进行前所未有的评估， 介导的血管舒张途径，并将提供一个独特的评估的相对重要性， PKG的每个主要组靶向胎儿、新生儿和成人的总体脑血管稳态。 该方法还应确定哪些cGMP依赖性机制可能最适合 危重新生儿的治疗药理学操作。

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.