CONTROL OF OXYGENATION DURING POSTNATAL LIFE

HD25256的持续目标是确定新生儿肠道循环的独特特征如何促进坏死性小肠结肠炎的发病机制，坏死性小肠结肠炎是新生儿最常见的获得性胃肠道疾病。在过去的资助周期中，我们确定新生儿受试者（即3天）机械减少进入肠道的流量会导致肠道血管阻力在数小时内逐渐增加。当内源性NO合成被阻断时，早期血管收缩（5-30分钟）不存在；同样，这种上升也可以通过阻断内皮素ETA受体而减弱。在这种情况下，晚期血管收缩可表现为血管收缩。我们假设新生儿肠道的基础血管张力是由本构活跃的扩张和收缩力之间的动态平衡决定的，而老年肠道的张力主要是由静态的建筑现象决定的。我们假设新生儿肠道的低静息张力反映了丰富的、血流介导的内皮来源的NO产生。我们进一步认为，本构收缩音也存在，由ETl介导；然而，由于三个原因，ET1的生理作用尚未被认识到：i)系统接近最大扩张，ii) NO降低了ETA受体的结合亲和力，iii)发育调节的ETB受体通过NO的产生产生抵偿性的血管舒张。我们假设流量减少迅速改变了这种情况：早期NO的产生减少，ETA结合亲和力增加，数小时后AT1表达增加。这种平衡的快速转变就像一个放大系统，因此局部肠道流动的微小扰动就会扩散和恶化。为了验证这一假设，该项目有四个具体目标：1)通过直接测量缓冲灌注肠系膜腔流出物中的NO，并通过测定肠系膜小动脉流量、剪应力和血管直径之间的关系，证明壁剪切应力的机械刺激与3-而非35日龄肠系膜NO的产生密切相关；2)通过选择性阻断内源性NO合成，证明本构性扩张力和收缩力在3-而非35日龄肠系膜内设定基础血管张力；ETA和ETB受体在肠系膜小动脉和血液灌注肠袢的各种血流动力学条件下，以及通过TaqMan系统确定内皮细胞和血管平滑肌细胞中ETA和ETB受体的个体发生，3)证明ETA受体的结合亲和力取决于与G蛋白的偶联，通过将培养的血管平滑肌细胞暴露于外源性NO供体，然后测量放射性配体结合动力学和KTPase活性，NO干扰这种偶联。4)通过测定体内持续低流量灌注的肠系膜动脉血管平滑肌中AT1受体和ACE活性的表达，表明体内持续低流量灌注的肠系膜动脉血管平滑肌中出现了AT1受体和ACE活性的上调；通过测定内皮细胞和血管平滑肌细胞中AT1和ACE活性的表达，表明体内持续低流量灌注的肠系膜动脉血管平滑肌中出现了ACE活性。这些数据为下一个更新周期的研究提供了理由，研究从NEC或非NEC原因的人类婴儿肠切除术中切除的组织中的受体生物化学。

The continuing goal of HD25256 is to determine how the unique characteristics of the newborn intestinal circulation contribute to the pathogenesis of necrotizing enterocolitis, the most common acquired GI disease of the newborn. In the past funding cycle, we determined that mechanical reduction of flow into the intestine of newborn subjects (i.e., 3- days) caused a progressive increase in gut vascular resistance that occurred over the course of hours. The early vasoconstriction (5-30 minutes) was absent when endogenous NO synthesis was blocked; as well, this rise could be attenuated by blockade of endothelin ETA receptors. The late vasoconstriction could exhibit vasoconstriction under these circumstances. We hypothesize that basal vascular tone in newborn intestine is determined by a dynamic balance between constitutively active dilator and constrictor force, whereas tone in older intestines is determined mainly by static, architectural phenomenon. We hypothesize that the low resting tone in newborn intestine reflects abundant, flow-mediated endothelium-derived NO production. We further contend that constitutively constrictor tone is also present, mediated by ETl; the physiologic effect of ET1 is not appreciated, however, for three reasons: i) the system is near maximal dilation, ii) NO decreases the binding affinity of ETA receptors, and iii) developmentally regulated ETB receptors produce a offsetting vasodilation via NO production We hypothesize that flow reduction rapidly changes this situation: NO production decreases and ETA binding affinity increases early, and AT1 expression increases hours later. This rapid shift in balance acts as an amplification system, so that otherwise minor perturbations in localized gut flow spread and worsen. To test this hypothesis, the project has four specific aims: 1) demonstrate that the mechanostimulus of wall shear stress is tightly linked to NO production in 3-, but not 35-day old intestine by directly measuring NO in the effluent of buffer-perfused mesenteric arcades and by determining the relationship between flow, shear stress and vascular diameter in mesenteric arterioles, 2) demonstrate that constitutive dilator and constrictor forces set basal vascular tone in 3-, but not 35-day old intestine by selectively blocking endogenous NO synthesis, ETA and ETB receptors under a variety of hemodynamic conditions in mesenteric arterioles and blood perfused gut loops, and also by determining the ontogeny of ETA and ETB receptors in endothelial and vascular smooth muscle cells using the TaqMan system, 3) demonstrate that the ETA receptor binding affinity is contingent upon coupling to G proteins and that NO interferes with this coupling by exposing cultured vascular smooth muscle cells to an exogenous NO donor and then measuring radioligand binding kinetics and KTPase activity, and 4) demonstrate that up-regulation of AT1 receptors and ACE activity occur in vascular smooth muscle from mesenteric arteries exposed in vivo to sustained low flow perfusion by determining expression of AT1 and ACE activity occur in vascular smooth muscle from mesenteric arteries exposed in vivo to sustained low flow perfusion by determining expression of AT1 and ACE activity in endothelial and vascular smooth muscle cells. These data should provide justification for moving the next renewal cycle of this work into studying receptor biochemistry in tissue removed from human infants undergoing bowel resection of NEC or non-NEC reasons.