CONTROL OF OXYGENATION DURING POSTNATAL LIFE

HD 25256的持续目标是确定新生儿肠循环的独特特征如何促成坏死性小肠结肠炎（新生儿最常见的获得性GI疾病）的发病机制。在过去的资助周期中，我们确定机械减少流入新生儿受试者的肠道（即，3-天）引起在数小时的过程中发生的肠血管阻力的进行性增加。当内源性NO合成被阻断时，早期血管收缩（5-30分钟）不存在;同时，这种升高可以通过阻断内皮素ETA受体而减弱。在这些情况下，晚期血管收缩可以表现出血管收缩。我们假设，新生儿肠道的基础血管张力是由组成性活动的扩张器和收缩力之间的动态平衡决定的，而在老年肠道的张力主要是由静态的，建筑现象。我们推测，低静息张力在新生儿肠道反映了丰富的，流量介导的内皮源性NO的生产。我们进一步主张，也存在由ET 1介导的组成性收缩张力;然而，由于三个原因，ET 1的生理作用未被认识到：i）系统接近最大扩张，ii）NO降低ETA受体的结合亲和力，和iii）发育调节的ETB受体通过NO产生产生抵消血管扩张。我们假设流量减少迅速改变这种情况：早期NO生成减少，ETA结合亲和力增加，数小时后AT 1表达增加。这种平衡的快速变化就像一个放大系统，使得局部肠道流动的微小扰动扩散和恶化。为了验证这一假设，该项目有四个具体目标：1）通过直接测量缓冲液灌注的肠系膜弓流出物中的NO并通过确定流量、切应力和血管之间的关系，证明壁切应力的机械刺激与3天龄肠道中NO的产生密切相关，但与35天龄肠道中NO的产生无关。肠系膜小动脉中的血管直径，2）证明组成性扩张和收缩力通过在肠系膜小动脉和血液灌注的肠环中的各种血液动力学条件下选择性地阻断内源性NO合成、ETA和ETB受体而在3天而不是35天龄的肠中设定基础血管张力，并且还通过使用TaqMan系统测定内皮和血管平滑肌细胞中ETA和ETB受体的个体发生，第三章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NO浓度，证明ETA受体结合亲和力取决于与G蛋白的偶联，并且NO干扰这种偶联。放射性配体结合动力学和KT 3活性，（4）证明了这一点，通过测定AT 1的表达，在体内暴露于持续低流量灌注的肠系膜动脉的血管平滑肌中发生AT 1受体和ACE活性的调节，内皮细胞和血管平滑肌细胞。这些数据应提供理由，将这项工作的下一个更新周期转移到研究接受NEC或非NEC原因肠切除术的人类婴儿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.