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有助于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，新生儿肠道流量的机械性减少(即3天)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，早期的血管收缩(5-30分钟)消失，这种升高可被内皮素ETA受体阻断而减弱。在这种情况下，晚期血管收缩可能表现为血管收缩。我们假设，新生儿肠道的基础血管张力是由结构性活跃的扩张器和收缩肌力之间的动态平衡决定的，而老年肠道的张力主要由静态的、构筑的现象决定。我们推测，新生儿肠道的低静息张力反映了大量的血流介导的内皮细胞衍生的NO的产生。我们进一步认为，也存在结构性收缩张力，由ETL介导；然而，ET1的生理作用尚未被认识，但原因有三：i)系统接近最大扩张，ii)NO降低ETA受体的结合亲和力，以及iii)发育调节的ETB受体通过NO的产生产生抵消性的血管扩张我们假设流量减少会迅速改变这种情况：NO的产生减少，ETA结合亲和力提前增加，AT1的表达在几小时后增加。这种平衡的快速变化起到了放大系统的作用，因此，在其他情况下，局部肠流的微小扰动会扩散并恶化。为了验证这一假说，该项目有四个具体目标：1)通过直接测量缓冲灌流的肠系膜弓流出物中的NO，并通过确定肠系膜小动脉的血流、剪应力和血管直径之间的关系，来证明壁切应力的机械刺激与3日龄而不是35日龄的小肠中NO的产生密切相关；2)证明结构性扩张器和缩窄力通过选择性地阻断各种血流动力学条件下肠系膜小动脉和血液灌流肠环的内源性NO合成、ETA和ETB受体，来设定3日龄的基础血管张力，而不是35日龄的肠管。还通过使用TaqMan系统确定内皮和血管平滑肌细胞中ETA和ETB受体的个体发育，3)证明ETA受体结合亲和力取决于与G蛋白的偶联，并且NO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活性来干扰这种偶联，4)通过检测血管内皮细胞和血管内皮细胞AT1的表达和ACE活性，证实在体肠系膜动脉血管内皮细胞和血管内皮细胞的AT1受体表达和ACE活性在持续低流量灌流条件下发生上调。这些数据应该为下一个更新周期的工作转移到研究从接受NEC或非NEC原因的肠切除的婴儿中取出的组织中的受体生物化学提供理由。

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.