INTESTINAL PERFUSION AND PERMEABILITY IN SEPSIS

描述：(改编自申请人的摘要)。的长期目标是 这一应用是为了阐明负责的基本机制 急性组织缺氧相关状态下的肠屏障功能障碍 和/或炎症。一个统一的假设是，细胞内的错乱 能量代谢引起或促成上皮屏障的改变 在危重疾病中发挥作用。目的研究细胞因子介导的阻遏作用。 缺氧诱导因子-1(HIF-1)依赖的适应性上皮细胞对 脓毒症期间的缺氧。HIF-1是一种转录因子，调节 与适应性细胞反应相关的一些基因的表达 缺氧。在初步研究中，他们证明了HIF-1DNA结合 当培养的肝细胞和肠细胞孵育时，活性增加 在常氧条件下应用干扰素-γ和肿瘤坏死因子的混合物。然而，这些细胞因子 未能诱导依赖于HIF-1的荧光素酶报告基因的表达。 最近，这些细胞因子抑制HIF-1依赖的报告活性 缺氧。申请人假设(1)HIF-1DNA结合活性将 败血症动物肝脏和肠道粘膜的增加；(2)适应性 细胞或组织对低氧的反应会受到损害 暴露在促炎环境中(如败血症时)； 干扰素和肿瘤坏死因子启动的信号转导抑制HIF-1诱导的基因表达 阻断CBP/p300在低氧诱导下的募集表达 推动者。目标二是评估一种潜在的方式，即 胞内游离钙离子浓度[Ca~(2+)]_i升高 肠上皮细胞旁通透性。他们之前的研究表明 ATP耗竭引起的上皮高通透性依赖于 由此产生的[Ca~(2+)]i升高的机制(S) 促进高渗透性是未知的。在AIM II中，他们将进行实验 为了检验[Ca~(2+)]i升高会导致A细胞活化的假设 钙依赖酶，肌球蛋白轻链激酶(MLCK)，从而 增加20 kDa细胞骨架蛋白肌球蛋白LIGH的磷酸化 链(MLC20)，导致细胞骨架收缩和上皮 在此基础上的高渗透性。目的三是调查 细胞因子、缺氧或对极化的代谢抑制 复合碳水化合物和其他亲水性物质的基侧到顶端的运输 肠道上皮中的化合物。这些研究是由以下因素推动的 他们获得的初步数据表明，各种各样的 化合物，包括右旋糖苷和各种阴离子染料，被输送到 大鼠结肠粘膜通过一种过程向浆膜到粘膜的方向 显然是能源依赖型的。基于这些发现，我们假设 脓毒症(或其他形式的急性疾病)的屏障功能受损可能 不仅反映了根尖到基底外侧被动渗透的增加 但也减少了相反方向的主动抽水(扫气) 方向。

DESCRIPTION: (Adapted from the applicant's abstract). The long- term goal of this application is to elucidate the fundamental mechanisms responsible for intestinal barrier dysfunction in states associated with acute tissue hypoxia and/or inflammation. One unifying hypothesis is that derangements in cellular energy metabolism cause or contribute to alterations in epithelial barrier function in critical illness. Aim I is to study cytokine mediated repression of hypoxia-inducible factor-1 (HIF-1)-dependent adaptive epithelial responses to hypoxia during sepsis. HIF-1 is a transcription factor that regulates the expression of a number of genes associated with adaptive cellular responses to hypoxia. In preliminary studies, they demonstrated that HIF-1 DNA-binding activity is increased when cultured hepatocytes and enterocytes are incubated with a mixture of IFN-gamma and TNF under normoxia. However, these cytokines fail to induce the expression of a HIF-1-dependent luciferase reporter gene. More recently, these cytokines inhibit HIF-1-dependent reporter activity during hypoxia. The applicants hypothesize that (1) HIF-1 DNA-binding activity will increase in the liver and intestinal mucosa of septic animals; (2) adaptive cellular responses to hypoxia will be impaired in cells or tissues that have been exposed to a pro-inflammatory milieu (such as occurs in sepsis); (3) signaling initiated by IFN-gamma and TNF suppresses HIF-1-induced gene expression by blocking the recruitment of CBP/p300 to hypoxia-inducible promoters. Aim II is to evaluate one potential way that an increase in cytosolic ionized calcium concentration, [Ca2+] i, could act to increase intestinal epithelial paracellular permeability. They previously showed that epithelial hyperpermeability caused by ATP depletion is dependent upon the resultant increase in [Ca2+] i. The mechanism(s) whereby increases in [Ca2+] i promote hyperpermeability are unknown. In Aim II they will conduct experiments to test the hypothesis that elevation of [Ca2+] i leads to activation of a calcium-dependent enzyme, myosin light chain kinase (MLCK), and thereby increases phosphorylation of the 20-kDa cytoskeletal protein, myosin light chain (MLC20), resulting in cytoskeletal contraction and epithelial hyperpermeability on that basis. Aim III is to investigate the effect of cytokines, hypoxia, or metabolic inhibition on the polarized basolateral-to-apical transport of complex carbohydrates and other hydrophilic compounds across the intestinal epithelium. These studies are prompted by preliminary data they have obtained, which indicate that a wide variety of compounds, including dextrans and various anionic dyes, are transported across rat colonic mucosa in the serosa-to-mucosa direction via a process that apparently is energy-dependent. Based on these findings, we hypothesize that that impaired barrier function in sepsis (or other forms of acute illness) may not just reflect increased passive permeation in the apical-to basolateral direction, but also decreased active pumping (scavenging) in the opposite direction.