INTESTINAL PERFUSION AND PERMEABILITY IN SEPSIS

脓毒症的肠道灌注和渗透性

• 批准号: 6197428
• 负责人: Mitchell P. Fink
• 金额: $ 32.52万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-04-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6197428
• 关键词: DNA binding protein; calcium flux; cytokine; gastrointestinal circulation; gastrointestinal epithelium; gastrointestinal toxin absorption; hypoxia; liver cells; membrane permeability; myosin light chain kinase; septic shock

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.

描述：（改编自申请人的摘要）。的长期目标 本申请旨在阐明 急性组织缺氧状态下肠屏障功能障碍 和/或炎症。一个统一的假设是， 能量代谢引起或促成上皮屏障的改变 在危重病中发挥作用。目的一是研究细胞因子介导的细胞因子抑制 缺氧诱导因子-1（HIF-1）依赖性适应性上皮细胞对 败血症时缺氧HIF-1是一种转录因子，它调节细胞凋亡。 许多与适应性细胞反应相关的基因表达， 缺氧在初步研究中，他们证明HIF-1 DNA结合 当培养的肝细胞和肠细胞孵育时， IFN-γ和TNF的混合物。然而，这些细胞因子 不能诱导HIF-1依赖性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-1 DNA结合活性将 脓毒症动物的肝脏和肠粘膜增加;（2）适应性 细胞对缺氧的反应将在具有以下特征的细胞或组织中受损： 暴露于促炎环境（如败血症）;（3） 由IFN-γ和TNF启动的信号传导抑制HIF-1诱导的基因 通过阻断CBP/p300向缺氧诱导的 发起人。目的二是评估一种可能的方式， 胞浆离子钙浓度[Ca ~（2+）] i可增加 肠上皮细胞旁通透性。他们先前表明， 由ATP耗竭引起的上皮细胞通透性过高依赖于 [Ca 2 +] i的增加。[Ca 2 +] i增加的机制 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升高导致A细胞活化的假设， 钙依赖性酶，肌球蛋白轻链激酶（MLCK），从而 增加20-kDa细胞骨架蛋白，肌球蛋白轻磷酸化 链（MLC 20），导致细胞骨架收缩和上皮细胞 在此基础上，目的三是研究 细胞因子、缺氧或对极化细胞的代谢抑制 复合碳水化合物和其他亲水性物质的基底侧向顶端转运 化合物穿过肠上皮。这些研究是由 他们获得的初步数据表明， 包括葡聚糖和各种阴离子染料在内的化合物被转运到 大鼠结肠粘膜在浆膜-粘膜方向通过一个过程， 显然是依赖于能量的。基于这些发现，我们假设， 脓毒症（或其他形式的急性疾病）中屏障功能受损可能 不仅反映了从顶侧到底侧的被动渗透增加 方向，但也减少了主动泵送（扫气）在相反的方向 方向