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：（根据申请人的摘要进行了改编）。长期目标的 该应用是为了阐明负责的基本机制 与急性组织缺氧有关的状态中的肠道屏障功能障碍 和/或炎症。一个统一的假设是细胞中的毁灭 能源代谢引起或导致上皮屏障的改变 在危急疾病中起作用。目的我是研究细胞因子介导的抑制 低氧诱导因子-1（HIF-1）依赖性自适应上皮反应 败血症期间缺氧。 HIF-1是调节的转录因子 表达与自适应细胞反应相关的许多基因 缺氧。在初步研究中，他们证明了HIF-1 DNA结合 培养的肝细胞和肠西细胞孵育时，活动会增加 在常氧症下与IFN-gamma和TNF的混合物。但是，这些细胞因子 无法诱导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依赖性报告基因活性 缺氧。申请人假设（1）HIF-1 DNA结合活性将 化粪池动物的肝脏和肠粘膜增加； （2）自适应 细胞对缺氧的反应将在具有的细胞或组织中受到损害 暴露于促炎的环境中（例如败血症发生）； （3） IFN-GAMMA和TNF发起的信号传导抑制了HIF-1诱导的基因 通过阻止CBP/P300募集到低氧诱导的表达 发起人。 AIM II是评估一种潜在方式的增加 胞质电离钙浓度[Ca2+] I可以起作用以增加 肠上皮细胞细胞通透性。他们以前表明 由ATP耗竭引起的上皮过度过敏性取决于 结果增加[Ca2+] i。在[Ca2+] i中增加的机制 促进过度过敏性尚不清楚。在AIM II中，他们将进行实验 为了测试[Ca2+] i的升高导致激活A的假设 依赖钙的酶，肌球蛋白轻链激酶（MLCK），从而 增加20 kDa细胞骨架蛋白，肌球蛋白光的磷酸化 链（MLC20），导致细胞骨架收缩和上皮 在此基础上过敏性。 AIM III是研究 对极化的细胞因子，缺氧或代谢抑制 复杂碳水化合物和其他亲水性的基底外侧运输 横跨肠上皮的化合物。这些研究是由 他们获得的初步数据，表明各种各样的数据 化合物，包括右旋球和各种阴离子染料，都在跨过 通过一个过程 显然是能量依赖的。基于这些发现，我们假设 败血症（或其他形式的急性疾病）中的障碍功能受损 不仅反映了顶端对基底外侧的被动渗透 方向，但也减少了相反的主动泵送（清除） 方向。