HEPATIC UREAGENESIS--STUDIES WITH 15-N GC/MS & 13C-NMR

肝脏尿生成——15-N GC/MS 研究

• 批准号: 2140911
• 负责人: ITZHAK NISSIM
• 金额: $ 16.26万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-04-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2140911
• 关键词: Krebs' cycle; acid base balance; acidosis; alkalosis; aminoacid metabolism; electrolyte balance; gas chromatography mass spectrometry; glutamine; hormone regulation /control mechanism; intracellular transport; laboratory rat; liver cells; liver metabolism; membrane transport proteins; nuclear magnetic resonance spectroscopy; phosphatidylinositols; protein kinase C; stable isotope; tissue /cell culture; urea cycle

The physiology of hepatic ureagenesis has posed one of the more complex problems in metabolic regulation. this proposal entails a comprehensive evaluation of the mechanism(s) regulating glutamine metabolism and urea production in isolated hepatocytes obtained from acute or chronically acidotic or alkalotic rats. The primary questions to be addressed are: 1) What is the role of TCA-cycle metabolism in the control of hepatic ureagenesis? 2) How do changes in HCO3 and/or PCO2 alter urea formation from glutamine? 3) How do changes in K+, Pi and Ca2+ homeostasis alter ureagenesis and what is the control site? 4) Do hormones (insulin, glucagon, catecholamine) enhance or inhibit changes in urea formation which occur in response to altered acid-base homeostasis? 5) What is the site(s) of the hormonal action? Is it linked to alterations of intracellular pH, Ca2+, Pi or to changes of phosphatidylinositol turnover, activation of protein kinase C and/or modulation of the Na+-H+- exchanger? Hypotheses to be explored include: (a) that the initial signal in evoking pH modulation of urea formation from glutamine is an alteration of TCA-cycle metabolism; (b) that in addition to H+- homeostasis changes in intracellular Ca2+ or Ca2+/Pi ratio modulate urea formation secondary to alteration of TCA-cycle metabolism; (c) that the hormonal regulation of ureagenesis is mediated through changes in intracellular Ca2+ and thereby, modulation of TCA-cycle metabolism and/or protein kinase C activity; (d) alternatively, hormones affect urea formation from glutamine secondary to their effect on the Na+-H+- exchanger and hence, changes in intracellular pH, and fluxes via the glutaminase and glutamate dehydrogenase pathways, and therefore, ammonia availability for urea synthesis. We will explore these themes by incubating isolated hepatocytes with 15N and/or 13C labeled substrates. Studies will be carried out during induction of acute acidosis and alkalosis or following induction of chronic acidosis and alkalosis in the presence and absence of metabolic modulators. We will define precursor-product relationships and flux rates using GC-MS and/or NMR. The proposed experiments are of scientific as well as clinical importance by deepening our understanding of hepatic glutamine metabolism and urea synthesis in response to perturbations of H+ associated with/or independent of other physiological factors. The results we obtain should provide a panoramic view of the mechanism(s) regulating hepatic ureagenesis.

肝性尿素生成的生理学已经成为一个比较复杂的 代谢调节的问题。 这一建议需要全面的 调节谷氨酰胺代谢和尿素的机制评价 在从急性或慢性感染中获得的分离肝细胞中的生产 酸中毒或缺氧大鼠。 需要解决的主要问题是： 1)TCA循环代谢在控制肝硬化中的作用是什么？ 尿素生成？ 2)HCO 3和/或PCO 2的变化如何改变尿素的形成 从谷氨酰胺？ 3)K+、Pi和Ca 2+稳态的变化如何改变 尿素生成和控制部位是什么？ 4)激素（胰岛素， 胰高血糖素、儿茶酚胺）增强或抑制尿素形成的变化 酸碱平衡改变时会发生什么 5)是什么 激素作用的部位？ 是否与 细胞内pH、Ca ~（2+）、Pi或磷脂酰肌醇的变化 周转、蛋白激酶C的活化和/或Na+-H+-的调节 交换器？ 需要探讨的假设包括：（a）最初的 在引起由谷氨酰胺形成尿素的pH调节中的信号是 TCA循环代谢的改变;（B）除了H+- 细胞内Ca ~（2+）或Ca ~（2+）/Pi比值调节尿素稳态变化 形成继发于TCA循环代谢的改变;（c） 尿素生成的激素调节是通过 细胞内Ca 2+，从而调节TCA循环代谢和/或 蛋白激酶C活性;（d）或者，激素影响尿素 谷氨酰胺的形成继发于它们对Na+-H+-的影响， 交换器，因此，细胞内pH值的变化，并通过通量 谷氨酰胺酶和谷氨酸脱氢酶途径，因此，氨 用于尿素合成。 我们将通过孵育分离的肝细胞与15 N 和/或13 C标记的底物。 研究将在 诱导急性酸中毒和酸中毒或在诱导 存在和不存在代谢性 调制器。 我们将定义供应商-产品关系和流量 速率使用GC-MS和/或NMR。 所提议的实验具有科学和临床重要性 通过加深我们对肝脏谷氨酰胺代谢和尿素的理解， 合成响应于与/或 与其他生理因素无关。 我们获得的结果应该 提供了调节肝脏功能的机制的全景图， 尿素生成