INTRACELLULAR PH HOMEOSTASIS IN MALPIGHIAN TUBULES

马氏管细胞内 pH 稳态

• 批准号: 2150434
• 负责人: DAVID H. PETZEL
• 金额: $ 4.91万
• 依托单位: CREIGHTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-05-01 至 1998-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2150434
• 关键词: Aedes; acid base balance; cyclic AMP; excretion; fluorimetry; hormone regulation /control mechanism; intracellular; invertebrate endocrinology; laboratory mouse

Fluid transport in insects is dependent on hydrogen ions. Vacuolar-type H+-ATPase pumps energize insect epithelial membranes in a manner similar to that of the ubiquitous Na/K-ATPase pump. Insects secrete fluid from hemolymph through cells into the lumen of the Malpighian tubule. The energy derived from the H + -ATPase pump allows for secondary transport that leads to this transport of fluid. The secretion of fluid is the first step in generation of urine in insects and regulation of the composition of their extracellular fluid. Despite the crucial role played by the H+-ATPase in urine formation, no information to date is available as to the nature of intracellular regulation of hydrogen ions in Malpighian tubules. Questions which arise are: What is the source of the hydrogen ions for the H+-ATPase? How is the pH of the Malpighian tubule cell cytoplasm maintained? Which acid/base transporter is activated during diuresis? The goals of the proposed research are to 1) determine the primary mechanism(s) of intracellular hydrogen ion homeostasis m isolated Mosquito Malpighian tubules and 2) determine the influence of intracellular and extracellular messenger molecules including cyclic AMP and diuretic hormones on intracellular hydrogen ion homeostasis. Preliminary data indicate that mosquito Malpighian tubules regulate their intracellular pH through at least two acid extrusion mechanisms, and two acid loading mechanism. Additionally, intracellular hydrogen and bicarbonate generation are under the control of carbonic anhydrase. Finally, cyclic AMP increases acid extrusion after an acute acid load suggesting a link between acid extrusion and cAMP-mediated diuresis. The proposed experiments will measure fluid secretion and hydrogen ion secretion rates in isolated mosquito Malpighian tubules in order to establish dose response relationships between inhibition of specific acid-base transporters and fluid and hydrogen ion secretion rates in insects. Intracellular pH will be measured using micro-fluorometric techniques which allow for real time recording of changes in quantities of cellular hydrogen ions. We will characterize the effects of inhibition and stimulation of the acid extruding and acid loading mechanism with respect to steady-state intracellular pH, recovery rates after acute acid loading, acid or base flux rates and Km and Vmax of the transporters. The results from the proposed experiments will, for the first time, describe in detail the underlying mechanism(s) for fluid secretion in Malpighian tubules. This body of knowledge will lead to potential insight into physiological processes that enable mosquitoes to be effective in resisting pesticide intervention and efficient vectors of pathogens.

昆虫体内的液体运输依赖于氢离子。 液泡型 H+-ATP酶泵以类似于 与普遍存在的Na/K-ATP酶泵相比。昆虫分泌液体， 血淋巴通过细胞进入马氏管腔。的 来自H + -ATP酶泵的能量允许二次运输 导致了液体的运输。液体的分泌是 昆虫产生尿液的第一步， 细胞外液的组成。尽管在这场战争中 通过尿形成中的H+-ATP酶，迄今为止没有可用的信息 至于细胞内调节氢离子的性质， 马氏管出现的问题是：什么是源 H+-ATP酶的氢离子？马氏管的pH值如何 细胞质保持不变？哪种酸/碱转运蛋白被激活 在利尿的时候吗 本研究的目的是：1）确定主要的 细胞内氢离子稳态的机制 苔藓马氏管和2）确定的影响 包括环AMP在内的细胞内和细胞外信使分子 和利尿激素对细胞内氢离子稳态的影响。 初步数据表明，蚊子的马氏管调节它们的 细胞内pH通过至少两种酸挤出机制，和两种 载酸机理此外，细胞内氢和 碳酸氢盐的生成受碳酸酐酶的控制。 最后，环AMP增加急性酸负荷后的酸排出 提示酸排出和cAMP介导的利尿之间的联系。 拟议的实验将测量液体分泌和氢离子 分泌速率在分离的蚊子马氏管， 建立特异性抑制之间的剂量反应关系 酸碱转运蛋白和液体和氢离子分泌率 昆虫将使用显微荧光测定法测量细胞内pH值 允许真实的时间记录数量变化的技术 细胞氢离子。我们将描述抑制的效果 并模拟了酸挤出和酸加载机理， 相对于稳态细胞内pH，急性酸中毒后的恢复率 负载、酸或碱通量率以及转运蛋白的Km和Vmax。 这些实验的结果将首次， 详细描述液体分泌的潜在机制， 马氏管这一知识体系将导致潜在的洞察力 使蚊子能够有效地 抗农药干扰和病原菌的有效载体。