INTRACELLULAR PH HOMEOSTASIS IN MALPIGHIAN TUBULES

昆虫体内的液体运输依赖于氢离子。液泡型 H+-ATPase泵以类似的方式为昆虫上皮膜提供能量 无处不在的Na/K-ATPase泵。昆虫从昆虫体内分泌液体 血淋巴通过细胞进入马氏管的管腔。这个 来自H+-ATPase泵的能量允许二次运输 这就导致了液体的运输。液体的分泌是 昆虫产生尿液的第一步及其调节 它们细胞外液的组成。尽管扮演了至关重要的角色 根据尿液中的H+-ATPase，到目前为止还没有任何信息 关于细胞内氢离子调节的性质 马氏管。由此产生的问题是：从何而来？ H+-ATPase的氢离子？马氏管的酸碱度如何？ 细胞质保持正常吗？哪个酸/碱转运体被激活 在利尿的时候？ 拟议研究的目标是：1)确定主要的 分离的细胞内氢离子动态平衡机制(S) 蚊子的马氏管和2)确定影响 细胞内和细胞外信使分子，包括环磷酸腺苷 利尿激素对细胞内氢离子动态平衡的影响。 初步数据表明，蚊子的马氏管调节它们的 细胞内pH通过至少两种排酸机制和两种 酸负荷机理。此外，胞内氢气和 碳酸氢酶控制着碳酸氢盐的生成。 最后，在急性酸负荷后，环状AMP增加了酸的排泄 提示排酸和cAMP介导的利尿之间存在联系。 拟议中的实验将测量液体分泌和氢离子。 在分离的蚊子马氏管中的分泌率 建立特异性抑制之间的剂量-反应关系 酸碱转运体与液体和氢离子的分泌速率 昆虫。将使用微量荧光法测量细胞内的pH值 允许实时记录量变化的技术 细胞内的氢离子。我们将描述抑制的影响 以及对酸挤压和酸负荷机制的刺激 关于细胞内稳态pH、急性酸后恢复率的研究 转运体的装载、酸或碱通量速率以及Km和Vmax。 拟议中的实验结果将首次， 详细描述黄氏腹水分泌的潜在机制(S) 马氏管。这一知识体系将带来潜在的洞察力 转化为生理过程，使蚊子能够有效地 抵抗农药干预和病原菌的有效媒介。

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.