Malpighian tubule Na/H exchange during development

发育过程中马氏小管 Na/H 交换

• 批准号: 6360609
• 负责人: DAVID H. PETZEL
• 金额: $ 14.25万
• 依托单位: CREIGHTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6360609
• 关键词: Aedes; adenylate cyclase; apical membrane; arthropod borne communicable disease; basolateral membrane; biological fluid transport; body fluids; communicable disease control; confocal scanning microscopy; cyclic AMP; disease vectors; fluorescent dye /probe; growth /development; hydrogen ions; immunocytochemistry; ion transport; membrane transport proteins; nutrient intake activity; nutrition related tag; polymerase chain reaction; protein kinase A; radioimmunoassay; second messengers; sodium ion; transport inhibitor; western blottings

DESCRIPTION (provided by the applicant): Fluid balance in insects is dependent on ion and fluid secretion across Malpighian tubules. The rate of fluid secretion by Malpighian tubules is dependent on the activity of a Na/H exchanger and the generation of the second messenger cAMP by adenylate cyclase. Inhibition of the Na/H exchanger results in a decrease in fluid secretion, a decrease in intracellular pH and a decrease in the extrusion of acid from the Malpighian tubule cells. Inhibition of the cAMP response by blocking the activity of the catalytic subunit of protein kinase A also decreases fluid secretion. These findings of a role for the Na/H exchanger and protein kinase A in regulating fluid secretion has been determined only in adult non-blood fed mosquitoes to date. We have extended these studies to Malpighian tubules from larval, pupal and blood fed female and determined that throughout development and after a blood meal that fluid secretion is dependent on Na/H exchange and cAMP. Additionally, we have cloned and sequenced the cDNA coding for Na/H exchanger from the adult mosquito allowing us to determine the molecular biological and immunological expression of the Na/H exchanger. The goals for the proposed research include a physiological, pharmacological, biochemical and immunological dissection of the role-played by the Na/H exchanger and cAMP during development and after a blood meal. The proposed experiments will investigate the temporal and spatial expression of the Na/H exchanger in individual Malpighian tubules from 1st instar larvae to adults five days after emergence. The role of the Na/H exchanger up to 48 hours after a blood meal will also be determined. The temporal studies will determine the amount of Na/H exchange activity, the effects of NaJH exchanger inhibitors on exchange activity and the amount of mRNA and protein at each developmental stage and after blood feeding. The spatial studies of the Na/H exchanger will immunologically determine the membrane (apical versus basolateral) location of the Na/H exchanger using confocal microscopy. These studies will be repeated in the presence of cAMP and inhibitors of protein kinase A to determine the key role-played by adenylate cyclase in regulating fluid secretion during development and after a blood meal. The results from the experiments will determine the role played by the Na/H exchanger during development and after a blood meal in mosquitoes. This body of knowledge will lead to potential sites for inhibition of fluid secretion during development and blood feeding. The inhibition of fluid secretion will disturb fluid balance in the mosquito and thereby render the mosquito in capable of spreading disease.

描述（由申请人提供）：昆虫的流体平衡取决于 在马皮亚小管之间的离子和液体分泌上。流体速率 马尔皮亚小管的分泌取决于Na/H的活性 交换器和通过腺苷酸环化酶产生的第二信使训练营的产生。 Na/H交换器的抑制导致流体分泌减少，A 减少细胞内pH和酸挤压从 马尔皮亚小管细胞。通过阻止营地反应的抑制 蛋白激酶A的催化亚基的活性也降低了流体 分泌。这些对Na/H交换剂和蛋白激酶A的作用的发现 在调节流体分泌方面仅在成年非血液中确定 迄今为止的蚊子。我们已经将这些研究扩展到马尔皮亚小管 幼虫，pupal和血液喂养女性，并确定在整个发育过程中 在一顿血餐后，流体分泌取决于Na/h的交换和 营。此外，我们已经克隆并测序了Na/H的cDNA编码 与成年蚊子交换器，使我们能够确定分子 Na/H交换器的生物学和免疫学表达。 拟议研究的目标包括生理，药理， Na/H扮演角色的生化和免疫学解剖 在发育期间和血液餐后的交换器和营地。提议 实验将研究Na/H的时间和空间表达 从第一龄幼虫到成年人的Malpighian小管中的交换器 出现五天后。 NA/H交换器的作用长达48小时 还将确定血液餐。临时研究将确定 Na/H交换活性的量，NaJH交换器抑制剂对 在每种发育中的交换活性以及mRNA和蛋白质的量 阶段和血液进食后。 NA/H交换器的空间研究将 从免疫学上确定膜（顶部与基底外侧）位置 使用共聚焦显微镜的Na/H交换器。这些研究将在 蛋白激酶A的cAMP和抑制剂的存在以确定钥匙 由腺苷酸环化酶在调节流体分泌过程中的角色扮演 发育和血液饭后。 实验的结果将确定NA/H的作用 在发育期间和蚊子的血液饭后交换器。这个身体 知识将导致潜在的部位抑制开发过程中的流体分泌 和血液喂养。抑制流体分泌会干扰流体平衡 在蚊子中，从而使蚊子能够传播 疾病。