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）：昆虫体内的液体平衡取决于 对离子和液体分泌的影响流体的速度 马氏管的分泌依赖于Na/H的活性， 交换和腺苷酸环化酶产生第二信使cAMP。 Na/H交换器的抑制导致液体分泌减少， 细胞内pH值的降低和酸从细胞内排出的减少， 马氏管细胞。通过阻断cAMP反应， 蛋白激酶A的催化亚基的活性也减少了流体 分泌物这些发现表明Na/H交换蛋白和蛋白激酶A 调节液体分泌的作用仅在非血喂养的成年人中得到确定 蚊子至今我们已经将这些研究扩展到马氏管， 幼虫、蛹和吸血雌性，并确定在整个发育过程中 在血液餐后，液体分泌依赖于Na/H交换， 营此外，我们还克隆了Na/H基因的cDNA序列， 使我们能够确定 Na/H交换器的生物和免疫表达。 拟议研究的目标包括生理学，药理学， Na/H所扮演角色的生化和免疫学剖析 交换器和cAMP在发展过程中和血餐后。拟议 实验将研究Na/H的时间和空间表达 1龄幼虫至成虫马氏管中的交换器 出现后5天。Na/H交换剂的作用长达48小时后， 还将确定血餐。时间研究将确定 Na/H交换活性的量，NaJH交换抑制剂对Na/H交换活性的影响， 各发育时期的蛋白质和mRNA含量 阶段和吸血后。钠/氢交换剂的空间研究将 免疫学确定膜（顶侧与基底侧）的位置， Na/H交换器的共聚焦显微镜。这些研究将在 cAMP和蛋白激酶A抑制剂的存在，以确定关键 腺苷酸环化酶在调节体液分泌中的作用 发展和血餐后。 实验结果将确定Na/H 蚊子在发育过程中和吸血后会产生一种交换器。这个身体 知识将导致在发育过程中抑制液体分泌的潜在位点 和吸血抑制体液分泌会扰乱体液平衡 从而使蚊子能够传播 疾病