Malpighian tubule Na/H exchange during development

发育过程中马氏小管 Na/H 交换

• 批准号: 6616162
• 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 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6616162
• 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(申请人提供)：昆虫体内的液体平衡依赖于 马氏管上的离子和液体分泌。液体的速率 马氏管的分泌依赖于Na/H的活性 交换器和腺苷环化酶产生第二信使cAMP。 抑制Na/H交换导致液体分泌减少，a 细胞内pH的降低和从细胞中排出的酸的减少 马氏管细胞。阻断cAMP反应抑制cAMP 蛋白激酶A催化亚单位的活性也减少了液体 分泌物。这些发现与Na/H交换和蛋白激酶A的作用有关 在调节体液分泌中的作用仅在成人非血液喂养中被确定 到目前为止都是蚊子。我们已经将这些研究扩展到马氏管 幼虫、蛹和血液喂养雌性，并决定了整个发育过程 血餐后体液分泌依赖于Na/H交换和 夏令营。此外，我们还克隆了Na/H的编码基因，并进行了序列测定 来自成年蚊子的交换器，让我们可以确定分子 Na/H交换器的生物学和免疫学表达 这项拟议研究的目标包括生理学、药理学、 Na/H所起作用的生化和免疫学剖析 在发育过程中和在一顿血餐之后，交换和露营。建议数 实验将研究Na/H的时间和空间表达 1龄幼虫至成虫个体马氏管的交换器 出现后五天。直到48小时后钠/氢交换器的作用 血餐也将被确定。时间研究将确定 Na/H交换活性，NaJH交换抑制剂的影响 各发育阶段的交换活性及mRNA和蛋白质的含量 进血期和喂血后。Na/H交换器的空间研究将 免疫学确定膜(根尖与基底外侧)的位置 共聚焦显微镜下的Na/H交换器。这些研究将在 CAMP和蛋白激酶A抑制剂的存在决定了关键 腺苷环化酶在体液分泌调节中的作用 发展和一顿血餐之后。 实验结果将确定Na/H所起的作用 蚊子在发育过程中和吃完血后的交换器。这具身体 知识将导致在发育过程中抑制体液分泌的潜在部位 和吸血。体液分泌受阻会扰乱体液平衡 从而使蚊子能够传播 疾病。