Control of transport by epithelia

上皮细胞对运输的控制

• 批准号: RGPIN-2017-05196
• 负责人: Marshall, William
• 金额: $ 2.04万
• 依托单位: St. Francis Xavier University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=745802
• 关键词: Control; transport; epithelia

We study mechanisms that animals use to regulate ion and water transport across epithelia. The main model system we use is a hardy teleost fish that frequents estuaries of N. America and is robust to environmental extremes (salinity, temperature, low oxygen, pollution), the mummichog (Fundulus heteroclitus), an important physiological and genomic model species and important as a bioindicator species. The models of the gill osmoregulatory function we use are flat epithelia rich in salt transporting ionocytes, the opercular epithelium in adults and the yolk sac epithelium of embryos. The research focuses on revealing regulatory proteins (kinases and phosphatases) that modulate the transport proteins directly or via kinase cascades. Aim 1. We will discover the regulatory components of the transport complexes that control the anion channel Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and the cotransporters Na,K,2Cl cotransporter (NKCC1) and NaCl cotransporter (NCC) which include many kinases as well as a calcium-dependent inhibitory pathway. These pathways are activated by osmotic stimuli, neurotransmitters and hormones. The overarching hypothesis is that membrane transport proteins have multiple regulatory components in apparently redundant pathways that regulate ion transport, but in hardy animals such as Fundulus and less hardy tropical fish (Nile tilapia,Oreochromis nilotica), the extreme variation in the environment determines which pathways are best-suited for operation in extreme environments. Aim 2. We will overlay the above with cellular dynamics of this highly regulated transport system using simple salinity changes and immunocytochemistry of transporter proteins to generate cellular succession vs. remodeling of existing ionocytes and their various phenotypic variations in freshwater, seawater and hypersaline conditions. Aim 3. We will use the yolk sac membrane of Fundulus embryos (also salt hardy and rich in ionocytes) to test regulatory pathways using short interfering mRNA (siRNA) injection to block specific kinase/phosphatase genes (confirmed by quantitative PCR) in the yolk sac and see how transport regulation (hypotonic, hypertonic shock, adrenergic agonists) are selectively disrupted to identify the critical components of the regulatory pathways. Importance: Understanding of ion transport and its regulation also informs fisheries and aquaculture and advances comparative physiology. The research will reveal new relationships among regulatory kinases controlling ion transport proteins also associated with disease, particularly CFTR (responsible for cystic fibrosis), NKCC (implicated in pseudohypoaldosteronism) and NCC (involved in Gitelman's syndrome). The work will contribute 19 HQP (BSc and MSc) to Canadian research.

我们研究了动物通过上皮调节离子和水运输的机制。我们使用的主要模型系统是一种耐寒硬骨鱼，它经常出现在北美洲的河口，对极端环境（盐度、温度、低氧、污染）有很强的抵抗力，mummichog （Fundulus heteroclitus）是一种重要的生理和基因组模型物种，也是一种重要的生物指示物种。我们使用的鳃渗透调节功能的模型是富含盐转运离子细胞的扁平上皮、成人的眼上皮和胚胎的卵黄囊上皮。研究的重点是揭示直接或通过激酶级联调节转运蛋白的调节蛋白（激酶和磷酸酶）。目的1。我们将发现控制阴离子通道囊性纤维化跨膜传导调节剂（CFTR）和共转运体Na，K，2Cl共转运体（NKCC1）和NaCl共转运体（NCC）的转运复合物的调控成分，其中包括许多激酶以及钙依赖性抑制途径。这些通路被渗透刺激、神经递质和激素激活。最重要的假设是，膜转运蛋白在调节离子运输的明显冗余途径中具有多种调节成分，但在耐寒动物（如底底鱼）和不那么耐寒的热带鱼（尼罗罗非鱼、尼罗河罗非鱼）中，环境的极端变化决定了哪种途径最适合在极端环境中运作。目标2。我们将利用简单的盐度变化和转运蛋白的免疫细胞化学，在淡水、海水和高盐条件下，对这种高度调节的转运系统进行细胞动力学研究，以产生细胞演替与重构，以及现有离子细胞的各种表型变化。目标3。我们将使用眼底胚胎的卵黄囊膜（也耐盐且富含离子细胞）来测试调节途径，使用短干扰mRNA （siRNA）注射来阻断卵黄囊中的特定激酶/磷酸酶基因（通过定量PCR证实），并观察运输调节（低渗、高渗休克、肾上腺素能激动剂）是如何被选择性破坏的，以确定调节途径的关键成分。重要性：对离子转运及其调控的理解也为渔业和水产养殖提供了信息，并促进了比较生理学的发展。该研究将揭示与疾病相关的控制离子转运蛋白的调节激酶之间的新关系，特别是CFTR（负责囊性纤维化），NKCC（涉及假性低醛固酮增多症）和NCC（涉及吉特曼综合征）。这项工作将为加拿大的研究贡献19个HQP（理学士和理学硕士）。