Regulation of Epithelia Solute Transport in Elasmobranch Fish

板鳃鱼上皮细胞溶质运输的调节

• 批准号: RGPIN-2015-05348
• 负责人: Anderson, WGary
• 金额: $ 2.4万
• 依托单位: University of Manitoba
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=613499
• 关键词: Regulation; Epithelia; Solute; Transport; Elasmobranch

The retention of urea for osmotic purposes in elasmobranch fish has been known for over 125 years yet we know little about solute transport across epithelia in this ancient group of fishes. Attempts have been made to understand urea transport mechanisms using gill and renal tissue, however, these studies are hampered by the complexity of the tissue used. Intestinal tissue lends itself well to transport physiology studies as it has a relatively flat surface and uniform epithelia. Recent advances have been made in establishing a viable preparation in elasmobranchs and data have consistently shown that feeding results in a net uptake of urea from the lumen of the intestine against a concentration gradient, and starvation leads to a net loss of urea to the intestinal lumen. These studies suggest significant repositioning of transport proteins in intestinal epithelia and/or remodeling of intestinal epithelia cell morphology in response to the presence of food. Either way such physiological responses would require substantial coordination through the actions of gastro-entero-pancreatic (GEP) hormones, many of which have been identified in the intestine of elasmobranch fish. While a number of studies have examined regulatory roles for these hormones in blood flow and smooth muscle contraction research investigating their role in the regulation of intestinal solute transport in elasmobranch fish is non-existent. Using isolated intestinal preparations, the proposed research will establish this tissue as a primary resource for understanding the mechanisms of solute transport in elasmobranchs. Using a classic Ussing chamber setup selective screening of specific inhibitors and agonists will identify key transport proteins involved in the movement of urea and other solutes across the intestinal epithelia. Molecular techniques will quantify mRNA expression of these proteins in fed and starved fish. These studies combined with immunohistochemical techniques will result in the development of a urea and solute transport model for elasmobranchs based on direct empirical evidence from a relatively homogeneous epithelia. Light, scanning and transmission microscopy will be used to identify morphological changes in the intestinal epithelia in response to feeding. The Ussing chamber preparation will be used to screen candidate GEP hormones for the control of urea and solute transport. Once identified, receptor expression for inhibitory and stimulatory hormones will be examined. Similarly a pancreatic in situ perfusion preparation will be established to determine if the hormones regulating solute movement across the intestine are also involved in regulating pancreatic exocrine and possibly endocrine activity. The overall goal of this proposed research program is to describe solute transport mechanisms in the elasmobranch intestine and ultimately functional evolution of vertebrate GEP hormones.

尿素在棘足目鱼类体内的渗透作用早在125年前就已为人所知，但我们对这类古老鱼类中的溶质跨上皮细胞的运输知之甚少。人们试图利用鳃和肾组织来了解尿素的转运机制，然而，这些研究因所用组织的复杂性而受到阻碍。肠道组织具有相对平坦的表面和均匀的上皮，非常适合运输生理学研究。最近在建立一种可行的制剂方面取得了进展，数据一直表明，喂食导致相对于浓度梯度的尿素从肠腔净吸收，饥饿导致尿素净损失到肠腔。这些研究表明，运输蛋白在肠道上皮细胞中的显著重新定位和/或肠道上皮细胞形态的重塑对食物的存在做出了反应。无论是哪种方式，这种生理反应都需要通过胃肠胰腺(GEP)激素的作用进行实质性的协调，其中许多激素已在棘鱼的肠道中被发现。虽然许多研究已经研究了这些激素在血流和平滑肌收缩中的调节作用，但还没有研究它们在板足鱼类肠道溶质运输调节中的作用。利用分离的肠道准备，这项拟议的研究将建立该组织作为了解弹簧肌中溶质运输机制的主要资源。使用经典的Ussing小室装置，选择性地筛选特定的抑制剂和激动剂，将识别涉及尿素和其他溶质在肠道上皮细胞中移动的关键运输蛋白。分子技术将量化这些蛋白质在喂食和饥饿的鱼中的mRNA表达。这些研究与免疫组织化学技术相结合，将基于来自相对均一的上皮细胞的直接经验证据，开发出弹肌的尿素和溶质运输模型。光学显微镜、扫描显微镜和透射显微镜将被用来识别肠道上皮细胞在摄食后的形态变化。Ussing小室制剂将用于筛选控制尿素和溶质运输的GEP候选激素。一旦确定，将检查抑制和刺激激素的受体表达。同样，将建立胰腺原位灌流制剂，以确定调节肠道内溶质运动的激素是否也参与调节胰腺外分泌和可能的内分泌活动。这项拟议的研究计划的总体目标是描述脊椎动物肠道中的溶质运输机制，并最终描述脊椎动物GEP激素的功能进化。