Acid/base sensing and regulation of multiple physiological processes in fish

所有生物体都必须能够感知和调节其体液中的pH值水平，以避免细胞故障，这种故障可能导致疾病，在极端情况下，甚至死亡。先前的工作发现鲨鱼和鱼的鳃中的一种可溶性酶是一种新型的分子传感器和血液高pH值调节器。目前的项目将探索这种可溶性酶在感知和调节鲨鱼鳃低血液酸碱度方面的假定作用，并将这些研究扩展到其他水生脊椎动物，硬骨鱼的鳃。此外，它还将探索鲨鱼和硬骨鱼的红细胞和心肌细胞的新功能。由于这两种细胞受pH的影响很大，这种可溶性酶可能在红细胞摄取和输送氧气以及心肌细胞收缩和心率等基本生理过程中发挥重要作用。该项目将开发生化、细胞和生理学工具，其中许多工具随后可能被修改以在其他生物中测试类似的假说。这些信息将有助于理解和预测生物如何应对环境和新陈代谢压力，并为在不断变化的海洋条件下管理野生和养殖种群提供信息。该项目将为本科生、研究生和博士后研究人员提供研究培训机会，并与当地的儿童和青少年海洋科学夏令营SeaCamp San Diego合作，为K-12学生开发现场和在线教育推广活动。酶溶腺苷酸环酶(SAC)可产生无处不在的信使分子环AMP，以响应二氧化碳、pH和HCO3-水平的变化。自1999年发现以来，SAC已被发现在从珊瑚到哺乳动物的多种动物门中调节不同的生理过程，因此被认为是进化上保守的酸/碱传感器。这项拟议的研究将：(1)继续对弹性鳃细胞进行研究，以阐明SAC是否调节酸的分泌；(2)对硬骨鱼(虹鱼)的SAC进行初步的分子和生化表征；(3)探索鲑鱼中存在多种SAC剪接变异体，以及它们可能存在的差异亚细胞定位；(4)表征鲑鱼鳃细胞中SAC在感知和调节血液酸碱状态方面的可能作用；(5)研究SAC在调节鲨鱼和鲑鱼红细胞的氧结合方面的潜在作用；(6)探讨SAC在心肌细胞中的作用，假设SAC调节心肌细胞的收缩。为了实现这些目标，将使用各种实验方法，包括原代细胞培养、培养的鲑鱼成纤维细胞中GFP标记的蛋白的表达、细胞内pH和钙的测量、红细胞氧结合曲线、肌节缩短和免疫细胞化学。鉴于酸/碱状态和cAMP对鳃细胞、红细胞和心肌细胞生理学的重要性，这项研究有可能发现新的SAC依赖机制，调节鱼类必不可少的生理功能，并有可能推广到其他动物。对硬骨鱼和硬骨鱼的同时研究增加了比较和进化成分。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

All organisms must be able to sense and regulate the pH level in their fluids to avoid cell malfunction that can lead to sickness and, in extreme cases, death. Prior work identified the a soluble enzyme in gills from sharks and rays as a novel molecular sensor and regulator of high blood pH. The current project will explore the putative role of this soluble enzyme in sensing and regulating low blood pH in shark gills, and will expand those studies to gills of other aquatic vertebrates, bony fishes. In addition, it will explore novel functions in red blood cells and heart muscle cells from both shark and bony fishes. Because those two cell types are greatly affected by pH, this soluble enzyme may play important roles in essential physiological processes such as oxygen uptake and delivery by red blood cells, and in contraction and heart beat rate by heart muscle cells. This project will develop biochemical, cellular, and physiological tools, many of which could be subsequently adapted to test similar hypotheses in other organisms. This information will help understand and predict how organisms respond to environmental and metabolic stress, as well as informing the management of wild and farmed populations in changing ocean conditions. This project will provide research training opportunities for undergraduate and graduate students and postdoctoral researchers and develop onsite and online educational outreach activities for K-12 students in collaboration with SeaCamp San Diego, a local marine science camp for kids and teenagers.The enzyme soluble adenylyl cyclase (sAC) produces the ubiquitous messenger molecule cyclic AMP in response to changes in CO2, pH and HCO3- levels. Since its discovery in 1999, sAC has been found to regulate diverse physiological processes in multiple animal phyla ranging from coral to mammals, and is therefore deemed an evolutionarily conserved acid/base sensor. The proposed research will: (1) continue research on elasmobranch gill cells to elucidate if sAC regulates acid secretion; (2) perform the initial molecular and biochemical characterization of sAC in a teleost fish (the rainbow trout); (3) explore the presence of multiple sAC splice variants in trout, and their putative differential subcellular localization; (4) characterize the putative role of sAC in trout gill cells in sensing and regulating blood acid/base status; (5) study a potential role of sAC in regulating oxygen binding in erythrocytes from shark and trout; (6) explore the role of sAC in cardiomyocytes, where it is hypothesized to regulate contractibility. To achieve those aims, a variety of experimental methods will be used including primary cell cultures, expression of GFP-tagged proteins in cultured trout fibroblasts, intracellular pH and Ca2+ measurements, erythrocyte oxygen-binding curves, sarcomere shortening, and immunocytochemistry. Given the established importance of acid/base status and cAMP on the physiology of gill cells, erythrocytes and cardiomyocytes, this research has the potential to find novel sAC-dependent mechanisms that regulate physiological functions essential to fish, and which potentially extend to other animals. The simultaneous study of elasmobranch and teleost adds a comparative and evolutionary component.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.