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

University Of California-San Diego Scripps Inst Of Oceanography, La Jolla CA

Investigators

Abstract

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.