| Researchers have been studying osmoregulation in fishes over the past 150 years. The one area that had received little attention is the role of paracrines/autocrines in the local regulation of ion transport, specifically in the teleost gill. The focus of my dissertation work was on the peptide, endothelin-1, and the components necessary for EDN1 signaling, including the enzyme that makes active EDN1, ECE1, and the sites of action, the EDN receptors (EDNRS). When I started this work, there were about a dozen papers on the effects of endogenous mammalian EDN1 on fish blood vessel tone or the effects of bolus injections of mammalian EDN1 on the cardiovascular system of fishes. The purposes of my dissertation work were to (1) localize the aforementioned components of EDN1 in the teleost gill; (2) determine if these components are regulated by environmental salinity, thus giving us some insight into whether or not EDN1 is involved in regulation of ion balance in fishes; (3) determine the evolutionary relationship among the protein sequences for the EDNs, ECEs, and EDNRs.;Through the use of molecular biology and immunohistochemistry, I determined that EDN1, ECE1 and the EDNRs are expressed in the euryhaline killifish ( Fundulus heteroclitus) and longhorn sculpin (Myoxocephalus octodecemspinosus) gills. From my localization studies, I modeled the putative functions of EDN1, and proposed it acts as a paracrine and autocrine in the fish gill. The localization of the EDNRs in the gill suggests that EDN1 signaling is involved in regulation of mitochondrion-rich cell functions, regulation of lamellar pillar cell tone (and ultimately perfusion of lamellae), and clearance of excess EDN1.;With the sequences obtained from the first part of my dissertation, I was able to determine the effects of environmental salinity on mRNA expression of each of these EDN1 signaling components using real-time quantitative PCR in both of these fishes. I was also able to measure protein level differences in these experiments. These mRNAs/proteins are regulated by hyperosmotic and hypo-osmotic stress, further suggesting that they are involved in not only ion balance, but also volume regulation. In addition, EDN1 signaling is postulated to be involved in cell survival during osmotic stress.;Finally, through the use of phylogenetics and bioinformatics, I determined that EDN1 and the EDNRs are vertebrate specific proteins, supporting the working hypothesis that EDN1 signaling was an important innovation leading to the development of the jaws and radiation of the vertebrates. Although ECE is found in Archaea, Bacteria and Eukarya, it is hypothesized to have shifted from being a monomer and general protease in all non-vertebrate organisms, to a dimer and a specific EDN1 protease in the vertebrates. Finally, the EDNRB2 was originally classified as an avian-specific EDNR; however, it is found in all non-therian gnathostomes, and I believe deleted from the therian genome 150 mya. From my Rate Shift Analysis of the EDNRA and EDNRB1, I hypothesize that therian EDNRA have different functions than the EDNRA in non-therian gnathostomes, but that the EDNRB1 is well conserved over gnathostome evolution. |
