Paracrine Control of Fish Gill Function

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The endothelial cells that line fish blood vessels secrete a suite of signaling molecules (termed paracrines) that control the tension in underlying vascular smooth muscle cells. Of these paracrines, endothelin (ET), nitric oxide (NO), and two prostanoids (PGI2 and PGE) are the best studied in fishes, as well as in mammals, where the same signaling systems are present. The fish gill has a complex vasculature that has been shown to play a vital role in regulating such important processes as gas exchange, ion and acid-base balance, and excretion of nitrogen. It has been demonstrated that ET can control the pattern of perfusion in the fish gill lamellae, suggesting that paracrines may play an important role in gill perfusion. It also has been shown that ET, NO, and PGI2/PGE can control the movement of ions across epithelial cells in mammals via interactions with specific transport proteins that mediate active transport. These proteins are also found in the fish gill epithelium and control the net extrusion of salt by various groups of marine fishes, including jawless fishes (lampreys and hagfish), sharks, and bony fishes. Preliminary studies in this laboratory have demonstrated that ET, NO, and PGE inhibit salt transport across the killifish opercular skin, a standard model for the salt-transporting epithelium of the marine fish gill. Thus, the principal investigator hypothesizes that ET, NO, and PGI2/PGE play a fundamental role in the control of fish gill function by regulating gill perfusion and ion transport. To test this hypothesis, an integrated series of physiological, pharmacological, and molecular techniques will be used in four species of fishes, representing the major fish groups. The objective is to describe the evolution of these gill paracrine control systems in the early vertebrates. Specifically, recordings will be made of blood flow changes in the gill during infusion of these paracrines in cannulated fishes, using videomicroscopy, and tests of their effects on gill ion transport will be performed, using the killifish opercular skin as a model system. In conjunction, the principal investigator will determine if the gill tissue itself generates ET, NO, and PGI2/PGE by chemical and immunoassays and localize the cellular sites of synthesis by immunohistochemical (light and confocal microscopy) techniques. Immunological (Western blot) and pharmacological techniques (radio-receptor analyses) will be used to confirm the presence of and to characterize the receptors for ET and PGI2/PGE that are expressed in the gill. Localization of ET and PGI2/PGE receptors in specific gill cells will be accomplished using immunohistochemical techniques. To provide protein sequence for future production of homologous antibodies and information on the evolution of the specific gene structure, the genes encoding fish nitric oxide synthase and ET as well as receptors for ET, PGI2, and PGE will be cloned in collaboration with the Molecular Services Core of the University of Florida's Interdisciplinary Center for Biotechnology Research. This integrated approach will provide clear evidence for the presence, evolution, and roles of these paracrine signaling pathways in fish gill function.