Ion transfer and control of pHi in pseudobranch cells

Support is requested to analyze the mechanisms of ion regulation and of pH regulation in teleost pseudobranch cells. Although the pseudobranch organ has attracted attention for more than a century, its physiological function remains a mystery. A recent hypothesis suggests that the pseudobranch may titrate the blood pH down to a level just above the threshold for the onset of the Root effect and thus, in cooperation with the choroid rete mirabile of the fish eye, facilitate oxygen supply to the retina. The present study therefore is designed to provide insight into the mechanisms of ion transfer through the membrane of pseudobranch cells. Especially the mechanisms of acid secretion and of pH regulation are of interest, because the ability of cells to adjust extracellular pH necessarily includes a sophisticated system for the control of acid release and intracellular pH. Based on our experience with cultured swimbladder gas gland cells, pseudobranch cells will be isolated and cultured. The rate of acid release in the face of changing extracellular pH levels will be analyzed, and pharmacological studies will elucidate which ion transport mechanisms (Na+/H+-exchange; V-type ATPase; anion exchange) are involved. Taking the functional and morphological polarity of pseudobranch cells into account, immunohistochemical studies to localize the transporters will be performed. The results will reveal whether pseudobranch cells are able to sense extracellular pH and to adjust it to a certain level as currently hypothesized. Combined with results on the mechanisms of acid secretion established for swimbladder gas gland cells in a previous project, the results will provide insight into the mechanisms of pH regulation in cells specialized for the production and the transport of acidic metabolites.

The pseudobranch is a gill-like hemibranch located within the subopercular cavity, and attached to the operculum of many teleost fishes. As indicated by the term hemibranch, the gill arch supports only one row of filaments, which carry a series of gill lamellae like the filaments of the regular gill arches. Although this structure has attracted attention for a long time, its physiological function remains a mystery. A recent hypothesis suggests that the pseudobranch may acidify the blood pH down to a level just above the threshold for the onset of the Root effect and thus, in cooperation with the choroid rete mirabile of the fish eye, facilitate oxygen supply to the retina. This study therefore was designed to develop a cell culture model for pseudobranch cells, and to provide insight into the mechanisms of ion transfer through the membrane of pseudobranch cells. After dissection pseudobranch cells were isolated either by enzymatic digestion or by homogenization in Ca2+-free medium. Microscopical inspection and fluorescence microscopical analysis revealed that the most stable preparation of pseudobranch cells was achieved by mechanical maceration of the tissue through a 70 m nylon cell strainer in Ca2+-free medium. Microscopical characterization of the cells revealed the typical features of pseudobranch cells, a large number of mitochondria, an extensive system of membrane invaginations (tubular system) and the presence of Na+ /K+ - ATPase. These cells could be kept in culture for a couple of days, but no cell differentiation or cell proliferation was observed. To confirm that these cells are indeed epithelial cells cytokeratin was used as an epithelial marker. The most intensive fluorescence signal was observed in small, spheroid cells of rather undifferentiated character. Presence of cytokeratin was also established in an intermediate cell type, which showed the typical mitochondrial arrangement of pseudobranch cells, but no tubular system, while in fully differentiated pseudobranch cells almost no cytokeratin staining was observed. These observations suggest that pseudobranch cells during differentiation abandon the expression of epithelial cytokeratin. Physiological experiments revealed that these cultured pseudobranch cells represent a very useful model for the analysis of the physiological functioning of these cells. Mechanisms of acid secretion were assessed by measuring the rate of acid secretion of cultured cells. Pharmacological studies revealed the presence of various pathways of acid secretion, e.g. Na+ /H+ -exchange, anion exchange and a V-ATPase. In addition, the production of CO2 in the aerobic metabolism and the diffusion of CO 2 also appear to contribute to the transfer of acid through cell membranes. A comparison with cultured gill pavement cells revealed distinct differences with respect to the characteristics of the anion exchange protein and the involvement of V-ATPase in acid secretion. In pseudobranch cells V-ATPase is involved in acid-base regulation under isosmotic conditions, while in gill cells this is not the case. In evolutionary terms the differentiation of the pseudobranch of the holobranchs therefore produced morphologically as well as physiologically different organs. With respect to the possible function of the pseudobranch the results show that the rate of acid secretion of pseudobranch cells is higher than in gill epithelial cells, and this would be in line with the idea that in the pseudobranch the blood is "preconditioned" by acidification in order to reduce the amount of acid that must be secreted by retina cells in order to switch on the Root effect.