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|Title:||Electrophysiological and Ion Transport Characteristics of Cultured Branchial Epithelia from Freshwater Rainbow Trout|
|Other Titles:||Studies on Cultured Freshwater Branchial Epithelia|
|Advisor:||O'Donnell, M. J.|
Wood, C. M.
|Keywords:||ion;transport;rainbow trout;trout;electrophysiology;branchial epithelia|
|Abstract:||Electrophysiological, morphological, and ion transport characteristics were examined in two cultured preparations of freshwater rainbow trout gills on permeable supports. The mitochondrial stain Rhodamine 123 (R123) revealed that single-seeded insert (SSI) preparations contained only pavement cells, and new double-seeded insert (DSI) preparations contained both pavement cells and mitochondrial-rich cells (MRCs). A series of physiological comparisons were made between the two preparations. Both preparations showed increases in transepithelial resistance (TER) with time in culture, reaching stable values (1500-25000 ohms.cm²) after 5-7 days in culture. Different transepithelial potentials (TEP) were observed in the two preparations with culture media (Leibowitz L15 supplemented with 5% foetal bovine serum (FBS)) on both surfaces of cultured preparations (symmetrical conditions). The TEP of SSI membranes prepared in Hamilton, Canada was negligible. The TEP of SSI membranes prepared in Uppsala, Sweden was 2.24 mV (serosal negative) and sensitive to vanadate and iodoacetate/KCN. DSI membranes under symmetrical conditions had mean TEP values of 1.87 mV (serosal positive). Replacing apical culture media with freshwater (asymmetrical conditions) mimicked the 𝘪𝘯 𝘷𝘪𝘷𝘰 environment of gills. Under these conditions TER increased and TEP was serosal negative in all membranes. TER recovered to initial values after culture media was returned to the apical side. Replacing culture media on the basolateral side with freshwater (opposite to 𝘪𝘯 𝘷𝘪𝘷𝘰 conditions) resulted in decreased TER which was irreversible and consistent with permanent damage. Taken together, TER measurements with freshwater on the apical side or basolateral side indicate the cultured cells of SSI and DSI membranes were polarized as 𝘪𝘯 𝘷𝘪𝘷𝘰. PEG-4000 fluxes were performed to assess paracellular permeability across DSI membranes. Results show that with freshwater on the apical side, paracellular permeability increased while conductance decreased in comparison to symmetrical conditions. In contrast, apical seawater exposure produced "leaky" DSI membranes with high paracellular permeability and high conductance. Na⁺K⁺ -ATPase activity of cells in primary culture (flasks) and on SSI membranes was retained (approximately 3uM PO₄²⁻/mgprotein/h) throughout culture conditions. Unidirectional Na⁺ and Cl⁻ fluxes in DSI membranes revealed negligible net fluxes under symmetrical conditions but large effluxes and small influxes under asymmetrical conditions, similar to previous results with SSI membranes. Ion transport was mainly conductive (indicated by linear relationships between conductance and fluxes) in DSI preparations. The Ussing flux criteria indicated active Na⁺ uptake and passive chloride movements in symmetrical conditions, and active Cl⁻ uptake and passive Na⁺ movements in asymmetrical conditions. SSI membranes also demonstrated active Cl⁻ uptake, however DSI membranes were the first 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 preparation of the gill to demonstrate active Na⁺ transport. This finding suggests MRCs may be involved in Na⁺ transport, and contradicts current models which suggest that pavement cells are the site of Na⁺ uptake. Unidirectional calcium fluxes revealed active Ca²⁺ transport in DSI branchial epithelia under both symmetrical and asymmetrical conditions, and no active Ca²⁺ transport in SSI epithelia. These results reinforce theories of calcium transport across chloride cells in the branchial epithelia.|
|Appears in Collections:||Digitized Open Access Dissertations and Theses|
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