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Abstract

A new double-seeded insert (DSI) technique is described for culture of branchial epithelial preparations from freshwater rainbow trout on filter supports. DSI epithelia contain both pavement cells and mitochondria-rich (MR) cells (15.7 (+ or -) 2.5% of total cell numbers). MR cells occur singly or in clusters, are voluminous, open apical to the 'external environment' and exhibit ultra-structural characteristics similar to those found in the 'chloride cells' of fresh water fish gills.

After 6-9 days in culture with Leibovitz's L-15 medium on both surfaces (symmetrical conditions), transepithelial resistance (TER) stabilized at values as high as 34 kOhms cm{2}, indicative of electrically 'tight' epithelia. The density of MR cells, the surface area of their clusters and transepithelial potential (TEP: up to +8 mV basolateral positive, mean +1.9 (+ or -) 0.2 mV) were all positively correlated with TER.

In contrast, preparations cultured using an earlier single-seeded insert (SSI) technique contained only pavement cells and exhibited a negligible TEP under symmetrical conditions. Na{+}/K{+} -ATPase activities of DSI preparations were comparable with those in gill filaments, but did not differ from those of SSI epithelia.

Replacement of the apical medium with fresh water to mimic the in viva situation (asymmetrical conditions) induced a negative TEP (-6 to -15 mV) and increased permeability to the paracellular marker PEG- 4000. Under symmetrical conditions, unidirectional Na+ and CI- fluxes were in balance, and there was no active transport by the Ussing flux ratio criterion. Under asymmetrical conditions, there were large effluxes, small influxes and evidence for active CI{-} uptake and Na{+} extrusion. Unidirectional Ca{2+} fluxes were only 0.5-1.0 % of Na{+} and CI{-} fluxes; active net Ca{2+} uptake occurred under symmetrical conditions and active net extrusion under asymmetrical conditions. Thus, DSI epithelia exhibit some of the features of the intact gill

Additional information

Authors: FLETCHER M, Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario, L8S 4K1 (CA);KELLY S, Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario, L8S 4K1 (CA);O'DONNELL M, Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario, L8S 4K1 (CA);WOOD C, Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario, L8S 4K1 (CA);PART P, Department of Environmental Toxicology, Uppsala University, Norbyvagen 18A, S-752 36 Uppsala (SE)
Bibliographic Reference: Article: The Journal of Experimental Biology 203, (2000) pp. 1523-1537
Record Number: 200013042 / Last updated on: 2001-01-24
Category: PUBLICATION
Original language: en
Available languages: en