Transport properties of cultured branchial epithelia from freshwater rainbow trout: A novel preparation with mitochondria-rich cells

A new double-seeded insert (DSI) technique is described for culture of bran chial epithelial preparations from freshwater rainbow trout on filter suppo rts, DSI epithelia contain both pavement cells and mitochondria-rich (MR) c ells (15.7+/-2.5 % of total cell numbers). MR cells occur singly or in clus ters, are voluminous, open apically to the 'external environment' and exhib it ultrastructural characteristics similar to those found in the 'chloride cells' of freshwater fish gills, After 6-9 days in culture with Leibovitz's L-15 medium on both surfaces (symmetrical conditions), transepithelial res istance (TER) stabilized at values as high as 34 k Ohm cm(2), indicative of electrically 'tight' epithelia, The density of MR cells, the surface area of their clusters and transepithelial potential (TEP; up to +8mV basolatera l positive, mean +1.9+/-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 un der symmetrical conditions, Na+/K+-ATPase activities of DSI preparations we re comparable with those in gill filaments, but did not differ from those o f SSI epithelia, Replacement of the apical medium with fresh water to mimic the in vivo situation (asymmetrical conditions) induced a negative TEP (-6 to -15mV) and increased permeability to the paracellular marker PEG-4000, tinder symmetrical conditions, unidirectional Na+ and Cl- fluxes were in ba lance, 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 Cl- uptake and Na+ extrusion, Unidirectional Ca2+ f luxes were only 0.5-1.0 % of Na+ and Cl- fluxes; active net Ca2+ uptake occ urred under symmetrical conditions and active net extrusion under asymmetri cal conditions, Thus, DSI epithelia exhibit some of the features of the int act gill, but improvements in culture conditions are needed before the MR c ells will function as true freshwater 'chloride cells'.