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

Mary Fletcher, Scott P. Kelly, Peter Pärt, Michael J. O'Donnell, Chris M. Wood

Research output: Contribution to journalArticle

68 Citations (Scopus)

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 ± 2.5% of total cell numbers). MR cells occur singly or in clusters, are voluminous, open apically to the 'external environment' and exhibit 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 resistance (TER) stabilized at values as high as 34 kΩ cm2, 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 ± 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 vivo 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 Cl- 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 Cl- uptake and Na+ extrusion. Unidirectional Ca2+ fluxes were only 0.5-1.0% of Na+ and Cl- fluxes; active net Ca2+ uptake occurred under symmetrical conditions and active net extrusion under asymmetrical conditions. Thus, DSI epithelia exhibit some of the features of the intact gill, but improvements in culture conditions are needed before the MR cells will function as true freshwater 'chloride cells'.

Original languageEnglish
Pages (from-to)1523-1537
Number of pages15
JournalJournal of Experimental Biology
Volume203
Issue number10
StatePublished - Jun 27 2000

Fingerprint

Oncorhynchus mykiss
transparent exopolymer particle
mitochondrion
Fresh Water
rainbow
Mitochondria
mitochondria
epithelium
Epithelium
extrusion
pavement
chloride
cells
gills
Chlorides
surface area
replacement
chlorides
permeability
filter

Keywords

  • Cell culture
  • Chloride cell
  • Filter insert
  • Gill
  • Mitochondria-rich cell
  • Na/K-ATPase activity, Ca transport
  • Pavement cell
  • Transepithelial potential
  • Transepithelial resistance

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Agricultural and Biological Sciences (miscellaneous)

Cite this

Transport properties of cultured branchial epithelia from freshwater rainbow trout : A novel preparation with mitochondria-rich cells. / Fletcher, Mary; Kelly, Scott P.; Pärt, Peter; O'Donnell, Michael J.; Wood, Chris M.

In: Journal of Experimental Biology, Vol. 203, No. 10, 27.06.2000, p. 1523-1537.

Research output: Contribution to journalArticle

Fletcher, Mary ; Kelly, Scott P. ; Pärt, Peter ; O'Donnell, Michael J. ; Wood, Chris M. / Transport properties of cultured branchial epithelia from freshwater rainbow trout : A novel preparation with mitochondria-rich cells. In: Journal of Experimental Biology. 2000 ; Vol. 203, No. 10. pp. 1523-1537.
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AU - Wood, Chris M.

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N2 - 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 ± 2.5% of total cell numbers). MR cells occur singly or in clusters, are voluminous, open apically to the 'external environment' and exhibit 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 resistance (TER) stabilized at values as high as 34 kΩ cm2, 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 ± 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 vivo 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 Cl- 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 Cl- uptake and Na+ extrusion. Unidirectional Ca2+ fluxes were only 0.5-1.0% of Na+ and Cl- fluxes; active net Ca2+ uptake occurred under symmetrical conditions and active net extrusion under asymmetrical conditions. Thus, DSI epithelia exhibit some of the features of the intact gill, but improvements in culture conditions are needed before the MR cells will function as true freshwater 'chloride cells'.

AB - 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 ± 2.5% of total cell numbers). MR cells occur singly or in clusters, are voluminous, open apically to the 'external environment' and exhibit 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 resistance (TER) stabilized at values as high as 34 kΩ cm2, 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 ± 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 vivo 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 Cl- 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 Cl- uptake and Na+ extrusion. Unidirectional Ca2+ fluxes were only 0.5-1.0% of Na+ and Cl- fluxes; active net Ca2+ uptake occurred under symmetrical conditions and active net extrusion under asymmetrical conditions. Thus, DSI epithelia exhibit some of the features of the intact gill, but improvements in culture conditions are needed before the MR cells will function as true freshwater 'chloride cells'.

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