Response of developing cultured freshwater gill epithelia to gradual apical media dilution and hormone supplementation

Abstract

We investigated gradual dilution of the apical medium (Leibovitz's L15 to fresh water [FW], analogous to gradual reduction in environmental salinity) and basolateral hormone support on the electrophysiological and ion-transporting properties of "developing" FW trout gill epithelia cultured on filter inserts. Epithelia were of the double-seeded type, containing both pavement cells and mitochondria-rich cells. In these experiments we were able to circumvent "symmetrical development" (typically L15 apical/L15 basolateral for 6-9 days) by commencing dilution of apical media (unchanged L15 basolateral, i.e., asymmetrical conditions) at culture-day 3, the time when transepithelial resistance (TER) and potential (TEP) would normally be increasing rapidly under symmetrical conditions. In Series 1 (without basolateral hormone support), epithelia were exposed to progressively diluted apical media (100%, 75%, 50% L15) at 24-hr intervals, thereafter cultured in 50% L15 apical media for 4 days, and then in apical FW. In Series 2, epithelia were exposed to progressively diluted apical media (100%, 75%, 50%, 25%, 12.5% L15, and FW) at 24-hr intervals with physiologically relevant doses of cortisol (500 ng ml(-1)), prolactin (50 ng ml(-1)), or cortisol + prolactin (500 ng ml(-1) + 50 ng ml(-1), respectively) added to basolateral media (100% L15). In Series 1, TER reached a plateau phase over 25 kohms cm2 under 50% L15/L15 culture conditions (after 4 days of culture) but fell to approximately 6 kohms cm2 after 24 hr in FW/L15 conditions. In Series 2, TER stabilized at 4-11 kohms cm2 depending on treatment. In general, apical media dilution during epithelial development was well tolerated. Preparations exhibited continued integrity right down to apical FW, indicated by only modest increases in net ion losses (i.e., basolateral to apical movement of ions), relatively stable TER values, and the expected changeover from positive to negative TEP in FW. Cortisol was clearly beneficial to FW adaptation, promoting greater TER, reduced unidirectional and net Na+ and Cl- flux rates, and elevated Na+, K+ -ATPase activity. Prolactin also offered some support, where its actions on TER were less than but additive to those of cortisol. There was no direct evidence that prolactin limited ion movements during gradual dilution. These in vitro studies demonstrate that "developing epithelia" were able to tolerate gradual dilution of apical media, the remarkable barrier properties of gill epithelia, and the importance of cortisol and prolactin in promoting integrity of this barrier during FW adaptation.