?(Fig.6a/b).6a/b). (i.e. three to four independent enzyme determinations from the same tissue sample, respectively). are significantly different (test, represents the mean??standard deviation of at least three independent preparations. test for apparent permeation coefficients in L-15/ex vs. L-15/FBS medium revealed GDC-0980 (Apitolisib, RG7422) nonsignificant differences (are the average apparent permeation coefficients calculated for the permeable membrane alone (without cells) Responses of cells to different stimuli To explore the response of the intestinal cell layer to a physiological stimulus, i.e. increased salinity as would be expected during seawater acclimation, 21-day-old cultures were exposed to a freshwater (FW) or a saltwater (SW) buffer on the apical side for up to 72?h. Despite varying levels GDC-0980 (Apitolisib, RG7422) of NKA mRNA abundance in the two independent cell preparations, which we show side by side, a significant upregulation in response to SW was consistently observed after 24?h (Fig. ?(Fig.6a/b).6a/b). No impact on mRNA levels occurred upon exposure to FW (Fig. ?(Fig.6a).6a). However, the NKA mRNA induction by SW was transient: comparable or lower levels to L-15/FBS or to FW were found for SW (Fig. ?(Fig.6a/b)6a/b) after 72?h of exposure. Similarly, TEER values were increased transiently after 24?h, to return to control levels at 72?h. This effect, however, was significant in only one of the two experiments performed (Fig. ?(Fig.66c/d). To test the response of the cells to a Rabbit polyclonal to LRCH4 toxicant and evaluate the capacity of RTgutGC cells to process intracellular silver, we exposed the cells for 24?h to silver ions in the form of AgNO3 and compared the impact on cell viability to that observed in cells grown and exposed the conventional way in micro-well plates. Based on the effect concentrations causing a 50% reduction of cell viability (EC50 values based on nominally added AgNO3), cells grown on inserts were about eightfold less sensitive (Fig. ?(Fig.7a).7a). Quantification of the intracellular silver on exposure to a nontoxic concentration (400?nM AgNO3) revealed that the cells in the inserts contain about threefold less silver/milligram protein than cells exposed in micro-well plates (Fig. ?(Fig.77b). Open in a separate window Fig. 7 Toxicity GDC-0980 (Apitolisib, RG7422) and accumulation of silver after 24?h exposure to RTgutGC cells grown as monolayer either on solid support in conventional micro-well plates or on permeable membranes. a Cell viability upon exposure to silver as measured by Alamar Blue. Exposure was done on confluent monolayers obtained after 48?h of cell culture in 12-well plates (denotes a significant difference in silver accumulation based on test (p?0.05) Discussion When presenting the first characterization of the RTgutGC cell line, Kawano et al. (2011) proposed that this intestinal cell line may have the potential to become the fish equivalent of the Caco-2 cell line, which is intensively used as human model of pharmacokinetics of drugs and other chemicals (Sun et al. 2008). Our results are in support of this proposal; moreover, in contrast to the Caco-2 cell line, which was derived from a human colon carcinoma, RTgutGC cells were initiated from a healthy rainbow trout. Establishment of the RTgutGC cell line as a single monolayer on permeable insert membranes leads to formation of a functional epithelium that accommodates transepithelial electrical resistance (TEER) levels of up to 50???cm2 (this study; Geppert et GDC-0980 (Apitolisib, RG7422) al. 2016). According to the classification presented by Claude and Goodenough (1973), these values are representative of a leaky epithelium. Indeed, fish intestinal epithelia have mostly been classified as leaky where resistance is seen as a measure of paracellular permeability (Loretz 1995). For Atlantic salmon (Salmo salar) adapted to freshwater, TEER values between 30 and 150???cm2 have been reported (Sundell et al. 2003); thus, the RTgutGC cell line appears to closely reflect the in vivo transepithelial resistance in salmonids. The cell line was initiated from the distal portion of a rainbow trout intestine where TEER is generally found to be higher.