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    • Next we investigated the accumulation of H

    2021-10-13

    Next, we investigated the accumulation of [3H]-ribavirin in fresh villous fragments of human placenta, which provide a physiologically relevant, well-described and validated model for assessing drug interactions with ENTs [32], [35], [69], [70]. The uptake of [3H]-ribavirin was time-dependent and significantly sensitive to treatment with 0.1 µM and 100 µM NBMPR, but not Na+ depletion alone (Fig. 3), suggesting that only ENTs are involved in its placental uptake. We propose that the dominantly expressed ENT1 [32] is responsible for this uptake. Moreover, the apparent lack of CNTs’ participation in placental uptake of ribavirin is consistent with our findings that functional CNTs are not expressed in placenta [32]. In analyses of [3H]-ribavirin accumulation in MVM vesicles we also detected contributions of ENT1 but not CNTs (Fig. 4), and inhibitory effects of uridine, presumably due solely to inhibition of ENTs (Fig. 4b, c). These results are consistent with findings from our experiments with villous fragments and previously reported studies with human and rat MVM vesicles [32], [33], [45]. In the next step, we employed an in situ model based on dually perfused rat placenta in open- and closed-circuit setups to evaluate the overall contribution of NTs and potentially ABC proteins to placental [3H]-ribavirin transfer at the organ level. Dual perfusion of rat term placenta is an established and well-justified method that has been used to investigate interactions of placental ABC and SLC transporters with various drugs, including antivirals [57], [58], [59], [88], [89], [90]. ENTs in the apical membrane of trophoblast layer of Wistar rats have been functionally characterized by analysing adenosine uptake from the maternal blood circulation into the foeto-placental unit and MVM vesicles prepared from rat placenta [45]. Analysis of placental M → F and F → M clearances of radiolabelled [3H]-adenosine is impossible, with current limitations, due to extensive adenosine placental 400 [34], so we could not directly validate this experimental system. With the open-circuit setup, we obtained the first evidence that transport of [3H]-ribavirin across rat placenta from mother to foetus and vice versa is low overall (Fig. 5a), especially relative to antipyrine clearance across the placenta driven by passive diffusion [91]. Both M → F and F → M clearances of [3H]-ribavirin were significantly reduced by presence of NBMPR (at 0.1 µM and 100 µM) or 5 mM uridine, suggesting a profound contribution of ENTs, most likely ENT1, not only to placental cell uptake but also to transport of ribavirin across rat syncytiotrophoblast. Effects of Na+ depletion could not be investigated using this model as it deleteriously affects rat term placenta, causing oedema and high pressure, during experiments. Lower M → F clearance than F → M clearance indicated that ribavirin is predominantly transferred in the F → M direction, therefore we performed dual perfusion experiments with a closed-circuit setup (Fig. 5b). We observed no changes in ribavirin concentrations on maternal or foetal sides, even after adding uridine to block potential masking activity of NTs, indicating no active efflux transport from foetus to mother (Fig. 5). These findings are consistent with our suggestions, based on results of in vitro experiments, that active efflux placental pumps do not recognize ribavirin as a substrate (Table 1). To further assess potential involvement of CNTs in ribavirin placental kinetics we quantified mRNA expression of slc28A1, slc28A2, and slc28A3 in our experimental animal model. We found that slc28A2 is by far the most strongly expressed CNT gene, slc28A1 expression is not detectable, in term placenta of our Wistar rat (Fig. 6), in accordance with previous reports [45], [81]. Nevertheless, as lack of CNT activity was observed in MVM vesicles derived from Wistar rat placenta [45], these transporters do not seem to be functionally expressed in the trophoblast apical membrane [45]. Therefore, we speculate that only CNT2/CNT3 embedded in the basal membrane may potentially contribute to the higher F → M clearances observed in rat placenta.