Abstract

Riboflavin (vitamin B₂) is essential for fetal development and must be acquired from maternal sources. The uptake mechanism of riboflavin and the major regulatory pathways involved were characterized in a model for the placental barrier, the human choriocarcinoma cell line, BeWo. Uptake of [³H]riboflavin was saturable (K_t = 1.32 ± 0.68 nM, J_max = 266.63 ± 26.89 fmol/mg of protein/20 min), and was significantly reduced at low temperature and in the presence of metabolic inhibitors (azide, 2-deoxyglucose) or structural analogs. Ouabain, amiloride, sodium-free buffers, and medium with pH values ranging from 3 to 8 did not affect uptake of [³H]riboflavin. In contrast, substitution of chloride with other monovalent anions significantly inhibited its uptake. Induced differentiation of BeWo cells into syncytiotrophoblasts by forskolin or 8-bromo-cyclic adenosine monophosphate introduced a time-dependent decrease of riboflavin uptake. Preincubation with activators of cyclic nucleotide-dependent protein kinase pathways (3-isobutyl-1-methylxanthine andp-chlorophenylthio-cyclic guanosine monophosphate) and calmodulin antagonists (calmidazolium and W-13) resulted in a concentration-dependent reduction of [³H]riboflavin uptake, whereas specific modulators of protein kinase C pathways did not have significant effects. 3-Isobutyl-1-methylxanthine exerted its regulatory effect on riboflavin uptake via decreasing bothK_t and J_max of the riboflavin uptake process (K_t = 6.32 ± 1.29 nM, J_max = 135.57 ± 10.42 fmol/mg of protein/20 min). In summary, we report the presence of high- affinity riboflavin transporter(s) on the microvillous membrane of BeWo cells that appears to be modulated by cellular cyclic nucleotide levels and calmodulin.