Riboflavin uptake in human trophoblast-derived BeWo cell monolayers: Cellular translocation and regulatory mechanisms

Riboflavin (vitamin B-2) is essential for fetal development and must be acq uired from maternal sources. The uptake mechanism of riboflavin and the maj or regulatory pathways involved were characterized in a model for the place ntal barrier, the human choriocarcinoma cell line, BeWo, Uptake of [H-3]rib oflavin was saturable (K-t = 1.32 +/- 0.68 nM, J(max) = 266.63 +/- 26.89 fm ol/mg of protein/20 min), and was significantly reduced at low temperature and in the presence of metabolic inhibitors (azide, 2-deoxyglucose) or stru ctural analogs. Ouabain, amiloride, sodium-free buffers, and medium with pH values ranging from 3 to 8 did not affect uptake of [H-3]riboflavin. In co ntrast, substitution of chloride with other monovalent anions significantly inhibited its uptake. Induced differentiation of BeWo cells into syncytiot rophoblasts by forskolin or 8-bromo-cyclic adenosine monophosphate introduc ed a time-dependent decrease of riboflavin uptake. Preincubation with activ ators of cyclic nucleotide-dependent protein kinase pathways (3-isobutyl-1- methylxanthine and p-chlorophenylthio-cyclic guanosine monophosphate) and c almodulin antagonists (calmidazolium and W-13) resulted in a concentration- dependent reduction of [H-3]riboflavin uptake, whereas specific modulators of protein kinase C pathways did not have significant effects. 3-lsobutyl-1 -methylxanthine exerted its regulatory effect on riboflavin uptake via decr easing both K-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 m icrovillous membrane of BeWo cells that appears to be modulated by cellular cyclic nucleotide levels and calmodulin.