Riboflavin uptake in human trophoblast-derived BeWo cell monolayers: cellular translocation and regulatory mechanisms.
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 (Kt = 1.32 +/- 0.68 nM, Jmax = 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 and p-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 both Kt and Jmax of the riboflavin uptake process (Kt = 6.32 +/- 1.29 nM, Jmax = 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.
Huang
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Division of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus, Ohio, USA.
Swaan
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PW
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects / Q000502:physiology; D018118:Chloride Channels / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport / Q000187:drug effects / Q000502:physiology; D010920:Placenta / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D012256:Riboflavin / Q000031:analogs & derivatives / Q000493:pharmacokinetics*; D014327:Trophoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000378:metabolism
D018118:Chloride Channels; D004791:Enzyme Inhibitors; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012256:Riboflavin