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 (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 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 bothKt 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.
Footnotes
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This work was supported by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK R01-DK56631).
- Abbreviations:
- PBS
- phosphate-buffered saline
- Kt
- Michaelis-Menten-type constant
- Jmax
- maximum uptake velocity
- cAMP
- cyclic adenosine monophosphate
- FMN
- flavin mononucleotide
- FAD
- flavin adenine dinucleotide
- DIDS
- 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid
- 8-Br-cAMP
- 8-bromo-cyclic adenosine monophosphate
- PKA
- protein kinase A
- IBMX
- 3-isobutyl-1-methylxanthine
- GMP
- guanosine monophosphate
- PKG
- protein kinase G
- pCPT-cGMP
- p-chlorophenylthio-cyclic guanosine monophosphate
- PKC
- protein kinase C
- CaM
- calmodulin
- Received January 31, 2001.
- Accepted March 30, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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