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Research ArticleENDOCRINE AND REPRODUCTIVE

Riboflavin Uptake in Human Trophoblast-Derived BeWo Cell Monolayers: Cellular Translocation and Regulatory Mechanisms

Se-Ne Huang and Peter W. Swaan
Journal of Pharmacology and Experimental Therapeutics July 2001, 298 (1) 264-271;
Se-Ne Huang
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Peter W. Swaan
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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

  • 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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Journal of Pharmacology and Experimental Therapeutics: 298 (1)
Journal of Pharmacology and Experimental Therapeutics
Vol. 298, Issue 1
1 Jul 2001
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Research ArticleENDOCRINE AND REPRODUCTIVE

Riboflavin Uptake in Human Trophoblast-Derived BeWo Cell Monolayers: Cellular Translocation and Regulatory Mechanisms

Se-Ne Huang and Peter W. Swaan
Journal of Pharmacology and Experimental Therapeutics July 1, 2001, 298 (1) 264-271;

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Research ArticleENDOCRINE AND REPRODUCTIVE

Riboflavin Uptake in Human Trophoblast-Derived BeWo Cell Monolayers: Cellular Translocation and Regulatory Mechanisms

Se-Ne Huang and Peter W. Swaan
Journal of Pharmacology and Experimental Therapeutics July 1, 2001, 298 (1) 264-271;
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