4-acetamido-4--isothiocyanatostilbene-2-2--disulfonic-acid and 5-methyltetrahydrofolate

The aim of this work was to investigate the effect of high glucose exposure on the absorption of folate by Caco-2 cells. We verified that apical high glucose did not affect the apical uptake of [(3)H]folate. Both different concentrations of glucose (10-45 mM) and different exposure times (10 min-24 h) were tested. Furthermore, apical high glucose (30 mM) did not affect the intracellular steady-state levels of [(3)H]folate, and simultaneous apical and basolateral high glucose (30 mM) did not change the apical-to-basolateral apparent permeability (P(app)) to [(3)H]folate. Both the apical uptake and the steady-state intracellular levels of [(3)H]folate were strongly reduced by 5-methyltetrahydrofolate, methotrexate, SITS (4-acetamido-4'-isothiocyanato-2,2'-stilbenedisulfonic acid), DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) and indomethacin, but were not affected or only hardly affected by p-aminohippuric acid and fumitremorgin C. Moreover, DIDS and indomethacin significantly reduced (by 50-60%) the apical-to-basolateral P(app) to [(3)H]folate, but [(3)H]folate present in the cells at the end of the experiment was higher in the case of indomethacin. Fumitremorgin C had no effect. The effect of the drugs tested was not changed or only hardly changed by high glucose. In conclusion, absorption of [(3)H]folate is not modulated by either apical or basolateral high glucose exposure in Caco-2 cells. Moreover, our results suggest that the apical uptake of [(3)H]folate by Caco-2 cells involves the Reduced Folate Transporter (but not the Organic Anion Transporter), and that Multidrug Resistance Protein and/or Organic Anion Transporter (but not Breast Cancer Resistance Protein) may mediate apical efflux of [(3)H]folate.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; ATP Binding Cassette Transporter, Subfamily B; Biological Transport; Caco-2 Cells; Cell Membrane Permeability; Dose-Response Relationship, Drug; Folic Acid; Glucose; Humans; Indoles; Indomethacin; Intestinal Absorption; Intestinal Mucosa; Membrane Transport Proteins; Methotrexate; Organic Anion Transporters; p-Aminohippuric Acid; Reduced Folate Carrier Protein; Tetrahydrofolates; Time Factors

Folates play a vital role in cellular processes that are essential for fetal growth and viability. Thus the human placenta, which contains high-affinity membrane-associated placental folate receptors (PFRs), maintains a concentrative maternal-to-fetal flux of the vitamin under conditions of minimal dependence on variations of maternal dietary intake. To define transplacental folate transport and the role of PFRs in this mechanism, we utilized the isolated perfused human placental cotyledon. In closed system perfusions with 10 nmol/L 5-methyltetrahydrofolate, placental binding was rapid and extensive (47%), with a gradual maternal-to-fetal transfer of 5-methyltetrahydrofolate. Although hydrophilic PFRs were released into the fetal perfusate, PFR-bound folates constituted only a fraction of net transplacental folate transport. Transfer was bidirectional, not saturable, not inhibited by anion channel blockers, and dependent on perfusate levels. Placental binding far exceeded transfer, and pulsing the maternal circuit with tritiated 5-methyltetrahydrofolate, followed by washout of unbound radiolabel and rechallenge with unlabeled 5-methyltetrahydrofolate or folate, led to release of bound tritiated 5-methyltetrahydrofolate, illustrating reversible binding. Perfusion with the N-hydroxysuccinimide ester of folic acid eliminated essentially all 5-methyltetrahydrofolate binding to PFRs, while increasing net maternal-to-fetal transfer of the vitamin. Finally, because it has been suggested that impaired placental transport of folate may be linked to the fetotoxic effects of ethanol, the effect of this compound on the above processes was examined. An acute 6-hour exposure to ethanol (2.5 to 3.1 mg/ml) had no effect (p > 0.05) on net maternal-to-fetal transfer of 5-methyltetrahydrofolate. These studies suggest that net maternal-to-fetal transfer is a process consisting of two steps. First is the concentrative component in which circulating 5-methyltetrahydrofolate is bound to (captured by) PFRs on the maternally facing chorionic surface. Although kinetics favor binding, there is a dynamic state wherein a gradual release of 5-methyltetrahydrofolate from this pool can add to incoming circulating folates to generate an intervillous blood level approximately 3 times that in the maternal blood. In the second step, folates are passively transferred to the fetal circulation along a downhill concentration gradient. This unique mechanism for transplacental folate transport ma

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Carrier Proteins; Ethanol; Female; Fetus; Folate Receptors, GPI-Anchored; Folic Acid; Humans; Maternal-Fetal Exchange; Perfusion; Placenta; Pregnancy; Probenecid; Receptors, Cell Surface; Tetrahydrofolates; Time Factors

Uptake of folic acid (PteGlu) was examined in remnant ileum of rats after resection of 65% of the small intestine with the brush border-membrane vesicle technique. The results were compared to that of sham-operated rats. In both rat groups transport of PteGlu was linear for approximately 40 s of incubation and was similar in the presence of a Na+ and a K+ gradient (out greater than in). In resected rats transport of PteGlu was inhibited by the structural analogues 5-methyltetrahydrofolate (5-CH3H4PteGlu) and methotrexate (MTX), by sulfasalazine, and by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and was saturable as a function of concentration (apparent Kt = 18.3 microM). In the ileum of sham-operated rats, on the other hand, transport of PteGlu was not affected by 5-CH3H4PteGlu, MTX, sulfasalazine, or DIDS and was linear with concentration. These results suggest that the PteGlu transport system is induced in remnant ileum of the rat after extensive intestinal resection.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Biological Transport; Dose-Response Relationship, Drug; Folic Acid; Ileum; Intestine, Small; Male; Methotrexate; Microvilli; Postoperative Period; Rats; Rats, Inbred Strains; Sulfasalazine; Tetrahydrofolates