Thiamine transport in human placental brush border membrane vesicles

Abstract

Pathways for transport of thiamine by the human placental epithelium were investigated using brush border membrane vesicles isolated by divalent cation precipitation. The presence of thiamine transport mechanisms mediating Na+-thiamine cotransport, proton/thiamine exchange and facilitated diffusion was assessed from [3H]-thiamine tracer flux measurements. The magnitude of intravesicular thiamine accumulation was unaffected by the imposition of an inwardly directed sodium gradient suggesting an absence of a mechanism mediating brush border membrane Na+-thiamine cotransport. Intravesicular thiamine accumulation was indistinguishable when measured in the presence and absence of conditions favoring the development of an inside-negative, potassium diffusion potential. The observed absence of conductive thiamine uptake suggests the absence of a mechanism mediating facilitated diffusion of thiamine in placental brush border membrane. The imposition of an inside-acid pH gradient was observed to induce concentrative accumulation of thiamine to levels exceeding equilibrium, suggesting the presence of a placental brush border membrane proton/thiamine exchange mechanism. Protonophore- induced dissipation of an imposed inside-acid pH gradient in the absence of membrane potential was observed to abolish concentrative accumulation of thiamine, suggesting a direct chemical coupling of protons and thiamine via a mediated exchange mechanism. Consistent with the functional properties expected for a mechanism mediating thiamine transport by organic cation exchange, the rate and magnitude of intravesicular [3H]-thiamine accumulation was increased when measured in the presence compared to the absence of an outwardly directed thiamine concentration gradient. Substrate specificity studies of the proton/thiamine exchange mechanism suggest that the amine at position four of the pyrimidine ring, but not the hydroxyethyl side chain or an unmodified thiazolium ring, is an important chemical determinant for interaction with the transporter substrate binding site(s). Substrate specificity studies further suggest the possible presence of three separate organic cation exchange mechanisms mediating transport of thiamine, guanidine and MIA across placental brush border membrane.

The effect of a series of organic cations upon the plasmalemmal serotonin transporter, SERT

“…Although we can also hypothesize that 5HT is not transported by the same mechanism that accepts thiamine as substrate (ThT1 (Dutta et al, 1999) or ThT2 (Rajgopal et al, 2001)), similarly to what was found with brush-border membrane vesicles from human placenta (Grassl, 1998), care must be taken with this last conclusion. This is because thiamine transport and thiamine transporters such as ThT1 or ThT2 were never identified in JAR cells (indeed, a lack of significant 3 H-thiamine uptake by JAR cells was recently observed (Lemos, personal communication, 2003)).…”

Cloning of the Human Thiamine Transporter, a Member of the Folate Transporter Family

“…Subsequent experiments involving the characterization of the transport function of ThT1 were done in the absence of Na ϩ . The transport of thiamine in placental and intestinal brush border membrane vesicles was stimulated by an inside-out proton gradient (5,8). We therefore examined the effect of extracellular pH on thiamine transport in vector-and cDNA-transfected cells (Fig.…”

“…Absorptive tissues such as placenta (13,14), kidney (15,16), and intestine (17) are known to express specific organic cation/H ϩ antiport systems that are capable of transporting both endogenous as well as exogenous organic cations. The presence of an outwardly directed proton gradient was shown to induce concentrative thiamine accumulation in brush border membrane vesicles isolated from human term placenta, suggesting a possible role of an organic cation/H ϩ antiport system in the uptake of thiamine into the placenta (5). Consistent with such an uptake mechanism, the thiamine uptake into placental brush border membrane vesicles was abolished in the presence of proton ionophores and unaffected by the presence of an inside-negative membrane potential.…”

“…The concentration of thiamine is more than 10-fold higher in fetal umbilical vein plasma compared with that present in maternal plasma (2), suggesting the presence of an efficient transport system for the vitamin in placenta. The underlying mechanism of transplacental transport of thiamine has been investigated using in vitro perfusion techniques (3,4) as well as isolated placental brush border membrane vesicles (5). Thiamine transport has also been functionally characterized in other tissues/ cells such as intestine (6 -9), erythrocytes (10), hepatocytes (11), and neuroblastoma cells (12).…”

Changes in fetal lamb arterial blood gas and acid-base status with advancing gestation

“…However, in the human placenta, there is evidence for the presence a Cl/HCO 3 Ϫ exchanger (25,40), and in the guinea pig, evidence for placental transfer of the HCO 3 Ϫ ion (28) . Further research in this area is required before the reason for the increased pH and HCO 3 Ϫ with increasing fetal number can be elucidated.…”