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 [ 3 H ]-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 [ 3 H ]-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.