Grassl S M
Department of Pharmacology, State University of New York Health Science Center, Syracuse, NY 13210, USA.
Biochim Biophys Acta. 1998 May 28;1371(2):213-22. doi: 10.1016/s0005-2736(98)00019-4.
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.
利用通过二价阳离子沉淀分离的刷状缘膜囊泡，研究了人胎盘上皮细胞转运硫胺素的途径。通过[³H] - 硫胺素示踪剂通量测量，评估了介导钠 - 硫胺素共转运、质子/硫胺素交换和易化扩散的硫胺素转运机制的存在情况。施加内向钠梯度对囊泡内硫胺素积累量没有影响，这表明不存在介导刷状缘膜钠 - 硫胺素共转运的机制。在存在和不存在有利于形成膜内负钾扩散电位的条件下测量时，囊泡内硫胺素积累情况没有差异。观察到不存在传导性硫胺素摄取，这表明不存在介导硫胺素在胎盘刷状缘膜中易化扩散的机制。观察到施加内向酸性pH梯度会诱导硫胺素浓缩积累至超过平衡的水平，这表明存在胎盘刷状缘膜质子/硫胺素交换机制。在不存在膜电位的情况下，质子载体诱导的施加内向酸性pH梯度的消散被观察到会消除硫胺素的浓缩积累，这表明质子和硫胺素通过介导的交换机制直接化学偶联。与通过有机阳离子交换介导硫胺素转运的机制预期的功能特性一致，与不存在外向硫胺素浓度梯度相比，在存在外向硫胺素浓度梯度时测量，囊泡内[³H] - 硫胺素积累的速率和量增加。质子/硫胺素交换机制的底物特异性研究表明，嘧啶环第4位的胺基，而非羟乙基侧链或未修饰的噻唑环，是与转运体底物结合位点相互作用的重要化学决定因素。底物特异性研究进一步表明，可能存在三种独立的有机阳离子交换机制介导硫胺素、胍和MIA跨胎盘刷状缘膜的转运。
