Grassl S M
Department of Pharmacology, State University of New York, Health Science Center at Syracuse 13210.
Biochim Biophys Acta. 1994 Aug 24;1194(1):203-13. doi: 10.1016/0005-2736(94)90221-6.
Pathways for transport of choline by human placental epithelia were investigated using brush border membrane vesicles isolated by divalent cation precipitation. The presence of choline transport mechanisms mediating Na(+)-choline cotransport, choline/H+ exchange and facilitated diffusion were assessed from [3H]choline tracer flux measurements. The rate and magnitude of intravesicular choline accumulation was unaffected by the imposition of an inwardly directed Na+ gradient suggesting an absence of a mechanism mediating brush border membrane Na(+)-choline cotransport. The imposition of inside-acid or inside-alkaline pH gradients was observed to have no significant effect on choline uptake suggesting choline is not a substrate for placental epithelial organic cation/H+ exchange. Conditions favoring the development of an inside-negative K+ diffusion potential was observed to induce a concentrative accumulation of choline to levels exceeding equilibrium suggesting the presence of a conductive uptake pathway for choline in placental brush border membrane. Evidence to suggest conductive choline uptake resulted from a mediated transport process includes a demonstration of the counterflow phenomena, the concentration-dependent inhibition by hemicholinium-3 (IC50 approximately equal to 100 microM) and the saturable rate of conductive choline uptake (Km approximately equal to 300 microM, Vmax approximately equal to 30 nmol/mg per min). Substrate specificity studies of the mechanism mediating conductive choline uptake suggest the interaction of choline with the transport protein occurs at a minimum of two sites: a site of negativity with the positively charged nitrogen group and a site of hydrogen bonding to the primary alcohol. Several commonly prescribed pharmaceuticals known to cross the placental barrier including imipramine, verapamil, propranolol, quinine, flurazepam, amiloride and ritodrin were observed to inhibit conductive choline uptake suggesting an interaction with the mechanism mediating conductive choline transport. Conductive choline uptake was unaffected by the presence of the basic amino acids lysine, arginine and histidine; the neurotransmitters serotonin, dopamine and histamine and the vitamins thiamine and carnitine which suggests the mechanism mediating conductive choline transport is not a pathway for placental uptake of these compounds.
利用通过二价阳离子沉淀分离得到的刷状缘膜囊泡，研究了人胎盘上皮细胞转运胆碱的途径。通过[3H]胆碱示踪通量测量，评估了介导Na(+)-胆碱共转运、胆碱/H+交换和易化扩散的胆碱转运机制的存在情况。囊泡内胆碱积累的速率和幅度不受内向Na+梯度施加的影响，这表明不存在介导刷状缘膜Na(+)-胆碱共转运的机制。观察到施加内向酸性或内向碱性pH梯度对胆碱摄取没有显著影响，这表明胆碱不是胎盘上皮有机阳离子/H+交换的底物。观察到有利于形成内向负性K+扩散电位的条件会诱导胆碱的浓缩积累，使其水平超过平衡状态，这表明胎盘刷状缘膜中存在胆碱的传导性摄取途径。表明传导性胆碱摄取是由介导转运过程导致的证据包括逆流现象的证明、半胱氨酸-3（IC50约等于100 microM）的浓度依赖性抑制以及传导性胆碱摄取的饱和速率（Km约等于300 microM，Vmax约等于30 nmol/mg每分钟）。对介导传导性胆碱摄取机制的底物特异性研究表明，胆碱与转运蛋白的相互作用至少发生在两个位点：带正电荷氮基团的负性位点和与伯醇的氢键位点。观察到几种已知可穿过胎盘屏障的常用药物，包括丙咪嗪、维拉帕米、普萘洛尔、奎宁、氟西泮、阿米洛利和利托君，会抑制传导性胆碱摄取，这表明它们与介导传导性胆碱转运的机制相互作用。传导性胆碱摄取不受碱性氨基酸赖氨酸、精氨酸和组氨酸、神经递质血清素、多巴胺和组胺以及维生素硫胺素和肉碱的存在的影响，这表明介导传导性胆碱转运的机制不是这些化合物胎盘摄取的途径。
