ROLE OF ALTERED MEMBRANE FUNCTION IN XENOBIOTIC TOXICITY

工作总结：有机阴离子（OA）的肾分泌转运和 有机阳离子（OC）控制着大多数外来化学物质的排泄 和/或其代谢物。 我们之前已经描述了 这两个过程的机制和能量。 我们目前的重点是 这些转运蛋白的生物化学;它们的发育，表达， 及其对外源性物质毒性的影响。 我们有 开始使用基因识别这些转运蛋白的编码基因， 在非洲爪蟾卵母细胞中表达克隆。 由于它们的体积大， 由于能够有效利用外源mRNA， 注射mRNA并测定每个卵中表达转运活性。 我们 现在已经克隆了一种OC转运蛋白和一种OA转运蛋白 也克隆的OC载体（OCT 2）已完全测序， 与其他最近克隆的OC转运蛋白（OCT 1）不同。 OCT 1具有 初步确定为基底外侧OC携带者。 功能 数据表明OCT 2与管腔OC/质子有许多共同特征， 交换器，包括质子依赖性传输以及类似的 底物特异性和动力学常数。 但也可以 介导电位驱动的OC转运，基底外侧的一种特性 载体 使用转染的上皮细胞单层培养的研究 已经启动了解决这一问题的小组。 克隆的OA转运蛋白 蛋白质也已测序。 功能研究表明， 是介导上坡进入的基底外侧OA/ -酮戊二酸交换剂 进入肾小管细胞。 它是第一个肾脏有机阴离子 转运蛋白被克隆，并已被指定为ROAT 1。 这是 特性包括约100 μ M的Km，多种OA的抑制 - 酮戊二酸的顺式抑制和反式刺激， 膜电位的独立性--基底外侧的所有特性 OA载体。 一项相关的研究评估了肾脏机制， 有助于观察到酚酞导致肾肿瘤。 这些研究，在分离的膜囊泡和完整的小管中， 显示母体分子的快速和有效的肾摄取， 其葡萄糖醛酸结合物通过OA系统;因此，显然使 肾脏特别容易受到这些化合物的毒性作用。 此外，母体药物和代谢产物均为肾毒性的强效抑制剂， 消除其他阴离子异生物质。

Summary of Work: Renal secretory transport of organic anions (OA) and organic cations (OC) controls the excretion of most foreign chemicals and/or their metabolites. We have previously characterized the mechanisms and energetics of both processes. Our current focus is the biochemistry of these transport proteins; their development, expression, and control; and their impact on the toxicity of xenobiotics. We have begun to identify the genes coding for these transport proteins using expression cloning in Xenopus oocytes. Because of their large size and ability to make efficient use of foreign mRNA, it is relatively easy to inject mRNA and assay for expressed transport activity in each egg. We have now cloned one of the OC transport proteins and one OA transporter as well. The cloned OC carrier (OCT2) has been fully sequenced and is distinct from the other recently cloned OC transporter (OCT1). OCT1 has been tentatively identified as the basolateral OC carrier. Functional data indicates that OCT2 shares many features with the luminal OC/proton exchanger, including proton-dependent transport as well as similar substrate specificity and kinetic constants. However, it can also mediate potential driven OC transport, a property of the basolateral carrier. Studies using monolayer cultures of transfected epithelial cells have been initiated to resolve this issue. The cloned OA transport protein has also been sequenced. Functional studies demonstrate that it is basolateral OA/ -ketoglutarate exchanger which mediates uphill entry of OA into the tubular cells. It is the first renal organic anion transporter to be cloned and has been designated as ROAT1. It's properties include a Km of about 100 muM, inhibition by a variety of OAs but not OCs, cis-inhibition and trans-stimulation by -ketoglutarate, and independence of membrane potential -- all properties of the basolateral OA carrier. A related study assessed the renal mechanisms which might contribute to the observation that phenolphthalein causes renal tumors. These studies, in both isolated membrane vesicles and intact tubules, demonstrated rapid and effective renal uptake of the parent molecule and its glucuronide conjugate via the OA system; thus, apparently rendering the kidney particularly at risk for the toxic effects of these compounds. In addition, both parent and metabolite were potent inhibitors of renal elimination of other anionic xenobiotics.