Hoet P H, Lewis C P, Demedts M, Nemery B
Laboratory of Pneumology, K.U. Leuven, Belgium.
Biochem Pharmacol. 1994 Aug 3;48(3):517-24. doi: 10.1016/0006-2952(94)90281-x.
Paraquat is accumulated into the lungs of various species by an active uptake system which also appears to mediate the uptake of endogenous polyamines, such as putrescine. The accumulation of putrescine in the human lung has been previously shown to be mainly located in the type II cells. In the present study, we have studied the mutually competitive inhibition of putrescine and paraquat in human lung slices and the inhibition of putrescine by paraquat or cystamine in isolated human type II pneumocytes. Peripheral lung tissue taken from patients undergoing pneumectomy or lobectomy was used. The initial steps of the cell isolation procedure differed from the literature in that the tissue was first sliced in 0.7 mm thick slices, which were washed in phosphate buffered saline without calcium and magnesium (PBS-), followed by incubations with trypsin. The type II cells were purified and isolated by differential adherence on plastic followed by Percoll gradient centrifugation. Uptake was determined 48 hr after cell isolation. The accumulation of radiolabelled putrescine showed saturation kinetics, with the following apparent kinetic parameters: Km 6.7 and 6.2-7.6 microM and Vmax 2.7 and 3.0-3.4 mumol/g prot/hr for slices and isolated cells, respectively. In the presence of paraquat, putrescine uptake was reduced, in both systems, in a manner compatible with competitive inhibition, with calculated inhibition constants (Ki) of 549-614 and 659-895 microM paraquat for slices and isolated cells, respectively. The accumulation of putrescine in isolated human pneumocytes was strongly reduced in the presence of cystamine, with calculated Ki of 3.7 microM cystamine. These data indicate that putrescine, paraquat and cystamine accumulate in the human lung by the same uptake system, but that the affinities for the three substrates differ. The presence of an uptake system for putrescine in cultured human pulmonary type II is probably useful as a functional viability test.
百草枯通过一种主动摄取系统在多种物种的肺中蓄积，该系统似乎也介导内源性多胺（如腐胺）的摄取。先前已表明，腐胺在人肺中的蓄积主要位于Ⅱ型细胞中。在本研究中，我们研究了腐胺和百草枯在人肺切片中的相互竞争性抑制作用，以及百草枯或胱胺对分离的人Ⅱ型肺细胞中腐胺摄取的抑制作用。使用了接受肺切除术或肺叶切除术患者的外周肺组织。细胞分离程序的初始步骤与文献不同之处在于，首先将组织切成0.7毫米厚的切片，在不含钙和镁的磷酸盐缓冲盐水（PBS-）中洗涤，然后用胰蛋白酶孵育。通过在塑料上的差异贴壁，随后进行Percoll梯度离心，对Ⅱ型细胞进行纯化和分离。在细胞分离48小时后测定摄取情况。放射性标记腐胺的蓄积呈现饱和动力学，其表观动力学参数如下：切片和分离细胞的Km分别为6.7和6.2 - 7.6微摩尔，Vmax分别为2.7和3.0 - 3.4微摩尔/克蛋白/小时。在百草枯存在的情况下，两个系统中腐胺的摄取均减少，其方式符合竞争性抑制，切片和分离细胞的计算抑制常数（Ki）分别为549 - 614和659 - 895微摩尔百草枯。在胱胺存在的情况下，分离的人肺细胞中腐胺的蓄积显著减少，胱胺的计算Ki为3.7微摩尔。这些数据表明，腐胺、百草枯和胱胺通过相同的摄取系统在人肺中蓄积，但三种底物的亲和力不同。培养的人肺Ⅱ型细胞中存在腐胺摄取系统可能作为一种功能活力测试是有用的。
