PUTRESCINE AND PARAQUAT UPTAKE IN HUMAN LUNG SLICES AND ISOLATED TYPE-II PNEUMOCYTES
PUTRESCINE AND PARAQUAT UPTAKE IN HUMAN LUNG SLICES AND ISOLATED TYPE-II PNEUMOCYTES
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DOI：
10.1016/0006-2952(94)90281-x
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发表时间：
1994-08-03
影响因子：
5.8
通讯作者：
NEMERY, B
中科院分区：
文献类型：
--
作者：
HOET, PHM;LEWIS, CPL;NEMERY, B
百草枯通过一个主动吸收系统积累到各种动物的肺部，该系统似乎也介导内源性多胺的吸收，如腐胺。腐胺在人体肺部的积累先前已被证明主要位于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抑制作用，以及百草枯或半胺在离体人II型肺细胞中的抑制作用。采用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，首先将组织切片成0.7 mm厚的薄片，用不含钙和镁的磷酸盐缓冲盐水（PBS-）洗涤，然后用胰蛋白酶孵育。II型细胞经塑料差别化贴壁纯化，Percoll梯度离心分离。细胞分离48小时后测定摄取。放射性标记腐胺的积累表现为饱和动力学，其表观动力学参数为：K-m为6.7和6.2-7.6 μ M， V-max为2.7和3.0- 3.4 μ mol/g prot/hr。在百草枯存在的情况下，腐胺的摄取在两种系统中都以一种与竞争性抑制相容的方式减少，计算出的抑制常数（K-i）分别为549-614 μ M百草枯对切片和离体细胞的抑制常数（K-i）为659-895 μ M百草枯。在存在半胱胺的情况下，腐胺在分离的人肺细胞中的积累大大减少，计算出的K-i为3.7 μ M半胱胺。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体肺中积累，但对三种底物的亲和力不同。在培养的人肺II型中腐胺摄取系统的存在可能作为功能活力测试有用。
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: K-m 6.7 and 6.2-7.6 mu M and V-max 2.7 and 3.0- 3.4 mu mol/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 (K-i) of 549-614 and 659-895 mu M 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 K-i of 3.7 mu M 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.