Hepatocyte membrane water permeability measured by silicone layer filtering centrifugation
Hepatocyte membrane water permeability measured by silicone layer filtering centrifugation
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DOI：
10.1006/abio.2001.5533
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发表时间：
2002-03-01
影响因子：
2.9
通讯作者：
Marinelli, RA
中科院分区：
文献类型：
--
作者：
Gradilone, SA;Ochoa, JE;Marinelli, RA
我们之前发现肝细胞能够通过调节表面水通道蛋白的数量来控制其渗透膜的水渗透性（P-f）。肝细胞P-f已通过相衬显微镜和细胞图像分析进行评估，这是一种已建立但相对费力的程序。我们在此报告了一种基于单硅胶层过滤离心系统评估肝细胞P-f的替代方法。分离的大鼠肝细胞在含(H2O)-H-3作为示踪剂的低渗或等渗缓冲液中孵育，然后通过硅树脂层快速离心过滤至裂解层。肝细胞内的渗透驱动放射性（即(H2O)-H-3）被计算为低渗和等渗条件下裂解介质中dpm的差值。在VC下，从放射性-时间曲线的初始斜率计算出的P-f为18妈/秒。二丁基环AMP处理肝细胞，通过水通道蛋白插入质膜增加P-f， P-f值增加37妈/秒。水通道蛋白阻滞剂二甲基亚砜选择性地阻止激动剂诱导的肝细胞P-f。这些数据与定量相衬显微镜测定的相应值吻合良好；因此，开发的方法可以快速可靠地测量肝细胞P-f。(C) 2002 Elsevier Science （USA）。
We previously found that hepatocytes are able to control their osmotic membrane water permeability (P-f) by regulating the number of surface aquaporin water channels. Hepatocyte P-f has been assessed by phase-contrast microscopy and cell image analysis, an established but relatively laborious procedure. We report here an alternative method to assess hepatocyte P-f based on a single silicone layer filtering centrifugation system. Isolated rat hepatocytes were incubated in hypotonic or isotonic buffers containing (H2O)-H-3 as a tracer and, then, were filtered by rapid centrifugation through a silicone layer down to a lysis layer. Osmotically driven radioactivity (i.e., (H2O)-H-3) within hepatocytes was calculated as the difference between the dpm in lysis media measured under hypotonic and isotonic conditions. The P-f calculated from the initial slope of the radioactivity-versus-time curve was 18 mum/s at VC. Hepatocytes treated with dibutyryl cyclic AMP, to increase P-f through the plasma membrane insertion of aquaporins, showed an increased P-f value of 37 mum/s. The aquaporin blocker dimethyl sulfoxide selectively prevented the agonist-induced hepatocyte P-f. These data are in good agreement with the corresponding values determined by quantitative phase-contrast microscopy; thus, the method developed allows the rapid and reliable measurement of hepatocyte P-f. (C) 2002 Elsevier Science (USA).