Translocator protein (18 kDa) ligand PK 11195 induces transient mitochondrial Ca2+ release leading to transepithelial Cl- secretion in HT-29 human colon cancer cells.
Translocator protein (18 kDa) ligand PK 11195 induces transient mitochondrial Ca2+ release leading to transepithelial Cl- secretion in HT-29 human colon cancer cells.
复制标题
易位蛋白 (18 kDa) 配体 PK 11195 诱导瞬时线粒体 Ca2 释放，导致 HT-29 人结肠癌细胞中的跨上皮 Cl 分泌。
DOI：
10.1042/bc20070048
复制
发表时间：
2007
影响因子：
2.7
通讯作者：
Lacapere,Jean-Jacques
中科院分区：
文献类型：
--
作者：
Ostuni,MarianoA;Ducroc,Robert;Péranzi,Gabriel;Tonon,Marie-Christine;Papadopoulos,Vassilios;Lacapere,Jean-Jacques
背景信息。 TSPO（转运蛋白），以前称为 PBR（外周型苯二氮卓受体），是一种在多种组织的线粒体膜中表达的 18 kDa 蛋白质。据报道，TSPO 在人类结直肠肿瘤和癌细胞系中过度表达，但其功能尚未得到很好的表征。结果。我们研究了 TSPO 在人结肠癌细胞 HT-29 中的表达和功能。免疫组织化学研究表明，TSPO 位于线粒体中，其内源性配体（多肽地西泮结合抑制剂）位于细胞质中。使用特异性高亲和力药物配体 [3H]PK 11195 和膜组分进行的放射性配体结合研究证明了可饱和结合，KddB 最大值分别为 13.5±1.5 nM 和 10.1±1.0 pmol/mg。 PK 11195 诱导细胞内 [Ca2+] 快速且短暂地剂量依赖性升高，该升高不受细胞外 Ca2+ 的影响，但会被 PTP（渗透性转变孔）抑制剂环孢菌素 A 和 TSPO 部分激动剂氟硝西泮阻断。使用形成细胞单层的 HT-29 克隆 19A 细胞系，我们证明 TSPO 配体刺激 Ca2+ 依赖性跨上皮 Cl- 分泌。这种分泌在以下情况下受到抑制：(i) 去除细胞外 Cl- 后； (ii) 通过顶部添加 Cl-通道阻滞剂 NPPB [5-硝基-2-(3-苯基丙氨基)-苯甲酸酯]； (iii) 通过基底外侧添加 Na+–K+–2Cl−协同转运蛋白抑制剂布美他尼。此外，细胞内 Ca2+ 螯合剂 BAPTA/AM [双-(邻氨基苯氧基)乙烷-N,N,N',N'-四乙酸四(乙酰氧基甲酯)]和环孢菌素 A 消除了 PK 11195 诱导的 Cl 分泌增加。结论。这些发现表明 TSPO 位于 HT-29 的线粒体膜中，并表明其激活会诱导胞质 Ca2+ 升高，从而刺激 Cl− 分泌。
Background information. TSPO (translocator protein), known previously as PBR (peripheral‐type benzodiazepine receptor), is a 18 kDa protein expressed in the mitochondrial membrane of a variety of tissues. TSPO has been reported to be over‐expressed in human colorectal tumours and cancer cell lines, but its function is not well characterized.Results. We investigated the expression and function of TSPO in the human colon cancer cells HT‐29. Immunohistochemical studies revealed that TSPO is localized in mitochondria, and its endogenous ligand, the polypeptide diazepam‐binding inhibitor, in the cytosol. Radioligand binding studies using the specific high‐affinity drug ligand [3H]PK 11195 and membrane fraction demonstrated saturable binding, withKdandBmaxvalues of 13.5±1.5 nM and 10.1±1.0 pmol/mg respectively. PK 11195 induced a rapid and transient dose‐dependent rise in intracellular [Ca2+], which was unaffected by extracellular Ca2+, but was blocked by the PTP (permeability transition pore) inhibitor, cyclosporin A, and by the TSPO partial agonist, flunitrazepam. Using HT‐29 clone 19A cell line, which forms cell monolayers, we demonstrated that TSPO ligand stimulated a Ca2+‐dependent transepithelial Cl−secretion. This secretion was inhibited: (i) after removal of extracellular Cl−; (ii) by apical addition of the Cl−channel blocker NPPB [5‐nitro‐2‐(3‐phenylpropylamino)‐benzoate]; and (iii) by basolateral addition of the Na+–K+–2Cl−co‐transporter inhibitor bumetanide. Furthermore, the intracellular Ca2+chelator BAPTA/AM [bis‐(o‐aminophenoxy)ethane‐N,N,N′,N′‐tetra‐acetic acid tetrakis(acetoxymethyl ester)] and cyclosporin A abolished the rise in PK 11195‐induced Cl−secretion.Conclusions. These findings indicate that TSPO is located in mitochondrial membranes of HT‐29 and reveal that its activation induces a rise in cytosolic Ca2+, leading to the stimulation of Cl−secretion.