Translocator protein (18 kDa) ligand PK 11195 induces transient mitochondrial Ca2+ release leading to transepithelial Cl- secretion in HT-29 human colon cancer cells.
BACKGROUND
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 [(3)H]PK 11195 and membrane fraction demonstrated saturable binding, with K(d) and B(max) values 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 [Ca(2+)], which was unaffected by extracellular Ca(2+), 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 Ca(2+)-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 Ca(2+) 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 Ca(2+), leading to the stimulation of Cl(-) secretion.
Ostuni
Mariano A
MA
Inserm U773, Centre de Recherche Biomédicale Bichat Beaujon CRB3, Université Paris 7 Denis Diderot, F-75018 Paris, France.
Ducroc
Robert
R
Péranzi
Gabriel
G
Tonon
Marie-Christine
MC
Papadopoulos
Vassilios
V
Lacapere
Jean-Jacques
JJ
D020533:Angiogenesis Inhibitors / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D045744:Cell Line, Tumor; D002712:Chlorides / Q000378:metabolism*; D003110:Colonic Neoplasms; D016572:Cyclosporine / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005445:Flunitrazepam / Q000494:pharmacology; D018757:GABA Modulators / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology*; D008024:Ligands; D008928:Mitochondria / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D009579:Nitrobenzoates / Q000494:pharmacology; D018079:Receptors, GABA / Q000096:biosynthesis*; D028021:Sodium-Potassium-Chloride Symporters
D020533:Angiogenesis Inhibitors; D000970:Antineoplastic Agents; D002712:Chlorides; D004791:Enzyme Inhibitors; D018757:GABA Modulators; D007546:Isoquinolines; D008024:Ligands; D024101:Mitochondrial Proteins; D009363:Neoplasm Proteins; D009579:Nitrobenzoates; D018079:Receptors, GABA; D028021:Sodium-Potassium-Chloride Symporters; C499229:TSPO protein, human; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; D005445:Flunitrazepam; D016572:Cyclosporine; D002118:Calcium; C037850:PK 11195