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 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 [(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.