CNS & Neurological Disorders - Drug Targets

Title:Concentration-Dependent Bimodal Effect of Specific 18 kDa Translocator Protein (TSPO) Ligands on Cell Death Processes Induced by Ammonium Chloride: Potential Implications for Neuropathological Effects Due to Hyperammonemia

VOLUME: 13 ISSUE: 4

Author(s):Beatriz Caballero, Leo Veenman, Julia Bode, Svetlana Leschiner and Moshe Gavish

Affiliation:Rappaport Family Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, Department of Molecular Pharmacology, Ephron Street, P.O. Box 9649, Bat-Galim, Haifa 31096, Israel;.

Keywords:Ammonium chloride, cell death, cell swelling, mitochondria, oxidative stress, 18 kDa Translocator Protein (TSPO), neuropathology.

Abstract:The role of the 18-kDa Translocator Protein (TSPO) in cell death induced by NH₄Cl (1-50 mM) for 24-72 hours to human glioblastoma U118MG cells was investigated. Cell death was already observed after 48 hours of treatment with NH₄Cl at 5 mM. Dose and time-responses curves indicated that 15 mM of NH₄Cl applied for 72 hours was the optimal condition for our viability assays. For example, 72 hours of 15 mM of NH₄Cl caused a 50.3% increase in propidium iodide uptake, and lactate dehydrogenase release was 41.2% of the positive control, indicating significant increases in cell death. Furthermore, compared to vehicle control, these experimental conditions resulted in a significant decrease of 44.9% of the mitochondrial activity, a 62.3% increase in incidence of collapse of mitochondrial membrane potential, and an increase of 49.0% of cardiolipin peroxidation. In addition, a significant 4.3 fold increase in the maximal binding capacity (B_max) of TSPO was found in NH₄Cl-exposed cells. Surprisingly, western blot analysis and real-time PCR did not demonstrate changes in TSPO expression. We also found that neither NH4Cl nor glutamine (a metabolic product of enhanced NH₄Cl levels) inhibited binding of the TSPO ligand [³H]PK 11195. Interestingly, we observed a bimodal effect of the TSPO ligands PK 11195, Ro5-4864, and FGIN-1-27 on the toxicity of NH₄Cl; such that 1-100 nM concentrations of TSPO ligands were protective, while concentrations above 1 μM enhanced NH₄Cl-induced cell death processes. In conclusion, TSPO takes part in a bimodal way in the lethal effects induced by NH₄Cl in glial type cells.