Induction of ATP Release, PPIX Transport, and Cholesterol Uptake by Human Red Blood Cells Using a New Family of TSPO Ligands

Abstract

Two main isoforms of the Translocator Protein (TSPO) have been identified. TSPO1 is ubiquitous and is mainly present at the outer mitochondrial membrane of most eukaryotic cells, whereas, TSPO2 is specific to the erythroid lineage, located at the plasma membrane, the nucleus, and the endoplasmic reticulum. The design of specific tools is necessary to determine the molecular associations and functions of TSPO, which remain controversial nowadays. We recently demonstrated that TSPO2 is involved in a supramolecular complex of the erythrocyte membrane, where micromolar doses of the classical TSPO ligands induce ATP release and zinc protoporphyrin (ZnPPIX) transport. In this work, three newly-designed ligands (NCS1016, NCS1018, and NCS1026) were assessed for their ability to modulate the functions of various erythrocyte's and compare them to the TSPO classical ligands. The three new ligands were effective in reducing intraerythrocytic Plasmodium growth, without compromising erythrocyte survival. While NCS1016 and NCS1018 were the most effective ligands in delaying sorbitol-induced hemolysis, NCS1016 induced the highest uptake of ZnPPIX and NCS1026 was the only ligand inhibiting the cholesterol uptake. Differential effects of ligands are probably due, not only, to ligand features, but also to the dynamic interaction of TSPO with various partners at the cell membrane. Further studies are necessary to fully understand the mechanisms of the TSPO's complex activation.

Keywords: ATP; TSPO; TSPO2; VDAC; ZnPPIX; erythrocyte; malaria; plasmodium; red blood cell.

Figure 1

TSPO ligands inhibited the P. falciparum growth. Infected RBCs were diluted to 1% parasitemia, and maintained at 1–5%, by dilution, for 48 h. Percentages of growth-inhibition were obtained, after two parasite cycles, by the flow cytometry analysis of parasitemia, and normalized to the control condition (solvent-treated cells). Data are presented as the mean ± SEM; N = 4. Differences between the control condition and the treatment were considered significant at ** p < 0.01; *** p < 001.

Figure 2

ZnPPIX uptake in healthy and infected RBCs induced by TSPO ligands. Healthy RBC (A) and infected RBC (B) at 2 to 5% parasitemia were incubated in the presence of 20 µM ZnPPIX and 50 µM TSPO ligands, and the uptake of ZnPPIX was assessed at different time points by flow cytometry. MFI values were normalized to background levels for the control and the ligand-treated conditions. Data are presented as the mean ± SEM; N = 7. Statistical analyses were performed for each ligand and the control condition. Differences were considered significant when * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 3

Reactive oxygen species (ROS) induced by TSPO ligands in the presence of ZnPPIX. Infected RBC at 2 to 5% parasitemia were incubated in the presence of 20 µM ZnPPIX and 50 µM TSPO ligands. At several time points samples were washed and incubated with DCFDA and analyzed by flow cytometry. MFI values were normalized to control background levels. No compensation was needed as monostained samples showed no interference between ZnPPIX and the ROS probes. Data are presented as the mean ± SEM; N = 8. Statistical analyses were performed for each ligand and control condition. Differences were considered significant when * p < 0.05; ** p < 0.01.

Figure 4

Sorbitol-induced hemolysis is modulated by the TSPO ligands in infected red blood cells. Infected RBCs at 2 to 5 % parasitemia were washed three times in a culture medium, without serum, and resuspended at 50% hematocrit in a media containing 10 μM (A) or 50 μM (B) TSPO ligands. Hemolysis was quantified at several time-points, by absorption at 540 nm wavelength. Data are presented as mean ± SEM; N = 4. Differences between each ligand and control condition (vehicle) were considered significant when p < 0.01.

Figure 5

Cholesterol uptake induced by TSPO ligands. Healthy (A) or infected red blood cells, at 2 to 5% parasitemia (B) were incubated in the presence of TopFluor Cholesterol and 50 µM of TSPO ligands. Cholesterol uptake was assessed at different time points by flow cytometry. MFI values were normalized to vehicle treated cells. Data are presented as mean ± SEM; N = 4. Differences between each ligand and control condition (vehicle) were considered significant when * p < 0.05.

Figure 6

TSPO ligand-induced ATP release. ATP release from human red blood cells was measured at 37 °C, 10% Hematocrit, as described in material and methods. Values are the ratio between extracellular ATP (ATPe) levels after and before the addition of TSPO ligands. Panel (A) shows the effect of 10 or 30 µM of PK 11195, Ro5-4864, NCS1016, and NCS1018. Panel (B) shows the effect of VDAC inhibitor Bcl-XLBH4 (10 µM), or Pannexin-1 inhibitors carbonexolone (CBX, 10 µM), or probenecid (PBC, 10 µM). Results are expressed as means ± SEM and considered different from basal (vehicle, unstimulated) values when * p < 0.05; ** p < 0.01 or from those incubated in the presence of Bcl-XLBH4 when ^# p < 0.05 and ^## p < 0.01; N = 7.

Figure 7

Chemical structure of the TSPO ligands. The six TSPO ligands used in this study belonged to the isoquinolines (PK 11195), benzodiazepines (Ro5-4864), pyridazinoindoles (SSR-180,575), and imidazoquinazolinone-derivative (NCS1016, NCS1018 and NCS1026) families.