3-(Fur-2-yl)-10-(2-phenylethyl)-[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one, a novel adenosine receptor antagonist with A(2A)-mediated neuroprotective effects

Abstract

In this study, compound FTBI (3-(2-furyl)-10-(2-phenylethyl)[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one) was selected from a small library of triazinobenzimidazole derivatives as a potent A(2A) adenosine receptor (AR) antagonist and tested for its neuroprotective effects against two different kinds of dopaminergic neurotoxins, 1-methyl-4-phenylpyridinium (MPP+) and methamphetamine (METH), in rat PC12 and in human neuroblastoma SH-SY5Y cell lines. FTBI, in a concentration range corresponding to its affinity for A(2A) AR subtype, significantly increased the number of viable PC12 cells after their exposure to METH and, to a similar extent, to MPP+, as demonstrated in both trypan blue exclusion assay and in cytological staining. These neuroprotective effects were also observed with a classical A(2A) AR antagonist, ZM241385, and appeared to be completely counteracted by the AR agonist, NECA, supporting A(2A) ARs are directly involved in FTBI-mediated effects. Similarly, in human SH-SY5Y cells, FTBI was able to prevent cell toxicity induced by MPP+ and METH, showing that this A(2A) AR antagonist has a neuroprotective effect independently by the specific cell model. Altogether these results demonstrate that the A(2A) AR blockade mediates cell protection against neurotoxicity induced by dopaminergic neurotoxins in dopamine containing cells, supporting the potential use of A(2A) AR antagonists in dopaminergic degenerative diseases including Parkinson's disease.

Keywords: A2A AR antagonists; PC12 cells; cell viability; human neuroblastoma cells; neuroprotection; neurotoxins.

Scheme 1. Chemical Synthesis of FTBI

Figure 1

Effect of FTBI on NECA-stimulated cAMP accumulation in A_2A CHO cells. Cells were treated with 100 nM NECA in the absence or in the presence of different FTBI concentrations (1 nM to 10 μM). Then the intracellular levels of cAMP were evaluated and expressed as percentage of NECA-stimulated cAMP levels set to 100%. Data represent means ± SEM of three separate experiments each performed in duplicate.

Figure 2

Dose-dependency of MPP+ (A) and METH (B) toxicity in PC12 cells. Cell counts were carried out following 72 h of MPP+ (A) or METH (B) exposure. Effects of DA toxins on cell viability were measured by using trypan blue staining. Results shown are expressed as means ± SEM of at least four independent experiments. *P < 0.05 compared with control; **P < 0.01 compared with control; ***P < 0.001 compared with control.

Figure 3

FTBI induced neuroprotection against MPP+ or METH toxicity. PC12 cells were treated for 72 h with 250 μM MPP+ (A) or METH (B) in the absence or in the presence of different FTBI concentrations. Effects of different treatments on cell viability were measured by using trypan blue staining. Results shown are expressed as means ± SEM of at least four independent experiments. ***P < 0.001 compared with control; ^§P < 0.05 compared with toxin alone; ^§§§P < 0.001 compared with toxin alone.

Figure 4

Representative pictures of protective effects of FTBI against METH and MPP+ toxicity: Papanicolau’s staining. PC12 cells were treated with either METH (250 μM) or MPP+ (250 μM), in the absence or in the presence of the adenosine antagonist FTBI (16 nM). (a) control; (b) FTBI (16 nM); (c) METH (250 μM); (d) METH (250 μM) plus FTBI (16 nM); (e) MPP+ (250 μM); (f) MPP+ (250 μM) plus FTBI (16nM). Scale bar: 12.5 μm.

Figure 5

ZM241385-induced neuroprotection against METH or MPP+ toxicity. PC12 cells were treated for 72 h with 250 μM METH or MPP+ in the absence or in the presence of 0.5 nM ZM241385. Effects of different treatments on cell viability were measured by using trypan blue (A) and H&E (B) staining. Results in (A) are expressed as means ± SEM of at least four independent experiments. **P < 0.01 compared with control; ***P < 0.001 compared with control; ^§§§P < 0.001 compared with METH (250 μM); ^###P < 0.001 compared with MPP+ alone. (B) Representative pictures of the protective effects of ZM241385 (a) control; (b) ZM241385 (0.5 nM); (c) METH (250 μM); (d) METH (250 μM) plus ZM241385 (0.5 nM); (e) MPP+ (250 μM); (f) MPP+ (250 μM) plus ZM241385 (0.5 nM). Scale bar: 8.3 μm.

Figure 6

Neuroprotection induced by the A_2A antagonist FTBI is lost by the coadministration of the adenosine agonist NECA. PC12 cells were treated for 72 h with 250 μM METH in the absence and in the presence of 32 nM FTBI and different concentrations of NECA ranging from 50 to 200 nM. Effects of different treatments on cell viability were measured by using trypan blue staining. Results shown are expressed as means ± SEM of at least four independent experiments. **P < 0.01 compared with control.

Figure 7

Representative pictures of protective effects of FTBI against METH and MPP+ toxicity in SH-SY5Y cells. SH-SY5Y cells were treated with either METH (2.5 mM) or MPP+ (1.5 mM) in the absence and in the presence of FTBI (16 and 32 nM). These pictures are representative of numerical data matching those obtained in PC12 cells. (a) Control; (b) FTBI (16 nM); (c) FTBI (32 nM); (d) METH (2.5 mM); (e) METH (2.5 mM) plus FTBI (16 nM); (f) MPP+ (1.5 mM); (g) MPP+ (1.5 mM) plus FTBI (32 nM). Scale bar: 40 μm.