Mitochondria-targeted antioxidant effects of S(-) and R(+) pramipexole

Abstract

Background: Pramipexole exists as two isomers. The S(-) enantiomer is a potent D3/D2 receptor agonist and is extensively used in the management of PD. In contrast, the R(+) enantiomer is virtually devoid of any of the DA agonist effects. Very limited studies are available to characterize the pharmacological spectrum of the R(+) enantiomer of pramipexole.

Results: Using differentiated SH-SY5Y neuroblastoma cells as an experimental model, here we show that S(-) and R(+) pramipexole are endowed with equipotent efficacy in preventing cell death induced by H2O2 and inhibiting mitochondrial reactive oxygen species generation. Both pramipexole enantiomers prevented mitochondrial ROS generation with a potency about ten times higher then that elicited for neuroprotection.

Conclusions: These results support the concept of both S(-) and R(+) pramipexole enantiomers as mitochondria-targeted antioxidants and suggest that the antioxidant, neuroprotective activity of these drugs is independent of both the chiral 6-propylamino group in the pramipexole molecule and the DA receptor stimulation.

Figure 1

Neuroprotective effects of pramipexole (PPX) enatiomers against H₂O₂-induced cell death. A) Differentiated SH-SY5Y cells were exposed to different concentrations of S(-) PPX (gray bars) and R(+) PPX (black bars) for 1 h before being exposed to 1 mM H₂O₂ for 10 min. Cell viability was evaluated 24 h after by MTT assay B) Cells were exposed to 50 μM μM S(-) PPX or R(+) PPX in the presence or absence of 10 μM haloperidol (H) or 10 μM (-) sulpiride (S). Data represent means ± SEM of at least three different experiments and are from three separate cell preparations. *, p < 0.01 vs H₂O₂ alone values.

Figure 2

Detection of mitochondrial ROS generation. A) Upper panel. Representative pictures from cells exposed to increasing laser intensity, as indicated. CM-DCF fluorescence intensity (green) was selectively recorded in mitochondria (red). Lower panel. Cells were preincubated with Vitamin E (2 ng/100 μl) for 30 min before the laser excitation (white bars in panel B). Fluorescence emission intensity was calculated as average grey level value per pixel and corrected for background. Bars in panel B represent the means ± SEM of at least three different experiments and are from three separate cell preparations. *, p < 0.01 vs the corresponding control values.

Figure 3

Inhibition of mitochondrial ROS generation by pramipexole (PPX) enantiomers. Cells were exposed to different concentrations, as indicated in the bottom, of S(-) PPX (upper panel) and R(+) PPX (lower panel) for 1 h before being exposed to laser. Mitochondrial ROS generation was evaluated as in figure 2. Data represent means ± SEM of at least three different experiments and are from three separate cell preparations. *, p < 0.05 and **, p < 0.001 vs the corresponding control values (black bars).

Figure 4

Lack of effect of DA receptor antagonists on the inhibition of mitochondrial ROS generation induced by pramipexole (PPX) enantiomers. Cells were exposed to 10 μM S(-) PPX (gray bars) and R(+) PPX (white bars) for 1 h in the absence or presence of 10 μM haloperidol (H) or 10 μM (-) sulpiride (S) before being exposed to laser. Mitochondrial ROS generation was evaluated as in figure 2. Data represent means ± SEM of at least three different experiments and are from three separate cell preparations. *, p < 0.01 vs the corresponding control values (black bars).