Effect of DL-nebivolol, its enantiomers and metabolites on the intracellular production of superoxide and nitric oxide in human endothelial cells.

Nebivolol, a third generation selective beta(1)-adrenoceptor (beta(1)-AR) antagonist, has been reported to reduce intracellular oxidative stress and to induce the release of nitric oxide (NO) from the endothelium. Nebivolol is also subjected to a complex metabolic process where glucuronidation, aromatic and alicyclic hydroxylation are the major pathways leading to several metabolites. We have studied the effect of nebivolol, its enantiomers and metabolites on intracellular oxidative stress and NO availability in human umbilical vein endothelial cells (HUVECs). Furthermore, since the receptors involved in this endothelial effect of nebivolol remain controversial, we have studied this matter by the use of antagonists of beta-AR. dl-Nebivolol, d-nebivolol and l-nebivolol significantly reduced the formation of reactive oxygen species (ROS) and superoxide induced by oxidized-low density lipoprotein (ox-LDL), although the racemic and l-form were significantly more active than d-nebivolol in this activity. A marked decrease in the availability of intracellular NO was found in HUVECs exposed to ox-LDL and this parameter was normalized by the prior incubation with dl-nebivolol, d-nebivolol and l-nebivolol; the effect of racemate was mainly mimicked by its l-enantiomer. eNOS activity significantly increased by a 5-min contact of HUVECs with dl-nebivolol and l-nebivolol, but not with d-nebivolol, and a similar pattern was observed when the intracellular calcium increase was measured. The metabolites A2, A3', A12 and A14 but not A1, A3 and R 81,928, derived from different metabolic pathways, retained the antioxidant activity of the parent racemic compound dl-nebivolol, reducing the intracellular formation of ROS and superoxide. The effects of dl-nebivolol on intracellular formation of NO, eNOS activity and intracellular Ca(2+) were partially antagonized by the antagonists of beta(1-2)-AR nadolol or by the beta(3)-AR antagonist SR59230A and further antagonized by their combination or by (beta(1-2-3)-AR antagonist bupranolol. In conclusion, this study shows that the NO releasing effect of nebivolol is mainly due to its l-enantiomer; the racemate and its enantiomers possess a remarkable antioxidant activity that contributes to its effect on the cellular NO metabolism and the activation of beta(3)-AR through a calcium dependent pathway is involved in the mechanisms leading to the NO release.