The favorable kinetics and balance of nebivolol-stimulated nitric oxide and peroxynitrite release in human endothelial cells

Background: Nebivolol is a third-generation beta-blocker used to treat hypertension. The vasodilation properties of nebivolol have been attributed to nitric oxide (NO) release. However, the kinetics and mechanism of nebivololstimulated bioavailable NO are not fully understood. Methods: Using amperometric NO and peroxynitrite (ONOO-) nanosensors, beta 3-receptor (agonist: L-755,507; antagonists: SR59230A and L-748,337), ATP efflux (the mechanosensitive ATP channel blocker, gadolinium) and P2Y-receptor (agonists: ATP and 2-MeSATP; antagonist: suramin) modulators, superoxide dismutase and a NADPH oxidase inhibitor (VAS2870), we evaluated the kinetics and balance of NO and ONOO-stimulated by nebivolol in human umbilical vein endothelial cells (HUVECs). NO and ONOO-were measured with nanosensors (diameter +/- 300 nm) placed 5 +/- 2 mu m from the cell membrane and ATP levels were determined with a bioluminescent method. The kinetics and balance of nebivolol-stimulated NO and ONOO-were compared with those of ATP, 2-MeSATP, and L-755,507. Results: Nebivolol stimulates endothelial NO release through beta 3-receptor and ATP-dependent, P2Y-receptor activation with relatively slow kinetics (75 +/- 5 nM/ s) as compared to the kinetics of ATP (194 +/- 10 nM/ s), L-755,507 (108 +/- 6 nM/ s), and 2-MeSATP (105 +/- 5 nM/ s). The balance between cytoprotective NO and cytotoxic ONOO-was expressed as the ratio of [NO]/[ONOO-] concentrations. This ratio for nebivolol was 1.80 +/- 0.10 and significantly higher than that for ATP (0.80 +/- 0.08), L-755,507 (1.08 +/- 0.08), and 2-MeSATP (1.09 +/- 0.09). Nebivolol induced ATP release in a concentrationdependent manner. Conclusion: The two major pathways (ATP efflux/P2Y receptors and beta 3 receptors) and several steps of nebivololinduced NO and ONOO-stimulation are mainly responsible for the slow kinetics of NO release and low ONOO-. The net effect of this slow kinetics of NO is reflected by a favorable high ratio of [NO]/[ONOO-] which may explain the beneficial effects of nebivolol in the treatment of endothelial dysfunction, hypertension, heart failure, and angiogenesis.