Characterization of Nitric Oxide Release by Nebivolol and Its Metabolites

AJH 2006; 19:579 –586 Blood Vessels Characterization of Nitric Oxide Release by Nebivolol and Its Metabolites Angelo Maffei, Carmine Vecchione, Alessandra Aretini, Roberta Poulet, Umberto Bettarini, Maria Teresa Gentile, Giuseppe Cifelli, and Giuseppe Lembo Methods: Conductance and resistance arteries from Wistar-Kyoto rats (WKY) (n ⫽ 33) incubated with the fluorescent probe diaminofluorescein-2 (DAF-2) were stimulated with increasing concentrations of nebivolol or its enantiomers and metabolites, and NO release was histologically evaluated. Results: Nebivolol induced a dose-dependent increase in NO levels in the endothelium of both arteries. Levels of NO were significantly increased at 10⫺6 mol/L and reached a plateau state at 10⫺5 mol/L. Induction of NO is not a general action of ␤-adrenoceptor antagonists, as atenolol had no effects. Nebivolol action on NO release was mainly caused by the D-isomer. Moreover NO production is also maintained after hepatic metabolism, as the three main metabolites of nebivolol were able to induce a significant increase in endothelial NO release. Finally, nebivolol-activated calcium mobilization is crucial to NO production. Conclusion: Our study shows the effects of D-nebivolol and its metabolites on endothelial NO pro- duction in both conductance and resistance arteries, and clarifies that this effect is realized through a calciumdependent mechanism. Am J Hypertens 2006;19: 579 –586 © 2006 American Journal of Hypertension, Ltd. Key Words: Nitric oxide, ␤-adrenoceptor antagonists, endothelium. ebivolol is a third-generation ␤-adrenoceptor antagonist that differs from earlier drugs in the same class, both because of its ability to antagonize selectively ␤1-adrenergic receptors and because of its hemodynamic properties contributing to its antihypertensive action.1 Although most ␤-adrenoceptor antagonists have the undesirable side effect of increasing peripheral vascular resistance, nebivolol has been shown to decrease this parameter both acutely, inasmuch as it determines vascular relaxation,2,3 and chronically, as it contrasts endothelial dysfunction.4 Several studies have attributed the vasorelaxant action of nebivolol, which is not mediated by its main pharmacodynamic profile as an adrenergic receptor antagonist, to the stimulation of nitric oxide (NO) release from vascular endothelium.5,6 However, the molecular mechanisms that could explain this proposed action of nebivolol on NO release have not been clarified. In this regard, NO release can be induced by two different N intracellular mechanisms. These involve the activation of the enzyme endothelial nitric oxide synthase (eNOS) either by its interaction with the Ca2⫹– calmodulin complex, or by its calcium-independent phosphorylation by the kinase Akt/ PKB. A previous study suggested that nebivolol may induce NO production in endothelial cells via a calciumdependent mechanism.7 On the other hand, another work suggests that nebivolol-induced vasodilatory activity could depend on certain calcium-independent pathways.8 Moreover another report9 has implied that a direct action of nebivolol on NO should be ruled out, suggesting that only some hepatic metabolites, and not the parent drug, should be accounted for in NO production by activating ␤2-adrenergic receptors. In contrast, activation of ␤-receptors has been excluded by other researchers,10 whereas both nebivolol and its 4-keto metabolite have been proposed to act on NO bioavailability only indirectly, by antagonizing its oxidant speciesinduced catabolism.11 Recently we measured NO production on vascular tis- Received June 13, 2005. First decision August 5, 2005. Accepted September 8, 2005. From the Department of AngioCardioNeurology (AM, CV, AA, RP, UB, MTG, GC, GL), IRCCS Neuromed, Pozzilli (IS), and Department of Experi- mental Medicine and Pathology (GL), La Sapienza University, Rome, Italy. Address correspondence and reprint requests to Dr. Giuseppe Lembo, IRCCS Neuromed, Località Camerelle, 86077 Pozzilli (IS), Italy; e-mail: © 2006 by the American Journal of Hypertension, Ltd. Published by Elsevier Inc. 0895-7061/06/$32.00 doi:10.1016/j.amjhyper.2005.09.021 Background: Nebivolol is a selective ␤1-adrenergic receptor antagonist that causes a direct vasodilator effect attributed to the action on vascular nitric oxide (NO). This study aimed to investigate whether nebivolol or its metabolites induces NO production and to explore the mechanisms underlying this pharmacologic effect. 580 AJH–June 2006 –VOL. 19, NO. 6 NEBIVOLOL AND NITRIC OXIDE sue through the use of a fluorescent probe.12 In this work we have used this technique, coupled with molecular biology and physiology studies, to characterize the effect on NO production by nebivolol or its hepatic metabolites, exploring the mechanisms underlying this pharmacologic effect. Methods Animals Evaluation of NO Production by DAF-2 Production of NO was assessed as previously reported.12 Briefly a large conduit artery such as thoracic aorta and a resistance artery such as the second branch of mesenteric artery were incubated for 2 h in the dark in aerated Krebs buffer containing DAF-2 diacetate (DAF-2DA, 10 ␮mol/L; Alexis Biochemicals, San Diego, CA). Nebivolol racemate and its enantiomers, nebivolol metabolites A4OH-nebivolol, A4=OH-nebivolol and A6H-nebivolol, atenolol (10⫺8 to 10⫺4 mol/L), or acetylcholine (10⫺9 to 10⫺6 mol/L) were administered in the last 30 min of DAF-2 incubation, alone and after 20 min exposure to N␻-nitro-L-arginine methyl ester (L-NAME) (300 ␮mol/L) or to the ␤1/␤2 antagonist propranolol (10⫺5 mol/L). To evaluate whether NO production by nebivolol was calcium dependent, some experiments were performed in presence of 1 mmol/L EGTA in calcium-free buffer. Vascular rings were cut in 10-␮m-thick sections and observed under a microscope with ⫻200 magnification. Colored pictures (24-bit) were taken at three different wavelengths and were merged so that the 515-nm emitted fluorescence caused by DAF appeared green above a purple all-wavelengths– emitted autofluorescence background derived from vascular fibers. Green fluorescence intensity was quantified using imaging software after subtraction of NO-independent autofluorescence from L-NAME–treated vessels. Plasmid Transfection Carotid arteries were used for these experiments, as the lack of collateral vessels departing from it blocks the plasmid inside the artery during pressure-induced transfection. To avoid damage to carotid arteries by decapitation, rats were anaesthetized with intraperitoneal tribromoethanol (300 mg/kg), and carotid arteries were Vascular Reactivity Vascular reactivity was evaluated by Mulvany myography as previously described.13 Briefly vessels were suspended in isolated tissue baths filled with Krebs solution at 37°C connected to an isometric force transducer. Passive tension was gradually increased to 1 g. Precontraction was elicited with phenylephrine or, in calcium-f (...truncated)