The Beta-1-Receptor Blocker Nebivolol Elicits Dilation of Cerebral Arteries by Reducing Smooth Muscle [Ca2+]i

Abstract

Rationale: Nebivolol is known to have beta-1 blocker activity, but it was also suggested that it elicits relaxation of the peripheral arteries in part via release of nitric oxide (NO). However, the effect of nebivolol on the vasomotor tone of cerebral arteries is still unclear.

Objective: To assess the effects of nebivolol on the diameter of isolated rat basilar arteries (BA) in control, in the presence of inhibitors of vasomotor signaling pathways of know action and hemolysed blood.

Methods and results: Vasomotor responses were measured by videomicroscopy and the intracellular Ca2+ by the Fura-2 AM ratiometric method. Under control conditions, nebivolol elicited a substantial dilation of the BA (from 216±22 to 394±20 μm; p<0.05) in a concentration-dependent manner (10-7 to 10-4 M). The dilatation was significantly reduced by endothelium denudation or by L-NAME (inhibitor of NO synthase) or by SQ22536 (adenylyl cyclase blocker). Dilatation of BA was also affected by beta-2 receptor blockade with butoxamine, but not by the guanylate cyclase blocker ODQ. Interestingly, beta-1 blockade by atenolol inhibited nebivolol-induced dilation. Also, the BKCa channel blocker iberiotoxin and KCa channel inhibitor TEA significantly reduced nebivolol-induced dilation. Nebivolol significantly reduced smooth muscle Ca2+ level, which correlated with the increases in diameters and moreover it reversed the hemolysed blood-induced constriction of BA.

Conclusions: Nebivolol seems to have an important dilator effect in cerebral arteries, which is mediated via several vasomotor mechanisms, converging on the reduction of smooth muscle Ca2+ levels. As such, nebivolol may be effective to improve cerebral circulation in various diseased conditions, such as hemorrhage.

Fig 1

Changes in basal diameter of basilar artery (BA) in response to endothelium denudation (Fig 1(A); n = 10; E+ endothelium intact; E- endothelium denuded), NO synthase inhibitor L-NAME (Fig 1(C); n = 5), adenylyl cyclase inhibitor SQ22536 (Fig 1(D); n = 9), guanylate cyclase inhibitor ODQ (Fig 1(E); n = 12) and increasing concentration (10⁻⁶ to 10⁻⁴ M) of beta-1 adrenoceptor blocker atenolol (Fig 1(G); n = 10). To assess the vascular function acetylcholine (Ach) and sodium nitroprusside (SNP) test-doses were applied before and after the endothelial denudation (Fig 1(B); n = 10; E+ endothelium intact; E- endothelium denuded). Summary data of diameter changes (normalized diameter [PD%]) of BA in response to ACh (10⁻⁴ M) and SNP (10⁻⁴ M) in the presence of guanylate cyclase inhibitor ODQ ((Fig 1(F); n = 7). Changes in basal diameter of BA (Fig 1(H)) in response to beta-2 adrenoceptor antagonist butoxamine (BTXN; n = 5), BK_Ca channel blocker iberiotoxin (IBTX; n = 6), and K_Ca channel blocker tetraethyl-ammonium chloride (TEA; n = 10). Data are mean ± S.E.M. (*significant compared to control; p<0.05).

Fig 2

Original record of diameter changes [μm] of a rat basilar artery in response to nebivolol 10⁻⁵ M as a function of time (Fig 2(A)). Summary data of diameter changes (as a % of passive diameter; [PD%]) of basilar artery (BA) in response to increased concentrations of nebivolol (10⁻⁷–10⁻⁴ M). EC₅₀ = 7.813 ± 0.19 x 10⁻⁶ M. Data are mean ± S.E.M. (*indicate significant changes compared to control; p<0.05; n = 9 (Fig 2(B)).

Fig 3. Summary data of diameter changes (normalized dilation; % of passive diameter; [PD%]) of BA in response to nebivolol (10⁻⁵ M) in the presence of guanylate cyclase inhibitor ODQ, beta-2 selective adrenoceptor antagonist butoxamine (BTXN), large conductance calcium-activated potassium channel antagonist iberiotoxin (IBTX), K_Ca channel antagonist tetraethyl-ammonium chloride (TEA),endothelium denudation, NO synthase inhibitor L-NAME, adenylyl cyclase inhibitor SQ22536 and beta-1 selective adrenoceptor antagonist atenolol.

Data are mean ± S.E.M. ($ indicate significant change compared to basal diameter, p<0.05; * indicate significant changes compared to nebivolol, p<0.05; # indicate significant change between nebivolol vs atenolol, p<0.05).

Fig 4

Representative pictures of Fura-2 AM fluorescence of ex vivo preparation of BA in absence (control; Fig 4(A)) and presence (Nebi; Fig 4B)) of nebivolol (10⁻⁵ M). Green color indicates low vascular Ca²⁺ level and red color indicates high vascular Ca²⁺ level. Summary data of changes in ratiometric signal (delta R) indicates decreasing vascular [Ca²⁺]_i of BA in response to increasing concentrations of nebivolol (10⁻⁸–10⁻⁵ M). Data are mean ± S.E.M. *indicate significant changes between nebivolol 10⁻⁷ M and 10⁻⁵ M; p<0.05); n = 9 (Fig 4(C)).

Fig 5. Original record shows the vasomotor function of perivascular hemolyzed blood (HB) and the vasomotor function of additional increased concentrations of nebivolol (from 10⁻⁷ M to 10⁻⁵ M) of basilar artery.

The diameter was not followed continuously in Fig 5(A). Summary data of changes in normalized diameter (% of passive diameter; [PD%]) of basilar arteries in response to hemolyzed blood (HB) and in response to nebivolol (from 10⁻⁷ to 10⁻⁵ M) in the presence of HB; n = 6. Data are mean ± S.E.M. (* indicate significant difference compared to basal diameter, p<0.05; # indicate significant difference between HB and nebivolol in the presence of HB, p<0.05); Fig 5(B).

Fig 6. Proposed mechanisms of action of nebivolol (NB)-induced dilation of cerebral arteries (E, endothelium; SMC, vascular smooth muscle cell).

These findings demonstrate that: 1) in isolated cerebral arteries nebivolol elicits significant dilations, 2) which may be—in part—due to beta-2 adrenoceptor (B2-R) mediated, endothelium-dependent NO and cAMP mechanisms resulting in either reduced [Ca²⁺]_i, and smooth muscle hyperpolarization. 3) On the other hand, its action seems to be mediated—in part—by beta-1 (B1-R) specific blocking ability connected with parallel induced vasodilation with endothelium derived hyperpolarization. 4) contribution of cGMP and other ion channels seem to be less important. These findings can contribute to a better understanding of the complex effects of this beta-1-receptor blocker on cerebral circulation and implicate important novel therapeutic potentials to improve cerebral blood flow in diseased conditions.