Introduction

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Nebivolol is a recently developed -blocker devoid of intrinsic sympathomimetic activity (Janssens et al., 1989) and provided with selectivity for ₁-adrenoceptors and vasodilator properties (Janssens et al., 1991). The drug is a mixture of equal amounts of two enantiomers, namely d-nebivolol (SRRR configuration) and l-nebivolol (RSSS configuration). Whereas the -adrenoceptor-blocking property resides in the d-enantiomer, its vasorelaxant activity is almost completely attributable to l-nebivolol (Janssens et al., 1991). Unlike most traditional -blockers, nebivolol induces in vivo an acute decrease in peripheral vascular resistance associated with a prompt reduction in blood pressure; this effect is mainly mediated by l-nebivolol (Van De Water et al., 1988a,b). Moreover, the l-form potentiates the hypotensive response to d-nebivolol (Xhonneux et al., 1990). Thus, the drug profile is characterized by a distinctive hemodynamic response that, in turn, is largely due to the presence of the l-form in the racemate. Experimental evidence suggests that its vasodilator effect is attributable to an endothelium-dependent mechanism involving nitric oxide (NO) generation by endothelial cells. This hypothesis stems from studies showing that the vasodilator response to the drug is blunted by endothelium removal and by NO synthase (NOS) inhibitor administration (Gao et al., 1991; Bowman et al., 1994; Cockcroft et al., 1995).

These studies were carried out mainly in isolated dog coronary arteries in vitro and in the human dorsal hand vein and forearm vasculature in vivo. Other preparations used include venous preparations and conduit arteries, such as the saphenous vein of the dog and the caudal artery and aorta of the rat (Janssens et al., 1991). However, studies on resistance vascular beds, characterized by a high neurogenic control of vascular tone, whose function can be more relevant for hemodynamic regulation, are not available. Therefore, in this study, we have characterized the vascular effects of nebivolol in an isolated resistance vascular bed, such as the mesenteric vascular bed preparation from the rat. Because the mechanism of the putative endothelium-dependent effect of this -adrenoceptor blocker has not been completely elucidated, the effects of nebivolol on cell messengers and on NOS activity have been investigated in cultured microvascular endothelial cells.

	Materials and Methods

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Indomethacin, 9,11-dideoxy-11,9-epoxymethano-prostaglandin F₂ (U46619), noradrenaline hydrochloride, cyclo--dextrin, N-nitro-L-arginine methyl ester hydrochloride (L-NAME), N-nitro-D-arginine methyl ester hydrochloride (D-NAME), bradykinin (BK), Dowex 50WX8-400 resin, Dulbecco's modified Eagle's medium (DMEM), lithium chloride, thapsigargin (TG), L-arginine, HEPES (sodium salt), HEPES (free acid), and acetylcholine were purchased from Sigma (St. Louis, MO). Bovine calf serum was purchased from Hyclone (Logan, UT). [³H]Arginine and myo-2-[³H]inositol were purchased from Amersham Life Science Ltd. (Buckinghamshire, UK). 1-(6((17-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122) and 1-(6-((17-3-methoxyestra-1,3,5(10)-trien-17-yl) amino)hexyl)-2,5-pyrrolidine-dione (U73343) were purchased from Biomol (Plymouth Meeting, PA). Anion exchange columns were prepared with AG-K8 (200-400 mesh, formate form) from Bio-Rad Laboratories (Richmond, CA). dl-Nebivolol, l-nebivolol, and d-nebivolol were gifts from Menarini Ricerche S.p.A. (Florence, Italy). Before dilutions, indomethacin was dissolved in ethanol; U73122, U73343, and TG were dissolved in dimethyl sulfoxide; and nebivolol and its enantiomers were dissolved in cyclo--dextrin solution. The other drugs were dissolved in double-distilled water.

Isolated Mesenteric Vascular Bed of the Rat. Mesenteric vascular beds were isolated from Wistar rats weighing 200 to 250 g, and the superior mesenteric artery was cannulated with a stainless steel cannula. To eliminate the blood from the vessels, the preparations were flushed with a modified Krebs' solution composed of 120 mM NaCl, 5 mM KCl, 1.2 mM MgSO₄, 25 mM NaHCO₃, 2.4 mM CaCl₂, and 5 mM glucose. The preparations were placed in an organ bath warmed to 37°C on a piece of titanic steel mesh and perfused with the same solution at a constant rate (4 ml/min) with a peristaltic pump. The solution (pH 7.3-7.4) was prewarmed and oxygenated with a gas mixture (5% CO₂, 95% O₂). Changes in the perfusion pressure were measured with a pressure transducer and recorded on a polygraph. To evaluate a drug-induced vasorelaxant effect, the preparation was precontracted by perfusion with concentrations of noradrenaline (30 µM) or of the thromboxane mimetic U46619 (0.3 µM) that was able to induce a vasoconstrictor response of similar degree. The presence of functional endothelium was assessed by testing the vasodilator effect of acetylcholine (ACh); the preparations in which ACh (1 µM) reduced the perfusion pressure by less than 30% were not used for the study. Cumulative concentration-response curves were obtained in preconstricted preparations; the effect of each concentration of nebivolol was followed for 10 min. In the experiments in which the influence of L-NAME on the response to nebivolol was tested, the NOS inhibitor was administered 30 min before testing the drug effect. The endothelium deprivation was performed by the method of perfusion with distilled water for 10 min, a procedure that is able to selectively destroy endothelial cells and induce effects similar to those obtained by rubbing off the endothelium (Bolton et al., 1984; Criscione et al., 1984). The lack of a relaxation response to ACh in preconstricted vessels indicated that the procedure was successful. The baseline perfusion pressure after perfusion with noradrenaline or U46619 was taken as the 100% value, and the reduction in perfusion pressure induced by relaxing agents was referred to this value.

Cell Culture. Bovine coronary postcapillary venular endothelial cells from bovine heart (CVEC) were obtained and maintained in culture as previously described (Schelling et al., 1988). Endothelial cells were characterized by immunofluorescent staining for factor VIII antigen and uptake of acetylated low density lipoproteins. Cells between passages 15 and 25 were used in the experiments.

NOS Activity. NOS activity was tested in CVEC monolayers according to the previously described method (Ghigo et al., 1995). The enzyme activity was evaluated by measuring the amount of L-[³H]citrulline produced after administration of L-[³H]arginine. Cells were seeded onto 60-mm culture dishes. Equilibration for 20 min at 37°C with HEPES buffer was followed by cell incubation for 30 min with 10 µM L-arginine and 20 min with 1 µCi of L-[³H]arginine. Cells were exposed to the test drug for 5 min at 37°C, then cold HEPES buffer was added to stop the reaction. After the addition of ethanol and 10 mM HEPES-Na at pH 5.5, the amount of L-[³H]citrulline produced was assayed by liquid scintillation counting after elution through a resin column (Dowex 50WX8-400 activated sodium-form).

Inositol Phosphate Metabolism. The method used in these experiments has been previously described (Ziche et al., 1993). Endothelial cells were seeded onto six multiwell plates (8 × 10⁴ cells/well) and, after overnight incubation, were labeled with myo-[³H]-inositol (2 µCi/ml) in DMEM containing 10% bovine calf serum, without cold inositol, for 48 h. Tritiated myo-inositol excess was removed by three washes with cold DMEM, followed by 4 h incubation with cold DMEM at 37°C. After washing, cells were incubated for 10 min with 20 mM LiCl to block myo-inositol-1-phosphatase and then was exposed to test compounds for the designated times. The reaction was stopped by adding ice-cold methanol for 30 min. Cells were scraped, and cell-associated inositols were recovered by chloroform-methanol (1:1) extraction. Water-soluble fractions were applied to anion exchange columns (Resin AG-K8, 200-400 mesh, formate form), and water-soluble inositols were eluted by successive washes with 60 mM CH₃NO₂ + 5 mM B₄Na₂O₇ · 10H₂O, 200 mM CH₅NO₂ + 0.1 M CH₂O₂, and 1.2 M CH₅NO₂ + 0.1 M CH₂O₂. Inositol monophosphate (IP₁) levels were measured as recovered radioactivity and expressed as dpm/well or in percent over basal. Each experiment was performed in duplicate.

Statistical Analysis. All results are means ± S.E. of n experiments. Statistical analyses were performed with Student's t tests for paired or unpaired data, with ANOVA followed by Fisher's least significant difference tests to evaluate the differences between groups. P < .05 was taken as significant.

	Results

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Effect of Nebivolol on the Preconstricted Rat Mesenteric Vascular Bed. The exposure of the preparations to noradrenaline (30 µM) and the thromboxane analog U46619 (0.3 µM) induced a stable increase in vascular tone. In fact, the perfusion pressure rose from 25.8 ± 6.1 to 86 ± 4.8 and 29.1 ± 6.1 to 88 ± 8.1 mm Hg after noradrenaline and U46619, respectively. The addition of increasing concentrations of dl-nebivolol to preconstricted preparations induced a vasorelaxant effect that was evident at concentrations above 3 µM and was concentration-dependent between 3 and 30 µM. Tachyphylaxis to the relaxant effect of nebivolol did not occur. The maximum relaxing effect observed with 30 µM dl-nebivolol consisted of a reduction in the perfusion pressure by 43.4 ± 2.8% (Fig. 1). The degree of vasodilator response induced by dl-nebivolol in noradrenaline-preconstricted preparations was smaller than that induced by ACh. In fact, the perfusion pressure was reduced by 17.2 ± 2.3% by 10 µM dl-nebivolol, and by 65.6 ± 2.3% by the same concentration of ACh (not shown in the figure). The maximum vasorelaxant effects obtained after dl-nebivolol and ACh consisted of reductions in perfusion pressure by 43.4 ± 2.8 and 65.6 ± 2.3%, respectively. The vasorelaxant response to dl-nebivolol developed slowly and reached the maximum value after 6 to 7 min. Drug concentrations of 1 and 3 µM had no effect on the vessel tone. Because the degree of relaxation induced by the drug was similar in the preparations preconstricted by noradrenaline and in those preconstricted by U46619, the successive experiments were carried out in preparations pretreated with the thromboxane analog. In preparations preconstricted by U46619, the pattern of the response to the l-enantiomer was not significantly different from that observed with the racemate; conversely, d-nebivolol was almost completely ineffective (Fig. 2). This observation prompted us to perform the successive investigations using the racemate.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Percent decrease in perfusion pressure induced by increasing concentrations of dl-nebivolol in preparations preconstricted by 30 µM noradrenaline and 0.3 µM U46619. Points represent the mean value of at least five experiments; vertical bars indicate S.E.M. values.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Percent decrease in perfusion pressure induced by increasing concentrations of dl-nebivolol (n = 8), l-nebivolol (n = 8), and d-nebivolol (n = 4) in preparations preconstricted by 0.3 µM U46619. Points represent the mean values; vertical bars indicate S.E.M. values. *P < .05 versus d-nebivolol; °P < .05 versus d-nebivolol.

Effect of Endothelium Deprivation, NOS Inhibition, U73122, and TG Administration on the Response to Nebivolol. In preparations denuded of endothelium, the vasorelaxant effect induced by nebivolol concentrations above 3 µM was changed to a potent concentration-dependent vasoconstrictor response that developed at drug concentrations between 3 and 30 µM (Fig. 3). The vasorelaxant effect of nebivolol in preparations preconstricted by U46619 (0.3 µM) was completely antagonized by treatment with a 100 µM concentration of the NOS inhibitor L-NAME (Fig. 4A). Pretreatment of the preparations for 30 min with the same concentration of the NOS inhibitor not only induced an inhibition of the relaxing response to nebivolol but also caused the appearance of a contractile response (Fig. 4B). This kind of response was not potentiated by an increase in L-NAME concentration up to 1 mM (not shown in the figure). The vasorelaxant response to nebivolol was not influenced by a pretreatment of the preparations with the cyclooxygenase inhibitor indomethacin at a concentration of 3 µM (data not shown). However, in preparations pretreated with both 100 µM L-NAME and indomethacin, nebivolol induced a concentration-dependent contractile response with a shape similar to that observed in endothelium-denuded preparations (Fig. 4B). The exposure of the preparations for 30 min to the phospholipase C inhibitor U73122 (1 µM) was able to completely block the vasorelaxant effect of nebivolol. Conversely, U73343, a molecule structurally close to U73122, which does not inhibit phospholipase C (Jin et al., 1994), did not significantly influence the pattern of response to nebivolol (Fig. 5A). Finally, the relaxant effect of nebivolol was completely antagonized by the endoplasmic reticulum Ca²⁺-ATPase inhibitor TG (1 µM, Fig. 5B). It is noteworthy that the concentrations of L-NAME, indomethacin, U73122, and TG used in these experiments were not able to significantly change the baseline tone of the preparations.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Changes in perfusion pressure induced by increasing concentrations of dl-nebivolol in intact preparations and in endothelium-denuded preparations. The preparations were preconstricted by exposure to 0.3 µM U46619. Points represent the mean value of at least four experiments; vertical bars indicate S.E.M. values, *P < .05 versus intact preparations.

View larger version (9K): [in this window] [in a new window]   Fig. 4.   A, typical experiment showing the relaxant effect of increasing concentrations of dl-nebivolol in a preparation preconstricted by 0.3 µM U46619 and the reversal induced by L-NAME (100 µM) administration. B, effects of 100 µM L-NAME and of 100 µM L-NAME plus 3 µM indomethacin on the relaxant response to increasing concentrations of dl-nebivolol in preparations preconstricted by 0.3 µM U46619. Points represent the mean value of at least four experiments; vertical bars indicate S.E.M. values.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   A, effect of a 30-min period of exposure to 1 µM U73122 or 1 µM U73343 on the relaxing response to increasing concentrations of dl-nebivolol in preparations preconstricted by 0.3 µM U46619. Points represent the mean value of four experiments. Vertical bars indicate S.E.M. values; when not indicated, they are included in the symbols, *P < .05 versus control. B, effect of a 30-min period of exposure to 1 µM TG on the relaxing response to increasing concentrations of dl-nebivolol in preparations preconstricted by 0.3 µM U46619. Points represent the mean value of four experiments. Vertical bars indicate S.E.M. values; when not indicated, they are included in the symbols, *P < .05 versus control.

Effect of Nebivolol on Inositol Phosphate Metabolism in Cultured Endothelial Cells. To identify the cellular events involved in nebivolol mechanism of action, the effect of the exposure of stabilized cultures of CVEC to the drug was investigated. CVEC were chosen among the available endothelial cell models, because these cells are isolated from a microcirculatory bed (Schelling et al., 1988), which may be relevant for the nebivolol pharmacological activity. Moreover, we have previously demonstrated that CVEC possess only the constitutive isoform of NO-synthase (cNOS), which is a Ca²⁺/calmodulin-dependent enzyme and is sensitive to endothelium-dependent vasorelaxant agents (Ziche et al., 1994; Parenti et al., 1998). The cellular level of the inositol triphosphate (IP₃) metabolite IP₁ was tested after endothelial cell contact with different concentrations of nebivolol for 15 min. The basal level of IP₁ was not affected by concentrations of nebivolol ranging from 100 nM to 1 µM; however, 10 and 30 µM concentrations of nebivolol significantly increased IP₁ levels by 78.2 ± 19.9 and 561 ± 124%, respectively. It is notable that the effect of the higher concentration of nebivolol tested (30 µM) was comparable with that induced by substances previously shown able to stimulate inositol phosphate metabolism in endothelial cells, such as BK (Ziche et al., 1993). In fact, a 100 nM concentration of the peptide increased the cellular level of IP₁ by 530 ± 104% (Fig. 6).

View larger version (33K): [in this window] [in a new window]   Fig. 6.   Effect of the exposure (15 min) to 10 and 30 µM dl-nebivolol on IP1 levels in cultured endothelial cells. The effect of 100 nM BK is shown for comparison. Columns indicate the mean values of four experiments in duplicate; vertical bars indicate S.E.M. values, *P < .05, **P < .01 versus control unstimulated cells.

Effect of Nebivolol on NOS Activity in Cultured Endothelial Cells. The cNOS activity of the cells was almost doubled by a 5-min contact with 10 µM nebivolol; this effect was almost identical with that induced by 100 nM BK (Fig. 7A). It is noteworthy that atenolol, at the same concentration (10 µM), was devoid of any stimulating effect on cNOS activity (data not shown). The exposure of endothelial cells to L-NAME (2 mM) or to TG (1 µM) impaired basal cNOS activity and significantly prevented cNOS activation in response to 10 µM nebivolol. Conversely, D-NAME (2 mM) did not affect either basal or nebivolol-stimulated cNOS activity (Fig. 7B).

View larger version (21K): [in this window] [in a new window]   Fig. 7.   A, effect of the exposure (5 min) to 10 µM dl-nebivolol on cNOS of cultured endothelial cells. The effect of 100 nM BK is shown for comparison. Columns indicate the mean values of two experiments in duplicate; vertical bars indicate S.E.M. value, *P < .05 versus control unstimulated cells. B, effect of 2 mM L-NAME, 2 mM D-NAME, and 1 µM TG on cNOS activity in untreated cultured endothelial cells and in cells treated with 10 µM dl-nebivolol for 5 min. Columns indicate the mean values of two experiments in duplicate; vertical bars indicate S.D. value, *P < .05 versus control untreated cells.

	Discussion

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The present findings obtained in an in vitro model of a resistance vascular bed, the rat mesenteric vascular beds, confirm that nebivolol has a vasorelaxant action in a range of micromolar concentrations identical with that previously shown in isolated canine coronary arteries (Gao et al., 1991). The relaxant effect of nebivolol is clearly dependent on the ability of endothelium to generate NO, because it is blunted by pretreatment of the preparations with the NOS inhibitor L-NAME, at a concentration (100 µM) that also effectively antagonizes the endothelium-dependent vasodilatory effect of ACh in the same kind of preparation (Mantelli et al., 1995). Moreover, both in endothelium-denuded preparations and in those pretreated with L-NAME plus indomethacin, the vasorelaxant effect of nebivolol changes to a concentration-dependent contractile response, with a behavior similar to that previously reported for ACh (Furchgott and Zawadzki, 1980). Therefore, to obtain more information about the mechanism involved in the drug effect on vascular tissue, we tested the influences of U73122 and TG on the relaxant effect of nebivolol. U73122 is an aminosteroid compound that has been shown able to inhibit calcium mobilization induced by phospholipase C stimulation in different cells (Bleasdale et al., 1990; Yule and Williams, 1992; Jin et al., 1994; Grierson and Meldolesi, 1995). TG is an inhibitor of the Ca²⁺-ATPase of the sarcoplasmic reticulum in various kinds of cells (Thastrup et al., 1990; Lytton et al., 1991; Dolor et al., 1992), which is able to inhibit the agonist-stimulated release of NO from endothelial cells and the relaxing response to ACh (Macarthur et al., 1993). The TG concentration used in this study (1 µM) was the same as that employed in other studies in which the drug was used to deplete the IP₃-sensitive calcium stores in sarcoplasmic reticulum (Low et al., 1991; Macarthur et al., 1993; Amerini et al., 1996). The observation that 1 µM TG completely blocked the vasodilator effect of nebivolol, after a 30-min pretreatment period previously shown sufficient to antagonize the relaxant response to ACh (Amerini et al., 1996), confirms that an endothelium-dependent mechanism is involved in the drug effect. In fact, it is widely accepted that the synthesis of endothelium-dependent relaxing factor is a calcium-dependent process (Luckhoff et al., 1988) and that in endothelial cells, constitutive NOS is a calcium/calmodulin-dependent enzyme (Berdeaux, 1993). Endothelium-dependent relaxing factor release induced by ACh, as well as by BK and by adenosine diphosphate and substance P, is triggered by an increase in cellular free calcium concentration (Busse et al., 1988; Berdeaux, 1993; Ziche et al., 1993). By emptying and preventing the refilling of IP₃-sensitive cellular calcium stores, TG is able to prevent the increase in intracellular free calcium levels induced by vasorelaxing endothelium-dependent agents (Macarthur et al., 1993; Amerini et al., 1996) and to block the agonist-induced release of NO by isolated vascular preparations, thus suppressing the vasodilator effect induced by ACh and other endothelium-dependent agents that act through phosphoinositide turnover stimulation (Guard and Watson, 1991; Jones, 1993; Tropea et al., 1993). Therefore, the findings that both U73122 and TG suppressed the vasorelaxant effect of nebivolol points to phospholipase C activation, and consequent inositol phosphate metabolism stimulation, as a possible mechanism to explain the vasorelaxant effect of nebivolol.

The results obtained in this study confirm this hypothesis by showing that nebivolol, at the same concentrations at which it induces a vasodilator response, is able to increase the cellular level of the IP₃ metabolite IP₁ in endothelial cells. In agreement with this observation, we also provide evidence that the activity of cNOS is increased by an intermediate concentration of nebivolol, such as 10 µM. It was beyond the scope of this study to investigate the mechanism by which phospholipase C activity is stimulated by nebivolol. However, two hypotheses seem possible, namely that the effect of nebivolol on inositol phosphate metabolism is due to a direct stimulation of phospholipase C or that it is mediated by an action on receptors linked to stimulation of the activity of this enzyme. The latter hypothesis seems particularly plausible, because it has been shown that micromolar concentrations of nebivolol have affinity for various kinds of receptors different from -adrenoceptors (Van De Water et al., 1988b).

In conclusion, the results of this study present evidence for the first time that the vasodilator effect of nebivolol is associated with activation of phosphoinositide turnover with consequent stimulation of cNOS activity in endothelial cells. It may be noted that these effects have been detected by the present in vitro study with a range of nebivolol concentrations higher than the nanomolar concentrations able to induce -adrenoceptor-blocking effects in vivo (Janssens et al., 1991).