protective effects of nebivolol and reversal of endothelial dysfunction in diabetes associated with...

gy 570 (2007) 149–158www.elsevier.com/locate/ejphar

European Journal of Pharmacolo

Protective effects of nebivolol and reversal of endothelial dysfunctionin diabetes associated with hypertension

Adriana Georgescu a,⁎, Doina Popov a, Emanuel Dragan a, Elena Dragomir a, Elisabeta Badila b

a Institute of Cellular Biology and Pathology “N. Simionescu”, Bucharest, Romaniab Emergency Hospital, Bucharest, Romania

Received 16 February 2007; received in revised form 5 May 2007; accepted 22 May 2007Available online 5 June 2007

Abstract

This study aims to decipher the potential effects of nebivolol in prevention and/or regression of renal artery dysfunction in diabetes associatedwith hypertension. Renal arteries were isolated from 80 male mice divided into four experimental groups: (i) group D: diabetics, at 2months sincestreptozotocin injection; (ii) group Din: mice that at the initiation of streptozotocin diabetes were treated with 10mg/kg b.w./day nebivolol for2months, to test for the potential prevention of vascular dysfunction; (iii) group Dfin: mice that after 2months of diabetes were treated daily with10mg/kg b.w./day nebivolol for additional 2months, in order to follow the possible regression of the dysfunction, and (iv) controls (C), age-matched healthy animals. The following measurements were performed: arterial blood pressure, plasma glucose concentration, and the vascularreactivity of the renal arteries in response to noradrenaline (10− 4M), acetylcholine (10− 4M) and sodium nitroprusside (10− 4M). To assess themolecular mechanisms involved in the reactivity of the renal artery, the contribution of mitogen-activated protein kinase (MAP kinase) pathwayand of L-type voltage gated Ca2+ channels (in the contractile response to noradrenaline), of nitric oxide (NO) and Ca2+ activated K+ channels (inthe endothelium-dependent vasodilator response), and of cGMP (in the endothelium-independent vasodilator response) was examined by exposingthe arteries to corresponding inhibitors, and by using myograph and patch-clamp techniques, immunoblotting and NO assays. Results showed that,group D was characterized by hyperglycemia (blood glucose concentration: 136.66 ± 4.96mg/dl, a value ∼ 65% increased compared to group C)and hypertension (systolic blood pressure: 145.66 ± 5.96mm Hg, a value ∼ 34% increased compared to group C). Compared to group D, groupDin was characterized by diminished blood glucose concentration (∼ 1.6 fold), reduced systolic and diastolic blood pressure (∼ 1.3 fold) and heartrate (∼ 1.6 fold), as well as by increased contractile response of the renal artery to noradrenaline (∼ 1.84 fold) and of the impeded vasodilatorresponse to acetylcholine (∼ 1.81 fold) and sodium nitroprusside (∼ 1.42 fold). Together, these effects demonstrate that administration of 10mg/kgb.w./day nebivolol at the moment of diabetes induction has preventive effects, ameliorating diabetes dysfunctions. Compared to group D, groupDfin was characterized by diminished glucose concentration (∼ 1.3 fold), reduced systolic and diastolic blood pressure and heart rate (both ∼ 1.2fold), and by augmentation of contractile response of the renal artery to noradrenaline (∼ 1.62 fold) and of vasodilator response to acetylcholine(∼ 1.13 fold) and sodium nitroprusside (∼ 1.19 fold). These effects assess that administration of 10mg/kg b.w./day nebivolol after 2months ofdiabetes contributes to regression of diabetes-associated dysfunctionalies. Nebivolol influenced the molecular mechanisms involved in renal arteryreactivity in diabetic and hypertensive mice: it increased the NO production and endothelial NO synthase (eNOS) protein expression, decreasedthe expression of ∝ protein in L-type calcium channels and Ca2+ activated K+ channels, and diminished the MAP kinase activity. The reporteddata suggest that nebivolol may offer additional vascular protection for treating diabetes associated with hypertension.© 2007 Elsevier B.V. All rights reserved.

Keywords: Renal artery; Nebivolol; Vasodilation; Endothelial dysfunction; Diabetes; Hypertension

⁎ Corresponding author. Institute of Cellular Biology and Pathology “NicolaeSimionescu”, 8, BP Hasdeu Street, PO Box 35-14, 050568-Bucharest, Romania.Tel.: +40 1 319 4518; fax: +40 1 319 4519.

E-mail address: [email protected] (A. Georgescu).

0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.ejphar.2007.05.031

1. Introduction

Prevention of end-stage renal disease by early detectionand treatment is an important tool to stop the growing need forrenal replacement therapy. The last decade has brought strongreasons for such an approach. The incidence of end-stage

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http://dx.doi.org/10.1016/j.ejphar.2007.05.031

150 A. Georgescu et al. / European Journal of Pharmacology 570 (2007) 149–158

renal disease has increased dramatically, and is mostlyattributed to type 2 diabetes and atherosclerotic vasculardisease. The progression of the renal function loss is dictatedby several classical risk factors such as hypertension,proteinuria, obesity, smoking, and hyper- or dyslipidemia,all of which being potentially modifiable (Stuveling et al.,2005).

The β-blockers are a well-established class of drugs fortreating hypertension, and there is a substantial amount ofevidence from randomized controlled trials demonstrating theirbenefit in reducing morbidity and mortality in patients with highblood pressure (Bonomo and Phillips, 1998; Tzemos et al.,2001; Kuroedov et al., 2004). Nebivolol is a third generation β-adrenergic blocker that exerts benefits in treatment of patientswith heart failure (Veverka et al., 2006). It appears to be aseffective as other antihypertensive drugs, and possessesdistinctive pharmacologic properties (compared to other agentsin its class) such as a high specificity for the β1 adrenoceptorand vasodilatory effects. Nebivolol differs from conventionalnonvasodilating-blockers, such as atenolol; thus, nebivolol, butnot atenolol, causes vasodilatation in the human forearmvascular bed, an effect blocked by nitric oxide synthase(NOS) inhibitors (McEniery et al., 2004). Human studieswhere nebivolol was infused into phenylephrine preconstrictedsuperficial hand veins (Bowman et al., 1994) or into the brachialartery of healthy volunteers (Cockcroft et al., 1995) haveconfirmed that nebivolol has nitric oxide (NO) mediatedvasodilator effects exerted via endothelial L-arginine–NOpathway (Cockcroft, 2004). Recently, it was reported thatcompared with placebo, nebivolol provides significant reduc-tion of blood pressure from baseline values (Sule and Frishman,2006).

The efficacy of nebivolol in diabetes and hypertension is atopic of few recent studies. Thus, nebivolol and metoprololwere reported to be effective and safe antianginal agents inpatients with ischemic heart disease and hypertension combinedwith type 2 diabetes, and doses of 5–7.5mg/day nebivolol (for8weeks) had favorable metabolic and hemodynamic effects(Makolkin et al., 2003). More recently it was reported thatnebivolol and metoprolol provide anti-ischemic effects inpatients with coronary heart disease and postinfarction leftventricular dysfunction, associated with type 2 diabetes mellitus(Tepliakov et al., 2005).

It is known that the renal vasculature plays an important rolein the control of the blood pressure and that endothelium-derived hyperpolarizing factor-mediated vasodilation is im-paired in the renal microcirculation of hypertensive and diabeticrats (De Vriese et al., 2000). Although the use of blockers in thetreatment of hypertension is widespread, there is no study onthe effects of nebivolol on the renal artery dysfunction indiabetes associated with hypertension, a vascular bed ofimportance in the pathogenesis of renal disease as well as inhypertension. This study aims to decipher the potential action ofnebivolol in the potential prevention and/or regression of therenal artery endothelial dysfunction in diabetes associated withhypertension, and to define the molecular mechanismsinvolved.

2. Materials and methods

2.1. Animals

The study was conducted on 80 RAP mice (4–6months old)divided into four experimental groups: (i) diabetics (D), at2months since a single i.p. streptozotocin injection (150mg/kgbody weight); (ii) Din: D animals that simultaneously withdiabetes induction received (by gavage) a daily dose of 10mgnebivolol/kg body weight; this group was designed to test forthe potential prevention of endothelial dysfunction; (iii) Dfin: Dmice that after 2months received by gavage a daily dose of10mg nebivolol/kg body weight for additional 2months; thisgroup was designed to follow the possible regression of theendothelial dysfunction, and (iv) controls (C), age-matchedhealthy animals.

The systolic and diastolic arterial blood pressure and theheart rate were recorded in the presence of 10U/ml heparinusing a Physiological Pressure Transducer (model MLT844/D)connected to a PowerLab data acquisition unit (ADInstruments,Sydney, Australia). Arterial blood pressure (blood pressure) wasmeasured invasively by placing a cannula into abdominal arteryand connecting it to an electronic pressure transducer. For allgroups of investigated animals, the plasma glucose concentra-tion was assayed using enzymatic kits (Sigma Chemical Co.,MO, USA). The renal arteries were excised and functional,structural, and biochemical studies were subsequently carriedout. The experiments on the animals were performed inaccordance with “Principles of laboratory animal care” (NIHpublication no. 83-25, revised 1985).

2.2. Renal arteries preparation for the myograph technique

2.2.1. Experimental procedure for renal artery isolation anduse in the myograph technique

Immediately after animal sacrification, a laparotomy wasperformed and both renal arteries were excised together with thekidney, and were pinned down on Sylgaard resin in a Petri dishcontaining saline solution. Segments (2mm long) of the renalarteries were dissected out, two stainless steel wires (Ø: 40μm)were threaded through the lumen, and arteries were mounted in asmall vessel myograph (Model 410A, J.P. Trading, Denmark), asdescribed by Mulvany and Halpern (1977). The myographchamber was filled with HEPES salt solution (HPSS) containing(in mM): 5 Hepes, 140 NaCl, 4.6 KCl, 1.17 MgSO4, 2.5 CaCl2,and 10 glucose (Chulia et al., 1995; Thurston et al., 1995)maintained at 37°C and continuously gassed with O2. After anequilibration period of 20min, the arterieswere set to a normalizedinternal circumference at which they give the maximum of theisometric response, estimated to be 0.9 times the circumferencethey would have when relaxed and subjected to a transmuralpressure of 100mm Hg. The mean internal diameter of the renalarteries used was in the range of 340 ± 50μm.

2.2.2. The vascular reactivity of the renal arteriesThe vascular function of the renal arteries isolated from all

experimental groups in response to noradrenaline (10− 4M),

Fig. 1. (A) A representative calibration trace showing the responses of theelectrode after an increase of the temperature; (B) NO sensor calibration: arepresentative calibration trace showing the responses of the electrode afterimmersion in 10 nM and 40 nM freshly prepared NO solution. The current (pA)was converted to concentration (nM).

151A. Georgescu et al. / European Journal of Pharmacology 570 (2007) 149–158

acetylcholine (10− 4M) and sodium nitroprusside (10− 4M) wasinvestigated using the myograph technique. To measure therelaxation, arteries with intact endothelium were contracted innoradrenaline for 5min, and then exposed to acetylcholine (anendothelium-dependent vasodilator agent) or sodium nitroprus-side (an endothelium-independent vasodilator agent) for 5min.

To assess the molecular mechanisms involved in the vascularreactivity, specific inhibitors were added to the organ bath of themyograph. Thus, the contribution of MAP kinase pathway and ofL-type voltage gated calcium channels to the contractile responseto noradrenalinewas assayed in the presence of 14μMPD098,059and of 10− 6M nifedipine, respectively. The arteries were exposedto inhibitors for 10min, afterwards noradrenaline was added, andthe force developed by artery wall was recorded after 5min (Cainet al., 2002). The organ chamber was protected from light whenusing theMAP kinase inhibitor, PD098,059. The roles of NO andof Ca2+ activated K+ channels in the endothelium-dependentvasodilator response, and of cGMP in the endothelium-independent vasodilator response were examined by exposingthe noradrenaline precontracted vessels to 10− 4M L-NAME (NG-nitro-L-arginine methylester, a NO synthase inhibitor), 10− 3Mtetraethylammonium (TEA), and 10− 6M methylene blue (MB),respectively, after 10min incubation. The measurement of therelaxation was performed after additional 5min incubation inacetylcholine or sodium nitroprusside, as described above.

2.3. Renal arteries preparation for the patch-clamp technique

For the patch-clamp experiments, the renal arteries wereisolated (as above), mounted in the myograph chamber, and cutalong the long axis of the vessel. The artery fragment waspinned on the Sylgaard resin with the adventitial layer upwards,as described by White and Hiley (1998). Recordings on thesmooth muscle cells were carried out essentially as described byYamamoto et al. (1998). Briefly, the adventitial layer wasremoved from the smooth muscle layer by a short incubation for15min at 35°C with 0.5mg/ml collagenase A in Ca2+-freesolution containing (in mM): 141.5 NaCl, 5.4 KCl, 1 MgCl2, 10HEPES, 5 glucose, pH 7.3.

2.3.1. Electrophysiological techniques applied on the renalarteries

The patch-clamp whole-cell configuration was used torecord the changes in current intensity of intact renal arteriesisolated from all groups of experimental animals. Theenzymatic treatment described above allows the access to thesmooth muscle cells layer. Preparations with a resting potentialbetween − 30mV and − 60mV were used for experiments.Changes in current intensity were recorded in the voltage-clampmode. The latter were performed using WPC-100 amplifier(ESF electronic, Göttingen, Germany) and borosilicate glasspipettes (GC150T, Harvard Apparatus, Edenbridge, Kent, UK)pulled with a microprocessor controlled vertical pipette pullerPUL-100 (WPI Inc., Sarasota, Florida, USA) and heat polishedto a resistance of 2–8MΩ. Controlled stimuli were delivered,and the digital recordings were captured with pClamp 8 software(Axon Instruments, Union City, CA, USA) and DigiData 1200

Series Interface (Axon Instruments). The experiments wereperformed at 37°C, the bath temperature being controlled with atemperature controller TC-202A (Harvard Apparatus). Thecurrent intensity changes were recorded in order to establisheffects induced by diabetes/hypertension as well as of thenebivolol administration.

The involvement of Ca2+ activated K+ channels and of L-type voltage gated calcium channels was established byexposing the preparations (for 10min) to the inhibitors TEA(10− 3M) and nifedipine (10− 6M). Afterwards, the changes inthe current intensity were recorded in the voltage-clamp mode.

2.3.2. Solutions for the patch-clamp techniqueThe composition of standard bath solution for the patch-

clamp experiments was (in mM): 141.5 NaCl, 5.4 KCl, 1.8CaCl2, 1 MgCl2, 10 HEPES, 5 glucose, pH 7.3. The pipettesolution contained 120μM amphotericin B supplemented to asolution containing (in mM): 10 NaCl, 35 KCl, 60K2SO4, 10HEPES, 3.44 NaOH, 20 sucrose, 1 EGTA, pH 7.3.

2.4. Renal arteries preparation for the NO sensor technique

2.4.1. Experimental procedureFor the direct measurement of NO, both intact and de-

endothelized arteries (2mm long) were mounted in themyograph chamber, cut along the long axis of the vessel, andthen pinned on Sylgard resin either with the endothelium orwith the smooth muscle cells layer upwards, using theprocedure described by White and Hiley, 1998 (to avoidelectrical noise). Removal of the endothelium was performed byair bubbling (for 2min) of the arteries lumen (Kristova et al.,


2000). To assess that endothelium was completely removed, theartery lumen was additionally gently rubbed with a stainlesssteel wire while mounting the vessel in the myograph (Akataet al., 1995).

2.4.2. The measurement of endothelial cells NO productionNO sensor (amiNO-700; Innovative Instruments, Inc.,

Tampa, FL, USA) was connected to inNO-T, an NO-measuringsystem including a current monitor and data acquisitionsoftware with a temperature compensation function thatautomatically compensates sample temperature fluctuation(Fig. 1A). Calibration of the sensor was performed beforeeach experiment using known concentrations of freshlyprepared NO-saturated solution; a calibration curve demon-strating changes in current or peak height as a function of NOconcentration was traced (Fig. 1B). The in situ generation of NOwas achieved by the addition of a standard nitrite solution to anacidified solution in the presence of a reducing agent such as theiodide ion, according to the following equation:

2NO−2 þ 2I− þ 4Hþ→2NOþ I2 þ 2H2O

We used as calibration solution 20ml potassium iodide (1mg/ml) prepared in 0.1M sulfuric acid. A nitrite standard solutionwith a concentration of 10nM, respectively 40nM was added.As shown above, the molar ratio of nitrite to NO is 1:1, andconsequently, the amount of NO generated equals to the amountof nitrite added. The NO generated in this reaction allows thesensor calibration. With the sample bathed in 2ml HEPES saltsolution (HPSS), the NO sensor was placed perpendicularly tothe luminal side of the artery (upwards), in the close vicinity ofthe endothelial cells layer, in order to minimize diffusion path ofNO, and to obtain a high signal. The continuous monitoring ofthe current (in pA) or of NO production (in nM) by intactendothelium of the renal arteries was recorded in the presence of10− 4M acetylcholine, an endothelium-dependent vasodilatorthat has M2 muscarinic receptors on endothelial cells only, at10min interval. The de-endothelized renal arteries were exposedto 10− 4M sodium nitroprusside, an endothelium-independentvasodilator agent, and NO recorded. In controls, NO productionwas recorded in the presence of 10− 4M acetylcholine ± 10− 4ML-NAME at 10min intervals.

2.5. Immunoblotting and dot-blotting techniques

To investigate eNOS expression, mice renal arteries weredissected out, collected on ice-cold PBS, the blood andconnective tissue removed, and the vascular wall was cut invery small pieces. The latter were homogenized in lysis buffercontaining: 10mM Trisma-base, 5mM EDTA, 10 % Triton X-100, 25μM PMSF (phenyl methyl sulfonyl fluoride), and 1μMbenzamidine, pH 7.4. The membrane proteins were pelleted bycentrifugation for 15min at 9600g. The protein concentrationwas assessed by a solid phase method for the quantitation ofprotein in the presence of sodium dodecyl sulphate (SDS) andother interfering substances (Sheffield et al., 1987), and proteinswere separated by 8% SDS-polyacrylamide gel electrophoresis

(SDS-PAGE). Equivalent amounts (40μg) of total protein in therenal arteries from all groups of the experimental animals weresubjected to SDS-PAGE, and the samples were run at 120V for2h. Gels were subsequently transferred to nitrocellulosemembrane, at 54mA, for 45min. The membranes were washedin 0.05% Tween-20 in PBS, and blocked in PBS supplementedwith 5% BSA overnight, at 4°C. Afterwards, the membraneswere incubated for 1h with rabbit IgG anti-eNOS, and then,washed and incubated for 1h with goat anti-rabbit IgG coupledwith peroxidase. A monoclonal mouse antibody raised againstβ-actin was used as a lane-loading control. The bound antibodywas detected by enhanced chemiluminescence (ECL) on an X-ray film. The density of immunoreactive bands was measuredusing Scion Image Program, a microcomputer imaging system.

To investigate the expression of KCa and CaL channel α-subunit, the renal arteries were dissected (as above) and placedin ice-cold buffer containing (in mM) 50 Tris–HCl (pH 7.7), 0.1EDTA, 1 EGTA, 250 sucrose, 0.1% 2-mercaptoethanol, 10%glycerol, 1 PMSF, 1 pepstatin A, 2 leupeptin, and 0.1%aprotinin (Dimitropoulou et al., 2002). The arteries were mincedinto small pieces and homogenized on ice. The volume ofhomogenizing buffer was 200μl and the final volume was ∼1ml. The large tissue debris and the nuclear fragments wereremoved by two centrifuge spins (1000g for 10min, and14,000g for 15min) at 4°C and the supernate was furtherpelleted at 100,000g for 1h (Liu et al., 1997) to sediment themembranary materials. The protein concentration was assayedusing the Amido Black staining, with bovine serum albumin asstandard. The protein expression was assayed by a dot-blottingtechnique, using as first antibodies, polyclonals against α-subunit of the KCa and CaL channels (anti-potassium channelBKCa developed in rabbit), and anti-calcium channel (α1csubunit of voltage gated L-type Ca2+ channel, developed inrabbit). The secondary antibody was goat anti-rabbit IgGcoupled with peroxidase.

2.6. Reagents

Noradrenaline, acetylcholine, sodiumnitroprusside, L-NAME,nifedipine, tetraethylammonium, HEPES, endothelial anti-nitricoxide synthase rabbit (eNOS, 596–609), goat anti-rabbit IgG-peroxidase, rabbit polyclonal anti α913–926, (a sequence-directedantibody raised against amino acids 913 to 926 of theα-subunit ofthe KCa channel), mouse monoclonal antibody against β-actin,and the antibody raised against α-subunit of the CaL channel,were purchased from Sigma Chemical Co (St. Louis, MO, USA).Clostridium histolyticum collagenase A was from Roche(Switzerland). Nebivolol was from Berlin— Chemie. All othersreagents used were of analytical grade.

2.7. Data analysis

The tension developed by the noradrenaline exposed arterieswas expressed as active wall tension (mN mm− 1 artery length)and the relaxation induced in the presence of acetylcholine andsodium nitroprusside was given as % of noradrenaline inducedcontraction. To quantify results, one-way ANOVA test (a one-

Fig. 2. Representative recordings of the arterial blood pressure and of the heart rate in experimental groups: (A) group C, control, (B) group D: at 2 months sincestreptozotocin injection, (C) group Din: mice that at the inception of streptozotocin diabetes were treated with 10 mg/kg b.w./day nebivolol for 2 months, to test forpotential preventive effects, (D) group Dfin: mice that after 2 months diabetes were treated daily with 10 mg/kg b.w./day nebivolol for additional 2 months, in order tofollow the possible regression.

Table 1The systolic and diastolic arterial blood pressure and the cardiac rhythm (BMP)in the experimental groups: Din+nebivolol, mice treated daily with nebivolol (toprevent vascular dysfunction); Dfin+nebivolol, D mice that after 2 months weretreated daily with nebivolol for 2 months (to follow the possible regression ofvascular dysfunction)

Experimentalgroups

Control Diabetic Din+nebivolol

Dfin+nebivolol

Systolic bloodpressure(mmHg)

108.78±7.43 145.66±5.96 110.99±7.9 120.67±11.13

Diastolic bloodpressure(mmHg)

85.23±8.82 125.86±8.49 90.81±11.84 101.0±9.69

BMP 290.91±13.24 440.31±17.07 333.09±15.60 340.99±8.16


way Analysis of Variance) was used. The data were consideredsignificant when P b 0.05. The patch-clamp data were analyzedusing the pClamp 8 (Axon Instruments) program. All valueswere expressed as mean ± S.E.M.

3. Results

3.1. The effect of nebivolol on the plasma glucoseconcentrations

In controls, plasma glucose concentration was 82.78 ±4.43mg/dl. In group D, glucose concentration was 136.66 ±4.96mg/dl, a value ∼ 65% augmented compared to group C.After nebivolol administration, in group Din glucose concen-tration was 86.99 ± 5.91mg/d, and in group Dfin, it was 105.67 ±6.13mg/dl. Thus, nebivolol administration in diabetic micecaused the diminishment of glucose concentration by ∼ 36.35%in group Din (prevention group), and by∼ 22.67% in group Dfin(regression group) (P b 0.05).

3.2. The effect of nebivolol on arterial blood pressure

Fig. 2 shows representative recordings of the arterial bloodpressure and of the heart rate in the experimental groups.

Quantitative evaluations showed that compared to C group, in Dgroup both systolic and diastolic arterial blood pressuresignificantly increased, i.e. by ∼ 1.3 fold and ∼ 1.4 fold,respectively, while the heart rate was ∼ 1.6 fold augmented(Table 1). Nebivolol administration reduced the increases ofboth systolic and diastolic blood pressure by∼ 1.3 fold in groupDin (prevention) and ∼ 1.2 fold in group Dfin (regression);additionally, it diminished the heart rate by ∼ 1.6 fold for

Fig. 3. The involvement of MAP kinase pathway and of L-type voltage gatedCa2+ channels in the contractile response of the renal arteries to 10−4 Mnoradrenaline (NA).


prevention group, and ∼ 1.2 fold for regression group (Table 1)(P b 0.05).

3.3. The effect of nebivolol on the renal artery dysfunction

The contractile response of the renal arteries to 10− 4Mnoradrenaline was measured by the myograph technique. Ingroup C, the artery wall developed a force of 3.2 ± 0.45mN/mm;in group D, the contractile force was 2.06 ± 0.33mN/mm,demonstrating an impeded functionality, while in groups inwhich nebivolol was administrated the contractile responsemeasured 3.9 ± 0.64mN/mm and 3.35 ± 0.29mN/mm in groupsDin and Dfin, respectively. These values were statisticallysignificant compared to those measured in group D (Fig. 3).

The contribution of MAP kinase pathway and of L-typevoltage gated calcium channels in the contractile response tonoradrenaline was assayed after 10 and 20min exposure of thearteries to the specific inhibitors 14μM PD098,05 and 10− 6Mnifedipine supplemented to the organ bath of the myograph.Thereafter, 10− 4M noradrenaline was added, and the forcerecorded after 5min. Experiments were performed in parallel at10min and at 20min incubation. Since the registered results

Fig. 4. The involvement of NO and of Ca2+ activated K+ channels in thevasodilator response of the renal arteries to 10−4 M acetylcholine (ACh).

were similar at both time points, and considering also the resultsreported byCain et al.(2002) we decided to select the 10minincubation period. The results showed that in group C thearterial wall tension measured 1.78 ± 0.37mN/mm, in thepresence of PD098,059 and 1.27 ± 0.52mN/mm in the presenceof nifedipine, demonstrating blockage of MAP kinase pathwayand of L-type voltage gated calcium channels, respectively (P b0.05). In group D, both inhibitors were ineffective, and thecontractile force developed by arteries was 2.25 ± 0.41mN/mmin the presence of PD098,059 and 2.20 ± 0.39mN/mm in thepresence of nifedipine. In group Din (prevention) the values ofthe arterial wall tension were 2.51. ± 0.81mN/mm in thepresence of PD098,059 and 2.99 ± 0.34mN/mm in the presenceof nifedipine (P b 0.05). In the group Dfin (regression) thevalues were 2.34 ± 0.66mN/mm and 2.28 ± 0.47mN/mm,respectively (P b 0.05) (Fig. 3).

The myograph technique was used also to measure therelaxation of the renal artery wall after precontraction in 10− 4Mnoradrenaline. Exposed to 10− 4M acetylcholine for 5min, renalarteries in C group showed a relaxation representing 13.93 ±1.39 % from the noradrenaline precontraction, while in D groupthe relaxation was 5.03 ± 2.32 % (P b 0.05). Nebivololadministration in Din group (prevention) increased relaxation to15.89 ± 4.29 %, and in Dfin group it represented 8.76 ± 0.25 %from the noradrenaline of precontaction (P b 0.05) (Fig. 4).

The involvement of NO and of Ca2+ activated K+ channels inthe endothelium-dependent vasodilator response was examinedby incubation of noradrenaline precontracted vessels with 10− 4ML-NAME and 10− 3M TEA, respectively for 10min. In C group,the vasodilator response of the arteries to acetylcholine decreasedto 2.3 ± 0.97% in the presence of L-NAME, and to 5.29 ± 2.11% inthe presence of TEA (P b 0.05). In D group, TEA did not modifythe arteries relaxation to acetylcholine, whereas L-NAME blockedit completely (Fig. 4). At the animals treated with nebivolol theeffect of the inhibitors (L-NAMEandTEA)was similar to that inCgroup, nomatter prevention or regression experiments performed.

To measure the endothelium-independent relaxation to 10− 4Msodium nitroprusside, arteries were contracted in 10− 4Mnoradrenaline for 5min, and then exposed to sodium nitroprussidefor additional 5min. In C group, the results indicated a relaxation of90.45 ± 5.89%, while in D group the relaxation measured 63.45 ±

Fig. 5. The involvement of cGMP in the vasodilator response of the renal arteriesto sodium nitroprusside (SNP).

Fig. 6. The membrane current intensity of smooth muscle cells in the absence and in the presence of 10−3 M TEA in: (A) group C; (B) group D; (C) group Din(prevention); (D) group Dfin (regression).

Fig. 7. (A) A representative immunoblot assessing protein expression of eNOSin all groups of experimental mice; (B) β-actin was run on the same transfers aspositive and internal control.


14.23% from the noradrenaline precontraction (P b 0.05)(Fig. 5). At diabetic mice treated daily with nebivolol, therelaxation to sodium nitroprusside increased to 90.53 ± 8.46%in Din group (prevention), and to 75.89 ± 25.39% in Dfingroup (regression) (P b 0.05) (Fig. 5).

The involvement of cGMP in the endothelium-independentvasodilator response to sodium nitroprusside was investigatedin noradrenaline precontracted arteries exposed to 10− 6M MB.After 10min incubation in the presence of MB, sodiumnitroprusside was added in the organ bath for 5min, and arteriesrelaxation was measured. The results showed that in group Crelaxation measured 53.90 ± 8.27% from noradrenalineprecontraction, in group D it represented 49.14 ± 28.58%, inDin group (prevention) it was 72.49 ± 26.24 %, and in Dfingroup (regression) the relaxation was 50.3 ± 30.27% (P b 0.05)(Fig. 5).

3.4. The effect of nebivolol on endothelial cells NO production

The continuous monitoring of NO production at the level ofendothelial layer of renal arteries was performed after 10minexposure to 10− 4M acetylcholine. The results showed that ingroup C, NO concentration produced by the endothelial layerrepresented 21.37 ± 0.31nM; in D group it was 14.23 ± 1.49nM,while after nebivolol administration in group Din NOconcentration was 19.97 ± 0.6nM, and in group Dfin it was17.1 ± 1.5nM. The results were statistically significant. Toprove that in the experiments performed endothelial layer was

intact, 10− 4M acetylcholine was supplemented with 10− 4M L-NAME, and no NO production was recorded. In separateexperiments, carried out on group C arteries have been de-endothelised and exposed to either acetylcholine (10− 4M) orsodium nitroprusside (10− 4M) for 10min. There was no NOreleased in the presence of acetylcholine, whereas in the

Fig. 8. A representative dot blot to compare the expression of∝ protein in L-type calcium channels and Ca2+ activated K+ channels in renal arteries of group C and ingroups D, Din (for testing potential prevention effects of nebivolol) and Dfin (for the potential regression effects).


presence of sodium nitroprusside concentration of NOwas 20.9 ±0.45nM.

3.5. The effect of nebivolol on the membrane current intensityof smooth muscle cells

Using patch-clamp technique in the whole-cell configura-tion, the changes in membrane current intensity of smoothmuscle cells were recorded in the voltage-clamp mode in groupsC, D, Din, and Dfin (Fig. 6). The contribution of Ca2+ activatedK+ channels and of L-type voltage gated calcium channels at themembrane current intensity was investigated using inhibitorsTEA (10− 3M) and nifedipine (10− 6M), respectively after10min exposure. Compared to C group, the smooth muscle cellsmembrane current intensity was significantly decreased for Dgroup, while at Din and Dfin groups it remained unchanged.The inhibitor effect of TEA on the membrane current wassignificantly pronounced for the arteries isolated from C grouponly (Fig. 6A). Nifedipine exposure induced similar effects withthose of TEA (data not shown).

3.6. The effect of nebivolol on the molecules in signalingpathways underlying renal artery dysfunction

The immunoblotting technique was used to investigateeNOS protein expression in renal arteries (Fig. 7). In D group,densitometry of eNOS protein expression showed mean valuesof 49.85 ± 0.76% (considering controls as 100%) (n = 8 distinctexperiments); thus, streptozotocin diabetes caused a significantdiminution of eNOS expression (Fig. 7A). Immunoblottingexperiments for β-actin assessed equal protein loading of the

gel (Fig. 7B). Nebivolol administration increased eNOS proteinexpression by ∼ 45% in group Din (prevention) and by ∼ 20%in group Dfin (regression). The dot blot technique was used tocompare the protein expression of ∝ protein in L-type calciumchannels and Ca2+ activated K+channels in renal arteries ofgroups C, D, Din and Dfin. The results in Fig. 8 showed thepresence of both subunits in the membrane fraction, and suggestapparent increases of the protein expressions in D group, anddecreases in both Din and Dfin groups (Fig. 8).

4. Discussion

As a general remark, renal arteries relaxation to acetylcholinewas small in the mouse experimental model used, representing14% of noradrenaline contraction, a value in agreement with10.43±0.7% recorded for the renal arteries of single transgenicnon-diabetic mice (Radu et al., 2004). In a previous study wereported that nebivolol had a reversible vasodilator effect onrenal arteries, and that this process involved endothelial β2-adrenoceptor ligation, with a subsequent rise in [Ca2+]i, eNOS-dependent NO and EDHF production; the latter passed throughmyoendothelial gap-junctions and activated Ca2+ activated K+

channels in smooth muscle cells (Georgescu et al., 2005). Wesuggested this may be an important mechanism underlyingnebivolol-induced arterial dilation.

There are no previous studies on the effect of nebivolol onrenal artery dysfunction in diabetes associated with hyperten-sion, an issue with potential consequences for renal disease. Toget a deeper insight into nebivolol effects, we investigated inthis study the potential efficiency of nebivolol in prevention andregression of the renal artery endothelial dysfunction in diabetes


associated with hypertension, and tried to disclose further themolecular mechanisms involved. The results show that in micewith streptozotocin diabetes associated with hypertension,nebivolol treatment lowers plasma glucose concentration,blood pressure and heart rate, and protects and partial reversesendothelial dysfunction. In rats, previous reports showed thatstreptozotocin injection produced not only the cardinalsymptoms of diabetes mellitus, like loss of body weight,hyperglycemia, and hypoinsulinemia, but also a significantelevation of blood pressure and renal dysfunctions (Muraliet al., 2003). In agreement with this, we demonstrate in thispaper that in mice streptozotocin injection produced increases insystolic and diastolic blood pressure, augmented the heart rate,and also increased plasma glucose concentration.

A new finding is that nebivolol administration, either at thevery moment of diabetes induction (group Din) or after 2 monthsof diabetes (group Dfin) reduced significantly the increases ofblood pressure and of heart rate. The blood pressure loweringeffects of nebivolol are due to its NO donor properties (Thuillezand Richard, 2005), to interaction with the endothelial NOpathway, increasing NO (Ignarro, 2004), and to ROS-scaveng-ing action of the molecule (de Groot et al., 2004). The beneficialeffects of nebivolol therapy were assessed in animal models ofhypertension (SHR rat) (Guerrero et al., 2003), as well as inhypertensive subjects (Thuillez and Richard, 2005).

Another result of this study is that nebivolol diminishedhyperglycemia (induced by streptozotocin) in the mice modelused. Although we did not investigate the mechanism(s)underlying this effect, Britt et al. (2006) reported recently thatnebivolol administration could participate in the reversion ofcardiovascular structural changes associated with the insulin-resistance syndrome.

The results in D group with associated hypertension (Fig. 2B),demonstrate a diminished reactivity of the mice renal arteries inresponse to 10−4 M noradrenaline, 10−4 M acetylcholine, and10−4 M sodium nitroprusside, and that oral administration of10mg/kg b.w./day nebivolol partially reversed these dysfunctions(Figs. 3–5). In another animal model such as SHR rat, it wasreported that the chronic antihypertensive effect of nebivolol wasaccompanied by an improvement in vascular structure andfunction, and in the cardiac hypertrophy index (Guerrero et al.,2003). In humans, short-term intrabrachial administration ofnebivolol evoked endothelial-dependent vasodilation (Daweset al., 1999). The antioxidant properties of nebivolol can explainits inhibitory effects on endothelial dysfunction, and thediminishment of reperfusion-induced myocardial injury (Kuroe-dov et al., 2004; Mason et al., 2006). In addition, it was reportedthat nebivolol possesses not only direct vasodilator properties, butalso augments the action of endothelium-dependent vasodilators,indicating nebivolol as a promising therapeutic tool for thetreatment of arterial hypertension and chronic heart failure(Kuroedov et al., 2004).

Related to the nebivolol intracellular pathway it was reportedthat this drug enhances NO bioavailability, eNOS andphospholipase C activities, and increases intracellular freecalcium concentrations (Parenti et al., 2000; Kuroedov et al.,2004). The precise mechanism by which nebivolol improves the

renal artery dysfunction in diabetes associated with hyperten-sion is less known. To clarify this, the contribution of MAPkinase pathway and of L-type voltage gated Ca2+ channels (inthe contractile response to noradrenaline), of nitric oxide (NO)and Ca2+ activated K+ channels (in the endothelium-dependentvasodilator response), and of cGMP (in the endothelium-independent vasodilator response) was examined by exposingthe arteries to inhibitors such as PD098,059, nifedipine, L-NAME, TEA, and MB, respectively. We are aware that TEAand MB are not selective blockers. However, in response toanother endothelium-dependent vasodilator, bradykinine, K+

channels in vascular smooth muscle cells and endothelial cellswere blocked by millimolar concentrations TEA, although thesechannels have a distinct pharmacology, (Jackson, 2005). Inaddition, it was reported that 5 μM MB significantly inhibitedaortic rings relaxations induced by peroxynitrite, supporting theconclusion that ONOO− triggered relaxation is mediated byelevation of cGMP levels (Li et al., 2005).

Taken together, the results of this study indicate that oraladministration of 10 mg/kg b.w./day nebivolol for 2 months indiabetic-hypertensive mice has potential benefic effects,regressing the renal artery dysfunctions: it increases the NOproduction and eNOS protein expression, decreases theexpression of ∝ protein in L-type calcium channels and Ca2+

activated K+ channels, and diminishes the MAP kinase activity.

Acknowledgements

We appreciate the dedicated work and the help of Sanda Nae(technician), who had a good care on the experimental animals aswell as of Marcela Toader and Marilena Isachi (biochemistry).

The electrophysiological experiments were performed underthe guidance of Professor Maria-Luiza Flonta at the “Multi-UserResearch Center in Molecular Physiology” of Faculty ofBiology, University of Bucharest.

This study was supported by grants from the RomanianAcademy and the “National Program VIASAN” of Ministry ofEducation and Research, Romania.

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