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The Effects of Calcium Channel Blockers are not Related to their Chemical Structure in the Collar Model of the Rabbit M Yasa, Z Kerry, B Reel, G Yetik Anacak, E Ertuna and A Ozer Journal of International Medical Research 2007 35: 59 DOI: 10.1177/147323000703500106 The online version of this article can be found at: http://imr.sagepub.com/content/35/1/59

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The Journal of International Medical Research 2007; 35: 59 – 71

The Effects of Calcium Channel Blockers are not Related to their Chemical Structure in the Collar Model of the Rabbit M Yasa, Z Kerry, B Reel, G Yetik Anacak, E Ertuna

and

A Ozer

Department of Pharmacology, Faculty of Pharmacy, Ege University, Izmir, Turkey Placing a silicone collar around the rabbit carotid artery induces intimal thickening, an early stage in atherosclerosis and restenosis. We investigated whether treatment with oral pranidipine, a new potent, longlasting dihydropyridine calcium channel blocker (CCB), inhibited collar-induced intimal thickening in addition to the changes in vascular reactivity usually observed in this model. Pranidipine treatment did not inhibit collar-induced intimal thickening. Placing the collar around the carotid artery resulted in the characteristic changes in vascular

reactivity, such as increased sensitivity to 5-hydroxytryptamine. Treatment with Nω-nitro-l-arginine (100 μM) and prani dipine, however, did not affect collarinduced changes in vascular reactivity. From results of this and previous studies, we conclude that pranidipine does not prevent collar-induced intimal thickening or collar-induced changes in vascular reactivity. Not all CCBs prevent collarinduced intimal thickening, suggesting that the effects of these agents are not related to their chemical structure and/or their calcium channel-blocking actions.

KEY WORDS: Pranidipine; Calcium channel blockers; Atherosclerosis; Rabbit collar model; Intimal thickening

Introduction

placement model, intimal thickening tends to occur within the collar and there is no involvement of the proximal or distal uncuffed artery. The underlying mechanism of the development of the formation of cuffinduced intimal thickening is still under investigation.3 – 5 Using this model, changes in vascular reactivity of the cuffed vessels to vasoconstrictor and vasodilator agents have been demonstrated, which occurred independently of the intimal thickening.6,7

Smooth muscle cell proliferation and migration are considered to play a major role in the development of intimal thickening, an early stage in the progression of atherosclerosis and re-stenosis. Experimental models in which intimal thickening has been induced using either transluminal (e.g. wire denudation)1 or extravascular (e.g. perivascular collar placement)2 arterial manipulation have been used widely. In the perivascular collar

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M Yasa, Z Kerry, B Reel et al. Pranidipine and the rabbit collar model With regard to the involvement of calcium in cellular processes such as proliferation and migration, the effects of calcium channel blockers (CCBs) in the development of atherosclerosis have always been considered a subject worthy of investigation.8,9 Although contradictory findings have been observed in a number of studies, the effects of CCBs on the development of intimal thickening and atherosclerosis are still being widely investigated.10 – 12 In this context, pranidipine (OPC13340; methyl 3-phenyl-2 [E]-propenyl 1,4-dihydro-2,6-dimethyl-4-[3-nitrophenyl]3,5-pyridinedicarboxylate) is a new potent and long-lasting dihydropyridine CCB that is being developed clinically as an antihypertensive and anti-anginal drug.13,14 The potential effectiveness of pranidipine on the atherosclerotic process has not been studied. The objective of this study was to investigate the effects of oral pranidipine (3 mg/kg per day)15 on collar-induced intimal thickening and the accompanying changes in vascular reactivity in the rabbit carotid artery.

food pellets (Yemta Tari¸s Yem Sanayii, Izmir, Turkey) and tap water, under standardized conditions of light (12 h light/12 h dark cycle) and temperature (22 – 25˚C).

RABBIT COLLAR MODEL After 7 days of treatment with pranidipine or placebo, the rabbits were anaesthetized with intravenous sodium pentobarbital (30 mg/kg). The left carotid artery was accessed surgically and a non-occlusive, flexible silicone collar 2 cm long was placed around it.2 The right carotid artery was sham-operated on by separating it from the surrounding connective tissue and vagus nerve, and stretching it to the same extent as the contralateral carotid artery. The carotid arteries were then returned to their original positions and the incisions were closed. After the animals had recovered from anaesthesia, they were kept in their individual cages for a further 2 weeks before tissue isolation.

MORPHOMETRY After anticoagulation with intravenous heparin (150 units/kg), the rabbits (n = 14) were killed with an overdose of sodium pentobarbital.11 Isolated vessel segments from sham-operated and collared arteries were cut into three pairs of rings, each one 4 mm long; one pair of rings was used for morphometry and two pairs were used for organ chamber experiments. The pair of rings to be used for morphometry was immediately placed in formalin fixative solution (4%) for 24 h, dehydrated in a graded series of isopropyl alcohol (60 – 100%) followed by toluol, before being embedded in paraffin. Transverse sections were cut and stained with Sirius red haematoxylin and eosin. Images of two randomly chosen transverse segments were recorded at × 4 magnification using a computer-assisted image analysis system that included a light

Materials and methods ANIMALS

The animal experiments were carried out in accordance with guidelines described by the Ethics Committee of the Faculty of Pharmacy, Ege University (Izmir, Turkey). White rabbits of both sexes of an inbred strain (weight 2 – 2.5 kg) (n = 16) were obtained from the Animal Breeding Facility of the Faculty of Pharmacy. The animals were randomly divided into two groups: the treatment group (n = 8) received oral pranidipine (3 mg/kg per day) and the placebo group (n = 8) received the vehicle orally (polyethyleneglycol, 2.5 ml/kg per day). Throughout the 3-week treatment period, each rabbit was kept in a separate cage and allowed free access to commercial

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M Yasa, Z Kerry, B Reel et al. Pranidipine and the rabbit collar model microscope (BH-2; Olympus, Tokyo, Japan) and video cameras (TK-890 E; JVC, Tokyo, Japan; and Sony, Tokyo, Japan). The images were processed using computer and image analysis software (CorelDRAW, 4.00.A5; Corel Corporation, Minneapolis, Minnesota, USA) and Autocad 12-c1; (Autodesk, San Rafael, California, USA).11 The images viewed on the monitor were outlined by drawing, and the areas of lumen, intima and media of the segments were measured. The ratio of intimal to medial cross-sectional area (index) was also calculated.11

at 37°C continuously oxygenated with 95% O2/5% CO2. Krebs solution contained (in mM): NaCl, 118; KCl, 4.7; CaCl2, 2.5; KH2PO4, 1.2; MgSO4, 1.2; NaHCO3, 25; glucose, 11.1.11,12 In order to examine the effects of the inhibition of nitric oxide (NO) synthesis on the contractile activities of the rings, Nω-nitro-l-arginine (LNA) (100 μM)7 was added to Krebs solution (one ring with LNA; one ring without LNA). Indomethacin was added to the Krebs solution (3 μM) to inhibit endogenous prostanoid synthesis.5 Isometric contractile force development was measured by means of a Grass FT3 force transducer and recorded by a Polywin 95 1.0 (Commat Ltd, Ankara, Turkey) by means of a microcomputer (IBM, PS/1). After 15 min of equilibration, tissues were gradually stretched to a tension of 7 g, a previously determined optimum resting tension based on the length–tension relationship, and left to equilibrate for 45 min.11 During this period, the bath solution was changed every 15 min. Acetylcholine (ACh)-induced endotheliumdependent vasorelaxant responses resulting from the release of NO were tested at the end of the equilibration period. For this purpose, arterial rings were contracted with phenylephrine (1 μM) and, during plateau contraction, ACh was added in a cumulative manner (0.001 – 100 μM). Tissues that relaxed by more than 40% of the initial contraction (indicative of the functional presence of endothelium) were washed three times with Krebs solution and contracted with increasing concentrations of 5-hydroxytryptamine (5-HT; 0.001 – 30 μM) and phenylephrine (0.001 – 100 μM). Each agonist was washed out by changing the bath solution three times during a 30-min period before the next agonist was added. The concentration–response relationships of 5-HT-, phenylephrine- and

BLOOD PRESSURE MEASUREMENTS Systolic and diastolic arterial blood pressures were measured before the application of the collar on day 8 and at the end of the treatment period on day 22. Arterial pressure was recorded by cannulation of the central ear artery in conscious rabbits. After insertion of the catheter, systolic and diastolic blood pressures (mmHg) were measured by means of a blood pressure transducer (Baxter Uniflow; Baxter Healthcare Group, Santa Ana, California, USA) and a transducer interface (May 9601; Commat Ltd, Ankara, Turkey) for 15 min using a computerized system. In this system, a direct blood pressure measurement program (Diasys; Commat Ltd) was used. Mean arterial pressure (MAP) was calculated using the following formula: MAP = (systolic pressure + 2 × diastolic pressure)/3.

ORGAN CHAMBER EXPERIMENTS The two remaining rings from the right (sham) and left (collared) carotid arteries were used in organ chamber experiments to study vascular reactivity. After careful removal of loose connective tissue, the rings were suspended in organ chambers filled with 25 ml physiological salt solution (Krebs)

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M Yasa, Z Kerry, B Reel et al. Pranidipine and the rabbit collar model ACh-induced responses were determined for each preparation. At the end of each experiment, the tissues were washed three times and contracted with 120 mM KCl (with equimolar replacement of NaCl) to determine the maximal contractility.

number of animals. Values of maximum effect (Emax) and 50% effective concentration (EC50) were derived for each cumulative concentration-response curve by nonlinear fitting (Polywin 95 1.0).5 The Emax values and pD2 (the negative logarithm of the EC50 value) were compared. The intima:media ratio (index) was calculated for statistical analyses. The means of the areas (mm2) and the index were compared. P-values < 0.05 were considered to be statistically significant.

DRUGS Phenylephrine hydrochloride, 5-HT creatinine sulphate, potassium chloride and indomethacin sodium were purchased from Merck (Darmstadt, Germany); sodium pentobarbital from Psyphac (Brussels, Belgium); heparin solution from Roche (Istanbul, Turkey); ACh chloride from Sigma Aldrich Chemie (Steinheim, Germany); silicone (MED–401) from Nusil Silicone Technology (Anglet, France) and polyethyleneglycol 400 from Riedel-de Haën (Seelze, Germany). Pranidipine was kindly provided by Otsuka Pharmaceutical Co. Ltd, (Tokyo, Japan). 5-HT was dissolved in an aqueous solution of ascorbic acid (0.01%) and diluted in distilled water. Pranidipine was dissolved in 40% polyethyleneglycol 400 solution (PEG400)16 to produce a concentration of 1.2 mg/ml and was administrated by gastric gavage. The other drug solutions were prepared in distilled water.

Results

SURVIVAL AND BODY WEIGHT One rabbit from each group died during the treatment period. Pranidipine did not appear to cause any visible side effects. The body weight of the animals in both groups did not change during the treatment period (data not shown).

MORPHOMETRY The intimal cross-sectional area and the ratio of the intimal area to the medial area (index) were significantly increased in collared arteries compared with those in sham-operated arteries in the placebotreated group (P < 0.01) (Fig. 1, Table 1). Pranidipine treatment did not affect the collar-induced changes in the intimal area and the index (Table 1, Fig. 1). Neither collaring nor pranidipine treatment altered the medial cross-sectional area (Table 1).

STATISTICAL ANALYSIS Statistical analysis of data obtained from morphometric measurements and vascular reactivity studies were evaluated with repeated measures analysis of variance (RM ANOVA). Analysis was designed for drug treatments (two levels: pranidipine or placebo) as the between-rabbit factor, collar (two levels: present or not) as the withinrabbit factor, and LNA (two levels: present or not) as the within-artery factor. Data are presented as the mean ± standard error of the mean (SEM) and n indicates the

BLOOD PRESSURE There was no significant difference in blood pressure measurements before and after collaring. Treatment with pranidipine did not alter the MAP (for placebo- and pranidipinetreated groups, 79 ± 3 and 76 ± 5 mmHg, respectively, before collaring, on day 8, n = 7; 82 ± 2 and 78 ± 4 mmHg, respectively, after collaring, on day 22, n = 7; RM ANOVA).

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M Yasa, Z Kerry, B Reel et al. Pranidipine and the rabbit collar model

A

C

B

D

500µm

FIGURE 1: Photomicrographs of paraffin transverse sections after staining of carotid arteries with haematoxylin and eosin. (A) Collared artery from the placebo-treated group; (B) collared artery from the pranidipine-treated group; (C) sham-operated artery from the placebo-treated group; (D) sham-operated artery from the pranidipine-treated group. I, tunica intima; M, tunica media; A, tunica adventitia

PRANIDIPINE AND VASCULAR REACTIVITY CHANGES

indicated by the absence of an interaction. Pranidipine treatment significantly increased the hypersensitivity to 5-HT in both shamoperated and collared arteries (P < 0.01) (Table 3). With regard to Emax values, collar placement significantly diminished the maximum contractile force development in response to 5-HT (P < 0.001). However, neither LNA nor pranidipine treatment altered the Emax values for 5-HT in either shamoperated or collared arteries (Table 3). Collar placement significantly decreased the sensitivity to phenylephrine (P < 0.001)

Contractions The Emax in response to KCl was significantly decreased in collared arteries (P < 0.001) (Table 2). Treatment with LNA and with pranidipine did not alter these collar-induced decreases in the response to KCl (Table 2). As indicated by the increased pD2 values in collared arteries, collaring increased the sensitivity to 5-HT (P < 0.01) (Table 3). The inhibition of NO synthesis by LNA did not affect the sensitivity to 5‑HT, as

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TABLE 1: Morphometric studies: effects of the perivascular collar and oral pranidipine (3 mg/kg per day) treatment on the intimal and medial cross-sectional areas and the intima:media ratio (index) in the perivascular collar model of intimal thickening in rabbits Intimal area (mm2)a Sham Collared Significance of factors in ANOVA Collar Pranidipine Interaction (pranidipine × collar)

Placebo group (n = 7)

Pranidipine group (n = 7)

0.004 ± 0.000 0.015 ± 0.006

0.003 ± 0.000 0.023 ± 0.005

P < 0.01 NS NS

Medial area (mm2)a Sham Collared Significance of factors in ANOVA Collar Pranidipine Interaction (pranidipine × collar)

0.110 ± 0.009 0.117 ± 0.023

0.109 ± 0.004 0.096 ± 0.008

NS NS NS

Index (intima:media ratio)a Sham Collared Significance of factors in ANOVA Collar Pranidipine Interaction (pranidipine × collar)

0.034 ± 0.004 0.159 ± 0.070

0.028 ± 0.006 0.263 ± 0.076

P < 0.01 NS NS

n, number of animals in each group; ANOVA, analysis of variance; NS, not significant. aValues are mean ± SEM.

(Table 4). Neither pranidipine nor LNA treatment affected the decreased pD2 values of this agent in sham-operated or collared arteries (Table 4). In terms of the maximum contractile force development in response to phenylephrine, Emax values of phenylephrine were significantly decreased by collar placement (P < 0.001). Treatment with pranidipine and with LNA did not influence the Emax values of this agent in collared and sham-operated arteries (Table 4).

sham-operated and collared arteries already contracted with 1 μM phenylephrine. Emax was decreased in the collared rings of both treatment groups (Emax in sham-operated and collared rings, 2.41 ± 0.37 and 1.06 ± 0.18 g, respectively, in the placebo-treated group; 2.52 ± 0.22 and 1.12 ± 0.16 g, respectively, in the pranidipine-treated group; n = 7; P < 0.001; RM ANOVA). Pranidipine did not alter the maximum relaxation in response to ACh in sham-operated or collared arteries. The collar placement significantly decreased the sensitivity to ACh both in placebo- and pranidipine-treated groups (pD2 for shamoperated and collared arteries, 6.71 ± 0.16,

Relaxations In the absence of LNA, ACh induced concentration-dependent relaxations in both

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TABLE 2: Organ chamber studies: effects of the perivascular collar, LNA and oral pranidipine (3 mg/ kg per day) treatment on the Emax values for 120 mM KCl-induced contractions of rabbit carotid arteries KCl, Emax (g) Sham –LNA +LNA Collared –LNA +LNA Significance of factors in ANOVA Collar LNA Pranidipine Interaction (pranidipine × collar × LNA)



5.07 ± 1.18 4.50 ± 1.30

5.32 ± 1.13 5.89 ± 0.76

1.32 ± 0.44 1.19 ± 0.54

1.56 ± 0.60 1.39 ± 0.42

P < 0.001 NS NS NS

n, number of animals in each group; NS, not significant; +LNA, LNA present; –LNA, LNA absent. Emax values are mean ± SEM.

6.26 ± 0.17, respectively, in the placebotreated group; 6.75 ± 0.09, 6.59 ± 0.15, respectively, in the pranidipine-treated group; n = 7; P < 0.05; RM ANOVA). Pranidipine did not alter the pD2 values in response to ACh in sham-operated or collared arteries.

effects of several CCBs on the formation of intimal thickening in the collar model.11,12,21 Nicardipine, a CCB of the dihydropyridine class, has been shown to inhibit the formation of intimal thickening in this animal model.12 However, pranidipine, another dihydropyridine CCB, was not found to inhibit the thickening of the intima in the present study. Similarly, in the same model, both verapamil, a phenylalkylamine-derived CCB,11 and CD-832,21 a dihydropyridine CCB, have been found to be ineffective. In this respect, the results of these studies demonstrate that the effects of CCBs on intimal thickening in the perivascular collar model are quite dissimilar. In the present study, the lack of an inhibitory effect of pranidipine on intimal thickening in the collar model could be due to an inadequate dose and/or an insufficient drug treatment period. The latter possibility seems unlikely since nicardipine, another dihydropyridine CCB, inhibited intimal

Discussion Calcium channel blockers are one of the most frequently used classes of drug used in the treatment of cardiovascular disorders. There are three main groups of CCBs: the phenylalkylamine derivatives (e.g. verapamil); the benzothiazepines (e.g. diltiazem); and the dihydropyridines (e.g. nifedipine).17 Several CCBs from different groups have been shown to reduce the extent of fatty streak lesions in animals fed a cholesterol-rich diet.18 However, the effects of CCBs on intimal thickening, an early stage of atherosclerosis, seem to be contradictory.11,12,19,20 During the last decade, our laboratory has investigated the

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TABLE 3: Organ chamber studies: effects of the perivascular collar, LNA and oral pranidipine (3 mg/ kg per day) treatment on the pD2 and Emax values for 5-HT-induced contractions of rabbit carotid arteries


5-HT, pD2a Sham –LNA 6.65 ± 0.05 +LNA 6.83 ± 0.09 Collared –LNA 6.85 ± 0.10 +LNA 7.01 ± 0.07 Significance of factors in ANOVA Collar P < 0.01 Pranidipine P < 0.01 LNA NS Interaction (pranidipine × collar × LNA) NS 5-HT, Emax (g)a Sham –LNA 4.20 ± 0.35 +LNA 4.60 ± 0.52 Collared –LNA 1.70 ± 0.28 +LNA 1.90 ± 0.39 Significance of factors in ANOVA Collar P < 0.001 Pranidipine NS LNA NS Interaction (pranidipine × collar × LNA) NS


7.03 ± 0.09 7.09 ± 0.11 7.43 ± 0.18 7.50 ± 0.17

4.80 ± 0.30 5.10 ± 0.48 2.40 ± 0.46 2.40 ± 0.55

n, number of animals in each group; NS, not significant; +LNA, LNA present; –LNA, LNA absent. a Values are mean ± SEM.

thickening in this model within the same 3-week treatment period.12 With regard to the dose, Nakayama et al.15 reported that pranidipine at a dose of 3 mg/kg exerted a hypotensive action in normotensive and hypertensive rats, indicating that the drug blocks calcium channels at this dose. Consequently, it is unlikely that the dose of pranidipine was insufficient in this study. The possible mechanism(s) of perivascular collar-induced intimal thickening are still under investigation.5,22 In this context, our previous study demonstrated that enhanced

NO production and superoxide anion were generated in response to collaring, resulting in oxidative stress within the segment enclosed by the collar. 23 Consistent with this finding, Arthur et al.24 showed that NO was produced by the inducible isoform of NO synthase in modified smooth muscle cells of the developing intimal thickening. Based on the experimental evidence described above, it is conceivable that oxidative stress may exist in the collar model. It has been shown that nicardipine prevents enhanced NO degradation23 and inhibits intimal

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TABLE 4: Organ chamber studies: effects of the perivascular collar, LNA and oral pranidipine (3 mg/ kg per day) on the pD2 and Emax values for phenylephrine-induced contractions of rabbit carotid arteries Phenylephrine, pD2a Sham –LNA +LNA Collared –LNA +LNA Significance of factors in ANOVA Collar Pranidipine LNA Interaction (pranidipine × collar × LNA) Phenylephrine, Emax (g)a Sham –LNA +LNA Collared –LNA +LNA Significance of factors in ANOVA Collar Pranidipine LNA Interaction (pranidipine × collar × LNA)



6.12 ± 0.07 6.08 ± 0.07

6.15 ± 0.12 6.15 ± 0.09

5.67 ± 0.09 5.61 ± 0.11

5.76 ± 0.13 5.89 ± 0.11

P < 0.001 NS NS NS

6.70 ± 0.51 6.20 ± 0.73

7.00 ± 0.33 7.30 ± 0.63

2.60 ± 0.23 2.20 ± 0.37

3.10 ± 0.51 3.00 ± 0.49

P < 0.001 NS NS NS

n, number of animals in each group; NS, not significant; +LNA, LNA present; –LNA, LNA absent. a Values are mean ± SEM.

thickening in response to collaring,12 possibly indicating an antioxidant activity of the drug.25 Pranidipine has been reported to upregulate superoxide dismutase, an antioxidant enzyme, in endothelial cells,26 and to enhance and/or prolong NO-dependent relaxations.27,28 However, pranidipine did not inhibit thickening of the intima in the collar model in our present study. In this respect, the assumption that the antioxidant activities of CCBs such as lacidipine10 and lercanidipine29 might be a factor in their anti-atherosclerotic activity needs further investigation.

With regard to the effectiveness of pranidipine in preventing cell proliferation,30 the lack of an inhibitory effect of pranidipine on intimal thickening in the collar model suggests the possibility that cell migration may play a dominant role in the formation of intimal thickening in this model. However, Bruijns and Bult31 reported that cytochalasin D, a fungal metabolite, suppressed in vitro collar-induced cell migration but did not reduce intimal thickening. In addition, cell proliferation was not inhibited by cytochalasin D.31 Our study did not

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M Yasa, Z Kerry, B Reel et al. Pranidipine and the rabbit collar model investigate the effect of pranidipine on either cell migration or proliferation in vivo or in vitro. In order to establish whether there is a connection between the inhibitory effect of CCBs on intimal thickening and their effects on cell migration and/or proliferation, the effect of each CCB on cell migration and/or proliferation should be tested separately in this perivascular collar model. As expected from the results of previous experiments,5,7,32 positioning the collar around the carotid artery did not affect arterial blood pressure. As a long-acting dihydropyridine CCB, pranidipine has been shown to lower blood pressure in normotensive and hypertensive rats15 and normotensive dogs16 within the dosage range tested in the present study. Moreover, in the chronically instrumented conscious rabbit model, pranidipine has been shown to exert a significant anti-ischaemic action, supported by normalization of the increase in heart rate.16 In contrast, we have demonstrated that pranidipine does not affect resting arterial pressure in this rabbit model of early atherosclerosis. The reasons underlying the different observations are not yet apparent but may possibly be related to the animal species in which the experiments are performed. It has been shown in a number of studies that the responsiveness to vasoconstrictor and vasodilator agents differs significantly during the early steps of lesion development in atherosclerotic vessels.33,34 Similarly, the effects of balloon angioplasty on vascular reactivity changes to vasoconstrictor and vaso dilator agents have also been reported.35 – 37 In the collar model, alterations in vascular reactivity in response to collaring have been well documented, independently of the development of intimal thickening. In the present study, consistent with previous results,7,11,12,38 the collar placement suppressed contractions induced by 120 mM

KCl in both the presence and the absence of NO inhibition, suggesting that NO does not modulate KCl-induced contractions in either sham-operated or collared rabbit carotid arteries. In the collar model, attenuation of KCl-induced contractions has been discussed in detail.7,39 In addition, this study confirms that inhibition of NO synthesis by LNA did not affect the reduced contractile responses to KCl in the collar model.7 As a long-acting CCB, pranidipine is likely to inhibit KCl-induced contractions.13 Similarly, the inhibitory effects of this agent on the contractions observed in vitro would also be expected to appear in the organ chamber, but this was not observed in this model. Moreover, in the same model, Kerry et al.12 reported that nicardipine did not prevent KCl-induced contractions. A conceivable explanation is that the results of the organ bath studies after the chronic 3-week treatment with CCBs and incubation directly with CCBs were not always carried out in parallel. In accordance with the findings of previous studies,6,7,11,12,32 collaring increased the pD2 values of 5-HT in collared arteries in both the presence and the absence of LNA, which is a generally accepted feature of the collar model. On the other hand, treatment with pranidipine increased the pD2 values of 5-HT in both collared and sham-operated arteries without affecting the Emax values of this agent, suggesting the possibility that chronic treatment with pranidipine (3 weeks) made the vessels over-reactive to supraphysiological concentrations of 5-HT in the organ chambers. As in previous experiments,11,38,40 the collar alone decreased both the sensitivity and the Emax values of phenylephrine. However, inhibition of NO synthesis did not affect the Emax values of phenylephrine in either the placebo- or the pranidipine-treated group. Unlike verapamil, a phenylalkylamine-

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M Yasa, Z Kerry, B Reel et al. Pranidipine and the rabbit collar model derived CCB,11 pranidipine did not affect phenylephrine-induced contractions in this model. Collaring decreased the sensitivity to ACh in both the placebo- and pranidipine-treated groups, a finding consistent with our previous experiments.38,41 In this model, pranidipine treatment did not affect the pD2 values of ACh, suggesting that the drug does not interfere with muscarinic receptor-mediated responses in the rabbit carotid artery. However, we are aware that pranidipine has been shown to augment ACh-induced and NO-dependent endothelium-dependent relaxations.27,28,42 Collaring decreased the Emax values of ACh in both the placebo- and pranidipine-treated groups. Regarding the fact that ACh-induced relaxations have been presented as grams of tension in this study, the effect of collaring on the Emax values of ACh-induced relaxation has been found to result in an increase,12,21 a decrease (present study), or no change.38 In general, it should be noted that, in rabbit vessels, in view of the methodological aspects of ACh-induced relaxations, contradictory findings have been reached in a number of studies.41,43 In conclusion, the combined data of previous studies and our present study demonstrate that pranidipine does not prevent collar-induced intimal thickening

and, in particular, does not affect the collarinduced changes in vascular reactivity in this model. Similarly, it can be inferred that not all CCBs prevent collar-induced intimal thickening, and thus the effects of CCBs are not related to their chemical structure and/or their calcium channel-blocking action in the perivascular collar model.

Acknowledgements We would like to thank Otsuka Pharmaceutical Co. Ltd, Tokyo, Japan, for kindly providing us with a sample of pranidipine for use in this study, and we would like to express our appreciation of Dr Toyoki Mori’s invaluable comments, which greatly assisted us in the preparation of the manuscript. This work was supported by a grant from Ege University Scientific Research Fund (98/ECZ/011). Dr Aslı Ozer would like to thank Dr Levent Ustunes for introducing members of the department to the collar model used in this study. The authors wish to thank Drs Necmettin Ozdemir and Yildiz Erhan of the Department of Pathology, Faculty of Medicine, Ege University, for their valuable contribution in the preparation of the transverse sections used in the morphometric analysis.

Conflicts of interest No conflicts of interest were declared in relation to this article.

• Received for publication 6 June 2006 • Accepted subject to revision 18 June 2006 • Revised accepted 4 October 2006 Copyright © 2007 Cambridge Medical Publications References 1 Fishman JA, Ryan GB, Karnovsky MJ: Endothelial regeneration in the rat carotid artery and the significance of endothelial denudation in the pathogenesis of myointimal thickening. Lab Invest 1975; 32: 339 – 351. 2 Booth RF, Martin JF, Honey AC, Hassall DG, Beesley JE, Moncada S: Rapid development of atherosclerotic lesions in the rabbit carotid artery induced by perivascular manipulation. Atherosclerosis 1989; 76: 257 – 268.

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Address for correspondence Dr Z Kerry Department of Pharmacology, Faculty of Pharmacy, Ege University, Bornova 35100, Izmir, Turkey. E-mail: [email protected]

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