in antiinflammatory effect of calcium channel blockers

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Role of adrenal gland hormones

in antiinflammatory effect of calcium channel blockers

Halis Suleyman, Zekai Halici, Ahmet Hacimuftuoglu, Fatma Gocer

Atatürk University, Faculty of Medicine, Department of Pharmacology, 25240 Erzurum, Turkey Correspondence: Halis Suleyman, e-mail: suleyman@atauni.edu.tr

Abstract:

Effects of amlodipine, lacidipine and nicardipine on acute phase of inflammation in the carregeenan model in intact rats were investigated in this study. In addition, the most effective dose of nicardipine that had the highest anti-inflammatory impact was investigated in carregeenan test in adrenalectomized rats. The effective dose of nicardipine was tested in the chronic phase of inflammation in the model of cotton pellet granuloma, and its efficiency was compared with diclofenac sodium. Amlodipine at 5 and 10 mg/kg doses showed 61%, 80%, lacidipine 73%, 34% and nicardipine 38%, 87% inhibition of carrageenan-induced inflammation, respectively. Nicardipine (10 mg/kg) and diclofenac sodium (25 mg/kg) showed 11.6% and 16.2% inhibition, respectively against carrageenan-induced edema. Diclofenac at 10 mg/kg showed 43% inhibition of the inflammation. In cotton pellet test, antiproliferative effects of nicardipine (10 mg/kg) and diclofenac sodium (10 mg/kg) were evaluated as 60% and 39.5%, respectively. The obtained results showed that calcium channel blockers and diclofenac sodium significantly blocked acute and chronic phases of inflammation in intact rats, but in adrenalectomized rats calcium channel blockers and diclofenac sodium had no significant antiinflammatory effect.

Key words:

Ca channel blockers, carrageenan, inflammation, adrenal glands, adrenalectomy

Introduction

Amlodipine, lacidipine and nicardipine are calcium channel antagonists. They are dihydropyridine derivatives. They have high affinity for L-type calcium channels [2, 43]. Calcium channel antagonists are useful in various diseases, such as angina pectoris, hypertension, hypertensive crisis, arrhythmia, left ven- tricular diastolic dysfunction, myocardial infarction, Raynaud’s phenomenon, progressive systemic sclero- sis, peripheral vascular diseases, chronic renal failure, Conn syndrome, pulmonary hypertension, migraine

and esophageal spasm [2, 38]. Amlodipine, lacidipine and nicardipine are vasoselective calcium channel blockers [44].

Calcium channel blockers generate their effects by coupling to L1 subtype of L-type calcium channels on the cell membrane [2, 39]. Calcium participates in many body functions and it has been shown to play an important role in the synthesis and release of chemical mediators of inflammation [1].

An increase in calcium ions in the tissues and cells can cause pain and inflammation. On the other hand, a decrease in calcium levels prevents or attenuates pain and inflammation [6]. In addition, it has been

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shown that intradermal injection of calcium causes acute inflammation [21]. As it is well known, polymorphonuclear leucocytes (PNL) play a role in en- hancing the inflammation [23]. Many experimental studies showed that calcium ions play an important role in PNL activation [15]. Calcium ions stimulate the synthesis of lipooxygenase (LO) products by activating the enzyme 5-LO in cells. Furthermore, Ca channel blockers enhances the synthesis of eicosanoids by activating cytosolic phospholipase A2 (cPA2) [9, 29]. The role of eicosanoids (the metabolites of arachidonic acid) in acute inflammation is known [32]. Also, the role of the metabolites of arachidonic acid in the pathogenesis of carrageenan-induced inflammation has been demonstrated [34]. Carrageenan-induced model of inflammation is used to investigate the effects of substances on acute phase of inflammation, while cotton pellet granuloma test is used to investigate the effect of substances on chronic phase of inflammation [4, 47].

Mechanisms of action of steroidal and non-steroidal antiinflammatory drugs are based on the inhibition of the synthesis of chemical mediators of inflammation [30]. Gluco- corticoids known as steroidal antiinflammatory drugs are synthesized in the adrenal cortex. They have been shown to play a role in the mechanism of action of non-steroidal antiinflammatory drugs [5, 48].

There are many articles which explain the mechanism of antiinflammatory action of calcium channel blockers in intact rats [1, 13, 16, 27, 28, 33]. But there is no study about the antiinflammatory effects of calcium channel blockers in adrenalectomized animals.

The aim of this study was to investigate the effects of amlodipine, lacidipine and nicardipine on acute phase of inflammation in the model of carrageenan- induced inflammation in intact rats, and then in the same model in adrenalectomized rats to investigate the role of adrenal gland hormones in antiinflammatory mechanisms of the most effective dose of the most powerful calcium channel blocker. In addition, we investigated the effects of this drug on chronic phase of inflammation using the cotton pellet granuloma test.

Materials and Methods

Animals

A total of 84 adult male Wistar albino rats weighing 200–215 g, obtained from the Department of Pharma-

cology, Experimental Animal Laboratory, Faculty of Medicine, Atatürk University, were used in this study.

The rats were fed standard laboratory chow and tap water before the experiment. Rats were housed at 22°C.

Chemicals

Amlodipine (Norvasc, 10 mg) was supplied by Pfizer Turkey, lacidipine (Lacipil, 4 mg) was purchased from GlaxoSmithKline Turkey, nicardipine (Loxen, 20 mg) was purchased from Sandoz Pharma Turkey, diclofenac sodium (Voltaren, 50 mg) was purchased from Novartis Turkey, and thiopental sodium (Pento- thal sodium, 1 g) was purchased from Abbott Turkey.

Carrageenan-induced inflammation model in intact rats

In this series of experiments, the antiinflammatory effects of amlodipine, lacidipine and nicardipine were investigated in inflamed paw edema [8]. We used a total of 48 rats divided into 8 groups. The mentioned drugs were given to animals perorally (po), with the aid of gavages, in 5 and 10 mg/kg doses. The antiinflammatory efficiency of drugs was compared with 10 mg/kg po diclofenac sodium. The same volume of distilled water was given as a solvent to the control group. One hour after the administration of the drugs, 0.1 ml of 1% carrageenan was injected into the paws of each rat. Before the carrageenan injection, foot volumes of animals had been measured with plethys- mometry from their toes to knees. Carrageenan- induced increase in paw volume (paw edema) was measured five times at 1 h intervals.

The anti-inflammatory effects of drugs were determined by comparing the results which were obtained with the control group.

Carrageenan-induced inflammation model in adrenalectomized rats

In this series of experiments, the effect of nicardipine, which was found the most effective drug in carrageenan-induced model of inflammation in intact rats, was investigated in carrageenan-induced inflammation test in adrenalectomized rats. For using this method, adrenals of 18 rats were taken out under the ketamine (25 mg/kg) anesthesia [4]. After surgery, rats were supported by 1% solution of sodium chlo- ride instead of water and pellet food during seven

Adrenal gland hormones and calcium channel blockers

Halis Suleyman et al.

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at a dose of 10 mg/kg was given (po) to another group of adrenalectomized rats. Distilled water was given by the same route to the control group of rats. One hour after the administration, 0.1 ml of 1% carrageenan was injected into the paw of each animal, and the antiinflammatory effects of drugs were determined by using the procedure which was mentioned above.

Cotton pellet granuloma test

In this part of experiment, we used 18 rats divided into 3 groups. In these series of experiments, the effects of nicardipine and diclofenac sodium on the proliferative phase of inflammation were examined [3].

Nicardipine at 10 mg/kg was given to one group of rats and 10 mg/kg of diclofenac sodium was given po with the aid of gavages to another group. Control group received equal volume of distilled water. Thirty minutes after the administration of drugs, rats were anesthetized by 25 mg/kg of ketamine. Then, cotton pellets, weighing 7 ± 1 mg and prepared under sterile conditions, were implanted subcutaneously (sc) in the interscapular area. Drugs were administered once a day for a period of 7 days. On the 8th day, rats were killed with high dose (50 mg/kg) of thiopental sodium. Cotton pellets with the granuloma tissue were removed and weighed.

Statistical analyses

All results were shown as the means ± SD. One-way analysis of variance was used to evaluate the results;

p < 0.05 was considered significant.

Animal experiments were performed in accordance with the national guidelines for the use and care for laboratory animals and were approved by the local animal care committee of Atatürk University.

Results

Carrageenan test in intact rats

After 4 h, 5 and 10 mg/kg doses of amlodipine showed 61%, 80%, lacidipine 73%, 34% and nicardipine 38%, and 87% inhibition of carrageenan- induced inflammation, respectively (Tab. 1). The dose of 10 mg/kg of diclofenac sodium showed 43% inhibition of the inflammation.

In control group, inflamed paw volume showed an increase of 0.42 ml in comparison with the normal value. The increases in paw volumes were 0.16 and 0.08, 0.11 and 0.27, 0.26 and 0.06 ml for the groups of amlodipine, lacidipine, nicardipine at 5 and 10 mg/kg,

Tab. 1. Effects of amlodipine, lacidipine, nicardipine and diclofenac sodium on carrageenan-induced inflammation (paw edema) in intact rats

Drugs Number of rats Dosage

(mg/kg)

Paw edema volume (ml) Increase in footvs.

normal (ml)

Antiinflammatory effect (%)

p Before

inflammation

Four hours after inflammation

Amlodipine 6 5 0.83 0.99 0.16 ± 0.02 61 p < 0.001

Amlodipine 6 10 0.79 0.87 0.08 ± 0.08 80 p < 0.0001

Lacidipine 6 5 0.89 1.00 0.11 ± 0.18 73 p < 0.0001

Lacidipine 6 10 0.82 1.09 0.27 ± 0.14 34 p < 0.05

Nicardipine 6 5 0.82 1.08 0.26 ± 0.09 38 p < 0.02

Nicardipine 6 10 0.83 0.89 0.06 ± 0.10 87 p < 0.0001

Diclofenac Sodium

6 10 0.82 1.06 0.24 ± 0.13 43 p < 0.01

Control 6 – 0.84 1.26 0.42 ± 0.10 – –

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respectively; and it was 0.24 ml for diclofenac sodium at 10 mg/kg.

Carrageenan test in adrenalectomized rats

In the 4th hour, nicardipine (10 mg/kg) and diclofenac sodium (25 mg/kg) showed 11.6% and 16.2% inhibition, respectively, against carrageenan-induced edema.

The increases in inflamed paw volume in the group of nicardipine, diclofenac sodium and control were 0.61, 0.58 and 0.69 ml, respectively in comparison with the normal value (Tab. 2).

Cotton-pellet granuloma test

On 8th day, mean weights of moist pellets taken out from the rat groups which were administered nicardipine (10 mg/kg), diclofenac sodium (10 mg/kg), and the control group were 178.8 ± 59 and 261.2 ± 17 mg and 447.5 ± 34 mg, respectively. According to these results, antiproliferative effects of nicardipine and diclofenac sodium were evaluated by 60% and 39.5%, respectively (Tab. 3).

Discussion

In this study, the effects of amlodipine, lacidipine and nicardipine on acute phase of inflammation were examined in the carrageenan model of inflammation in intact rats. In addition, the most effective dose of nicardipine that had the highest anti-inflammatory impact was investigated in carrageenan test in adrenalectomized rats. The effective dose of nicardipine was tested in the chronic phase of inflammation in the model of cotton pellet granuloma, and its efficiency was compared with diclofenac sodium. Our studies on rats showed that all Ca channel blockers which we tested significantly prevented the carrageenan-induced inflammation in intact rats. Amlodipine (in 5 and 10 mg/kg doses) decreased the carrageenan induced inflammation significantly. The antiinflammatory effects of amlodipine 10 mg/kg were higher than those observed for 5 mg/kg dose of amlodipine, but the difference found between these two groups was not statistically significant. The antiinflammatory power of amlodipine was dose-dependent, but the

Tab. 2. Effects of nicardipine and diclofenac sodium on carrageenan-induced inflammation (paw edema) in adrenalectomized rats

Drugs Number

of rats

Dosage (mg/kg)

Paw edema volume (ml) Increase in foot volumevs.

normal (ml)

Antiinflammatory effect (%)

p Before

inflammation

Four hours after inflammation

Nicardipine 6 10 0.85 1.46 0.61 ± 0.12 11.6 p > 0.05

Diclofenac Sodium

6 25 0.86 1.44 0.58 ± 0.11 16.2 p > 0.05

Control 6 – 0.82 1.51 0.69 ± 0.25 – –

Tab. 3. Effects of nicardipine and diclofenac sodium on the proliferative phase of inflammation (cotton pellet granuloma test)

Drugs Number of rats Dosage

(mg/kg)

Initial weight of cotton pellets (mg)

Weight of cotton pellets which were removed after 8 days

(mg)

Antiproliferative effect (%)

p

Nicardipine 6 10 7 ± 1 178.8 ± 59 60.0 p < 0.03

Diclofenac Sodium

6 10 7 ± 1 261.2 ± 17 39.5 p < 0.05

Control 6 – 7 ± 1 447.5 ± 34 – –

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cidipine was statistically significant. The antiinflammatory effect of 5 and 10 mg/kg doses of nicardipine and the difference between the doses were statistically significant. The most effective dose of nicardipine was 10 mg/kg. The results showed that nicardipine was the most effective Ca channel blocker in the carrageenan test. It is known that Ca channel blockers inhibit the entrance of extracellular calcium to intracellular space. Many studies have shown that calcium ions play an important role in the synthesis and release of the chemical mediators of inflammation [1, 21, 25]. It is known that histamine, serotonin, bradykinin, nitric oxide (NO), LO products, cytokines, free oxygen radicals, lysosomal enzymes, prostaglandins, etc. are implicated in development of inflammation [8, 22, 42, 49]. It has been shown that these mediators also have a role in carrageenan-induced inflammatory reaction [19, 34, 51].

Verapamil, a calcium channel blocker, was shown to antagonize the destructive effects of histamine on the gastric tissue [45]. An increase in intracellular calcium ion in mast cell caused activation of these cells and release of histamine, which plays an important role in inflammation [10]. In addition, many studies have shown that verapamil had an antiinflammatory effect [13, 33]. Also in another study it was determined that a T-type calcium channel blocker, mibefra- dil blocked more strongly histamine-induced paw edema in rats in comparison with indomethacin [7]. It is known that dihydropyridine derivatives (nifedipine, nisoldipine, nicardipine) decrease the production of mediators of inflammation; these mediators are pros- taglandin, oxygen anion, elastase, etc., and they take part in the pathogenesis of inflammation [28]. It is thought that antiatherogenic effect of calcium channel blockers may result from the anti-inflammatory effect of these drugs. Antiinflammatory effect of verapamil relies on inhibition of the synthesis of cytokines and NO, which are known as mediators. It also produces its antiinflammatory effect by blocking the migration and adhesion of PNL [13, 33]. The antiatherogenic effect of amlodipine was reported to depend on its anti-inflammatory activity by the inhibition of NO synthesis [26].

Amlodipine decreased the production of superoxide radical and increased the level of superoxide dis- mutase in angiotensin-induced oxidative stress. In ad-

tokines (TNF-a, IL-6 etc.), which increase in inflam- matory events [35]. The previous studies and investi- gations have shown that the antiatherogenic effect of dihydropyridine may arise from the anti-inflammatory and antioxidant effects [27].

Amlodipine, lacidipine and nicardipine that were used in our study, markedly decreased the carrageenan-induced inflammation. It is known that inflammatory reaction which is induced by carrageenan has two phases. They are called early (till 1 h) and late phases (1–4 h) [11]. It has been shown that early phase is dependent on the release of histamine, serotonin and bradykinin, while the late phase is dependent on the formation of prostaglandins [41]. In addition, hydrogen peroxide, superoxide and hydroxyl radicals which originate from neutrophils also play a role in infiltration of neutrophils in the late phase of carrageenan-induced inflammation [34, 37]. It is known that cyclooxygenase (COX) and LO play a role in formation of carrageenan-induced edema [17].

There is some evidence that NO can contribute to the increased permeability of vessels in carrageenan- induced edema [36]. By increasing the production of bradykinin in the early phase of carrageenan-induced inflammation, it elicits the activation of L and T type calcium channels and increases the level of intracellular calcium level [14]. Vajja and coworkers have shown that the release of IL-1b increases with the increase in intracellular calcium level [50].

Antiinflammatory effect of amlodipine, lacidipine and nicardipine which were used in this study reached the highest level in the fourth hour after injection of carrageenan. This time period is accepted as the late phase of carrageenan inflammation which was mentioned above. All the knowledge from literature and from the studies we have conducted shows that calcium ions play a principal role in synthesis and release of inflammatory mediators which cause inflammation.

In our study, 10 mg/kg dose of nicardipine was determined to be the most powerful inhibiting drug in carrageenan-induced inflammation in intact animals.

Antiinflammatory effect was investigated in carregeenan test in adrenalectomized rats, as well. Nicar- dipine in adrenalectomized rats decreased carrageenan inflammation by 11.6% in comparison with control group, which was found statistically insignificant. Nicardipine decreased carrageenan induced in-

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flammation in intact animals by 75.4%. The percent- age was statistically significant compared with the adrenalectomized rats. Antiinflammatory effects of nicardipine in adrenalectomized rats were lower in the early phase of inflammation elicited by carregeenan.

Like in intact rats, also in adrenalectomized rats, the increases in paw volumes were measured five times at 1h intervals. The antiinflammatory effects of nicardipine in adrenalectomized rats were found insignificant at all times measured. Moreover, increment of inflamed paw volume in intact animals in control group was calculated as 0.42 ± 0.1 in comparison with the normal value. This increment was 0.61 ± 0.12 in adrenalectomized rats, which were given 10 mg/kg dose of nicardipine. These data show that inflammation induced by carrageenan in adrenalectomized rats is stronger than in intact rats. In the control group of adrenalectomized rats, inflammation was increased by 60.8% as compared with the control group of intact rats.

There are many articles which explain the mechanism of antiinflammatory action of calcium channel blockers in intact rats [12, 13, 16, 27, 28, 33], but there is no study about the antiinflammatory effects of calcium channel blockers in adrenalectomized animals. Is it possible that calcium channel blockers can- not influence its own receptors in adrenalectomized rats? It is possible that the decrease in antiinflammatory effect of calcium channel blockers in adrenalectomized rats results from the decreased sensitivity of nicardipine binding to a-1 subtype of calcium chan- nel. However, diclofenac sodium which had no action on calcium channels had an insignificant antiinflammatory effect. These results show that adrenal gland hormones are endogenous factors which may play a central role in blocking inflammation. The results of our experiments have shown that the blockage of calcium channels with nicardipine and inhibition of inflammatory mediators with diclofenac sodium insignificantly suppressed the inflammation in adrenalectomized animals.

The levels of glucocorticoids are increased in inflammation [46]. This increase can be seen as a defen- sive mechanism of the body in response to inflammation. It was shown that the death in adrenalectomized animals depended on the decreased resistance against infections and inflammations [20].

As it is well known, glucocorticoids exert their antiinflammatory effect by inhibiting the synthesis of mediators of inflammation and suppressing the genes of proinflammatory cytokines [18]. It is known that

the increased NO production in inflammation is inhib- ited by glucocorticoids [30]. The increased synthesis and release of corticotropin-releasing hormone (CRH) in adrenalectomized animals is natural. CRH gener- ates inflammation by stimulating the production of proinflammatory cytokines in macrophages and it has been reported that antalarmine which is a CRH recep- tor inhibitor, suppressed this inflammation [3].

The effects of nicardipine and diclofenac sodium on chronic phase of inflammation in cotton pellet granuloma test of intact rats were investigated, either.

The cotton pellet test is a chronic inflammation model which is used for evaluating the antiproliferative effects of drugs [40].

Nicardipine at 10 mg/kg decreased the weight of cotton pellets, which were placed subcutaneously, by 60% compared to the control. The antiproliferative power of nicardipine was found to be by 65.8%

higher than that of diclofenac sodium at the same doses. The results which were obtained from cotton pellet granuloma test showed that the antiproliferative effect of nicardipine was more significant than that of diclofenac sodium. The difference between the antiin- flamatory effects of nicardipine and diclofenac sodium was found to be statistically significant. The most effective dose of nicardipine in carrageenan- induced inflammation in intact rats was investigated in chronic inflammation. After a short time from the beginning of acute inflammation, proliferative cells developed and inflammation became chronic. These cells were spread or granuloma was formed.

Prevention of the collagen fiber formation and sup- pression of mucopolysaccharids are indicators of the antiproliferative effect of antiinflammatory agents [50].

In the nicardipine group, the volume of cotton pellets was smaller, and the extent of hyperemia was lesser when compared with the control group macro- scopically. In control group, the extent of hyperemia of tissues around cotton pellets was profound. Hyperemia was less intense in the nicardipine group compared with the diclofenac sodium and control groups.

Monocyte infiltration and fibroblast proliferation rather occur in chronic inflammation than neutrophil infiltration and exudation [24]. Activated monocyte-macrophages are blood cells which have antitu- mor and antimicrobial functions. Also they have phagocytotic function against pathogens [36].

In summary, we can say that carrageenan produced by 60.8% stronger inflammation in adrenalectomized rats than in intact rats. It should be possible that the

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carrageenan in adrenalectomized rats may have caused cell membrane damage. Violent inflammation with cell membrane damage in adrenalectomized rats may have depended on the blockade of the production of natural glucocorticoids and on the increase in the level of CRH which has proinflammatory effect. Cal- cium channel blockers may have suppressed the inflammation evoked by carrageenan in intact rats by blocking L-type calcium channels.

Blocking calcium channels by nicardipine was not enough for elimination of the inflammation induced by carrageenan in adrenalectomized rats. In the light of this observation, we can say that adrenal gland hormones play an important role in protecting the en- tirety of cell membrane. In addition, it is possible that nicardipine may have depressed the chronic inflammation by decreasing the proliferation of fibroblasts and infiltration of monocytes, by blocking the production of collagen fibers and by suppressing mucopoly- saccharides as classic non-steroidal anti-inflamatory drugs. Further studies are required for clarification of the mechanism of anti-inflammatory action of calcium channel blockers.

Acknowledgments:

We would like to express our thanks to Prof. Fahri Yavuz, Bahadir Süleyman, M.Erdem Sagsoz, Ilker Angin, Assoc.Prof. Mustafa Gül, Elif Cadirci and Yilmaz Yigit for their contribution to this work.

References:

1. Abdollahi M, Nikfar S, Abdoli N: Potentiation by nitric oxide synthase inhibitor and calcium channel blocker of aspartame-induced antinociception in the mouse formalin test. Fundam Clin Pharmacol, 2001, 15, 117–123.

2. Abernethy DR, Schwartz JB: Calcium-antagonist drugs.

N Engl J Med, 1999, 341, 1447–1457.

3. Agelaki S, Tsatsanis CH, Gravanis A, Margioris AN:

Corticotropin-Releasing Hormone augments proinflam- matory cytokin production from macrophages in vitro and in lipopolisaccharide-induced endotoxin shock in mice. Infect Immun, 2002, 70, 6068–6074.

4. Al-Tuwaijri AS, Mustafa AA: Verapamil enhances the inhibitory effect of diclofenac on the chemiluminescence of human polymorphonuclear leucocytes and

carrageenan-induced rat’s paw eadema. Int J Immuno- pharmacol, 1992, 14, 83–93.

vated calcium and calcium antagonists on 6, 7-benzomorfan- induced analgesia. Eur J Pharmacol, 1983, 75, 385–390.

7. Bilici D, Akpinar E, Gürsan N, Özbakis Dengiz G, Bilici S, Altas S: Protective effect of T-type calcium channel blocker in histamine-induced paw inflammation in rat.

Pharmacol Res, 2001, 44, 527–531.

8. Campos MM, Mata LV, Calixto JB: Expression of Bl ki- nin receptors mediating paw eadema and formalin- induced nociception. Modulation by glucocorticoids.

Can J Physiol Pharmacol, 1995, 73, 812–819.

9. Carnevale KA, Cathcart MK: Calcium-independent phospholipase A2 is required for human monocyte che- motaxis to monocyte chemoattractant protein 1. J Immu- nol, 2001, 167, 3414–3421.

10. Chand N, Pillar J, Diamantis W, Sofia RD: In vitro in- hibiton of allergic histamine release by calcium antagonists. Eur J Pharmacol, 1985, 107, 353–358.

11. Chaudhuri AKN, Karmakar S, Rey D, Pal S, Pal M, Sen T: Anti-inflammatory activity of Indian black tea (Sik- kim variety). Pharmacol Res, 2005, 51, 169–175.

12. Chow FS, Jusko WJ: Immunosuppresive interactions among calcium channel antagonists and selected corticosteroids and macrolides using human whole blood lymphocytes. Drug Metab Pharmacokinet, 2004, 19, 413–121.

13. De Souza AP, Tanowitz HB, Chandra M, Shtutin V, Weiss LM Morris SA, Factor SM, Huang H et al: Effects of early and late verapamil administration on the development of cardiomyopathy in experimental chronic try- panosoma cruzi (Brazil strain) infection. Parasitol Res, 2004, 92, 496–501.

14. El-Bizri N, Bkaily G, Wang S, Jacques D, Regoli D, D’Orleans-Juste P, Sukarieh R: Bradykinin induced a positive chronotropic effect via stimulation of T-and L-type calcium currents in heart cells. Can J Physiol Pharmacol, 2003, 81, 247–258.

15. Elferink JG, Boonen GJ, Koster BM: The role of calcium in neutrophil migration: the effect of calcium and calcium-antagonists in electroporated neutrophils. Bio- chem Biophys Res Commun, 1992, 182, 864–869.

16. Fansa I, Gol MK, Nisanoglu V, Yavas S, Iscan Z, Tasde- mir O: Does diltiazem inhibit the inflammatory response in cardiopulmonary bypass? Med Sci Monit, 2003, 9, 48–54.

17. Gamache DA, Povlishock JT, Ellis EF: Carragenan induced brain inflammation. J Neurosurg, 1986, 65, 679–685.

18. Goulding NJ: The molecular complexity of glucocorti- coid actions in inflammation – a four-ring circus. Curr Opin Pharmacol, 2004, 4, 629–636.

19. Gupta M, Mazumdar UK, Sivakumar T,Vamsi MLM, Karki SS, Sambathkumar R, Manikandan L: Evaluation of anti-inflammatory activity of chloroform extract of Bryonia Laciniosa in experimental animal models. Biol Pharm Bull, 2003, 26, 1342–1144.

20. Guyton AC, Hall JE: Textbook of Medical Physiology, 10th edn. WB Saunders, Philadelphia, 2001, 869–884.

21. Gürdal H, Sara Y, Tulunay FC: Effect of calcium channel blokers on formalin-induced nociception and inflammation in rats. Pharmacology, 1992, 44, 290–296.

(8)

22. Habashy RR, Abdel-Naim AB, Khalifa AE, Al-Azizi MM: Anti-inflammatory effects of jojoba liquid wax in experimental models. Pharmacol Res, 2005, 51, 95–105.

23. Higgins AJ: The biology pathophysiology and control of eicosanoids in inflammation. J Vet Pharmacol Ther, 1985, 8, 1–8.

24. Hosseinzadeh H, Ramezani M, Salmani G: Antinocicep- tive, antiinflammatory and acute toxicity effects of Zataria Multiflora Boiss extracts in mice and rats. J Eth- nopharmacol, 2000, 73, 379–385.

25. Ishikawa J, Ohga K, Yoshino T, Takezawa R, Ichikawa A, Kubota H, Yamada T: A pyrazole derivative, YM-58483, potently inhibits store-operated sustained Ca influx and IL-2 production in T lymphocytes. J Immunol, 2003, 4441–4449.

26. Jukema JW van der Hoorn JW: Amlodipin and atorvasta- tin in atherosclerosis. Exp Opin Pharmacother, 2004, 5, 459–468.

27. Kataoka C, Egashira K, Ishibashi M, Inoue S, Ni W, Hi- asa K, Kitamoto S et al: Novel anti-inflammatory actions of amlodipine in a rat model of arteriosclerosis induced by long-term inhibition of nitric oxide synthesis Am J Physiol Heart Circ Physiol, 2004, 286, 768–774.

28. Kouoh F, Gressier B, Dine T, Luyckx M, Brunet C, Ball- ester L, Cazin JC: Antioxidant effects and anti-elastase activity of the calcium antagonist nikardipine on activated human and rabbit neutrophils – a potential antia- therosclerotic property of calcium antagonists? Cardio- vasc Drugs Ther, 2002, 16, 515–520.

29. Leslie CC: Proporties and regulation of cytosolic phospholipase A2. J Biol Chem, 1997, 272, 16709–16712.

30. Linehan JD, Kolios G, Valatas V, Robertson DA, West- wick J: Effect of corticosteroids on nitric oxide production in inflammatory bowel disease: are leucocytes the site of action? Am J Physiol Gastrointest Liver Physiol, 2005, 288, 261–267.

31. Livingston A: Mechanism of action of nonsteroidal anti- inflamatory drugs. Vet Clin North Am Small Anim Pract, 2000, 30, 773–781.

32. Maling HM, Webster ME, Williams MA, Saul W, Ander- son W Jr: Inflammation induced histamine, serotonin, bradykinin and compound 48–80 in the rat: antagonist and mechanisms of action. J Pharmacol Exp Ther, 1974, 191, 300–310.

33. Martinez LL, Aparecida De Oliveira M, Fortes ZB: In- fluence of verapamil and diclofenac on leukocyte migration in rats. Hipertension, 1999, 34, 997–1001.

34. Marzocco S, Di Paola R, Serraino I, Sorrentino R, Meli R, Mattaceraso G, Cuzzocrea S et al: Effect of methyl- guanidine in carrageenan-induced acute inflammation in the rats. Eur J Pharmacol, 2004, 484, 341–350.

35. Mohler III ER, Sorensen LC, Ghali LK, Schocken DD, Willis PW, Bowers JA et al.: Role of cytokines in the mechanism of action of amlodipine: the PRAISE heart failure trial. Prospective Randomized Amlodipine Sur- vival Evaluation. J Am Coll Cardiol, 1997, 30, 35–41.

36. Nacife VP, Soeiro Mde N, Gomes RN, D’Avila H, Castro-Faria Neto HC, Meirelles Mde N: Morphological and biochemical characterization of macrophages

activated by carrageenan and lipopolysaccharide in vivo.

Cell Struct Funct. 2004, 29, 27–34.

37. Neto AG, Costa JMLC, Belati CC, Vinholis AHC, Pos- sebom LS, Da Silva Filho AA, Cunha WR et al.: Analge- sic and antiinflammatory activity of a crude root extract of Pfaffia glomerata (Spreng) Pedersen. J Ethnopharma- col, 2005, 96, 87–91.

38. Opie LH: Pharmacological differences between calcium antagonists. Eur Heart J, 1997, 18, 71–79.

39. Pitt B: Diversity of calcium antagonists. Clin Ther, 1997, 19, 3–17.

40. Panthong A, Kanjanapothi D, Taesotikul T, Phankum- moon A, Panthong K, Reutrakul V: Antiinflammatory activity of methanolic extracts from Ventilago harmandi- ana Pierre. J Ethnopharmacol, 2004, 91, 237–242.

41. Ramprasath VR, Shanthi P, Sachdanandam P: Anti- inflammatory effect of Semecarpus anacardium Linn.

Nut extract in acute and chronic inflammatory conditions. Biol Pharm Bull, 2004, 27, 2028–2031.

42. Serhan CN: Novel omega-3 derived local mediators in anti-inflammation and resolution. Pharmacol Ther, 2005, 105, 7–21.

43. Seth SD, Seth S: Calcium channels and calcium channel blockers. Indian J Physiol Pharmacol, 1991, 35, 217–231.

44. Singh BN, Josephson MA: Clinical pharmacology, phar- macokinetics and hemodynamic effects of nicardipine.

Am Heart J, 1990, 119, 427–434.

45. Sirmagul B, Kilic FS, Batu O, Erol K: The effects of verapamil on stres-and histamine-induced gastric lesions in rats.

Methods Find Exp Clin Pharmacol, 2004, 26, 763–767.

46. Slowik A, Turaj W, Pankiewicz J, Dziedzic T, Szermer P, Szczudlik A: Hypercortisolemia in acute stroke is related to the inflammatory response. J Neurol Sci, 2002, 196, 27–32.

47. Suleyman H, Demircan B, Karagoz Y, Oztasan N, Suley- man B: Anti-inflammatory effects of selective cox-2 in- hibitors. Pol J Pharmacol, 2004, 56, 775–780.

48. Suleyman H, Demirezer LO, Kuruuzum A, Banoglu ZN, Gocer F, Ozbakis G, Gepdiremen A: Antiinflammatory effect of the aqueous extract from Rumex patientia L.

roots. J Ethnopharmacol, 1999, 65, 141–148.

49. Takeuchi A, Kobayashi K, Yukiyama Y, Chihara T, Mat- suta K, Hashimoto A: Role of protease, protease inhibitor complexes in inflammation. Int Tissue React, 1984, 6, 1–8.

50. Vajja BN, Juluri S, Kumari M, Kole L, Chakrabarti R, Joshi VD: Lipopolysaccharide-induced paw edama model for detection of cytokine modulating anti-inflammatory agent. Int Immunopharmacol, 2004, 4, 901–909.

51. Weissmann G: Prostaglandins as modulators rather than mediators of inflammation. J Lipid Mediat, 1993, 6, 275–286.

52. Zhou MS, Maimes EA, Raij L: Inhibiton of oxidative stress and improvement of endothelial function by amlodipine in angiotensin II-infused rats. Am J Hypertens, 2004, 17, 167–171.

Received:

February 21, 2006; in revised form: June 6, 2006