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In This Article
 »  Abstract
 »  Introduction
 »  Material and Methods
 »  Results
 »  Discussion
 »  Acknowledgements
 »  References

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Year : 2004  |  Volume : 36  |  Issue : 6  |  Page : 351-354

Calcium channel blockers reduce inflammatory edema in the rat: Involvement of the hypothalamus-pituitary-adrenal axis

Department of Physiology and Pharmacology, Faculty of Medicine, Kerman, Iran

Date of Submission26-Feb-2003
Date of Decision04-Mar-2004
Date of Acceptance10-May-2004

Correspondence Address:
Department of Physiology and Pharmacology, Faculty of Medicine, Kerman, Iran
[email protected]

 » Abstract 

OBJECTIVE: To evaluate the role of calcium channel blockers and their mechanisms of action on acute inflammation of rat paw. MATERIAL AND METHODS: The study was conducted using carrageenan-induced rat paw inflammation model. Two different doses of nifedipine and verapamil (25 and 400 µg/kg, i.p.) were used. Edema was assessed by calculating the volume changes and by extravasation of Evans blue dye. RESULTS: Nifedipine reduced edema dose-dependently, whereas verapamil was effective only at low dose. Adrenalectomy prevented the effect of nifedipine and verapamil. With low dose of nifedipine 66% of antiinflammatory effect was observed. Pretreatment with -helical corticotropin releasing factor (CRF 9-41), a corticotropin-releasing hormone (CRH) receptor antagonist, had the same effect as that of adrenalectomy for either doses of verapamil, but only the effect of low-dose nifedipine was prevented completely. CONCLUSION: Our data suggest that verapamil and nifedipine exerts a potent antiinflammatory action possibly through pituitary adrenocortical activation.

How to cite this article:
Khaksari M, Mahani S E, Mahmoodi M. Calcium channel blockers reduce inflammatory edema in the rat: Involvement of the hypothalamus-pituitary-adrenal axis. Indian J Pharmacol 2004;36:351-4

How to cite this URL:
Khaksari M, Mahani S E, Mahmoodi M. Calcium channel blockers reduce inflammatory edema in the rat: Involvement of the hypothalamus-pituitary-adrenal axis. Indian J Pharmacol [serial online] 2004 [cited 2023 Jan 27];36:351-4. Available from: https://www.ijp-online.com/text.asp?2004/36/6/351/13504

 » Introduction Top

Calcium movement is an important factor in the activation of cells responsible for inflammation.[1] Calcium does this by releasing the inflammatory mediators[2],[3],[4] or by the activation of the plasma membrane or intracellular enzymes.[5] It has been reported that calcium activates the nitric oxide (NO) synthase enzyme,[5] phospholipase A2 and phospholipase C. This results in the activation of the release of arachidonic acid, with resultant formation of prostaglandins, leukotrienes, and thromboxanes.

One-way to test the role of calcium in inflammation would be by preventing voltage-dependent calcium influx into cells using calcium channel blockers (CCB), e.g. verapamil and nifedipine. CCBs have been shown to possess non-cardiovascular effects. These drugs have an effect on smooth muscles and secretory cells in the gastrointestinal tract and kidney;[4] they also prevent the action of neutrophils and lymphocytes in vitro and increase the production and secretion of IL-6 and IL-8, in vitro.[1]

We have shown in our previous study, that these two CCBs inhibit the carrageenan-induced paw edema.[6] It has been demonstrated that some of the CCBs can stimulate the hypothalamus-pituitary-adrenal (HPA) axis by acting on pituitary and hypothalamic levels.[7],[8] The present study was aimed to investigate the possible involvement of the HPA axis in the antiinflammatory action of CCBs.

 » Material and Methods Top


Male albino rats (200-250 g) allocated to 26 groups (eight in each) were used. The animals were allowed free access to food and water.

Drugs and solutions

Verapamil, nifedipine or ibuprofen (Rose Daro, Iran) were administered intraperitoneally (i.p.) just before the injection of 0.1 ml of 0.5% carrageenan (Sigma. Co. UK) or saline (0.1 ml) into the subplantar tissue of the hind paw.[9] Nifedipine and ibuprofen were dissolved in ethanol and verapamil in saline. Verapamil and nifedipine were given in one low and one high dose (25 and 400 g/kg) while ibuprofen was 12 mg/kg.[6]


Bilateral adrenalectomy was performed through a dorsal incision under thiopental (40 mg/kg) anesthesia. After surgery, the rats were returned to their cages with free access to food and normal saline[10] (instead of water). A control group was sham-operated with free access to food and water. One week was allowed for recovery from operative procedure.

Injection of anticorticotropin-releasing hormone (CRH)

A polyethylene cannula was placed in the right lateral ventricle under thiopental anesthesia for intracerebro-ventricular (i.c.v.) administration of corticotropin-releasing hormone receptor antagonist, -helical corticotropin releasing factor (CRF) (AntiCRF; 9-41, Sigma Co. UK). AntiCRF was dissolved in sterile pyrogen-free water and injected at the dose of 20 g/rat in volume of 10 l (i.c.v.); the control animals were treated with 10 l of sterile pyrogen-free water.[11]

Induction and measurement of the inflammation

Inflammatory edema was induced by subcutaneous injection of 0.1 ml of 0.5% carrageenan solution in the hind paw. The hind paw volume was measured by a plethysmometer, 4 h after carrageenan injection[9] and the algebraic difference between the treated and untreated hind paw volumes was taken as the edema volume.

Another method, involving the spectrophotometry technique,[12] was also used to measure the inflammation. Here  Evans blue More Details dye (Sigma, Co., UK) was injected (20 g/kg, i.v.) to evaluate the rate of albumin leakage as an indicator of inflammation.

Statistical analysis

Data were expressed as mean±SEM. The results were analyzed by analysis of variance (ANOVA) followed by Tukey's test or the Student's 't ' test. P<0.05 was considered as a significant difference.

 » Results Top

Carrageenan injection increased the hind paw volume compared to normal group (0.47±0.04 Vs 0.03±0.01) and both CCBs caused a significant reduction in inflammatory edema (P<0.01). The effect of nifedipine was dose-dependent, nifedipine (400 g/kg) and verapamil (25 g/kg) had the same effect as ibuprofen (12 mg/kg) ([Table - 1]).

As shown in [Table - 1], adrenalectomy blocked the antiedema effect of verapamil in both doses and of nifedipine (only 400 g/kg), but the administration of a dose of 25 g/kg nifedipine and 12 mg/kg ibuprofen caused significant inhibition of inflammation (P<0.01 and P<0.001 respectively). [Table - 1] also shows that pretreatment with CRH receptor antagonist blocked the antiedema effect of verapamil but not of nifedipine (400 g/kg), and ibuprofen (12 mg/kg), i.e. they reduced the hind paw edema significantly (P<0.05 and P<0.01 respectively) in spite of pretreatment with CRH receptor antagonist. Measurement of  Evans blue More Details in the hind paw tissue showed that in an intact animal the dye in nifedipine (400 g/kg) and ibuprofen (12 mg/kg) groups was significantly lower in content than in the carrageenan (untreated) groups (P<0.001) ([Table - 2]).

In the adrenalectomized (ADX) animals, only with 25 g/kg nifedipine, the dye content was significantly decreased compared to the carrageenan group (P<0.05) ([Table - 2]).

In the group which received CRH receptor antagonist plus nifedipine (400 g/kg), the content of  Evans blue More Details dye was significantly decreased compared to the carrageenan group (P<0.05) ([Table - 2]).

 » Discussion Top

The results of the present study indicate that CCBs (verapamil and nifedipine), that selectively block L-type calcium channel, exerted a potent antiedema effect in rat paw. These effects are similar to the report of De Vries et al (1995), though they used different topical CCBs and a different inflammatory model (skin inflammation).[1] The present results are also consistent with the reports on the reduction of acute pancreatitis.[2]

The possible mechanisms involved in the antiinflammatory activity of CCBs may be through (1) a reduction of the Ca2+ concentration in blood, causing a decrease in the vessel resistance, and consequent reduction of hydrostatic pressure in the capillaries, (2) inhibition of the release of pro-inflammatory mediators,[2],[3] (3) reduction of Ca2+ leading to inhibition of the activity of PLA2 and/or PLC, the enzymes responsible for the synthesis of eicosanoids and leukotrienes,[13] and (4) stabilization of the cell membrane integrity (by inhibiting Ca2+ influx), thus preventing tissue injury and inflammation.[5]

To find out the possible mechanism(s) of the antiinflammatory activity of CCBs, we studied the effect of verapamil and nifedipine in adrenalectomized, and CRH receptor antagonist pretreated rats. The observations from both experiments showed that CCBs inhibit inflammation. Both CCBs can activate the HPA axis. The activation of the HPA axis by verapamil can explain the antiinflammatory effect of this drug, but the inhibition of the HPA axis only tended to antagonize the effect of nifedipine. We cannot explain the discrepancy of results between nifedipine and verapamil. So we suggest an involvement of the hypothamic-pituitary-adrenal (HPA) axis in the action of these drugs.

It is conceivable that since these CCBs have diverse molecular structures, they have different modes and sites of action in the calcium system.[14] The difference between the effects of these CCBs has also been reported by other researchers who stated that verapamil has predominant effect on the heart and nifedipine on the vessels.[14] It has also been demonstrated that there is a significant suppressive effect on skin sensitivity by these CCBs.[15] The dose-dependent suppressive effect was seen with nifedipine but not with verapamil.[15] It has been reported that nicotine enhances the nifedipine-induced analgesia.[16] Rodler et al have reported that verapamil can increase the inflammatory cytokines like IL-6 in a high dose but not in a low dose.[17]

Ibuprofen (positive control for antiinflammatory study) inhibited the increase of paw volume by up to 83%, and the antiinflammatory effect of verapamil (25 g/kg) and nifedipine (400 g/kg), was similar to ibuprofen. Reduction of the efficacy of ibuprofen on reducing the paw volume in adrenalectomized and antiCRF treated rats, suggest participation of the adrenal gland and CRF receptors in the antiinflammatory activity of ibuprofen.

The  Evans blue More Details content of the inflammated paw decreased to 58% by ibuprofen. This was significantly more compared to both the doses of verapamil and 25 g/kg of nifedipine. Adrenalectomy and antiCRH treatment of rats resulted in the elimination of the inhibitory effect of ibuprofen on the dye extravasation. It can be claimed that ibuprofen needs an intact HPA for its action. The effects of CCBs on  Evans blue More Details content showed that only nifedipine is effective, whereas the water content in control, ADX and antiCRH groups decreased, by 52%, 41% and 37% by nifedipine respectively. These findings suggest that verapamil may increase vascular permeability to proteins through secretion of interleukins[17] or CCBs (verapamil and nifedipine), like other antiinflammatory drugs, may effect on protein and fluid leakage via different mechanisms involving arachidonic acid metabolites.[18]

The present study has shown that nifedipine and verapamil reduced acute carrageenan-induced paw edema in the rats. It is probable that the HPA axis mediates the antiinflammatory effects of verapamil. However, it is likely that the antiedema effect of nifedipine may involve both peripheral and HPA mechanisms. Further study to determine the relevance of this finding in humans should be undertaken.

 » Acknowledgements Top

This work was supported by Rafsanjan Faculty of Medicine. 

 » References Top

1.De Vries GW, Mc Laughlin A, Wenzel MB, Perez J, Harcourt D, Lee G, et al. The antiinflammatory activity of topically applied novel calcium channel antagonists. Inflammation 1995;19:261-75.  Back to cited text no. 1    
2.Lake BG, Lyubsky S. Dose dependent effect of continuous subcutaneous verapamil infusion on experimental acute pancreatitis in mice. Dig Dis Sci 1995;40:2349-55.  Back to cited text no. 2    
3.Rodler S, Roth M, Nauck M, Tamm M, Block LH. Ca2+ channel blockers modulate the expression of interleukin 6 and interleukin 8 genes in human vascular smooth muscle cell. J Mol Cell Cardiol 1995;27:2295-302.  Back to cited text no. 3  [PUBMED]  
4.Warren JB. Vascular control of inflammatory oedema. Clin Sci (Lond) 1993;84:581-4.  Back to cited text no. 4  [PUBMED]  
5.Southan GJ, Szabo C. Selective pharmacological inhibition of distinct nitric oxide synthase isoforms. Biochem Pharmacol 1996;51:383-94.  Back to cited text no. 5  [PUBMED]  [FULLTEXT]
6.Khaksari M, Sajjadi MA. Effect of verapamil and nifedipine on carrageenin-induced inflammation in the rat paw. J Kerman University Med Sci 1999;6:191-8.  Back to cited text no. 6    
7.Bianchi M, Panerai AE. CRH and the noradrenergic system mediate the antinociceptive effect of central interleukin-1 alpha in the rat. Brain Res Bull 1995;36:113-7.  Back to cited text no. 7  [PUBMED]  [FULLTEXT]
8.Le Beau AP, Mason DR. The effect of chemically diverse range of calcium channel antagonists on the AVP-stimulated ACTH responses in ovine corticotrophs. Cell Calcium 1994;16:47-58.  Back to cited text no. 8  [PUBMED]  
9.Bianchi M, Rossoni G, Sacerdote P, Panerai AE, Berti F. Effect of clomipramine and fluoxetine on subcutaneous carrageenan-induced inflammation in the rat. Inflamm Res 1995;44:466-9.  Back to cited text no. 9  [PUBMED]  
10.Waynforth MB, Flecknell PA. Experimental and surgical technique in rat. New York: Academic Press; 1992.  Back to cited text no. 10    
11.Bianchi M, Sacerdote P, Panerai AE. Fluoxetine reduces inflammatory edema in the rat: Involvement of the pituitary-adrenal axis. Eur J Pharmacol 1994; 263:81-4.  Back to cited text no. 11  [PUBMED]  
12.Scott DT, Lam FY, Ferrell WR. Acute inflammation enhances substance P-induced plasma protein extravasation in the rat knee joint. Regul Pept 1992;39: 227-35.  Back to cited text no. 12  [PUBMED]  
13.Craven PA, De Rubertis FR. Ca2+-calmodulin-dependent release of arachidonic acid for renal medullary prostaglandin synthesis: Evidence for involvement of phospholipase A2 and C. J Biol Chem 1983;258:4814-23.  Back to cited text no. 13    
14.Katzung BG. Basic and clinical pharmacology. 8th Ed. New York: Lange Medical Book; 2001.  Back to cited text no. 14    
15.Corteza Q, Shen S, Revie D, Chretien P. Effect of calcium channel blockers on in vivo cellular immunity in mice. Transplantation 1989;47:339-42.  Back to cited text no. 15  [PUBMED]  
16.Zbuzek VK, Cohen B, Wu W. Antinociceptive effect of nifedipine and verapamil tested on rats chronically exposed to nicotine and after its withdrawal. Life Sci 1997;60:1651-8.  Back to cited text no. 16  [PUBMED]  [FULLTEXT]
17.Rodler R, Roth M, Nauck M, Tamm M, Block LH. Ca2+ channel blockers modulate the expression of interleukin 6 and interleukin 8 genes in human vascular smooth muscle cell. J Mol Cell Cardiol 1995;27:2295-302.  Back to cited text no. 17    
18.Alexander F, Mathieson M, Teoh KH, Huval WV, Lelcuk S, Valeri CR, et al. Arachidonic acid metabolites mediate early burn edema. J Trauma 1984;24:709-12.  Back to cited text no. 18  [PUBMED]  
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