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DOI: 10.7860/JCDR/2013/6286.3249 Original Article Study on Neuromuscular Blockade Section

Pharmacology Action of Verapamil in Albino Rats

JAYASHREE NAGARAL1, SHASHIKALA GH2, JAGADEESH K3, SHARATH KUMAR K4, JAYANTH GS5, CHENNAVEERAPPA PK6, RAJANI PATIL7 ­ ABSTRACT Group 3-Verapamil (2.5mg/kg), Background: Channel Blockers (CCBs) are now Group 4-given Verapamil (10mg/kg). widely employed in the treatment of cardiovascular diseases The time of onset of hind limb and total duration of and peri-operative hypertension. It has been reported that recovery are noted using inclined screen method. calcium channel blockers inhibit neuromuscular transmission. Results: Analysis of the results of group 3 that was received They have been shown to increase the neuromuscular blockade 2.5mg/kg of Verapamil, there was no onset of paralysis, in produced by neuromuscular blocking agents in in-vitro muscle group 4 that received injection Verapamil 10mg/kg, showed nerve preparations. The present study is undertaken to neuromuscular blockade activity. The mean onset of hind limb demonstrate the effect of , verapamil paralysis was delayed compared to standard group and the on neuromuscular transmission in albino rats. mean duration of hind limb paralysis was shorter than standard Objectives: To study the neuromuscular blockade action of group. It was statistically significant (P≤ 0.05). verapamil in albino rats. Interpretation and conclusion: It is generally held that external Methods: Twenty four albino rats of either sex weigh 150- calcium is not necessary for the contraction of mammalian 250gms are selected and are randomly divided into 4 equal , the demonstration of inward calcium currents groups. The experimental rats are divided into four groups of 6 that can be abolished by CCBs in these muscles prompted rats each and they are given the following treatment. to re-examine the effect of Verapamil on the neuromuscular Group 1(Control) - Normal saline (1ml/ kg), transmission. The present study allows us to determine the Group 2 (Standard) - Pancuronium (0.04 mg/kg) neuromuscular blockade activity of Verapamil.

Key words: Calcium channel blockers, Neuromuscular blocking agents, Hind limb Paralysis, Verapamil, and Pancuronium

Introduction In the light of the aforementioned developments, this study has The neuromuscular blocking have revolutionized the practice been undertaken to evaluate the neuromuscular blockade action of . Skeletal muscle relaxation and paralysis can occur of verapamil. interruption of function at several different sites, that includes the central , myelinated somatic nerves, unmyelinated Material & Methods motor nerve terminals, nicotinic receptors, the motor The present study was conducted in the Department of end plate and the muscle membrane or intra-cellular contractile , JJM Medical College, Davangere, India, with the apparatus itself [1,2]. approval of institutional ethical committee. Calcium Channel Blockers (CCBs) are chemically heterogeneous Wistar albino rats of either sex weighing 150-250g were selected. group of drugs that inhibit the ionic current carried through the The animals were housed under standard conditions with free slow calcium channel in cardiac and vascular . access to water and food. The animals were screened for normal This action is the basis of their usefulness in the treatment of motor activity before initiating the procedure. cardiovascular disorders [3]. While studying the new property of it had to be compared with The excitation – contraction coupling in skeletal muscle is thought the existing standard reference drug already in use. Pancuronium to depend mainly on intra-cellular stores of calcium. The calcium is the ideal non–depolarizing blocker which is commonly used entry blockers might not be expected to exert any significant effects taken as standard drug to compare the neuromuscular blockade on voluntary muscle contraction. However, recent studies have action of verapamil. suggested that the calcium entry blocker Verapamil, can inhibit Inclusion Criteria: neuromuscular transmission in anaesthetized cats and dogs [4]. • Animals weighing >150 and <250g Anesthesiologists may have to deal with the interaction of • More than 21 days of prior use of any experimental purpose. calcium entry blockers with muscle relaxants in patients who • Age < 4 months are on prolonged calcium antagonist therapy before or • Healthy with normal behavior others giving one or two doses of verapamil IV for treatment of supraventricular paroxysmal tachycardia or reduction of ventricular Exclusion Criteria: rate associated with atrial fibrillation or flutter during anesthesia • Animals weighing < 150g and > 250g. [5]. • Within 21 days of prior use for any experimental purpose. There are many studies showing potentiation or interaction of • Age > 4 months Neuromuscular Blockers with CCBs but, very few in vivo studies to • All visible diseases demonstrate the neuromuscular blockade action of CCBs alone. A total number of 24 rats of either sex were divided into four

Journal of Clinical and Diagnostic Research. 2013 Aug, Vol-7(8): 1617-1619 1617 Jayashree Nagaral et al., Neuromuscular Blockade Action of Verapamil www.jcdr.net

groups of six rats each. in minutes in each group has been provided in [Table/Fig-1 and 2]. Group 1 (Control): Normal saline 1ml/ kg There was no hind limb paralysis seen with Group 3 Verapamil 2.5 mg/kg [Table/Fig-1 and 3]. Neuromuscular blockade action was Group 2 (Standard): Pancuronium 0.04 mg/kg seen with Group 4 Verapamil 10mg/kg., but the onset of action Group 3 (Test Group-1): Verapamil 2.5mg/kg was delayed (26.3 min ±4.3 min ) compared to Group 2 Standard Group 4 (Test Group-2): Verapamil 10mg/kg treated with Pancuronium (5.5 min ± 1.5 min). Group 4 Verapamil Drugs were injected by a 24-25 gauge needle attached to a 10mg/kg showed shorter duration of hind limb paralysis. (25.8 min 1ml tuberculin syringe into the intraperitonial cavity of rats. After ± 19.4 min) compared to Group 2 Standard (37.3 min ± 6.8 min) injecting the drug, animals, the neuromuscular blockade activity [Table/Fig-2 and 4).The results of intra and inter group comparisons was observed over a period of 1 hour using inclined screen were statistical significant (p≤0.05). method. Statistical Analysis Inclined screen method Results were presented as Mean ± SD. One way ANOVA was An inclined screen was used for testing -like agents for used for multiple comparisons followed by Bonferroni’s post–hoc testing compounds for muscle relaxant activity [6]. The rats were test for comparison between groups. For all the tests a ‘p’ value of placed at the upper part of the inclined screen and were given 30 0.05 or less was considered for statistical significance seconds to hang on or to fall off. The following parameters were observed. Discussion 1. Time of Onset of hind limb paralysis (in minutes) The effects of calcium channel blockers have been extensively 2. Total duration of paralysis (in minutes) investigated in the , smooth vascular, respiratory and intestinal, there are few studies on the action of these drugs Results on neuromuscular activity. The skeletal muscle calcium channel The study includes total number of 24 rats divided into four groups contains slow type, similar to that observed in cardiac and of six rats each and received the drugs accordingly. The time of vascular smooth muscles, with special binding sites for calcium onset of hind limb paralysis & total duration of paralysis recorded channel blockers. These channels in cardiac and vascular smooth

GROUPS SERIAL NO.OF RATS MEAN SD

1 2 3 4 5 6 Group 1 Control ------* (Normal saline) Group 2 Standard 7 5 5 7 3 6 5.5 1.5 (Pancuronium) Group 3 ------* Verapamil 2.5mg/kg Group 4 20 30 27 25 32 24 26.3 4.3 Verapamil 10mg/kg

[Table/Fig-1]: Time taken for onset of hind limb paralysis following injection (in minutes). (* No hind limb paralysis)

GROUPS SERIAL NO.OF RATS MEAN SD

1 2 3 4 5 6 Group 1 Control ------* (Normal saline) Group 2 Standard 45 30 28 40 42 39 37.3 6.8 (Pancuronium) Group 3 ------* Verapamil 2.5mg/kg Group 4 15 20 65 18 15 22 25.8 19.4 Verapamil 10mg/kg

[Table/Fig-2]: Total duration of hind limb paralysis (in minutes). (* No hind limb paralysis)

[Table/Fig-3]: Bar diagram showing mean duration of onset of hind limb paralysis [Table/Fig-4]: Bar diagram showing mean duration of hind limb paralysis

1618 Journal of Clinical and Diagnostic Research. 2013 Aug, Vol-7(8): 1617-1619 www.jcdr.net Jayashree Nagaral et al., Neuromuscular Blockade Action of Verapamil

muscles are very sensitive to CCBs, where as skeletal muscles are such potential neuromuscular blockade action in patients receiving less sensitive to CCBs [7]. Because of these peculiarities in some long term therapy with calcium channel blockers physiological processes in the muscle or may be affected by these drugs. Limitations of study The study has several limitations. It has been carried out only in The results of the present study demonstrate that Verapamil one species of animals like rats and needs to be extended to other showed neuromuscular blockade activity at 10mg/kg. In a animals as well and experiment was conducted using only one previous study done by Patel BG et al., demonstrating the screening method. The comparison other CCBs on neuromuscular interaction of verapamil with neuromuscular blocker gallamine in transmission could not be done as there are very in vivo studies. rats, three graded doses of verapamil 2.5mg/kg, 5mg/kg & 10 mg/ kg potentiated the neuromuscular blockade action of gallamine [8]. References As compared to the earlier study, where 2.5 mg/kg can potentiate [1] Thandla Raghavendra. Neuromuscular blocking drugs: Discovery and the action but in this study, at 2.5 mg/kg Verapamil could not show development. J R Soc Med. 2002; 95(7):363–67. the neuromuscular blockade property alone. [2] Paul F. White, Bertram G. Skeletal muscle relaxants. In: Katzung BG, editor. Basic and clinical Pharmacology. 10thed. Singapore: McGraw – Hill companies. Although it is not designed primarily to investigate the underlying 2007.p.424-41. neuromuscular action of calcium channel blocker vera- [3] Godfraind T, Millar R, Wibo M. Calcium antagonism and calcium entry blockers. pamil, several mechanisms have been proposed. Calcium is essential Pharmacology Review. 1986; 38:321-417. for the release of acetylcholine at neuromuscular junction. It has been [4] Kraynack BJ, Lawson NW, Gintautes J, Tjay HT. Effects of verapamil on indirect muscles twitch responses. Anesth Analg. 1983; 62:827. postulated that verapamil inhibits conductance of the presynaptic [5] Bikhaji GB, Leung I, Flores C, Mikati HM. Foldes FF. Potentiation of membrane to calcium. Some studies suggest that verapamil affects neuromuscular blocking agents by calcium channel blockers in rats. Anaest transmitter release or may interfere with neuromuscular transmission Analg. 1988; 67:1-8. [6] Verney RE, Linegar LR, Holadey HA. The assay of curare by the rabbit head by blocking the action of acetylcholine [9]. drop method. J Pharmacol Exp Ther. 1949; 97:72-83. [7] Nayler WG - Tissue-selectivity. In: WG Nayler - Calcium antagonists, 2 nd Ed, Conclusion London: Harcourt Brace Jovanovich. 1989: 113-29. In the present study, verapamil demonstrated the neuromuscular [8] Patel BG, Chotai NP, Shah NN. An interaction study of verapamil with the neuromuscular blocker gallamine. Ind J Pharmacol. 1997; 29: 266-68 blockade activity. In order to specify the mechanisms involved [9] Lawson NW, Kraynack BJ, Gintautas J. Neuromuscular and electrocardiographic in neuromuscular blockade activity of verapamil more extensive responses to verapamil in dogs.1983; 62:50-54. studies are required. Whatever the actual mechanism(s) involves, the present data suggests that the clinicians should be aware of

PARTICULARS OF CONTRIBUTORS: 1. Assistant Professor, Department of Pharmacology, Hassan Institute of Medical Sciences, Hassan, India. 2. Professor, Department of Pharmacology, JJM Medical College, Davangere, India. 3. Professor & Head, Department of Pharmacology, Shivamogga Institute of Medical Sciences, Shivamogga, India. 4. Assistant Professor, Department of Pharmacology, AJ Institute of Medical Sciences, Manglore, India. 5. Assistant Professor, Department of Pharmacology, Subbaiah Medical College Hospital and Research Centre, Shivamogga, India. 6. Associate Professor, Department of Pulmonary Medicine, Hassan Institute of Medical Sciences, Hassan, India. 7. Assistant Professor, Department of Pharmacology, Karnataka Institute of Medical Sciences, Hubli, India.

NAME, ADDRESS, E-MAIL ID OF THE CORRESPONDING AUTHOR: Dr. Jayashree Nagaral, Assistant Professor, Department of Pharmacology, Hassan Institute of Medical Sciences, Hassan-573201, Karnataka., India. Phone: 91 9481981405, E-mail: [email protected] Date of Submission: Apr 23, 2013 Date of Peer Review: Jun 25, 2013 Financial OR OTHER COMPETING INTERESTS: None. Date of Acceptance: Jul 10, 2013 Date of Online Ahead of Print: Jul 15, 2013 Date of Publishing: Aug 01, 2013

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