Evaluation and Comparison Study of Acetylcholine and Histamine on Isolated Chick, Rat, Guinea pig and Rabbit Ileum with Special Reference to PD₂ Value in Pharmacology Practical

 

Susanta Kumar Rout*, Saptarshi Dutta, Mukul Sengupta and Srimanta Das

L.B.Rao Inst of Pharm. Edu. and Res. Bethak Road, Khambhat Anand, Gujarat

*Corresponding Author E-mail: uhumwangho@uniben.edu

ABSTRACT:

In the present investigation sought to examine and compare the suitability of the easily available and economical Chick ileum with rat, guinea pig and rabbit ileum preparation as a teaching aid for pharmacology teachers for the purpose of Pharmacology Practicals. In this study the different preparations were screened against the common practical’s like PD₂ value of different drugs. All the isolated tissue preparation showed a significant response to most of the common spasmogens. It was observed chick ileum is particularly suitable for cholinergic receptors as comparison to histaminergic receptors. Rat ileum was suitable for cholinergic receptors as comparison to histaminergic receptors. Rabbit ileum produces good effect against both drugs, but histaminergic drugs produces better response. In this study it was found that students feel more comfort with chick ileum than the rat ileum, after chick ileum Students prefer rabbit ileum for Histaminergic practical. It was found chick and rabbit ileum suitable, and meant for UG Practicals for both Medical and pharmacy students in basic and preliminary practical. However, more studies required to compare all the effect in different isolated tissues and comparison study between chick and goat ileum though that are easily available from the slaughter house, and animals need not be killed just for practical purposes.

 

 

 

INTRODUCTION:

Acetylcholine, Physostigmine, Neostigmine, Bariumchloride, histamine, adrenaline, pheniramine maleate and atropine has been commonly used drugs used in practical pharmacology for different stream. These drugs produce different effects on isolated smooth muscle in dose dependent manner¹. Investigated done by various workers for the effects of various drugs on smooth muscle in in-vitro design. In spite of the extensive use of spasmogenic drugs, it produced contraction on isolated smooth muscles. The smooth muscle preparations are used for bioassay of drugs, characterization of specific receptor or its subtypes, to determine concentration response curve of an agonist, to study antagonism of drug and in new drug discovery¹ Isolated ileum of rat, rabbit and guinea pig are the three commonly used preparations for 2^nd year and 3^rd year undergraduate (UG) B-Pharm as well as medical students in Pharmacology practical.

 

Due to certain limitations of CPCSEA and IAEC, In pharmacology practical this has driven us to conduct alternative isolated tissue experiments. Ileum consists of a number of receptors including muscarinic, histaminic, GABAergic, serotonergic, and adrenoreceptors. Guinea pig ileum preparations respond to most of the common spasmogens and are particularly suitable for the study of histaminergic receptors². Chick ileum preparations also respond to most of the spasmogens. However, it requires some skill and is less expensive for routine practical purposes. Isolated ileum preparations from other species like rabbitts and rats are being characterised for interactions between specific receptors and drugs^3,4. These preparations are widely used in researches to identify new receptors or to determine the variation in species produced by anticholinergic, antiadrenergic drugs. To use these preparations for teaching purposes, the laboratory animal species has to be sacrificed. However, chick ileum is the tissue that is easily available from the slaughter house, and animals need not be killed just for practical purposes.

 

The present investigations were carried out on isolated smooth muscle preparations of different animals with different drugs to define the nature of action on isolated tissue preparations with respect to study the effects of drugs on specific type of receptors. The aim of this study is to examine and compare the suitability of the easily available and economical Chick ileum as a teaching aid for pharmacology teachers for the purpose of Pharmacology Practicals, with the isolated ileum preparation from laboratory animals. This ileum, it found suitable, then it was meant for the entire practical in Pharmacy and medical students and also meant for minor research purpose.

 

Methods-:

Fresh ileum of healthy chicken was obtained from a slaughterhouse in Tyrode solution. The tissues were transported with suitable environments (under ice) to the laboratory. The intestinal contents were removed by washing with Tyrode solution through syringes, and the mesenteric residues were dissected out. About 2 to 3 cm of ileum was mounted similar to rat, rabbit and guinea pig ileum preparation¹.  For chick ileum, the preparation is made with few modifications². In the organ bath, inner organ containing 25 ml of Tyrode solution. Experiments were performed using -:

§     Tyrode solution at 37 ± 1 C)

§     bubbled with Oxygen (I bubble/sec)

§     Magnification more than 10 times

§     Tension 500 mg

 

Tissues were maintained at 37±1 C, aerated with air and resting load of 500mg during an equilibration of 45 min. Tension adjustments were made as necessary. The tissues were washed every 15 min interval in the stabilization period^5-6. Same procedure was followed for the chick ileum preparation. Contractions were recorded using frontal writing lever on kymograph.

 

Concentration response curve (CRC) to ACh and histamine (laboratory supplied) were constructed with a contact time of 60 sec. This was followed by three times washing with tyrode solution at an interval of 1 min. The concentrations of ACh (1 microgram to 1000 migrogram, in a fixed volume) were added in geometric progression⁷. The CRC was constructed till ceiling effect to Ach was obtained. In a separate set of experiments, the concentrations of Histamine (1 microgram to 1000 microgram, in a fixed volume) were added in geometric progression after completing the CRC of Ach and histamine, two to three contractile responses of barium chloride were recorded. The obtained responses of Ach and histamine were interpolated on the standard graph. To obtain the concentration of the different spasmogen same procedure was repeated for different tissues.

 

Separate tissues of both were put for 30 min with Tyrode solution. It was allowed to equilibrate for 20 min in the presence of atropine the antagonistic effect of atropine was determined by constructing the CRC of ACh in the presence of atropine.

Responses to ACh were recorded as changes in height from baseline and expressed as percent of maximum response of the ACh. The EC₅₀ values for ACh were calculated in the absence or in the presence of atropine using Graph Paper. The apparent value of PD₂ was calculated using the formula -:

1)  PD₂  = -log [EC₅₀]

 

Observations -: In this we documents the comparative study of different drugs of spasmogenic and spasmolytic with different tissues. In this study we observed ACh (0.1 ml to 0.16ml) produces a dose-dependent-Response, increase in contraction/dose of other spasmogenic drugs also produceses contraction on different isolated tissue preparation. [Figure-1, 2] From these results we calculated the EC50. After that we calculate PD₂ value. The response of ACH is compared with different tissues.

 

Figure-1

 

Figure-2

 

Histamine (0.2 ml to 0.32ml) produced a dose-dependent response on different isolated tissue preparations. Increase in contraction on isolated rat ileum and chick ileum produces significant response. [Figure 5,6] The EC₅₀ value of histamine was calculated.

 

Same procedure was followed for the guineapig (trachea) and rabbit ileum preparation. After getting the result of different tissue and different drugs we compare the results.

 

Table 1

Isolated Tissue Preparation	PD2 Value of Acetylcholine	PD2 Value of Histamine
Ret ileum Preparation	6.8	5.6
Chick ileum Preparation	5.7	5.2
Guineapig tracheal chain Preparation	6.4	5.8
Rabbit ileum Preparation	5.9	-----

 

 

Figure-3

 

Figure-4

 

Figure-5

 

Figure-6

 

Figure-7

 

RESULTS AND DISCUSSION -:

In rat ileum, Ach, Histamine and adrenaline produced concentration-dependent contractions and relaxation respectively^6-7. In Chick ileum Ach and Histamine produces response. In guineapig and rabbit these drugs also produces dose dependent contraction but in rabbit the response of adrenaline is significant than rat and chicken. Guineapig trachea gives significant response on Ach as well as Histamine. [Figure-1 to figure-7]

 

In order to study the action of spasmogenic drugs Ach and His on the chick ileum produces good response unlike rat, guineapig and rabbit in a concentration-dependent manner. Individual PD₂ value of the two drugs on different isolated tissues are shown in table-1. Results indicate that PD₂ value of Ach on chick ileum is quite similar to the rat, guineapig isolated tissue preparation.

 

CONCLUSIONS-:

At last we conclude that, chick ileum preparations can be used to demonstrate spasmogenic activity of cholinergic and histaminergic drugs. Though the cholinergic and histaminergic receptors are present in different isolated tissue preparation and produceses spasmogenic activity. Spasmolytic activity of anticholinergic and antihistaminergic drugs. We also study on the same tissue.Furthermore, it can be helpful for starting practical in isolated tissue in pharmacology for medical and pharmacy students. It is also used to teach the basic concepts or preliminary idea regarding PA₂ and value and study of bioassay if rat, rabbit and guineapig are not available. In this study the limitation is this preparation we cannot be used as a tool to discover new drugs and less sensitive drugs.

 

REFERENCES:

1.       Kela AK, Srivastava AK, Tripathi CD, Mehta SC. Effect of alcohols and their potentiating responses on acetylcholine induced contractures on frog rectus abdominis. Indian J Exp Biol 1991; 29:849- 51.

2.       Kulkarni SK, editor. Hand book of Experimental Pharmacology. 3^rd ed. New Delhi: Vallabh Prakashan; 2004.

3.       Burn JH. Practical pharmacology. Oxford: Blackwell Scientific Publications, 1952.

4.       Ghosh MN, editor. Fundamentals of Experimental Pharmacology. 3^rd ed. Kolkata: Hilton and Company; 2005.

5.       Unno T, Matsuyama H, Sakamoto T, Uchiyama M, Izumi Y, Okamoto H, et al. M2 and M3 muscarinic receptor-mediated contractions in longitudinal smooth muscle of the ileum studied with receptor knockout mice. Br J Pharmacol 2005; 146:98-108.

6.       Isaacs PE, Whitehead JS, Kim YS. Muscarinic acetylcholine receptors of the small intestine and pancreas of the rat: Distribution and the effect of vagotomy. Clin Sci (Lond) 1982; 62:203-7.

7.       Experimental Pharmacology, Goyel R.K. 4^th ed, Ahmedabad.

 

 

 

 

Received on 20.07.2010       Modified on 05.08.2010

Accepted on 11.08.2010      © RJPT All right reserved