The Effect Of Agonist And Antagonist On Guinea Pig Ileum

Introduction Neuronal cell bodies synthesize neurotransmitters like acetylcholine and stored in neuronal vesicles unless they are required to get released into the synapse. Membrane depolarization and excess calcium entry into the cell enables the release of these neurotransmitters stored in the vesicles. The exocytosis pathway by which these neurotransmitters are released into the synapse includes the fusion of vesicle membrane and the plasma membrane (Brownlee & Johnson, 1965), facilitated by the interaction between vesicle proteins such as synaptobrevin & synaptotagmin and proteins in plasma membrane such as syntaxin & neurexin (Craver & Barrett, 1951). On exocytosis of these neurotransmitters, they activate both presynaptic and postsynaptic receptors, until they are taken back by the presynaptic neurons or degraded by the enzymes. When all different neurotransmitters are released from different types of neurons, altogether they form a pool of neurotransmitters called chemical milieu. The influence of this chemical milieu is depended on the concentrations of agonists and antagonists that are capable of acting at a particular synapse (Clark et.al, 2012). When agonists are more in concentration in chemical milieu, then more amounts of neurotransmitters are released. When antagonists are more in concentration in chemical milieu, then the production of neurotransmitters are inhibited (Craver & Barrett, 1951). Acetylcholine the major neurotransmitter: Acetylcholine is the major neurotransmitter studied so far. The enzyme Choline acetyltransferase catalyzes the synthesis of acetylcholine from choline and acetate in the neuronal cytoplasm. This acetylcholine formed is then stored in vesicles. Under stimulation of parasympathetic nerves, the action potential occurs that results in the influx of more calcium ions into the neuron cells. This releases the vesicles containing acetylcholine through exocytosis. Once the activation of post synaptic acetylcholine receptors, the acetylcholine is then hydrolyzed to form choline and acetate back by the enzyme acetylcholinesterase. Therefore, choline is taken back by the presynaptic neuron through a membrane protein embedded in the plasma membrane. Acetylcholine also activates presynaptic autoreceptors thereby inhibiting excess release of acetylcholines further by neurons (Clark et.al, 2012) (Brownlee & Johnson, 1965). There are five different types of muscarinic receptors which differ in their target acting site and function. Muscarinic M3 type of cholinergic receptors is the ones which are involved in the contraction of smooth molecules of Guinea Pig Ileum (Camille Georges Wermuth, 2003). On binding of acetylcholine with this receptor initiates the G protein coupled receptor mediated signal transduction pathway with the drastic increase in the concentration of Phospholipase C that increases the concentration of Inositol triphosphates and Diacylglycerol. This increased concentration of IP3 and DAG results in the excess influx of calcium ions into the cytosol of the cells including the release of calcium ions from sarcoplasmic reticulum. This increased concentration of calcium ions in the cytoplasm of the

ileum cells indicate the cells to contract, thereby the muscles are contracted which is the major event happen during digestion. Other biological processes activated by other receptors binding to acetylcholine include the following: controlling the thoughts, learning processes and memory processes. For learning and memory, this neurotransmitter acetylcholine is highly important in sharpening the concentration and increasing the perception (Moghaddam et.al, 2013). Atropine is a naturally occurring alkaloid that is a potential anti-cholinergic agent. It is an antagonist for acetylcholine receptors competing with acetylcholine. It is a tertiary amine alkaloid with high affinity for muscarinic receptors. These antagonists can act both centrally and peripherally (Author name unknown, article taken from Light, 2007). EC50: EC50 is the concentration of agonist that provides half of the maximum response. pA2: It is a measure of affinity of the antagonist for its receptor, which is also referred as equilibrium dissociation constant, which can be calculated using Schild plot analysis. Schild Plot analysis is the compilation of different graphs showing the dose response curves that can be compared between the conditions in the presence of agonist only without antagonist and the condition where different antagonist concentrations are used along with agonist. (http://facpub.stjohns.edu/~yoburnb/pages/dictimages/schild1.html Objectives: The objectives of the experiment are as follows: 1. To simulate the effect of an agonist (Acetylcholine) and three different concentrations of an antagonist (Atropine) on Guinea Pig Ileum. 2. Dose response curves of all the four conditions (use of only agonist, the use of an agonist with three different concentrations of an antagonist) need to be drawn to determine EC50 values. 3. With the help of EC50 values, Schild plot need to be drawn to find out the pA2 value for the antagonist. Method The whole experimental setup was a simulated model using organ bath contains the Guinea Pig ileum sample in it. To the first set of the experiment, 10-5 M concentration of Acetylcholine (Agonist) was used in the absence of Antagonists and the responses in terms of contractions were tabulated. To the next three sets, 10-1 M, 1M and 10-3 M concentrations of Acetylcholine (Agonist) were used in the presence of 4 X 10-6 M, 5 X 10-5M and 2 X 10-8M concentrations of Atropine (Antagonist). The contractions were observed and the values were tabulated. Dose response graph for all the four conditions (agonist only and three conditions of agonist and antagonists in different concentrations) were compiled using Schild analysis. Schild graph was constructed by plotting log (agonists) vs log (antagonist) where dose-ration was

the ratio of equiactive agonist concentrations meansured as EC50 in the absence and presence of antagonist concentrations. Schild graph was used to determine the dissociation constant of a given antagonist in fixed conditions (Golan et.al, 2012). From this dissociation constant only, the value of pA2 could be calculated using the relationship, pA2 = -log Kd. From the graph, the values for EC50 and pA2 were calculated. Results Tables for each condition: Table 1: Drug concentration and response of only agonist (Acetylcholine) Table 2: Drug concentration Vs response of Acetylcholine (Agonist) in the presence of 4 X 10-6M of Atropine (Antagonist) Table 3: Drug concentration Vs response of Acetylcholine (Agonist) in the presence of 5 X 10-5M of Atropine (Antagonist) Table 4: Drug concentration Vs response of Acetylcholine (Agonist) in the presence of 2 X 10-8M of Atropine (Antagonist) [From PDF, the table is not copied here, please copy the table for values under corresponding table numbers]
70 60 50 40 30 20 10 0 0 2

X axis: Dose Y axis: Response

Only Agonist Agonist with 4 X 106M Atropine Agonist with 5X10-5M Atropine Agonist with 2X10-8M Atropine

10

Figure shows the Dose Response curve of all the four conditions used in this experiment. [A refers to EC50 of Agonist and the three A refers to EC50 of Antagonists respectively] Calculation for EC50 and pA2: EC50: The maximum response is 60 when Acetylcholine an agonist is provided to the organ bath. The concentration of agonist corresponding to half the maximum response (which is 30) is 3.32 X 10-7M. Therefore, EC50 is 3.32 X 10-7M.

pA2: For the below table, pA2 can be calculated. Antagonist Dose 1.42 X 10-4 1.58 X 10-3 6.66 X 10-7 A/A 4.27 4.75 0.2 -log B -0.61 -0.60 -5.76 Log{(A/A)-1} 0.51 0.57 -0.1

Using the calculations seen in the above table, the pA2 value can be calculated by drawing a graph with Log B in X-axis and Log{(A/A)-1} in Y axis as follows:

The point at which the linear line meets the X axis provides the value of pA2 and therefore, pA2 is -5. Therefore, the contraction of the Guinea pig ileum is observed and the values for EC 50 and pA2 are also obtained Discussion Through this experiment, the role of Atropine as inhibitor for muscarinic receptor type 3 (M3) is clearly understood. In the near future, this molecule can serve as potential drug to control cholinergic neurotransmission diseases that are associated with this M3 receptor. The following table illustrates the use of molecules including Atropine as potential drug for some of the diseases associated with Cholinergic neurotransmission.

Cholinergic Pharmacological Agents Drug Action Clinical Use Relaxes muscle in the eye causing the pupil to dilate. Used when the eye is inflamed and during eye examinations. Atropine (and other anticholinergics) Blocks muscarinic receptors Slows the activity of the stomach and intestinal track and reduces acid secretion. Therefore, used for stomach cramps, diarrhea, diverticulitis, pancreatitis, bed wetting, motion sickness. There has been some indication of this drug for Parkinsons disease. Scopolamine Blocks CNS muscarinic receptors Blocks muscarinic receptors Mimics ACh Blocks ACh breakdown Blocks ACh breakdown Used topically to prevent dizziness, nausea and other aspects of motion sickness. Antidyskinetics used to treat Parkinsons disease and the dyskinesia associated with antipsychotic drugs Used to treat urinary retention, and stimulate movement of intestinal tract. Treat Alzheimers disease Reduces pressure in the eye and is used to treat glaucoma Used to diagnose and treat myasthesia gravis

Amantadine (Symmetrel) Bethanechol Tacrine (Cognex) Eserine or physostigmine

[Source of this table : http://neuroscience.uth.tmc.edu/s1/chapter11.html]

References 1. Brownlee & Johnson (1965). The release of Acetylcholline from the isolated ileum of the Guinea pig induced by 5-hydroxytryptamine and Dimethylphenylpiperazinium. Brit. J. Pharmacol. Vol. 24, pp. 689-700. 2. Camille Georges Wermuth (2003). The Practice of Medicinal Chemistry. Second Edition, Elsevier publication. 3. Clark et.al (2012). Pharmacology. Lippincott Williams & Wilkins publication. 4. Craver & Barrett (1951). Responses of the Guinea pigs ileum to histamine, acetylcholine, antigen, and electrical stimulation as influenced by progressive decreases in the concentrations of calcium, magnesium, or potassium separately or in pairs, The American Journal of Digestive Diseases. Vol. 18, Issue 5, pp. 163-165. 5. Golan et.al (2012). Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy. Third Edition. Lippincott Williams & Wilkins publication. 6. Greengard et.al (1972). Role of muscarinic cholinergic receptors in regulation of guanosine 3:5 cyclic monophosphate content in mammalian brain, heart muscle, and intestinal smooth muscle. Proc. Nat. Acad. Sci. USA. Vol.69, No.11, pp. 3287-3291. 7. http://facpub.stjohns.edu/~yoburnb/pages/dictimages/schild1.html 8. Moghaddam et.al (2013). Cholinergic and Histaminergic Effects of the Aqueous Fraction of Rosa damascene Extract in Guinea Pig Ileum and Rabbit Jejunum, Asian Journal of Biological Sciences. 9. The effect of Acetylcholine, Histamine, Atropine and Chlorpheniramine on the contractility of the Guinea pig ileum in vitro. Published in the journal Light. 2007. 10. Weiwei. Effects of drugs on isolated ileum motility in guinea pig, Department of Pharmacology. Available from: http://www.google.co.in/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&ved=0CD QQFjAB&url=http%3A%2F%2Fbmc.zju.edu.cn%2FAdmin%2FUploadFiles%2FFile %2Fpe%2Fexp4.pdf&ei=P4WEUu2SNYPqrAel5YCoDg&usg=AFQjCNFO9mHwP EotNXGuvLnZvmA1dQVEDQ&sig2=uaDbb07UDyjjHfuVAHpsDg&bvm=bv.5634 3320,d.bmk 11. http://neuroscience.uth.tmc.edu/s1/chapter11.html