An Examination of the Selectivity of Antagonist Drugs - Essay Example

www.academia-research.com Sumanta Sanyal d: 13.01.2006 An Examination of the Selectivity of Antagonist Drugs (Pharmacology Practical) Introduction (Pharmacology) To better understand the full implications of the interpretations of the results of this practical it is first deemed necessary to define a few important concepts dealt with herein. The definitions are as below. A Drug is defined herein as a chemical substance that interacts with a biological system to produce a physiologic effect. (Piascik, M.T., 2005) A Receptor is any cellular macromolecule that a drug binds to initiate its effect. (Piascik, M.T., 2005) The ability of a drug to bind to a particular receptor on a cellular environment is determined by the chemical structure of the drug that allows it to interact with complementary surfaces on the receptor. Drugs that successfully interact with such complementary surfaces on a particular receptor are either classified as Agonists or Antagonists. (Piascik, M.T., 2005) An Agonist binds to a receptor and activates or enhances cellular activities. The binding often initiates a series of biochemical events that ultimately leads to the alteration of function within the cell. The biochemicals that initiate such change are also known as Second Messengers. Antagonists also bind to receptors but they do not have the ability of agonists to initiate changes in cellular functions. Instead, their prime characteristics are that they tend to occupy receptor surfaces thereby blocking agonist bindings on the same surface. This diminishes or even totally inhibits agonist functions that initiate changes in cellular functions. That is why they are termed as antagonists or blockers. There are two kinds of antagonists: competitive and irreversible. Competitive antagonists bind reversibly with receptors and high dosage of agonists may displace them and reinstate agonist interaction. On the other hand, irreversible antagonists bind irreversibly with receptors and do not allow displacement with increasing dosage of agonists. (Piascik, M.T., 2005) Experimental Introduction Two sets of experiments were conducted on the cells of Guinea Pig ileum. In the first half of the first set of experiments acetylcholine (Ach) was used in isolation in 8 varyingly increasing concentrations to separate segments of the guinea pig ileum tissue. The response times to the dosages were noted and recorded. Ach is a known agonist and binds with receptors in smooth muscle cells to cause contraction. In the second half of the first set two separate segments of the ileum tissue were first treated with atropine and diphenhydramine, both of which are known to be antagonists to muscular contractile activity, albeit to different sets of agonists. The separate tissues treated with either atropine or diphenhydramine were next treated with Ach and the response times were noted and recorded. In the second set of experiments, in the first half as control, 8 separate segments of the ileum tissue were treated with increasingly concentrations of histamine, another known agonist. The response times in each case were noted and recorded. In the second half of the experiments two separate segments of the tissue were treated with atropine and diphenhydramine prior to being treated with histamine. The response times in each case after treatment with histamine were noted and recorded. There are two hypotheses. Hypothesis 1: Treatment with Acetylcholine alone in increasingly concentrated dosages will increase response times to a certain dosage concentration. Thereafter, any increase in concentration will not elicit any significant increase in response time. The same effect will be observed for increasingly concentrated dosages by histamine alone. Hypothesis 2: In the second hypothesis it is proposed that it will be found in the second half of the first set of experiments that there will be significant decrease in response time after Ach dosage is applied on tissue treated first with atropine. This will not be so with Ach dosage applied to tissue first treated with diphenhydramine. This is so because atropine is specifically antagonistic to Ach interaction on the tissue while diphenhydramine is not. For the second half of the second set of experiments it will be found that the response time will decrease significantly for treatment with histamine on tissue first treated with diphenhydramine but not for the tissue first treated with atropine. This is so because diphenhydramine is known to be antagonistic specifically to histamine interaction while atropine is not. Methodology and Results Experiment 1a: 8 separate segments of the guinea pig ileum tissue are treated with increasingly concentrated doses of acetylcholine ranging from 1-3000 nanomolar strengths, concentrations taken at Final Bath Concentration (FBC). The FBCs and response times are tabulated in Table 1.1 of the appendix. Experiment 1b: One segment of tissue is treated with acetylcholine at FBC 1 micromolar strength. Two separate segments of the ileum tissue are treated with atropine and diphenhydramine respectively, both at FBCs of 3 micromolar strength, before being treated with acetylcholine at FBC 1 micromolar strength. The response times for treatment with Ach alone and treatment after dosage with the antagonists atropine and diphenhydramine are tabulated below in Table 1.2. Table 1.2: Ach (mm) Atropine (mm) Diphenhydramine (mm) 81 3 47 Graph 1.1 in the appendix demonstrates the FBC to corresponding response time curve for treatment of the 8 segments with Ach alone. Experiment 2a: 8 separate segments of the ileum tissue are treated with histamine alone at increasing concentrations ranging from 1-3000 nM FBC. The concentrations and corresponding response times are tabulated in Table 2.1 of the appendix. Experiment 2b: Three segments of the tissue are taken. One is treated with histamine alone at FBC 1 micromolar strength. The other two are treated respectively with atropine and diphenhydramine at FBC 3 micromolar strength FBCs. These are next treated with histamine at FBC 1 micromolar strength and the response times for the three segments are after full treatment are tabulated in Table 2.2 below. Table 2.2: Histamine (mm) Atropine (mm) Diphenhydramine (mm) 70 68 7 Graph 2.1 demonstrates the concentration-response time curve for the experiments of experiment 2a. Discussion Acetylcholine is a neurotransmitter found at the nerve endings of animals. It is produced in the axons of nerve cells located in the nervous system and the neuromuscular junctions. The biochemical is released across the synaptic cleft and attaches to receptors on the muscle cells’ plasma membranes. The receptors change shape allowing calcium ions to diffuse into the cells. The calcium ions then attach themselves to a calcium-binding protein calmodulin. This bonded protein then activates a kinase enzyme – myosin kinase – that in turn adds a phosphate group to the muscle protein myosin. This causes the muscle cell to contract. Removal of the phosphate group relaxes the muscle cell again (Discussion, 2003). From both Table 1.1 and Graph 1.1 in the appendix it can be construed that increase in concentrations from 10-100 nM is rewarded with corresponding increase in response time. Thereafter, from 100-3000 nM FBC, any increase in concentration is not correspondingly complemented by increase in response time. A threshold is reached at 90 mm for both concentrations 1000 nM and 3000 nM. The explanation for this is simple. Up to 100 nM there are extra receptors that remain unoccupied and increase in concentrations only allows for their subsequent occupancy. The maximal obtainable effect is reached for acetylcholine at 1000nM when all available receptors are occupied leaving no scope for increase in response time with increase in concentration. This phenomenon bears out one part of the first hypothesis that a threshold will be reached for increasing concentrations of acetylcholine. In the second half of the first set of experiments it must be stated at the beginning that atropine is a known antagonist to the Ach interaction in smooth muscle cells. The biochemical is a known depressant of the parasympathetic nervous system. It has known affinity for the same receptors Ach binds to in muscle cells. This binding previous to Ach action interferes with Ach neurotransmission and inhibits the contraction of the muscle cell by Ach. This maintains the relaxes state of the muscle cell and continual stimulation of the muscle cell is possible as atropine prevents Ach from binding to the receptors and thus prevents the biochemical from breaking down and initiating its contraction action (Discussion, 2003). This bears out the results tabulated in Table 1.2 where it is found that segment treated with atropine first and treated with Ach later shows a very low response time in comparison to the one first treated with diphenhydramine. This is because atropine is specifically antagonistic to Ach interaction with affinity for the same receptors Ach itself binds to while diphenhydramine is not and has affinity for other sets of receptors that Ach does not bind to. This bears out the first part of the second hypothesis. In the second set of experiments segments of the guinea pig ileum were treated with histamine taken at 8 varying concentrations. Histamine is a known agonist. It is a biochemical produced and released by mast cells in the peritoneal cavity and connective tissues. Histamine mediates two important reactions due to antibody/antigen interactions inducing anaphylaxis and allergic responses. In this particular context of it being applied to guinea pig ileum, it binds to receptors in smooth muscle cells in the intestine. It initiates a contractile reaction that is much similar in context to relaxation of the plasma membrane of the cells allowing calcium ions to migrate inwards. Protonation of histamine is an important feature for the binding with its receptors and its agonist interactions (Messer, W.S., 2000). Using Table 2.1 and Graph 2.1 from the appendix and using the same logic as postulated in the case for Ach it is found that histamine has a maximal obtainable effect at concentration 1000 nM where the response time peaks and does not increase at all with any increase in concentration. All receptors are occupied at this maximal concentration and a threshold for response is reached. This also bears out the first hypothesis. In the second half of the second set of experiments segments of the guinea pig ileum tissue were first treated respectively with atropine and diphenhydramine and then treated with histamine. In this context it is necessary to mention that diphenhydramine (a medically-administered drug available under various brand names) is a known antagonist against interaction by histamine. It is an aminoalkyl etherantagonist and is primarily used to relax bronchoconstriction as histamine is known to cause intestinal and bronchial smooth muscle constriction. Its sedative side-effects (CNS depressant activity) may be due to blockade of receptors involved in procuring attention (Messer, W.S., 2000). The Table 2.2 shows that the segment first treated with diphenhydramine and subsequently with histamine gives very low response time value compared to the one first treated with atropine. This also bears out the second hypothesis as diphenhydramine is antagonistic specifically to histamine interaction and has affinity for the same receptors as histamine. Conclusion Comparing the potency of the two drugs acetylcholine and histamine from the two sets of tables and graphs it is found that Ach is slightly more potent than histamine as it induces higher response time values at the lower concentrations more consistently than histamine though histamine gives a higher response time value at concentration 10 nM for both drugs. The potency difference is very low and this cannot be easily discerned from the curves in the two graphs though the tabulated values of the response times are better indicators of this. Also, as further recommendation, it can be stated that experimentation with the paired sets Ach-atropine and histamine-diphenhydramine can be done in the following sense. Since both atropine and diphenhydramine are reversible antagonists treatments with increasing concentrations of Ach and histamine on segments treated first with atropine and diphenhydramine respectively can reveal at what concentration of the agonist drugs that antagonists are overcome and removed and allow initiation of the agonistic interactions. The effective dosage of drugs are usually 50 % of the maximal obtainable effect and in both these cases will lie in the rising parts of the two plotted curves in the range 3-100 nM for Ach and 1-100 nM for histamine. This in itself shows that the potency of the two drugs are similar though they do have different mechanisms for causing the same physiologic phenomenon – muscular contraction. More experiments in these two ranges can possibly accurately pinpoint the effective dosage using the concentration formulae. It is also noteworthy that atropine as a drug against the action of acetylcholine and diphenhydramine as a drug against histamine action are extremely effective though finding at what concentration of the agonists the antagonists are displaced from the respective set of receptors will yield more useful information of the interactive properties of these two sets of agonists-antagonists. List of References Discussion 11-13-03, Microsoft Word document. Extracted on 11th Jaunary, 2006, from: http://people.wwc.edu/student/andeba/Discussion%2011-13-03.doc Messer, W.S., University of Toledo, Last updated: 2000. Extracted on 11th January, 2006, from: http://www.neurosci.pharm.utoledo.edu/MBC3320/histamine.htm#synthesis Piascik, M.T., 2000, University of Kentucky Chandler Medical Center, Last updated: 2005. Extracted on 12th January, 2006, from: C:\WINDOWS\Desktop\Subpharma\Receptor Workbook.htm Appendix Table 1.1: Control Dose/Response Effects of Acetylcholine Control Dose (nM) Log CD Response Time (mm) 1 0 0 3 0.4771 0 10 1.0 16 30 1.4771 50 100 2.0 85 300 2.4771 94 1000 3.0 96 3000 3.4771 96 Table 2.1: Control Dose/Response Effects of Histamine Control Dose (nM) Log CD Response Time (mm) 1 0 0 3 0.4771 6 10 1.0 23 30 1.4771 39 100 2.0 71 300 2.4771 86 1000 3.0 90 3000 3.4771 90 Graph 1.1: X-axis values correspond one-to-one with the log of concentration values tabulated in Table 1.1 for easier working. Graph 2.1: X-axis values correspond one-to-one with the log of concentration values tabulated in Table 2.1 for easier working. Read More