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BS2013: Physiology & Pharmacology
Practical Preparation Exercise 1
Practical preparation: a computer-based ‘dry’ practical. Introduction and exercise: understanding and modelling responses to agonists. This document contains an introduction to the Guinea pig ileum experimental set up used to generate the data you will be plotting, a guide on how to plot a graph of the data provided, a table to fill in and questions for you to answer (i) – (vii). This exercise can be completed after watching the Panopto video called ‘Practical preparation exercise 1’. The aim to complete exercise 1 by 14 th^ October and bring any queries to the question and answer live session scheduled for 4pm on Friday 14 th^ October. Please post any questions ahead of time on the discussion board so we can collate your queries and answer frequently asked questions in the live session. You can also ask questions live in the Q + A session.
Assessments Assessment of the practical component forms 40% of the module marks. The MCQ assessment based on the practical preparation material (‘dry practical’) is worth 10 %. This will be given on Friday 28 th^ October. There is 3 hours given to complete this. Although it is a multiple choice style, please keep in mind that you will have to plot graphs, analyse and interpret data in order to answer the questions. It is estimated that it will take you approximately 3 hour to complete so allow plenty of time. The practical report, based on the computer simulation of the experiments on Guinea pig ileum (‘wet practical’) is worth 30 % of the module marks. This will be given to you at the laboratory or simulation practical class. Exercise 1 and 2 will help prepare you for both assessments.
Drugs that affect gut motility (1) Acetylcholine. Acetylcholine acts at both muscarinic (G-protein coupled) receptors and nicotinic (ligand gated ion channel) receptors. Atropine is a selective muscarinic receptor antagonist. Nicotine is a selective agonist at the nicotinic acetylcholine receptor (present in enteric ganglia) and hexamethonium is a nicotinic acetylcholine receptor antagonist. (2) Histamine. Histamine is released locally in the gut and leads to contraction through the activation of H 1 histamine (G-protein coupled) receptors. Mepyramine in a selective antagonist of the H 1 mediated response. (3) Opioids. Primarily prescribed as analgesics (pain killers) e.g. morphine, they have a number of side effects including a reduction in gut motility (constipation). Opioid receptors are G-protein coupled receptors and in some neurons their activation leads to membrane hyperpolarisation (due to opening of potassium channels) and inhibition of the opening of voltage dependent calcium channels. Naloxone is an antagonist at opioid receptors and DAMGO is an agonist ( 4 ) Tetrodotoxin (TTX). A potent and selective blocker of voltage gated sodium channels and blocks action potential propagation in nerves (effective concentration 0.3 M). An Agonist - a drug that binds to a receptor and activates it producing a response. An Antagonist - a drug that has no effect on its own but attenuates the effect of an agonist. This needs to be an online pre activity that JW will go through at his first session Complete the table Selective agonist / activator Selective antagonist / blocker G-protein coupled receptor/ ligand gated ion channel Muscarinic ACh receptor Nicotinic ACh receptor Histamine H receptor Opioid receptor Voltage gated sodium channel
Experimental set-up
A piece of ileum (lumen flushed to clear gut contents) is threaded at either end, mounted in an organ bath and set under tension/load of 0.5 g (this should make the preparation fully relaxed). The tissue is bathed at 37oC in physiological solution (Ringer’s) bubbled with carbogen (a mix of 95% oxygen and 5% carbon dioxide). The effects of drugs (agonists and antagonists) applied to the organ bath on ileum contraction can be measured in this system. In addition, responses to nerve stimulation can also be determined. Nerves can be stimulated electrically by field stimulating electrodes in the organ bath. Short duration electrical pulses (< 1 ms) can be used to selectively activate nerves. Typical trace showing the contractile responses of the guinea-pig ileum
Plotting the data using Excel: Data presentation is an important skill; these instructions highlight key issues when analysing and displaying the data.
1. Open a blank Excel spread sheet and input data as shown below (alternatively on Blackboard under “Information for the Practical” open the Excel file “Tables.xlsx”). IMPORTANT! Concentrations must be input as shown below: i.e. 1X10-^9 M 1E- 9 2. Then plot the data using Excel: a. Highlight both columns of data (use shift + arrow keys to select more than one column or cell) then select the Insert tab and go to Charts and select the Scatter icon (on some versions of Excel, this may be labelled Insert Scatter (X,Y) or Bubble chart ).
b. Click Scatter with Smooth Lines and Markers and a graph will be generated
j. Double-click on the horizontal axis, to open a panel named Format Axis on the right. k. Click the graph icon (three vertical bars) so it appears in green l. Click on Tick Mark and select Major type “ Outside ” m. Click on Number , select Scientific and Decimal place “ 0 ” n. Double-click on the horizontal axis. Select Logarithmic scale and Base = 10 from the panel named Format Axis. o. Then for Vertical axis crosses select Axis Value and change it to 1.E- 10.
p. Click on the graph, then on the green cross located on its right. Click on Gridlines and add Primary Minor Vertical to the selection. q. Click on the horizontal axis, then in Axis Options , in the section Units, subsection Major add “ 10 ” and press the key ENTER on the keyboard.
