Isolated Ileum Experiment 2021 - Instructions Ob Sim PDF

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notes for a lab week 4...

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PHPY 308

Isolated Ileum Experiment

Dr. K. Lebedeva

Isolated Ileum Experiment (2021) Background: Responses to various contractile agonists and their inhibition by competitive as well non-competitive antagonists were discovered using in vitro preparations. Different isolated tissue preparations were discovered to have different receptors, which proved useful to identify agonists/antagonists for those receptors. Thus, different types of isolated preparations were used to identify unknown substances in extracts (characterized by the response they produced in isolated tissue preparations), a method known as bioassay. Agonist/antagonist interactions at the receptors can be used to identify the type of receptor present in different tissues, as well as to determine the nature of agonist, antagonist and their interaction. Aims: 1. Students will use Organ Bath Simulator guinea pig ileum as a smooth muscle preparation that responds to cholinergic agonists, such as ACh to generate a cumulative concentration-response (C-R) curve. 2. Students will determine the type of antagonism exerted by atropine on the isolated ileal tissue by  constructing the CR curves following the addition of increasing concentrations of atropine (100 nM, 1µM, 10 µM)  calculating dose-ratios and creating the Schild plot. -----------------------------------------------------------------------------------------------------------Materials and Instructions: Organ Bath Simulator (Obsim) can be accessed at the University Virtual Lab. Use this link: https://vlab.usask.ca 1. Log in with your NSID. 2. Go to Common U of S folder and locate ObSim. 3. Once the application is loaded, click on “New Experiment”, choose Guinea Pig Ileum in the drop-down menu. 4. Leave the stimulation setting OFF. 5. Under Agonists, choose Acetylcholine. For the calculations purposes we’ll set the organ bath volume to 30 mL. The Buffer in the organ bath is Krebs. Experimental Procedures: Part A. Control ACh - concentration response curve (CRC ) 1. Start the recording (“Record” button at the bottom of the chart). 1

PHPY 308

Isolated Ileum Experiment

Dr. K. Lebedeva

2. Generate the ACh CRC by using the following concentration steps below. Please remember: all concentrations are cumulative, do not wash (or change buffer solution) in between ACh additions. It is not necessary to go through all 14 steps, you should stop the ACh additions when the ileal contraction (observed in the ObSim Chart) is not increasing any further (i.e. reached the peak).

Effective Bath Concentration

10 nM

ACh Additions to Organ Bath (remember to do the unit conversions in ObSim)

1. Add 30 μl of the 10 μM ACh

Always wait until the muscle response plateaus before adding the next dose.

30 nM 100 nM 300 nM 1 μM 3 μM 10 μM 30 μM 100 μM 300 μM 1 mM 3 mM 10 mM 14 mM

2. Add 60 μl of the 10 μM ACh 3. Add 21 μl of the 100 μM ACh. 4. Add 60 μl of the 100 μM ACh. 5. Add 21 μl of the 1 mM ACh. 6. Add 60 μl of the 1 mM ACh. 7. Add 21 μl of the 10 mM ACh. 8. Add 60 μl of the 10 mM ACh. 9. Add 21 μl of the 100 mM ACh. 10. Add 60 μl of the 100 mM ACh 11. Add 21 μl of the 1 M stock ACh 12. Add 60 μl of the 1 M stock ACh 13. Add 21 μl of the 1 M stock ACh 14. Add 120 μl of the 1 M stock ACh

After completing the control ACh CRC, wash the ileum tissue by clearing the drugs from reservoir and flushing the reservoir to bath, 2-3 times. After these washes, the ileum should relax and return to its baseline tension (0 g).

Part B: CRC in presence of Atropine (100 nM effective concentration): 1. Add 30 μl of Atropine from the 100 μM solution to the organ bath. In ObSim, you can find Atropine under the “Antagonists” Tab. 2. Complete the ACh CRC as in Part A, you should be able to go past the concentration you used to achieve the Emax in Part A. 3. Once the CRC is complete, wash the ileum tissue by clearing the drugs from Reservoir and flushing the reservoir to bath, 2-3 times. After these washes, the ileum should relax and return to its baseline tension (0 g). 2

PHPY 308

Isolated Ileum Experiment

Dr. K. Lebedeva

Part C: CRC in presence of Atropine (1 μM effective concentration): 1. Add 30 μl of Atropine from the 1 mM solution to organ bath. 2. Complete the ACh CRC as in Part A, you should be able to go past the concentration you used to achieve the Emax in Part B. 3. Once the CRC is complete, wash the ileum tissue by clearing the drugs from Reservoir and flushing the reservoir to bath, 2-3 times. After these washes, the ileum should relax and return to its baseline tension (0 g). Part D: CRC in presence of Atropine (10 μM effective concentration): 1. Add 30 μl of Atropine from the 10 mM stock to the organ bath. 2. Complete the ACh CRC as in Part A, you should be able to go past the concentration you used to achieve the Emax in Part C. 3. Stop the Recording. How to save your data and chart: 1. Optional: You can save your Experiment in .obs format, which you can open with ObSim and work on it later. File – Save Experiment. 2. Required: You need to take a screenshot of your chart and include it in your lab report appendix. Please make sure to condense your chart’s window size (click arrows on the bottom right corner), so that the entire experiment with 4 CRCs is visible on screen, before taking the screenshot. Alternatively, you can take 4 separate screenshots of 4 CRCs. How to collect data: 1. Once your experiment is completed, you can collect your data from the chart. The ileum response is measured in grams. You need to take a reading of the contractile response to each dose you added. 2. Use the green vertical bar to take the exact reading of the response trace. You can drag the green bar to any location on your chart. Make sure your window size is adjusted properly to see the detailed changes in the ileal contraction. 3. In Module 7, you can find the video showing some features of ObSim.

Graphing your data in GraphPad Prism: 1. Once your data is collected from Obsim, you can standardize it, i.e. convert contraction responses to percent of maximal contraction. NOTE: In this experiment, the maximal contraction achieved in the control CRC must be used in calculating percentages for ALL four CRCs. 2. Transfer data to Prism GraphPad and prepare all four CRCs on one graph. 3

PHPY 308

Isolated Ileum Experiment

Dr. K. Lebedeva

Creating the Schild Plot: A Schild plot helps to determine the pA2 value of an antagonist, based on at least three (or more) EC50 values of the agonist obtained in the presence of three different concentrations of the antagonist. You will have three EC50 values of ACh for three concentrations of atropine. Calculate the dose ratios for each EC50 value using the formula: Dose ratio = EC50 value in presence of antagonist / EC 50 value of agonist in absence of antagonist (control). Then calculate DR-1 values followed by Log (DR-1) values. Example: If the control EC50 is 8 µM and EC50 in presence of 10 nM atropine is 38 µM, then Dose Ratio = 38/8 = 4.75 Log (DR-1) = Log (4.75 -1) = Log 3.75 = 0.574 Plot the negative log molar concentrations of atropine (antagonist) on the X-axis and the Log (DR-1) values for ACh (agonist) on the Y-axis. Analyze using linear regression (straight line graph). The X-axis value where the graph intersects is the pA2 value of antagonist (atropine). NOTE:  

you can use either GraphPad or Excel for graphing the Schild Plot. calculations must be included in the Appendix.

Directions for your laboratory report: 1. Discuss competitive, non-competitive and physiological antagonism. 2. Include your graph with 4 CRCs. Obtain and discuss the EC50 values, Emax, pEC50 Discuss the meaning of these pEC50. 3. Include your Schild plot with the marked X-axis intercept, - the pA2 value. Define and discuss pA2. Determine and discuss the type of antagonism exerted by atropine, and how you concluded it. 4. Include your ObSim experiment chart screenshot(s) (with response traces from Parts A-D visible), calculations of DRs, and your calculation of pEC50.

Supplemental material Calculation of pEC50 (formerly pD2) values: Find the EC50 value, convert the EC50 into a molar concentration (if not already in molar form). Calculate the log of the molar concentration and take its negative value. 4

PHPY 308

Isolated Ileum Experiment

Dr. K. Lebedeva

E.g. EC50 is 1 µM. What is the pEC50? 1 µM = 0.000001 M. Log (0.000001) = -6. pEC50 = -(log EC50) = -(-6) = 6 A higher pEC50 indicates lower concentrations of an agonist are required for a halfmaximal response, which means greater potency. A lower pEC50 would indicate a lower potency. The potency is determined by the affinity as well the intrinsic activity of an agonist.

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