PHA2022 Organ bath simulation worksheet_final (1)

PHA2022 Workshop 2: Organ bath simulation Organ bath simulation worksheet In this session you will use a computer simulation of a guinea-pig isolated ileum experiment to investigate the effect of an antagonist on the concentration-response curves to two agonists acting at different receptors – acetylcholine and histamine. The simulation will be open on the lab class computers. Work through this exercise in groups of 3 and complete the included worksheet. Get your responses checked by your teaching associate before you leave the class (you are not required to submit the worksheet). How to use the simulation Below are some general instructions for using the simulation program. Take some time to familiarise yourself with these before starting the Protocol. Tissue preparation Tissue Type: Guinea Pig Ileum ( see the Experimental Setup for further information) To start a new experiment:  Select Guinea Pig Ileum from the Tissue Type list. ( see the Experimental Setup for further information about the tissue setup )  Click the New Experiment button.  Click the Record button to start the chart recorder running.  Select and apply drugs from the list of available Agonist and Antagonists. 1 Adding agonists: To add agonists to the organ bath  Select the agonist to be applied from the list of available Agonists.  Select the concentration of the drug solution to be applied from the Stock Soln . list.  Enter the volume (between 0 and 1 ml) of the stock solution to be added into the Volume box.  Click Add to Organ Bath to inject the selected volume of the agonist stock solution into the bath.  When the tissue response on the chart recording reaches a steady state (or after 30 seconds if no response has occurred) click Flush Reservoir to Bath to wash out the agonist of the organ bath. Note: In this set up the bath volume is 10 ml . We suggest you work with the smallest volume of addition as 0.01ml (=10  l). To achieve a bath [agonist] of 1x10 -8 M, you will need to add 10  l of a 1x10 -5 M drug solution

Adding Antagonists: Antagonists are typically studied by observing their effects on different concentrations of a chosen agonist. While antagonists can be added to the organ bath just as the agonists are, to avoid the necessity of repeatedly applying the antagonist to the organ bath before each agonist, but ensure the receptors are continually exposed to the antagonist, antagonists can be added to the reservoir of bathing solution used to flush the organ bath between agonist applications. To add an antagonist drug to the Krebs’ solution reservoir :  Select the Antagonist to be applied from the list of available antagonists. PHA2022 Workshop 2: Organ bath simulation 2 Calculating Final Bath Concentration (FBC) of drugs added to the Organ Bath: The final bath concentration (FBC), in M (Moles/litre), is related to the stock solution concentration [stock] (in M), the volume of stock added to the bath (in l or l) and the tissue bath volue (l) by the forula: The organ volue is 10 l. Therefore, adding 0.01l (10  l) of a 10 -4 M stock solution gives a final bath concentration of 10 -7 M . The forula can be rearranged to work out the volue you need to add to achieve a required FBC: Ai to add volues between of 0.01 – 0.10 l to the tissue bath [FBC] x Bath volume Volume added = --------------------------- [stock] [stock] x Volume added [FBC] = -------------------------------- Bath volume Calculating Final Bath Concentration by adding to the Reservoir When applying drugs to the Krebs’ solution reservoir, the resulting concentration within the reservoir [RC], in M (Moles/litre), is related to the stock solution concentration [stock] (in M), the volume of stock added to the reservoir (in ml) and the reservoir volume (ml) by the formula: The reservoir volume is 1000 ml (1L). Therefore, adding 0.1 ml of a 10 -3 M stock solution gives a final bath concentration of 10 -7 M. [stock] x Volume added [RC] = -------------------------------- Reservoir volume

PHA2022 Workshop 2: Organ bath simulation 1 A half-log unit = 3 x Table 1: Concentration-response curve to ACh in the absence and presence of unknown antagonist Bath [ACh] RESPONSE (M) Control (ie before antagonist) (g) In the presence of antagonist (g) 1 x 10 -10 0 0.13 3 x 10 -10 0.29 0.82 1 x 10 -9 0.48 2.70 3 x 10 -9 1.74 5.97 1 x 10 -8 4.63 9.88 3 x 10 -8 8.43 12.52 1 x 10 -7 11.62 13.35 3 x 10 -7 12.99 13.85 1 x 10 -6 13.58 14.02 3 x 10 -6 13.73 14.05 1 x 10 -5 13.83 3 x 10 -5 1 x 10 -4 3 x 10 -4 1 x 10 -3 Table 2: Concentration-response curve to HA in the absence and presence of unknown antagonist Bath [HA] RESPONSE (M) Control (ie before antagonist) (g) In the presence of antagonist (g) 1 x 10 -10 0 0 3 x 10 -10 0.13 0.41 1 x 10 -9 0.50 1.41 3 x 10 -9 1.68 3.55 1 x 10 -8 4.90 10.76 3 x 10 -8 8.73 12.83 1 x 10 -7 11.97 13.63 3 x 10 -7 13.28 13.75 1 x 10 -6 13.71 13.80 3 x 10 -6 13.95 13.9 1 x 10 -5 13.94 3 x 10 -5 1 x 10 -4 3 x 10 -4 1 x 10 -3 4

PHA2022 Workshop 2: Organ bath simulation Comparing the concentration-response curves to acetylcholine and histamine  did you obtain a maximal response to ACh? Yes / No  did you obtain a maximal response to histamine? Yes / No For each agonist, determine the concentration that caused a response that was 50% of the maximum (ie determine the EC 50 (M)). 2See reading log scales below . ACh: EC 50 2E-8 M histamine: EC 50 2E-8 M  Which is more potent, ACh or HA? Explain your response. They were both as potent as each other, both recorded a reading of 2.00E-08 Effects of the antagonist on the CR curves to ACh and HA  Calculate the EC 50 for each agonist in the presence of the antagonist. ACh + antag: EC 50 4.00E-9 M histamine + antag: EC 50 5.00E-9M Discussion  Describe the effect of the antagonist on each agonist CR curve . The antagonist caused both the acetylcholine and the histamine to reach the maximum concentration at a slightly faster rate than without the antagonist.  Can you identify if the unknown is reversible or irreversible? Justify your answer. The unknown is reversible as they reached the same maximum concentration. If the antagonist was irreversible than the maximum concentration would be lower. 5