Prac: Pig bladder organ bath

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From the 20-01-14 and 03-02-14 Human Physiology practicals.

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Question Answer
What type of tissue was used in this organ bath? Pig detrusor muscle.
What type of muscle is found in the bladder? How is its function controlled? The detrusor muscle is a smooth muscle which is innervated by the autonomic nervous system, as well as by hormonal control.
Why is it important that smooth muscle does not exhibit the 'twitch' function seen in skeletal muscle? Its function is in acting as a sphincter for hollow organs, so it needs to contract and relax slowly to allow these organs to fill. One example is the bladder, which must be capable of holding up to 300mL of liquid while other more important activities such as running away from predators (!) and other sympathetic responses occur.
Which receptor type and neurotransmitter is involved in the control of the detrusor muscle? Acetylcholine acting on cholinergic muscarinic receptors.
What is a common muscarinic receptor antagonist? Atropine.
What type of relationship can be measured between the concentration of the drug added to the organ bath and the response recorded? A proportional, quantitative relationship.
Did you use a PowerLab in this experimental setup? YES
Why did you have to calibrate the force transducer? Because its raw output is millivolts, but we wanted to measure the number of grams of force exerted by the bladder tissue so we calibrated the force transducer using a 1g weight.
What proportions of O2 and CO2 did you supply the tissue with in the organ bath? 95% O2, 5% CO2.
At what rate did you supply the tissue with O2/CO2? Approximately 1 bubble per second.
How did you equilibrate the tissue? What was the point of this? The tissue was left to equilibrate under 1-1.5g of tension for 30 minutes, and the saline was replaced by emptying and refilling the organ bath several times in this period to ensure the tissue was submerged in the saline at all times. This was to allow the tissue to adjust biochemically to the new conditions so that these adjustments did not affect the measurements taken in the assay.
How did you prepare your dilutions from the 10^-2 M solution of acetylcholine? We added 9ml of Ringer Tyrode solution to 4 test tubes. To the first we added 1ml of 10^-2 M acetylcholine - this produced a 10^-3 M solution, our first dilution. We added 1 ml of the 10^-3M solution to 9ml of Ringer Tyrode, producing our 10^-4M solution. We continued this until we had the following solutions: 10^-3 M, 10^-4 M, 10^-5 M, and 10^-6 M.
How did you add half measures of each dilution to the organ bath e.g. 5x10^-6 M? We added 0.5ml of the more concentrated solution. As soon as we added any of our dilutions into the Ringer Tyrode in the organ bath, it became further diluted, so in order to produce a 5x10^-6 M solution of acetylcholine in the organ bath, we removed 0.5ml of Ringer Tyrode from the organ bath, and added 0.5ml of the 10^-4 M acetylcholine solution. We were more interested in this final bath concentration than the dilution of the acetylcholine that we had prepared as the final bath concentration is what the tissue experiences and relates to the response recorded.
Why does pig bladder tissue react more slowly to acetylcholine than the rat ilium used in the organ bath prac last year? Because its functional is to slowly fill and to store, as well as to eventually excrete (which it cannot do too rapidly without causing damage), whereas the rat ilium tissue has these functions as well as that of peristalsis to further mechanically break down and propel food.
What was the timing schedule for the assay (just acetylcholine)? 0-60s: prepare the syringe to remove the Ringer Tyrode and the syringe to add the acetylcholine, prepare the label to add to the graph on PowerLab so that you can later analyse the data from the point of addition. 60-300s: Add the drug and record for 3 minutes/ until plateau. 300-580s: Wash tissue by emptying and refilling organ bath twice. Leave tissue alone for 3 minutes. 550-580s: Prepare syringes and label for next drug concentration. 580s: remove Ringer Tyrode and add drug and label and record...repeat as necessary until all dilutions have had responses recorded.
What differed between the method in the first practical (just acetylcholine) and the second? In the second practical, different antagonists (for the M1, M2, and M3 subtypes of the muscarinic receptors in the detrusor muscle tissue) were added to the Ringer Tyrode solution (one solution per group) before the assay was carried out so that different concentration-response curves could be drawn in order to determine the predominate receptor type that controls muscle contraction in the bladder tissue. Ringer Tyrode was the control.
How was the second practical a double-blind experiment? We used letters rather than names for solution so that those leading the lab did not know which antagonist we were being given and we did not know which we were using. Only the lab techs knew that D = Ringer Tyrode, for instance.
Which muscarinic receptor antagonists were used and which receptor subtype did they antagonise? M1-selective antagonist: 1mM pirenzipine. M2-selective antagonist: 1 mM AF-DX-116. M3-selective antagonist: 40μM 4-DAMP. Control: Ringer Tyrode.
What were the acetylcholine solutions used in the second practical? 5x10^-4M for the three initial acetylcholine additions to ensure that the tissue was responsive and the set up worked, and then: 5x10^-5, 10^-4, 5x10^-4, 10^-3, 5x10^-3, 10^-2.
What were the main activities in parts 1 and 2 of the second practical? Part 1: set up the organ bath without antagonist, establish that the tissue is responsive to acetylcholine and make any appropriate adjustments. Part 2: add antagonist to the Ringer Tyrode reservoir and bathe the tissue in it, allow to acclimatise to the new conditions for 10 minutes, add increasing concentrations of acetylcholine to the tissue to initiate a response.
What is the major subtype in the muscarinic receptor population found in the pig detrusor muscle (the largest proportion, not necessarily the largest contributor to function)? M2 constitutes ~75% of muscarinic receptors in the pig detrusor muscle.
What is the EC50? The molar concentration of the agonist that produces 50% of the maximal response.
Affinity constants (pKb) can be calculated in order to give pharmacologists an idea of how selective an antagonist/agonist is for a certain receptor or receptor subtype. How is pKb calculated? pKb = log (agonist concentration ratio - 1) - log (concentration of antagonist) Concentration ratio: shift in the concentration response curve in response to an antagonist. This is likely the difference between the EC50 values for the concentration response curves.
What was the main finding of the double blind second practical? The dose-response curve for bladder contractions induced by acetylcholine in the presence of 4-DAMP was significantly shifted to the right. This meant that it required a greater concentration to reach 50% of the maximal response i.e. its EC50 was higher. 4-DAMP was specific for the M3 subtype muscarinic receptor, and the data shows that these are the main receptors involved in detrusor muscle contraction. The other receptor antagonists did not significantly change their dose-response curves, showed that their antagonism was not affecting detrusor muscle contraction.
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