Competitive antagonism of acetylcholine by atropine and determination of pA2 value in experiments
The purpose of this experiment is to grasp the method of determining the pA2 value and its significance while also focusing on the observation of the competitive antagonism of atropine on acetylcholine. pA2 is an antagonism parameter that indicates the strength of the competitive antagonist effect, and the larger the value, the stronger the effect of the antagonist.
Operation method
Scott's ratio method
Principle
Acetylcholine acts on M receptors in the guinea pig ileum to cause intestinal muscle contraction. When the M receptor antagonist atropine is added, if increasing the concentration of acetylcholine still achieves the maximal response before the addition of the antagonist and shifts the dose-response curve parallel and to the right, then it indicates that atropine exhibits a competitive antagonism against acetylcholine. Acetylcholine is expressed as a molar concentration, and according to the law of mass action, the quantity-effect relationship is a straight-sided hyperbola, conforming to the linear relationship of the Clark equation, which can be calculated by straight-line regression using the Scott Ratio method. pA2 is the antagonism parameter (antagonism parameter), which expresses the strength of the action of a competitive antagonist, and the larger the value indicates that the antagonist is more potent. pA2 is a parameter that can make the agonist more potent than the agonist. pA2 is a parameter that can make the agonist more potent than the agonist. The concept of pA2 is the negative logarithm of the molar concentration of antagonist required to produce the original effect (i.e., 50% of the maximal effect, or 50% of the receptor occupied by the agonist) when the concentration of the agonist is increased to a factor of two. pA2 = -log[I] = -logKI.
Materials and Instruments
Guinea pig Move First, debug the BL-420 biological acquisition and processing system, so that the state of dystonia measurement. Table 1 Concentration of Ach Caveat 1. Since the binding of small molecule drugs to large molecule receptors is an intermolecular interaction, receptor kinetic studies of agonists (ACh)and blocking agent (atropine) should be used with gram molecular concentration (mol/L). 2. Body tissue experiments should pay special attention to the following links： （1） Tissue specimens should be prepared gently, avoiding pulling and pressing;(2) Physiological solutions should be prepared very accurately； （3） The temperature in the bath should be kept constant, and the bath should be continuously gassed with 95 % O2and 5% CO2O 2and 5% CO 2 .(4) Allow the tissue contraction reaction to equilibrate before starting the experiment.(3) Administer the drug in a cumulative manner and do not rinse the specimen after each administration. 4. Do not arbitrarily change the experimental parameter settings of the biosignal processing acquisition system. Common Problems I. Describe the effect of competitive and noncompetitive receptor antagonists on the quantitative effect profile of agonists? For more product details, please visit Aladdin Scientific website.
M Atropine M ACh
BL-420 Biological Acquisition and Processing System Tension Sensor Insulated Macrothermal Bath Super Thermostatic Bath "L" Ventilation Hooks High-Level Flasks Measuring Cylinders Beakers Incubators Oxygen Cylinders Surgical Scissors Ophthalmic Scissors Ophthalmic Tweezers Suture Needles Cotton Threads Syringes Benchtop Nutrient Dispensers
1. In the main menu, use the mouse to select 〈Experiment〉 in the 〈Digestion〉, select the system to display the <Smooth Muscle Physiological Properties>.
2. in the interface on the right side of the control parameters area select 〈tension〉, 〈select 3 g〉, scanning speed of 1.0 s/div, determine the signal input, select 〈channel 2〉, in the tools menu select fast zero (no load when the hook on nothing).
3. Select 〈Oscilloscope〉 in the main menu to enter the oscilloscope state.
4. in the main menu toolbar select 〈Grid switching 〉 on the background grid to switch to a fine grid, and select the waveform color red in the options.
Second, adjust the instrument:
Mai bath with 50 ml of Tait's solution, adjust the temperature 38 ℃ ± 0.5 ℃; oxygen 2 ~ 3 bubbles / second, and prepare guinea pig ileum specimens.
Third, formal experiments:
1. the two ends of the intestinal tube specimen with a sewing needle each through a line, one end of an empty knot (about l cm small set), the other end of the long line through the knot, with ophthalmic forceps pincers fixed in the empty knot on the ventilator hooks, put into the Mai bath, the other end of the end of the long line to tie an empty knot, hanging in the tension transducer on the small hooks, adjusting the height of the transducer, so that the preload of 1 g, stabilizing the specimen for 20 minutes.
2. Press the button under the main menu to select 〈Recorded State〉, first trace a normal curve, and then administer the drug in the following order.
(1) Add 0.1 ml of 3×10-3 M ACh to the McClintock bath, observe the curve change, check the intestinal tubes for excitation, and then flush the intestinal tubes to return them to normal.
(2) Add acetylcholine cumulatively according to the dosage given in Table 1, and make the cumulative dose-effect curve of acetylcholine. Specific practice: first add a small dose of ACh, if there is a reaction, when the reaction reaches the highest peak, immediately add the next dose of ACh, in every addition to the drug need to be labeled, only labeled with the serial number of the addition of the drug, do not play the name of the drug, until the curve rises to the highest peak is no longer elevated until. Turn the system into the "oscillometric state", rinse the intestinal tubes three times with TES and stabilize the specimen for 15 minutes to return to normal.
(3) In the main menu, select 〈Record Status〉, add 3×10-3M Atropine 0.1 ml to the bath, and mark the name of the drug, and then make the cumulative concentration of Ach and the quantitative and quantitative effect curve once again after 1 minute.
3. Select "End Experiment" from the main menu. Activate the toolbar and the system enters the analysis state.
IV. Results
1. In the main menu under the analysis item can choose the area measurement, mark query, start inversion, mouse capture, after measurement, press the toolbar to cancel the mark line, respectively, measured the normal curve and each time after the addition of tension size, and finally to acetylcholine caused by the maximum contractile force as 100%, respectively, calculated the percentage of each dose response
2. The dose-response curves were plotted with the percentage of each dose-response as the vertical coordinate and the negative logarithm of the dose as the horizontal coordinate.
3. Then find the pA2 value according to the formula. pA2= log (B/A -1)-logC
A: the concentration of acetylcholine required to elicit 50% of the maximum response in the absence of atropine.
B: the concentration of acetylcholine required to elicit 50% of the maximum response in the presence of atropine.
C: Concentration for the antagonist atropine.
II.Briefly explain the concept and significance of pA2.