DETERMINATION OF PA2 VALUE OF PRAZOSIN ON RAT ANOCOCCYGEUS MUSCLE
To determine the pA₂ value of prazosin on the rat anococcygeus muscle by assessing its competitive antagonism against adrenergic agonists. This study aims to evaluate the potency and receptor affinity of prazosin by constructing dose-response curves in the presence and absence of the antagonist, thereby providing insights into its pharmacological characteristics and inhibitory effects on α₁-adrenoceptors. The two anococcygeal muscles emerge from upper coccygeal vertebrae that are close together in the middle of the pelvic cavity.
EQUIPMENT REQUIRED
Animal :- Rat
Drug:- Prazosin (1mg/ml)
Instrument:- Student Organ Bath, kymograph.
Physiological salt solution:- Krebs solution or Tyrode solution
PRINCIPLE
The pA₂ value is a pharmacological parameter used to determine the potency of a competitive antagonist by measuring its ability to shift the dose-response curve of an agonist. In this study, prazosin, a selective α₁-adrenoceptor antagonist, is tested on the rat anococcygeus muscle, a smooth muscle known for its adrenergic responsiveness.
The experiment involves generating cumulative dose-response curves for an adrenergic agonist (e.g., norepinephrine or phenylephrine) in the absence and presence of prazosin at different concentrations. A rightward parallel shift of the dose-response curve without a change in maximal response indicates competitive antagonism. The Schild plot is then constructed to determine the pA₂ value, which represents the negative logarithm of the antagonist concentration required to double the agonist EC₅₀ (effective concentration for 50% maximal response).
This approach provides insight into the receptor-binding affinity and inhibitory potency of prazosin, contributing to a better understanding of its pharmacodynamics on α₁-adrenoceptors in smooth muscle tissues.
When simply and swiftly dissected, the two muscles are roughly 3cm long, 0.5cm wide at the widest point, and just 150-300mm thick. Tissue preparation provides numerous advantages for common procedures, including:
1) Parallel bundles of smooth muscle cells form a thin sheet. This design decreases diffusion for drug access and ion exchange experiments. 2) The muscle is bilateral, allowing for control and test preparations from the same animal. Additionally, it is densely adrenergically innervated. 3) Pre- and post-ganglionic fibers are found. Other agonists, such as acetylcholine and adenosine, cause smooth muscle contractions based on concentration.
PROCEDURE:
1. Preparation of Solutions:
· Prepare physiological salt solution Krebs solution or Tyrode solution and maintain it at pH 7.4.
· Prepare stock solutions of prazosin (α₁-adrenoceptor antagonist)
· Serially dilute the agonist and antagonist to obtain working concentrations.
2. Animal Preparation:
· Humanely sacrifice a rat following ethical guidelines.
· Dissect and isolate the anococcygeus muscle under a dissecting microscope.
· Transfer the muscle to a petri dish containing aerated Krebs or tyrode solution.
3. Mounting of Tissue:
· Attach one end of the anococcygeus muscle to a tissue holder and the other end to an isometric force transducer in an organ bath containing Krebs solution or Tyrode solution (maintained at 37°C and continuously aerated with 95% O₂ and 5% CO₂).
· Maintain a resting tension of approximately 1 g and allow the tissue to equilibrate for 30–45 minutes, with regular washing every 10 minutes.
5. Antagonism Study:
· Wash the tissue thoroughly and allow it to re-equilibrate.
· Repeat the dose-response experiment in the presence of a fixed concentration of prazosin.
· Observe the shift in the dose-response curve and repeat with additional higher concentrations of prazosin.
6. Data Analysis:
· Plot dose-response curves of the agonist in the absence and presence of prazosin.
· Determine the EC₅₀ values for each curve.
· Calculate dose-ratios and construct a Schild plot (log(dose-ratio -1) vs. log [prazosin]).
· Determine the pA₂ value, which is the negative logarithm of the antagonist concentration that produces a twofold shift in EC₅₀.
7. Interpretation:
· A parallel rightward shift of the dose-response curve without a change in maximum response confirms competitive antagonism.
· The pA₂ value indicates the potency of prazosin as an α₁-adrenoceptor antagonist.
CALCULATION & INTERPRETATION:
Amount in μg = conc. Of Prazosin (0.1 μg/mL) x Amount added in ml
Concentration of Prazosin in Organ bath contains 20ml solution = Amount in μg/20mL
Molar conc. Of Prazosin in micromole/ml = (Conc. Of Prazosin in organ bath contains 20ml solution) / Molecular weight of Prazosin (383.4g/mol)
PA2 = - (Log molar conc. Of Antagonist)
PA2= -(-6.10)
PA2= 6.10
CONCLUSION
The determination of the pA₂ value of prazosin on the rat anococcygeus muscle provides insights into its potency as a competitive α₁-adrenoceptor antagonist. The rightward shift of the dose-response curve in the presence of prazosin, without a change in maximal response, confirms its competitive antagonism.
The calculated pA₂ value represents the affinity of prazosin for α₁-adrenoceptors, allowing a quantitative comparison with other antagonists. This study contributes to understanding the pharmacodynamic properties of prazosin, which is clinically used for conditions such as hypertension and benign prostatic hyperplasia by inhibiting α₁-mediated smooth muscle contraction.
IDEAL OBSERVATION
Sr. No
Conc. Of Ach
(μg/mL)
Amount Added in Organ Bath
Conc. In Organ bath
In Absence of Prazosin
In mL
In μg
μg/mL
Log Conc.
Response
(in mm)
%Response
1.
10
0.1
1
0.05
-1.301
5
22.7
2.
0.2
2
-1
9
40.9
3.
0.4
4
-0.69
14
63
4.
0.8
8
-0.39
18
81
5.
1.6
16
-0.09
22
100
6.
3.2
32
Sr. No.
Vol. of Norepinephrine (Agonist)
Increasing dose of prazosin (Antagonist)
Response height (mm)
% Response
Molar Conc. Of Antagonist
Log Molar Conc. Of Antagonist
12
85.71
2.6 x 10-6
-6.58
71.42
5.2 x 10-6
-6.28
3
0.6
7
50
7.82 x 10-6
-6.10
RESULT:
The results of the study on the determination of the pA₂ value of prazosin on the rat anococcygeus muscle showed a dose-dependent inhibition of the contractile response induced by an α₁-adrenoceptor agonist (e.g., noradrenaline or phenylephrine).
The Schild plot analysis yielded a pA₂ value, indicating the potency of prazosin as a competitive antagonist at α₁-adrenoceptors.
The calculated pA₂ value was within the expected range for prazosin, confirming its high affinity for α₁-adrenoceptors and competitive inhibition mechanism. These findings validate the use of prazosin as a standard α₁-adrenoceptor antagonist in pharmacological studies.
DISCUSSION:
The study confirmed prazosin as a potent competitive α₁-adrenoceptor antagonist in the rat anococcygeus muscle.
The obtained pA₂ value aligns with reported data, indicating its high affinity and competitive inhibition mechanism.
The rightward shift in the dose-response curve without altering the maximum response supports its mode of action.
These findings reinforce prazosin’s pharmacological role in smooth muscle relaxation, relevant to its clinical use in hypertension and benign prostatic hyperplasia.
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