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Rat Ileum Organ Bath

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    Introduction

    Small animals have intestines that exhibit a longer time response when kept in suitable solutions. In order to establish a good reference point by which comparisons in tensional changes can be examined for a rat ileum there is need to use a Tyrode solution that is aerated with Carbogen (95% O2, 5% CO2), the tissue should be maintained at 32  under a tension of 10 mN. Concentration responses of animals to drugs are greatly influenced by quite a number of factors but the most important factor is the drug dosage, thus forming the basis of this practical experiment. Generally it has been established that the higher the concentration the higher the response, though this is not well defined for all concentrations or even all responses. It is possible to observe the shapes of response curves by plotting response against agonist concentration. This experiment examines the response as a contractile response of the longitudinal smooth muscle in the rat ileum. Comparisons done graphically for drugs are simpler to interpret if the response is plotted against concentration resulting to a hyperbolic curve. Nevertheless if the response is linear then the plot becomes a linear scale plot against log concentration of agonist where in this case the plotted curve is sigmoidal in nature. Some of the experimental objectives are:

    ·         To recognize and understand the roles of isolated tissues in any drug research processes.

    ·         To determine potencies of acetylcholine and 5-hydroxytryptamine using the rat ileum preparation.

    ·         To demonstrate how response to drugs can be antagonized by other drugs with different mechanisms of action.

    ·         Interpretation of the agonistic and antagonistic properties in terms of the relevant pharmacological mechanisms.

    ·         To perform precise drug dilutions by using the organ birth apparatus and rat isolated ileum in order to study the drug mechanisms.

    Materials and methods

    Preparation of drug dilutions

    100 mM stock solutions of acetylcholine and 10 mM stock solutions of 5-hydroxytryptamine were provided. A series of dilutions were made of each stock solution in order to achieve specified concentrations. Each Eppendorff tube was clearly labeled with the name of the drug and the concentration of the working stock solution indicated. For each dilution accuracy was observed when measuring the volumes using the formula C1V1 = C2V2.

    Synchronizing ileum tissue

    Synchronization of the tissue was done by adding 25 l of 1 mM agonist stock and immediately washing. This is done to verify the responsiveness of the tissue to the agonist.

    Obtaining a concentration-response curve

    This was done by using the lowest concentration of each agonist at 1nM in the organ bath. This was done by a series of at least 3 or 4 sub maximal concentrations in order to plot a log concentration response curve. Calculation of the response to each concentration as a percentage of the maximum response was done. Using a semi-log graph paper the log agonist concentration against contractile response was plotted. A concentration of each agonist that gives an approximate response of 50% of its maximum response was chosen. The addition of acetylcholine and 5-hydroxytryptamine was repeated until responses were reproducible.

    Adrenaline effect

    From the stock of 1mM, a concentration of 1 M of adrenaline was added in the organ birth and left for 1 minute. A selected concentration of (EC50) of 5-hydroxytryptamine was added in the presence of adrenaline. Adrenaline was added again after washout and after 1 minute a selected concentration (EC50) of acetylcholine was added in the presence of adrenaline and response recorded. The same procedure was performed on flouxetine and atropine effects.

    Using PRISM software, concentration response curves were plotted having both agonists on one graph with clearly labeled axes as presented in the results below.

    Results

    Table for EC50

    ACH
    Carbachol
    Histamine
    HTMA
    EC50
    9 x E-8
    9 x E-7
    9 x E-6
    1.5 x E-4

    Table for schild plot

    Molar Concentration Mepyramine

    [B](M)
    Log Mepyramine Concentration

    Ie. Log[B]
    EC50 Control

    [A]

    (M)
    EC50 in Presence of Mepyramine [A1](M)
    Concentration Ratio (Cr)

    [A1]/[A]
    (Cr-1)
    Log (Cr-1)
    1 x E-8
    -8
    2 x E-8
    2.5 x E-7
    12.5
    11.5
    1.06
    1 x E-7
    -7
    2 x E-8
    2.5 x E-6
    125
    124
    2.09
    1 x E-6
    -6
    2 x E-8
    2.5 x E-5
    1500
    1499
    3.18

    2nd Table Agonist : 5-Hydroxytryptamine  EC50 = 2.569 x E-7 M

    Antagonist Drug

    Absolute size of contraction to agonist (mN)

    5-Hydroxytryptamine

    change in response

    (+ or – mN)

    Before adding

    the antagonist

    In the presence of

    the antagonist

    Adrenaline[1 μM]

    9.41
    0
    -9.41

    Fluoxetine [1 μM]

    6.88
    6.06
    -0.82

    Atropine [1 μM]

    12.34
    8.08
    -4.26

    Discussion

     

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