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Procedure: a. Preliminary investigation The jejunum preparation is equilibrated in the bath fluid for at least 10 minutes to stabilize the base-line value. Every 5 minutes it is washed. All washes consist of 3 replacements of bath fluid. To determine a log dose-response curve, acetylcholine chloride is pipetted into the organ bath in a cumulative way, starting with a bath concentration of 10-8 mol/l. After equilibration to stabilize the response the concentration is raised by a factor of 3,2. This is continued until the maximum contraction is obtained. The jejunum preparation is washed with bath fluid and this wash is repeated every 5 minutes until the zero-level is stabilized. Then a second curve for acetylcholine is made. This procedure is repeated until 2 consecutive, identical cumulative log dose response curves for acetylcholine have been obtained. After the last curve for acetylcholine the jejunum preparation is washed with bath fluid every 5 minutes until the zero-level is stabilized. Test compound is pipetted into the organ bath and the jejunum preparation is equilibrated for at least 15 minutes (the response must be stable) and the agonistic activity is evaluated. To evaluate antagonistic activity a log dose-response curve is made for acetylcholine in the presence of test compound as described above. To check the reversibility of the interactions the jejunum preparation is washed with bath fluid and equilibrated for at least 10 minutes (a wash every 5 minutes). Then another log dose-response curve for acetylcholine is made. If the last curve is identical with the 2 initial curves the jejunum preparation can be used for another experiment. b. Determination of agonistic activity For test compounds showing agonistic activity in the preliminary investigation a cumulative log dose-response curve is made. After the 2 consecutive, identical cumulative log dose-response curves for acetylcholine, the curves for the test compound are made, alternated with one for acetylcholine. The curves are made in at least 2 different jejunum preparations. To check that the effect is mediated via the cholinergic receptors a cumulative log dose-response curve is made for the test compound in the presence of atropine sulphate, a specific cholinergic receptor blocker. c. Determination of antagonistic activity For test compounds showing antagonistic activity in the preliminary investigation, cumulative log dose-response curves for acetylcholine are made in the presence of at least 3 different doses of test compound (usually in the range 1:3,2:10). After the 2 consecutive, identical cumulative log dose-response curves for acetylcholine, test compound is added to the organ bath and after equilibration for at least 15 minutes the curves for acetylcholine are made again. The curves for each dose of test compound are made in at least 2 different jejunum preparations. For test compounds showing potentiating activity in the preliminary investigation, cumulative log dose-response curves for acetylcholine are made in the presence of higher doses of test compound in order to detect a dose dependency or a change to an antagonistic effect. The curves are made in at least 2 different jejunum preparations. Compound doses If no relevant data from other tests are available, the usual initial concentration is 3 x 10-5 mol/l bath fluid. Evaluation of responses: The heights of deflection on the recording paper are measured in mm, using the response just before addition of test compound or acetylcholine as zero level. Each response is expressed as a percentage based on the maximum response of the preceding curve for acetylcholine (= 100%). Cumulative log dose-response curves are made for each series of measurements by plotting the percentage change (ordinate) against the log concetration (abscissa), as described by Van Rossum. Agonistic activity Agonistic activity of a test compound is characterized by the pD2 -value and the intrinsic activity α (figure 1). pD2 is a measure for the dissociation constant of the equilibrium: receptor agonist. It is the negative logarithm of the concentration of agonist which induces a contraction of 50% of the maximum response. This value can be read from the cumulative log dose-respons curve. The final result for a test compound is expressed as the mean pD2 -value of all corresponding curves, together with the s.e. mean. The pD 2 -value of acetylcholine is approx. 6 - 7. The intrinsic activity α is a measure for the potency of an agonist in comparison to acetylcholine, defined as the ratio of the maximum responses induced by the test compound and acetylcholine. It can be easily calculated with the help of the cumulative log dose-response curves. The final result for a test compound is expressed as the mean α -value of all corresponding curves. Antagonistic activity Competitive antagonistic activity of a test compound (it induces a parallel shift of the log dose-response curve for acetylcholine to the right) is characterized by the pA2 -value (figure 2). pA 2 is a measure for the dissociation constant of the equilibrium: cholinergic receptor-antagonist. It is the negative logarithm of the concentration of antagonist which produces a log dose-response curve for acetylcholine, identical to the original one when twice as much acetylcholine is used (a shift of log 2 to the right measured at the 50% response level). This value can be calculated using the formula: pA 2 = pAx + log (x - 1) where: pA x = negative logarithm of the concentration of antagonist x = antilogarithm of the shift of the log dose-response curve for acetylcholine, measured at the 50% response level, due to the presence of antagonist (pAx ). It can be easily calculated with the help of the cumulative log dose-response curves. The final result for a test compound is expressed as the mean pA 2 -value of all curves made in the presence of test compound, together with the s.e. mean. Non-competitive antagonistic activity of a test compound (it induces no shift of the log dose-response curve for acetylcholine, but all responses are decreased by the same ratio) is characterized by the pD2 ' -value (figure 3). pD 2 ' is a measure for the blocking of cholinergic receptors for interaction with acetylcholine, by irreversible binding of antagonist to the receptor or by any other process. It is the negative logarithm of the concentration of antagonist which decreases the maximum response of acetylcholine by 50%. This value can be calculated using the formula: 100 - Δ pD 2' = pA x + log --------------- D where: pA x = negative logarithm of the concentration of antagonist Δ = maximum response of acetylcholine in the presence of antagonist (pAx ), expressed as a percentage based on the maximum response of the preceding curve for acetylcholine (= 100%). This value can be read from the log dose-response curve. The final result for a test compound is expressed as the mean pD2 '-value of all curves made in the presence of test compound, together with the s.e. mean. Mixed competitive and non-competitive (dualistic) antagonistic activity of a test compound (it induces a shift of the log dose-response curve for acetylcholine and all responses are decreased by the same ratio) is characterized by the pA2 - and pD 2 '-values (figure 4). These values can be calculated using the formulas: pA 2 = pAx + log (x-1) and 100 - Δ pD 2' = pA x + log --------------- D The final result for a test compound is expressed as the mean pA2 - and pD 2 '-values of all curves made in the presence of test compound, together with the s.e. means. Potentiating activity of a test compound (it induces a parallel shift of the log dose-response curve for acetylcholine to the left) is not evaluated. Interpretation of results : Test compounds showing agonistic activity, which is inhibited by atropine, have cholinergic activity; they are cholinergic compounds. Compounds having a low intrinsic activity and an antagonistic activity are called "partial agonists". The pA2 -value and the pD 2 '-value are measures for the competitive and the non-competitive anticholinergic activities of the test compound, respectively. The interpretation of the results is as follows:
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Compounds having both competitive
and non-competitive antagonistic activity are called "dualistic
antagonists. 
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