One common feature of all these experiments is that tissues are not stimulated continuously but only for 2-min periods separated by periods in which no electrical stimuli are applied (see Note 7). These assays can be used to construct agonist concentration-response curves in the absence (Subheading 3.3.1.) or presence of an antagonist (Subheading 3.3.2.) or to follow the time course of the response to a single dose of agonist (see Note 8). Typical concentrations or concentration ranges used for a selection of cannabinoid receptor ligands can be found in Table 3. Only one cannabinoid concentration-response curve is constructed per experiment (see Note 9). How data from such experiments are analyzed is discussed in Subheading 3.3.3.
Ideally the time required for the agonist under investigation to produce its full effect should be predetermined (see Note 8). For the construction of a concentration-response curve, the tissue is electrically stimulated to produce contractions 2 min prior to the addition of the lowest dose of agonist. The stimulator is then turned off and the tissue allowed to rest for the time, Y, required for the agonist to produce its full effect. After this time has elapsed, the tissue is once again electrically stimulated for 2 min prior to the addition of the second dose of agonist. This cycle is repeated until the concentration-response curve has been completed. This protocol is illustrated in Fig. 2. Concentration-
response curves are constructed without bath wash-out between successive additions (see Note 9 for explanation). Table 3 shows dose cycles employed in this laboratory for some commonly used cannabinoid receptor agonists.
3.3.2. Agonist Concentration-Response Curves in the Presence of an Antagonist
Each tissue is electrically stimulated 2 min prior to the addition of the antagonist or its vehicle. After sufficient time, X, has elapsed for the antagonist to reach its pharmacological target (e.g., 30 min for the CB1 receptor antagonist, SR141716A), the tissue is once again stimulated and the heights of the con tractions recorded for 2 min before a concentration-response curve of the agonist is constructed as described in Subheading 3.3.1. and portrayed in Fig. 2.
3.3.3. Data Analysis for Assays Using Electrically Stimulated Tissues
Analysis of the recorded data involves measuring the height of the last six contractions produced during each 2-min stimulation period. This includes the period immediately before the first addition of agonist, antagonist, or vehicle is made. For experiments with antagonists (Subheading 3.3.2.), any change in contraction height produced by the antagonist is monitored to establish whether the antagonist itself has any direct effect on the amplitude of electrically evoked contractions. SR141716A and cannabidiol are both lig-ands that can significantly enhance the height of electrically evoked contractions in the mouse vas deferens (9,12). The contraction amplitudes measured just prior to the addition of the first (lowest) dose of agonist serve as the baseline amplitudes with which each subsequent set of contraction heights is compared when calculating the percentage change in contraction height produced by the agonist.
For agonists, values for EC50, for maximal effects (Emax) and for the SEM or 95% confidence limits of these values can be calculated by nonlinear regression analysis using the equation for a sigmoid concentration-response curve (GraphPad Prism; GraphPad Software, San Diego, CA). It is usually sufficient to replicate each concentration-response curve six times. The dissociation constant (KB) of a competitive surmountable antagonist can be obtained by constructing agonist dose response curves in the presence of more than one concentration of the antagonist and then determining the slope (1/Kb) of the best-fit straight line of a plot of (x-1) against B, constructed by linear regression analysis and constrained to pass through the origin (GraphPad Prism) (15). The equation for this graph is (x-1) = B/KB, where x (the concentration ratio) is the concentration of a twitch inhibitor that produces a particular degree of inhibition in the presence of a competitive reversible antagonist at a concentration, B, divided by the concentration of the same twitch inhibitor that produces an identical degree of inhibition in the absence of the antagonist. KB values of competitive surmountable antagonists can also be calculated by substituting a single concentration ratio value into the above equation. Values of the concentration ratio and its 95% confidence limits can be determined by symmetrical (2 + 2) dose parallel line assays (16), using responses to pairs of agonist concentrations located on the steepest part of each log concentration-response curve. This method can also be used to establish whether 2-point log concentration-response plots deviate significantly from parallelism.
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