Figure 7.

Simulations. Pre-incubation of receptors with a fast-dissociating competitor followed by a wash-step: effect of the release of adsorbed competitor in solution on subsequent radioligand binding. Competitor and radioligand parameters, radioligand concentration and incubation times are given in Table 1. Calculations of k₄ (given below as the corresponding t_1/2) were based on Equation 5 (A) and 4 (B). (A) For simulating the TSC data, receptors are pre-incubated for 30 min with different concentrations of competitor, washed and further incubated for 30 min with a fixed concentration (2 nM) of radioligand either in the absence (0%) or presence of still remaining competitor at the indicated fractions of its initial concentration. Radioligand binding is expressed in percent of control, i.e. binding to receptors pretreated with medium only (i.e. [LR]_c=0). [LR]_cmax/[LR]_c=0 ratios obtained by non-linear regression analysis varied little: that is, 0.866 (red curve, for 1-site competition), 0.867 (blue curve, for 2-site competition) and 0.863 (green curve, for 2-site competition). The calculated competitor dissociation t_1/2 values (8.9–9.1 min) according to Equation 5 were close to the input value (9.0 min). The lower potency component of the green and blue TSC curves were shifted 17- and 360-fold to the right of the reference (black) curve. (B) For simulating the ‘delayed association’ data, receptors are pre-incubated for 30 min in medium only or with a fixed concentration (3.10⁻⁸ M) of competitor (open symbols) and further incubated for increasing time periods with a fixed concentration (2 nM) of radioligand either in the absence or presence of competitor released at 0.5 or 10% of its initial concentration. Radioligand binding is expressed in percent of control, i.e. binding after 30 min to receptors pretreated with medium only. The calculated competitor dissociation t_1/2 and initial receptor occupancy ([CR]_t=0) values according to Equation 4 were identical to the input values (9.0 min and 97.5% of [R_tot]) in case of no competitor release (red curve) but diverged more and more with increasing competitor release (blue curve for 0.5% release: 11.3 min and 90.3%; green solid curve for 10% release: 82 min and 78.2%). In case of 10% competitor release, the data fitted better with a mono-exponential association paradigm (dotted green curve) in where only 44.5% of the control radioligand binding was recoverable. TSC, two-step competition.