Electrophysiological perspectives on the therapeutic use of nicotinic acetylcholine receptor partial agonists

Abstract

Partial agonist therapies rely variously on two hypotheses: the partial agonists have their effects through chronic low-level receptor activation or the partial agonists work by decreasing the effects of endogenous or exogenous full agonists. The relative significance of these activities probably depends on whether acute or chronic effects are considered. We studied nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus laevis oocytes to test a model for the acute interactions between acetylcholine (ACh) and weak partial agonists. Data were best-fit to a basic competition model that included an additional factor for noncompetitive inhibition. Partial agonist effects were compared with the nAChR antagonist bupropion in prolonged bath application experiments that were designed to mimic prolonged drug exposure typical of therapeutic drug delivery. A primary effect of prolonged application of nicotine was to decrease the response of all nAChR subtypes to acute applications of ACh. In addition, nicotine, cytisine, and varenicline produced detectable steady-state activation of α4β2* [(α4)(2)(β2)(3), (α4)(3)(β2)(2), and (α4)(2)(β2)(2)α5)] receptor subtypes that was not seen with other test compounds. Partial agonists produced no detectable steady-state activation of α7 nAChR, but seemed to show small potentiation of ACh-evoked responses; however, "run-up" of α7 ACh responses was also sometimes observed under control conditions. Potential off-target effects of the partial agonists therefore included the modulation of α7 responses by α4β2 partial agonists and decreases in α4β2* responses by α7-selective agonists. These data indicate the dual effects expected for α4β2* partial agonists and provide models and insights for utility of partial agonists in therapeutic development.

Fig. 1.

Model applied to the activation of nAChR by simultaneous coapplications of the full agonist (a) and a partial agonist (p). The terms are defined as follows: [a], concentration of ACh, concentration of the partial agonist (either S 24795 or cytisine in Fig. 2, A or B, respectively); [a + p], the sum of the concentrations; n_a, the Hill coefficient for ACh agonist activity; n_p, the Hill coefficient for partial agonist activity; R_maxP, the maximal agonist activity for the partial agonist relative to ACh; EC_50a, the EC₅₀ for ACh; EC_50p, the EC₅₀ for the partial agonist; In_maxP, the maximal inhibitory activity of the partial agonist; IC_50p, the IC₅₀ for the inhibitory effects of the partial agonist; n_i, the Hill coefficient for inhibition by the partial agonist. The I_Max of the full agonist is assigned a value of 1. The first and second terms of the equation represent the activation produced by the full and partial agonists, respectively, scaled by their potencies and weighted by receptor availability. The third term allows noncompetitive effects of the partial agonist. If such noncompetitive activity is inhibitory, then n_i is assigned a negative value.

Fig. 2.

Experimental (points) and predicted (lines) data for full and partial agonist interactions. A, the observed experimental data for the net charge responses of α7-expressing cells to the coapplications of ACh and S 24795 across a range of concentrations are compared with the responses predicted by the model at the same concentrations. For clarity of presentation, half of the data representing responses obtained in progressively increasing concentration of S 24795 is shown on the left (from 0.1 μM to 1 mM, by factors of 10), and the other half of the data is shown on the right (from 0.3 to 300 μM, by factors of 10). B, the experimental data for the peak current responses of cells expressing the (α4)₃(β2)₂ form of α4β2 to coapplications of ACh and cytisine across a range of concentrations are compared with the responses predicted by the model at the same concentrations. For clarity of presentation, half of the data representing responses obtained in progressively increasing concentration of cytisine is shown on the left (from 0.1 μM to 1 mM, by factors of 10), and the other half of the data set is shown on the right (from 0.3 to 300 μM, by factors of 10). For an alternative presentation of these data as pseudo three-dimensional plots, see Supplemental Fig. 1.

Fig. 3.

Effects of bath-applied nicotine on the ACh-evoked responses of α4β2 and α7 nAChR. Pure populations of (α4)₃(β2)₂ and (α4)₂(β2)₃ nAChRs were obtained by coexpressing the β2–6-α4 concatamer with monomeric α4 or β2, respectively. After measuring two baseline ACh-evoked responses, the α4β2 partial agonists were added to the bath solution, and the cells were repeatedly probed for their ACh responses. The ranges of nicotine concentrations tested were 1 nM to 3 μM and 0.3 nM to 3 μM for (α4)₃(β2)₂ and (α4)₂(β2)₃, respectively, and 1 nM to 10 μM for α7. All points represent an average of at least four oocytes (±S.E.M.) for each condition. The summary at the bottom displays the average of the last three responses in the upper graphs, plotted as functions of the bath concentrations applied.

Fig. 4.

Effects of bath-applied cytisine and varenicline on the ACh-evoked responses of α4β2 and α7 nAChR. Pure populations of (α4)₃(β2)₂ and (α4)₂(β2)₃ nAChR were obtained by coexpressing the β2–6-α4 concatamer (Zhou et al., 2003) with monomeric α4 or β2, respectively. After measuring two baseline ACh-evoked responses, the α4β2 partial agonists were added to the bath solution, and the cells were repeatedly probed for their ACh responses at intervals of 223 s (α7) or 277 s (non-α7). The ranges of cytisine concentrations tested were: 0.03 nM to 3 μM for both (α4)₃(β2)₂ and (α4)₂(β2)₃ and 1 nM to 1 μM for α7. The ranges of varenicline concentrations tested were 1 nM to 19 μM and 0.01 nM to 3 μM for (α4)₃(β2)₂ and (α4)₂(β2)₃, respectively, and 1 nM to 10 μM for α7. All points represent an average of at least four oocytes (±S.E.M.) for each condition. The summaries at the bottom display the average of the last three responses in the upper graphs, plotted as functions of the bath concentrations applied.

Fig. 5.

Summary of effects obtained with α4β2α5 nAChR. Pure populations of α4β2α5 nAChR were obtained by coexpressing the β2–6-α4 concatamer with monomeric α5 (Kuryatov et al., 2008). Bath application experiments were conducted as described for the (α4)₂(β2)₃ and (α4)₃(β2)₂ receptors (Fig. 4). This summary shows the average of the three responses obtained after nicotine or the cytisine-related compounds were in the bath for 30 ± 4 min.

Fig. 6.

Effects of mecamylamine on steady-state baseline current of α4β2α5 receptors stimulated by bath-applied varenicline. A, before the addition of varenicline to the bath, application of 3 μM ACh (black bar) produced a large transient current, as illustrated by the representative response. B, the bath application of 1 μM varenicline (gray bar) stimulated a response that appeared as a sustained shift in baseline current. The presence of 1 μM varenicline in the bath also had the effects of suppressing the transient response to an application of 3 μM ACh (black bar). C, after 30 min of continuous varenicline bath application, the baseline current remained elevated and ACh-evoked responses were suppressed. D, after 30 min of continuous varenicline bath application, 100 μM mecamylamine (Mec) was applied (open bar), which reduced the baseline current to the original control level, indicating that the baseline shift was caused by steady-state activation of α4β2α5 nAChR.

Fig. 7.

Effects of bupropion on the ACh-evoked responses of nAChR. A, bupropion was coapplied with ACh, and the evoked responses were calculated relative to the responses to ACh applied alone. B, the effects of bath-applied bupropion on the ACh-evoked responses of α4β2 and α7 nAChR. Pure populations of (α4)₃(β2)₂ and (α4)₂(β2)₃ and α4β2α5 nAChR were obtained by coexpressing the β2–6-α4 concatamer (Zhou et al., 2003) with monomeric α4, β2, or α5, respectively. After measuring two baseline ACh-evoked responses, bupropion was added to the bath solution, and the cells were repeatedly probed for their ACh responses. The plot displays the average of three responses obtained after bupropion had been in the bath for 30 min, plotted as functions of the bath concentrations applied. All points represent an average of at least four oocytes (±S.E.M.) for each condition.

Fig. 8.

Summary of the effects of bath-applied 3-pyr-Cyt on (α4)₃(β2)₂, (α4)₂(β2)₃, and α7 nAChR. Bath application experiments were conducted as described for cytisine, varenicline, and nicotine. This summary shows the average of the three response obtained after 3-pyr-Cyt was in the bath for 30 ± 4 min.

Fig. 9.

Effects of bath-applied GTS-21 on the ACh-evoked responses of α4β2 and α7 nAChR. Pure populations of (α4)₃(β2)₂ and (α4)₂(β2)₃ nAChR were obtained by coexpressing the β2–6-α4 concatamer (Zhou et al., 2003) with monomeric α4 or β2, respectively. After measuring two baseline ACh-evoked responses, the α7-selective partial agonist was added to the bath solution, and the cells were repeatedly probed for their ACh responses. The ranges of GTS-21 concentrations tested were 1 nM to 30 μM and 1 nM to 10 μM for (α4)₃(β2)₂ and (α4)₂(β2)₃, respectively, and 1 nM to 30 μM for α7. All points represent an average of at least four oocytes (±S.E.M.) for each condition. The summary at the bottom displays the average of the last three responses in the upper graphs, plotted as functions of the bath concentrations applied.

Fig. 10.

Effects of bath-applied S 24795 on the ACh-evoked responses of α4β2 and α7 nAChRs. Pure populations of (α4)₃(β2)₂ and (α4)₂(β2)₃ nAChRs were obtained by coexpressing the β2–6-α4 concatamer (Zhou et al., 2003) with monomeric α4 or β2, respectively. After measuring two baseline ACh-evoked responses, the α7-selective partial agonist was added to the bath solution, and the cells were repeatedly probed for their ACh responses. The ranges of S 24795 concentrations tested were 1 nM to 100 μM for (α4)₃(β2)₂ and (α4)₂(β2)₃ and 1 nM to 30 μM for α7. All points represent an average of at least four oocytes (±S.E.M.) for each condition. The summary at the bottom displays the average of the last three responses in the upper graphs, plotted as functions of the bath concentrations applied.