Evaluation of benzyltetrahydroisoquinolines as ligands for neuronal nicotinic acetylcholine receptors

Abstract

Effects of derivatives of coclaurine (C), which mimic the 'eastern' or the nonquaternary halves of the alkaloids tetrandrine or d-tubocurarine, respectively, both of which are inhibitors of nicotinic acetylcholine receptors (nACh), were examined on recombinant, human alpha7, alpha4beta2 and alpha4beta4 nACh receptors expressed in Xenopus oocytes and clonal cell lines using two-electrode voltage clamping and radioligand binding techniques. In this limited series, Cs have higher affinity and are most potent at alpha4 subunit-containing-nACh receptors and least potent at homomeric alpha7 receptors, and this trend is very marked for the N-unsubstituted C and its O,O'-bisbenzyl derivative. 7-O-Benzyl-N-methylcoclaurine (BBCM) and its 12-O-methyl derivative showed the highest affinities and potencies at all three receptor subtypes, and this suggests that lipophilicity at C7 and/or C12 increases potency. Laudanosine and armepavine (A) were noncompetitive and voltage-dependent inhibitors of alpha7, alpha4beta2 or alpha4beta4 receptors, but the bulkier C7-benzylated 7BNMC (7-O-benzyl-N-methylcoclaurine) and 7B12MNMC (7-O-benzyl-N,12-O-dimethyl coclaurine) were voltage-independent, noncompetitive inhibitors of nACh receptors. Voltage-dependence was also lost on going from A to its N-ethyl analogue. These studies suggest that C derivatives may be useful tools for studies characterising the antagonist and ion channel sites on human alpha7, alpha4beta2 or alpha4beta4 nACh receptors and for revealing structure-function relationships for nACh receptor antagonists.

Figure 1

Structure of BTHIQ. (a) Structures of tetrandrine, C (R^N=R⁷=R¹¹=R¹²=H) and derivatives, and d-tubocurarine. (b) C and its congeners used in this study.

Figure 2

Effects of BTHIQs on binding of [¹²⁵I]α-BgTx to SH-SY5Y-hα7 membrane homogenates. (a) Displacement of [¹²⁵I]α-BgTx binding to SH-SY5Y-hα7 cells by BTHIQ. SH-SY5Y-hα7-membrane homogenates were incubated with 1 nM [¹²⁵I]α-BgTx for 90 min at room temperature in the presence of various concentrations of BTHIQ. Data are the mean±s.e.m. of 4–5 experiments. (b) Saturation analysis of the specific binding of [¹²⁵I]α-BgTx to SH-SY5Y-hα7-membrane homogenates in the absence and presence of IC₅₀ concentrations of BTHIQ. Data points are the means of triplicate samples±s.e.m. of eight experiments.

Figure 3

Functional effects of BTHIQs on human α7 nACh receptors. (a) Concentration–response curve for antagonist effects of the indicated BTHIQs on function of human α7 nACh receptors. The data were normalised to the responses elicited by 100 μM ACh (approx. EC₅₀ of ACh at α7 nACh receptors) and then fitted to a single site Hill equation. Data points represent the mean±s.e.m. of 6–10 experiments. Where no error bars are shown, they are smaller than the symbols. (b) Concentration–response curve for ACh responses in the absence or presence of IC₅₀ concentrations of BTHIQ. Oocytes were first exposed to ACh to obtain control responses, then to BTHIQ for 2 min, and finally to both ACh and BTHIQ. Data were normalised to responses elicited by 1 mM ACh (maximal ACh response) and represent the mean±s.e.m. of 8–10 experiments. (c) Data show the inhibition of α7 receptor function by concentrations of BTHIQ close to their respective IC₅₀ values at a range of membrane potentials. Inhibition by NEA, 7BNMC and 7B12MNMC was equivalent at all potentials, but inhibition by L or A was dependent on holding potential.

Figure 4

Effects of BTHIQ on [³H]cytisine binding to human α4β2 and α4β4 nACh receptors. (a) Displacement of [³H]cytisine binding to SHEP-hα4β2 or SHEP-hα4β4 membrane homogenates by BTHIQ. SH-EP1-hα4β2 or SHEP-hα4β4 membrane homogenates were incubated with 1 nM [³H]cytisine for 75 min at 4°C in the presence of various concentrations of BTHIQ. Data are the mean±s.e.m. of 10 experiments. (b) Saturation analysis of the specific binding of [³H]cytisine to SH-EP1-hα4β2 or SHEP-hα4β4 membrane homogenates in the absence and presence of IC₅₀ concentrations of BTHIQ. Data points are the means of triplicate samples±s.e.m. of 10 (α4β2) or 4–5 (α4β4) experiments.

Figure 5

Functional effects of BTHIQ on human α4β2 nACh receptors. (a) Concentration–response curve for the antagonist effects of the test BTHIQ on human α4β2 nACh receptors. The data were normalised to the responses elicited by 100 μM ACh (approximate low affinity EC₅₀ of ACh at α4β2 nACh receptors) and then fitted to a two-component Hill equation. Data points represent the mean±s.e.m. of 8–10 independent experiments. Where no error bars are shown, they are smaller than the symbols. (b) Concentration–response curve for ACh responses in the absence or presence of IC₅₀ concentrations of BTHIQ. After control EC₅₀ ACh responses were elicited, oocytes were first superfused with BTHIQ alone for 2 min and then with EC₅₀ ACh and BTHIQ. Data were normalised to responses elicited by 1 mM acetylcholine (maximal ACh response) and represent the mean±s.e.m. of 10 experiments. (c) Inhibition of α4β2 receptor function by IC₅₀ concentrations of BTHIQ at a range of holding potentials. Inhibition by 7BNMC, 7B12MNMC, BBC, NEA and C was equivalent at all potentials, but inhibition by L or A was dependent on holding potential (n=10, Anova test).

Figure 6

Functional effects of BTHIQ on human α4β4 nACh receptors. (a) Concentration–response curve for the antagonist effects of the test BTHIQ on human α4β4 nACh ceptors. The data were normalised to the responses elicited by 30 μM ACh (approximate EC₅₀ of the ACh response at α4β4 nACh receptors) and then fitted to a single component Hill equation. Data points represent the mean±s.e.m. of 3–4 experiments. Error bars are not shown when they are smaller than the symbols. (b) Concentration–response curve for ACh responses in the absence or presence of IC₅₀ concentrations of BTHIQ. After control EC₅₀ ACh responses were elicited, oocytes were first superfused with BTHIQ alone for 2 min and then with EC₅₀ ACh and BTHIQ. Data were normalised to responses elicited by 1 mM ACh (maximal ACh response) and represent the mean±s.e.m. of 6–10 experiments. (c) Inhibition of α4β4 receptor function by concentrations of BTHIQ close to their respective IC₅₀ concentrations at a range of holding potentials. Inhibition by 7BNMC, 7B12MNMC, BBC, C and NEA was equivalent at all potentials at which ACh responses could be elicited, but inhibition by L or A was dependent on holding potential (n=10, Anova test).