N-desmethylclozapine, an allosteric agonist at muscarinic 1 receptor, potentiates N-methyl-D-aspartate receptor activity

Abstract

The molecular and neuronal substrates conferring on clozapine its unique and superior efficacy in the treatment of schizophrenia remain elusive. The interaction of clozapine with many G protein-coupled receptors is well documented but less is known about its biologically active metabolite, N-desmethylclozapine. Recent clinical and preclinical evidences of the antipsychotic activity of the muscarinic agonist xanomeline prompted us to investigate the effects of N-desmethylclozapine on cloned human M1-M5 muscarinic receptors. N-desmethylclozapine preferentially bound to M1 muscarinic receptors with an IC50 of 55 nM and was a more potent partial agonist (EC50, 115 nM and 50% of acetylcholine response) at this receptor than clozapine. Furthermore, pharmacological and site-directed mutagenesis studies suggested that N-desmethylclozapine preferentially activated M1 receptors by interacting with a site that does not fully overlap with the acetylcholine orthosteric site. As hypofunction of N-methyl-d-aspartate (NMDA) receptor-driven neuronal ensembles has been implicated in psychotic disorders, the neuronal activity of N-desmethylclozapine was electrophysiologically investigated in hippocampal rat brain slices. N-desmethylclozapine was shown to dose-dependently potentiate NMDA receptor currents in CA1 pyramidal cells by 53% at 100 nM, an effect largely mediated by activation of muscarinic receptors. Altogether, our observations provide direct evidence that the brain penetrant metabolite N-desmethylclozapine is a potent, allosteric agonist at human M1 receptors and is able to potentiate hippocampal NMDA receptor currents through M1 receptor activation. These observations raise the possibility that N-desmethylclozapine contributes to clozapine's clinical activity in schizophrenics through modulation of both muscarinic and glutamatergic neurotransmission.

Fig. 1.

M1 agonist activity of N-desmethylclozapine. (A) FLIPR experiments on M1-CHONFAT cells revealed that N-desmethylclozapine (•) is a partial M1 agonist compared with acetylcholine (Ach) (▪). Clozapine (▵) and its metabolite clozapine-N-oxide (▴) weakly stimulated intracellular calcium mobilization. Note that the activity of N-desmethylclozapine was blocked by 1 μM atropine (○). (B) Haloperidol (▴) and olanzapine (□) did not potently activate M1 receptor compared with acetylcholine (▪) and N-desmethylclozapine (•). Results shown are from a representative experiment (points represent the mean ± SEM of quadruplicate determinations) that was repeated at least three times with comparable results (mean ± SEM given in Table 2).

Fig. 2.

Effect of the M1 allosteric potentiator brucine (100 μM) on N- desmethylclozapine. Dose-response curves for N-desmethylclozapine without (•) and with (○) brucine and for acetylcholine (Ach) without (▪) and with (□) brucine showed that brucine shifted the acetylcholine concentration-response curve to the left by a factor of 4.5 but shifted the N-desmethylclozapine concentration-response curve to the right by a factor of 3.5. Results shown are from a representative experiment (points represent the mean ± SEM of quadruplicate determinations) that was repeated at least three times with comparable results (mean ± SEM given in the text).

Fig. 3.

Effect of mutation at M1 orthosteric site on N-desmethylclozapine and acetylcholine (Ach)-induced functional response measured by using FLIPR. The Y381A mutation (□ and ○) caused a shift of acetylcholine (▪) and N-desmethylclozapine (•) concentration-response curves to the right by a factor of 315 and to the left by a factor of 16, respectively. Results shown are from a representative experiment (points represent the mean ± SEM of quadruplicate determinations) that was repeated at least three times with comparable results (mean ± SEM given in the text).

Fig. 4.

N-desmethylclozapine potentiates NMDA-evoke currents in CA1 pyramidal cells by activation of muscarinic receptors. (A₁ and A₂) Representative traces of NMDA-evoked currents before (predrug), during, and 15 min after application of 100 nM N-desmethylclozapine in the absence (A₁) and presence (A₂) of 1 μM atropine. (B) Average time course of the effect of 100 nM N-desmethylclozapine on NMDA-evoked currents; each point represents the mean ± SEM of data from four cells. (C) Dose-response relationship of the N-desmethylclozapine-induced potentiation of NMDA-evoked currents; each point represents the mean ± SEM of data from three to six cells. (D) Bar graph showing the average maximum effect of 100 nM N-desmethylclozapine on NMDA-evoked currents in the absence and presence of 1 μM atropine. Each bar represents the mean ± SEM of data from five and four cells, respectively. ^*, P < 0.05, Student's paired t test.