Elsevier

Brain Research

Volume 822, Issues 1–2, 20 March 1999, Pages 72-79
Brain Research

Research report
Modulation of the neuronal nicotinic acetylcholine receptor-channel by the nootropic drug nefiracetam

https://doi.org/10.1016/S0006-8993(99)01077-XGet rights and content

Abstract

The effects of nefiracetam (DM-9384) on the neuronal nicotinic acetylcholine (ACh) receptor-channel were studied by the whole-cell patch clamp technique using PC12 cells. Nefiracetam had a dual effect on ACh-induced currents: it augmented the currents induced by low concentrations (10–30 μM) of ACh and suppressed those induced by high concentrations (100–1000 μM) of ACh. These effects were reversible after washing with drug-free solution. The stimulating effect of nefiracetam was clearly observed at a concentration of 10 μM, and slight increases in currents were detected even at 0.1 μM or 1 μM. Nefiracetam at 100 μM suppressed the currents induced by a low concentration (10 μM) of ACh. The rate of desensitization of ACh-induced current was greatly accelerated by nefiracetam, and this effect could not be reversed by washing with drug-free solution. When added to the internal pipette solution, the protein kinase A inhibitor KT 5720 (0.6 μM), but not the protein kinase C inhibitor calphostin C (0.5 μM), abolished the nefiracetam stimulation of the ACh receptor. Pre-incubation of cells with 200 ng/ml pertussis toxin for 24 h also abolished the nefiracetam action. Thus, the nefiracetam modulation of the neuronal nicotinic ACh receptor-channel is exerted via G proteins and protein kinase A. The stimulation of the ACh receptor may be directly related to the cognitive enhancing action of nefiracetam.

Introduction

Nootropic drugs may be classified into two groups, cholinergic agonists and pyrrolidone derivatives. The latter includes oxiracetam, aniracetam, and piracetam. Despite a number of studies that have been undertaken in an attempt to understand the mechanism of action of nootropic drugs, our knowledge about their cognitive enhancing action is far from satisfactory. The biochemical effects of nootropic drugs that have been disclosed include, but are not limited to, the enhancement of dopamine release, choline uptake, cholinergic transmission, AMPA receptor function, phosphatidylinositol turnover, and phosphatase A2 activity 2, 8, 10, 27, 33, 38. Nootropic drugs also facilitate long-term potentiation [39]and enhance synaptic transmission by increasing the neurotransmitter release from presynaptic nerve terminals 6, 25or by increasing the postsynaptic response to neurotransmitters 13, 14, 43. Ca2+ influx into postsynaptic cells may be increased by nootropic drugs through the potentiation of the AMPA-sensitive glutamate receptors [3], leading to facilitation of long-term potentiation.

Nefiracetam (DM-9384), a pyrrolidone derivative, is a nootropic drug being developed for the treatment of dementia [27]. It has been shown to ameliorate various types of amnesia in rodents 18, 28, 36, 44. Our previous study has clearly demonstrated that nefiracetam modulates the activity of the γ-aminobutyric acid (GABA)A receptor-channel via interactions with G proteins and protein kinase A (PKA) [11]. Nefiracetam interactions with G proteins and PKA predict that it acts on other receptors and ion channels which are regulated by these systems. In fact, this was already shown to be the case in voltage-gated L/N-type calcium channels [51]. Along this line, one of the most likely receptors/channels that would be modulated by nefiracetam is the neuronal nicotinic acetylcholine (ACh) receptor-channel, since there are structural similarities between the ACh and GABAA receptors 21, 23, 26, 40. A variety of nootropic drugs structurally related to nefiracetam are known to affect the cholinergic system (reviewed by Gouliaev and Senning [10]; Pepeu and Spignoli [33]). Furthermore, the neuronal nicotinic ACh receptor, which is also regulated by G proteins and protein kinases, is known to be altered in various neurological disorders including Alzheimer's disease [1]. Thus, we have undertaken a patch clamp study of the nefiracetam modulation of the neuronal nicotinic ACh receptor-channel. It was found that the response of the ACh receptor-channel to nefiracetam was very similar to that of the GABAA receptor-channel. Nefiracetam had a dual effect on the ACh receptor-channel, current potentiation and suppression depending on the ACh concentration, and the potentiating effect was exerted via G proteins and PKA, but not PKC.

Section snippets

Materials

PC12 cells were used as a model for the neuronal nicotinic ACh receptors which are known to be different from the muscle-type nicotinic ACh receptors with respect to the molecular structure, physiology and pharmacology 7, 17, 21, 26. The PC12 cell line was kindly provided by Drs. Edson X. Albuquerque and Edna F.R. Pereira of the University of Maryland School of Medicine in Baltimore. Cells were cultured in Dulbecco's modified Eagles' medium containing fetal bovine serum (0.1 mg/ml, Sigma, St.

Mechanism of dual action of nefiracetam

Nefiracetam was found to cause a dual action on ACh-induced currents depending on the concentration of ACh. At a low (10 μM) ACh applied for 2 s, 10 μM nefiracetam applied to the bath clearly augmented the current, and the effect was reversed after washing with nefiracetam-free solution (Fig. 1). Little or no suppression of current was observed during the application of nefiracetam. Since ACh-induced currents in PC12 cells have been shown to be blocked by hexamethonium or d-tubocurarine 29, 30,

Discussion

Nefiracetam has been found to modulate the neuronal nicotinic ACh receptor-channel in a manner very similar to the modulation of the GABAA receptor-channel reported previously [11]. It has a dual action, augmentation of the currents induced by low concentrations of ACh and suppression of the currents induced by high concentrations of ACh. Currents induced by a low concentration (10 μM) of ACh are reversibly augmented by 0.1 μM, 1 μM, and 10 μM nefiracetam, although only the effect of 10 μM

Acknowledgements

This work was supported in part by Daiichi Pharmaceutical and by a grant from the National Institutes of Health (NS 14144). The authors wish to thank Drs. Edson X. Albuquerque and Edna F.R. Pereira of the University of Maryland School of Medicine for providing us with PC12 cell line, Nayla Hasan for technical assistance and Julia Irizarry for secretarial assistance.

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