Introduction

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Numerous studies have shown that piracetam-like nootropics (or cognition-enhancing agents) can improve various neurotransmissions in the brain, those in the dopaminergic (Funk and Schmidt, 1984), cholinergic (Spignoli and Pepeu, 1987), glutamatergic (Marchi et al., 1990) and -aminobutyric acid-ergic (Watabe et al., 1993) systems. Nootropics also facilitate long-term potentiation (Satoh et al., 1986), a model system of memory and learning. These actions are explained by an increase in the release of neurotransmitters from presynaptic terminals (Funk and Schmidt, 1984; Marchi et al., 1990) or an enhancement in neurotransmitter receptor responses at postsynaptic sites (Isaascon and Nicoll, 1991; Ito et al., 1990; Tang et al., 1991). The underlying regulatory mechanism, however, is unknown. We identified the intracellular signal transduction pathways responsible for the nootropic actions by monitoring currents through expressed native and mutant Torpedo californica nACh receptors. The results of the present study suggest that nefiracetam is involved in the activation of PKA and PKC, leading to inhibition and potentiation of ACh-evoked currents, respectively.

	Materials and Methods

Top Summary Introduction Materials & Methods Results & Discussion References

In vitro transcription and translation in Xenopus laevis oocytes

Construction of the plasmids containing the T. californica nicotinic ACh receptor subunits has been described previously (Sumikawa and Miledi, 1989). The , , ,  subunit constructs were transcribed in vitro using SP6 RNA polymerase as previously described (Sumikawa and Miledi, 1989). T. californica nACh receptors are known to have PKA phosphorylation sites on the  and  subunits (Huganir and Greengard, 1990) and PKC phosphorylation sites on the  and  subunits (Huganir, 1987). The  and  subunit mutants that lack PKA phosphorylation sites were constructed using site-directed mutagenesis (Gehle and Sumikawa, 1991); Ser353,354 on the  subunit and Ser361,362 on the  subunit were replaced by Ala, and Ser333 on the  subunit and Ser377 on the  subunit were replaced by Ala for the  and  subunit mutants that lacked PKC phosphorylation sites. After surgical removal from female X. laevis frogs and manual separation from the ovary, the isolated oocytes were incubated in Barth's solution (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO₃, 0.82 mM MgSO₄, 0.33 mM Ca(NO₂)₂, 0.41 mM CaCl₂, and 7.5 mM Tris, pH 7.6). One day before microinjection, collagenase treatment (0.5 mg/ml) of oocytes was performed. Oocytes were injected (approximately 40 nl) with combinations of normal (, , , ) and mutant (mPKA/Ser353,354 mPKA/Ser361, 362) or (mPKC/Ser333 mPKC/Ser377) subunit mRNAs and incubated at 18°.

Two-electrode voltage-clamp recording

The injected oocytes were transferred to the recording chamber 24 to 48 hr after incubation and continuously superfused at room temperature (20 to 22°) in a standard frog Ringer's solution (115 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, and 5 mM HEPES, pH 7.0). Ca²⁺-free extracellular solution consisted of 115 mM NaCl, 2 mM KCl, 5 mM MgCl₂, 5 mM HEPES, and 1 mM EGTA, pH 7.0. To remove the effect of the muscarinic ACh receptor, 1 µM atropine was added to the extracellular solution. ACh-activated currents were recorded using two-electrode, voltage-clamp techniques with a GeneClamp-500 amplifier (Axon Instruments, Burlingame, CA) (Nishizaki and Ikeuchi, 1995). The currents were analyzed on a microcomputer using pClamp software (version 6; Axon Instrument). ACh was bath-applied to oocytes. Nefiracetam [DM-9384; N-(2,6-dimethylphenyl)-2-(2-oxo-pyrrolidinyl)acetamide] (Daiichi Pharmaceutical, Tokyo, Japan) was dissolved in distilled water at 1 mM for stock solution and diluted into concentrations required with the extracellular solution.

Assay of [Ca²⁺]_i

Oocytes were injected with Calcium Green-1 (15 µM final concentration; Molecular Probes, Eugene, OR) and were incubated at 18° for 30 min. The oocytes were transferred to the recording chamber onto the stage of a Nikon DIAPHOT 300 microscope and were bathed at room temperature (20-22°) in standard or Ca²⁺-free frog Ringer's solution superfused continuously. The oocytes were viewed with a ×4 UV fluor Nikon objective lens and the images were acquired at 2-sec intervals with a xenon confocal laser-scanning microscope (Nikon Xenon Power Supply XPS-100; Nikon, Tokyo, Japan) attached to an intensified charge-coupled device camera (ARGUS-50/CA; Hamamatsu Photonics, Japan). The Calcium Green signal was long pass-filtered (490 nm). Images were analyzed with ARGUS-50/CA software (version 3.0). To compensate different levels of dye loading between oocytes, I (intensity of Ca²⁺ signal after application of ACh  basal intensity) and the ratio of the I to the basal intensity were calculated; to compensate different levels of functional receptor expression, ACh-evoked currents were recorded in Ca²⁺-free extracellular solution (ACh-gated channel currents). Ca²⁺ mobilizations therefore were normalized by the I/basal intensity/ACh-gated channel current.

	Results and Discussion

Top Summary Introduction Materials & Methods Results & Discussion References

In our X. laevis oocyte expression system for the wild-type nACh receptor, 100 µM ACh evoked inward membrane currents (Fig. 1A). Currents were recorded at 10-min intervals in which spontaneous attenuation of the currents was within 5% during experiments (data not shown). Lower (submicromolar) concentrations of the nootropic nefiracetam reduced ACh-evoked currents to 30 ± 9% (0.01 µM) and 38 ± 11% (0.1 µM) of control after a 10-min treatment (Fig. 1A). The inhibitory effect was slowly recovered; the currents reached the control level (104 ± 19 and 103 ± 20% for 0.01 and 0.1 µM nefiracetam, respectively) 70 min after treatment (Fig. 1A). In contrast, higher (micromolar) concentrations of nefiracetam enhanced the currents in a time-dependent manner during treatment and continued to do so after washing-out of the drug (Fig. 1A). The current potentiating effect was long-lasting, the currents reaching 243 ± 20 and 255 ± 18% at 1 and 10 µM, respectively, 90 min after treatment (Fig. 1A). These results indicates that nefiracetam exerted dose-dependent biphasic effects on ACh-evoked currents: a short-term depression at submicromolar concentrations and a long-term enhancement at micromolar concentrations. When 1 µM nefiracetam was applied in the process of the current suppression by 0.01 µM nefiracetam, the drug action was switched from suppression to enhancement (Fig. 1B). This suggests that at least two different signal transduction pathways were involved in such a biphasic action of nefiracetam.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Effects of nefiracetam on ACh-evoked currents. A, 100 µM ACh was applied to a single oocyte expressing the normal ACh receptors at 10-min intervals. The oocyte was treated with nefiracetam at 0.01, 0.1, 1 and 10 µM for 10 min. The illustrated currents were recorded at the times indicated. The holding potential was 30 mV. Inward currents correspond to inward deflections. The time-course effects of nefiracetam on the currents are summarized in lower column. Points, mean percent ± standard deviation of the original amplitude (10 min) from seven oocytes. B, An oocyte was treated with 0.01 µM followed by 1 µM nefiracetam (seven oocytes).

T. californica nACh receptors form nonselective cation channels; ACh-evoked currents in X. laevis oocytes that express the ACh receptors are composed of ACh-gated channel currents and Ca²⁺-dependent chloride currents that are evoked by Ca²⁺ entry through the ACh receptor channels (Miledi and Parker, 1984). To ascertain the site of the nefiracetam action, Ca²⁺-sensitive chloride currents were induced by activation of the intrinsic muscarinic ACh receptors. Nefiracetam had no effect on the Cl currents (Fig. 2A), which suggests that nefiracetam modulated ACh-gated channel currents but not chloride currents. To obtain further evidence for this, [Ca²⁺]_i was assayed. ACh increased [Ca²⁺]_i in the presence of Ca²⁺-containing extracellular solution, whereas no increase was observed in Ca²⁺-free extracellular solution (Fig. 2B), which indicated that the ACh receptor channels permeated calcium and that the source of the [Ca²⁺]_i increase was extracellular. Nefiracetam, however, did not induce further rise in [Ca²⁺]_i, although it potentiated ACh-evoked currents (Fig. 2B), which implies that nefiracetam did not affect Ca²⁺ permeability; in other words, nefiracetam potentiated ACh-evoked currents by modulating ACh receptor currents, possibly Na⁺ currents, but not by the secondary effect of Ca²⁺-sensitive chloride currents as a consequence of intracellular Ca²⁺ rise.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Effect of nefiracetam on Ca2+-dependent chloride currents and Ca2+ influx through the normal nicotinic ACh receptors. A, 100 µM ACh was applied to an oocyte without expression of the ACh receptors in atropine-free frog Ringer's solution. The evoked Cl currents were recorded 10 min before, and 0 and 30 min after treatment with nefiracetam (1 µM). The holding potential was 30 mV. Inward currents correspond to inward deflections. B, Two-electrode voltage-clamp was made to a Calcium Green-loaded oocyte expressing the normal ACh receptors. Intracellular Ca2+ mobilizations were monitored in the presence and absence of nefiracetam (1 µM). Simultaneously, 100 µM ACh-evoked currents (IA) were recorded in Ca2+-containing (1) and Ca2+-free extracellular solution (extCa2+ (); 2). The holding potential was 30 mV. Inward currents correspond to inward deflections. The Ca2+ rise was normalized by  increase of intensity/basal intensity/current amplitude (2). Data are presented as the mean ± standard deviation of seven independent experiments.

Subsequently, an attempt was made to assess the intracellular signals that mediate the nefiracetam-induced current depression and potentiation. H-89, a selective inhibitor of PKA, blocked the inhibitory action of nefiracetam at submicromolar concentrations on ACh-evoked currents; however, it potentiated the currents to 172 ± 19% of control 30 min after treatment with 0.01 µM nefiracetam (Fig. 3A), which suggests that nefiracetam at lower concentrations reduced the currents via PKA activation. ACh-evoked currents were inhibited drastically in the presence of the PKA activator forskolin (21 ± 8% of control) (Fig. 3A), providing indirect evidence that nefiracetam interacts with a PKA pathway, leading to inhibition of the currents. Nefiracetam (0.01 µM) enhanced ACh receptor currents in oocytes treated with PTX, a G protein (G_i/o) inhibitor, to the same level as observed in H-89 (Fig. 3A), which suggests that the inhibitory action of nefiracetam was mediated by PKA under the regulation of PTX-sensitive G proteins. In addition, the facilitatory action of 1 µM nefiracetam was more enhanced in the presence of H-89, the currents reaching 236 ± 18% (five oocytes) 30 min after treatment (data not shown). This suggests strongly that higher concentrations of nefiracetam can still activate PKA and decrease the currents, although such inhibitory effects will be masked by the apparent current potentiation.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Regulation of ACh-evoked currents by nefiracetam-mediated PKA and PKC activation. A, 100 µM ACh was applied to an oocyte expressing the normal or mutant ACh receptors lacking PKA phosphorylation sites in the presence and absence of H-89 (1 µM). Some oocytes were treated with 0.1 µg/ml PTX for 24 hr before experiments. The illustrated currents were recorded 10 min before and 30 min after treatment with 0.01 µM nefiracetam. In other experiments, ACh was applied to a single oocyte expressing the normal ACh receptors before and after 15 min treatment with forskolin (20 µM) (five oocytes). The holding potential was 30 mV. Inward currents correspond to inward deflections. Points, mean percent ± standard deviation of the original amplitude (10 min) from seven oocytes; bars, standard deviation. B, 100 µM ACh was applied to an oocyte expressing the normal or mutant ACh receptors lacking PKC phosphorylation sites in the presence and absence of 100 nM GF109203X, 5 µM staurosporine, or in Ca2+-free extracellular solution. The illustrated currents were recorded 10 min before and 30 min after treatment with nefiracetam (1 µM). The holding potential was 30 mV. Inward currents correspond to inward deflections. Points, mean percent ± standard deviation of the original amplitude (10 min) from seven oocytes.

To further examine whether the current depression by nefiracetam is caused by PKA phosphorylation of the receptors, mutant ACh receptors lacking potent PKA phosphorylation sites on the  and  subunits (mPKA/Ser353, 354 mPKA/Ser361, 362) were expressed in oocytes. There, nefiracetam (0.01 µM) did not decrease ACh-evoked currents in the mutant ACh receptors; conversely, it increased them to 159 ± 20% of control 30 min after treatment (Fig. 3A). These results indicate that nefiracetam inhibited ACh receptor currents by PKA activation and the subsequent PKA phosphorylation of the receptors.

In an earlier study using NG108-15 cells, nefiracetam enhanced the activity of neuronal L-type calcium channels in a fashion that mimics the effect of dibutyryl cAMP, and the enhancement was inhibited by PTX (Yoshii and Watabe, 1994), supporting the idea that the action of nefiracetam is associated with PTX-sensitive G proteins and their regulation of PKA activation. No evidence, however, has been provided for the mechanism by which PTX-sensitive G proteins (G_i/o-proteins) can facilitate adenylyl cyclase, not even for any well characterized receptor site as a target of nootropics (Gouliaev and Senning, 1994). How, then, could nefiracetam activate PKA? It might stimulate unknown cytosolic PTX-sensitive G proteins involving PKA activation.

On the other hand, the selective PKC inhibitor, GF109203X (Heikkila et al., 1993) or staurosporine inhibited potentiation of ACh-evoked currents by 1 µM nefiracetam: the currents reaching 60 ± 16 or 63 ± 12% of control at 30 min washing (Fig. 3B). This suggests that nefiracetam potentiated ACh receptor currents via PKC activation. The potentiation was not observed in Ca²⁺-free media; instead, the currents were reduced to the same extent as achieved in the presence of the PKC inhibitors (Fig. 3B). The finding that ACh never increased [Ca²⁺]_i in Ca²⁺-free media (Fig. 2B) suggests that nefiracetam interacted with a Ca²⁺-dependent PKC pathway. Furthermore, 1 µM nefiracetam exhibited no current potentiation in the mutant ACh receptors that lacked potent PKC phosphorylation sites on the  and  subunits (mPKC/Ser333mPKC/Ser377) (Fig. 3B). These results indicate that nefiracetam potentiated ACh-gated channel currents by activation of Ca²⁺-dependent PKC and the subsequent PKC phosphorylation of the receptors. Nefiracetam, therefore, seems to act on two different signal transduction pathways; one is responsible for PTX-sensitive G protein-regulated PKA activation and the other for Ca²⁺-dependent PKC activation.

At present, the type of PKC pathways with which nefiracetam interacts remains unknown. Of PKCs discovered, only the conventional PKC (cPKC) isozymes (, I, II, ) are activated in the presence of Ca²⁺ and diacylglycerol after the activation of phospholipase C (Exon, 1994; Liscovitch and Cantley, 1994). There is evidence for a significant role of cis-unsaturated free fatty acids in sustained PKC activation, possibly by binding to the C1 domain of PKC (Nishizuka, 1995). It is also suggested that the nACh receptor is capable of activating phospholipase C-mediated PKC (Eusebi et al., 1987). Taken together, nefiracetam may sustain nACh receptor-mediated cPKC activation as cis-unsaturated free fatty acids do, leading in turn to a prolonged potentiation of ACh-evoked currents as a result of PKC phosphorylation of the receptors.

Previous studies have shown that nefiracetam modulates -aminobutyric acid_A receptor currents (Huang et al., 1996) or L-type calcium channel-operated currents (Yoshii and Watabe, 1994) by interacting with a PKA pathway. In addition to a PKA pathway, the present study identified a nefiracetam-mediated PKC pathway. Lines of evidence suggest that nootropic agents serve as cognition enhancers by facilitating a variety of neurotransmissions, including cholinergic systems (Funk and Schmidt, 1984; Spignoli and Pepeu, 1987; Marchi et al., 1990; Watabe et al., 1993). The results presented here demonstrate that the nootropic nefiracetam can influence two signal transduction pathways linked to PKA and PKC activation. This may explain that nootropics potentially have multiple downstream targets, providing a clue to understand the cellular mechanism for modification of various synaptic transmissions.