Tamotsu Nomura

The anti-dementia drug nefiracetam facilitates hippocampal synaptic transmission by functionally targeting presynaptic nicotinic ACh receptors.

Nefiracetam, a pyrrolidone derivative developed as an anti-dementia drug, persistently potentiated currents through neuronal nicotinic acetylcholine (ACh) receptors (alpha7, alpha4beta2) expressed in Xenopus oocytes, and the potentiation was blocked by either the selective protein kinase C (PKC) inhibitors, GF109203X and staurosporine, or co-expressed active PKC inhibitor peptide. In primary cultures of rat hippocampal neurons, nefiracetam increased the rate of nicotine-sensitive miniature excitatory postsynaptic currents, without affecting the amplitude, and the increase was inhibited by GF109203X. In addition, the drug caused a marked increase in the glutamate release from electrically stimulated guinea pig hippocampal slices, and the effect was abolished by the nicotinic ACh receptor antagonists, alpha-bungarotoxin and mecamylamine. Nefiracetam induced a long-lasting facilitation of synaptic transmission in both the CA1 area and the dentate gyrus of rat hippocampal slices, and the facilitation was inhibited by alpha-bungarotoxin and mecamylamine. Such facilitatory action was still found in the hippocampus with selective cholinergic denervation. The results of the present study, thus, suggest that nefiracetam enhances activity of nicotinic ACh receptors by interacting with a PKC pathway, thereby increasing glutamate release from presynaptic terminals, and then leading to a sustained facilitation of hippocampal neurotransmission. This may represent a cellular mechanism underlying the cognition-enhancing action of nefiracetam. The results also provide the possibility that nefiracetam could be developed as a promising therapeutic drug for senile dementia or Alzheimers disease.

Modulation of ACh receptor currents by arachidonic acid

The present study investigated the effects of arachidonic acid on Torpedo (αβγδ) and neuronal nicotinic acetylcholine (ACh) receptors (chick α7; rat α7, α3β2, α3β4, α4β2, and α4β4). Arachidonic acid (10 μM) depressed currents through normal Torpedo ACh receptors during treatment and afterward, persistently (≥30 min) potentiated the currents. The potentiation was blocked by the selective protein kinase C (PKC) inhibitor, GF109203X or PKC inhibitor peptide (PKCI). The depression was not inhibited by any protein kinase inhibitor examined here, but greater in Ca2+-free extracellular solution. Arachidonic acid also potentiated currents through mutant Torpedo ACh receptors lacking PKC phosphorylation sites at Ser333 on the α subunit and Ser377 on the δ subunit without depression, but otherwise, it depressed currents through mutant receptors replacing of each Ser by negatively charged amino acid residue, possibly that mimics PKC phosphorylation of the receptors. These results suggest that the depression was due to the direct blocking effect on Ca2+-modulatory sites, which was accelerated under conditions of the receptors phosphorylated by PKC, and that the potentiation was caused by PKC activation, independently of PKC phosphorylation of the receptors. Arachidonic acid reduced currents through chick α7 receptors by a mechanism independent of protein kinase activation. In contrast, arachidonic acid potentiated currents through rat α7, α3β2, α4β2, and α4β4 receptors, perhaps by the same mechanism as the potentiation observed in Torpedo ACh receptors, although it had no effect on rat α3β4 receptors. The results of the present study thus demonstrate that arachidonic acid exerts diverse actions on nicotinic ACh receptors by different mechanisms.

Nefiracetam facilitates hippocampal neurotransmission by a mechanism independent of the piracetam and aniracetam action

Nefiracetam, a nootropic (cognition-enhancing) agent, facilitated neurotransmission in the dentate gyrus of rat hippocampal slices in a dose-dependent manner at concentrations ranged from 1 nM to 1 microM, being evident at 60-min washing-out of the drug. The facilitatory action was blocked by the nicotinic acetylcholine (ACh) receptor antagonists, alpha-bungarotoxin and mecamylamine. A similar facilitation was induced by the other nootropic agents, piracetam and aniracetam, but the facilitation was not inhibited by nicotinic ACh receptor antagonists and it did not occlude the potentiation induced by nefiracetam. In the Xenopus oocyte expression systems, nefiracetam potentiated currents through a variety of neuronal nicotinic ACh receptors (alpha 3beta 2, alpha 3beta 4, alpha 4 beta 2, alpha 4 beta 4, and alpha 7) to a different extent. In contrast, neither piracetam nor aniracetam had any potentiating action on alpha 7 receptor currents. While aniracetam delayed the decay time of currents through the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor, GluR1, -2, -3, expressed in oocytes, nefiracetam or piracetam had no effect on the currents. Nefiracetam, thus, appears to facilitate hippocampal neurotransmission by functionally targeting nicotinic ACh receptors, independently of the action of piracetam and aniracetam.

Arachidonic acid potentiates currents through Ca2+-permeable AMPA receptors by interacting with a CaMKII pathway

The present study investigated the effect of arachidonic acid on the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, presumably heteromeric receptors formed of GluR1, GluR2, and GluR3, expressed in Xenopus oocytes. Arachidonic acid (10 microM) potentiated currents through receptors expressing GluR1 and 3 (GluR1,3) to 170% of basal level during initial 20 min following application, being still evident at 60-min washing-out of the drug, while it never or little enhanced currents through receptors expressing GluR1 and 2 (GluR1,2) or GluR1, 2, and 3 (GluR1,2,3) (110% 30 min after treatment). The effect of arachidonic acid on GluR1,3 currents was not observed in Ca2+-free extracellular solution, and the potentiation was blocked by either KN-93, a selective Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor, or NP217, an active CaMKII inhibitor peptide, when co-expressed with the receptors. In contrast, the protein synthesis inhibitor, cycloheximide, the selective inhibitor of cAMP-dependent protein kinase (PKA), H-89, the selective inhibitors of protein kinase C (PKC), PKCI and GF109203X, the mitogen-activated protein (MAP) kinase kinase inhibitor, PD98059, or the inactive CaMKII inhibitors, KN-92 and NP218, had no effect on the currents. In the assay of intracellular calcium mobilizations, Ca2+ influx in response to receptor activation was greatest with receptors formed in oocytes expressing GluR1,3. The results of the present study indicate that arachidonic acid induces a long-lasting potentiation of GluR1,3 currents, possibly as a result of the interaction with a CaMKII pathway.

Sustained facilitatory action of FK960 on hippocampal neurotransmission.

The present study investigated the effect of N-(4-acetyl-1-piperazinyl)-p-fluorobenzamide monohydrate (FK960), a piperazine derivative developed as antidementia drug, on hippocampal neurotransmission. FK960 increased the amplitude of population spikes (PSs) from the granular cell layer in rat hippocampal slices in a bell-shaped dose-dependent manner at concentrations ranged from 10 nM to 10 microM. A similar potentiation was found in the intact mouse hippocampus, the action being still evident 120 min after injection; a maximal effect was obtained with 3 mg/kg intraperitoneal injection of FK960 at concentrations ranged from 0.3 to 5 mg/kg. The facilitatory action induced by FK960 did not occlude the potentiation induced by tetanic stimulation. The results of the present study indicate that FK960 induces a long-lasting facilitation of hippocampal neurotransmission, but by a mechanism independent of the tetanic long-term potentiation. This may account for the memory enhancing action of FK960.

ATP produces potassium currents via P3 purinoceptor in the follicle cell layer of Xenopus oocytes.

Purinoceptor agonists produced potassium currents with the order of potency: ATP > adenosine = ADP = AMP > beta,gamma-methylene ATP, while a small response or no response was induced by 2-methylthio ATP, UTP, or alpha,beta-methylene ATP. The response induced by beta,gamma-methylene ATP was completely inhibited in the presence of alpha,beta-methylene ATP, suggesting that the relevant receptor for these agonists was a P3 purinoceptor. ATP induced currents with a latency of 24 s and the currents were not induced in defolliculated oocytes. The currents were not affected by either the Gi/o-protein inhibitor, pertussis toxin (PTX), or the selective cAMP-dependent protein kinase inhibitor, H-89, or the phospholipase C (PLC) inhibitor, neomycin, or the phospholipase A2 (PLA2) inhibitor, 4-bromophenacyl bromide. The currents were enhanced by the selective protein kinase C (PKC) inhibitor, GF109203X, but otherwise, they were reduced by the potent PKC activator, 12-O-tetradecanoylphorbol-13-acetate. The results of the present study suggest that a P3 purinoceptor in the follicle cell layer of oocytes is involved in activation of potassium channels and that the evoked currents are regulated by PLC/PLA2-independent PKC activation.

Methamphetamine modulates ACh-evoked currents in Xenopus oocytes expressing the rat α7 receptors

Abstract Although the cholinergic system is proposed to be involved in methamphetamine (MeAMPH)-induced abnormal behaviors, no direct evidence has been provided yet. The present study investigated the effects of MeAMPH on acetylcholine (ACh)-evoked currents in the neuronal nicotinic ACh receptors ( α 7) expressed in Xenopus oocytes. MeAMPH enhanced the currents in a time- and dose-dependent manner at concentrations ranged from 1 nM to 3 μ M, reaching a maximum of 150% 30 min after treatment. Lesser potentiation was observed at higher concentrations (>3 μ M) and instead, the currents were inhibited at more than 10 μ M MeAMPH with a slow reverse after washing-out of the drug. The current potentiation or depression was caused via a pathway independent of G-protein, protein kinase C or cAMP-dependent protein kinase. The ACh dose-response curve was shifted to the left and to the right after treatment with 1 and 100 μ M MeAMPH, respectively, suggesting that MeAMPH potentiated or inhibited ACh-evoked currents by a change in the affinity for ACh. The actions of MeAMPH on the neuronal nicotinic ACh receptors may represent a cellular mechanism for MeAMPH-induced abnormal behaviors and sensitization.