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Profilin catalyzes the exchange of actin-bound ADP to ATP. The present study investigated the role of profilin in PKCε activation. Profilin associated with PKCε in differentiated PC-12 cells under the basal conditions, which was inhibited by the PKC inhibitor GF109203X. The selective PKCε activator DCP-LA markedly increased the association, which was clearly prevented by GF109203X. The basal PKC activity in PC-12 cells was attenuated by knocking-down profilin, while the basal activities of PKA and CaMKII were not affected. DCP-LA enhanced the PKC activity to approximately 3.5 folds of the basal levels, and the effect was suppressed by knocking-down profilin. In the cell-free system, PKCε was not activated by profilin alone. DCP-LA activated PKCε in an ATP concentration (2-500 μM)-dependent manner, and addition of profilin shifted the ATP concentration/DCP-LA-induced PKCε activity relation curve to the left (the direction of lower ATP concentrations). Taken together, the results of the present study indicate that profilin binds to activated PKCε and facilitates PKCε activation by accelerating ATP supply to PKCε., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Amyloid-β peptide 1-42 (Aβ1-42) reduced PC-12 cell viability in a concentration (1-10 μM)- and treatment time (48-72 h)-dependent manner. Nicotine prevented Aβ1-42-induced PC-12 cell death, but conversely, the α7 ACh receptor antagonist α-bungarotoxin enhanced Aβ1-42-induced cell toxicity. Extracellularly applied Aβ1-42 significantly increased cell surface localization of α7 ACh receptor in PC-12 cells as compared with that for non-treated control cells. Cell surface localization of α7 ACh receptor in the brain of 5xFAD mouse, an animal model of Alzheimer's disease (AD), apparently increased in an age (1-12 months)-dependent manner in association with increased accumulation of Aβ1-42 in the plasma membrane component. Taken together, these results indicate that Aβ1-42 promotes translocation of α7 ACh receptor towards the cell surface and that α7 ACh receptor rescues neuronal cells from Aβ1-42-induced damage., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Accumulating evidence has pointed to the direct inhibitory action of lithium, an anti-depressant, on GSK-3β. The present study investigated further insight into lithium signaling pathways. In the cell-free assay Li2CO3 significantly inhibited phosphoinositide 3-kinase (PI3K)-mediated phosphorylation of Akt1 at Ser473, but Li2CO3 did not affect PI3K-mediated PI(3,4,5)P3 production and 3-phosphoinositide-dependent protein kinase 1 (PDK1)-mediated phosphorylation of Akt1 at Thr308. This indicates that lithium could enhance GSK-3β activity by suppressing Akt-mediated Ser9 phosphorylation of GSK-3β in association with inhibition of PI3K-mediated Akt activation. There was no direct effect of Li2CO3 on Akt1-induced phosphorylation of GSK-3β at Ser9, but otherwise Li2CO3 significantly reduced GSK-3β-mediated phosphorylation of β-catenin at Ser33/37 and Thr41. This indicates that lithium directly inhibits GSK-3β in an Akt-independent manner. In rat hippocampal slices Li2CO3 significantly inhibited phosphorylation of Akt1/2 at Ser473/474, GSK-3β at Ser9, and β-catenin at Ser33/37 and Thr41. Taken together, these results indicate that lithium exerts its potentiating and inhibiting bidirectional actions on GSK-3β activity., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Extracellular adenosine is well reported to suppress tumor growth by induction of apoptosis. However, in this study we found that adenosine treatment results in cellular senescence in A549 lung cancer cells both in vitro and in vivo; adenosine induces cell cycle arrest and senescence in a p53/p21 dependent manner; adenosine elevates the level of phosphor-γH2AX, pCHK2 and pBRCA1, the markers for prolonged DNA damage response which are likely responsible for initiating the cellular senescence. Our study first demonstrates that adenosine suppresses growth of cancer cells by inducing senescence and provides additional evidence that adenosine could act as an effective anticancer agent for targeted cancer therapy., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The present study investigated proliferation of MKN28 and MKN45 human gastric cancer cells regulated by the N-methyl-d-aspartate (NMDA) receptor subunit. The NMDA receptor antagonist dl-2-amino-5-phosphonovaleric acid (AP5) inhibited proliferation of MKN45 cells, but not MKN28 cells. Of the NMDA subunits such as NR1, NR2 (2A, 2B, 2C, and 2D), and NR3 (3A and 3B), all the NMDA subunit mRNAs except for the NR2B subunit mRNA were expressed in both MKN28 and MKN45 cells. MKN45 cells were characterized by higher expression of the NR2A subunit mRNA and lower expression of the NR1 subunit mRNA, but MKN28 otherwise by higher expression of the NR1 subunit mRNA and lower expression of the NR2A subunit mRNA. MKN45 cell proliferation was also inhibited by silencing the NR2A subunit-targeted gene. For MKN45 cells, AP5 or knocking-down the NR2A subunit increased the proportion of cells in the G(1) phase of cell cycling and decreased the proportion in the S/G(2) phase. The results of the present study, thus, suggest that blockage of NMDA receptors including the NR2A subunit suppresses MKN45 cell proliferation due to cell cycle arrest at the G(1) phase; in other words, the NR2A subunit promotes MKN45 cell proliferation by accelerating cell cycling.

In the assay of glutamate and gamma-aminobutyric acid (GABA) with a high-performance liquid chromatography, spontaneous release of glutamate and GABA from rat hippocampal slices was significantly enhanced by mecamylamine, an inhibitor of non-alpha7 ACh receptors, or alpha-bungarotoxin, an inhibitor of alpha7 ACh receptors in the absence of tetrodotoxin (TTX), but not in the presence of TTX. Nicotine significantly enhanced glutamate and GABA release in the absence of TTX, that is abolished by mecamylamine or alpha-bungarotoxin, while it had no effect on the release in the presence of TTX. In the recording of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-mediated excitatory postsynaptic currents (AMPA-EPSCs) and GABA(A) receptor-mediated inhibitory postsynaptic currents (GABA(A)-IPSCs) from CA1 pyramidal neurons of rat hippocampal slices, nicotine did not affect the rate and amplitude of AMPA-EPSCs and AMPA-miniature EPSCs. In contrast, nicotine significantly increased the rate of GABA(A)-IPSCs, without affecting the amplitude, but such effect was not obtained with GABA(A)-miniature IPSCs. The collective results suggest that alpha7 and non-alpha7 ACh receptors expressed in the hippocampus, activated under the basal conditions, inhibit release of glutamate and GABA controlled through multi-synaptic relays, but that otherwise, those receptors, highly activated by nicotine, stimulate both the release, with a part of GABA released from interneurons transmitting to CA1 pyramidal neurons. Furthermore, the results also suggest that alpha7 and non-alpha7 ACh receptors do not have potency sufficiently to modulate glutamate and GABA release controlled by single synapses.

The glutamate transporter inhibitor, L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) reversibly enhanced hippocampal neuronal activity in the rat and mouse dentate gyrus. The PDC action was still found in mice lacking the glial glutamate transporter GLT-1. PDC did not influence the rate of spontaneous miniature excitatory postsynaptic currents and spontaneous inhibitory postsynaptic currents, ionotropic glutamate receptor currents, or GABA-evoked currents in cultured rat hippocampal neurons. PDC increased glutamate released from cultured hippocampal astrocytes from normal rats, normal mice, and GLT-1 knock-out mice, that is not inhibited by deleting extracellular Na(+), while the drug had no effect on the release from cultured rat hippocampal neurons. The results of the present study thus suggest that PDC stimulates glial glutamate release by a mechanism independent of inhibiting glutamate transporters, which perhaps causes an increase in synaptic glutamate concentrations, in part responsible for the enhancement in hippocampal neuronal activity.

The present study investigated the effects of amyloid-beta peptides on nicotinic ACh receptors (Torpedo, alpha 4 beta 2, and alpha 7 receptors) and AMPA receptors expressed in Xenopus oocytes by monitoring whole-cell membrane currents. Ten-minutes treatment with amyloid-beta(1-42) (1 microM) inhibited Torpedo ACh receptor currents, reaching 53% of original levels 30 min after treatment. Amyloid-beta(1-40) inhibited the currents in a dose-dependent manner (0.1-10 microM) during treatment, gradually reversing after treatment. Amyloid-beta(1-40) and amyloid-beta(1-42) (0.1 microM) depressed alpha 4 beta 2 receptor currents to each 69% and 62% of original levels at 10-min treatment and lesser depression was obtained with alpha 7 receptors. Amyloid-beta(1-42) (0.1 microM) did not significantly inhibit AMPA receptor currents, but amyloid-beta(1-40) (0.1 microM) potentiated the currents to 145-191% of original levels. Amyloid-beta peptides, thus, exert their diverse actions on nicotinic ACh receptors and AMPA receptors, and the inhibitory actions on nicotinic ACh receptors may account for the deterioration of learning and memory in Alzheimer's disease.

NMDA produced whole-cell membrane currents in cultured human astrocytes. The currents were not inhibited by the selective NMDA receptor antagonist, APV, while they were partially inhibited by the broad G-protein inhibitor, GDPbetaS. NMDA-induced currents were enhanced by either the microsomal Ca2+/ATPase inhibitors, thapsigargin and cyclopiazonic acid, or the ATP-uncoupler, dinitrophenol (DNP). In the Ca2+ assay, NMDA increased intracellular calcium concentration. The increase was inhibited by 26% in Ca2+-free extracellular solution, and it was not inhibited by APV. The results of the present study suggest that NMDA responses in human astrocytes are regulated by store Ca2+ depletion-associated signal., (Copyright 1999 Academic Press.)

Arachidonic acid is suggested to play a role in the expression of long-term potentiation (LTP), a synaptic analog of memory and learning. However, it is unknown whether this free fatty acid is actually released during LTP or not. To address this question, we assayed arachidonic acid with an HPLC system using 9-anthryldiazomethane (ADAM) as a fluorescent probe. High frequency stimulation (tetanic stimulation) to a hippocampal slice from the guinea pig brain caused a huge increase in the release of glutamate from presynaptic terminals and in turn, a gradual increase in the release of arachidonic acid. A similar increase in the release of arachidonic acid was induced by application of glutamate and the increase was inhibited by either the selective AMPA/kainate receptor antagonist, DNQX, or to a lesser extent by the selective NMDA receptor antagonist, APV. These findings suggest that arachidonic acid is produced by activation of ionotropic glutamate receptors involving expression of LTP. Arachidonic acid exerted a long-lasting facilitatory action on synaptic transmission in the CA1 region of rat hippocampal slices and the facilitation occluded the tetanic LTP. Arachidonic acid, thus, appears to be a significant factor for the expression of LTP., (Copyright 1999 Academic Press.)

The present study investigated the ion channel responsible Ca2+ influx in cultured smooth muscle cells from bovine brain arteries by monitoring Ba2+ currents. Voltage pulses at a range between -100 and +100 mV from a holding potential of 0 mV induced currents and the current/voltage (I/V) relations were linear with a reversal potential of +/- 0 mV. The currents were increased by elevating extracellular Ba2+ concentrations, suggesting that the voltage-sensitive non-selective cation channel, which favors Ca2+ influx, is expressed in brain vascular smooth muscle cells. In contrast, when voltage pulses at a range between -50 to +50 mV from a holding potential of -80 mV were applied to carotid smooth muscle cells, inward currents were evoked by depolarization to > or = -10 mV and the I/V relations were bell-shaped, typical for the L-type calcium channels. The dihydropyridine derivatives, efonidipine and nicardipine, inhibited the L-type Ca2+ channel-operated currents in carotid smooth muscles, and further efonidipine had an inhibitory effect also on non-selective cation currents in brain vascular smooth muscle cells. These results suggest that the voltage-dependent non-selective cation channel expressed in brain vascular smooth muscle cells is sensitive to a kind of the dihydropyridine derivatives and regulates Ca2+ influx.

A serum factor is recognized to interact with a protein kinase C (PKC) pathway. Indeed, treatment with fetal bovine serum enhanced ACh-evoked currents by PKC activation in the neuronal nicotinic ACh receptors (alpha7) and Torpedo ACh receptors expressed in Xenopus oocytes. In addition, potentiation of ACh-evoked currents induced by fetal bovine serum was observed also in the mutant Torpedo ACh receptors lacking potent PKC phosphorylation sites at Ser333 on the alpha subunit and Ser377 on the delta subunit; the potentiation was inhibited by the PKC inhibitor, PKC inhibitor peptide (PKCI), indicating that ACh receptor currents were enhanced by PKC activation but not by PKC phosphorylation of the receptors. On the other hand, fetal bovine serum enhanced kainate-evoked currents in oocytes expressing the alpha-amino3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, GluR1,3. The enhancement was not affected by the PKC inhibitors, PKCI or GF109203X, and instead, was inhibited by the Ca2+/calmodulin-dependent kinase II (CaMKII) inhibitor, KN-62. These results suggest that serum is not only involved in PKC activation but in CaMKII activation, and that thereby ACh receptor currents and AMPA receptor currents are each potentiated., (Copyright 1997 Academic Press.)

NT2 cells, a human teratocarcinoma cell line, are shown to be differentiated in neuron-like cells (NT2-N cells) by treatment with retinoic acid. The present study identified the neurotransmitter receptors expressed in NT2-N cells using patch-clamp recording. Voltage-sensitive Na+ currents, which are specific for neurons, were observed in NT2-N cells but not in NT2 cells, suggesting that NT2-N cells actually function as neurons. Glutamate receptor agonists, N-methyl-D-aspartate (NMDA) and kainate, evoked whole-cell currents. In addition, gamma-aminobutyric acid (GABA) evoked currents and the currents were inhibited by the selective GABAA receptor antagonist, bicuculline. In outside-out patches, GABA elicited single channel currents with two classes of the slope conductance (26 and 50 pS). No current, however, was induced by ACh, serotonin, or dopamine NT2-N cells, thus, express at least two types of the major excitatory and inhibitory neurotransmitter receptor in the central nervous system, the glutamate and GAGAA receptors, suggesting that these receptors have a crucial role in neurotransmission from the earlier stage of the brain development.

The effects of arachidonic acid on ACh-gated channel currents were examined using Torpedo nicotinic ACh receptors expressed in Xenopus oocytes. Arachidonic acid decreased ACh-evoked currents during treatment, to a greater extent in Ca(2+)-free extracellular solution. The currents were enhanced for more than 30 min after washing, reaching 150 and 170% in Ca(2+)-containing and -free extracellular solutions, respectively. The current enhancement was inhibited by the selective protein kinase C (PKC) inhibitor, GF109203X, whereas the current depression was not affected. Furthermore, arachidonic acid-evoked current depression was blocked in mutant ACh receptors with PKC phosphorylation site deletions on the alpha and delta subunits, but the long-lasting potentiation effect remained. These results indicate that arachidonic acid may decrease ACh receptor currents by a direct binding to PKC phosphorylation sites of the ACh receptors and may potentiate the currents via a novel pathway related to arachidonic acid-regulated PKC activation, but not via PKC phosphorylation of the ACh receptor itself.

Fetal bovine serum evoked Ca(2+)-dependent chloride currents with two components in Xenopus oocytes. The evoked currents were inhibited by GDP beta S, but not by pertussis toxin (PTX). An inositol 1,4,5-triphosphate (IP3) receptor antagonist, heparin completely inhibited the currents, although a phospholipase C inhibitor, neomycin had no effect. The serum-activated currents were enhanced to 171% by a selective protein kinase C (PKC) inhibitor, GF109203X. By contrast, a potent PKC activator, TPA, abolished the initial component of the currents and arachidonic acid enhanced this effect. The effects of TPA and/or arachidonic acid on the currents inhibited by GF109203X. These results indicate that the receptor for serum is linked to a PTX-insensitive G-protein involving cytosolic Ca2+ release through IP3 and PKC activation by a mechanism independent of a phospholipase C-mediated phospholipid signaling. Furthermore, the evoked currents are regulated by PKC and arachidonic acid appears to potentiate its effect.

A potent P2Y purinoceptor agonist, 2-methylthio ATP (2-MeSATP), produced whole-cell potassium currents through a purinoceptor linked to a pertussis toxin (PTX)-insensitive G-protein in rat hippocampal neurons. The currents were not affected by a selective protein kinase C or A inhibitor. Single channel recordings demonstrated that the potassium channel is activated without latency even in outside-out patches. These suggest that the channel may be regulated directly by the beta gamma subunits of a G-protein. In addition, 2-MeSATP enhanced intracellular free Ca2+ concentration ([Ca2+]i) with a very rapid initiation time. The [Ca2+]i increase was inhibited by a broad G-protein inhibitor, but not by a phospholipase C (PLC) inhibitor or an IP3 receptor antagonist. These indicate that this Ca2+ mobilization may be regulated by a mechanism independent of a PLC-mediated phosphatidylinositol signaling pathway.

P2 purinoceptor agonists produced whole-cell potassium currents in cerebellar neurons with the order of potency 2-methylthio ATP (2-MeSATP) > ADP > ATP > adenosine > alpha,beta- methylene ATP > AMP > UTP. In the outside-out patch clamp configuration, 2-MeSATP evoked single channel currents with two major classes of slope conductances without latency. The currents were blocked by a G-protein inhibitor, GDP beta S, although they were not affected by a phospholipase C inhibitor, a selective protein kinase C or A inhibitor. In contrast, a potent G-protein activator, GTP gamma S, produced single channel currents with same conductances as those of the currents induced by 2-MeSATP. These provide an indication that the P2 purinoceptor-operated potassium channel is regulated by the beta gamma subunits of a G-protein.

In whole-cell patches of inferior colliculus neurons, ADP evoked outwardly rectifying potassium currents with a latency of < 1 sec via P2Y purinoceptor. These currents were blocked by GDP beta S, while not by pertussis toxin (PTX). Additionally, a selective protein kinase C inhibitor, GF109203X, or a selective phospholipase A2 inhibitor, BPB, had no effect on the currents. In outside-out patches, ADP elicited single channel currents with same slope conductances as those obtained in cell-attached patches and the currents were again blocked by GDP beta S. The results presented here indicate that the P2Y purinoceptor-operated potassium channel in inferior colliculus neurons is activated only by a plasma membrane component, most likely by a direct coupling to the beta gamma subunits of a PTX-insensitive G-protein.