Youji Ikeuchi

ATP-evoked potassium currents in rat striatal neurons are mediated by a P2 purinergic receptor.

The effect of ATP on cultured striatal neurons was examined by whole cell voltage clamp recordings. ATP produced outwardly rectifying currents that reversed near the expected equilibrium potential for the potassium ion and the currents were blocked by intracellular Cs+. Purinergic receptor agonists such as ADP, AMP adenosine, and 2-methylthio ATP (2-MeSATP) also evoked similar outward currents. The order of their potencies was ATP >> 2-MeSATP > or = ADP > adenosine > AMP, corresponding to a P2 purinergic receptor. ATP-evoked currents were blocked by a specific protein kinase C (PKC) inhibitor, GF109203X. In addition, the intracellular perfusion of a G-protein inactivator, GDP beta S abolished ATP-induced currents, whereas pertussis toxin (PTX) had no effect on the currents. These results suggest that ATP activates a potassium channel in striatal neurons, which is regulated by protein kinase C (PKC) activation through a P2 purinergic receptor linked to PTX-insensitive G protein.

Activation of endogenous protein kinase C enhances currents through α1 and α2 glycine receptor channels

Abstract The effects of Ca2+/phospholipid dependent (PKC) phosphorylation on the current amplitudes of α1 and α2 glycine receptors expressed in Xenopus oocytes were examined by whole cell voltage clamp recording. In studies using phorbol esters, PKC phosphorylation has been shown to reduce glycine-induced currents. Endogenous PKC activation by pretreatment with serum, however, enhanced the currents to around 140% in both α1 and α2 glycine receptors. This effect was completely blocked by a specific PKC inhibitor, GF109203X. Instead, treatment with a potent PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA) revealed a decrease in glycine-gated channel currents. Thus, the present results demonstrate that glycine receptor phosphorylation mediated by endogenous pathway of PKC activation potentiates glycine-induced currents and phorbol esters may have a direct action on glycine receptor channels independent of PKC activation.

Short-term depression and long-term enhancement of ACh-gated channel currents induced by linoleic and linolenic acid.

The effects of cis-unsaturated free fatty acids such as linoleic and linolenic acid on ACh-evoked currents were examined using normal and mutant nicotinic acetylcholine (ACh) receptors lacking protein kinase C (PKC) phosphorylation sites on the alpha and delta subunits expressed in Xenopus oocytes. These free fatty acids reduced ACh-gated channel currents during treatment and to a greater extent in Ca2+-free extracellular solution. After treatment, the currents were enhanced as the drug was washed out, but this effect was not observed in the absence of extracellular Ca2+. Linolenic acid was more potent of the current enhancement (300% of the control) than linoleic acid (190% of the control). The current enhancement induced by these free fatty acids was inhibited by the selective PKC inhibitor, GF109203X, while the current depression was not affected. Furthermore, these lipids decreased ACh-evoked currents in mutant ACh receptors to the same extent as in normal ACh receptors, but never enhanced the currents. These results indicate that linoleic and linolenic acid have biphasic actions on ACh receptor currents; a short-term depression and a long-term enhancement. The short-term depression may be due to an interaction with the ACh receptor channels, presumably at Ca2+ binding sites. The long-lasting enhancement appears to result from Ca2+-dependent PKC activation followed by PKC phosphorylation of the ACh receptors.

Differential changes in glutamatergic transmission via N -methyl- D -aspartate receptors in the hippocampus and striatum of rats behaviourally sensitized to methamphetamine

We searched for changes in glutamatergic transmission via N-methyl-D-aspartate (NMDA) receptors in the hippocampus and striatum of rats behaviourally sensitized to methamphetamine (Meth). Prior to being given a challenge dose of Meth (2 mg/kg, s.c.), the rats were given Meth (4 mg/kg, s.c.) five times a week for 3 wk. Seven days after the challenge test, we examined glutamate (Glu) release from hippocampal and striatal slices evoked by 30 mm KCl, and NMDA-evoked dopamine (DA) release from striatal slices. We further immunoquantified NMDAR1, R2A and R2B receptors as well as the fodrin alpha-subunit, a 240 kDa cytoskeletal protein that is cleaved to form 150 kDa limited proteolytic fragments by NMDA receptor stimulation. In the study of KCl-evoked Glu release, Glu release from the hippocampus was 31% lower in the Meth-sensitized rats than in the control rats, while Glu release from the striatum was 34% higher in the Meth-sensitized rats. NMDAR1, R2A and R2B immunoreactivities in the striatum were significantly lower in the Meth-sensitized rats (by 12, 13 and 12%, respectively) than those in the control rats. However, no differences in the immunoreactivities were found for the hippocampus. Immunoquantification of the fodrin alpha-subunit in the hippocampus revealed that 150 kDa fragments were significantly lower (by 10%) in the Meth-sensitized rats than in the control rats. In contrast to the control rats, NMDA-evoked DA release from the striatum was diminished in the Meth-sensitized rats. These results indicate that the activity of the Glu system is functionally decreased in the hippocampus of Meth-sensitized rats, whereas the Glu system in the striatum of Meth- sensitized rats shows adaptive and functional changes in the receptors in response to the increased Glu release.

ATP-regulated K+ channel and cytosolic Ca2+ mobilization in cultured rat spinal neurons

ATP activated the K+ channel responsible for outwardly rectifying currents via a P2Y purinoceptor linked to a pertussis toxin-insensitive G-protein in cultured rat spinal neurons. The evoked currents were inhibited by a selective protein kinase C inhibitor, GF109203X, whereas a phospholipase C inhibitor, neomycin had no effect. These indicate that the currents are regulated by phospholipase C-independent protein kinase C activation. In addition, ATP enhanced intracellular free Ca2+ concentration. The increase in intracellular free Ca2+ concentration was inhibited by a broad G-protein inhibitor, GDP beta S, but not affected by neomycin or an inositol 1,4,5-triphosphate receptor antagonist, heparin, suggesting that the cytosolic Ca2+ mobilization is regulated by a mechanism independent of a phospholipase C-mediated phosphatidylinositol signaling. These results, thus, demonstrate that ATP has dual actions on the coupled K+ channel and cytosolic Ca2+ release.

Long-lasting enhancement of ACh receptor currents by lysophospholipids.

Lysophosphatidylcholine (LysoPtdCho) and lysophosphatidylethanolamine (LysoPtdEtn), which are formed by phospholipase A2-catalyzed hydrolysis of phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn), respectively, are proposed to be involved in protein kinase C (PKC) activation. Their physiological significance, however, remains unclear. We examined the effects of lysoPtdCho and lysoPtdEtn on acetylcholine (ACh) receptor currents using oocytes expressing Torpedo nicotinic ACh receptors. LysoPtdCho enhanced the currents in a washing time- and dose-dependent manner (10 nM-1 microM), reaching a maximum of 191% at 20 min after treatment. The currents were enhanced to a lesser extent at higher concentrations, and instead, inhibited to 81% at 10 microM. Likewise, lysoPtdEtn also potentiated the currents to 200% at 10 microM, although its dose-dependent curve shifted to right as compared with that of lysoPtdCho. The current potentiation was blocked by a PKC inhibitor, PKC inhibitor peptide (PKCI), or removal of extracellular Ca2+. In addition, lysoPtdCho and lysoPtdEtn enhanced the currents in mutant ACh receptors lacking PKC phosphorylation sites on the alpha and delta subunits. These results suggest that lysophospholipids such as lysoPtdCho and lysoPtdEtn potentiated ACh receptor currents by Ca2+-dependent PKC activation, but that this effect did not require PKC phosphorylation of the ACh receptor.

Repetitive applications of ATP potentiate potassium current by Ca2+/calmodulin kinase in cultured rat hippocampal neurons.

ATP evoked whole-cell potassium currents in hippocampal neurons. The second application of ATP to the same cell potentiated the current amplitude to around 140% and the current potentiation was maintained by further applications. A calmodulin inhibitor, W-7, or a selective Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor, KN-62, inhibited the current potentiation, although a selective protein kinase C inhibitor, GF109203X, or a selective cAMP-dependent protein kinase inhibitor, H-89, had no effect. In addition, ATP enhanced intracellular free Ca2+ concentration, which may activate CaMKII, but this enhancement was blocked by repetitive applications. These results provide an indication that CaMKII may be involved in the current potentiation by repetitive applications of ATP.

Adenosine evokes potassium currents by protein kinase C activated via a novel signaling pathway in superior colliculus neurons

Adenosine evoked whole‐cell potassium currents and enhanced intracellular free Ca2+ concentration ([Ca2+]i) in superior colliculus neurons through a P2Y purinoceptor linked to a pertussis toxin‐insensitive G‐protein, possibly Gq‐protein, which is involved in a protein kinase C(PKC) activation pathway. The [Ca2+]i increase was inhibited by a phospholipase C(PLC) inhibitor, whereas the evoked currents were not affected by a PLC inhibitor or a phospholipase A2(PLA2) inhibitor. Adenosine elicited single channel currents via PKC activation in cell‐attached patches and furthermore, those currents with conductances of the same slope were induced even in excised patches, suggesting that PKC can be activated only by cell membrane factors without intracellular components. These results thus indicate that the P2Y purinoceptor‐coupled potassium channel is regulated via a novel PKC activation pathway independent of PLC or PLA2.

Adenosine activates the potassium channel via a P2 purinoceptor but not via an adenosine receptor in cultured rat superior colliculus neurons

The effect of adenosine on superior colliculus neurons was examined by whole-cell patch clamp recording. Adenosine elicited whole-cell potassium currents. A selective A1 or A2a adenosine receptor agonist induced no current and furthermore, adenosine-evoked currents were not inhibited by selective A1 or A2a adenosine receptor antagonists or a non-selective adenosine receptor (P1 purinoceptor) antagonist, indicating that the currents are not mediated by adenosine receptors. In contrast, P2 purinoceptor agonists, such as 2-methylthio ATP (2-MeSATP), ATP, ADP, and AMP, produced similar potassium currents, whereas alpha,beta-methylene ATP, beta,gamma-metylene ATP, or UTP had no response. The order of their potencies for the current amplitudes was 2-MeSATP > ADP > adenosine > ATP >> AMP and this order corresponds to that for the P2Y purinoceptor. These results, thus, suggest that adenosine exerts an inhibitory effect at the postsynaptic site by activating the potassium channel via a P2Y purinoceptor in superior colliculus neurons.

Excitatory and inhibitory effects of toluene on neural activity in guinea pig hippocampal slices

To investigate the effect of toluene and its derivatives on neural activity, postsynaptic field potential (population spike, PS) of granule cells as well as antidromic potential (AP) and presynaptic fiber potential (FP) (perforant path) were recorded in the guinea pig hippocampal slices. Toluene at the concentration of 0.2 ng/ml to 20 micrograms/ml in the perfusion medium increased the amplitude of PS to 109-150%. Toluene also increased the amplitude of FP and AP, although the most remarkable enhancement was observed in the PS. However, toluene at the concentrations over 1000 micrograms/ml completely depressed the PS, whereas it increased the amplitude of AP to 130% of the original level. These results indicate that toluene has excitatory and inhibitory biphasic effects on neurotransmission in the hippocampal slices according to concentration applied.