In this study, we examine whether an anti-inflammatory thiourea derivative, compound #326, actions on ion channels. The effects of compound #326 on Ca -activated K channels were evaluated by patch-clamp recordings obtained in cell-attached, inside-out or whole-cell configuration. In pituitary GH cells, compound #326 increased the amplitude of Ca -activated K currents (I ) with an EC value of 11.6 μM, which was reversed by verruculogen, but not tolbutamide or TRAM-34. Under inside-out configuration, a bath application of compound #326 raised the probability of large-conductance Ca -activated K (BK ) channels. The activation curve of BK channels was shifted to less depolarised potential with no modification of the gating charge of the curve; consequently, the difference of free energy was reduced in the presence of this compound. Compound #326-stimulated activity of BK channels is explained by a shortening of mean closed time, despite its inability to alter single-channel conductance. Neither delayed-rectifier nor erg-mediated K currents was modified. Compound #326 decreased the peak amplitude of voltage-gated Na current with no clear change in the overall current-voltage relationship of this current. In HEK293T cells expressing α-hSlo, compound #326 enhanced BK channels effectively. Intriguingly, the inhibitory actions of compound #326 on interleukin 1β in lipopolysaccharide-activated microglia were significantly reversed by verruculogen, whereas BK channel inhibitors suppressed the expressions of inducible nitric oxide synthase. The BK channels could be an important target for compound #326 if similar in vivo results occur, and the multi-functionality of BK channels in modulating microglial immunity merit further investigation.

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Pioglitazone (PIO), a thiazolidinedone, was reported to stimulate peroxisome proliferator-activated receptor-γ (PPAR-γ) with anti-inflammatory, anti-proliferative, anti-diabetic, and antidepressive activities. However, whether this compound exerts any perturbations on Ca2+-activated K+ and M-type K+ currents in central neurons remains largely unresolved. In this study, we investigated the effects of PIO on these potassium currents in hippocampal neurons (mHippoE-14). In whole-cell current recordings, the presence of PIO (10 μM) increased the amplitude of Ca2+-activated K+ current [IK(Ca)] in mHippoE-14 cells. PIO-induced stimulation of IK(Ca) observed in these cells was reversed by subsequent addition of paxilline, yet not by TRAM-39 or apamin. In inside-out current recordings, PIO applied to the bath concentration-dependently increased the activity of large-conductance Ca2+-activated K+ (BKCa) channels with an EC50 value of 7.6 μM. Its activation of BKCa channels in mHippoE-14 cells was voltage-dependent and accompanied by both a lengthening in mean open time and a shortening in slow component of mean closed time. The activation curve of BKCa channels after addition of PIO was shifted to less depolarized potential without any change in the gating charge. PIO also suppressed the amplitude of M-type K+ currents inherently in mHippoE-14 neurons. Taken together, in addition to its agonistic action on PPAR-γ, PIO-induced perturbation of these potassium channels may be responsible for its widely pharmacological actions on hippocampal neurons.

OD-1, a scorpion toxin, has been previously recognized as an activator of voltage-gated Na+ currents. To what extent this agent can alter hippocampal neuronal Na+ currents and network excitability and how it can be applied to neuronal hyperexcitability research remains unclear. With the aid of patch-clamp technology, it was revealed that, in mHippoE-14 hippocampal neurons, OD-1 produced a concentration-, time-, and state-dependent rise in the peak amplitude of INa. It shifted the INa inactivation curve to a less negative potential and increased the frequency of spontaneous action currents. Further characterization of neuronal excitability revealed higher excitability in the hippocampal slices treated with OD-1 as compared with the control slices. A stereotaxic intrahippocampal injection of OD-1 generated a significantly higher frequency of spontaneous seizures and epileptiform discharges compared with intraperitoneal injection of lithium-pilocarpine- or kainic acid-induced epilepsy, with comparable pathological changes. Carbamazepine significantly attenuated OD-1 induced seizures and epileptiform discharges. The OD-1-mediated modifications of INa altered the electrical activity of neurons in vivo and OD-1 could potentially serve as a novel seizure and excitotoxicity model.

Perampanel (PER) is a selective blocker of AMPA receptors showing efficacy in treating various epileptic disorders including brain tumor-related epilepsy and also potential in treating motor neuron disease. However, besides its inhibition of AMPA-induced currents, whether PER has any other direct ionic effects in different types of neurons remains largely unknown. We investigated the effects of PER and related compounds on ionic currents in different types of cells, including hippocampal mHippoE-14 neurons, motor neuron-like NSC-34 cells and U87 glioma cells. We found that PER differentially and effectively suppressed the amplitude of voltage-gated Na+ currents (INa) in mHippoE-14 cells. The IC50 values required to inhibit peak and late INa were 4.12 and 0.78 μM, respectively. PER attenuated tefluthrin-induced increases in both amplitude and deactivating time constant of INa. Importantly, PER also inhibited the amplitude of M-type K+ currents (IK(M)) with an IC50 value of 0.92 μM. The suppression of IK(M) was attenuated by the addition of flupirtine or ZnCl2 but not by L-quisqualic acid or sorafenib. Meanwhile, in cell-attached configuration, PER (3 μM) decreased the activity of M-type K+ channels with no change in single-channel conductance but shifting the activation curve along the voltage axis in a rightward direction. Supportively, PER suppressed IK(M) in NSC-34 cells and INa in U87 glioma cells. The inhibitory effects of PER on both INa and IK(M), independent of its antagonistic effect on AMPA receptors, may be responsible for its wide-spectrum of effects observed in neurological clinical practice.

Summary:Purpose: Perinatal hypoxia is an important cause of brain injury in the newborn and has consequences that are potentially devastating and life-long, such as an increased risk of epilepsy in later life. The postsynaptic density (PSD) is a cytoskeletal specialization involved in the anchoring of neurotransmitter receptors and in regulating the response of postsynaptic neurons to synaptic stimulation. The postsynaptic protein PSD-95 binds to the N-methyl-D-aspartate receptor (NMDAR) subunit, and hence activates cascades of NMDAR-mediated events, such as cyclic adenosine monophosphate (cAMP)-responsive element binding protein phosphorylation at serine-133 (pCREB ). Here we studied the effect of perinatal hypoxia on protein interactions involving PSD-95 and the NM-DAR, as well as pCREB Ser−133 expression at an age when the animals show increased seizure susceptibility.Methods: Rats were assigned randomly to the control rats or the rats exposed to transient global hypoxia at postnatal day 10 (P10). At P45, some rats from both groups were treated with pentylenetetrazol (PTZ) intraperitoneally to test the seizure threshold, and others were studied for neuronal loss, pCREB Serine−133 , PSD-95, and NMDAR expressions in the midbrain, temporal cortex, and hippocampal CA1 subfield by using immunohistochemistry, co-immunoprecipitation, and immunoblotting techniques, respectively. Results:The rats with prior exposure to perinatal hypoxia exhibited increased seizure susceptibility to PTZ, compared with the control rats. Associated with this long-term change in seizure susceptibility, selective neuronal loss was observed in the midbrain region while pCREB Ser−133 expression was reduced in the midbrain, temporal cortex, and hippocampal CA1 subfield. Perinatal hypoxia led to a decrease in PSD-95 expression in the both midbrain and hippocampal CA1 subfield, with the exception of temporal cortex. Furthermore, the association between PSD-95 and NMDAR subunits (NR1, NR2A, and NR2B) in the hippocampal CA1 was also markedly altered by perinatal hypoxia.Conclusions: This study demonstrates that the decrease in several protein complexes that are essential components of the postsynaptic apparatus is associated with the observed increase in seizure susceptibility in adult rats with prior exposure to perinatal hypoxia. The results indicate that reductions in PSD-95 expression, PSD-95 binding of NMDAR subunits, and subsequent NMDAR-mediated CREB phosphorylation, particularly in hippocampal CA1, are long-term consequences of perinatal hypoxia and may, at least in part, contribute to perinatal hypoxia-induced reduction in seizure threshold. Key Words: CREB-Longterm encephalopathy-NMDA receptor-PentylenetetrazolPerinatal hypoxia-PSD-95.Hypoxic encephalopathy is the most common cause of neonatal seizures and is associated with an increased risk of epilepsy in the later life (1). Neonatal rats exposed to transient global hypoxia on postnatal days 10-12 (P10-12) exhibited spontaneous electrographic and behavioral seizures that resulted in ...

How sodium metabisulfite (SMB; NaSO), a popular food preservative and antioxidant, interacts with excitable membrane and induces excitotoxicity is incompletely understood. In this study, the patch-clamp technique was used to investigate and record the electrophysiological effect of SMB on electrically excitable HL-1 cardiomyocytes and NSC-34 neurons, as well as its relationship to pilocarpine-induced seizures and neuronal excitotoxicity in rats. We used Western blotting, to analyze sodium channel expression on hippocampi after chronic SMB treatment. It was found that voltage-gated Na current (I ) was stimulated, and current inactivation and deactivation were slowed in SMB-treated (30 μM) HL-1 cardiomyocytes. SMB-induced increases of I were attenuated in cells treated with ranolazine (10 μM) or eugenol (30 μM). The current-voltage relationship of I shifted to slightly more negative potentials in SMB-treated cells, the peak I with an EC value of 18 μM increased, and the steady-state inactivation curve of I shifted to a more positive potential. However, the tail component of the rapidly activating delayed-rectifier K current (I ) was dose-dependently inhibited. Cell-attached voltage-clamp recordings in SMB-treated cells showed that the frequency of action currents and prolonged action potential were higher. In SMB-treated NSC-34 neurons, the peak I was higher; however, neither the time to peak nor the inactivation time constant (I ) changed. Pilocarpine-induced seizures were exacerbated, and acute neuronal damage and chronic mossy fiber sprouting increased in SMB-treated rats. Western blotting showed higher expression of the sodium channel in cells after chronic SMB treatment. We conclude that SMB contributes to the sodium channel-activating mechanism through which it alters cellular excitability and excitotoxicity in wide-spectrum excitable cells.

Telmisartan (TEL), a non-peptide blocker of the angiotensin II type 1 receptor, is a widely used antihypertensive agent. Nevertheless, its neuronal ionic effects and how they potentially affect neuronal network excitability remain largely unclear. With the aid of patch-clamp technology, the effects of TEL on membrane ion currents present in hippocampal neurons (mHippoE-14 cells) were investigated. For additional characterization of the effects of TEL on hippocampal neuronal excitability, we undertook in vivo studies on Sprague Dawley (SD) rats using pilocarpine-induced seizure modeling, a hippocampal histopathological analysis, and inhibitory avoidance testing. In these hippocampal neurons, TEL increased the peak amplitude of I Na , with a concomitant decline in the current inactivation rate. The TEL concentration dependently enhanced the peak amplitude of depolarization-elicited I Na and lessened the inactivation rate of I Na. By comparison, TEL was more efficacious in stimulating the peak I Na and in prolonging the inactivation time course of this current than tefluthrin or (-)-epicatechin-3-gallate. In the continued presence of pioglitazone, the TEL-perturbed stimulation of I Na remained effective. In addition, cell exposure to TEL shifted the steady-state inactivation I Na curve to fewer negative potentials with no perturbations of the slope factor. Unlike chlorotoxin, either ranolazine, eugenol, or KMUP-1 reversed TEL-mediated increases in the strength of non-inactivating I Na. In the cell-attached voltage-clamp recordings, TEL shortened the latency in the generation of action currents. Meanwhile, TEL increased the peak I Na , with a concurrent decrease in current inactivation in HEKT293T cells expressing SCN5A. Furthermore, although TEL did not aggravate pilocarpine-induced chronic seizures and tended to preserve cognitive performance, it significantly accentuated hippocampal mossy fiber sprouting. Collectively, TEL stimulation of peak I Na in combination with an apparent retardation in current inactivation could be an important mechanism through which hippocampal neuronal excitability is increased, and hippocampal network excitability is accentuated following status epilepticus, suggesting further attention to this finding.