Effects of ketamine and its metabolites on ion currents in differentiated hippocampal H19-7 neuronal cells and in HEK293T cells transfected with α-hslo subunit.

Ketamine (KT), a dissociative anesthetic, is known to induce schizophrenia-like psychosis. The percentage of KT abuse has recently grown fast despite KT being a controlled drug. The mechanism of KT actions is related to the inhibition of NMDA receptors. Whether KT produces other effects on ion currents in hippocampal neurons remains unclear. In this study, we attempted to evaluate the possible effects of KT and other related compounds on ion currents in hippocampal neuron-derived H19-7 cells. This drug exerted an inhibitory effect on Ca(2+)-activated K(+) current (IK(Ca)) in these cells with an IC(50) value of 274 µM. Pimaric acid (30 µM) or abietic acid (30 µM), known to stimulate large-conductance Ca(2+)-activated K(+) channels, reversed KT-induced inhibition of I(K)(Ca). In HEK293T cells expressing a-humans low poke, KT-induced inhibition of I(K)(Ca) still existed. Dehydronorketamine (300 µM) had little or no effect on the IK(Ca) amplitude, while norketamine (300 µM) slightly but significantly suppressed it. In inside­out configuration, KT applied to the intracellular face of the membrane did not alter single channel conductance of large-conductance Ca(2+)-activated K(+) (BKCa) channels; however, it did significantly reduce the probability of channel openings. Addition of KT was effective in depressing the peak amplitude of voltage-gated Na(+) current. Moreover, the presence of KT was noted to enhance the amplitude of membrane electroporation-induced inward currents (IMEP) in differentiated H19-7 cells. KT-stimulated IMEP was reversed by further application of LaCl(3) (100 µM), but not by NMDA (30 µM). The modulations by this compound of ion channels may contribute to the underlying mechanisms through which KT and its metabolites influence the electrical behavior of hippocampal neurons if similar findings occur in vivo.

Contribution of non-inactivating Na+ current induced by oxidizing agents to the firing behavior of neuronal action potentials: experimental and theoretical studies from NG108-15 neuronal cells.

The effects of chemical injury with oxidizing agents on voltage-gated Na+ current (I(Na)) in differentiated NG108-15 neuronal cells were investigated in this study. In whole-cell patch-clamp recordings, the challenge of these cells with t-butyl hydroperoxide (t-BHP; 1 mM) decreased the peak amplitude of I(Na) with no modification in the current-voltage relationship. It caused a slowing of current inactivation, although there was no alteration in the activation time course of I(Na). Cell exposure to t-BHP also increased a non-inactivating I(Na) (I(Na(NI)) elicited by long-lasting ramp pulses. The t-BHP-induced increase of I(Na(NI)) was reversed by a further application of riluzole (10 microM) or oxcarbazepine (10 microM). When I(Na) was elicited by simulated waveforms of action potentials (APs), during exposure to t-BHP, the amplitude of this inward current was diminished, accompanied by a reduction in inactivation/deactivation rate and an increase in current fluctuations. Under current-clamp recordings, addition of t-BHP (0.3 mM) enhanced AP firing in combination with clustering-like activity and sub-threshold membrane oscillations. In the simulation study, when the fraction of non-inactivating Na(v) channels was elevated, the simulated window component of I(Na) in response to a long-lasting ramp pulse was reduced; however, the persistent I(Na) was markedly enhanced. Moreover, when simulated firing of APs was generated from a modeled neuron, changes of AP firing caused by the increased fraction of non-inactivating Na(v) channels used to mimic the t-BHP actions were similar to the experimental observations. Taken together, it is anticipated that the effects of oxidizing agents on I(Na(NI)) could be an important mechanism underlying their neurotoxic actions in neurons or neuroendocrine cells occurring in vivo.

Inhibitory action of methadone and its metabolites on erg-mediated K+ current in GH3 pituitary tumor cells.

Methadone (Mtd) is a widely used opioid drug associated with the side effect of hyperprolactinemia. The mechanism of how Mtd induces prolactin secretion remains unclear. The effects of Mtd and its two main metabolites (EDDP: (±)-2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolinium percholarate and EMDP: 2-ethyl-5-methyl-3,3-dipnehyl-1-pyrroline) on ion currents were investigated in GH3 pituitary tumor cells. Hyperpolarization-elicited K+ currents in GH3 cells bathed in a high-K(+), Ca(2+)-free solution were studied to evaluate the effects of Mtd and other related compounds on the ether-à-go-go-related-gene (erg) K(+) current (I(K(erg))). Mtd suppressed the amplitude of I(K(erg)) in a concentration-dependent manner with an IC(50) value of 10.4 µM. With the aid of a minimal binding scheme, the inhibitory action of Mtd on I(K(erg)) was estimated with a dissociation constant of 8.2 µM. Mtd tended to increase the rate of I(K(erg)) deactivation in a voltage-dependent fashion. EDDP (10 µM) had no effect on I(K(erg)), while EMDP (10µM) slightly suppressed it. In GH3 cells incubated with naloxone (30 µM), the Mtd-induced inhibition of I(K(erg)) remained unaltered. Under cell-attached voltage-clamp recordings, Mtd increased the frequency of spontaneous action currents with no change in current amplitude. Similarly, Mtd can suppress I(K(erg)) in differentiated NG108-15 cells; dynorphin A(1-13) did not reverse Mtd-induced inhibition of I(K(erg)). This study shows that Mtd has a depressant effect on I(K(erg)), and suggests its ability to affect membrane excitability and prolactin secretion. The cyclization of Mtd, in which EDDP and EMDP are formed, tends to be critical in removal of the Mtd binding to erg K+ channel.

Thymol-evoked Ca+ mobilization and ion currents in pituitary GH3 cells.

In this study, an attempt was made to elucidate the effects of thymol, a monocyclic phenolic compound, on Ca2+ mobilization and ion currents in pituitary GH3 cells with the aid of fura-2 fluorimetry and the whole-cell voltage-clamp technique. Thymol increased intracellular Ca2+ concentrations ([Ca2+]i) in GH3 cells loaded with Ca2+-sensitive dye fura-2. Removing extracellular Ca2+ reduced the thymol-induced [Ca2+]i rise. In Ca2+ -free solution, thymol-evoked [Ca2+]i rise was unchanged by depleting the Ca2+ store with thapsigargin (1 microM), while the thapsigargin-induced [Ca2+]i rise was reduced by pretreatment with thymol. These results imply that the Ca2+ stores depleted by thymol comprise thapsigargin-sensitive and thapsigargin-insensitive pools. In addition, after depletion of the internal Ca2+ store with 100 microM thymol in Ca2+ -free solution, a subsequent application of Ca2+ greatly induced a [Ca2+]i increase. The results indicate that, similar to thapsigargin, 100 microM thymol may activate the capacitative calcium entry (CCE) channel. However, thymol (100 microM) had a slight depressant action in L-type calcium current (I(CaL)). The stimulatory actions of thymol on Ca2+ signaling may partly be responsible for the underlying cellular mechanisms through which it affects neuroendocrine functions.

Inhibition of intermediate-conductance Ca2+-activated K+ channel and cytoprotective properties of 4-piperidinomethyl-2-isopropyl-5-methylphenol.

The ionic mechanisms and cytoprotective activities of 4-piperidinomethyl-2-isopropyl-5-methylphenol (THPI), an analogue of thymol, were investigated in HL-60 granulocytes and in human erythrocytes, respectively. THPI inhibited K+ outward current (I(K)) in a concentration-dependent manner in HL-60 leukocytes, with an IC50 value of 4 microM. Neither iberiotoxin (200 nM) nor paxilline (1 microM) suppressed the amplitude of I(K), whereas clotrimazole (5 microM) significantly inhibited it. In the inside-out configuration of single channel recordings, application of THPI (5 microM) into the bath medium did not alter the single-channel conductance of intermediate-conductance Ca2+-activated K+ (IK(Ca)) channels (i.e K(Ca)3.1 channels), but it suppressed the channel activity significantly. THPI-induced inhibition of IK(Ca) channels was reversed by a further application of 1-ethyl-2-benzimidazolinone (10 microM). THPI-induced reduction in IK(Ca)-channel activity in these cells was primarily due to a decrease in mean open time. These results provide direct evidence that THPI is capable of suppressing the activity of IK(Ca) channels in HL-60 cells. The antioxidant action of THPI also revealed a beneficial cytoprotective effect against mitomycin C-mediated haemolytic effect in human erythrocytes. The results of this study suggest that blockade of IK(Ca) channels and the membrane-protecting activity of THPI would combine to have beneficial effects in lessening the severity of haemolytic crisis and reducing anaemia in sickle cell disease.

Antioxidant and antiplatelet effects of dang-gui-shao-yao-san on human blood cells.

Dang-Gui-Shao-Yao-San (DGSYS) is a mixture of medicinal herbs, which has long been used in traditional Chinese medicine for treating anemia and ovulary disorders. Its preparation comprises Angelicae sinensis (Oliv.) Diels, Ligustucum chuanxiong Hort, Paeonia lactiflora pall, Poria cocos (Schw.) Wolf, Atractylodis macrocephala Koidz and Alisma orientalis (Sam.) Juzep. The present study examined the anti-superoxide formation, free radical scavenging and anti-lipid peroxidation activities of DGSYS by xanthine oxidase inhibition, cytochrome C system with superoxide anion released by the fMLP or PMA activating pathway in human neutrophils, and FeCl2 ascorbic acid-induced lipid peroxidation effects on lipids in rat liver homogenate, respectively. DGSYS showed anti-superoxide formation and free radical scavenging activity in a concentration-dependent manner. It also inhibited PMA- but not fMLP-induced superoxide anion released from human neutrophils. These antioxidant actions of DGSYS showed beneficial cytoprotective effects against lipid peroxidation in rat liver homogenate, human platelet aggregation induced by arachidonic acid (AA) and adenosine diphosphate (ADP) and mitomycin C-mediated hemolytic in human erythrocytes.

Effects of 4-piperidinomethyl-2-isopropyl-5-methylphenol on oxidative stress and calcium current.

4-Piperidinomethyl-2-isopropyl-5-methylphenol (THPI) was synthesized by reaction of thymol with piperidine and formaldehyde. The biological effect of THPI on superoxide anion scavenging activity, antiplatelet activity and calcium current inhibition were investigated. THPI (50 microM) was shown to be a scavenger of superoxide radicals in human neutrophils stimulated with N-formyl-Met-Leu-Phe (66% inhibition). Since superoxide anions are essential for platelet aggregation and L-type Ca2+-channel activity, we further found that THPI inhibited platelet aggregation induced by arachidonic acid (IC50 46.80+/-6.88 microM). The effect of THPI on Ca2+ current in NG108-15 cells was investigated using the whole-cell voltage-clamp technique. THPI inhibited voltage-dependent L-type Ca2+ current (ICa, L). The IC50 value of THPI-induced inhibition of ICa, L was 3.60+/-0.81 microM. THPI caused no change in the overall shape of the current-voltage relationship of ICa, L. This indicates that THPI is an inhibitor of ICa, L in NG108-15 cells. Therefore, the channel-blocking properties of THPI may contribute to the underlying mechanism by which it affects neuronal or neuroendocrine function. Furthermore, no significant cytotoxic effects of THPI (0.3-50 microM) were observed in NG108-15 cells. The results indicate that THPI is a potential reactive oxygen species scavenger and may prevent platelet aggregation or inhibit L-type Ca2+-channel activity, possibly by scavenging reactive oxygen species.

Stimulatory actions of thymol, a natural product, on Ca(2+)-activated K(+) current in pituitary GH(3) cells.

Drugs that influence the opening of potassium (K(+)) channels and as a consequence cause hyperpolarization of cell membrane possess clinical potential. The large conductance Ca(2+)-activated K(+) (BK) channel is highly selective for K(+). Activation of this channel is Ca(2+)- and voltage-dependent. We have investigated the effects of thymol, a natural product, on ion currents in pituitary GH(3) cells. The patch-clamp technique was used to investigate the effect of thymol (100 microM) in these cells. Thymol reversibly stimulated the Ca(2+)-activated K(+) current with an EC (50) value of 75 microM. In a cell-attached configuration, application of thymol to the bath increased the activity of BK channels. BAPTA (1 mM) attenuated thymol-stimulated channel activity. In an experiment using the inside-out configuration, thymol exposed to the intracellular face of excised patches did not modify the single-channel conductance of these channels whereas it enhanced the channel activity. Neither menthol (100 microM) nor zingerone (100 microM) had an effect on BK-channel activity while AAPH (100 microM) suppressed it significantly. The stimulatory actions of thymol on Ca(2+)-activated K(+) currents may be associated with the underlying cellular mechanisms through which it affects neuronal or neuroendocrine functions.

Differential effects of quercetin, a natural polyphenolic flavonoid, on L-type calcium current in pituitary tumor (GH3) cells and neuronal NG108-15 cells.

The effects of quercetin, a natural polyphenolic compound, on voltage-dependent L-type Ca(2+) current (I(Ca,L)) in rat pituitary GH(3) cells were investigated with the aid of the whole-cell voltage-camp technique. Quercetin (0.5-200 microM) stimulated I(Ca,L) in a concentration-dependent manner. The current-voltage (I-V) relationship of I(Ca,L) was slightly shifted to more negative potentials in the presence of quercetin. The EC(50) value of the quercetin-induced stimulation of I(Ca,L) was about 7 microM. The presence of quercetin (5 microM) shifted the steady state inactivation curve of I(Ca,L) to a more negative potential by approximately -10 mV. Although quercetin might increase intracellular cyclic AMP, sp-cAMPS did not affect I(Ca,L). In addition, neither flavone nor wortmannin had any effect on the amplitude of I(Ca,L), while epicatechin and genistein slightly suppressed it. Quercetin (50 microM) decreased the amplitude of tetrodotoxin-sensitive Na(+) current in GH(3) cells. Under current-clamp configuration, quercetin could increase the firing frequency of actions potentials. Conversely, in NG108-15 neuronal cells, quercetin suppressed the amplitude of I(Ca,L). The quercetin-induced inhibition of I(Ca,L) was abolished in NG108-15 cells preincubated with t-butyl hydroperoxide (1 mM). Quercetin-mediated stimulation of I(Ca,L) in GH(3) cells was presumably not associated with the level of intracellular cyclic AMP, or with the activity of tyrosine or phosphoinositide 3-kinases. Therefore, the effects of quercetin on ion currents may, at least in part, contribute to the underlying mechanisms through which it affects neuronal or neuroendocrine function.

Inhibition of Ca2+-activated and voltage-dependent K+ currents by 2-mercaptophenyl-1,4-naphthoquinone in pituitary GH3 cells: contribution to its antiproliferative effect.

Quinones have been shown to possess antineoplastic activity; however, their effects on ionic currents remain unclear. The effects of 2-mercaptophenyl-1,4-naphthoquinone (2-MPNQ), menadione (MD) and 1,4-naphthoquinone (1,4 NQ) on cell proliferation and ionic currents in pituitary GH3 lactotrophs were investigated in this study. 2-MPNQ was more potent than menadione or 1,4-naphthoquinone in inhibiting the growth of GH3 cells. 2-MPNQ decreased cell proliferation in a concentration-dependent manner with an IC50 value of 3 microM. In whole-cell recording experiments, 2-MPNQ reversibly caused an inhibition of Ca2+-activated K+ current (I(K(Ca)) in a concentration-dependent manner. The IC50 value for 2-MPNQ-induced inhibition of I(K(Ca)) was 7 microM. In the inside-out configuration of single channel recording, 2-MPNQ (30 microM) applied intracellularly suppressed the activity of large-conductance Ca2+-activated K+ (BK(Ca)) channels but did not modify single channel conductance. Menadione (30 microM) had no effect on the channel activity, whereas 1,4-naphthoquinone (30 microM) suppressed it by about 26%. Both 2-MPNQ and thimerosal suppressed the dithiothreitol-stimulated channel activity. 2-MPNQ also blocked voltage-dependent K+ currents, but it produced a slight reduction of L-type Ca2+ inward current. However, unlike E-4031, 2-MPNQ (30 microM) did not suppress inwardly rectifying K+ current present in GH3 cells. Under the current clamp configuration, the presence of 2-MPNQ (30 microM) depolarized the cells, and increased the frequency and duration of spontaneous action potentials. The 2-MPNQ-mediated inhibition of K+ currents would affect hormone secretion and cell excitability. The blockade of these ionic channels by 2-MPNQ may partly explain its inhibitory effect on the proliferation of GH3 cells.