Effects of rosiglitazone, an antidiabetic drug, on Kv3.1 channels

Rosiglitazone is a thiazolidinedione-class antidiabetic drug that reduces blood glucose and glycated hemoglobin levels. We here investigated the interaction of rosiglitazone with Kv3.1 expressed in Chinese hamster ovary cells using the wholecell patch-clamp technique. Rosiglitazone rapidly and reversibly inhibited Kv3.1 currents in a concentration-dependent manner (IC 50 = 29.8 µM) and accelerated the decay of Kv3.1 currents without modifying the activation kinetics. The rosiglitazonemediated inhibition of Kv3.1 channels increased steeply in a sigmoidal pattern over the voltage range of –20 to +30 mV, whereas it was voltage-independent in the voltage range above +30 mV, where the channels were fully activated. The deactivation of Kv3.1 current, measured along with tail currents, was also slowed by the drug. In addition, the steady-state inactivation curve of Kv3.1 by rosiglitazone shifts to a negative potential without significant change in the slope value. All the results with the use dependence of the rosiglitazone-mediated blockade suggest that rosiglitazone acts on Kv3.1 channels as an open channel blocker.

The antidiabetic drug rosiglitazone blocks Kv1.5 potassium channels in an open state

An antidiabetic drug, rosiglitazone is a member of the drug class of thiazolidinedione. Although restrictions on use due to the possibility of heart toxicity have been removed, it is still a drug that is concerned about side effects on the heart. We here examined, using Chinese hamster ovary cells, the action of rosiglitazone on Kv1.5 channels, which is a major determinant of the duration of cardiac action potential. Rosiglitazone rapidly and reversibly inhibited Kv1.5 currents in a concentrationdependent manner (IC 50 = 18.9 µM) and accelerated the decay of Kv1.5 currents without modifying the activation kinetics. In addition, the deactivation of Kv1.5 current, assayed with tail current, was slowed by the drug. All of the results as well as the usedependence of the rosiglitazone-mediated blockade indicate that rosiglitazone acts on Kv1.5 channels as an open channel blocker. This study suggests that the cardiac side effects of rosiglitazone might be mediated in part by suppression of Kv1.5 channels, and therefore, raises a concern of using the drug for diabetic therapeutics.

Antidepressant drug paroxetine blocks the open pore of Kv3.1 potassium channel

In patients with epilepsy, depression is a common comorbidity but difficult to be treated because many antidepressants cause pro-convulsive effects. Thus, it is important to identify the risk of seizures associated with antidepressants. To determine whether paroxetine, a very potent selective serotonin reuptake inhibitor (SSRI), interacts with ion channels that modulate neuronal excitability, we examined the effects of paroxetine on Kv3.1 potassium channels, which contribute to highfrequency firing of interneurons, using the whole-cell patch-clamp technique. Kv3.1 channels were cloned from rat neurons and expressed in Chinese hamster ovary cells. Paroxetine reversibly reduced the amplitude of Kv3.1 current, with an IC₅₀ value of 9.43 µM and a Hill coefficient of 1.43, and also accelerated the decay of Kv3.1 current. The paroxetine-induced inhibition of Kv3.1 channels was voltage-dependent even when the channels were fully open. The binding (k₊₁) and unbinding (k₋₁) rate constants for the paroxetine effect were 4.5 µM⁻¹s⁻¹ and 35.8 s⁻¹, respectively, yielding a calculated K(D) value of 7.9 µM. The analyses of Kv3.1 tail current indicated that paroxetine did not affect ion selectivity and slowed its deactivation time course, resulting in a tail crossover phenomenon. Paroxetine inhibited Kv3.1 channels in a usedependent manner. Taken together, these results suggest that paroxetine blocks the open state of Kv3.1 channels. Given the role of Kv3.1 in fast spiking of interneurons, our data imply that the blockade of Kv3.1 by paroxetine might elevate epileptic activity of neural networks by interfering with repetitive firing of inhibitory neurons.

Blockade of Kv1.5 by paroxetine, an antidepressant drug

Paroxetine, a selective serotonin reuptake inhibitor (SSRI), has been reported to have an effect on several ion channels including human ether-a-go-go-related gene in a SSRI-independent manner. These results suggest that paroxetine may cause side effects on cardiac system. In this study, we investigated the effect of paroxetine on Kv1.5, which is one of cardiac ion channels. The action of paroxetine on the cloned neuronal rat Kv1.5 channels stably expressed in Chinese hamster ovary cells was investigated using the whole-cell patch-clamp technique. Paroxetine reduced Kv1.5 whole-cell currents in a reversible concentration-dependent manner, with an IC50 value and a Hill coefficient of 4.11 microM and 0.98, respectively. Paroxetine accelerated the decay rate of inactivation of Kv1.5 currents without modifying the kinetics of current activation. The inhibition increased steeply between -30 and 0 mV, which corresponded with the voltage range for channel opening. In the voltage range positive to 0 mV, inhibition displayed a weak voltage dependence, consistent with an electrical distance delta of 0.32. The binding (k(+1)) and unbinding (k(-1)) rate constants for paroxetine-induced block of Kv1.5 were 4.9 microM(-1)s(-1) and 16.1 s-1, respectively. The theoretical K(D) value derived by k(-1)/k(+1) yielded 3.3 microM. Paroxetine slowed the deactivation time course, resulting in a tail crossover phenomenon when the tail currents, recorded in the presence and absence of paroxetine, were superimposed. Inhibition of Kv1.5 by paroxetine was use-dependent. The present results suggest that paroxetine acts on Kv1.5 currents as an open-channel blocker.

Blockade of Kv1.5 channels by the antidepressant drug sertraline

Sertraline, a selective serotonin reuptake inhibitor (SSRI), has been reported to lead to cardiac toxicity even at therapeutic doses including sudden cardiac death and ventricular arrhythmia. And in a SSRI-independent manner, sertraline has been known to inhibit various voltage-dependent channels, which play an important role in regulation of cardiovascular system. In the present study, we investigated the action of sertraline on Kv1.5, which is one of cardiac ion channels. The eff ect of sertraline on the cloned neuronal rat Kv1.5 channels stably expressed in Chinese hamster ovary cells was investigated using the whole-cell patch-clamp technique. Sertraline reduced Kv1.5 whole-cell currents in a reversible concentration-dependent manner, with an IC50 value and a Hill coefficient of 0.71 microM and 1.29, respectively. Sertraline accelerated the decay rate of inactivation of Kv1.5 currents without modifying the kinetics of current activation. The inhibition increased steeply between -20 and 0 mV, which corresponded with the voltage range for channel opening. In the voltage range positive to +10 mV, inhibition displayed a weak voltage dependence, consistent with an electrical distance delta of 0.16. Sertraline slowed the deactivation time course, resulting in a tail crossover phenomenon when the tail currents, recorded in the presence and absence of sertraline, were superimposed. Inhibition of Kv1.5 by sertraline was use-dependent. The present results suggest that sertraline acts on Kv1.5 currents as an open-channel blocker.

Constitutive stabilization of hypoxia-inducible factor alpha selectively promotes the self-renewal of mesenchymal progenitors and maintains mesenchymal stromal cells in an undifferentiated state

With the increasing use of culture-expanded mesenchymal stromal cells (MSCs) for cell therapies, factors that regulate the cellular characteristics of MSCs have been of major interest. Oxygen concentration has been shown to influence the functions of MSCs, as well as other normal and malignant stem cells. However, the underlying mechanisms of hypoxic responses and the precise role of hypoxia-inducible factor-1alpha (Hif-1alpha), the master regulatory protein of hypoxia, in MSCs remain unclear, due to the limited span of Hif-1alpha stabilization and the complex network of hypoxic responses. In this study, to further define the significance of Hif-1alpha in MSC function during their self-renewal and terminal differentiation, we established adult bone marrow (BM)-derived MSCs that are able to sustain high level expression of ubiquitin-resistant Hif-1alpha during such long-term biological processes. Using this model, we show that the stabilization of Hif-1alpha proteins exerts a selective influence on colony-forming mesenchymal progenitors promoting their self-renewal and proliferation, without affecting the proliferation of the MSC mass population. Moreover, Hif-1alpha stabilization in MSCs led to the induction of pluripotent genes (oct-4 and klf-4) and the inhibition of their terminal differentiation into osteogenic and adipogenic lineages. These results provide insights into the previously unrecognized roles of Hif-1alpha proteins in maintaining the primitive state of primary MSCs and on the cellular heterogeneities in hypoxic responses among MSC populations.

Inhibitory Actions of HERG Currents by the Immunosuppressant Drug Cyclosporin A

The effect of cyclosporin A (CsA), an immunosuppressant, on human ether-a-go-go-related gene (HERG) channel as it is expressed in human embryonic kidney cells was studied using a whole-cell, patch-clamp technique. CsA inhibited the HERG channel in a concentration-dependent manner, with an IC50 value and a Hill coefficient of 3.17 microM and 0.89, respectively. Pretreatment with cypermethrine, a calcineurin inhibitor, had no effect on the CsA-induced inhibition of the HERG channel. The CsA-induced inhibition of HERG channels was voltage-dependent, with a steep increase over the voltage range of the channel opening. However, the inhibition exhibited voltage independence over the voltage range of fully activated channels. CsA blocked the HERG channels predominantly in the open and inactivated states rather than in the closed state. Results of the present study suggest that CsA acts directly on the HERG channel as an open-channel blocker, and it acts independently of its effect on calcineurin activity.

Octyl Gallate Inhibits ATP-induced Intracellular Calcium Increase in PC12 Cells by Inhibiting Multiple Pathways

Phenolic compounds affect intracellular free Ca2+ concentration ([Ca2+]i) signaling. The study examined whether the simple phenolic compound octyl gallate affects ATP-induced Ca2+ signaling in PC12 cells using fura-2-based digital Ca2+ imaging and whole-cell patch clamping. Treatment with ATP (100 micrometer) for 90 s induced increases in [Ca2+]i in PC12 cells. Pretreatment with octyl gallate (100 nM to 20 micrometer) for 10 min inhibited the ATP-induced [Ca2+]i response in a concentration-dependent manner (IC50=2.84 micrometer). Treatment with octyl gallate (3 micrometer) for 10 min significantly inhibited the ATP-induced response following the removal of extracellular Ca2+ with nominally Ca2+-free HEPES HBSS or depletion of intracellular Ca2+ stores with thapsigargin (1 micrometer). Treatment for 10 min with the L-type Ca2+ channel antagonist nimodipine (1 micrometer) significantly inhibited the ATP-induced [Ca2+]i increase, and treatment with octyl gallate further inhibited the ATP-induced response. Treatment with octyl gallate significantly inhibited the [Ca2+]i increase induced by 50 mM KCl. Pretreatment with protein kinase C inhibitors staurosporin (100 nM) and GF109203X (300 nM), or the tyrosine kinase inhibitor genistein (50 micrometer) did not significantly affect the inhibitory effects of octyl gallate on the ATP-induced response. Treatment with octyl gallate markedly inhibited the ATP-induced currents. Therefore, we conclude that octyl gallate inhibits ATP-induced [Ca2+]i increase in PC12 cells by inhibiting both non-selective P2X receptor-mediated influx of Ca2+ from extracellular space and P2Y receptor-induced release of Ca2+ from intracellular stores in protein kinase-independent manner. In addition, octyl gallate inhibits the ATP-induced Ca2+ responses by inhibiting the secondary activation of voltage-gated Ca2+ channels.

Effects of Apigenin on Glutamate-induced Ca2+i Increases in Cultured Rat Hippocampal Neurons

Flavonoids have been shown to affect calcium signaling in neurons. However, there are no reports on the effect of apigenin on glutamate-induced calcium signaling in neurons. We investigated whether apigenin affects glutamate-induced increase of free intracellular Ca2+concentration ([Ca2+]i) in cultured rat hippocampal neurons, using fura-2-based digital calcium imaging and microfluorimetry. The hippocampal neurons were used between 10 and 13 days in culture from embryonic day 18 rats. Pretreatment of the cells with apigenin (1micrometerto 100micrometer for 5 min inhibited glutamate (100 micrometer 1 min) induced [Ca2+]i increase, concentration-dependently. Pretreatment with apigenin (30micrometer for 5 min significantly decreased the [Ca2+]i responses induced by two ionotropic glutamate receptor agonists, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA, 10 micrometer 1 min) and N-methyl-D-aspartate (NMDA, 100 micrometer 1 min), and significantly inhibited the AMPA-induced peak currents. Treatment with apigenin also significantly inhibited the [Ca2+]i response induced by 50 mM KCl solution, decreased the [Ca2+]i responses induced by the metabotropic glutamate receptor agonist, (S)-3,5-dihydroxyphenylglycine (DHPG, 100micrometer 90 s), and inhibited the caffeine (10 mM, 2 min)-induced [Ca2+]i responses. Furthermore, treatment with apigenin (30micrometer significantly inhibited the amplitude and frequency of 0.1 mM [Mg2+o-induced [Ca2+]i spikes. These data together suggest that apigenin inhibits glutamate-induced calcium signaling in cultured rat hippocampal neurons.

NMDA Receptor-dependent Inhibition of Synaptic Transmission by Acute Ethanol Treatment in Rat Corticostriatal Slices

The effects of ethanol on corticostriatal synaptic transmission were examined, using extracellular recording and analysis of population spike amplitudes in rat brain slices, to study how acute ethanol intoxication impairs striatal function. Ethanol caused a decrease in population spike amplitudes in a dose dependent manner (50~200 mM). Pretreatment with picrotoxin, a gamma-amino butyric acid (GABA)A receptor antagonist, increased the population spikes but ethanol (100 mM) was still effective in decreasing the population spikes under this condition. In the presence of (DL)-2-amino-5-phosphonovaleric acid (APV), N-methyl-D-aspartate (NMDA) receptor antagonist, the inhibitory action of ethanol on population spikes was not shown. These results suggest that ethanol inhibits the glutamatergic corticostriatal synaptic transmission through blockade of NMDA receptors.

Open Channel Block of Kv3.1 Currents by Genistein, a Tyrosine Kinase Inhibitor

The goal of this study was to analyze the effects of genistein, a widely used tyrosine kinase inhibitor, on cloned Shaw-type K+ currents, Kv3.1 which were stably expressed in Chinese hamster ovary (CHO) cells, using the whole-cell configuration of patch-clamp techniques. In whole-cell recordings, genistein at external concentrations from 10 to 100 micrometer accelerated the rate of inactivation of Kv3.1 currents, thereby concentration-dependently reducing the current at the end of depolarizing pulse with an IC50 value of 15.71+/-0.67 micrometer and a Hill coefficient of 3.28+/-0.35 (n=5). The time constant of activation at a 300 ms depolarizing test pulses from -80 mV to +40 mV was 1.01+/-0.04 ms and 0.90+/-0.05 ms (n=9) under control conditions and in the presence of 20 micrometer genistein, respectively, indicating that the activation kinetics was not significantly modified by genistein. Genistein (20 micrometer) slowed the deactivation of the tail current elicited upon repolarization to -40 mV, thus inducing a crossover phenomenon. These results suggest that drug unbinding is required before Kv3.1 channels can close. Genistein-induced block was voltage-dependent, increasing in the voltage range (-20 mV~0 mV) for channel opening, suggesting an open channel interaction. Genistein (20 micrometer) produced use-dependent block of Kv3.1 at a stimulation frequency of 1 Hz. The voltage dependence of steady-state inactivation of Kv3.1 was not changed by 20 micrometer genistein. Our results indicate that genistein blocks directly Kv3.1 currents in concentration-, voltage-, time-dependent manners and the action of genistein on Kv3.1 is independent of tyrosine kinase inhibition.

The Effect of Epicatechin on the High Glucose-induced TSP-1 Expression and MMP-2 Activity in Rat Vascular Smooth Muscle Cells / 대한내분비학회지

BACKGROUND: The incidence of atherosclerosis is well correlated with the progression of type 2 diabetes mellitus. High plasma glucose in uncontrolled diabetic patients evokes many vascular complications such as atherosclerosis. Specifically, high glucose was reported to induce thrombospondin-1 (TSP-1), which activates matrix metalloproteinase-2 (MMP-2) and leads to the invasion of vascular smooth muscle cells (VSMCs) into the intima. Catechins with antioxidant effects are known to inhibit MMP-2 activity. Therefore, this study was aimed at revealing the effect of epicatechin, one of catechins, on high glucose-induced TSP-1 and the invasiveness of VSMCs. METHODS: VSMCs were primarily isolated from Sprague-Dawley rat aorta. The VSMCs were incubated with different doses (30, 100 and 300 micrometer) of epicatechin under high glucose concentration (30 mM). The TSP-1 protein and mRNA expressions were analyzed by performing Western blotting and Northern blot analyses, respectively. RT-PCR was performed to observe the MMP-2 mRNA expression. Gelatin zymography was performed for the measurement of MMP-2 activity. Invasion assays were performed to evaluate the invasiveness of VSMCs. RESULTS: Epicatechin inhibited the high glucose-induced TSP-1 expression and the MMP-2 activity in a dose-dependent manner. Also, epicatechin inhibited the high glucose-induced invasiveness of VSMCs across the matrix barrier in a dose-dependent fashion. CONCLUSION: Collectively, epicatechin may prevent the high glucose-induced proliferation and invasion of VSMCs by inhibiting the TSP-1 expression and the MMP-2 activity. Therefore, epicatechin appears to play a protective role in the development of atherosclerosis.

The Effect of Epicatechin on the High Glucose-induced TSP-1 Expression and MMP-2 Activity in Rat Vascular Smooth Muscle Cells / 대한내분비학회지

BACKGROUND: The incidence of atherosclerosis is well correlated with the progression of type 2 diabetes mellitus. High plasma glucose in uncontrolled diabetic patients evokes many vascular complications such as atherosclerosis. Specifically, high glucose was reported to induce thrombospondin-1 (TSP-1), which activates matrix metalloproteinase-2 (MMP-2) and leads to the invasion of vascular smooth muscle cells (VSMCs) into the intima. Catechins with antioxidant effects are known to inhibit MMP-2 activity. Therefore, this study was aimed at revealing the effect of epicatechin, one of catechins, on high glucose-induced TSP-1 and the invasiveness of VSMCs. METHODS: VSMCs were primarily isolated from Sprague-Dawley rat aorta. The VSMCs were incubated with different doses (30, 100 and 300 micrometer) of epicatechin under high glucose concentration (30 mM). The TSP-1 protein and mRNA expressions were analyzed by performing Western blotting and Northern blot analyses, respectively. RT-PCR was performed to observe the MMP-2 mRNA expression. Gelatin zymography was performed for the measurement of MMP-2 activity. Invasion assays were performed to evaluate the invasiveness of VSMCs. RESULTS: Epicatechin inhibited the high glucose-induced TSP-1 expression and the MMP-2 activity in a dose-dependent manner. Also, epicatechin inhibited the high glucose-induced invasiveness of VSMCs across the matrix barrier in a dose-dependent fashion. CONCLUSION: Collectively, epicatechin may prevent the high glucose-induced proliferation and invasion of VSMCs by inhibiting the TSP-1 expression and the MMP-2 activity. Therefore, epicatechin appears to play a protective role in the development of atherosclerosis.

Direct Block of Cloned K+ Channels, Kv1.5 and Kv1.3, by Cyclosporin A, Independent of Calcineurin Inhibition

The interaction of cyclosporine A (CsA), an immunosuppressant, with rat brain Kv1.5 (Kv1.5) channels, which were stably expressed in Chinese hamster ovary cells, was investigated using the whole-cell patch-clamp technique. CsA reversibly blocked Kv1.5 currents at +50 mV in a reversible concentration- dependent manner with an apparent IC50 of 1.0microM. Other calcineurin inhibitors (cypermethrin, autoinhibitory peptide) had no effect on Kv1.5 and did not prevent the inhibitory effect of CsA. Fast application of CsA led to a rapid and reversible block of Kv1.5, and the onset time constants of the CsA-induced block were decreased in a concentration-dependent manner. The CsA-induced block of Kv1.5 channels was voltage-dependent, with a steep increase over the voltage range of channel opening. However, the block exhibited voltage independence over the voltage range in which channels were fully activated. The rate constants for association and dissociation of CsA were 7.0microM-1s-1 and 8.1 s-1, respectively. CsA slowed the deactivation time course, resulting in a tail crossover phenomenon. Block of Kv1.5 by CsA was use-dependent. CsA also blocked Kv1.3 currents at +50 mV in a reversible concentration-dependent manner with an apparent IC50 of 1.1microM. The same effects of CsA on Kv1.3 were also observed in excised inside-out patches when applied to the internal surface of the membrane. The present results suggest that CsA acts directly on Kv1.5 currents as an open-channel blocker, independently of the effects of CsA on calcineurin activity.

Role of Protein kinase C in Desensitization of Somatostatin-induced Calcium Signalling in NG108-15 Cells / 대한내분비학회지

BACKGROUND: Activation of G-protein coupled-somatostatin receptors induces the release of calcium from inositol 1, 4, 5-trisphosphate-sensitive intracelluar stores. G-protein-coupled receptor signaling decreases with prolonged exposure to an agonist. SEBJECTS and METHODS: Fura-2-based digital Ca2+ imaging was used to study the effects of prolonged exposure to an agonist on the somatostatin-induced intracellular Ca2+ concentration([Ca2+]i) increases in NG108-15 cells, which were differentiated with CO2-independent medium and 10micrometer forskolin. RESULTS: Exposure to somatostatin(1micrometer) for 30 min completely desensitized the NG108-15 cells to a second somatostatin-induced response. The cells recovered gradually over 20 min following washout of the somatostatin. The desensitization was not due to depletion of the intracellular Ca2+ stores, and pretreatment for 30 min with bradykinin(100nM), which activates phospholipase C, or DADLE(D-Ala2-D-Leu5 enkephalin, 1microM), which activates phospholipase C, failed to cross-desensitize the somatostatin-evoked [Ca2+]i increases. Treatment with 8-cpt-cAMP(0.1mM) for 30min did not influence the somatostatin-induced[Ca2+]i increases. Phorbol 12, 13-dibutyrate(PdBu, 1microM) blocked the response completely. Down-regulation of PKC due to 24 h exposure of PdBu (1microM) inhibited the somatostatin-induced desensitization. CONCLUSION: Prolonged exposure of somatostatin to NG108-15 cells desensitized the somatostatin-induced release of Ca2+ from the intracelluar store, with protein kinase C also involved in the desensitization.

Effect of Fluoxetine on the Induction of Long-term Potentiation in Rat Frontal Cortex

Serotonin (5-hydroxytroptamine, 5-HT) has been shown to affect the induction of long-term potentiation (LTP) in the cortex such as the hippocampus, the visual cortex and the prefrontal cortex. Fluoxetine, as a selective serotonin reuptake inhibitor, is used in the management of a wide variety of psychological diseases. To study the effect of fluoxetine on the induction of LTP, we recorded the field potential in layer II/III of the frontal cortex from 3-wk-old. LTP was induced in horizontal input by theta burst stimulation (TBS). TBS with two-folds intensity of the test stimulation induced LTP, which was blocked by application of D-AP5 (50microM), an NMDA receptor antagonist. Whereas bath application of 5-HT (10microM) inhibited the induction of LTP, treatment with the 5-HT depleting agent, para-chloroamphetamine (PCA, 10microM), for 2hr did not affect the induction of LTP. Bath application of fluoxetine (1, 3, and 10microM) suppressed the induction of LTP in concentration-dependent manner, however, fluoxetine did not inhibit the induction of LTP in 5-HT-depleted slices. These results indicate that fluoxetine may inhibit the induction of LTP by modulating serotonergic mechanism in the rat frontal cortex.

Efffects of Fluoxetine on ATP-induced Calcium Signaling in PC12 Cells

Fluoxetine, a widely used anti-depressant compound, has several additional effects, including blockade of voltage-gated ion channels. We examined whether fluoxetine affects ATP-induced calcium signaling in PC12 cells by using fura-2-based digital calcium imaging and assay for [3H]-inositol phosphates (IPs). Treatment with ATP (100microM) for 2 min induced [Ca2+]i increases. The ATP-induced [Ca2+]i increases were significantly decreased by removal of extracellular Ca2+ and treatment with the inhibitor of endoplasmic reticulum Ca2+ ATPase thapsigargin (1microM). Treatment with fluoxetine for 5 min blocked the ATP-induced [Ca2+]i increase concentration-dependently. Treatment with fluoxetine (30microM) for 5 min blocked the ATP-induced [Ca2+]i increase following removal of extracellular Ca2+ and depletion of intracellular Ca2+ stores. While treatment with the L-type Ca2+ channel antagonist nimodipine for 10 min inhibited the ATP-induced [Ca2+]i increases significantly, treatment with fluoxetine alone blocked the ATP-induced responses. Treatment with fluoxetine also inhibited the 50 mM K+-induced [Ca2+]i increases completely. However, treatment with fluoxetine did not inhibit the ATP-induced [3H]-IPs formation. Collectively, we conclude that fluoxetine inhibits ATP-induced [Ca2+]i increases in PC12 cells by inhibiting both an influx of extracellular Ca2+ and a release of Ca2+ from intracellular stores without affecting IPs formation.

Inhibition by Norfluoxetine, the Major Metabolite of Fluoxetine, of Voltage-Gated K+ Channels in Primary Cultured Rat Hippocampal Neurons / 대한마취과학회지

BACKGROUND: Fluoxetine (Prozac), a selective serotonin reuptake inhibitor, has been shown to be effective in the treatment of depression. We investigated the effects of norfluoxetine, the major active metabolite of fluoxetine, on voltage-gated K+ currents in primary cultured hippocampal neurons, and determined the potency and modes of actions of norfluoxetine. METHODS: Voltage-gated K+ currents were studied in primary cultured rat hippocampal neurons using the whole-cell configuration of the patch-clamp technique. Electrophysiological recordings were done in hippocampal neurons between 5-10 days in culture. Transient A-type K+ currents (KA) and delayed-rectifier K+ (KDR) currents were isolated from whole-cell K+ currents using a pulse protocol. RESULTS: Norfluoxetine accelerated the decay rate of whole-cell K+ currents, and thus decreased the current amplitude at the end of a pulse in a concentration-dependent manner. Norfluoxetine inhibited KA and KDR currents in a concentration-dependent manner with IC50's of 0.93 and 0.70micro M, respectively. Norfluoxetine also reduced the areas of KA currents and the steady-state KDR current over the range of test potentials, and the reduction was voltage-dependent (greater increase at more positive potentials). From the onset of the fractional block of KA currents by norfluoxetine during the initial 40 ms of a clamp step, we calculated k1 = 53.26/micro M.s for the association rate constant, and k2 = 70.24/s for the dissociation rate constant. The resulting apparent KD was 1.32micro M, which is similar to the IC50 value obtained from the concentration-response curve. CONCLUSIONS: Our results indicate that norfluoxetine, the major metabolite of fluoxetine, at therapeutic levels, produces a concentration- and voltage-dependent inhibition of KA and KDR currents in primary cultured hippocampal neurons. These effects could perturb the neuronal excitability in the hippocampus, and may contribute to the therapeutic antidepressant action of fluoxetine.

Electrophysiological and Morphological Classification of Inhibitory Interneurons in Layer II/III of the Rat Visual Cortex

Interneuron diversity is one of the key factors to hinder understanding the mechanism of cortical neural network functions even with their important roles. We characterized inhibitory interneurons in layer II/III of the rat primary visual cortex, using patch-clamp recording and confocal reconstruction, and classified inhibitory interneurons into fast spiking (FS), late spiking (LS), burst spiking (BS), and regular spiking non-pyramidal (RSNP) neurons according to their electrophysiological characteristics. Global parameters to identify inhibitory interneurons were resting membrane potential (> -70 mV) and action potential (AP) width ( 200 M omega) and the shorter P-T time (< 20 msec) than those of regular spiking pyramidal neurons. Confocal reconstruction of recorded cells revealed characteristic morphology of each subtype of inhibitory interneurons. Thus, our results provide at least four subtypes of inhibitory interneurons in layer II/III of the rat primary visual cortex and a classification scheme of inhibitory interneurons.

Comparative Analysis of Phospholipase D2 Localization in the Pancreatic Islet of Rat and Guinea Pig

To examine the localization pattern of phospholipase D2 (PLD2) in the pancreatic islet (the islet of Langerhans) depending on species, we conducted a morphological experiment in the rat and guinea pig. Since individual islets display a typical topography with a central core of B cell mass and a peripheral boundary of A, D, and PP cells, double immunofluorescent staining with a panel of antibodies was performed to identify PLD2-immunoreactive cells in the islets PLD2 immunoreactivity was mainly present in A and PP cells of the rat pancreatic islets. And yet, in the guinea pig, PLD2 immunoreactivity was exclusively localized in A cells, and not in PP cells. These findings suggest a possibility that PLD2 is mainly located in A cells of rodent pancreatic islets, and that the existence of PLD2 in PP cells is not universal in all species. Based on these results, it is suggested that PLD2 may play a significant role in the function of A and/or PP cells via a PLD-mediated signaling pathway.