The level of nitric oxide regulates lipocalin-2 expression under inflammatory condition in RINm5F beta-cells.

We previously reported that proinflammatory cytokines (interleukin-1ß and interferon-γ) induced the expression of lipocalin-2 (LCN-2) together with inducible nitric oxide synthase (iNOS) in RINm5F beta-cells. Therefore, we examined the effect of nitric oxide (NO) on LCN-2 expression in cytokines-treated RINm5F beta-cells. Additionally, we observed the effect of LCN-2 on cell viability. First, we found the existence of LCN-2 receptor and the internalization of exogenous recombinant LCN-2 peptide in RINm5F and INS-1 beta-cells. Next, the effects of NO on LCN-2 expression were evaluated. Aminoguanidine, an iNOS inhibitor and iNOS gene silencing significantly inhibited cytokines-induced LCN-2 expression while sodium nitroprusside (SNP), an NO donor potentiated it. Luciferase reporter assay showed that transcription factor NF-κB was not involved in LCN-2 expression. Both LCN-2 mRNA and protein stability assays were conducted. SNP did not affect LCN-2 mRNA stability, however, it significantly reduced LCN-2 protein degradation. The LCN-2 protein degradation was significantly attenuated by MG132, a proteasome inhibitor. Finally, the effect of LCN-2 on cell viability was evaluated. LCN-2 peptide treatment and LCN-2 overexpression significantly reduced cell viability. FACS analysis showed that LCN-2 induced the apoptosis of the cells. Collectively, NO level affects LCN-2 expression via regulation of LCN-2 protein stability under inflammatory condition and LCN-2 may reduce beta-cell viability by promoting apoptosis.

Cholinergic induction of input-specific late-phase LTP via localized Ca2+ release in the visual cortex.

Acetylcholine facilitates long-term potentiation (LTP) and long-term depression (LTD), substrates of learning, memory, and sensory processing, in which acetylcholine also plays a crucial role. Ca(2+) ions serve as a canonical regulator of LTP/LTD but little is known about the effect of acetylcholine on intracellular Ca(2+) dynamics. Here, we investigated dendritic Ca(2+) dynamics evoked by synaptic stimulation and the resulting LTP/LTD in layer 2/3 pyramidal neurons of the rat visual cortex. Under muscarinic stimulation, single-shock electrical stimulation (SES) inducing ∼20 mV EPSP, applied via a glass electrode located ∼10 µm from the basal dendrite, evoked NMDA receptor-dependent fast Ca(2+) transients and the subsequent Ca(2+) release from the inositol 1,4,5-trisphosphate (IP(3))-sensitive stores. These secondary dendritic Ca(2+) transients were highly localized within 10 µm from the center (SD = 5.0 µm). The dendritic release of Ca(2+) was a prerequisite for input-specific muscarinic LTP (LTPm). Without the secondary Ca(2+) release, only muscarinic LTD (LTDm) was induced. D(-)-2-amino-5-phosphopentanoic acid and intracellular heparin blocked LTPm as well as dendritic Ca(2+) release. A single burst consisting of 3 EPSPs with weak stimulus intensities instead of the SES also induced secondary Ca(2+) release and LTPm. LTPm and LTDm were protein synthesis-dependent. Furthermore, LTPm was confined to specific dendritic compartments and not inducible in distal apical dendrites. Thus, cholinergic activation facilitated selectively compartment-specific induction of late-phase LTP through IP(3)-dependent Ca(2+) release.

Exendin-4 inhibits iNOS expression at the protein level in LPS-stimulated Raw264.7 macrophage by the activation of cAMP/PKA pathway.

Glucagon-like peptide-1 (GLP-1) and its potent agonists have been widely studied in pancreatic islet ß-cells. However, GLP-1 receptors are present in many extrapancreatic tissues including macrophages, and thus GLP-1 may have diverse actions in these tissues and cells. Therefore, we examined the mechanism by which exendin-4 (EX-4), a potent GLP-1 receptor agonist, inhibits lipopolysaccharide (LPS)-induced iNOS expression in Raw264.7 macrophage cells. EX-4 significantly inhibited LPS-induced iNOS protein expression and nitrite production. However, Northern blot and promoter analyses demonstrated that EX-4 did not inhibit LPS-induced iNOS mRNA expression and iNOS promoter activity. Electrophoretic mobility shift assay (EMSA) showed that EX-4 did not alter the binding activity of NF-κB to the iNOS promoter. Consistent with the result of EMSA, LPS-induced IκBα phosphorylation and nuclear translocation of p65 were not inhibited by EX-4. Also, actinomycin D chase study and the promoter assay using the construct containing 3'-untranslated region of iNOS showed that EX-4 did not affect iNOS mRNA stability. Meanwhile, cycloheximide chase study demonstrated that EX-4 significantly accelerated iNOS protein degradation. The EX-4 inhibition of LPS-induced iNOS protein was significantly reversed by adenylate cyclase inhibitors (MDL-12330A and SQ 22536), a PKA inhibitor (H-89) and PKAα gene silencing. These findings suggest that EX-4 inhibited LPS-induced iNOS expression at protein level, but not at transcriptional mechanism of iNOS gene and this inhibitory effect of EX-4 was mainly dependent on cAMP/PKA system.

Induction mechanism of lipocalin-2 expression by co-stimulation with interleukin-1ß and interferon-γ in RINm5F beta-cells.

Lipocalin-2 (LCN-2) was known to play a role in obesity and insulin resistance, however, little is known about the expression of LCN-2 in pancreatic islet ß-cells. We examined the molecular mechanisms by which proinflammatory cytokines interleukin-1ß (IL-1ß) and interferon-γ (IFN-γ) induce LCN-2 expression in RINm5F ß-cells. IL-1ß significantly induced LCN-2 expression while IFN-γ alone did not induce it. IFN-γ significantly potentiated IL-1ß-induced LCN-2 protein and mRNA expression. However, promoter study and EMSA showed that IFN-γ failed to potentiate IL-1ß-induced LCN-2 promoter activity and binding activity of transcription factors on LCN-2 promoter. Furthermore, LCN-2 mRNA stability and transcription factors NF-κB and STAT-1 were not involved in the stimulatory effect of IFN-γ on IL-1ß-induced LCN-2 expression. Meanwhile, Western Blot and promoter analyses showed that NF-κB was a key factor in IL-1ß-induced LCN-2 expression. Collectively, IL-1ß induces LCN-2 expression via NF-κB activation in RINm5F ß-cells. IFN-γ potentiates IL-1ß-induced LCN-2 expression at mRNA and protein levels, but not at promoter level and the stimulatory effect of IFN-γ is independent of NF-κB and STAT-1 activation. These data suggest that LCN-2 may play a role in ß-cell function under an inflammatory condition.

Molecular mechanisms of early growth response protein-1 (EGR-1) expression by quercetin in INS-1 beta-cells.

Early growth response-1 (EGR-1), one of immediate early response genes, is involved in diverse cellular response. We recently reported that quercetin increased catalytic subunit of γ-glutamylcysteine ligase (GCLC) via the interaction of EGR-1 to GCLC promoter in INS-1 beta-cells. Therefore, this study investigated molecular mechanisms of quercetin-induced EGR-1 expression in INS-1 cells. Quercetin significantly induced EGR-1 protein and its mRNA expressions. This induction of EGR-1 was completely blocked by pretreatment with a PKA inhibitor, H89 and partially blocked by a p38 inhibitor, SB203580. Additionally, the siRNA-mediated inhibition of PKAα and p38 resulted in significant reduction of quercetin-induced EGR-1 promoter activity. Also, quercetin-induced EGR-1 protein expression was significantly decreased in the cells transfected with PKAα siRNA. Study using truncated EGR-1 promoter constructs showed that serum response element (SRE) sites, not cAMP response element site, were essential for EGR-1 transcription. However, electrophoretic mobility shift assay showed that quercetin did not affect the band intensity of DNA-protein complex on SRE site of EGR-1 promoter. Also, immune-shift assay using serum response factor (SRF) and phospho-SRF antibodies showed no difference between control and quercetin-treated groups. Collectively, quercetin-induced EGR-1 expression is largely dependent on PKA and partly on p38 MAPK pathway, and SRE sites of EGR-1 promoter are involved in quercetin-induced EGR-1 transcriptional activity.

Grape seed proanthocyanidin extract inhibits glutamate-induced cell death through inhibition of calcium signals and nitric oxide formation in cultured rat hippocampal neurons.

BACKGROUND: Proanthocyanidin is a polyphenolic bioflavonoid with known antioxidant activity. Some flavonoids have a modulatory effect on [Ca²âº]i. Although proanthocyanidin extract from blueberries reportedly affects Ca²âº buffering capacity, there are no reports on the effects of proanthocyanidin on glutamate-induced [Ca²âº]i or cell death. In the present study, the effects of grape seed proanthocyanidin extract (GSPE) on glutamate-induced excitotoxicity was investigated through calcium signals and nitric oxide (NO) in cultured rat hippocampal neurons. RESULTS: Pretreatment with GSPE (0.3-10 µg/ml) for 5 min inhibited the [Ca²âº]i increase normally induced by treatment with glutamate (100 µM) for 1 min, in a concentration-dependent manner. Pretreatment with GSPE (6 µg/ml) for 5 min significantly decreased the [Ca²âº]i increase normally induced by two ionotropic glutamate receptor agonists, N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). GSPE further decreased AMPA-induced response in the presence of 1 µM nimodipine. However, GSPE did not affect the 50 mM K+-induced increase in [Ca²âº]i. GSPE significantly decreased the metabotropic glutamate receptor agonist (RS)-3,5-Dihydroxyphenylglycine-induced increase in [Ca²âº]i, but it did not affect caffeine-induced response. GSPE (0.3-6 µg/ml) significantly inhibited synaptically induced [Ca²âº]i spikes by 0.1 mM [Mg²âº]o. In addition, pretreatment with GSPE (6 µg/ml) for 5 min inhibited 0.1 mM [Mg²âº]o- and glutamate-induced formation of NO. Treatment with GSPE (6 µg/ml) significantly inhibited 0.1 mM [Mg²âº]o- and oxygen glucose deprivation-induced neuronal cell death. CONCLUSIONS: All these data suggest that GSPE inhibits 0.1 mM [Mg²âº]o- and oxygen glucose deprivation-induced neurotoxicity through inhibition of calcium signals and NO formation in cultured rat hippocampal neurons.

Quercetin-induced upregulation of human GCLC gene is mediated by cis-regulatory element for early growth response protein-1 (EGR1) in INS-1 beta-cells.

The catalytic subunit of gamma-glutamylcysteine ligase (GCLC) catalyses the rate-limiting step in the de novo synthesis of glutathione (GSH), which is involved in maintaining intracellular redox balance. GSH is especially important for antioxidant defense system since beta-cells show intrinsically low expression of antioxidant enzymes. In the present study, we investigated the regulatory mechanisms by which quercetin, a flavonoid, induces the expression of the GCLC gene in rat pancreatic beta-cell line INS-1. Promoter study found that the proximal GC-rich region (from -90 to -34) of the GCLC promoter contained the quercetin-responsive cis-element(s). The quercetin-responsive region contains consensus DNA binding site for early growth response 1 (EGR1) at -67 (5'-CGCCTCCGC-3') which overlaps with a putative Sp1 binding site. Electrophoretic mobility shift assay showed that an oligonucleotide containing the EGR1 site was bound to nuclear factors EGR1, Sp1, and Sp3. In the promoter analysis, mutation of EGR1 site significantly reduced the quercetin response, whereas mutation of Sp1 site decreased only the basal activity of the GCLC promoter. Additionally, the transient overexpression of EGR1 significantly increased basal activity of the GCLC promoter. Finally, we showed that quercetin potently induced both EGR1 mRNA and its protein levels without affecting the expression of Sp1 and Sp3 proteins. Therefore, we concluded that EGR1 was bound to GC-rich region of the GCLC gene promoter, which was prerequisite for the transactivation of the GCLC gene by quercetin.

CCAAT box is required for the induction of human thrombospondin-1 gene by trichostatin A.

Histone deacetylase (HDAC) inhibitors have been reported to inhibit angiogenesis as well as tumor growth. Thrombospondin-1 (TSP1) has been recognized as a potent inhibitor of angiogenesis. Such an action of TSP1 may account for the effect of HDAC inhibitors. In the present study, we investigated the molecular mechanism by which trichostatin A, a HDAC inhibitor, induces the expression of TSP1 gene. Trichostatin A increased both mRNA and protein levels of TSP1 in HeLa cells. Promoter and actinomycin D chase assays showed that trichostatin A-induced TSP1 expression was regulated at the transcriptional level without changing mRNA stability. CCAAT box on the TSP1 promoter was found to primarily mediate the trichostatin A response by deletion and mutation analyses of the TSP1 promoter. Electrophoretic mobility shift assay indicated that CCAAT-binding factor (CBF) was specifically bound to the CCAAT box of TSP1 promoter. Moreover, chromatin immunoprecipitation assay showed that trichostatin A increased the binding of acetylated form of histone H3 to the CCAAT box region of TSP1 promoter. Taken together, these results strongly suggest that trichostatin A activates the transcription of TSP1 gene through the binding of transcription factor CBF to CCAAT box and the enhanced histone acetylation. Thus, the present study provides the clue that the inhibition of angiogenesis by trichostatin A is accomplished through the upregulation of TSP1, the anti-angiogenic factor.

Exendin-4 induction of Egr-1 expression in INS-1 beta-cells: interaction of SRF, not YY1, with SRE site of rat Egr-1 promoter.

Glucagon-like peptide-1 (GLP-1) induces several immediate early response genes such as c-fos, c-jun, and early growth response-1 (Egr-1), which are involved in cell proliferation and differentiation. We recently reported that exendin-4 (EX-4), a potent GLP-1 agonist, upregulated Egr-1 expression via phosphorylation of CREB, a transcription factor in INS-1 beta-cells. This study was designed to investigate the role of another transcription factors, serum response factor (SRF) and Yin Yang-1 (YY1), in EX-4-induced Egr-1 expression. EX-4 significantly increased Egr-1 mRNA and subsequently its protein level. EX-4-induced Egr-1 expression was inhibited by pretreatment with a PKA inhibitor, H-89, and an MEK inhibitor, PD 98059. The siRNA-mediated inhibition of PKA and ERK1 resulted in significant reduction of EX-4-induced Egr-1 expression. Promoter analyses showed that SRE clusters were essential for Egr-1 transcription, and YY1 overexpression did not affect Egr-1 promoter activity. EMSA results demonstrated that EX-4-induced transient increase in DNA-protein complex on SRE site, and that both SRF and phospho-SRF were bound to this site. Treatment of either YY1 consensus oligonucleotide or YY1 antibody did not effect the change of density or migration of the DNA-protein complex. Collectively, EX-4-induced Egr-1 expression is largely dependent on cAMP-mediated extracellular signal-regulated kinase activation, and EX-4 induces Egr-1 transcription via the interaction of SRF and phospho-SRF to SRE sites.

Transmodulation between phospholipase D and c-Src enhances cell proliferation.

Phospholipase D (PLD) has been implicated in the signal transduction pathways initiated by several mitogenic protein tyrosine kinases. We demonstrate for the first time that most notably PLD2 and to a lesser extent the PLD1 isoform are tyrosine phosphorylated by c-Src tyrosine kinase via direct association. Moreover, epidermal growth factor induced tyrosine phosphorylation of PLD2 and its interaction with c-Src in A431 cells. Interaction between these proteins is via the pleckstrin homology domain of PLD2 and the catalytic domain of c-Src. Coexpression of PLD1 or PLD2 with c-Src synergistically enhances cellular proliferation compared with expression of either molecule. While PLD activity as a lipid-hydrolyzing enzyme is not affected by c-Src, wild-type PLDs but not catalytically inactive PLD mutants significantly increase c-Src kinase activity, up-regulating c-Src-mediated paxillin phosphorylation and extracellular signal-regulated kinase activity. These results demonstrate the critical role of PLD catalytic activity in the stimulation of Src signaling. In conclusion, we provide the first evidence that c-Src acts as a kinase of PLD and PLD acts as an activator of c-Src. This transmodulation between c-Src and PLD may contribute to the promotion of cellular proliferation via amplification of mitogenic signaling pathways.

Open channel block of Kv1.3 by rosiglitazone and troglitazone: Kv1.3 as the pharmacological target for rosiglitazone.

The effects of rosiglitazone and troglitazone were examined on cloned Kv1.3 channels stably expressed in Chinese hamster ovary cells using the whole-cell configuration of the patch-clamp technique. Rosiglitazone decreased the Kv1.3 currents and accelerated the decay rate of current inactivation in a concentration-dependent manner with an IC(50) of 18.6 microM. These effects were reversible after washout of the drug. Troglitazone caused the block of Kv1.3 with a similar pattern but was five times more potent than rosiglitazone with an IC(50) of 3.5 microM. The block of Kv1.3 by rosiglitazone and troglitazone was voltage-dependent at a membrane potential coinciding with the activation of the channels. Both drugs decreased the tail current amplitude and slowed the deactivation process of Kv1.3, resulting in a tail crossover phenomenon. These results indicate that rosiglitazone and troglitazone block the open state of Kv1.3 channels, suggesting that it is an important pharmacological target for rosiglitazone as a potent blocker of Kv1.3 channels.

Spatial profile of back-propagating action potential-evoked Ca2+ transients in basal dendrites.

Calcium influx in basal dendrites evoked by back-propagating action potentials is important for understanding the input-specific modulation of synaptic transmission. Calcium transients evoked by back-propagating action potentials were measured for the basal dendrites of layer II/III pyramidal neurons in visual cortex slices. In contrast to apical dendrites, the peak calcium transients evoked by a single somatic action potential increased with distance from the soma. The peak calcium transients evoked by bursts of back-propagating action potentials (three or five pulses, 20 Hz) were greater than those evoked by a single back-propagating action potential. These different spatial profiles for calcium transients triggered by back-propagating action potentials between apical and basal dendrites suggest that the segregated inputs from brain areas might be modulated in different ways, according to their synaptic location.

Desensitization of somatostatin-induced inhibition of low extracellular magnesium concentration-induced calcium spikes in cultured rat hippocampal neurons.

Neuronal excitability is inhibited by somatostatin, which might play important roles in seizure and neuroprotection. The possibility of whether the effect of somatostatin on neurotransmission is susceptible to desensitization was investigated. We tested the effects of prolonged exposure to somatostatin on 0.1 mM extracellular Mg(2+) concentration ([Mg(2+)](o))-induced intracellular free Ca(2+) concentration ([Ca(2+)](i)) spikes in cultured rat hippocampal neurons using fura-2-based microfluorimetry. Reducing [Mg(2+)](o) to 0.1 mM elicited repetitive [Ca(2+)](i) spikes. These [Ca(2+)](i) spikes were inhibited by exposure to somatostatin-14. The inhibitory effects of somatostatin were blocked by pretreatment with pertussis toxin (PTX, 100 ng/ml) for 18-24 h. Prolonged exposure to somatostatin induced a desensitization of the somatostatin-induced inhibition of [Ca(2+)](i) spikes in a concentration-dependent manner. The somatostatin-induced desensitization was retarded by the nonspecific protein kinase C (PKC) inhibitor staurosporin (100 nM) or chronic treatment with phorbol dibutyrate (1 microM) for 24 h, but not by the protein kinase A inhibitor KT5720. The desensitization was significantly retarded by the novel PKCepsilon translocation inhibitor peptide (1 microM). In addition, suramin (3 microM), an inhibitor of G-protein-coupled receptor kinase 2 (GRK2), caused a reduction in the desensitization. After tetrodotoxin (TTX, 1 microM) completely blocked the low [Mg(2+)](o)-induced [Ca(2+)](i) spikes, glutamate-induced [Ca(2+)](i) transients were slightly inhibited by somatostatin and the inhibition was desensitized by prolonged exposure to somatostatin. These results indicate that the prolonged activation of somatostatin receptors induces the desensitization of somatostatin-induced inhibition on low [Mg(2+)](o)-induced [Ca(2+)](i) spikes through the activation of GRK2 and partly a novel PKCepsilon in cultured rat hippocampal neurons.

Serotonin inhibits the induction of NMDA receptor-dependent long-term potentiation in the rat primary visual cortex.

An increase in serotonin [5-hydroxytryptamine (5-HT)] levels in the rat visual cortex is correlated with the developmental decrease in long-term potentiation (LTP), and 5-HT may play an important role in the closure of the critical period by regulating LTP. The effect of 5-HT on the induction of N-methyl-D-aspartate receptor (NMDAR)-dependent and metabotropic glutamate receptor (mGluR)-dependent LTP in visual cortex slices from young rats was investigated. The field potential in layer II/III was recorded by stimulating the underlying layer IV. NMDAR-dependent LTP was induced in slices from 3-week-old rats by theta-burst stimulation (TBS) but not in slices from 5-week-old rats. However, LTP was induced in 5-HT-depleted slices from 5-week-old rats by incubation with para-chloroamphetamine (10 microM, 2 h), a 5-HT-depleting agent. The reinstated LTP in 5-HT-depleted slices was inhibited by the application of D-aminopentanoate, an NMDAR antagonist (50 microM) and 5-HT (10 and 30 microM). In contrast, the induction of mGluR-dependent LTP by weak TBS in disinhibited slices with picrotoxin (1 microM) in the bath was not affected by 5-HT application. The coapplication of 5-HT1A and 5-HT2 receptor agonists inhibited the induction of NMDAR-dependent LTP in 5-HT-depleted slices. 5-HT levels in the visual cortex increased with age. Based on these findings, we conclude that NMDAR-dependent LTP is specifically inhibited by coactivation of 5-HT1A and 5-HT2 receptors with the increase in 5-HT levels in the rat visual cortex at the end of the critical period.

Fluoxetine inhibits ATP-induced [Ca(2+)](i) increase in PC12 cells by inhibiting both extracellular Ca(2+) influx and Ca(2+) release from intracellular stores.

Fluoxetine, a widely used antidepressant, has additional effects, including the blocking of voltage-gated ion channels. We examined whether fluoxetine affects ATP-induced calcium signaling in PC12 cells using fura-2-based digital calcium imaging, an assay for [3H]-inositol phosphates (IPs) and whole-cell patch clamping. Treatment with ATP (100 microM) for 2 min induced increases in intracellular free Ca(2+) concentrations ([Ca(2+)](i)). Treatment with fluoxetine (100 nM to 30 microM) for 5 min inhibited the ATP-induced [Ca(2+)](i) increases in a concentration-dependent manner (IC(50) = 1.85 microM). Treatment with fluoxetine (1.85 microM) for 5 min significantly inhibited the ATP-induced responses following the removal of extracellular Ca(2+) or depletion of intracellular Ca(2+) stores. Whereas treatment for 10 min with nimodipine (1 microM) significantly inhibited the ATP-induced [Ca(2+)](i) increase, treatment with fluoxetine further inhibited the ATP-induced response. Treatment with fluoxetine significantly inhibited [Ca(2+)](i) increases induced by 50 mM K(+). In addition, treatment with fluoxetine markedly inhibited ATP-induced inward currents in a concentration-dependent manner. However, treatment with fluoxetine did not inhibit ATP-induced [3H]-IPs formation. Therefore, we conclude that fluoxetine inhibits ATP-induced [Ca(2+)](i) increases in PC12 cells by inhibiting both the influx of extracellular Ca(2+) and the release of Ca(2+) from intracellular stores without affecting IPs formation.

Mechanism of block by fluoxetine of 5-hydroxytryptamine3 (5-HT3)-mediated currents in NCB-20 neuroblastoma cells.

The effect of fluoxetine (Prozac) on 5-hydroxytryptamine(3) (5-HT(3))-mediated currents in NCB-20 neuroblastoma cells was examined using the whole-cell patch-clamp technique. Fluoxetine produced a significant reduction of peak amplitude without altering the activation time course of 5-HT(3)-mediated currents. These effects were concentration-dependent, with an IC(50) value of 4.15 microM. No voltage dependence was evident in fluoxetine's block of 5-HT(3)-mediated currents over the entire voltage range tested. The extent of block by pre-application of fluoxetine was significantly greater than that by co-application. Fluoxetine also increased the apparent rate of current desensitization to 5-HT application. Using a first-order kinetics analysis, the open-channel blocking rate constants were 0.06 microM(-1)s(-1) (k(+1), association rate constant) and 0.05 s(-1) (k(-1), dissociation rate constant), with an apparent K(d) (=k(-1)/k(+1)) of 0.83 microM. This value is close to an IC(50) of 1.11 microM obtained from the reduction in tau, the time constant of desensitization. Intracellular application of fluoxetine for long durations had no effect on the amplitude or kinetics of 5-HT(3)-mediated currents. Similarly, norfluoxetine, the major metabolite of fluoxetine, reduced the peak current, and enhanced the rate of current desensitization in a concentration-dependent manner with an IC(50) of 2.66 microM, indicating that norfluoxetine is more potent than fluoxetine in blocking 5-HT(3)-mediated currents. These results indicate that, at clinically relevant concentrations, fluoxetine and its metabolite, norfluoxetine, block 5-HT(3)-mediated currents in NCB-20 neuroblastoma cells.

Inhibitory effects of epicatechin on interleukin-1beta-induced inducible nitric oxide synthase expression in RINm5F cells and rat pancreatic islets by down-regulation of NF-kappaB activation.

Cytokines that are released by infiltrating inflammatory cells around the pancreatic islets are involved in the pathogenesis of type 1 diabetes mellitus. Specifically, interleukin-1beta (IL-1beta) stimulates inducible nitric oxide synthase (iNOS) expression and nitric oxide overproduction, leading to the beta-cell damage. In activating this pathway, nuclear factor-kappaB (NF-kappaB) plays a crucial role, and many of the IL-1beta-sensitive genes contain NF-kappaB binding sites in their promoter regions. We have recently shown that epicatechin, which is a flavonoid, had a protective effect on pancreatic beta-cells in both streptozotocin-treated rats and islets. In the present study, the effects of epicatechin on IL-1beta-induced beta-cell damage were examined. RINm5F cells and islets were pretreated with epicatechin and next incubated with IL-1beta. The released nitrite, iNOS protein and mRNA expression levels were then measured. IkappaBalpha protein, nuclear translocation of NF-kappaB, and NF-kappaB DNA binding activity were also determined. Following the transient transfection of an iNOS promoter into the cells, the iNOS promoter activity was measured. ATP- or D-glucose-induced insulin release was measured in RINm5F cells and islets, respectively. Epicatechin significantly reduced IL-1beta-induced nitrite production, iNOS protein and mRNA expressions, and it also inhibited IL-1beta-induced IkappaBalpha protein degradation, NF-kappaB activation, and iNOS promoter activity. Epicatechin partly restored the IL-1beta-induced inhibition of insulin release. These results suggest that epicatechin inhibits the IL-1beta-induced iNOS expression by down-regulating NF-kappaB activation, and protecting beta-cells from IL-1beta.

Fluoxetine blocks cloned neuronal A-type K+ channels Kv1.4.

The effects of fluoxetine were studied on cloned K+ channel Kv1.4 stably expressed in Chinese hamster ovary (CHO) cells using the whole-cell configuration of the patch-clamp technique. Extracellular application of various concentrations of fluoxetine inhibited the amplitude of the peak current of Kv1.4 and accelerated its inactivation time course in a concentration-dependent manner. Thus, fluoxetine decreased Kv1.4 (the integral of the outward current) in a concentration-dependent manner; the IC50 was 33.1 +/- 2.5 microM. The inhibitory effect of fluoxetine was time-dependent. The apparent association (k) and dissociation (l) rate constants measured at +40 mV were 3.5 +/- 0.7 microM-1s-1 and 132.5 +/- 13.3 s-1, respectively. The Kd (= l/k) was 37.9 microM, which was close to the value obtained from the concentration-response curve. The block produced by fluoxetine increased steeply between -30 and 0 mV, which corresponded with the voltage range for channel opening. The fluoxetine block was constant at more depolarized potentials, suggesting that the block by fluoxetine was not voltage dependent. Our data indicate that fluoxetine blocks Kv1.4 channels by preferentially binding to open state.

Endogenous somatostatin receptors mobilize calcium from inositol 1,4,5-trisphosphate-sensitive stores in NG108-15 cells.

Somatostatin receptors are members of the G-protein-coupled receptor superfamily and exert their principal effects by coupling to inhibitory G-proteins. We used fura-2-based digital calcium imaging and assayed for [3H]inositol phosphates (IPs) to study the effects of somatostatin on intracellular calcium signaling in neuroblastomaxglioma NG108-15 cells. Both somatostatin-14 and octreotide induced concentration-dependent increases in intracellular Ca(2+) concentration ([Ca(2+)](i)). Thirty-four percent of the cells responded to treatment with 100 nM somatostatin-14. Somatostatin-induced responses were not blocked by the removal of extracellular calcium; instead, they were abolished by pretreatment with 100 nM thapsigargin, an agent that depletes and prevents refilling of intracellular Ca(2+) stores. Pretreatment with the inositol 1,4,5-trisphosphate (IP(3)) receptor antagonist xestospongin C (10 microM) for 20 min inhibited markedly the somatostatin-induced response. Somatostatin (100 nM) increased [3H]IPs formation. U73122 (1 microM), an inhibitor of phospholipase C (PLC), completely blocked the somatostatin-induced [Ca(2+)](i) increases and the formation of [3H]IPs. Pretreatment with pertussis toxin (PTX, 200 ng/ml) for 24 h blocked the somatostatin-induced responses. Thus, we conclude that activation of endogenous somatostatin receptors in NG108-15 cells induces the release of calcium from IP(3)-sensitive intracellular stores through PTX-sensitive G-protein-coupled PLC.

Fluoxetine inhibits A-type potassium currents in primary cultured rat hippocampal neurons.

The effects of fluoxetine (Prozac) on the transient A-currents (IA) in primary cultured hippocampal neurons were examined using the whole-cell patch clamp technique. Fluoxetine did not significantly decrease the peak amplitude of whole-cell K+ currents, but it accelerated the decay rate of inactivation, and thus decreased the current amplitude at the end of the pulse. For further analysis, IA and delayed rectifier K+ currents (IDR) were isolated from total K+ currents. Fluoxetine decreased IA (the integral of the outward current) in a concentration-dependent manner with an IC50 of 5.54 microM. Norfluoxetine, the major active metabolite of fluoxetine, was a more potent inhibitor of IA than was fluoxetine, with an IC50 of 0.90 microM. Fluoxetine (3 microM) inhibited IA in a voltage-dependent manner over the whole range of membrane potentials tested. Analysis of the time dependence of inhibition gave estimates of 34.72 microM(-1) s(-1) and 116.39 s(-1) for the rate constants of association and dissociation, respectively. The resulting apparent Kd was 3.35 microM, similar to the IC50 value obtained from the concentration-response curve. In current clamp configuration, fluoxetine (3 microM) induced depolarization of resting membrane potential and reduced the rate of action potential. Our results indicate that fluoxetine produces a concentration- and voltage-dependent inhibition of IA, and that this effect could affect the excitability of hippocampal neurons.