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.

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.

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.

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.

Effects of physiological quercetin metabolites on interleukin-1ß-induced inducible NOS expression.

Cytokines released by inflammatory cells around the pancreatic islets are implicated in the pathogenesis of diabetes mellitus. Specifically, interleukin-1ß (IL-1ß) is known to be involved in islet ß-cell damage by activation of nuclear factor-κB (NF-κB)-mediated inducible nitric oxide synthase (iNOS) gene expression. Though most flavonoids are shown to have various beneficial effects, little is known about the anti-inflammatory effects of their metabolites. Therefore, we investigated the effects of quercetin and its metabolites quercetin 3'-sulfate, quercetin 3-glucuronide and isorhamnetin 3-glucuronide on IL-1ß-stimulated iNOS gene expression in RINm5F ß-cells. The nitrite level, iNOS protein and its mRNA expression levels and iNOS promoter activity were measured. In addition, IκBα protein phosphorylation, nuclear translocation of nuclear factor-κB (NF-κB) and NF-κB DNA binding activity were determined. Adenosine 5'-triphosphate disodium salt-induced insulin release was also measured. Quercetin significantly reduced IL-1ß-induced nitrite production, iNOS protein and its mRNA expression levels, and it also inhibited IL-1ß-induced IκBα phosphorylation, NF-κB activation and iNOS promoter activity. Additionally, quercetin significantly restored the inhibition of insulin secretion by IL-1ß. Meanwhile, quercetin metabolites did not show any effect on IL-1ß-induced iNOS gene expression and also on insulin secretion. Therefore, in terms of iNOS expression mechanism, dietary ingestion of quercetin is unlikely to show anti-inflammatory effects in rat islet ß-cells exposed to IL-1ß.

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.

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

Phenolic compounds affect intracellular free Ca(2+) concentration ([Ca(2+)](i)) signaling. The study examined whether the simple phenolic compound octyl gallate affects ATP-induced Ca(2+) signaling in PC12 cells using fura-2-based digital Ca(2+) imaging and whole-cell patch clamping. Treatment with ATP (100 microM) for 90 s induced increases in [Ca(2+)](i) in PC12 cells. Pretreatment with octyl gallate (100 nM to 20 microM) for 10 min inhibited the ATP-induced [Ca(2+)](i) response in a concentration-dependent manner (IC(50)=2.84 microM). Treatment with octyl gallate (3 microM) for 10 min significantly inhibited the ATP-induced response following the removal of extracellular Ca(2+) with nominally Ca(2+)-free HEPES HBSS or depletion of intracellular Ca(2+) stores with thapsigargin (1 microM). Treatment for 10 min with the L-type Ca(2+) channel antagonist nimodipine (1 microM) significantly inhibited the ATP-induced [Ca(2+)](i) increase, and treatment with octyl gallate further inhibited the ATP-induced response. Treatment with octyl gallate significantly inhibited the [Ca(2+)](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 microM) 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 [Ca(2+)](i) increase in PC12 cells by inhibiting both non-selective P2X receptor-mediated influx of Ca(2+) from extracellular space and P2Y receptor-induced release of Ca(2+) from intracellular stores in protein kinase-independent manner. In addition, octyl gallate inhibits the ATP-induced Ca(2+) responses by inhibiting the secondary activation of voltage-gated Ca(2+) channels.

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.

Exendin-4 inhibits interleukin-1beta-induced iNOS expression at the protein level, but not at the transcriptional and posttranscriptional levels, in RINm5F beta-cells.

Cytokines such as interleukin-1beta (IL-1beta) stimulate inducible nitric oxide synthase (iNOS) expression and nitric oxide overproduction leading to beta-cell damage. Meanwhile, glucagon-like peptide-1 (GLP-1) and its potent analog exendin-4 (EX-4) were well known for beta-cell proliferation. However, the protective mechanisms of GLP-1 in beta-cells exposed to cytokines were not fully elucidated. Therefore, the effects of EX-4 on the IL-1beta-induced iNOS gene expression were investigated employing RINm5F beta-cells. EX-4 inhibited IL-1beta-induced iNOS protein expression and nitrite production. However, northern blot and promoter analyses showed that EX-4 failed to inhibit IL-1beta-induced iNOS mRNA expression and iNOS promoter activity. By electrophoretic mobility shift assay (EMSA), EX-4 did not alter the binding activity of NF-kappaB to the iNOS promoter. Consistent with the EMSA result, EX-4 did not inhibit nuclear translocation of p65. We also tested the effect of EX-4 on iNOS mRNA stability. Actinomycin D chase experiments showed that EX-4 did not affect the decay rate of iNOS mRNA and the promoter assay using the construct containing 3'-untranslated region of iNOS showed that EX-4 did not alter the stability of iNOS mRNA. Meanwhile, forskolin significantly inhibited IL-1beta-induced iNOS protein, which was reversed by H-89, a protein kinase A (PKA) inhibitor. Moreover, EX-4 pretreatment restored IL-1beta-induced decrease in cAMP toward control level. Additionally, the cycloheximide chase study demonstrated that EX-4 significantly accelerated iNOS protein degradation. We therefore concluded that EX-4 inhibited IL-1beta-induced iNOS protein and nitrite production via cAMP/PKA system irrespective of both transcriptional and posttranscriptional mechanisms of iNOS gene, and this inhibitory effect of EX-4 appears to be regulated at posttranslational level.

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.

Differential cholinergic modulation of Ca2+ transients evoked by backpropagating action potentials in apical and basal dendrites of cortical pyramidal neurons.

The effect of the cholinergic agonist carbachol (CCh) on backpropagating action potential (bAP)-evoked Ca2+ transients in distal apical and basal dendrites of layer 2/3 pyramidal neurons in the primary visual cortex of rats was studied using whole cell recordings and confocal Ca2+ imaging. In the presence of CCh (20 microM), initial bAP-evoked Ca2+ transients were followed by large propagating secondary Ca2+ transients that were restricted to proximal apical dendrites < or =40 microm from the soma. In middle apical dendrites (41-100 microm from the soma), Ca2+ transients evoked by AP bursts at 20 Hz, but not by single APs, were increased by CCh without secondary transients. CCh failed to increase the bAP-evoked Ca2+ transients in distal apical dendrites (101-270 microm from the soma). In contrast, in basal dendrites, CCh increased Ca2+ transients evoked by AP bursts, but not by single APs, and these transients were relatively constant over the entire length of the dendrites. CCh further increased the enhanced bAP-evoked Ca2+ transients in the presence of 4-aminopyridine (200 microM), an A-type K+ channel blocker, in basal and apical dendrites, except in distal apical dendrites. CCh increased large Ca2+ transients evoked by high-frequency AP bursts in basal dendrites, but not in distal apical dendrites. CCh-induced increase in Ca2+ transients was mediated by InsP3-dependent Ca2+-induced Ca2+-release. These results suggest that cholinergic stimulation differentially increases the bAP-evoked increase in [Ca2+]i in apical and basal dendrites, which may modulate synaptic activities in a location-dependent manner.

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.

Effects of Apigenin on Glutamate-induced [Ca](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 Ca(2+) concentration ([Ca(2+)](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 (1 microM to 100 microM) for 5 min inhibited glutamate (100 microM, 1 min) induced [Ca(2+)](i) increase, concentration-dependently. Pretreatment with apigenin (30 microM) for 5 min significantly decreased the [Ca(2+)](i) responses induced by two ionotropic glutamate receptor agonists, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA, 10 microM, 1 min) and N-methyl-D-aspartate (NMDA, 100 microM, 1 min), and significantly inhibited the AMPA-induced peak currents. Treatment with apigenin also significantly inhibited the [Ca(2+)](i) response induced by 50 mM KCl solution, decreased the [Ca(2+)](i) responses induced by the metabotropic glutamate receptor agonist, (S)-3,5-dihydroxyphenylglycine (DHPG, 100 microM, 90 s), and inhibited the caffeine (10 mM, 2 min)-induced [Ca(2+)](i) responses. Furthermore, treatment with apigenin (30 microM) significantly inhibited the amplitude and frequency of 0.1 mM [Mg(2+)](o)-induced [Ca(2+)](i) spikes. These data together suggest that apigenin inhibits glutamate-induced calcium signaling in cultured rat hippocampal neurons.

Effect of KAI1/CD82 on the beta1 integrin maturation in highly migratory carcinoma cells.

The KAI1/CD82 protein has been documented as the tumor metastasis suppressor in many types of human cancers. KAI1/CD82 regulates cell motility and invasiveness; however, the mechanism by which this occurs remains to be fully established. Several studies have shown that KAI1/CD82 modulates integrin-dependent signaling. It was suggested that KAI1/CD82 might function to attenuate the beta1 integrin function of inducing cellular migration. A wound-healing and modified Boyden chamber assays were performed to investigate the mechanism of the KAI1/CD82-mediated inhibition of cell migration. It was found that the migratory ability of H1299/CD82 was inhibited. The immunoblotting and biotinylation assays revealed that H1299/CD82 showed significantly decreased expression of the mature form of beta1, which was functional at the cell surface. It was confirmed that KAI1/CD82 regulates the maturation of the beta1 integrin using CD82-specific si-RNA. These results support a model in which KAI1/CD82 attenuates the maturation of the beta1 integrin precursor and thereby suppresses cell migration.

Open channel block of A-type, kv4.3, and delayed rectifier K+ channels, Kv1.3 and Kv3.1, by sibutramine.

The effects of sibutramine on voltage-gated K+ channel (Kv)4.3, Kv1.3, and Kv3.1, stably expressed in Chinese hamster ovary cells, were investigated using the whole-cell patch-clamp technique. Sibutramine did not significantly decrease the peak Kv4.3 currents, but it accelerated the rate of decay of current inactivation in a concentration-dependent manner. This phenomenon was effectively characterized by integrating the total current over the duration of a depolarizing pulse to +40 mV. The IC50 value for the sibutramine block of Kv4.3 was 17.3 microM. Under control conditions, the inactivation of Kv4.3 currents could be fit to a biexponential function, and the time constants for the fast and slow components were significantly decreased after the application of sibutramine. The association (k+1) and dissociation (k-1) rate constants for the sibutramine block of Kv 4.3 were 1.51 microM-1s-1 and 27.35 s-1, respectively. The theoretical KD value, derived from k-1/k+1, yielded a value of 18.11 microM. The block of Kv4.3 by sibutramine displayed a weak voltage dependence, increasing at more positive potentials, and it was use-dependent at 2 Hz. Sibutramine did not affect the time course for the deactivating tail currents. Neither steady-state activation and inactivation nor the recovery from inactivation was affected by sibutramine. Sibutramine caused the concentration-dependent block of the Kv1.3 and Kv3.1 currents with an IC50 value of 3.7 and 32.7 microM, respectively. In addition, sibutramine reduced the tail current amplitude and slowed the deactivation of the tail currents of Kv1.3 and Kv3.1, resulting in a crossover phenomenon. These results indicate that sibutramine acts on Kv4.3, Kv1.3, and Kv3.1 as an open channel blocker.

Proximal cyclic AMP response element is essential for exendin-4 induction of rat EGR-1 gene.

Glucagon-like peptide-1 and its potent agonist exendin-4 induce several immediate early response genes (IEGs) that code for transcription factors implicated in cell proliferation, differentiation, and apoptosis. We recently observed that early growth response factor-1 (EGR-1), an IEG product, was required for transcriptional activation of Ccnd1 (cyclin D1) gene by exendin-4. Herein, the regulatory mechanism whereby exendin-4 activates the transcription of EGR-1 gene was investigated in the pancreatic beta-cell line INS-1. Deletion analysis of rat EGR-1 promoter identified a critical region between -73 and -46 for the activation of EGR-1 in response to exendin-4. Mutation of the proximal putative cAMP response element (CRE, 5'-GTACGTCA-3') located at -69 resulted in a significant decrease in the EGR-1 transcription, whereas the mutation of the distal putative CRE at -139 was without such an effect. In immune supershift assays using exendin-4-treated cells, binding of cAMP response element-binding protein (CREB) phosphorylated on Ser(133) to the proximal CRE was increased. Employment of a CREB mutant containing Ala substitution at Ser(133) or a dominant negative CREB mutant that inhibits the binding of endogenous CREB to DNA significantly decreased the exendin-4-induced EGR-1 transcription. In experiments using specific protein kinase inhibitors, the effect of H-89 was more prominent than PD-98059, indicating the predominance of the PKA signaling over the MEK/ERK in induction of EGR-1. Therefore, it appears that the proximal CRE site is critical and the binding with CREB phosphorylated on Ser(133) is necessary for induction of the EGR-1 transcription by exendin-4.

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.

Calcineurin-independent inhibition of KV1.3 by FK-506 (tacrolimus): a novel pharmacological property.

The interaction of FK-506 with K(V)1.3, stably expressed in Chinese hamster ovary cells, was investigated with the whole cell patch-clamp technique. FK-506 inhibited K(V)1.3 in a reversible, concentration-dependent manner with an IC(50) of 5.6 microM. Rapamycin, another immunosuppressant, produced effects that were similar to those of FK-506 (IC(50) = 6.7 microM). Other calcineurin inhibitors (cypermethrin or calcineurin autoinhibitory peptide) alone had no effect on the amplitude or kinetics of K(V)1.3. In addition, the inhibitory action of FK-506 continued, even after the inhibition of calcineurin activity. The inhibition produced by FK-506 was voltage dependent, increasing in the voltage range for channel activation. At potentials positive to 0 mV (where maximal conductance is reached), however, no voltage-dependent inhibition was found. FK-506 exhibited a strong use-dependent inhibition of K(V)1.3. FK-506 shifted the steady-state inactivation curves of K(V)1.3 in the hyperpolarizing direction in a concentration-dependent manner. The apparent dissociation constant for FK-506 to inhibit K(V)1.3 in the inactivated state was estimated from the concentration-dependent shift in the steady-state inactivation curve and was calculated to be 0.37 microM. Moreover, the rate of recovery from inactivation of K(V)1.3 was decreased. In inside-out patches, FK-506 not only reduced the current amplitude but also accelerated the rate of inactivation during depolarization. FK-506 also inhibited K(V)1.5 and K(V)4.3 in a concentration-dependent manner with IC(50) of 4.6 and 53.9 microM, respectively. The present results indicate that FK-506 inhibits K(V)1.3 directly and that this effect is not mediated via the inhibition of the phosphatase activity of calcineurin.

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.