The mineralocorticoid receptor and extra-synaptic NMDA receptor in the lateral habenula involve in the vulnerability to early life stress in the maternal separation model.

The lateral habenula (LHb) plays a pivotal role in regulating emotional responses during stress reactions, and its hyperactivity has been associated with depression. Recently it has been demonstrated that chronic early-life stress results in individual differences in stress vulnerability among rodents. However, how synaptic function in the LHb varies between susceptibility and resilience to early life stress remains elusive. In this study, we used a maternal separation model to assign animals with different stress vulnerabilities into groups and investigated the synaptic responses in the LHb. Our findings indicate that synaptic long-term depression (LTD) was impaired and extra-synaptic LTD was enhanced in the LHb of the susceptible group. To mimic the synaptic alteration in stress situations, when administered corticosterone, a stress hormone, the intervention appeared to impair synaptic LTD in the LHb of the control group, through the activation of mineralocorticoid receptors (MR). Indeed, there was an up-regulation of MR mRNA observed in the susceptible group. Following there was an up-regulation of both NR2A and NR2B subunits in the LHb. These results indicated that MR and extra-synaptic NMDA receptors in LHb are critically engaged in the susceptibilities to stress. Furthermore, our findings propose potential therapeutic targets for alleviating stress-related symptoms.

NMDA receptor-dependent long-term depression in the lateral habenula: implications in physiology and depression.

Abnormally increased neuronal activity in the lateral habenula (LHb) is closely associated with depressive-like behavior. Despite the emphasis on the pathological importance of NMDA receptor (NMDAR)-dependent long-term depression (LTD) and the involvement of calcium permeable AMPA receptor (CP-AMPAR) as major Ca2+ source, the functions of NMDAR and CP-AMPAR on LTD modulation in the LHb still have not been fully investigated. Here, we found that NMDAR-dependent LTD by low frequency stimulation was induced in both synaptic and extrasynaptic regions in the LHb. In addition, CP-AMPAR was necessary for the activation of NMDAR in the induction phase of NMDAR-dependent LTD. The acute stress, which induced depressive behavior, had a blocked effect on synaptic NMDAR-dependent LTD but left extrasynaptic NMDAR-dependent LTD intact. These findings show that NMDAR-dependent LTD in LHb plays an important role in regulating neuronal activity, which is probable to be excessively increased by repeated stress, via maintaining homeostasis in both synaptic and extrasynaptic regions of the LHb. Moreover, NMDAR and CP-AMPAR may serve as a depression-related modulator and be regarded as a promising therapeutic target for treatment of psychopathology such as depression.

Modulation of Dopaminergic Neuronal Excitability by Zinc through the Regulation of Calcium-related Channels.

Depending on the intracellular buffering of calcium by chelation, zinc has the following two apparent effects on neuronal excitability: enhancement or reduction. Zinc increased tonic activity in the depolarized state when neurons were intracellularly dialyzed with EGTA but attenuated the neuronal activity when BAPTA was used as an intracellular calcium buffer. This suggests that neuronal excitability can be modulated by zinc, depending on the internal calcium buffering capacity. In this study, we elucidated the mechanisms of zinc-mediated alterations in neuronal excitability and determined the effect of calcium-related channels on zinc-mediated alterations in excitability. The zinc-induced augmentation of firing activity was mediated via the inhibition of small-conductance calcium-activated potassium (SK) channels with not only the contribution of voltage-gated L-type calcium channels (VGCCs) and ryanodine receptors (RyRs), but also through the activation of VGCCs via melastatin-like transient receptor potential channels. We suggest that zinc modulates the dopaminergic neuronal activity by regulating not only SK channels as calcium sensors, but also VGCCs or RyRs as calcium sources. Our results suggest that the cytosolic calcium-buffering capacity can tightly regulate zinc-induced neuronal firing patterns and that local calcium-signaling domains can determine the physiological and pathological state of synaptic activity in the dopaminergic system.

Repeated nicotine exposure in adolescent rats: Reduction of medial habenular activity and augmentation of nicotine preference.

Adolescence is a critical period for the initiation of tobacco use. Nicotine not only stimulates brain reward circuits to establish and maintain the tobacco smoking habit, but also produces aversive reactions to nicotine after initial exposure, due to its noxious properties. Although new insights into the mechanisms that regulate nicotine avoidance could result in an advantageous treatment strategy for addiction, little is known about the mechanism of nicotine aversion in adolescence. Because growing evidences suggest that the habenula to interpeduncular nucleus circuitry plays a critical role in nicotine aversion, we investigated the effects of repeated nicotine exposure on the electrical activity of medial habenular neurons in adolescent rats, using extracellular recordings. Nicotine strongly increased the frequency of spontaneous spike activity in the medial habenula of naïve rats. In repeated nicotine-injected rats, we found a reduction in nicotine-induced spontaneous spike frequency, such that these neurons displayed a significantly lower basal activity and reduced spontaneous activity upon re-exposure to nicotine. Moreover, nicotine intake preference in repeated nicotine-injected rats is significantly more increased than that in saline-injected rats. These results demonstrate that repeated phases of nicotine exposure induce a functional switch in the activity of medial habenular neurons in adolescent rats and suggest that medial habenular activity is one of mediators for an inhibitory motivational signal that limits nicotine consumption.

5HT(1B) receptor-mediated pre-synaptic depression of excitatory inputs to the rat lateral habenula.

Accumulating lines of evidence indicate that the lateral habenula (LHb), which reciprocally interacts with raphe nuclei (RN), displays hyperactivity including synaptic potentiation of excitatory inputs to the LHb during a depressed state. Despite the potential importance of glutamatergic excitatory synapses in depression-like behavior, modulation of these LHb synapses by monoamines such as serotonin (5HT) is not fully understood at the cellular and molecular level. Therefore, we used whole cell voltage-clamp recording to examine the molecular mechanisms by which 5HT modulates glutamatergic transmission in the LHb. The present study provides the first evidence that glutamatergic transmission of LHb synapses is inhibited by activation of the 5HT(1B) receptor at the pre-synapse in both acute depression (5HT-AD) and long-term depression (5HT-LTD). We further show that 5HT-AD results from the activation of Shaker-type K(+) channels whereas 5HT-LTD depends on inhibition of the adenylyl cyclase-cAMP (AC-cAMP) pathway with an increase in pre-synaptic Ca(2+) release from ryanodine-sensitive internal stores in an NO-dependent manner.

Attenuated effects of Neu2000 on hypoxia-induced synaptic activities in a rat hippocampus.

Neu2000 (NEU; 2-hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethyl-benzylamino)-benzoic acid), a recently developed derivative of acetylsalicylic acid and sulfasalazine, potently protects against neuronal cell death following ischemic brain injury by antagonizing NMDA receptor-mediated neuronal toxicity and oxidative stress. However, it has yet to be determined whether NEU can attenuate hypoxia-induced impairment of neuronal electrical activity. In this study, we carried out extracellular recordings of hippocampal slices in order to investigate the effects of NEU on the electrical activity of neurons exposed to a hypoxic insult (oxygen and glucose deprivation). NEU prominently suppressed hypoxia-induced impairment of neuronal activity in a concentration-dependent manner. NEU, at a low dose (1 µM), competently depressed the hypoxia-induced convulsive activity in a manner similar to trolox. Furthermore, high concentrations of NEU (50 µM) markedly abolished all hypoxia-mediated impairment of neuronal activity and accelerated the slow recovery of neuronal activity more efficiently than ifenprodil and APV. These results suggest that NEU attenuates hypoxia-induced impairment of neuronal activity more potently than the antioxidant, trolox, and the NMDA receptor antagonists, ifenprodil and APV. We propose that NEU is a striking pharmacological candidate for neuroprotection against hypoxia because of its defensive action on hypoxia-mediated impairment of electrical neurotransmission as well as its neuroprotective action against neuronal cell death induced by exposure to pathological hypoxic conditions.

Neu2000 potentiates a kainate response in mouse cortical neurons.

Neu2000, acting as an antioxidant with N-methyl-d-aspartate-receptor antagonism, demonstrates excellent protection against ischemic insults in rodents. In this study, we report that Neu2000 also dramatically enhances the activity of kainate (KA) receptors. Neu2000 non-competitively and reversibly potentiated KA-evoked responses in a voltage-independent manner, mainly by increasing the open probability of KA receptor channels.

HYP-1, a novel diamide compound, relieves inflammatory and neuropathic pain in rats.

In the present study, we investigated whether a novel compound, 2-(2-(4-((4-chlorophenyl)(phenyl)methyl) piperazin-1-yl)-2-oxoethylamino)-N-(3,4,5-trimethoxybenzyl)acetamide (HYP-1), is capable of binding to voltage-gated sodium channels (VGSCs) and evaluated both its inhibitory effect on Na+ currents of the rat dorsal root ganglia (DRG) sensory neuron and its in vivo analgesic activity using rat models of inflammatory and neuropathic pain. HYP-1 showed not only high affinity for rat sodium channel (site 2), but also potent inhibitory activity against the TTX-R Na+ currents of the rat DRG sensory neuron. HYP-1 co-injected with formalin (5%, 50 µl) under the plantar surface of rat hind paw dose-dependently reduced spontaneous pain behaviors during both the early and late phases. This result was confirmed by c-Fos immunofluorescence in the L4-5 spinal segments. A large number of c-Fos-positive neurons were observed in rat injected with a mixture of formalin and vehicle, but not in rat treated with a mixture of formalin and HYP-1. In addition, the effectiveness of HYP-1 (6 and 60 mg/kg, i.p.) in suppression of neuropathic pain, such as mechanical, cold and warm allodynia, induced by rat tail nerve injury was investigated. HYP-1 showed limited selectivity over hERG, N-type and T-type channels.Our present results indicate that HYP-1, as a VGSC blocker, has potential analgesic activities against nociceptive, inflammatory and neuropathic pain.

Neu2000, an NR2B-selective, moderate NMDA receptor antagonist and potent spin trapping molecule for stroke.

Excess activation of ionotropic glutamate receptors, primarily N-methyl-D-aspartate (NMDA) receptors and free radicals, evoke nerve cell death following hypoxic-ischemic brain injury in various animal models. However, clinical trials in stroke patients using NMDA receptor antagonists have failed to show efficacy primarily due to the limited therapeutic time window for neuroprotection and a narrow therapeutic index. In comparison, antioxidants prolonged the time window for neuroprotection in animal models of ischemic stroke and showed greater therapeutic potential in clinical trials for ischemic stroke. Excess activation of NMDA receptors and free radicals mediate the two separate pathways of nerve cell death in stroke and a safe and multifunctional drug that can block both routes in the brain will likely provide a better therapeutic outcome in patients with stroke. Derivatives of the lead structures of sulfasalazine and aspirin have led to the discovery of a new molecule, Neu2000, that has demonstrated excellent neuroprotection against NMDA- and free radical-induced cell death. Neu2000 is an NR2B-selective, moderate NMDA receptor antagonist with potent cell-permeable, spin trapping antioxidant action even at nanomolar concentrations. Nonclinical and human phase I studies demonstrated that Neu2000 can be translated to treat patients with stroke with better efficacy and therapeutic time window.

Zn2+ enhances the intrinsic bursting activity of a rat thalamic relay neuron.

Although zinc ion (Zn2+) reduced the low-threshold T-type Ca2+ current of a rat thalamic relay neuron (TRN), we observed that Zn2+ increased a bursting activity of TRN by altering the generation and maintenance of low-threshold spike (LTS). Interestingly and importantly, Zn2+ shifted dramatically the voltage-dependence of both steady-state inactivation and activation of the transient A-type K+ current (I(A)) to a depolarizing direction. As I(A) is one of the main factors in shaping thalamic LTS, such alterations of gating properties of I(A) would contribute to the enhancement of TRN excitability under Zn2+.

A novel mechanism of zinc block on alpha1G-like low-threshold T-type Ca2+ channels in a rat thalamic relay neuron.

To elucidate biophysical mechanisms underlying the Zn2+ block on the low-threshold T-type Ca2+ current (I(T)), we examined the Zn(2+)-induced alterations of gating properties of I(T) of a rat thalamic relay neuron and of alpha1G channels expressed in HEK-293 cells, using a whole-cell voltage clamp technique. The effect of Zn2+ block depended upon holding potentials but not test potentials, indicating that, the greater the inactivation, the less Zn2+ blocked I(T). Except for the inactivation near the activation threshold of I(T), no significant changes in the kinetics of activation and inactivation were induced by Zn2+. In contrast, the rates of both de-inactivation and deactivation were dramatically increased by Zn2+, and moreover the channels were rapidly re-blocked upon re-polarization under Zn2+. Furthermore, the outward current via alpha1G channel was almost insensitive to Zn2+. All these results imply that Zn2+ alters the gating properties of I(T) mainly by accelerating its deactivation process.

The novel NMDA receptor antagonist, 2-hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethyl-benzylamino)-benzoic acid, is a gating modifier in cultured mouse cortical neurons.

Neu2000 [NEU, 2-hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethyl-benzylamino)-benzoic acid], a derivative of sulfasalazine, attenuates NMDA-induced neuronal toxicity. Here we investigated the effects of NEU on the NMDA receptor (NMDAR) using whole-cell patch clamp technique to determine the molecular mechanisms underlying its neuroprotective role. NEU reversibly suppressed NMDA responses in an uncompetitive manner with fast binding kinetics. Its inhibition of NMDAR activity depended on both the concentration and the use of agonist but not on the membrane potential. NEU accelerated NMDA desensitization without affecting the binding affinity of NMDAR for its agonists and stabilized the closed state of NMDAR. Therefore, NEU should effectively alleviate disorders that are a result of glutamate excitoxicity with fewer side effects because it is a low-affinity gating modifier that antagonizes NMDAR in an uncompetitive manner. Moreover, in the presence of ifenprodil (an NR2B antagonist) but not NVP-AAM077 [(R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydro-quinoxalin-5-yl)-methyl]-phosphonic acid, an NR2A antagonist], the extent of NEU block was decreased, suggesting that NEU is an NR2B-specific antagonist.

Extracellular ATP mediates necrotic cell swelling in SN4741 dopaminergic neurons through P2X7 receptors.

Extracellular ATP has recently been identified as an important regulator of cell death in response to pathological insults. When SN4741 cells, which are dopaminergic neurons derived from the substantia nigra of transgenic mouse embryos, are exposed to ATP, cell death occurs. This cell death is associated with prominent cell swelling, loss of ER integrity, the formation of many large cytoplasmic vacuoles, and subsequent cytolysis and DNA release. In addition, the cleavage of caspase-3, a hallmark of apoptosis, is induced by ATP treatment. However, caspase inhibitors do not overcome ATP-induced cell death, indicating that both necrosis and apoptosis are associated with ATP-induced cell death and suggesting that a necrotic event might override the apoptotic process. In this study we also found that P2X(7) receptors (P2X(7)Rs) are abundantly expressed in SN4741 cells, and both ATP-induced swelling and cell death are reversed by pretreatment with the P2X(7)Rs antagonist, KN62, or by knock-down of P2X(7)Rs with small interfering RNAs. Therefore, extracellular ATP release from injured tissues may act as an accelerating factor in necrotic SN4741 dopaminergic cell death via P2X(7)Rs.

Cholecystokinin-8S-induced intracellular calcium signaling in acutely isolated periaqueductal gray neurons of the rat.

Many behavior studies indicate that cholecystokinin (CCK) is related to nociception and anxiety/panic actions in the midbrain periaqueductal gray (PAG). We previously reported that a sulfated form of CCK octapeptide (CCK-8S) produced excitatory effects at both pre- and postsynaptic loci in PAG neurons using slice preparations and whole-cell patch-clamp recordings. Here, we further examined the detailed mechanism of CCK-8S in acutely isolated PAG neurons of the rat using fura-2-based imaging of intracellular Ca2+ concentration ([Ca2+]i) and whole-cell patch-clamp recordings. Application of 1 microM CCK-8S produced an increase of [Ca2+]i, and its effect did not desensitize. This CCK-8S-induced [Ca2+]i increase was inhibited by the CCK2 receptor antagonist L-365260 but not by the CCK1 receptor antagonist L-364718. In addition, the effect of CCK-8S was eliminated by removing extracellular Ca2+, but not by an addition of the intracellular Ca2+ reuptake inhibitor thapsigargin. When simultaneous recordings of [Ca2+]i imaging and whole-cell patch-clamp were performed, CCK-8S-induced [Ca2+]i increase was significantly reduced at a membrane holding potential of -60 mV while CCK-8S-induced inward current was still observed. Current-voltage plots revealed that CCK-8S-induced inward current reversed near the equilibrium potential for K+ ions with a decreased membrane conductance. However, CCK-8S produced a significant inhibition on high-voltage-activated Ca2+ channel currents. These results suggest that CCK-8S can excite PAG neurons by inhibiting K+ channels, and CCK-8S-induced [Ca2+]i increase occurs secondary to depolarization. The evidence presented here expands our understanding of cellular mechanisms for CCK-mediated anti-analgesic and anxiogenic actions in the PAG.

Neuroprotective effects of ginsenoside Rg3 against homocysteine-induced excitotoxicity in rat hippocampus.

We previously demonstrated that ginsenoside Rg(3) (Rg(3)), one of the active ingredients in Panax ginseng, attenuates NMDA receptor-mediated currents and NMDA-induced neurotoxicity (Kim, S., Kim, T., Ahn, K., Park, W.K., Nah, S.Y., Rhim, H., 2004. Ginsenoside Rg(3) antagonizes NMDA receptors through a glycine modulatory site in rat cultured hippocampal neurons. Biochem. Biophys. Res. Commun. 323, 416-424). Accumulating evidence suggests that homocysteine (HC), a metabolite of methionine, exerts its excitotoxicity through NMDA receptor activation. In the present study, we examined the neuroprotective effects of Rg(3) on HC-induced hippocampal excitotoxicity in vitro and in vivo. Our in vitro studies using rat cultured hippocampal neurons revealed that Rg(3) treatment significantly and dose-dependently inhibited HC-induced hippocampal cell death, with an EC(50) value of 28.7+/-7.5 muM. Rg(3) treatment not only significantly reduced HC-induced DNA damage, but also dose-dependently attenuated HC-induced caspase-3 activity in vitro. Our in vivo studies revealed that intracerebroventricular (i.c.v.) pre-administration of Rg(3) significantly and dose-dependently reduced i.c.v. HC-induced hippocampal damage in rats. To examine the mechanisms underlying the in vitro and in vivo neuroprotective effects of Rg(3) against HC-induced hippocampal excitotoxicity, we examined the effect of Rg(3) on HC-induced intracellular Ca(2+) elevations in cultured hippocampal cells and found that Rg(3) treatment dose-dependently inhibited HC-induced intracellular Ca(2+) elevation, with an IC(50) value of 41.5+/-17.5 muM. In addition, Rg(3) treatment dose-dependently inhibited HC-induced currents in Xenopus oocytes expressing the NMDA receptor, with an IC(50) of 47.3+/-14.2 muM. These results collectively indicate that Rg(3)-induced neuroprotection against HC in rat hippocampus might be achieved via inhibition of HC-mediated NMDA receptor activation.

Marked prevention of ischemic brain injury by Neu2000, an NMDA antagonist and antioxidant derived from aspirin and sulfasalazine.

Excitotoxicity and oxidative stress mediate neuronal death after hypoxic-ischemic brain injury. We examined the possibility that targeting both N-methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity and oxidative stress would result in enhanced neuroprotection against hypoxic-ischemia. 2-Hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethyl-benzylamino)-benzoic acid (Neu2000) was derived from aspirin and sulfasalazine to prevent both NMDA neurotoxicity and oxidative stress. In cortical cell cultures, Neu2000 was shown to be an uncompetitive NMDA receptor antagonist and completely blocked free radical toxicity at doses as low as 0.3 micromol/L. Neu2000 showed marked neuroprotection in a masked fashion using histology and behavioral testing in two rodent models of focal cerebral ischemia without causing neurotoxic side effects. Neu2000 protected against the effects of middle cerebral artery occlusion, even when delivered 8 h after reperfusion. Single bolus administration of the drug prevented gray and white matter degeneration and spared neurologic function for over 28 days after MACO. Neu2000 may be a novel therapy for combating both NMDA receptor-mediated excitotoxicity and oxidative stress, the two major routes of neuronal death in ischemia, offering profound neuroprotection and an extended therapeutic window.

Cellular action of cholecystokinin-8S-mediated excitatory effects in the rat periaqueductal gray.

The peptide cholecystokinin (CCK) is one of the major neurotransmitters modulating satiety, nociception, and anxiety behavior. Although many behavioral studies showing anti-analgesic and anxiogenic actions of CCK have been reported, less is known about its cellular action in the central nervous system (CNS). Therefore, we examined the action of CCK in rat dorsolateral periaqueductal gray (PAG) neurons using slice preparations and whole-cell patch-clamp recordings. Application of CCK-8S produced an inward current accompanied by increased spontaneous synaptic activities. The CCK-8S-induced inward current (I(CCK)) was recovered after washout and reproduced by multiple exposures. Current-voltage plots revealed that I(CCK) reversed near the equilibrium potential for K(+) ions with a decreased membrane conductance. When several K(+) channel blockers were used, application of CdCl(2), TEA, or apamin significantly reduced I(CCK). I(CCK) was also significantly reduced by the CCK(2) receptor antagonist, L-365,260, while it was not affected by the CCK(1) receptor antagonist, L-364,718. Furthermore, we examined the effects of CCK-8S on miniature excitatory postsynaptic currents (mEPSCs) in order to determine the mechanism of CCK-mediated increase on synaptic activities. We found that CCK-8S increased the frequency of mEPSCs, but had no effect on mEPSC amplitude. This presynaptic effect persisted in the presence of CdCl(2) or Ca(2+)-free bath solution, but was completely abolished by pre-treatment with BAPTA-AM, thapsigargin or L-365,260. Taken together, our results indicate that CCK can excite PAG neurons at both pre- and postsynaptic loci via the activation of CCK(2) receptors. These effects may be important for the effects of CCK on behavior and autonomic function that are mediated via PAG neurons.

Underlying mechanism for NMDA receptor antagonism by the anti-inflammatory drug, sulfasalazine, in mouse cortical neurons.

Sulfasalazine (SULFA), of anti-inflammatory drugs, shows a protective action against NMDA-induced neuronal toxicity. Here, we used an electrophysiological study of the pharmacological effects of SULFA on NMDA receptors to examine the molecular mechanisms underlying the neuroprotective role of SULFA. The drug acted as a typical noncompetitive inhibitor with neither agonist- nor use-dependency, and antagonized NMDA-evoked responses in a voltage-independent manner, suggesting that SULFA is not an open channel blocker. Noise and single channel analyses showed that SULFA-blocked NMDA responses by reducing the number of NMDA channels available for activation, and also reduced the channel open probability without changing single channel conductance. Moreover, SULFA accelerated NMDA desensitization without affecting the affinity of the receptor for NMDA or glutamate. Taken together, these data indicate that SULFA blocks the NMDA response by reducing the number of NMDA channels available for activation. This appears to occur via a SULFA-induced decrease in the channel open probability, and a concomitant acceleration of the desensitization response, which is likely associated with a reduced affinity for glycine. SULFA indeed decreased the glycine-potentiated NMDA response without binding directly to the glycine site. Our results suggest that SULFA acts as a noncompetitive NMDA receptor antagonist with an allosteric glycine modulation.

Iron enhances NGF-induced neurite outgrowth in PC12 cells.

Rat pheochromocytoma 12 (PC12) cells undergo neuronal differentiation in response to nerve growth factor (NGF). NGF-induced differentiation involves a number of protein kinases, including extracellular signal-regulated kinase (ERK). We studied the effect of iron on neuronal differentiation, using as model the neurite outgrowth of PC12 cells triggered by NGF when the cells are plated on collagen-coated dishes in medium containing 1% serum. The addition of iron enhanced NGF-mediated cell adhesion, spreading and neurite outgrowth. The differentiation-promoting effect of iron seems to depend on intracellular iron, since nitrilotriacetic acid (an efficient iron-uptake mediator) enhanced the response to iron. In agreement with this, intracellular, but not extracellular, iron enhanced NGF-induced neurite outgrowth in pre-spread PC12 cells, and this was correlated with increased ERK activity. Taken together, these data suggest that intracellular iron promotes NGF-stimulated differentiation of PC12 cells by increasing ERK activity.

[Deoxycholic acid-induced signal transduction in HT-29 cells: role of NF-kappa B and interleukin-8].

BACKGROUND/AIMS: Deoxycholic acid (DCA) has been appeared to be an endogenous colon tumor promoter. In this study, we investigated whether DCA induces nuclear factor-kappa B (NF-kappa B) activation and IL-8 expression, and tauroursodeoxycholic acid (TUDC) inhibits this signaling in HT-29 cells. METHODS: After DCA treatments, time courses of NF-kappa B binding activity were determined by electrophoretic mobility shift assay (EMSA). Also, we performed Western blotting of I kappa B alpha to confirm NF-kappa B activation. Time and concentration courses of DCA-induced secretion of IL-8 were measured with ELISA in supernatants of cultured media from the cells. To evaluate the role of NF-kappa B, IL-8 levels were assessed after pretreatment with using phosphorothioate-modified anti-sense oligonucleotides (ODN). Moreover, DCA-induced secretions of IL-8 were measured after pretreatment with TUDC. RESULTS: DCA dose-dependently induced prominent NF-kappa B binding complexes from 30 min to 8 hr and degradation of I kappa B alpha. The secretions of IL-8 were increased with DCA (50-200 micro M) treatment in a time and dose-dependent manner. Pre-incubation of the cells with TUDC (0.1-10 micro M) for 2 hours caused significant decreases in DCA induced IL-8 secretion. However, transient transfection using p50 or p65 AS-ODN showed no effect on IL-8 secretion. CONCLUSIONS: DCA may play as a colonic tumor promoter through anti-apoptotic effect of NF-kappa B activation and IL-8 expression, and DCA-induced NF-kappa B independent IL-8 expression is inhibited by TUDC.