Sites and Regulation of L-Type Ca2+ Channel Cav1.2 Phosphorylation in Brain.

Cav1.2 channel phosphorylation plays an important role in regulating neuronal plasticity by action potential-dependent Ca2+ entry. Most studies of Cav1.2 regulation by phosphorylation have been reported in heart and muscles. Here, we identified phosphorylation sites of neuronal Cav1.2 channel protein purified from rat brain using mass spectrometry. The functional characterization of these phosphorylation sites showed altered voltage-dependent biophysical properties of the channel, without affecting current density. These results show that neuronal Cav1.2 channel is regulated by phosphorylation in a complex mechanism involving multiple phosphorylation sites.

Melatonin modulates nitric oxide-regulated WNK-SPAK/OSR1-NKCC1 signaling in dorsal raphe nucleus of rats.

The sleep-wake cycle is regulated by the alternating activity of sleep- and wake-promoting neurons. The dorsal raphe nucleus (DRN) secretes 5-hydroxytryptamine (5-HT, serotonin), promoting wakefulness. Melatonin secreted from the pineal gland also promotes wakefulness in rats. Our laboratory recently demonstrated that daily changes in nitric oxide (NO) production regulates a signaling pathway involving with-no-lysine kinase (WNK), Ste20-related proline alanine rich kinase (SPAK)/oxidative stress response kinase 1 (OSR1), and cation-chloride co-transporters (CCC) in rat DRN serotonergic neurons. This study was designed to investigate the effect of melatonin on NO-regulated WNK-SPAK/OSR1-CCC signaling in wake-inducing DRN neurons to elucidate the mechanism underlying melatonin's wake-promoting actions in rats. Ex vivo treatment of DRN slices with melatonin suppressed neuronal nitric oxide synthase (nNOS) expression and increased WNK4 expression without altering WNK1, 2, or 3. Melatonin increased phosphorylation of OSR1 and the expression of sodium-potassium-chloride co-transporter 1 (NKCC1), while potassium-chloride cotransporter 2 (KCC2) remained unchanged. Melatonin increased the expression of tryptophan hydroxylase 2 (TPH2, serotonin-synthesizing enzyme). The present study suggests that melatonin may promote its wakefulness by modulating NO-regulated WNK-SPAK/OSR1-KNCC1 signaling in rat DRN serotonergic neurons.

Amomum tsao-ko fruit extract suppresses lipopolysaccharide-induced inducible nitric oxide synthase by inducing heme oxygenase-1 in macrophages and in septic mice.

Amomum tsao-ko Crevost et Lemarié (Zingiberaceae) has traditionally been used to treat inflammatory and infectious diseases, such as throat infections, malaria, abdominal pain and diarrhoea. This study was designed to assess the anti-inflammatory effects and the molecular mechanisms of the methanol extract of A. tsao-ko (AOM) in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages and in a murine model of sepsis. In LPS-induced RAW 264.7 macrophages, AOM reduced the production of nitric oxide (NO) by inhibiting inducible nitric oxide synthase (iNOS) expression, and increased heme oxygenase-1 (HO-1) expression at the protein and mRNA levels. Pretreatment with SnPP (a selective inhibitor of HO-1) and silencing HO-1 using siRNA prevented the AOM-mediated inhibition of NO production and iNOS expression. Furthermore, AOM increased the expression and nuclear accumulation of NF-E2-related factor 2 (Nrf2), which enhanced Nrf2 binding to antioxidant response element (ARE). In addition, AOM induced the phosphorylation of extracellular regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) and generated reactive oxygen species (ROS). Furthermore, pretreatment with N-acetyl-l-cysteine (NAC; a ROS scavenger) diminished the AOM-induced phosphorylation of ERK and JNK and AOM-induced HO-1 expression, suggesting that ERK and JNK are downstream mediators of ROS during the AOM-induced signalling of HO-1 expression. In LPS-induced endotoxaemic mice, pretreatment with AOM reduced NO serum levels and liver iNOS expression and increased HO-1 expression and survival rates. These results indicate that AOM strongly inhibits LPS-induced NO production by activating the ROS/MAPKs/Nrf2-mediated HO-1 signalling pathway, and supports its pharmacological effects on inflammatory diseases.

Fulgidic Acid Isolated from the Rhizomes of Cyperus rotundus Suppresses LPS-Induced iNOS, COX-2, TNF-α, and IL-6 Expression by AP-1 Inactivation in RAW264.7 Macrophages.

To identify bioactive natural products possessing anti-inflammatory activity, the potential of fulgidic acid from the rhizomes of Cyperus rotundus and the underlying mechanisms involved in its anti-inflammatory activity were evaluated in this study. Fulgidic acid reduced the production of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-induced RAW264.7 macrophages. Consistent with these findings, fulgidic acid suppressed the LPS-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the protein level, as well as iNOS, COX-2, TNF-α, and IL-6 at mRNA levels. Fulgidic acid suppressed the LPS-induced transcriptional activity of activator protein-1 (AP-1) as well as the phosphorylation of c-Fos and c-Jun. On the other hand, fulgidic acid did not show any effect on LPS-induced nuclear factor κB (NF-κB) activity. Taken together, these results suggest that the anti-inflammatory effect of fulgidic acid is associated with the suppression of iNOS, COX-2, TNF-α, and IL-6 expression through down-regulating AP-1 activation in LPS-induced RAW264.7 macrophages.

Inhibitory effects of ß-chamigrenal, isolated from the fruits of Schisandra chinensis, on lipopolysaccharide-induced nitric oxide and prostaglandin E2 production in RAW 264.7 macrophages [corrected].

Much is known about the bioactive properties of lignans from the fruits of Schisandra chinensis. However, very little work has been done to determine the properties of sesquiterpenes in the fruits of S. chinensis. The aim of the present study was to investigate the anti-inflammatory potential of new sesquiterpenes (ß-chamigrenal, ß-chamigrenic acid, α-ylangenol, and α-ylangenyl acetate) isolated from the fruits of S. chinensis and to explore their effect on macrophages stimulated with lipopolysaccharide. Of these four sesquiterpenes, ß-chamigrenal most significantly suppressed lipopolysaccharide-induced nitric oxide and prostaglandin E2 production in RAW 264.7 macrophages (47.21 ± 4.54 % and 51.61 ± 3.95 % at 50 µM, respectively). Molecularly, the inhibitory activity of ß-chamigrenal on nitric oxide production was mediated by suppressing inducible nitric oxide synthase activity but not its expression. In the prostaglandin E2 synthesis pathway, ß-chamigrenal prevented the upregulation of inducible microsomal prostaglandin E synthase-1 expression after stimulation with lipopolysaccharide. Conversely, ß-chamigrenal had no effect on the expression and enzyme activity of cyclooxygenase-2. In addition, the expression of early growth response factor-1, a key transcription factor of microsomal prostaglandin E synthase-1 expression, was inhibited by ß-chamigrenal. These results may suggest a possible anti-inflammatory activity of ß-chamigrenal which has to be proven in in vivo experiments.

T-type Ca2+ channel blocker, KYS05090 induces autophagy and apoptosis in A549 cells through inhibiting glucose uptake.

It has been reported that [3-(1,1'-biphenyl-4-yl)-2-(1-methyl-5-dimethylamino-pentylamino)-3,4-dihydroquinazolin-4-yl]-N-benzylacetamide 2hydrochloride (KYS05090), a selective T-type Ca2+ channel blocker, reduces tumor volume and weight in the A549 xenograft model, but the molecular mechanism of cell death has not yet been elucidated. In this study, KYS05090 induced autophagy- and apoptosis-mediated cell death in human lung adenocarcinoma A549 cells. Although KYS05090 decreased intracellular Ca2+ levels, it was not directly related with KYS05090-induced cell death. In addition, KYS05090 generated intracellular reactive oxygen species (ROS) and reduced glucose uptake, and catalase and methyl pyruvate prevented KYS05090-induced cell death. These results indicate that KYS05090 can lead to autophagy and apoptosis in A549 cells through ROS generation by inhibiting glucose uptake. Our findings suggest that KYS05090 has potential chemotherapeutic value for the treatment of lung cancer.

Regulatory Effect of 25-hydroxyvitamin D3 on Nitric Oxide Production in Activated Microglia.

Microglia are activated by inflammatory and pathophysiological stimuli in neurodegenerative diseases, and activated microglia induce neuronal damage by releasing cytotoxic factors like nitric oxide (NO). Activated microglia synthesize a significant amount of vitamin D3 in the rat brain, and vitamin D3 has an inhibitory effect on activated microglia. To investigate the possible role of vitamin D3 as a negative regulator of activated microglia, we examined the effect of 25-hydroxyvitamin D3 on NO production of lipopolysaccharide (LPS)-stimulated microglia. Treatment with LPS increased the production of NO in primary cultured and BV2 microglial cells. Treatment with 25-hydroxyvitamin D3 inhibited the generation of NO in LPS-activated primary microglia and BV2 cells. In addition to NO production, expression of 1-α-hydroxylase and the vitamin D receptor (VDR) was also upregulated in LPS-stimulated primary and BV2 microglia. When BV2 cells were transfected with 1-α-hydroxylase siRNA or VDR siRNA, the inhibitory effect of 25-hydroxyvitamin D3 on activated BV2 cells was suppressed. 25-Hydroxyvitamin D3 also inhibited the increased phosphorylation of p38 seen in LPS-activated BV2 cells, and this inhibition was blocked by VDR siRNA. The present study shows that 25-hydroxyvitamin D3 inhibits NO production in LPS-activated microglia through the mediation of LPS-induced 1-α-hydroxylase. This study also shows that the inhibitory effect of 25-hydroxyvitamin D3 on NO production might be exerted by inhibiting LPS-induced phosphorylation of p38 through the mediation of VDR signaling. These results suggest that vitamin D3 might have an important role in the negative regulation of microglial activation.

6,7-Dimethoxy-3-(3-methoxyphenyl)isoquinolin-1-amine induces mitotic arrest and apoptotic cell death through the activation of spindle assembly checkpoint in human cervical cancer cells.

Previously, we reported that 6,7-dimethoxy-3-(3-methoxyphenyl)isoquinolin-1-amine (CWJ-082) has potent cytotoxic effects on various cancer cells, but the underlying molecular mechanism responsible was not determined. In the present study, CWJ-082 caused cervical cancer cell cycle arrest at the G2/M phase and subsequent caspase-dependent apoptosis. The mitotic arrest caused by CWJ-082 found to be due to increases in the activation of cyclin-dependent kinase 1/cyclin B1 complex and the phosphorylation of histone H3. In addition, CWJ-082 induced the phosphorylation of BubR1 and the association between mitotic arrest deficient 2 (Mad2) and cell division cycle protein 20. These findings suggested that CWJ-082 activated the mitotic spindle checkpoint. Furthermore, knockdown of the spindle checkpoint proteins BubR1 or Mad2 using specific small interfering RNAs significantly reduced CWJ-082-induced mitotic cell accumulation and apoptosis. In addition, CWJ-082 induced the appearance of spindle abnormalities by inducing α-tubulin polymerization. In BALB/c(nu/nu) mice bearing a HeLa xenograft, CWJ-082 significantly inhibited tumor growth. Taken together, these results suggest that CWJ-082 inhibits cell growth via mitotic arrest by activating the mitotic spindle checkpoint and by inducing α-tubulin polymerization and that these events ultimately lead to the apoptosis of human cervical cancer cells and inhibit tumor growth in HeLa xenograft mice.

Proteomic analysis of primary cultured rat cortical neurons in chemical ischemia.

To elucidate the molecular events involved in early ischemic neuronal death, we performed two-dimensional proteome profiling of primary cultures of rat cortical neurons following chemical ischemia induced by the administration of sodium azide under glucose-free conditions. Using a lactic dehydrogenase assay and Western blot analysis of dephosporylation of the voltage-gated potassium channel Kv2.1, we determined duration of chemical ischemia of 2 h to be the relevant time-point for early ischemic neuronal death. Sixty-one proteins were differentially expressed, and 26 different proteins were identified by MALDI-TOF with Mascot database searching. The proteome data indicated that chemical ischemia altered the expression of 20 proteins that are involved in stress response/chaperone, brain development, cytoskeletal/structural proteins, metabolic enzymes, and calcium ion homeostasis. Western blotting and immunocytochemical studies of the 6-most functionally significant proteins showed that, in the ischemia-treated group, the expression of glucose-related protein 78, heat shock protein 90 alpha, and α-enolase was significantly increased, while the expression of inositol triphosphate receptor 1 and ATP synthase beta subunit was decreased. In addition, the expression of dihydropyrimidinase-like 3 showed a truncated pattern in the ischemia group. The changes in the expression of these proteins might be significant indicators of early ischemic neuronal death.

Fimasartan, anti-hypertension drug, suppressed inducible nitric oxide synthase expressions via nuclear factor-kappa B and activator protein-1 inactivation.

Since inhibition of angiotensin II type 1 (AT1) receptor reduces chronic inflammation associated with hypertension, we evaluated the anti-inflammatory potential and the underlying mechanism of fimasartan, a Korean Food and Drug Administration approved anti-hypertension drug, in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Fimasartan suppressed the expressions of inducible nitric oxide synthase (iNOS) by down-regulating its transcription, and subsequently inhibited the productions of nitric oxide (NO). In addition, fimasartan attenuated LPS-induced transcriptional and DNA-binding activities of nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1). These reductions were accompanied by parallel reductions in the nuclear translocation of NF-κB and AP-1. Taken together, our data suggest that fimasartan down-regulates the expression of the iNOS in macrophages via NF-κB and AP-1 inactivation.

Relative bioavailability of levodropropizine 60 mg capsule and syrup formulations in healthy male Korean volunteers: a singledose, randomized-sequence, open-label, two-way crossover study.

BACKGROUND: Levodropropizine is an oral non-opioid anti-tussive drug used in treatment of cough. A new generic 60 mg capsule formulation of levodropropizine has recently been developed. OBJECTIVES: The aim of this study was to assess the pharmacokinetics and bioequivalence of the test (capsule) formulation and reference (syrup) formulation of levodropropizine (60 mg) in healthy, fasted, male Korean volunteers. METHODS: This was a single-dose, randomized sequence, open-label, 2-period crossover study conducted in healthy male Korean volunteers in the fasted state at Kyung Hee University Medical Center (Seoul, Republic of Korea). A single oral dose of the test or reference formulation was followed by a 1-week washout period, after which subjects received the alternative formulation. Blood samples were collected at 0 (predose), 0.17, 0.33, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, and 12 hours after study drug administration. Plasma concentration of levodropropizine was determined using a validated liquid chromatography tandem mass spectrometry (LCMS/ MS) method. The formulations were considered bioequivalent if the 90% CIs for C(max), AUC(0-12h) and AUC(0-∞) were within the predetermined bioequivalence range (80 - 125%, according to the guidelines of the Korea Food and Drug Administration (Korea FDA)). Tolerability was evaluated throughout the study based on vital sign measurements, laboratory analysis (blood biochemistry, hematology, hepatic function and urinalysis) and subject interviews concerning adverse events (AEs). RESULTS: A total of 36 male Korean subjects (mean (SD) age, 23.9 (2.4) years (range 19 - 30 years); height, 176.2 (6.1) cm (range 161 - 190 cm); weight, 69.8 (9.1) kg (range 54.0 - 92.2 kg); body mass index, 22.4 (2.1) kg/m2 (range 19.1 - 28.3 kg/m2)) was enrolled and completed the study. The mean values for C(max), t(max), AUC(0-12h), and AUC(0-∞) with the test formulation of levodropropizine were 331.51 ng/ml, 0.60 hours, 784.32 ng×h/ml, and 825.82 ng×h/ml, respectively; for the reference formulation, the values were 332.81 ng/ml, 0.44 hours, 726.46 ng×h/ml, and 769.46 ng×h/ ml, respectively. The 90% CIs for the logtransformed ratios of C(max) (92.74 - 111.24), AUC(0-12h) (104.31 - 113.67) and AUC(0-∞) (103.87 - 113.57) were within the predetermined range for the assumption of bioequivalence. No serious adverse events were reported. CONCLUSIONS: This single-dose (60 mg) study found that the test (capsule) and reference (syrup) formulations of levodropropizine met the regulatory criterion for assuming bioequivalence in these healthy, fasted, male Korean subjects. Both formulations were well tolerated in the population studied. Korea FDA registration number: BED-1784.

Echinocystic acid isolated from Eclipta prostrata suppresses lipopolysaccharide-induced iNOS, TNF-α, and IL-6 expressions via NF-κB inactivation in RAW 264.7 macrophages.

In this study, we aimed to identify the compounds in Eclipta prostrata responsible for its anti-inflammatory effects using an in vitro bioassay. Three triterpenoids, eclalbasaponin I, eclalbasaponin II, and echinocystic acid, were isolated from an EtOAc fraction of the 70 % EtOH extract of E. prostrata by activity-guided fractionation based on the inhibition of nitric oxide release from lipopolysaccharide-induced RAW 264.7 macrophages. Of these three triterpenoids, echinocystic acid inhibited lipopolysaccharide-induced production of nitric oxide and cytokines such as tumor necrosis factor-α and interleukin-6. Consistent with these observations, echinocystic acid concentration-dependently inhibited lipopolysaccharide-induced inducible nitric oxide synthase expression at the protein level and inducible nitric oxide synthase, tumor necrosis factor-α, and interleukin-6 expression at the mRNA level, and inhibited lipopolysaccharide-induced iNOS promoter binding activity. In addition, echinocystic acid suppressed the lipopolysaccharide-induced transcriptional activity of nuclear factor-κB by blocking the nuclear translocation of p65.

Rapid and sensitive LC-MS/MS method for determination of megestrol acetate in human plasma: application to a human pharmacokinetic study.

A rapid, simple and fully validated LC-MS/MS method was developed and validated for the determination of megestrol acetate in human plasma using tolbutamide as an internal standard (IS) after one-step liquid-liquid extraction with methyl-tert-butyl-ether. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the transitions m/z 385.5 → 267.1 for megestrol acetate and m/z 271.4 → 155.1 for IS. Chromatographic separation was performed on a YMC Hydrosphere C18 column with an isocratic mobile phase, which consisted of 10 mm ammonium formate buffer (adjusted to pH 5.0 with formic acid)-methanol (60:40, v/v) at a flow rate of 0.4 mL/min. The achieved lower limit of quantitation (LLOQ) was 1 ng/mL (signal-to-noise ratio > 10) and the standard calibration curve for megestrol acetate was linear (r > 0.99) over the studied concentration range (1-2000 ng/mL). The proposed method was fully validated by determining its specificity, linearity, LLOQ, intra- and inter-day precision and accuracy, recovery, matrix effect and stability. The validated LC-MS/MS method was successfully applied for the evaluation of pharmacokinetic parameters of megestrol acetate after oral administration of a single dose 800 mg of megestrol acetate (Megace™) to five healthy Korean male volunteers under fed conditions.

Expression and activity of the na-k ATPase in ischemic injury of primary cultured astrocytes.

Astrocytes are reported to have critical functions in ischemic brain injury including protective effects against ischemia-induced neuronal dysfunction. Na-K ATPase maintains ionic gradients in astrocytes and is suggested as an indicator of ischemic injury in glial cells. Here, we examined the role of the Na-K ATPase in the pathologic process of ischemic injury of primary cultured astrocytes. Chemical ischemia was induced by sodium azide and glucose deprivation. Lactate dehydrogenase assays showed that the cytotoxic effect of chemical ischemia on astrocytes began to appear at 2 h of ischemia. The expression of Na-K ATPase α1 subunit protein was increased at 2 h of chemical ischemia and was decreased at 6 h of ischemia, whereas the expression of α1 subunit mRNA was not changed by chemical ischemia. Na-K ATPase activity was time-dependently decreased at 1, 3, and 6 h of chemical ischemia, whereas the enzyme activity was temporarily recovered to the control value at 2 h of chemical ischemia. Cytotoxicity at 2 h of chemical ischemia was significantly blocked by reoxygenation for 24 h following ischemia. Reoxygenation following chemical ischemia for 1 h significantly increased the activity of the Na-K ATPase, while reoxygenation following ischemia for 2 h slightly decreased the enzyme activity. These results suggest that the critical time for ischemia-induced cytotoxicity of astrocytes might be 2 h after the initiation of ischemic insult and that the increase in the expression and activity of the Na-K ATPase might play a protective role during ischemic injury of astrocytes.

Chronic hyperosmotic stress converts GABAergic inhibition into excitation in vasopressin and oxytocin neurons in the rat.

In mammals, the increased secretion of arginine-vasopressin (AVP) (antidiuretic hormone) and oxytocin (natriuretic hormone) is a key physiological response to hyperosmotic stress. In this study, we examined whether chronic hyperosmotic stress weakens GABA(A) receptor-mediated synaptic inhibition in rat hypothalamic magnocellular neurosecretory cells (MNCs) secreting these hormones. Gramicidin-perforated recordings of MNCs in acute hypothalamic slices prepared from control rats and ones subjected to the chronic hyperosmotic stress revealed that this challenge not only attenuated the GABAergic inhibition but actually converted it into excitation. The hyperosmotic stress caused a profound depolarizing shift in the reversal potential of GABAergic response (E(GABA)) in MNCs. This E(GABA) shift was associated with increased expression of Na(+)-K(+)-2Cl(-) cotransporter 1 (NKCC1) in MNCs and was blocked by the NKCC inhibitor bumetanide as well as by decreasing NKCC activity through a reduction of extracellular sodium. Blocking central oxytocin receptors during the hyperosmotic stress prevented the switch to GABAergic excitation. Finally, intravenous injection of the GABA(A) receptor antagonist bicuculline lowered the plasma levels of AVP and oxytocin in rats under the chronic hyperosmotic stress. We conclude that the GABAergic responses of MNCs switch between inhibition and excitation in response to physiological needs through the regulation of transmembrane Cl(-) gradients.

Anti-inflammatory and anti-arthritic effects of new synthetic 3-(4-hydroxyphenyl)-4-(4-thiomethoxyphenyl)-1H-pyrrole-2,5-dione.

We previously reported that 3-(4-hydroxyphenyl)-4-(4-thiomethoxyphenyl)-1H-pyrrole-2,5-dione (1, HMP) has a strong inhibitory effect on prostaglandin E(2) (PGE(2)) production. In this study, the anti-inflammatory and anti-arthritic effects of HMP were evaluated on LPS-induced RAW 264.7 macrophages and rats with carrageenan-induced paw edema and adjuvant-induced arthritis (AIA). The attenuation of PGE(2) production by HMP was found to be caused by the inhibition of cyclooxygenase-2 (COX-2) activity, but not COX-1 activity. However, HMP did not affect COX-2 at the protein or mRNA levels, whereas it suppressed the releases and expressions of inflammatory cytokines, such as, interleukin-1ß (IL-1ß) and IL-6 in LPS-induced macrophages. Furthermore, HMP suppressed LPS-induced nitric oxide (NO) production by down regulating the protein and mRNA expressions of inducible nitric oxide synthase (iNOS). In rats with carrageenan-injected acute inflammation, oral administration of HMP (25 or 50mg/kg, po) reduced paw swelling, and PGE(2) release and myeloperoxidase (MPO) activity in tissue. Furthermore, HMP (25 or 50mg/kg, po) significantly reduced paw swelling, arthritic indices and plasma PGE(2) concentrations in rat with AIA. These results show that HMP reduces swelling in a model acute inflammation and inhibits arthritic responses in a model of chronic inflammation via the inhibition of PGE(2) production. These results suggest that HMP is a potential therapeutic agent for the treatment of arthritis and associated disorders.

T-type Ca2+ channel blocker, KYS05047 induces G1 phase cell cycle arrest by decreasing intracellular Ca2+ levels in human lung adenocarcinoma A549 cells.

In a previous study, we found that the 3,4-dihydroquinazoline derivative, 4-(Benzylcarbamoylmethyl)-2-(biphenyl-4-ylamino)-3-(5-tert-butyloxycarbamoyl-1-pentyl)-3,4-dihydroquinazoline (KYS05047), was a selective T-type Ca(2+) channel blocker with anti-proliferative effects against various cancer cells. However, the mechanism responsible for its effects has not been studied. In this study, we investigated the effect of KYS05047 on cell cycle arrest and the mechanisms involved in human lung adenocarcinoma A549 cells. Among the G(1) phase cell cycle-related proteins examined, the levels of cyclin-dependent protein kinase (Cdk2) and Cdk4 were reduced by KYS05047 (7 µM), whereas the steady-state levels of cyclin D1 and E were unaffected. In addition, KYS05047 increased the protein level of p27(KIP1) and suppressed the kinase activities of Cdk2 and Cdk4. In addition, pretreatment with KCl, which increases intracellular Ca(2+) levels, prevented KYS05047-induced intracellular Ca(2+) decreases and cell cycle arrest. Furthermore, the administration of KYS05047 (2 or 10 mg/kg, po) for 21 days was also found to significantly inhibit tumor growth in an A549 xenograft nude mice model. In conclusion, our results suggested that KYS05047 induced G(1) phase cell cycle arrest in A549 cells associated with a decrease in intracellular Ca(2+) concentrations and inhibited the in vivo tumor growth of A549 xenograft mice.

Anti-inflammatory effect of pelubiprofen, 2-[4-(oxocyclohexylidenemethyl)-phenyl]propionic acid, mediated by dual suppression of COX activity and LPS-induced inflammatory gene expression via NF-κB inactivation.

Pelubiprofen is a non-steroidal anti-inflammatory drugs (NSAIDs) that is related both structurally and pharmacologically to ibuprofen. Anti-inflammatory properties of ibuprofen are due to its ability to both decrease prostaglandin synthesis by inhibiting the activities of cyclooxygenases (COXs) and IκB kinase-ß (IKK-ß). However, the exact mechanisms that accounts for the anti-inflammatory effects of pelubiprofen are not reported. In this study, we investigated the molecular mechanisms how pelubiprofen modulates the inflammatory mediators in LPS-induced macrophages and carrageenan-induced acute inflammatory rat model. Pelubiprofen potently diminished PGE(2) productions through inhibition of COX enzyme activity (IC(50) values for COX-1 and COX-2 are 10.66 ± 0.99 and 2.88 ± 1.01 µM, respectively), but also reduced the expressions of COX-2, inducible nitric oxide (iNOS), tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and IL-6 at transcriptional level in LPS-induced RAW 264.7 cells. In addition, pelubiprofen attenuated the LPS-induced transcription activity and the DNA binding activity of NF-κB, which was accompanied by a parallel reduction of degradation and phosphorylation of inhibitory kappa B-α (IκB-α) and consequently by decreased nuclear translocation of NF-κB. Furthermore, pelubipofen inhibited the LPS-induced phosphorylation of IKK-ß and transforming growth factor-ß activated kinase-1 (TAK1). In acute inflammatory rat model, pretreatment with pelubiprofen inhibited carrageenan-induce edema, neutrophil migration, PGE(2) production, and p65, a subunit of NF-κB, nuclear translocation in inflamed paw. Taken together, our data indicated that pelubiprofen is involved in the dual inhibition of COX activity and TAK1-IKK-NF-κB pathway, revealing molecular basis for the anti-inflammatory properties of pelubiprofen.

Roxatidine suppresses inflammatory responses via inhibition of NF-κB and p38 MAPK activation in LPS-induced RAW 264.7 macrophages.

Roxatidine is a novel, specific, competitive H(2) -receptor antagonist that is used to treat gastric and duodenal ulcers, and which is known to suppress the growth of several tumors by reducing vascular endothelial growth factor (VEGF) expression. Nevertheless, it remains unclear whether roxatidine has anti-inflammatory effects. In this study, we the authors investigated the anti-inflammatory effect of roxatidine in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. It was found that roxatidine dose-dependently inhibited the productions of prostaglandin E(2) (PGE(2)), nitric oxide (NO), and histamine, and the protein and mRNA expressions of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and histidine decarboxylase (HDC). In addition, roxatidine reduced the productions and expressions of VEGF-1 and pro-inflammatory cytokines, including those of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6). Electrophoretic mobility shift assays (EMSA) and reporter gene assays revealed that treatment with roxatidine attenuated the LPS-induced DNA-binding and transcriptional activity of nuclear factor kappa B (NF-κB). In addition, it was found that pretreatment with roxatidine significantly inhibited the nuclear translocations of the p65 and p50 subunits of NF-κB, and these inhibitions were not found to be associated with decreases in the phosphorylation or degradation of inhibitory kappa B-α (IκBα). Furthermore, roxatidine suppressed the phosphorylation of p38 MAP kinase, but not of IκB kinase-α/ß (IKKα/ß), c-Jun NH(2) -terminal kinase (JNK), or extracellular signal-regulated kinase (ERK). Taken together, these results indicate that the anti-inflammatory properties of roxatidine in LPS-treated RAW 264.7 macrophages are mediated by the inhibition of NF-κB transcriptional activity and the p38 MAP kinase pathway.

GABAergic synaptic response and its opioidergic modulation in periaqueductal gray neurons of rats with neuropathic pain.

BACKGROUND: Neuropathic pain is a chronic and intractable symptom associated with nerve injury. The periaqueductal gray (PAG) is important in the endogenous pain control system and is the main site of the opioidergic analgesia. To investigate whether neuropathic pain affects the endogenous pain control system, we examined the effect of neuropathic pain induced by sacral nerve transection on presynaptic GABA release, the kinetics of postsynaptic GABA-activated Cl- currents, and the modulatory effect of µ-opioid receptor (MOR) activation in mechanically isolated PAG neurons with functioning synaptic boutons. RESULTS: In normal rats, MOR activation inhibited the frequency of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) to 81.3% of the control without any alteration in their amplitude. In neuropathic rats, the inhibition of mIPSC frequency by MOR activation was 82.4%. The frequency of GABAergic mIPSCs in neuropathic rats was 151.8% of normal rats without any difference in the mIPSC amplitude. Analysis of mIPSC kinetics showed that the fast decay time constant and synaptic charge transfer of mIPSCs in neuropathic rats were 76.0% and 73.2% of normal rats, respectively. CONCLUSIONS: These results indicate that although the inhibitory effect of MOR activation on presynaptic GABA release is similar in both neuropathic and normal rats, neuropathic pain may inhibit endogenous analgesia in the PAG through an increase in presynaptic GABA release.