Search

Biogenic Mn oxides are reactive and ubiquitous in many Earth surface environments, yet their role in radionuclide sequestration at U mill-tailings sites still require an improved understanding at the nano- and molecular-scales. This study concerns the uptake of Ba, utilized as a safe and chemically appropriate surrogate for radioactive Ra, by biogenic Mn oxides produced by a fungal species, Coprinopsis urticicola, isolated from the mine water of the Ningyo-toge U mine, Okayama, Japan. The biogenic Mn oxides were identified as birnessite nanocrystals O binding to the newly overlying MnO6 layer during coprecipitation. Static desorption experiments for 7 days reveal that the steady-state release rate of adsorbed Ba is ∼1.4 times faster than that of the coprecipitated Ba, when the Ba concentration in the initial loading solution was ∼10–8 mol/L, indicating that the release of intercalated (coprecipitation) Ba to solution is retarded. The present study demonstrates the importance of fungus-generated Mn oxides as an efficient absorber of Ba2+, and likely Ra2+, among soil compounds in U mill-tailings. This may be applicable to other contaminated sites due to the ubiquitous occurrence of fungi in the environment.

Biogenic manganese (Mn) oxides occur ubiquitously in the environment including the uranium (U) mill tailings at the Ningyo-toge U mine in Okayama, Japan, being important in the sequestration of radioactive radium. To understand the nanoscale processes in Mn oxides formation at the U mill tailings site, Mn

Ferrihydrite, a hydrous ferric oxide, is ubiquitous in silica-rich mine drainage sites, although some uncertaity remains concerning its nanoscale texture and colloidal properties such as its association with amorphous silica and reactions with contaminant metals. To understand the mechanisms of natural attenuation of contaminants governed by ferrihydrite and amorphous silica aggregations, mine tailings material from the Ningyo-toge uranium deposit, Japan, has been investigated in detail utilizing many traditional solution and mass spectroscopy methods, as well as synchrotron-based methods the latest in FIB- and analytic TEM-based technologies. At this site, the past mining activity results in slightly acidic mine drainage and the contamination of a mill tailings pond with relatively high concentrations of toxic elements, including U, As, and Ra. We have systematically investigated the geochemistry of water, colloids, and sediments from the groundwater draining the former mine site (now remediated), down gradient to the mill tailings pond where the water pH remains ∼6.2. Groundwater with 0.18 mg/L dissolved oxygen (DO) is oxygenated rapidly to levels of 1.65 mg/L DO when exposed to the atmosphere along this flow path. Dissolved Fe2+ is oxidized instantly to form ferrihydrite nanoparticle aggregates > 200 nm in diameter, adsorbing ∼67% of the total As (3.36 µmol/L) in solution. For solutions and particles/aggregates passing through 200-nm filters, ∼17% of the 1.17 µmol/L As is sorbed on these smaller ferrihydrite aggregates. Simultaneously, varying amounts of amorphous silica with particle sizes as small as several nanometers were observed to adsorb onto ferrihydrite aggregates. Arsenic is present predominantly as arsenate, As(V), and preferentially adsorbs to ferrihydrite within these ferrihydrite–amorphous silica aggregates. Arsenate adsorption is not inhibited by silica adsoption on ferrihydrite when the Si/Fe composition is ∼0.25. Uranium is also adsorbed onto aggregates of colloids. After 24 h of static exposure to the ambient atmosphere, the zeta potential of the aggregate that formed in oxygenated water decreases from −16 to −24 mV, near the zeta potential of pure silica, potentially leading to increased colloidal stability. Some large-sized aggregates are deposited at the bottom of the collecting well and slowly adsorb minor amounts of Mn (1.45 wt.%); other aggregates are transported to the mill tailings pond downsteam, where the water is further oxidized (DO = 3.09 mg/L), and deposited onto the sediment together with birnessite, a hydrated Mn-oxide that appears to be biogenic in this case. During sedimentation, associations of amorphous silica within the aggregate play a key role in retaining the ferrihydrite structure that adsorbs toxic elements such as As. Consequently, the mechanism of natural attenuation at the nanoscale constrained by the chemical and physical properties of ferrihydrite–amorphous silica aggregates is of critical importance in the Ningyo-toge mill tailing site. It is shown that it is also important in other mine drainage and mill tailings sites due to the ubiquitous and dominant occurrence of both Si and Fe in these types of settings.

Sepsis is a major clinical challenge with unacceptably high mortality. The signal transducers and activators of transcription (STAT) family of transcription factors is known to activate critical mediators of cytokine responses, and, among this family, STAT3 is implicated to be a key transcription factor in both immunity and inflammatory pathways. We investigated whether in vivo introduction of synthetic double-stranded STAT3 decoy oligodeoxynucleotides (ODNs) can provide benefits for reducing organ injury and mortality in mice with cecal ligation and puncture (CLP)-induced polymicrobial sepsis. We found that STAT3 was rapidly activated in major end-organ tissues following CLP, which was accompanied by activation of the upstream kinase JAK2. Transfection of STAT3 decoy ODNs downregulated pro-inflammatory cytokine/chemokine overproduction in CLP mice. Moreover, STAT3 decoy ODN transfection significantly reduced the increases in tissue mRNAs and proteins of high mobility group box 1 (HMGB1) and strongly suppressed the excessive elevation in serum HMGB1 levels in CLP mice. Finally, STAT3 decoy ODN administration minimized the development of sepsis-driven major end-organ injury and led to a significant survival advantage in mice after CLP. Our results suggest a critical role of STAT3 in the sepsis pathophysiology and the potential usefulness of STAT3 decoy ODNs for sepsis gene therapy.

Plutonium association and transport by ferrihydrite has been confirmed in the groundwater system of Lake Karachai at the Mayak Production Association, Russia (Novikov et al. 2006). Since this initial description, there has been substantial progress in understanding the mechanisms that control the concentrations and distribution of Pu at the Mayak site. This chapter is a concise review of work during the past 10 years that utilizes to advantage recent advances in nanoscale, analytical techniques. The structure of ferrihydrite (P63mc) was determined using a pair-distribution function analysis. Scanning transmission electron microscopy of Mayak colloids resolved the occurrence of mixed silica nano-colloids and ferrihydrite. Thermodynamic properties for aqueous Pu-species have been updated. Inner-sphere surface complexation was determined for adsorbed Pu using synchrotron-based X-ray absorption spectroscopy. The adsorbed Pu can transform into Pu-dioxide nanoparticles as revealed by high-resolution transmission electron microscopy. Plutonium polymerization can also proceed by aggregation of cubic Pu-dihydroxy building blocks of Pu(OH)22+•6H2O. Plutonium desorption occurs associated with re-adsorption at neutral pH. To establish comprehensive model of Pu transport at the Mayak site, the detailed mechanisms described above must be considered. Rigorous characterization of colloids is necessary and these results must be compared with results obtained from experimental studies. The state-of-the-art knowledge on the Pu association with Fe hydroxides is now well enough understood that safety assessments no longer need to rely on the classical Kd approach.

Levosimendan and milrinone may be used in place of dobutamine to increase cardiac output in septic patients with a low cardiac output due to impaired cardiac function. The effects of the two inotropic agents on cardiac inflammation and left ventricular (LV) performance were examined in mice with cecal ligation and puncture (CLP)-induced sepsis. CLP mice displayed significant cardiac inflammation, as indicated by highly increased pro-inflammatory cytokines and neutrophil infiltration in myocardial tissues. When continuously given, levosimendan prevented but milrinone exaggerated cardiac inflammation, but they significantly reduced the elevations in plasma cardiac troponin-I and heart-type fatty acid-binding protein, clinical markers of cardiac injury. Echocardiographic assessment of cardiac function showed that the effect of levosimendan, given by an intravenous bolus injection, on LV performance was impaired in CLP mice, whereas milrinone produced inotropic responses equally in sham-operated and CLP mice. A lesser effect of levosimendan on LV performance after CLP was also found in spontaneously beating Langendorff-perfused hearts. In ventricular myocytes isolated from control and CLP mice, levosimendan, but not milrinone, caused a large increase in the L-type calcium current. This study represents that levosimendan and milrinone have cardioprotective properties but provide different advantages and drawbacks to cardiac inflammation/dysfunction in sepsis.

Sepsis-associated encephalopathy (SAE), characterized as diffuse brain dysfunction and neurological manifestations secondary to sepsis, is a common complication in critically ill patients and can give rise to poor outcome, but understanding the molecular basis of this disorder remains a major challenge. Given the emerging role of G protein-coupled receptor 2 (GRK2), first identified as a G protein-coupled receptor (GPCR) regulator, in the regulation of non-G protein-coupled receptor-related molecules contributing to diverse cellular functions and pathology, including inflammation, we tested the hypothesis that GRK2 may be linked to the neuropathogenesis of SAE. When mouse MG6 microglial cells were challenged with lipopolysaccharide (LPS), GRK2 cytosolic expression was highly up-regulated. The ablation of GRK2 by small interfering RNAs (siRNAs) prevented an increase in intracellular reactive oxygen species generation in LPS-stimulated MG6 cells. Furthermore, the LPS-induced up-regulation of inducible nitric-oxide synthase expression and increase in nitric oxide production were negated by GRK2 inhibitor or siRNAs. However, GRK2 inhibition was without effect on overproduction of tumor necrosis factor-α, interleukin (IL)-6, and IL-1β in LPS-stimulated MG cells. In mice with cecal ligation and puncture-induced sepsis, treatment with GRK2 inhibitor reduced high levels of oxidative and nitrosative stress in the mice brains, where GRK2 expression was up-regulated, alleviated neurohistological damage observed in cerebral cortex sections, and conferred a significant survival advantage to CLP mice. Altogether, these results uncover the novel role for GRK2 in regulating cellular oxidative and nitrosative stress during inflammation and suggest that GRK2 may have a potential as an intriguing therapeutic target to prevent or treat SAE.

This study was designed to clarify effects of postoperative adjuvant chemotherapy for high-risk Stage Ⅱ colorectal cancer.The subjects were 99 patients with high-risk Stage Ⅱcolorectal cancer who underwent surgery at our department from October 2013 to March 2018. Patients were classified into adjuvant chemotherapy group and nonadjuvant chemotherapy group. Overall survival(OS)and recurrence-free survival(RFS)were analyzed between the 2 groups.Thirty six patients(36.4%)underwent adjuvant chemotherapy. Adjuvant chemotherapy group were younger(p0.010), had a better ASA-PS(p0.010), good preoperative Hb(p0.010), and preoperative Alb(p0.010)compared to non-adjuvant chemotherapy group. There was no difference between the 2 groups in the high-risk factors for recurrence. Most patient had an oral medication as for adjuvant chemotherapy. There was no difference in OS and RFS between the 2 groups.Postoperative adjuvant chemotherapy for high-risk Stage Ⅱ colorectal cancer did not significantly improve the OS and RFS. Further study is necessary to asses the suitable regimen and patients eligible for chemotherapy.

Objectives:The calcium sensitizer levosimendan is used in treatment of decompensated heart failure and may also exhibit anti-inflammatory properties. We examined whether treatment with levosimendan is substantially beneficial in mice with cecal ligation and puncture–induced polymicrobial sepsis, and

Background Epigenetic programming, dynamically regulated by histone acetylation, may play a key role in the pathophysiology of sepsis. We examined whether histone deacetylase (HDAC) can contribute to sepsis-associated inflammation and apoptosis. Materials and methods Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in BALB/c mice. An intraperitoneal injection of CG200745 (10 mg/kg), a novel broad-spectrum HDAC inhibitor, or valproic acid (500 mg/kg), a predominant inhibitor of class I HDACs, was given 3 h before surgery. Results HDAC1, HDAC2, and HDAC3 protein levels were decreased in lungs after CLP. Furthermore, CLP-induced sepsis increased both histone H3 and H4 acetylation levels in lungs. When CG200745 was given, apoptosis induction was strongly suppressed in lungs and spleens of septic mice. This antiapoptotic effect of CG200745 was not accompanied by upregulation of antiapoptotic and downregulation of proapoptotic Bcl-2 family member proteins. Treatment with CG200745 failed to inhibit elevated levels of serum cytokines and prevent lung inflammation in septic mice. Valproic acid also showed antiapoptotic but not anti-inflammatory effects in septic mice. Conclusions These findings imply that HDAC inhibitors are a unique agent to prevent cell apoptosis in sepsis at their doses that do not improve inflammatory features, indicating that septic inflammation and apoptosis may not necessarily be essential for one another's existence. This study also represents the first report that CLP-induced sepsis downregulates HDACs. Nevertheless, the data with HDAC inhibitors suggest that imbalance in histone acetylation may play a contributory role in expression or repression of genes involved in septic cell apoptosis.

Olprinone, a specific phosphodiesterase III inhibitor, and corforsin daropate, a direct adenylate cyclase activator, are now being used in critical conditions. We investigated whether their therapeutic use provides protection against septic acute lung injury (ALI) and mortality. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in BALB/c mice. Olprinone or colforsin daropate was continuously given through an osmotic pump that was implanted into the peritoneal cavity immediately following CLP. These treatments prevented the ALI development in CLP mice, as indicated by the findings that severe hypoxemia, increased pulmonary vascular permeability, and histological lung damage were strikingly remedied. Furthermore, continued administration of olprinone or colforsin daropate suppressed apoptosis induction in septic lungs and improved the survival of CLP mice. Olprinone and corforsin daropate enhanced Akt phosphorylation in septic lungs. Wortmannin, which inhibits the Akt upstream regulator phosphatidylinositol 3-kinase, abrogated the protective effects of olprinone and corforsin daropate on sepsis-associated lung inflammation and apoptosis. In vivo transfection of cyclic AMP response element binding protein (CREB) decoy oligodeoxynucleotide failed to negate the abilities of these agents to increase Akt phosphorylation and to inhibit IκBα degradation in septic lungs. These results demonstrate for the first time that CREB-independent Akt-mediated signaling is a critical mechanism contributing to the therapeutic effects of olprinone and corforsin daropate on septic ALI. Moreover, our data also suggest that these cyclic AMP-related agents, by blocking both nuclear factor-κB activation and apoptosis induction, may represent an effective therapeutic approach to the treatment of the septic syndrome.

There is growing evidence that the HMG-CoA reductase inhibitors (statins) provide some of the beneficial effects that are independent of their lipid-lowering effects. Recent animal experiments and clinical trials suggest that statin use may limit the development of sepsis and associated systemic inflammation. The aim of this study was to explore the potential role of statins in the prevention treatment of sepsis-induced acute lung injury (ALI). Mice were rendered septic by cecal ligation and puncture (CLP). An intraperitoneal injection of 3 mg/kg per day of pitavastatin was initiated 4 days before surgery and was maintained for life support afterward, which significantly improved the survival of CLP mice. Treatment with pitavastatin prevented the ALI development in CLP mice, as indicated by the findings that severe hypoxemia, increased pulmonary vascular permeability, and histological lung damage, including inflammatory cell infiltrate, were greatly remedied. This was associated with down-regulation of increased activity of nuclear factor-κB (NF-κB) in septic lungs. Although plasma cortisol showed a sharp rise, glucocorticoid receptor (GCR) expression in the lungs was strikingly reduced after the onset of CLP-induced sepsis. It is noteworthy that pitavastatin increased GCR expression with an increase in alveolar macrophages in which GCRs are localized, without modifying the sepsis-associated rise in plasma cortisol. These results confirm significant protection by pitavastatin on septic ALI and demonstrate that down-regulated NF-κB activation associated with the GCR expression increase consequent to the increased number of alveolar macrophages may explain, in part, the mechanisms responsible for favorable effects of statins on the ALI management.

Background and purpose: Hyperglycaemia is known to reduce nitric oxide (NO) bioavailability by modulating endothelial NO synthase (eNOS) activity, and polyphenols are believed to have cardiovascular benefit. One possible mechanism could be through interaction with eNOS.

Sepsis remains the leading cause of death in critically ill patients. A major problem contributing to sepsis-related high mortality is the lack of effective medical treatment. Thus, the key goal in critical care medicine is to develop novel therapeutic strategies that will impact favorably on septic patient outcome. While it is generally accepted that sepsis is an inflammatory state resulting from the systemic response to infection, apoptosis is implicated to be an important mechanism of the death of lymphocytes, gastrointestinal and lung epithelial cells, and vascular endothelial cells associated with the development of multiple organ failure in sepsis. The pivotal role of cell apoptosis is now highlighted by multiple studies demonstrating that prevention of cell apoptosis can improve survival in clinically relevant animal models of sepsis. In this review article, we address the scientific rationale for remedying apoptotic cell death in sepsis and propose that therapeutic efforts aimed at blocking cell signaling pathways leading to apoptosis may represent an attractive target for sepsis therapy. Keywords:: sepsis, apoptosis, death receptor, acute lung injury, endothelial cell, gene therapy

OBJECTIVE Insulin-resistant hyperglycemia is commonly observed in septic patients and may actually lead to some of adverse outcomes. We examined the changes in insulin signaling and glucose uptake regulation in sepsis and the involvement of the nuclear factor-kappaB pathway. DESIGN Controlled animal study. SETTING University research laboratory. SUBJECTS One hundred fifty-four BALB/c mice (8-12 wks of age). INTERVENTIONS The following four experimental groups were studied: sham-operated control, cecal ligation and puncture-induced sepsis, sepsis + nuclear factor-kappaB decoy oligodeoxynucleotide treatment, and sepsis + scrambled decoy oligodeoxynucleotide treatment. MEASUREMENTS AND MAIN RESULTS Septic mice were markedly hyperinsulinemic with apparently normal blood glucose levels in the fasted state, suggesting they are insulin-resistant. In fact, glucose clearance in response to insulin was markedly impaired in septic mice. They had impaired GLUT4 membrane translocation resulting from impaired insulin signaling as indicated by the decreased amount of insulin receptor substrate protein and the reduced activation of phosphatidylinositol 3-kinase and Akt. Interestingly, injection of nuclear factor-kappaB decoy oligodeoxynucleotide into the skeletal muscle dramatically improved all of the changes, including glucose clearance and insulin signaling. We also found that the Cbl-associated protein to TC10 pathway, another pathway regulating GLUT4 translocation, was up-regulated in septic mice in a nuclear factor-kappaB-dependent manner. This pathway may be one of the compensatory mechanisms to translocate GLUT4 because silencing of the individual components of the pathway with small interfering RNAs further reduced GLUT4 translocation in muscles of septic mice. CONCLUSIONS In sepsis, skeletal muscle GLUT4 translocation is impaired as a result of the reduced phosphatidylinositol 3-kinase/Akt pathway associated with insulin receptor substrate down-regulation through nuclear factor-kappaB activation.

Our previous study showed that treatment of cultured bovine adrenal chromaffin cells with phorbol 12, 13-dibutyrate (PDBu) or 12-O-tetradecanoylphorbol 13-acetate (TPA) caused a rapid ( 15 h) translocation of both conventional (c) protein kinase C-α (PKC-α) and novel PKC-e (but not atypical PKC-ζ) from cytosol to membranes, whereas thymeleatoxin (TMX) increased the similar but selective membrane association of only cPKC-α. In the present study, chronic (≥12 h) treatment of chromaffin cells with PDBu raised cell surface 125I-insulin binding without altering the KD value ; it developed in a concentration (EC50 = 1.9 nM)-and time (t1/2 = 14.6 h)-dependent manner, reaching its maximum 115% increase at 48 h. Either TPA (30 nM) or TMX (EC50 = 6.4 nM) also increased 125I-insulin binding by 97 or 88%, whereas the biologically inactive 4α-TPA had no effect. The increasing effect of PDBu (30 nM for 24 h) on 125I-insulin binding was significantly blocked, even when H7, an inhibitor of PKC, was added at 8 h after the initiation of PDBu treatment. Concurrent treatment with brefeldin A, an inhibitor of vesicular transport from the trans-Golgi network, cycloheximide, an inhibitor of protein synthesis, or 5,6-dichlorobenzimidazole riboside, an inhibitor of RNA synthesis, abolished the PDBu-induced increment of 125I-insulin binding. Western blot analysis, using antibody against the β-subunit of the insulin receptor, showed that treatment with PDBu (30 nM) or TMX (EC50 = 2.3 nM) increased levels of insulin receptor precursor (~190 kDa ; t1/2 = 7.1 h) and insulin receptor β-subunit (t1/2 = 15.4 h), causing their almost maximum 52 and 59% rises, respectively, at 24 h. Northern blot analysis revealed that PDBu or TMX increased levels of insulin receptor mRNAs by ~35% as soon as 3 h, producing its monophasic peak ~76% increases at 24 h. All of these increasing effects of PDBu and TMX on 125I-insulin binding and insulin receptor β-subunit and insulin receptor mRNA levels were entirely prevented by simultaneous treatment with Go6976, a selective inhibitor of cPKC. These results suggest that long-term activation of cPKC-α up-regulates the density of the cell surface insulin receptor via transcriptional/translational events.

In cultured bovine adrenal chromaffin cells, 48 h-treatment with 20 mmol/L LiCl, 1 mmol/L valproic acid, 30 micromol/L SB216763, 30 micromol/L SB415286, or 100 nmol/L insulin, a condition that inhibits constitutive active glycogen synthase kinase-3 (GSK-3), decreased cell surface (125)I-insulin binding capacity by approximately 39%, without altering the K(d) value; LiCl, SB216763 or insulin decreased insulin receptor (IR) and IR precursor levels, attenuating insulin-induced Tyr-autophosphorylation of IR. LiCl increased inhibitory Ser9-phosphorylation of GSK-3beta at 6 h, decreasing (125)I-insulin binding at 24 h. SB216763-induced (125)I-insulin binding reduction (IC(50) = 3 micromol/L) was preceded by beta-catenin level increase by SB216763 (EC(50) = 11 micromol/L), a hallmark of GSK-3 inhibition. Insulin-induced rapid (> 1 min) Ser9-phosphorylation of GSK-3beta (Nemoto et al. 2006) was followed by approximately 48% decrease of IR level. LiCl did not stimulate endocytosis, nor proteolysis of IR. LiCl destabilized IR mRNA (t(1/2) = 9.3 vs. 6.5 h), decreasing IR mRNA level by approximately 47%, without altering IR gene transcription. Decreases of (125)I-insulin binding and IR level, as well as increased Ser9-phosphorylation of GSK-3beta were restored to the control levels by washing the test compound-treated cells. Thus, GSK-3beta regulates IR level via controlling IR mRNA stability.

Water homeostasis in the brain is essential for brain function. We have studied how aquaporin (AQP) 1 expression in GP8 immortalized rat brain microvascular endothelial cells is regulated by glucocorticoid. AQP1 protein level was raised by dexamethasone treatment in a time- and concentration-dependent manner. The up-regulation of AQP1 protein by dexamethasone was associated with an increase of AQP1 mRNA level, with no change in the degradation rate of AQP1 mRNA. AQP1 was concentrated in detergent-insoluble fractions in the cells treated with or without dexamethasone, suggesting that function/trafficking of AQP1 may be regulated via the interaction with lipid rafts. Since glucocorticoid therapy has well known beneficial effects in the treatment of brain edema, the induction of AQP1 by dexamethasone raises a possibility that AQP1 plays a role in ameliorating brain edema.

Photoreactions of 2-halotropones with the excited 9,10-dicyanoanthracene gave a meta-adduct and substitution products occurred at the C-2 position of troponoids. The mechanism of the meta-adduct was proved by the product analysis of the reaction of 3,7-dideuterio-2-bromo-5-isopropyltropone and 9,10-dicyanoanthracene. In the photoreaction of 2-chloro-5-isopropyltropone and 9,10-dicyanoanthracene in a mixed solvent of benzene and methanol, a benzaldehyde with a dibenzo-2-oxabicyclo[3.2.2]nonane system was obtained to support occurrence of an [8+4] cycloaddition reaction between them.

Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) are multi-functional peptides derived from the same precursor, proadrenomedullin. We have studied the regulatory mechanism of expression of these peptides during neuronal differentiation of rat pheochromocytoma PC12 cells by nerve growth factor (NGF). The cellular levels of the peptides increased slightly, and then progressively decreased below the control by NGF. Immunoreactive (ir)-AM in the medium was transiently increased by NGF. Cytochemical staining showed that ir-AM and ir-PAMP were abundantly present in cytoplasm in the undifferentiated cells, and were decreased during culture with NGF. There was no preferential localization of ir-AM or ir-PAMP in neurites in comparison with in cytoplasm in the differentiated cells. Northern blot analysis showed that mRNA encoding these peptides, as detected as a band of 1.6 kb, increased more than three-fold at 1 h after the addition of NGF and then progressively decreased to one fifth of the control during 72 h. Degradation rate of the mRNA was slowed by NGF even when mRNA level is decreased after 72 h of NGF treatment. The transcription rate of their gene increased transiently and then decreased by the long-term treatment with NGF. These results demonstrate that expression of AM and PAMP is regulated by NGF along with time-dependent differentiation: AM gene transcription is transiently activated by NGF, whereas it was suppressed during neuronal differentiation of the cells.

Water homeostasis of the brain is essential for its neuronal activity. Changes in water content in the intra- and extra-cellular space affect ionic concentrations and therefore modify neuronal activity. Aquaporin (AQP) water channels may have a central role in keeping water homeostasis in the brain. Among AQP subtypes cloned in mammalian, only AQP1, AQP4, and AQP9 were identified in the brain. Changes in AQP expression may be correlated with edema formation of the brain. In this review, we describe the physiological function of AQPs and the regulatory mechanism of their expression in the brain. Keywords:: aquaporin, brain, edema, glucocorticoid, lipid raft

In cultured bovine adrenal chromaffin cells, (+/-)-bupivacaine inhibited veratridine-induced 22Na(+) influx (IC(50) 6.8 microM). The IC(50) of (+)-bupivacaine (2.8 microM) was 6.2-, 7.4-, and 17.1-fold lower than those of (-)-bupivacaine (17.3 microM), (-)-ropivacaine (20.6 microM), and lidocaine (47.8 microM). Chronic (i.e. 3-h) treatment of cells with (+/-)-bupivacaine increased cell surface [3H]saxitoxin ([3H]STX) binding capacity by 48% (EC(50) of 233 microM; t(1/2)=7.4 h), without changing the K(d) value. Treatment for 24 h with either (+)- or (-)-bupivacaine, or (-)-ropivacaine elevated [3H]STX binding, whereas 24-h treatment with lidocaine had no effect. The rise of [3H]STX binding by (+/-)-bupivacaine was prevented by cycloheximide, an inhibitor of protein synthesis, or brefeldin A, an inhibitor of cell surface vesicular exit from the trans-Golgi network; however, (+/-)-bupivacaine did not increase Na(+) channel alpha- and beta(1)-subunit mRNA levels. In cells subjected to (+/-)-bupivacaine treatment (1 mM for 24 h) followed by 3-h washout, veratridine-induced 22Na(+) influx was enhanced, even when measured in the presence of ouabain, an inhibitor of Na(+),K(+)-ATPase. Ptychodiscus brevis toxin-3 potentiated veratridine-induced 22Na(+) influx by 2.3-fold in the (+/-)-bupivacaine-treated cells, as in non-treated cells. These results suggest that lipophilic bupivacaine enantiomers or (-)-ropivacaine acutely inhibit Na(+) channel gating, whereas its chronic treatment up-regulates cell surface expression of Na(+) channels via translational and externalization events.

Treatment (/=24 h) of cultured bovine adrenal chromaffin cells with ketoacidosis-related concentrations (/=3 mM) of acetoacetate (but not beta-hydroxybutyrate, acetone, and acidic medium) caused a time- and concentration-dependent reduction of cell surface (125)I-insulin binding by ~38%, with no change in the K(d) value. The reduction of (125)I-insulin binding returned to control nontreated level at 24 h after the washout of acetoacetate-treated cells. Acetoacetate did not increase the internalization rate of cell surface insulin receptor (IR), as measured in the presence of brefeldin A, an inhibitor of cell surface vesicular exit from the trans-Golgi network. Acetoacetate (10 mM for 24 h) lowered cellular levels of the immunoreactive IR precursor molecule (approximately 190 kDa) and IR by 22 and 28%, respectively. Acetoacetate decreased IR mRNA levels by approximately 23% as early as 6 h, producing their maximum plateau reduction at 12 and 24 h. The half-life of IR mRNA was shortened by acetoacetate from 13.6 to 9.5 h. Immunoprecipitation followed by immunoblot analysis revealed that insulin-induced (100 nM for 10 min) tyrosine-phosphorylation of insulin receptor substrate-1 (IRS-1) was attenuated by 56% in acetoacetate-treated cells, with no change in IRS-1 level. These results suggest that chronic treatment with acetoacetate selectively down-regulated the density of cell surface functional IR via lowering IR mRNA levels and IR synthesis, thereby retarding insulin-induced activation of IRS-1.

The density and electrical activity of cell surface voltage-dependent Na(+) channels are key determinants regulating the neuronal plasticity including development, differentiation, and regeneration. Abnormalities of Na(+) channels are associated with various neurological diseases. In this paper, we review the regulatory mechanisms of cell surface Na(+) channel expression mediated by Ca(2+) signaling pathways in cultured bovine adrenal chromaffin cells. Sustained, but not transient, elevation of intracellular Ca(2+) concentration reduced the number of cell surface Na(+) channels. The reduction of Na(+) channels was suppressed by an inhibitor of calpain, a Ca(2+)-dependent protease, and by an inhibitor of protein kinase C (PKC). The activation of conventional PKC-alpha and novel PKC-epsilon reduced cell surface Na(+) channels by the acceleration of internalization of the channels and by the increased degradation of Na(+) channel alpha-subunit mRNA, respectively. On the contrary, the activation of PKC-epsilon increased Na(+) channel beta(1)-subunit mRNA level. The inhibition of calcineurin, a Ca(2+)/calmodulin-dependent protein phosphatase 2B, by immunosuppressants upregulated cell surface Na(+) channels by both stimulating externalization and inhibiting internalization of the channels without changing Na(+) channel alpha- and beta(1)-subunit mRNA levels. Thus, the signal transduction pathways mediated by intracellular Ca(2+) modulate cell surface Na(+) channel expression via multiple Ca(2+)-dependent events, and the changes in the intracellular vesicular trafficking are the important mechanisms in the regulation of Na(+) channel expression.

Vascular endothelial cells can absorb higher radiation doses than any other tissue in the body, and post-radiation impaired endothelial nitric oxide synthase (eNOS) function may be developed as a potential contributor to the pathogenesis of vascular injury. In this study, we investigated early alterations of eNOS signaling in human umbilical venous endothelial cells (HUVECs) exposed to X-ray radiation. We found that ionizing radiation increased eNOS phosphorylation at Ser-1177 and dephosphorylation at Thr-495 in HUVECs in a dose-dependent (≤ 20 Gy) and time-dependent (6-72 h) manner. The total expression levels of eNOS were unchanged by radiation. Although a transient but significant increase in extracellular signal-regulated protein kinase 1/2 (ERK1/2) phosphorylation and a biphasic decline in Akt phosphorylation were observed after irradiation, these inhibitors were without effect on the radiation-induced changes in eNOS phosphorylation. There was an increase in protein kinase C-βII (PKC-βII) expression and the ablation of PKC-βII by small interfering RNA (siRNA) negated the radiation effect on the two eNOS phosphorylation events. Furthermore, when the radiation-induced increase in reactive oxygen species (ROS) generation was prevented by the anti-oxidant N-acetyl-L-cysteine, eNOS Ser-1177 phosphorylation and Thr-495 dephosphorylation in irradiated HUVECs were significantly reduced. However, transfection of PKC-β siRNA did not alter ROS production after irradiation, and NAC failed to block the radiation-induced increase in PKC-βII expression. Taken together, our results suggest that ionizing radiation-induced eNOS activation in human vascular endothelial cells is attributed to both the up-regulation of PKC-βII and the increase in ROS generation which were independent of each other.

The middle ear cavity is an air-filled space that must be maintained for effective sound transmission to the inner ear. To examine the mechanisms of water homeostasis in the middle ear, we investigated whether aquaporins (AQPs), a family of water-permeable channels, were expressed in the middle ear. Reverse transcription-polymerase chain reaction and immunoblot analyses revealed that mRNAs encoding AQP1, 4 and 5 (but not 2 or 3) subtypes were expressed in rat middle ear epithelium; AQP1, 4 and 5 were detected as 28-, 30- and 30-kDa proteins, respectively. Immunohistochemical analysis showed that AQP1 was localized at capillary endothelial cells and fibroblasts in lamina propria mucosae; AQP4 was present solely at the basolateral membrane of ciliated cells, whereas AQP5 was on the apical surface of ciliated cells as well as of flat and columnar epithelial cells. The characteristic different localizations of AQP1, 4 and 5 subtypes in the middle ear suggest that middle ear water homeostasis requires the coordinated operation of these AQPs.

1. Long-term (> or = 12 h) treatment of cultured bovine adrenal chromaffin cells with A23187 (a Ca(2+) ionophore) or thapsigargin (TG) [an inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)] caused a time- and concentration-dependent reduction of cell surface [(3)H]-saxitoxin (STX) binding capacity, but did not change the K:(D:) value. In A23187- or TG-treated cells, veratridine-induced (22)Na(+) influx was reduced (with no change in veratridine EC(50) value) while it was enhanced by alpha-scorpion venom, beta-scorpion venom, or Ptychodiscus brevis toxin-3, like in nontreated cells. 2. The A23187- or TG-induced decrease of [(3)H]-STX binding was diminished by BAPTA-AM. EGTA also inhibited the decreasing effect of A23187. A23187 caused a rapid, monophasic and persistent increase in intracellular concentration of Ca(2+) ([Ca(2+)](i)) to a greater extent than that observed with TG. 2,5-Di-(t-butyl)-1,4-benzohydroquinone (DBHQ) (an inhibitor of SERCA) produced only a rapid monophasic increase in [Ca(2+)](i), without any effect on [(3)H]-STX binding. 3. Reduction in [(3)H]-STX binding capacity induced by A23187 or TG was attenuated by Go6976 (an inhibitor of conventional protein kinase C) or calpastatin peptide (an inhibitor of calpain). When the internalization rate of cell surface Na(+) channels was measured in the presence of brefeldin A (an inhibitor of vesicular exit from the trans-Golgi network), A23187 or TG accelerated the reduction of [(3)H]-STX binding capacity. 4. Six hours treatment with A23187 lowered Na(+) channel alpha- and beta(1)-subunit mRNA levels, whereas TG had no effect. 5. These results suggest that elevation of [Ca(2+)](i) caused by A23187, TG or DBHQ exerted differential effects on down-regulation of cell surface functional Na(+) channels and Na(+) channel subunit mRNA levels.

Long-term (or =12 h) treatment of cultured bovine adrenal chromaffin cells with thapsigargin (TG), an inhibitor of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), caused a time (t(1/2)=16.3 h)- and concentration (IC50=37.8 nM)-dependent decrease of cell surface 125I-insulin binding by 35%, but did not change the Kd value. TG caused a sustained increase of cytoplasmic concentration of Ca2+ ([Ca2+]c) in a biphasic manner, and the effect of TG on 125I-insulin binding was abolished by BAPTA-AM. Western blot analysis showed that TG lowered insulin receptor (IR) beta-subunit level in membrane, but did not alter total cellular levels of IR precursor and IR beta-subunit. Internalization of cell surface IR, as measured by using brefeldin A, an inhibitor of vesicular exit from the trans-Golgi network (TGN), was not changed by TG. These results suggest that inhibition of SERCA by TG and the subsequent increase of [Ca2+]c down-regulates cell surface IR by retarding externalization of IR from the TGN.

We studied whether a novel proadrenomedullin derived peptide was present and what was its physiological function in cultured bovine adrenal chromaffin cells. We found a high level of proadrenomedullin N-terminal 12 peptide (PAMP-12) which consists of a peptide from 9th amino acid to 20th amino acid of proadrenomedullin N-terminal 20 peptide (PAMP-20). PAMP-12 was released from the cells along with catecholamine upon stimulation of nicotinic cholinergic receptors. When PAMP-12 was added in the incubation medium, this peptide inhibited nicotinic receptor-mediated catecholamine release and influx of Na + and Ca 2+ into the cells. PAMP-12 did not affect catecholamine release evoked by histamine or by depolarization by high concentration of potassium. PAMP-12 also inhibited synthesis of catecholamines as well as the activation of tyrosine hydroxylase by nicotinic stimulation. Thus, PAMP-12 is an endogenous peptide that regulates release and synthesis of catecholamines by acting on nicotinic cholinergic receptors in an autocrine manner in adrenal chromaffin cells.

Long-term (>/=3 h) treatment of cultured bovine adrenal chromaffin cells with cyclosporin A (CsA) decreased cell surface (125)I-insulin binding by 62% in a concentration (IC(50)=18 microM)- and time (t(1/2)=16 h)-dependent manner, but did not change the K(d) value. FK506 (1 microM) or rapamycin (3 microM) treatment reduced (125)I-insulin binding. Western blot analysis showed that CsA treatment decreased insulin receptor (IR) beta-subunit level (t(1/2)=15 h) in membrane fraction, but did not alter total cellular levels of IR precursor and IR beta-subunit. Internalization rate of cell surface IR measured by using brefeldin A, an inhibitor of vesicular exit from the trans-Golgi network, was comparable between non-treated and CsA-treated cells. Thus, CsA, FK506 and rapamycin inhibit peptidyl prolyl cis-trans isomerase activities of cyclophilin and FK506-binding protein, and down-regulate IR presumably by reducing cell surface externalization of IR.

In cultured bovine adrenal chromaffin cells, NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride], a newly-synthesized neuroprotective drug, inhibited veratridine-induced 22Na+ influx via voltage-dependent Na+ channels (IC50=11.4 μM). The inhibition by NS-7 occurred in the presence of ouabain, an inhibitor of Na+,K+ ATPase, but disappeared at higher concentration of veratridine, and upon the washout of NS-7. NS-7 attenuated veratridine-induced 45Ca2+ influx via voltage-dependent Ca2+ channels (IC50=20.0 μM) and catecholamine secretion (IC50=25.8 μM). Chronic (⩾12 h) treatment of cells with NS-7 increased cell surface [3H]-STX binding by 86% (EC50=10.5 μM; t1/2=27 h), but did not alter the KD value; it was prevented by cycloheximide, an inhibitor of protein synthesis, or brefeldin A, an inhibitor of vesicular transport from the trans-Golgi network, but was not associated with increased levels of Na+ channel α- and β1-subunit mRNAs. In cells subjected to chronic NS-7 treatment, 22Na+ influx caused by veratridine (site 2 toxin), α-scorpion venom (site 3 toxin) or β-scorpion venom (site 4 toxin) was suppressed even after the extensive washout of NS-7, and veratridine-induced 22Na+ influx remained depressed even at higher concentration of veratridine; however, either α- or β-scorpion venom, or Ptychodiscus brevis toxin-3 (site 5 toxin) enhanced veratridine-induced 22Na+ influx as in nontreated cells. These results suggest that in the acute treatment, NS-7 binds to the site 2 and reversibly inhibits Na+ channels, thereby reducing Ca2+ channel gating and catecholamine secretion. Chronic treatment with NS-7 up-regulates cell surface Na+ channels via translational and externalization events, but persistently inhibits Na+ channel gating without impairing the cooperative interaction between the functional domains of Na+ channels. Keywords: Neuroprotective drugs, sodium channels, up-regulation, [3H]-saxitoxin binding, Northern blot, veratridine, 22Na+ influx, 45Ca2+ influx, catecholamine secretion, adrenal chromaffin cells Introduction It has become increasingly evident that noninactivating Na+ currents (Taylor, 1993) via voltage-dependent Na+ channels (Catterall, 1992) may initiate the detrimental cascade of hypoxia/ischaemia-induced cell injury, such as the massive overflow of glutamate and catecholamines (Toner & Stamford, 1997), the intracellular Ca2+ overload via reversed operation of Na+-Ca2+ exchanger (Obrenovitch & Richards, 1995; Urenjak & Obrenovitch, 1996), as well as cytotoxic formation of nitric oxide (NO) (Strijbos et al., 1996). A few studies have revealed that during hypoxia, the steady-state inactivation of Na+ currents was shifted to a more hyperpolarizing potential in human cortical neurons (Cummins et al., 1993) and rat hippocampus (O'Reilly et al., 1997), presumably as the compensatory defensive response against hypoxia-induced neuronal injury. Also, density of cell surface Na+ channels was fluctuated in brain during hypoxia (Perez-Pinzon et al., 1992; Xia & Haddad, 1994; 1999). NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride] is a newly-synthesized neuroprotective agent. Previous in vivo and in vitro studies in cerebral cortex have shown that NS-7 attenuated hypoxia-induced degradation of cytoskeletal protein fodrin (Takagaki et al., 1997) and cell injury (Tatsumi et al., 1998b) at 10–30 μM, and the cellular mechanisms of NS-7 for neuroprotection are postulated to be attributed to the blockade of voltage-dependent Na+ and Ca2+ channels. In brain, NS-7 bound with a Ki value of 1 μM (Shimidzu et al., 1997) to the toxin binding site 2 of the Na+ channel α-subunit, a major subunit forming the ion-pore and the toxin binding sites 1–5 (Catterall, 1992); NS-7 at 10–30 μM diminished the overflow of glutamate (Shimidzu et al., 1997) and dopamine (Itoh et al., 1998) caused by veratridine, a toxin that interacts with the site 2 in the transmembrane segment 6 of domain I (IS6) of the Na+ channel α-subunit (Trainer et al., 1996) and activates Na+ channels (Catterall, 1992). NS-7 suppressed depolarization-elicited Na+ currents with an IC50 of 7.8 μM in NG108-15 cells (Suma et al., 1997). Also, in NG108-15 cells, NS-7 decreased Ca2+ currents via L-, N-, and T-type Ca2+ channels with IC50 values of 7.3, 4.5, and 17.1 μM, respectively (Suma et al., 1997). In cerebrocortical neurons, NS-7 attenuated high K+- or veratridine-induced activation of NO synthase at 10–30 μM presumably by inhibiting Ca2+ influx via L- and P/Q-type Ca2+ channels (Tatsumi et al., 1998a; Oka et al., 1999). NS-7 inhibited high K+-induced dopamine secretion at 10 μM (Itoh et al., 1998), but did not affect high K+-induced glutamate release in brain (Shimidzu et al., 1997). Na+ channel α-subunits arise from multiple genes and their alternative splicing (Dietrich et al., 1998), whereas the β1-subunits are structurally similar among various tissues (Makita et al., 1994). In adrenal chromaffin cells (embryologically derived from the neural crest), Na+ channel α-subunit (Yamamoto et al., 1997) is homologous to the tetrodotoxin (TTX)/saxitoxin (STX) (site 1 toxin)-sensitive human neuroendocrine type Na+ channel α-subunit (hNE-Na) (Klugbauer et al., 1995). Previous studies showed that veratridine-induced Na+ influx via Na+ channels, and the subsequent depolarization increases Ca2+ influx via voltage-dependent Ca2+ channels (Wada et al., 1985a,1985b; Lopez et al., 1995), thereby triggering veratridine-induced exocytic secretion of catecholamines (Ito et al., 1980). We found that chronic treatment of chromaffin cells with the antiepileptic drug valproic acid raised Na+ channel α- and β1-subunit mRNA levels, as well as cell surface [3H]-STX binding, thus enhancing veratridine-induced 22Na+ influx, 45Ca2+ influx and catecholamine secretion (Yamamoto et al., 1997). Similar observations were made in long-term treatment of chromaffin cells with the anticonvulsant carbamazepine (Yoshimura et al., 1998). Also, chronic in vivo and in vitro treatment with the antiepileptic drug phenytoin (Sashihara et al., 1994) and the antiarrhythmic drug mexiletine (Kang et al., 1997) caused up-regulation of Na+ channels, thereby modifying neuronal and cardiac pathophysiologies. Our present study examined whether/how short- and long-term treatment of chromaffin cells with NS-7 might alter 22Na+ influx, 45Ca2+ influx and catecholamine secretion, using veratridine, α-scorpion venom (site 3 toxin), β-scorpion venom (site 4 toxin) and Ptychodiscus brevis toxin-3 (PbTx-3) (site 5 toxin) (Wada et al., 1987; 1992; Catterall, 1992). Also, the effects of chronic treatment with NS-7 on cell surface [3H]-STX binding, Na+ channel α- and β1-subunit mRNA levels were evaluated.

To identify alpha(1)-adrenergic receptor subtypes involved in the regulation of cerebral microcirculation, we studied alpha(1)-adrenergic receptor subtypes expressed in the rat cerebral microvessels. The microvessels were prepared from rat cerebral cortex by albumin flotation and glass bead filtration techniques. [(125)I]HEAT binding to the cerebral microvessels was displaced by low concentrations of 5-methylurapidil, a selective antagonist for alpha(1A)-receptors, and modified Scatchard analysis of the data revealed that half of alpha(1)-receptors is alpha(1A)-subtype, and that alpha(1B)- and/or alpha(1D)-receptors are also present. The K(i) value of the high-affinity component for 5-methylurapidil was 3.90+/-1.08 nM, which is comparable with the value obtained in the rat cerebral cortex (2.17+/-0.88 nM). Reverse transcription and polymerase chain reaction experiments showed that mRNAs of alpha(1A)- and alpha(1B)-receptors, but not alpha(1D)-receptors, were expressed in the cerebral microvessels. These results suggest that alpha(1)-receptors involved in the regulation of cerebral microvessel function are alpha(1A)- and alpha(1B)-receptor subtypes.

In cultured bovine adrenal chromaffin cells, NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride], a newly-synthesized neuroprotective drug, inhibited nicotine-induced 22 Na + influx via nicotinic receptors (IC 50 =15.5 μM); the suppression by NS-7 was observed in the presence of ouabain, an inhibitor of Na + ,K + -ATPase, and was not attenuated upon the washout of NS-7. NS-7 decreased nicotine-induced maximum influx of 22 Na + without altering the EC 50 value of nicotine. Also, NS-7 diminished nicotine-induced 45 Ca 2+ influx via nicotinic receptors and voltage-dependent Ca 2+ channels (IC 50 =14.1 μM) and catecholamine secretion (IC 50 =19.5 μM). These results suggest that NS-7 produces noncompetitive and long-lasting inhibitory effects on neuronal nicotinic receptors in adrenal chromaffin cells, and interferes with the stimulus-secretion coupling.

We examined the effects of riluzole, a neuroprotective drug, on voltage-dependent Na channels, nicotinic receptors, and voltage-dependent Ca channels, as well as catecholamine secretion, in comparison with those of verapamil and nicardipine, in primary cultures of bovine adrenal chromaffin cells. Riluzole inhibited veratridine-induced 22Na influx via voltage-dependent Na channels even in the presence of ouabain, an inhibitor of Na,K-ATPase. Blockade of Na channels by riluzole was concentration-dependent with an IC50 of 5.3 microM. It was associated with a similar concentration-related reduction of veratridine-induced 45Ca influx via voltage-dependent Ca channels, and of catecholamine secretion. Riluzole had no effect on 45Ca influx caused by high K, which directly activates voltage-dependent Ca channels, and on nicotine-induced 22Na influx, which passes through the nicotinic receptors. Verapamil and nicardipine attenuated 22Na influx caused by veratridine or nicotine at the same concentrations as they suppressed high K-induced 45Ca influx. The inhibitory effect of riluzole on veratridine-induced 22Na influx disappeared at high concentrations of veratridine. A potentiation of veratridine (site 2 toxin)-induced 22Na influx caused by alpha-scorpion venom (site 3 toxin), beta-scorpion venom (site 4 toxin), or brevetoxin PbTx-3 (site 5 toxin), occurred in the presence of riluzole in the same manner as in control cells. These results suggest that riluzole binds to the veratridine site in voltage-dependent Na channels. It does not impair the cooperative interaction between the functional peptide segments of Na channels, but selectively inhibits gating of Na channels, thereby reducing Ca influx via Ca channels and catecholamine secretion. In contrast, verapamil and nicardipine suppress Na influx both Na channels and nicotinic receptors.

心電図R-R間隔の周波数解析において,高周波成分は副交感神経活動の,低周波成分は交感神経と副交感神経の活動性の指標になるとされる。脊椎麻酔施行時に高位脊椎麻酔(

In the present study, we investigated whether activation of the MAPK family could regulate the cell surface expression of Na channels in cultured bovine adrenal chromaffin cells. The results suggest that constitutively activated ERK (but not p38 or JNK), by various extracellular stimuli, down-modulates the density of cell surface Na channels, which was mediated via the destabilization of Na channel alpha-subunit mRNA.

Background High glucose can induce apoptosis in vascular endothelial cells, which may contribute to the development of vascular complications in diabetes. We evaluated the role of the death receptor pathway of apoptotic signaling in high glucose-induced apoptosis in human coronary artery endothelial cells (HCAECs). Methods HCAECs were treated with media containing 5.6, 11.1, and 16.7 mM of glucose for 24 h in the presence or absence of tumor necrosis factor (TNF)-α. For detection of apoptosis, DNA fragmentation assay was used. HCAEC expression of death receptors were analyzed by the PCR and flow cytometry methods. Also, using immunohistochemical techniques, coronary expression of death receptors was assessed in streptozotocin-nicotinamide-induced type 2 diabetic mice. Results Exposure of HCAECs to high glucose resulted in a significant increase in TNF-R1 and Fas expression, compared with normal glucose. High glucose increased TNF-α production by HCAECs and exogenous TNF-α up-regulated TNF-R1 and Fas expression in HCAECs. High glucose-induced up-regulation of TNF-R1 and Fas expression was undetectable in the presence of TNF-α. Treatment with TNF-R1 neutralizing peptides significantly inhibited high glucose-induced endothelial cell apoptosis. Type 2 diabetic mice displayed appreciable expression of TNF-R1 and Fas in coronary vessels. Conclusions In association with increased TNF-α levels, the death receptors, TNF-R1 and Fas, are up-regulated in HCAECs under high glucose conditions, which could in turn play a role in high glucose-induced endothelial cell apoptosis.

Hyperglycaemia is known to reduce nitric oxide (NO) bioavailability by modulating endothelial NO synthase (eNOS) activity, and polyphenols are believed to have cardiovascular benefit. One possible mechanism could be through interaction with eNOS.The effects of the oligomerized polyphenol oligonol on eNOS phosphorylation status and activity were examined in porcine aortic endothelial cells cultured in high glucose concentrations.Exposure to high glucose concentrations strongly inhibited eNOS phosphorylation at Ser-1177 and dephosphorylation at Thr-495 in bradykinin (BK)-stimulated cells. These inhibitory effects of high glucose were significantly prevented by treatment with oligonol. Akt and p38 mitogen-activated protein kinase (MAPK) were activated in BK-stimulated cells. High glucose inhibited Akt activation but enhanced p38 MAPK activation, both of which were reversed by oligonol treatment. The phosphatidylinositol 3-kinase inhibitor wortmannin blocked the reversal by oligonol of phosphorylation at Ser-1177, but not dephosphorylation at Thr-495, in BK-stimulated cells exposed to high glucose. The effect of oligonol on BK dephosphorylation under high glucose was mimicked by protein kinase C (PKC) epsilon-neutralizing peptides. These data suggest that the effects of oligonol on high glucose-induced attenuation of eNOS Ser-1177 phosphorylation and Thr-495 dephosphorylation may be regulated by Akt activation and PKCepsilon inhibition respectively. Oligonol also prevented high glucose-induced attenuation of BK-stimulated NO production.Oligonol prevented the impairment of eNOS activity induced by high glucose through reversing altered eNOS phosphorylation status. This mechanism may underlie the beneficial cardiovascular health effects of this oligomerized polyphenol.

The use of glucocorticoids for treatment of sepsis has waxed and waned during the past several decades, and recent randomized controlled trials have evoked a reassessment of this therapy. Most glucocorticoid actions are mediated by its specific intracellular receptors (GRs). Thus we initially evaluated whether sepsis and high-dose corticosteroid therapy can regulate guinea pig pulmonary expression of GRs: active receptor, GRalpha, and dominant negative receptor, GRbeta. Sepsis induction by LPS injection (300 mug/kg ip) decreased mRNA and protein levels of GRalpha and increased protein expression of GRbeta in lungs. High-dose methylprednisolone (40 mg/kg ip), administered simultaneously with LPS, markedly potentiated the decrease in GRalpha expression but slightly affected the increase in GRbeta expression. Consequently, this led to a significant reduction in GRalpha nuclear translocation. Nevertheless, methylprednisolone treatment strongly eliminated LPS induction of NF-kappaB activity, as determined by NF-kappaB nuclear translocation and by gel mobility shift assays. Furthermore, the LPS-induced increase in inflammatory cells in bronchoalveolar lavage fluid was blunted by administration of the corticosteroid. On the other hand, immunofluorescent staining for cleaved caspase-3 showed a marked increase in this proapoptotic marker in lung sections, and terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) represented an enhanced appearance of cell apoptosis in lungs and spleen when methylprednisolone was given together with LPS. Cell apoptosis is now considered to play a role in the pathogenesis of septic syndrome. We thus suggest that the action of glucocorticoids at high doses to accelerate sepsis-induced cell apoptosis may overwhelm their therapeutic advantages in septic shock.