UNC93B1 Mediates the Effects of cGAS-STING on ER Stress and Iron Death, and Reduces Renal Toxicity in Cadmium-Exposed Rats.

BACKGROUND: Long-term exposure to cadmium can induce renal toxicity in rats, leading to endoplasmic reticulum (ER) stress and iron death. Notably, in cadmium-exposed rats, there is an increased expression of UNC93B1 (unc-93 homolog B1). Consequently, our investigation aims to determine the impact of UNC93B1 on ER stress and iron death in cadmium-exposed rats by modulating the cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) pathway. METHODS: A cadmium-exposed rat model was established by intrabacally injecting chromium chloride (5 mg/kg, once a day for 4 weeks), and the levels of UCd (urine cadmium), UNAG (urine N-acetyl-ß-D-glucosaminidase), and UCr (urine creatinine) in urine were assessed. A silent UNC93B1 lentivirus was constructed, and STING agonists were procured and administered to the rats. Subsequently, kidney tissues were extracted post-mortem, and pathological changes in renal tissue were observed through hematoxylin and eosin (HE) staining. The expression and mRNA levels of UNC93B1, cGAS, and STING were examined using western blot (WB) and polymerase chain reaction (PCR). Autophagy proteins (light chain 3 (LC3), Beclin-1, p62) were also assessed by WB. Additionally, iron concentration was determined using a kit, while oxidative stress markers (cytochrome oxidase subunit 2 (COX2), glutathione peroxidase 4 (GPX4), superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH)) were measured through enzyme-linked immunosorbent assay (ELISA). Furthermore, endoplasmic reticulum stress proteins (protein kinase RNA-like ER kinase (PERK), CCAAT enhance-binding protein homologous protein (CHOP), activating transcription factor-4 (ATF4)) were analyzed by WB. RESULTS: Wstaining, WB, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), ELISA, and HE staining collectively revealed a heightened expression of UNC93B1, cGAS, and STING, accompanied by increased levels of autophagy, oxidative stress, and ER stress in cadmium-exposed rats (p < 0.05). Nephrotoxicity exhibited a reduction following the inhibition of UNC93B1, leading to decreased levels of oxidative stress, autophagy, and ER stress (p < 0.05). Notably, this observed phenomenon was reversed upon the addition of STING agonists, suggesting that UNC93B1 might exert a nephroprotective effect in cadmium-exposed rats through modulation of the cGAS-STING pathway. CONCLUSIONS: The inhibition of UNC93B1 mitigates nephrotoxicity in cadmium-exposed rats, and this protective effect is mechanistically linked to the cGAS-STING pathway.

AD-DETR: DETR with asymmetrical relation and decoupled attention in crowded scenes.

Pedestrian detection in crowded scenes is widely used in computer vision. However, it still has two difficulties: 1) eliminating repeated predictions (multiple predictions corresponding to the same object); 2) false detection and missing detection due to the high scene occlusion rate and the small visible area of detected pedestrians. This paper presents a detection framework based on DETR (detection transformer) to address the above problems, and the model is called AD-DETR (asymmetrical relation detection transformer). We find that the symmetry in a DETR framework causes synchronous prediction updates and duplicate predictions. Therefore, we propose an asymmetric relationship fusion mechanism and let each query asymmetrically fuse the relative relationships of surrounding predictions to learn to eliminate duplicate predictions. Then, we propose a decoupled cross-attention head that allows the model to learn to restrict the range of attention to focus more on visible regions and regions that contribute more to confidence. The method can reduce the noise information introduced by the occluded objects to reduce the false detection rate. Meanwhile, in our proposed asymmetric relations module, we establish a way to encode the relative relation between sets of attention points and improve the baseline. Without additional annotations, combined with the deformable-DETR with Res50 as the backbone, our method can achieve an average precision of 92.6%, MR$ ^{-2} $ of 40.0% and Jaccard index of 84.4% on the challenging CrowdHuman dataset. Our method exceeds previous methods, such as Iter-E2EDet (progressive end-to-end object detection), MIP (one proposal, multiple predictions), etc. Experiments show that our method can significantly improve the performance of the query-based model for crowded scenes, and it is highly robust for the crowded scene.

Circ_0003356 suppresses gastric cancer growth through targeting the miR-668-3p/SOCS3 axis.

BACKGROUND: Circular RNAs (circRNAs) have attracted extensive attention as therapeutic targets in gastric cancer (GC). Circ_0003356 is known to be downregulated in GC tissues, but its cellular function and mechanisms remain undefined. AIM: To investigate the role of circ_0003356 in GC at the molecular and cellular level. METHODS: Circ_0003356, miR-668-3p, and SOCS3 expression were assessed via quantitative real time-polymerase chain reaction (qRT-PCR). Wound healing, EdU, CCK-8, flow cytometry and transwell assays were used to analyze the migration, proliferation, viability, apoptosis and invasion of GC cells. The subcellular localization of circ_0003356 was monitored using fluorescence in situ hybridization. The interaction of circ_0003356 with miR-668-3p was confirmed using RIP-qRT-PCR, RNA pull-down, and dual luciferase reporter assays. We observed protein levels of genes via western blot. We injected AGS cells into the upper back of mice and performed immunohistochemistry staining for examining E-cadherin, N-cadherin, Ki67, and SOCS3 expressions. TUNEL staining was performed for the assessment of apoptosis in mouse tumor tissues. RESULTS: Circ_0003356 and SOCS3 expression was downregulated in GC cells, whilst miR-668-3p was upregulated. Exogenous circ_0003356 expression and miR-668-3p silencing suppressed the migration, viability, proliferation, epithelial to mesenchy-mal transition (EMT) and invasion of GC cells and enhanced apoptosis. Circ_0003356 overexpression impaired tumor growth in xenograft mice. Targeting of miR-668-3p by circ_0003356 was confirmed through binding assays and SOCS3 was identified as a downstream target of miR-668-3p. The impacts of circ_0003356 on cell proliferation, apoptosis, migration, invasion and EMT were reversed by miR-668-3p up-regulation or SOCS3 down-regulation in GC cells. CONCLUSION: Circ_0003356 impaired GC development through its interaction with the miR-668-3p/SOCS3 axis.

Recent Progress in Electrochemical Nano-Biosensors for Detection of Pesticides and Mycotoxins in Foods.

Pesticide and mycotoxin residues in food are concerning as they are harmful to human health. Traditional methods, such as high-performance liquid chromatography (HPLC) for such detection lack sensitivity and operation convenience. Efficient, accurate detection approaches are needed. With the recent development of nanotechnology, electrochemical biosensors based on nanomaterials have shown solid ability to detect trace pesticides and mycotoxins quickly and accurately. In this review, English articles about electrochemical biosensors in the past 11 years (2011-2022) were collected from PubMed database, and various nanomaterials are discussed, including noble metal nanomaterials, magnetic metal nanoparticles, metal-organic frameworks, carbon nanotubes, as well as graphene and its derivatives. Three main roles of such nanomaterials in the detection process are summarized, including biomolecule immobilization, signal generation, and signal amplification. The detection targets involve two types of pesticides (organophosphorus and carbamate) and six types of mycotoxins (aflatoxin, deoxynivalenol, zearalenone, fumonisin, ochratoxin A, and patulin). Although significant achievements have been made in the evolution of electrochemical nano-biosensors, many challenges remain to be overcome.

Identification of qnrVF in a Multidrug-Resistant Vibrio furnissii Clinical Strain.

We found a new qnr gene, qnrVF1, carried by a multidrug resistance plasmid in a clinical Vibrio furnissii isolate. QnrVF1 exhibits 44.6% to 72.5% similarity in identity with other Qnr family proteins. QnrVF alleles are mainly encoded by chromosomes of V. furnissii and Vibrio fluvialis. Phylogenic analysis showed that QnrVF1 and QnrVF2 form a distinct clade in Qnr proteins. Thus, qnrVF represents a new qnr family. In addition, the qnrVF1 gene is often flanked by the mobile element ISCR1. Thus, it is likely that qnrVF1 is mobilized by ISCR1 from chromosome to plasmid in V. furnissii. IMPORTANCE Quinolones are widely used drugs. Bacteria contain a quinolone resistance gene, which mediates resistance to quinolones. Currently, seven families of Qnr proteins, QnrVC, QnrA, QnrB, QnrC, QnrD, QnrE, and QnrS, have been identified. However, it is unclear whether there are any other qnr families. In this study, we identified a new qnr family, qnrVF. We found many V. furnissii and V. fluvialis strains that possess chromosomal qnrVF alleles, suggesting that V. furnissii and V. fluvialis are the reservoirs of qnrVF. We also found that QnrVF1 confers low-level resistance to quinolones. ISCR1 may facilitate the spread of qnrVF1. The emergence and spread of qnrVF may pose a considerable threat to public health.

Effect of Different Soft Segment Contents on the Energy Storage Capacity and Photo-Thermal Performance of Polyurethane-Based/Graphene Oxide Composite Solid-Solid Phase Change Materials.

A series of polyurethane/graphene oxide (PU/GO) solid-solid phase change materials (SSPCMs) were synthesized by using GO as a light-absorbing filler and polyethylene glycol (PEG) as a phase change matrix. The effects of PEG content on the energy storage capacity, thermal stability and photo-thermal conversion performance of PU were investigated. The results show that the form-stability of PU/GO decreases while the phase change enthalpy and photo-thermal conversion efficiency of PU/GO increases with the increasing PEG content. The introduction of a very low content of GO can maintain comparable energy storage density and greatly improve light absorption by reasonably modulating the soft segment contents. The PU/GO composite with 92 wt% PEG has a phase change enthalpy of 138.12 J/g and a high photo-thermal conversion efficiency (87.6%). The composite solid-solid PCMs have great potential for effective energy storage and solar energy utilization.

Umbelliferone and scopoletin target tyrosine kinases on fibroblast-like synoviocytes to block NF-κB signaling to combat rheumatoid arthritis.

Rheumatoid arthritis (RA) is a complex autoimmune condition primarily affecting synovial joints, which targeted synthetic drugs have damaging safety issues. Saussurea laniceps, a reputed anti-rheumatic medicinal herb, is an excellent place to start looking for natural products as safe, effective, targeted therapeutics for RA. Via biomimetic ultrafiltration, umbelliferone and scopoletin were screened as two anti-rheumatic candidates with the highest specific affinities towards the membrane proteomes of rheumatic fibroblast-like synoviocytes (FLS), the pivotal effector cells in RA. In vitro assays confirmed that the two compounds, to varying extents, inhibited RA-FLS proliferation, migration, invasion, and NF-κB signaling. Network pharmacology analysis and molecular docking analysis jointly revealed that umbelliferone and scopoletin act on multiple targets, mostly tyrosine kinases, in combating RA. Taken together, our present study identified umbelliferone and scopoletin as two major anti-rheumatic components from SL that may bind and inhibit tyrosine kinases and subsequently inactivate NF-κB in RA-FLSs. Our integrated drug discovery strategy could be valuable in finding other multi-target bioactive compounds from complex matrices for treating multifactorial diseases.

Effects of Bonding Materials on Optical-Thermal Performances and High-Temperature Reliability of High-Power LED.

The die-bonding layer between chips and substrate determinates the heat conduction efficiency of high-power LED. Sn-based solder, AuSn20 eutectic, and nano-Ag paste were widely applied to LED interconnection. In this paper, the optical-thermal performances and high-temperature reliability of LED with these bonding materials have systematically compared and studied. The thermal conductivity, electrical resistivity, and mechanical property of these bonding materials were characterized. The LED module packaged with nano-Ag has a minimum working temperature of 21.5 °C. The total thermal resistance of LED packaged with nano-Ag, Au80Sn20, and SAC305 is 4.82, 7.84, and 8.75 K/W, respectively, which is 4.72, 6.14, and 7.84 K/W higher after aging for 500 h. Meanwhile, the junction temperature change of these LEDs increases from 2.33, 3.76, and 4.25 °C to 4.34, 4.81, and 6.41 °C after aging, respectively. The thermal resistance of the nano-Ag, Au80Sn20 and SAC305 layer after aging is 1.5%, 65.7%, and 151.5% higher than before aging, respectively. After aging, the LED bonded with nano-Ag has the better optical performances in spectral intensity and light output power, which indicates its excellent heat dissipation can improve the light efficiency. These results demonstrate the nano-Ag bonding material could enhance the optical-thermal performances and high-temperature reliability of high-power LED.

Sevoflurane-induced delayed neuroprotection involves mitoK(ATP) channel opening and PKC ε activation.

There is an increasing body of evidence that a brief exposure to anesthesia induces ischemic tolerance in rat brain (anesthetic preconditioning). However, it is unknown whether preconditioning with sevoflurane, a commonly used volatile anesthetic in current clinical practice, produces a delayed window of neuroprotection against ischemia and what the mechanisms are for this protection. To address these issues, adult male Sprague-Dawley rats were subjected to middle cerebral arterial occlusion (MCAO) for 2 h. Sevoflurane preconditioning was induced 24 h before brain ischemia by exposing the animals to sevoflurane at 1.0 minimum alveolar concentration (2.4%) in oxygen for 60 min. Animals preconditioned with sevoflurane had lower neurological deficit scores and smaller brain infarct volumes than animals with brain ischemia at 6 and 24 h after MCAO, respectively. Application of a selective antagonist for mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel, 5-hydroxydecanoate (5-HD, 40 mg/kg i.p.) 30 min before sevoflurane exposure attenuated this beneficial effect. Moreover, protein kinase C ε (PKC ε) was translocated to the membrane fraction at 6 h, but not 24 h, after brain reperfusion in animals preconditioned with sevoflurane and this effect was also abolished by 5-HD. We concluded that sevoflurane preconditioning induces a delayed neuroprotection and that mitochondrial K(ATP) channels and PKC ε may be involved in this neuroprotection.

Inhibition of isoflurane-induced increase of cell-surface redistribution and activity of glutamate transporter type 3 by serine 465 sequence-specific peptides.

Excitatory amino acid transporters (EAAT) transport glutamate into cells to regulate glutamate neurotransmission and to maintain nontoxic extracellular glutamate levels for neurons. We showed previously that the commonly used volatile anesthetic isoflurane increases the transporting activity of EAAT3, the major neuronal EAAT. This effect requires a protein kinase C (PKC) α-mediated and S465-dependent EAAT3 redistribution to the plasma membrane. Thus, we hypothesize that specific peptides can be designed to block this effect. We conjugated a 10-amino acid synthetic peptide with a sequence identical to that of EAAT3 around the S465 to a peptide that can facilitate permeation of the plasma membrane. This fusion peptide inhibited the isoflurane-increased EAAT3 activity and redistribution to the plasma membrane in C6 cells and hippocampus. It did not affect the basal EAAT3 activity. This peptide also attenuated isoflurane-induced increase of PKCα in the immunoprecipitates produced by an anti-EAAT3 antibody. A scrambled peptide that has the same amino acid composition as the S465 sequence-specific peptide but has a random sequence did not change the effects of isoflurane on EAAT3. The S465 sequence-specific peptide, but not the scrambled peptide, is a good PKCα substrate in in vitro assay. These peptides did not affect cell viability. These results, along with our previous findings, strongly suggest that PKCα interacts with EAAT3 to regulate its functions. The S465 sequence-specific peptide may interrupt this interaction and is an effective inhibitor for the regulation of EAAT3 activity and trafficking by PKCα and isoflurane.

Critical role of s465 in protein kinase C-increased rat glutamate transporter type 3 activity.

Glutamate transporters, also called excitatory amino acid transporters (EAATs), uptake extracellular glutamate and regulate neurotransmission. Activation of protein kinase C (PKC) increases the activity of EAAT type 3 (EAAT3), the major neuronal EAAT. We designed this study to determine which amino acid residue(s) in EAAT3 may be involved in this PKC effect. Selective potential PKC phosphorylation sites were mutated. These EAAT3 mutants were expressed in the Xenopus oocytes. Phorbol 12-myristate 13-acetate, a PKC activator, significantly increased wild-type EAAT3 activity. Mutation of serine 465 to alanine or aspartic acid, but not the mutation of threonine 5 to alanine, abolished PKC-increased EAAT3 activity. Our results suggest a critical role of serine 465 in the increased EAAT3 activity by PKC activation.

Opioid preconditioning induces opioid receptor-dependent delayed neuroprotection against ischemia in rats.

We have shown that exposure of neurons to opioid immediately before ischemia induces ischemia tolerance. This phenomenon is called acute opioid preconditioning. In this study, we test the hypothesis that opioids induce delayed neuropreconditioning (from hours to days after opioid exposure). Exposure to morphine, an agonist for delta-, mu-, and kappa-opioid receptors, or Tan-67, a selective delta1-receptor agonist, for 30 minutes at 24 hours before a 35-minute oxygen-glucose deprivation (OGD, to simulate ischemia in vitro) dose-dependently reduced the OGD-induced neuronal death in the CA1 region of the rat organotypic hippocampal slice cultures. The morphine preconditioning-induced neuroprotection was inhibited by beta-funaltrexamine, a mu-opioid receptor antagonist, but not by 7-benzylidenenaltrexone, a delta1-receptor antagonist, or nor-binaltorphimine, a kappa-receptor antagonist. The Tan-67 preconditioning-induced neuroprotection was inhibited by 7-benzylidenenaltrexone. The combination of morphine and Tan-67 did not induce a better preconditioning effect than did morphine or Tan-67 alone. Application of morphine and Tan-67 at 24 hours before permanent right middle cerebral arterial occlusion reduced brain infarct volume and improved neurologic functional outcome assessed 24 hours after the occlusion in adult male rats. These results suggest that morphine and Tan-67 induce a delayed preconditioning effect in the brain under in vivo and in vitro conditions. Whereas the delayed phase of morphine preconditioning may involve mu-opioid receptors, Tan-67 preconditioning may be mediated by delta1-opioid receptors. Morphine and Tan-67 may activate a shared intracellular signaling pathway to induce the delayed preconditioning effects in the brain.

Critical role of serine 465 in isoflurane-induced increase of cell-surface redistribution and activity of glutamate transporter type 3.

Glutamate transporters (also called excitatory amino acid transporters, EAATs) bind extracellular glutamate and transport it to intracellular space to regulate glutamate neurotransmission and to maintain extracellular glutamate concentrations below neurotoxic levels. We previously showed that isoflurane, a commonly used anesthetic, enhanced the activity of EAAT3, a major neuronal EAAT. This effect required a protein kinase C (PKC) alpha-dependent EAAT3 redistribution to the plasma membrane. In this study, we prepared COS7 cells stably expressing EAAT3 with or without mutations of potential PKC phosphorylation sites in the putative intracellular domains. Here we report that mutation of threonine 5 or threonine 498 to alanine did not affect the isoflurane effects on EAAT3. However, the mutation of serine 465 to alanine abolished isoflurane-induced increase of EAAT3 activity and redistribution to the plasma membrane. The mutation of serine 465 to aspartic acid increased the expression of EAAT3 in the plasma membrane and also abolished the isoflurane effects on EAAT3. These results suggest an essential role of serine 465 in the isoflurane-increased EAAT3 activity and redistribution and a direct effect of PKC on EAAT3. Consistent with these results, isoflurane induced an increase in phosphorylation of wild type, T5A, and T498A EAAT3, and this increase was absent in S465A and S465D. Our current results, together with our previous data that showed the involvement of PKCalpha in the isoflurane effects on EAAT3, suggest that the phosphorylation of serine 465 in EAAT3 by PKCalpha mediates the increased EAAT3 activity and redistribution to plasma membrane after isoflurane exposure.

Isoflurane preconditioning protects human neuroblastoma SH-SY5Y cells against in vitro simulated ischemia-reperfusion through the activation of extracellular signal-regulated kinases pathway.

It has been reported that a prior exposure of isoflurane, a commonly used volatile anesthetic in clinical practice, reduces brain cell death after ischemia. This isoflurane preconditioning-induced neuroprotection has been shown in rat in vivo and in vitro brain ischemia models. To investigate the mechanisms of this protection, we used the human neuroblastoma SH-SY5Y cells and simulated ischemia in vitro by oxygen-glucose deprivation. We found that isoflurane exposure for 30 min at 24 h before a 5-h oxygen-glucose deprivation dose-dependently reduced cell death. Isoflurane exposure induced phosphorylation/activation of extracellular signal-regulated kinase (ERK). Inhibition of the phospho-ERK expression abolished the isoflurane preconditioning-induced protection. Isoflurane exposure also increased the expression of early growth response gene 1 (Egr-1) and Bcl-2, proteins downstream of ERK. Egr-1 is a transcription factor and plays a role in cell survival. Bcl-2 is an anti-apoptotic protein. The increased expression of Egr-1 and Bcl-2 by isoflurane was inhibited by ERK inhibition. Thus, our results suggest a role of ERK/Egr-1/Bcl-2 pathway in the isoflurane preconditioning-induced protection in the human neuroblastoma SH-SY5Y cells.

Enhancement of substrate-gated Cl- currents via rat glutamate transporter EAAT4 by PMA.

Glutamate transporters (also called excitatory amino acid transporters, EAAT) are important in extracellular homeostasis of glutamate, a major excitatory neurotransmitter. EAAT4, a neuronally expressed EAAT in cerebellum, has a large portion (approximately 95% of the total L-aspartate-induced currents in human EAAT4) of substrate-gated Cl(-) currents, a distinct feature of this EAAT. We cloned EAAT4 from rat cerebellum. This molecule was predicted to have eight putative transmembrane domains. L-glutamate induced an inward current in oocytes expressing this EAAT4 at a holding potential -60 mV. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, significantly increased the magnitude of L-glutamate-induced currents but did not affect the apparent affinity of EAAT4 for L-glutamate. This PMA-enhanced current had a reversal potential -17 mV at extracellular Cl(-) concentration ([Cl(-)](o)) 104 mM with an approximately 60-mV shift per 10-fold change in [Cl(-)](o), properties consistent with Cl(-)-selective conductance. However, PMA did not change EAAT4 transport activity as measured by [(3)H]-L-glutamate. Thus PMA-enhanced Cl(-) currents via EAAT4 were not thermodynamically coupled to substrate transport. These PMA-enhanced Cl(-) currents were partially blocked by staurosporine, chelerythrine, and calphostin C, the three PKC inhibitors. Ro-31-8425, a PKC inhibitor that inhibits conventional PKC isozymes at low concentrations (nM level), partially inhibited the PMA-enhanced Cl(-) currents only at a high concentration (1 microM). Intracellular injection of BAPTA, a Ca(2+)-chelating agent, did not affect the PMA-enhanced Cl(-) currents. 4alpha-Phorbol-12,13-didecanoate, an inactive analog of PMA, did not enhance glutamate-induced currents. These data suggest that PKC, possibly isozymes other than conventional ones, modulates the substrate-gated Cl(-) currents via rat EAAT4. Our results also suggest that substrate-gated ion channel activity and glutamate transport activity, two EAAT4 properties that could modulate neuronal excitability, can be regulated independently.

Carbamazepine enhances the activity of glutamate transporter type 3 via phosphatidylinositol 3-kinase.

Glutamate transporters (also called excitatory amino acid transporters, EAAT) participate in maintaining extracellular homeostasis of glutamate, a major excitatory neurotransmitter, and regulating glutamate neurotransmission. EAAT3, the major neuronal EAAT, may also regulate gamma-aminobutyric acid-mediated inhibitory neurotransmission. Dysfunction of EAAT3 has been shown to induce seizure in rats. We hypothesize that carbamazepine, a commonly used antiepileptic agent, enhances EAAT3 activity. We tested this hypothesis using oocytes artificially expressing EAAT3 and C6 rat glioma cells expressing endogenous EAAT3. In oocytes, carbamazepine dose-dependently enhanced EAAT3 activity. The EC50 of this carbamazepine effect was 12.2muM. The concentrations of carbamazepine to significantly enhance EAAT3 activity were within the therapeutic serum levels (17-51muM) of carbamazepine for the antiepileptic effect. Carbamazepine decreased the Km but did not change the maximal response of EAAT3 to glutamate. Carbamazepine-increased EAAT3 activity was inhibited by wortmannin or LY-294002, phosphatidylinositol 3-kinase (PI3K) inhibitors, but was not affected by staurosporine, chelerythrine or calphostin C, protein kinase C inhibitors. In C6 cells, carbamazepine also enhanced the endogenous EAAT3 activity. However, carbamazepine did not affect the activity of EAAT4 expressed in Cos7 cells. These results suggest that carbamazepine at clinically relevant concentrations specifically enhances the affinity of EAAT3 for glutamate to increase EAAT3 activity via a PI3K-dependent pathway. EAAT3 may be a therapeutic target for carbamazepine in the central nervous system.

Isoflurane induces a protein kinase C alpha-dependent increase in cell-surface protein level and activity of glutamate transporter type 3.

Glutamate transporters regulate extracellular concentrations of glutamate, an excitatory neurotransmitter in the central nervous system. We have shown that the commonly used anesthetic isoflurane increased the activity of glutamate transporter type 3 (excitatory amino acid transporter 3, EAAT3) possibly via a protein kinase C (PKC)-dependent pathway. In this study, we showed that isoflurane induced a time- and concentration-dependent redistribution of EAAT3 to the cell membrane in C6 glioma cells. This redistribution was inhibited by staurosporine, a pan PKC inhibitor, or by 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (Go6976) at a concentration that selectively inhibits conventional PKC isozymes (PKC alpha, -beta, and -gamma). This isoflurane-induced EAAT3 redistribution was also blocked when the expression of PKC alpha but not PKC beta proteins was down-regulated by the respective antisense oligonucleotides. The isoflurane-induced increase of glutamate uptake by EAAT3 was abolished by the down-regulation of PKC alpha expression. Immunoprecipitation with an anti-EAAT3 antibody pulled down more PKC alpha in cells exposed to isoflurane than in control cells. Isoflurane also increased the phosphorylated EAAT3 and the redistribution of PKC alpha to the particulate fraction of cells. Consistent with the results in C6 cells, isoflurane also increased EAAT3 cell-surface expression and enhanced the association of PKC alpha with EAAT3 in rat hippocampal synaptosomes. Our results suggest that the isoflurane-induced increase in EAAT3 activity requires an increased amount of EAAT3 protein in the plasma membrane. These effects are PKC alpha-dependent and may rely on the formation of an EAAT3-PKC alpha complex. Together, these results suggest an important mechanism for the regulation of glutamate transporter functions and expand our understanding of isoflurane pharmacology at cellular and molecular levels.

Hypothermic preconditioning increases survival of purkinje neurons in rat cerebellar slices after an in vitro simulated ischemia.

BACKGROUND: A period of hypothermia before ischemia (hypothermic preconditioning) induces a delayed phase of ischemic tolerance in rat brain. However, whether hypothermic preconditioning induces an acute phase (within a few hours after the hypothermia) of ischemic tolerance remains unknown. This study was designed to determine the time window of the hypothermic preconditioning-induced acute phase of neuroprotection, which is useful information for situations during surgery with anticipated ischemic episodes, and its involved mechanisms. METHODS: Survival of Purkinje cells in rat cerebellar slices was evaluated after a 20-min oxygen-glucose deprivation (OGD, in vitro simulated ischemia) followed by a 4-h recovery. Mild hypothermia (33 degrees C) for 20 min was applied at various times before the OGD. RESULTS: The hypothermia applied immediately to 3 h before the OGD equally effectively reduced OGD-induced Purkinje cell death/injury. Glibenclamide, a selective KATP channel blocker; 8-cyclopentyl-1,3-dipropylxanthine, a selective adenosine A1 receptor antagonist; and farnesyl protein transferase inhibitor III, a selective inhibitor to reduce Ras farnesylation, abolished hypothermic preconditioning-induced neuroprotection when applied during the hypothermia. OGD increased the expression of high-mobility group I(Y) proteins, which are nuclear transcription factors to enhance the expression of putatively damaging proteins such as cyclooxygenase-2, in cerebellar slices. This increase was attenuated by hypothermic preconditioning. CONCLUSIONS: Hypothermic preconditioning induces an acute phase of neuroprotection. This neuroprotection depends on activation of the signaling molecules, adenosine A1 receptors, KATP channels, and Ras. Inhibition of putatively damaging proteins via the effects of hypothermic preconditioning on high-mobility group I(Y) expression may also be involved in hypothermic preconditioning-induced neuroprotection.

Isoflurane enhances the expression and activity of glutamate transporter type 3 in C6 glioma cells.

BACKGROUND: Glutamate transporters play an important role in maintaining extracellular glutamate homeostasis. Volatile anesthetics have been shown to affect glutamate transporter activity acutely (within minutes after the exposure). It is not known whether volatile anesthetics affect the expression of glutamate transporters. METHODS: Rat cultured C6 glioma cells that express excitatory amino acid transporter type 3 (EAAT3) were exposed to isoflurane at various concentrations (0.5-4.0%) or for different periods (1-24 h) at 37 degrees C. EAAT3 mRNA, proteins, and activity were quantified. RESULTS: Isoflurane induced a time- and concentration-dependent increase in the mRNA and protein levels of EAAT3 in C6 cells. The maximal increase was induced by 2% isoflurane, and the cells incubated with 2% isoflurane for 3 and 7 h expressed the highest levels of EAAT3 mRNA and proteins, respectively. Similarly, glutamate uptake was higher in C6 cells exposed to 2% isoflurane for 7 h than in control cells. Actinomycin D and cycloheximide, inhibitors for mRNA and protein synthesis, respectively, did not affect the isoflurane-induced increase in EAAT3 mRNA and protein expression. Phorbol 12-myristate 13-acetate, a protein kinase C activator, also enhanced EAAT3 expression. The combination of 2% isoflurane and phorbol 12-myristate 13-acetate caused a higher level of EAAT3 expression than that induced by 2% isoflurane alone. Neither staurosporine, a protein kinase C inhibitor, nor wortmannin, a phosphatidylinositol 3 kinase inhibitor, inhibited the isoflurane-induced increase in EAAT3 expression. CONCLUSIONS: The results of this study suggest that isoflurane increases the expression and activity of EAAT3 by stabilizing EAAT3 mRNA and proteins via protein kinase C- and phosphatidylinositol 3 kinase-independent pathways.

Effects of local and intravenous anesthetics on the activity of glutamate transporter type 2.

Glutamate transporters may be important targets for anesthetic action in the central nervous system. The authors investigated the effects of the intravenous anesthetics, thiopental and ketamine, and the local anesthetics, lidocaine and bupivacaine, on the activity of glutamate transporter type 2, EAAT2. EAAT2 was expressed in Xenopus oocytes by injection of its mRNA. By using two-electrode voltage clamping, membrane currents were recorded after the application of L-glutamate (30 microM) in the presence or absence of various concentrations of anesthetics. Lidocaine and bupivacaine did not change glutamate-induced inward currents at the tested concentrations (1-1000 microM). Thiopental and ketamine also did not affect the activity of EAAT2 at the tested concentrations (0.3-300 microM). Our results suggest that the two commonly used local anesthetics (lidocaine and bupivacaine) and intravenous anesthetics (thiopental and ketamine) do not affect the activity of EAAT2 expressed in oocytes. EAAT2 may not be a target for these anesthetics.