Inhibition Of Propofol Research Articles

Influence of Anesthetic Regimes on Extracellular Vesicles following Remote Ischemic Preconditioning in Coronary Artery Disease.

Remote ischemic preconditioning (RIPC) reduces ischemia-reperfusion injury in aortocoronary bypass surgery, potentially via extracellular vesicles (EVs) and their micro-RNA content. Clinical data implicate that propofol might inhibit the cardioprotective RIPC effect. This prospective, randomized study investigated the influence of different anesthetic regimes on RIPC efficacy and EV micro-RNA signatures. We also assessed the impact of propofol on cell protection after hypoxic conditioning and EV-mediated RIPC in vitro. H9c2 rat cardiomyoblasts were subjected to hypoxia, with or without propofol, and subsequent simulated ischemia-reperfusion injury. Apoptosis was measured by flow cytometry. Blood samples of 64 patients receiving anesthetic maintenance with propofol or isoflurane, along with RIPC or sham procedures, were analyzed, and EVs were enriched using a polymer-based method. Propofol administration corresponded with increased Troponin T levels (4669 ± 435.6 pg/mL), suggesting an inhibition of the cardioprotective RIPC effect. RIPC leads to a notable rise in miR-21 concentrations in the group receiving propofol anesthesia (fold change 7.22 ± 6.6). In vitro experiments showed that apoptosis reduction was compromised with propofol and only occurred in an EV-enriched preconditioning medium, not in an EV-depleted medium. Our study could clinically and experimentally confirm propofol inhibition of RIPC protection. Increased miR-21 expression could provide evidence for a possible inhibitory mechanism.

Propofol rescues voltage-dependent gating of HCN1 channel epilepsy mutants.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels1 are essential for pacemaking activity and neural signalling2,3. Drugs inhibiting HCN1 are promising candidates for management of neuropathic pain4 and epileptic seizures5. The general anaesthetic propofol (2,6-di-iso-propylphenol) is a known HCN1 allosteric inhibitor6 with unknown structural basis. Here, using single-particle cryo-electron microscopy and electrophysiology, we show that propofol inhibits HCN1 by binding to a mechanistic hotspot in a groove between the S5 and S6 transmembrane helices. We found that propofol restored voltage-dependent closing in two HCN1 epilepsy-associated polymorphisms that act by destabilizing the channel closed state: M305L, located in the propofol-binding site in S5, and D401H in S6 (refs. 7,8). To understand the mechanism of propofol inhibition and restoration of voltage-gating, we tracked voltage-sensor movement in spHCN channels and found that propofol inhibition is independent of voltage-sensor conformational changes. Mutations at the homologous methionine in spHCN and an adjacent conserved phenylalanine in S6 similarly destabilize closing without disrupting voltage-sensor movements, indicating that voltage-dependent closure requires this interface intact. We propose a model for voltage-dependent gating in which propofol stabilizes coupling between the voltage sensor and pore at this conserved methionine-phenylalanine interface in HCN channels. These findings unlock potential exploitation of this site to design specific drugs targeting HCN channelopathies.

Propofol-Induced miR-493-3p Inhibits Growth and Invasion of Gastric Cancer through Suppression of DKK1-Mediated Wnt/β-Catenin Signaling Activation.

Gastric cancer (GC) is one of the most common malignant tumors and also one of the most deadly tumors. In recent years, studies have shown that propofol can inhibit the proliferation and metastasis of many tumor cells. In the present study, we aimed to investigate the underlying mechanism of propofol inhibition of the growth and invasion of GC cells. Human gastric cancer cell line SGC-7901 and human normal gastric epithelial cell GES-1 were cultured in high-glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum at 37°C with 5% CO2. Propofol of different concentrations (0, 2, 5, and 10 μg/mL) was used to treat SGC-7901, and miR-493-3p inhibitor was transfected into SGC-7901. The cell proliferation of SGC-7901 was analyzed by MTT as well as colony formation assay. The qRT-PCR was used to assess the expression of mRNA for key genes. We examined the protein expression of DKK1 and relative markers with western blot. Putative binding places of miR-493-3p on the 3'-untranslated area of DKK1 were predicted using bioinformatics and dual-luciferase method. Propofol prohibited phenotypic features of GC, according to our findings. Furthermore, research into the underlying mechanisms of propofol's suppressive effects in GC cell proved that propofol therapy improved the degrees of expression of the potential tumor suppressor miR-493-3p. The inhibiting properties of propofol on GC cell development, migration, and invasion were abolished when propofol-induced miR493-3p was silenced with anti-miR-493-3p. We also found that this drug reversed epithelial-mesenchymal transformation in SGC-7901 cells via inducing miR-493-3p. Propofol-induced miR-493-3p decreases GC cell development via targeting DKK1 and hence inhibits Wnt/β-catenin signaling, according to these findings. Propofol-induced miR-493-3p decreased GC cell development via targeting DKK1 and hence inhibited Wnt/β-catenin signaling, according to these findings.

Propofol suppresses proliferation, migration, invasion, and tumor growth of liver cancer cells via suppressing cancer susceptibility candidate 9/phosphatase and tensin homolog/AKT serine/threonine kinase/mechanistic target of rapamycin kinase axis.

Propofol is a commonly used drug for sedation and general anesthesia during cancer surgery. Previous studies indicate that propofol exerts anti-tumor effect in various cancers. The aim of this study was to investigate the underlying molecular mechanism of propofol in liver cancer. The effects of propofol on liver cancer cells were evaluated by cell viability assay, colony formation assay, and tumor xenograft model. Dysregulated lncRNAs of propofol-treated liver cancer cells were evaluated by transcriptome RNA sequencing. The underlying molecular mechanisms of lncRNA cancer susceptibility candidate 9 (CASC9) in propofol-induced anti-tumor effects were evaluated by western blot, quantitative real-time polymerase chain reaction (qRT-PCR), wound scratch healing assay, transwell cell migration and invasion assay, TUNEL staining, fluorescence in situ hybridization, RNA immunoprecipitation (RIP), and chromatin immunoprecipitation (ChIP). We found that propofol suppressed proliferation, migration, invasion, and tumor xenograft growth of liver cancer cells in a dose-dependent manner. Exosomes transfer from propofol-treated cells inhibited proliferation, migration, and invasion and promoted apoptosis of liver cancer cells. Transcriptional profiling of propofol-treated liver cancer cells identified CASC9 as significantly downregulated lncRNA in cells and exosomes. Enforced CASC9 expression partially rescued the inhibitory effects of propofol on liver cancer cells. Furthermore, CASC9 was found to interact directly with EZH2 and epigenetically regulated PTEN expression. Restoration of CASC9 partially abrogated the inhibition of propofol on Akt/mTOR signaling. Our results indicated that propofol exerted anti-tumor effects by downregulating CASC9, and subsequently suppressed Akt/mTOR signaling. Our findings provided a novel insight into propofol-induced anti-tumor effects in liver cancer.

Propofol inhibited gastric cancer proliferation via the hsa-miR-328-3p/STAT3 pathway.

The aim of the present study was to elucidate the functional role of hsa-miR-328-3p/STAT3 pathway in the effects of propofol on gastric cancer proliferation. Bioinformatics was used to analyze the molecular expression differences of hsa-miR-328-3p/STAT3 axis in stomach adenocarcinoma (n = 435) and normal samples (n = 41) from TCGA database. The expression of the above molecules in gastric cancer cells SGC-7901 and normal gastric mucosal cells GES-1 was verified via qPCR. The dual-luciferase assay was carried out to confirm the interaction between hsa-miR-328-3p and STAT3. Subsequently, the cell proliferation and the expression of the above molecules in SGC-7901 and GES-1 cells were evaluated after 10μM propofol treatment. Finally, we analyzed whether propofol still inhibited the proliferation of gastric cancer by suppressing STAT3 pathway after hsa-miR-328-3p down-regulation. Compared with normal samples, the expression of hsa-miR-328-3p was significantly down-regulated in stomach adenocarcinoma samples, while the expression of STAT3 and downstream target genes (MMP2, CCND1 and COX2) was up-regulated. The results were consistent with those in GES-1 and SGC-7901 cell lines. Meanwhile, we found that hsa-miR-328-3p can bind to the 3'-UTR of the potential target gene STAT3. Furthermore, propofol significantly inhibited the proliferation of gastric cancer cell line SGC-7901, where hsa-miR-328-3p was up-regulated and the expression of STAT3 and downstream proliferation-related target genes were down-regulated. However, the growth inhibition of propofol on SGC-7901 cell was significantly reversed after the inhibition of hsa-miR-328-3p. To sum up, propofol suppressed the STAT3 pathway via up-regulating hsa-miR-328-3p to inhibit gastric cancer proliferation.

Impact of soman and acetylcholine on the effects of propofol in cultured cortical networks

Patients intoxicated with organophosphorous compounds may need general anaesthesia to enable mechanical ventilation or for control of epileptiform seizures. It is well known that cholinergic overstimulation attenuates the efficacy of general anaesthetics to reduce spontaneous network activity in the cortex. However, it is not clear how propofol, the most frequently used intravenous anaesthetic today, is affected. Here, we investigated the effects of cholinergic overstimulation induced by soman and acetylcholine on the ability of propofol to depress spontaneous action potential activity in organotypic cortical slices measured by extracellular voltage recordings.Cholinergic overstimulation by co-application of soman and acetylcholine (10 μM each) did not reduce the relative inhibition of propofol (1.0 μM; mean normalized action potential firing rate 0.49 ± 0.06 of control condition, p < 0.001, Wilcoxon signed rank test) but clearly reduced its efficacy. Co-application of atropine (10 nM) did not improve the efficacy.Propofol preserved its relative inhibitory potential but did not produce a degree of neuronal depression which can be expected to assure hypnosis in humans. Since a combination with atropine did not improve its efficacy, an increase in dosage will probably be necessary when propofol is used in victims suffering from organophosphorous intoxication.

Propofol Induces Postoperative Depression and Inhibits Microglial Function in Mice.

Many patients experience excellent physical recoveries after surgery; however, there are some of them who from suffer mood fluctuation, even depression. Postoperative depression may be resulted from cognitive dysfunction, pain, and a compromised immune system during the surgery. But there is a higher possibility that general anaesthesia may be responsible for the development of depression. Here, we employed one of the most used anaesthetics, propofol, in a mouse model to investigate whether this intravenous anaesthetic compound could cause depressive-like behavioural performance in mice. We found a single dose of propofol caused significant abnormal behavioural performance in tail suspension, forced swimming, and open field tests. We also examined the brain section of these mice and revealed that there was significant reduced expression of the CD11b protein, which demonstrated an inhibition of propofol on microglial function. We investigated the effect of propofol on synaptic protein, SYP, and found there was no notable influence on the protein expression. These above results suggested that propofol treatment might promote the depressive-like behaviours in mice via influencing the microglial cell function. Furthermore, we found the level of the IL-6 cytokine was significantly increased in the brain tissue, which might subsequently cause the activation of the transcriptional factor, STAT3. Our finding may provide a new perspective of further understanding the mechanism of anaesthetic drugs and deciphering the underlying mechanism of postoperative depression.

Propofol-induced miR-219-5p inhibits growth and invasion of hepatocellular carcinoma through suppression of GPC3-mediated Wnt/β-catenin signalling activation.

Propofol is one of the most extensively used intravenous anaesthetic agents, which has been found to improve the surgical intervention outcome of several types of cancer, including hepatocellular carcinoma (HCC). Additionally, in vitro and in vivo experiments have also indicated that propofol affects the biological behaviour of HCC. However, the underlying mechanisms of the surgical resection of HCC with propofol have not been fully understood. In the present study, we aimed to investigate the underlying mechanism of propofol inhibition of the growth and invasion of HCC cells. Our results showed that treatment with propofol suppressed the proliferation, invasion and migration of HCC in vitro. The subcutaneous xenograft tumour and orthotopic xenograft tumour experiments in nude mice showed that propofol significantly decreased tumour volumes, growth rates and the liver orthotopic xenograft tumour in vivo. Furthermore, the underlying mechanism investigations of the suppressive effects of propofol on HCC cells revealed that propofol treatment upregulated the expression levels of the candidate tumour suppressor miR-219-5p. Silencing of propofol-induced miR-219-5p using anti-miR-219-5p abrogated the inhibitory effects on the proliferation, migration and invasion of HCC cells exerted by propofol treatment. Additionally, we demonstrated that propofol reversed the epithelial-mesenchymal transition of Huh7 and SMMC7721 cells via miR-219-5p induction. The molecular mechanism behind these findings is that propofol-induced miR-219-5p inhibits HCC cell progression by targeting glypican-3 and subsequently resultsin the inhibition of Wnt/β-catenin signalling. Taken together, our study provides new insights into the advantages of the surgical intervention of HCC with propofol anaesthetization.

Propofol Reduced Mammosphere Formation of Breast Cancer Stem Cells via PD-L1/Nanog In Vitro.

Several researches revealed that propofol, a hypnotic intravenous anesthesia agent, could inhibit the cancer cell proliferation and tumor formation, which might affect cancer recurrence or metastasis and impact patients' prognosis. Cancer stem cells (CSCs) comprised a tiny fraction of tumor bulk and played a vital role in cancer recurrence and eventual mortality. This study investigates the effect of propofol on breast cancer stem cells (BCSCs) in vitro and the underlying molecular mechanisms. Tumor formation of CSCs was measured by mammosphere culture. Cultured BCSCs were exposed to different concentrations and durations of propofol. Cell proliferation and self-renewal capacity were determined by MTT assays. Expressions of PD-L1 and Nanog were measured using western blotting and real-time PCR. We knocked down the PD-L1 expression in MDA-MB-231 cells by lentivirus-mediated RNAi technique, and the mammosphere-forming ability of shControl and shPD-L1 under propofol treatment was examined. Mammosphere culture could enrich BCSCs. Compared with control, cells exposed to propofol for 24 h induced a larger number of mammosphere cells (P = 0.0072). Levels of PD-L1 and Nanog were downregulated by propofol. Compared with shControl stem cells, there was no significant difference in the inhibitory effect of propofol on the mammosphere-forming ability of shPD-L1 stem cells which indicated that the inhibition of propofol could disappear in PD-L1 knockdown breast stem cells. Propofol could reduce the mammosphere-forming ability of BCSCs in vitro. Mechanism experiments indicated that the inhibition of propofol in mammosphere formation of BCSCs might be mediated through PD-L1, which was important to maintain Nanog.

HCN channels contribute to the sensitivity of intravenous anesthetics in developmental mice.

It is widely accepted that the induction dose of anesthetics is higher in infants than in adults, although the relevant molecular mechanism remains elusive. We previously showed neuronal hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels contribute to hypnotic actions of propofol and ketamine. Interestingly, the expression of HCN channels in neocortex significantly changes during postnatal periods. Thus, we postulated that changes in HCN channels expression might contribute to sensitivity to intravenous anesthetics. Here we showed the EC50 for propofol- and ketamine-induced loss-of-righting reflex (LORR) was significantly lower for P35 than for P14 mice. Cerebrospinal fluid concentrations of propofol and ketamine were significantly higher in P14 mice than in P35 mice, with similar propofol- and ketamine-induced anesthesia at the LORR EC50. Western blotting indicated that the expression of HCN channels in neocortex changed significantly from P14 to P35 mice. In addition, the amplitude of HCN currents in the neocortical layer 5 pyramidal neurons and the inhibition of propofol and ketamine on HCN currents dramatically increased with development. Logistic regression analysis indicated that the changes of HCN channels were correlated with the age-related differences of propofol- and ketamine-induced anesthesia. These data reveal that the change of HCN channels expression with postnatal development may contribute to sensitivity to the hypnotic actions of propofol and ketamine in mice.

Sites Contributing to TRPA1 Activation by the Anesthetic Propofol Identified by Photoaffinity Labeling

In addition to inducing anesthesia, propofol activates a key component of the pain pathway, the transient receptor potential ankyrin 1 ion channel (TRPA1). Recent mutagenesis studies suggested a potential activation site within the transmembrane domain, near the A-967079 cavity. However, mutagenesis cannot distinguish between protein-based and ligand-based mechanisms, nor can this site explain the complex modulation by propofol. Thus more direct approaches are required to reveal potentially druggable binding sites. Here we apply photoaffinity labeling using a propofol derivative, meta-azipropofol, for direct identification of binding sites in mouse TRPA1. We confirm that meta-azipropofol activates TRPA1 like the parent anesthetic, and identify two photolabeled residues (V954 and E969) in the S6 helix. In combination with docking to closed and open state models of TRPA1, photoaffinity labeling suggested that the A-967079 cavity is a positive modulatory site for propofol. Further, the photoaffinity labeling of E969 indicated pore block as a likely mechanism for propofol inhibition at high concentrations. The direct identification of drug-binding sites clarifies the molecular mechanisms of important TRPA1 agonists, and will facilitate drug design efforts to modulate TRPA1.

Role of mitochondrial complex I and protective effect of CoQ10 supplementation in propofol induced cytotoxicity.

Propofol (2,6-diisopropylphenol) is an anaesthetic widely used for human sedation. Due to its intrinsic antioxidant properties, rapid induction of anaesthesia and fast recovery, it is employed in paediatric anaesthesia and in the intensive care of premature infants. Recent studies have pointed out that exposure to anaesthesia in the early stage of life might be responsible of long-lasting cognitive impairment. The apoptotic neurodegeneration induced by general anaesthetics (GA) involves mitochondrial impairment due to the inhibition of the OXPHOS machinery. In the present work, we aim to identify the main mitochondrial respiratory chain target of propofol toxicity and to evaluate the possible protective effect of CoQ10 supplementation. The propofol effect on the mitochondrial functionality was assayed in isolated mitochondria and in two cell lines (HeLa and T67) by measuring oxygen consumption rate. The protective effect of CoQ10 was assessed by measuring cells viability, NADH-oxidase activity and ATP/ADP ratio in cells treated with propofol. Our results show that propofol reduces cellular oxygen consumption rate acting mainly on mitochondrial Complex I. The kinetic analysis of Complex I inhibition indicates that propofol interferes with the Q module acting as a non-competitive inhibitor with higher affinity for the free form of the enzyme. Cells supplemented with CoQ10 are more resistant to propofol toxicity. Propofol exposure induces cellular damages due to mitochondrial impairment. The site of propofol inhibition on Complex I is the Q module. CoQ10 supplementation protects cells against the loss of energy suggesting its possible therapeutic role to minimizing the detrimental effects of general anaesthesia.

Common Anesthetic-binding Site for Inhibition of Pentameric Ligand-gated Ion Channels.

Identifying functionally relevant anesthetic-binding sites in pentameric ligand-gated ion channels (pLGICs) is an important step toward understanding the molecular mechanisms underlying anesthetic action. The anesthetic propofol is known to inhibit cation-conducting pLGICs, including a prokaryotic pLGIC from Erwinia chrysanthemi (ELIC), but the sites responsible for functional inhibition remain undetermined. We photolabeled ELIC with a light-activated derivative of propofol (AziPm) and performed fluorine-19 nuclear magnetic resonance experiments to support propofol binding to a transmembrane domain (TMD) intrasubunit pocket. To differentiate sites responsible for propofol inhibition from those that are functionally irrelevant, we made an ELIC-γ-aminobutyric acid receptor (GABAAR) chimera that replaced the ELIC-TMD with the α1β3GABAAR-TMD and compared functional responses of ELIC-GABAAR and ELIC with propofol modulations. Photolabeling showed multiple AziPm-binding sites in the extracellular domain (ECD) but only one site in the TMD with labeled residues M265 and F308 in the resting state of ELIC. Notably, this TMD site is an intrasubunit pocket that overlaps with binding sites for anesthetics, including propofol, found previously in other pLGICs. Fluorine-19 nuclear magnetic resonance experiments supported propofol binding to this TMD intrasubunit pocket only in the absence of agonist. Functional measurements of ELIC-GABAAR showed propofol potentiation of the agonist-elicited current instead of inhibition observed on ELIC. The distinctly different responses of ELIC and ELIC-GABAAR to propofol support the functional relevance of propofol binding to the TMD. Combining the newly identified TMD intrasubunit pocket in ELIC with equivalent TMD anesthetic sites found previously in other cationic pLGICs, we propose this TMD pocket as a common site for anesthetic inhibition of pLGICs.

Propofol attenuates lipopolysaccharide-induced reactive oxygen species production through activation of Nrf2/GSH and suppression of NADPH oxidase in human alveolar epithelial cells.

Propofol is a widely used intravenous anesthetic. The aim of this study was to investigate the roles of nuclear factor erythroid-2-related factor 2 (Nrf2) and NADPH oxidase (NOX) in propofol protection in inflammatory conditions induced by lipopolysaccharide (LPS). Human alveolar epithelial cells (A549 cell line) were incubated with propofol (10, 25, and 50 μmol/L) for 1 h and then treated with LPS (100 ng/mL) for 24 h. Results indicated that propofol not only attenuated LPS-induced expression of iNOS, NOX, and COX2, but decreased the production of ROS, NO, and PGE2 as well. Propofol also increased the GSH levels and the mRNA and protein levels of Nrf2. Notably, Nrf2 siRNA and the inhibitors of COX-2 and NOX attenuated the inhibition of propofol on ROS production. In conclusion, propofol reduced LPS-induced ROS production via inhibition of inflammatory factors and enhancement of Nrf2-related antioxidant defense, providing its cytoprotective evidence under inflammatory conditions.

Inhibition of propofol on single neuron and neuronal ensemble activity in prefrontal cortex of rats during working memory task

Working memory (WM) refers to the temporary storage and manipulation of information necessary for performance of complex cognitive tasks. There is a growing interest in whether and how propofol anesthesia inhibits WM function. The aim of this study is to investigate the possible inhibition mechanism of propofol anesthesia from the view of single neuron and neuronal ensemble activities. Adult SD rats were randomly divided into two groups: propofol group (0.9mgkg−1min−1, 2h via a tail vein catheter) and control group. All the rats were tested for working memory performances in a Y-maze-rewarded alternation task (a task of delayed non-matched-to-sample) at 24, 48, 72h after propofol anesthesia, and the behavior results of WM tasks were recorded at the same time. Spatio-temporal trains of action potentials were obtained from the original signals. Single neuron activity was characterized by peri-event time histograms analysis and neuron ensemble activities were characterized by Granger causality to describe the interactions within the neuron ensemble. The results show that: comparing with the control group, the percentage of neurons excited and related to WM was significantly decreased (p<0.01 in 24h, p<0.05 in 48h); the interactions within neuron ensemble were significantly weakened (p<0.01 in 24h, p<0.05 in 48h), whereas no significant difference in 72h (p>0.05), which were consistent with the behavior results. These findings could lead to improved understanding of the mechanism of anesthesia inhibition on WM functions from the view of single neuron activity and neuron ensemble interactions.

Opposing Effects of the Anesthetic Propofol at Pentameric Ligand-Gated Ion Channels Mediated by a Common Site

Propofol is an intravenous general anesthetic that alters neuronal excitability by modulating agonist responses of pentameric ligand-gated ion channels (pLGICs). Evidence suggests that propofol enhancement of anion-selective pLGICs is mediated by a binding site between adjacent subunits, whereas propofol inhibition of cation-selective pLGICs occurs via a binding site contained within helices M1-M4 of individual subunits. We considered this idea by testing propofol modulation of homomeric human glycine receptors (GlyRs) and nematode glutamate-gated chloride channels (GluCls) recombinantly expressed in Xenopus laevis oocytes with electrophysiology. The Haemonchus contortus AVR-14B GluCl was inhibited by propofol with an IC50 value of 252 ± 48 μM, providing the first example of propofol inhibition of an anion-selective pLGIC. Remarkably, inhibition was converted to enhancement by a single I18'S substitution in the channel-forming M2 helix (EC50 = 979 ± 88 μM). When a previously identified site between adjacent subunits was disrupted by the M3 G329I substitution, both propofol inhibition and enhancement of GluCls were severely impaired (IC50 and EC50 values could not be calculated). Similarly, when the equivalent positions were examined in GlyRs, the M2 S18'I substitution significantly altered the maximum level of enhancement by propofol, and the M3 A288I substitution abolished propofol enhancement. These data are not consistent with separate binding sites for the opposing effects of propofol. Instead, these data suggest that propofol enhancement and inhibition are mediated by binding to a single site in anion-selective pLGICs, and the modulatory effect on channel gating depends on the M2 18' residue.

A general anaesthetic propofol inhibits aquaporin-4 in the presence of Zn2+

AQP4 (aquaporin-4), a water channel protein that is predominantly expressed in astrocyte end-feet, plays an important role in the brain oedema formation, and is thereby considered to be a potential therapeutic target. Using a stopped-flow analysis, we showed that propofol (2,6-diisopropylphenol), a general anaesthetic drug, profoundly inhibited the osmotic water permeability of AQP4 proteoliposomes in the presence of Zn²⁺. This propofol inhibition was not observed in AQP1, suggesting the specificity for AQP4. In addition, the inhibitory effects of propofol could be reversed by the removal of Zn²⁺. Other lipid membrane fluidizers also similarly inhibited AQP4, suggesting that the modulation of protein-lipid interactions plays an essential role in the propofol-induced inhibition of AQP4. Accordingly, we used Blue native PAGE and showed that the profound inhibition caused by propofol in the presence of Zn²⁺ is coupled with the reversible clustering of AQP4 tetramers. Site-directed mutagenesis identified that Cys²⁵³, located at the membrane interface connecting to the C-terminal tail, is responsible for Zn²⁺-mediated propofol inhibition. Overall, we discovered that propofol specifically and reversibly inhibits AQP4 through the interaction between Zn²⁺ and Cys²⁵³. The findings provide new insight into the functional regulation of AQP4 and may facilitate the identification of novel AQP4-specific inhibitors.

Propofol Binding to the Resting State of the Gloeobacter violaceus Ligand-gated Ion Channel (GLIC) Induces Structural Changes in the Inter- and Intrasubunit Transmembrane Domain (TMD) Cavities

General anesthetics exert many of their CNS actions by binding to and modulating membrane-embedded pentameric ligand-gated ion channels (pLGICs). The structural mechanisms underlying how anesthetics modulate pLGIC function remain largely unknown. GLIC, a prokaryotic pLGIC homologue, is inhibited by general anesthetics, suggesting anesthetics stabilize a closed channel state, but in anesthetic-bound GLIC crystal structures the channel appears open. Here, using functional GLIC channels expressed in oocytes, we examined whether propofol induces structural rearrangements in the GLIC transmembrane domain (TMD). Residues in the GLIC TMD that frame intrasubunit and intersubunit water-accessible cavities were individually mutated to cysteine. We measured and compared the rates of modification of the introduced cysteines by sulfhydryl-reactive reagents in the absence and presence of propofol. Propofol slowed the rate of modification of L240C (intersubunit) and increased the rate of modification of T254C (intrasubunit), indicating that propofol binding induces structural rearrangements in these cavities that alter the local environment near these residues. Propofol acceleration of T254C modification suggests that in the resting state propofol does not bind in the TMD intrasubunit cavity as observed in the crystal structure of GLIC with bound propofol (Nury, H., Van Renterghem, C., Weng, Y., Tran, A., Baaden, M., Dufresne, V., Changeux, J. P., Sonner, J. M., Delarue, M., and Corringer, P. J. (2011) Nature 469, 428-431). In silico docking using a GLIC closed channel homology model suggests propofol binds to intersubunit sites in the TMD in the resting state. Propofol-induced motions in the intersubunit cavity were distinct from motions associated with channel activation, indicating propofol stabilizes a novel closed state.

Reversal of ion-charge selectivity renders the pentameric ligand-gated ion channel GLIC insensitive to anaesthetics.

pLGICs (pentameric ligand-gated ion channels) are a family of structurally homologous cation and anion channels involved in neurotransmission. Cation-selective members of the pLGIC family are typically inhibited by general anaesthetics, whereas anion-selective members are potentiated. GLIC is a prokaryotic cation pLGIC and can be inhibited by clinical concentrations of general anaesthetics. The introduction of three mutations, Y221A (Y-3'A), E222P (E-2'P) and N224R (N0'R), at the selectivity filter and one, A237T (A13'T), at the hydrophobic gate, converted GLIC into an anion channel. The mutated GLIC (GLIC4) became insensitive to the anaesthetics propofol and etomidate, as well as the channel blocker picrotoxin. MD (molecular dynamics) simulations revealed changes in the structure and dynamics of GLIC4 in comparison with GLIC, particularly in the tilting angles of the pore-lining helix [TM2 (transmembrane helix 2)] that consequently resulted in different pore radius and hydration profiles. Propofol binding to an intra-subunit site of GLIC shifted the tilting angles of TM2 towards closure at the hydrophobic gate region, consistent with propofol inhibition of GLIC. In contrast, the pore of GLIC4 was much more resilient to perturbation from propofol binding. The present study underscores the importance of pore dynamics and conformation to anaesthetic effects on channel functions.

Inhibition of propofol on HMGB1 expression and releasing from mouse macrophage cells induced by lipopolysaccharide

Objective To explore the inhibition effect of propofol on HMGB1 expression and releasing from mouse macrophage cell RAW264.7 induced by LPS. Methods Detect the time course of HMGB1 expression of RAW264.7 cells using RT-PCR and western blot. Then cells were divided into three groups: control, 250μg/L LPS stimulated, 250μg/L LPS stimulated simultaneously with 50μmol/L propofol. HMGB1 expression and releasing were detected using RT-PCR and western blot when cell were stimulated until the time the HMGB1 expression got to the highest level. Results 16 h after stimulated by LPS, the HMGB1 expression got to the highest level. When LPS stimulated with 50μmol/L propofol, the expression and releasing of HMGB1 by RAW264.7 were inhibited (P<0.05);16 h after stimulated by LPS, the HMGB1 mRNA expression got to the highest level,When LPS stimulated with 50μmol/L propofol, the expression and releasing of HMGB1 mRNA by RAW264.7 were inhibited Significantly,in each group ,the changes of HMGB1 Protein are the same to that of HMGB1 mRNA. Conclusion Propofol can inhibit the expression and releasing of HMGB1 by RAW264.7, which may be play a role in protect of sepsis by LPS. Key words: Propofol ; High mobility group proteins ; Lipopolysaccharide ; RAW 264.7 cells ;