Shared structural mechanisms of general anaesthetics and benzodiazepines.

Most general anaesthetics and classical benzodiazepine drugs act through positive modulation of γ-aminobutyric acid type A (GABAA) receptors to dampen neuronal activity in the brain1-5. However, direct structural information on the mechanisms of general anaesthetics at their physiological receptor sites is lacking. Here we present cryo-electron microscopy structures of GABAA receptors bound to intravenous anaesthetics, benzodiazepines and inhibitory modulators. These structures were solved in a lipidic environment and are complemented by electrophysiology and molecular dynamics simulations. Structures of GABAA receptors in complex with the anaesthetics phenobarbital, etomidate and propofol reveal both distinct and common transmembrane binding sites, which are shared in part by the benzodiazepine drug diazepam. Structures in which GABAA receptors are bound by benzodiazepine-site ligands identify an additional membrane binding site for diazepam and suggest an allosteric mechanism for anaesthetic reversal by flumazenil. This study provides a foundation for understanding how pharmacologically diverse and clinically essential drugs act through overlapping and distinct mechanisms to potentiate inhibitory signalling in the brain.

Propofol improves survival in a murine model of sepsis via inhibiting Rab5a-mediated intracellular trafficking of TLR4.

BACKGROUND: Propofol is a widely used anesthetic and sedative, which has been reported to exert an anti-inflammatory effect. TLR4 plays a critical role in coordinating the immuno-inflammatory response during sepsis. Whether propofol can act as an immunomodulator through regulating TLR4 is still unclear. Given its potential as a sepsis therapy, we investigated the mechanisms underlying the immunomodulatory activity of propofol. METHODS: The effects of propofol on TLR4 and Rab5a (a master regulator involved in intracellular trafficking of immune factors) were investigated in macrophage (from Rab5a-/- and WT mice) following treatment with lipopolysaccharide (LPS) or cecal ligation and puncture (CLP) in vitro and in vivo, and peripheral blood monocyte from sepsis patients and healthy volunteers. RESULTS: We showed that propofol reduced membrane TLR4 expression on macrophages in vitro and in vivo. Rab5a participated in TLR4 intracellular trafficking and both Rab5a expression and the interaction between Rab5a and TLR4 were inhibited by propofol. We also showed Rab5a upregulation in peripheral blood monocytes of septic patients, accompanied by increased TLR4 expression on the cell surface. Propofol downregulated the expression of Rab5a and TLR4 in these cells. CONCLUSIONS: We demonstrated that Rab5a regulates intracellular trafficking of TLR4 and that propofol reduces membrane TLR4 expression on macrophages by targeting Rab5a. Our study not only reveals a novel mechanism for the immunomodulatory effect of propofol but also indicates that Rab5a may be a potential therapeutic target against sepsis.

Medium- and long-chain triglyceride propofol activates PI3K/AKT pathway and inhibits non-alcoholic fatty liver disease by inhibiting lipid accumulation.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease. The mechanism by which medium- and long-chain triglyceride (MCT/LCT) propofol plays a role in promoting NAFLD remains unclear. In this study, we investigated the effect of MCT/LCT propofol on NAFLD progression and its mechanism of action. In Huh-7 and HepG3 cells induced by free fatty acids (FFA), propofol downregulated the expression levels of TG and lipid metabolism-related proteins by promoting the activation of the PI3K/AKT pathway and suppressing FFA-induced lipid metabolic disorders. In a high-fat diet (HFD) -induced NAFLD mouse model, we demonstrated that propofol significantly inhibited liver steatosis, inflammatory cell infiltration, and fibrosis. In conclusion, our results suggest that MCT/LCT propofol reduces liver lipid accumulation by activating the PI3K/AKT pathway and further suppressing the NAFLD process.

Dexmedetomidine attenuates postoperative spatial memory impairment after surgery by reducing cytochrome C.

BACKGROUND: Anesthesia and surgery can induce perioperative neurocognitive disorders (PND). Mitochondrial dysfunction has been proposed to be one of the earliest triggering events in surgery-induced neuronal damage. Dexmedetomidine has been demonstrated to attenuate the impairment of cognition in aged rats induced by surgery in our previous study. METHODS: Male Sprague-Dawley rats underwent hepatic apex resection under anesthesia with propofol to clinically mimic human abdominal surgery. The rats were divided into three groups: Control group, Model group and Dexmedetomidine (Dex) group. Cognitive function was evaluated with the Morris water maze (MWM), Open Field Test (OFT)and Novel object recognition task (NOR). Ultrastructural change in neuronal mitochondria was measured by transmission electron microscopy. Mitochondrial function was measured by mitochondrial membrane potential and activities of mitochondrial complexes. Neuronal morphology was observed with H&E staining and the activation of glial cells was observed by immunohistochemistry in the hippocampus. Protein levels were measured by Western blot (WB) and immunofluorescence at 3 and 7 days after surgery. RESULTS: Surgery-induced cognitive decline lasts three days, but not seven days after surgery in the model group. Transmission electron microscope showed the mitochondrial structure damage in the model group, similar changes were not induced in the Dex group. Dexmedetomidine may reverse the decrease in mitochondrial membrane potential and mitochondrial complex activity. Compared with the Control group, the expression of cytochrome c was significantly increased in model group by Western blot and immunofluorescence on days 3, but not day 7. Rats from the Model group expressed significantly greater levels of Iba-1 and GFAP compared with the Control group and the Dex group. CONCLUSION: Dexmedetomidine appears to reverse surgery-induced behavior, mitigate the higher density of Iba-1 and GFAP, reduce the damage of mitochondrial structure and function by alleviating oxidative stress and protect mitochondrial respiratory chain, thus increasing cytochrome c oxidase (COX) expression and downregulate the expression of cytochrome c protein in the hippocampus of rats.

Subanesthetic Dose of Propofol Activates the Reward System in Rats.

BACKGROUND: Propofol has addictive properties, even with a single administration, and facilitates dopamine secretion in the nucleus accumbens (NAc). Activation of the dopaminergic circuits of the midbrain reward system, including the ventral tegmental area (VTA) and NAc, plays a crucial role in addiction. However, the effects of propofol on synaptic transmission and biochemical changes in the VTA-NAc circuit remain unclear. METHODS: We investigated the effects of subanesthetic doses of propofol on rat VTA neurons and excitatory synaptic transmission in the NAc using slice patch-clamp experiments. Using immunohistochemistry and western blot analyses, we evaluated the effects of intraperitoneal propofol administration on the expression of addiction-associated transcription factor ΔFosB (truncated form of the FBJ murine osteosarcoma viral oncogene homolog B protein) in the NAcs in 5-week-old rats. RESULTS: In the current-clamp mode, a subanesthetic dose (0.5-5 µmol/L) of propofol increased the action potential frequency in about half the VTA neurons (excited neurons: control: 9.4 ± 3.0 Hz, propofol 0.5 µmol/L: 21.5 ± 6.0 Hz, propofol 5 µmol/L: 14.6 ± 5.3 Hz, wash: 2.0 ± 0.7 Hz, n = 14/27 cells; unchanged/suppressed neurons: control: 1.68 ± 0.94 Hz, propofol 0.5 µmol/L: 1.0 ± 0.67 Hz, propofol 5 µmol/L: 0.89 ± 0.87 Hz, wash: 0.16 ± 0.11 Hz, n = 13/27 cells). In the voltage-clamp mode, about half the VTA principal neurons showed inward currents with 5 µmol/L of propofol (inward current neurons: control: -20.5 ± 10.0 pA, propofol 0.5 µmol/L: -62.6 ± 14.4 pA, propofol 5 µmol/L: -85.2 ± 18.3 pA, propofol 50 µmol/L: -17.1 ± 39.2 pA, washout: +30.5 ± 33.9 pA, n = 6/11 cells; outward current neurons: control: -33.9 ± 14.6 pA, propofol 0.5 µmol/L: -29.5 ± 16.0 pA, propofol 5 µmol/L: -0.5 ± 20.9 pA, propofol 50 µmol/L: +38.9 ± 18.5 pA, washout: +40.8 ± 32.1 pA, n = 5/11 cells). Moreover, 0.5 µmol/L propofol increased the amplitudes of evoked excitatory synaptic currents in the NAc, whereas >5 µmol/L propofol decreased them (control: 100.0 ± 2.0%, propofol 0.5 µmol/L: 118.4 ± 4.3%, propofol 5 µmol/L: 98.3 ± 3.3%, wash [within 10 min]: 70.7 ± 3.3%, wash [30 minutes later]: 89.9 ± 2.5%, n = 13 cells, P < .001, Dunnett's test comparing control and propofol 0.5 µmol/L). Intraperitoneally administered subanesthetic dose of propofol increased ΔFosB expression in the NAc, but not in VTA, 2 and 24 hours after administration, compared with the Intralipid control group (propofol 2 hours: 0.94 ± 0.15, 24 hours: 0.68 ± 0.07; Intralipid 2 hours: 0.40 ± 0.03, 24 hours: 0.37 ± 0.06, P = .0002 for drug in the 2-way analysis of variance). CONCLUSIONS: Even a single administration of a subanesthetic dose of propofol may cause rewarding change in the central nervous system. Thus, there is a potential propofol rewarding effect among patients receiving anesthesia or sedation with propofol, as well as among health care providers exposed to propofol.

The effects of thiamine pyrophosphate on propofol-induced oxidative liver injury and effect on dysfunction.

Propofol may cause an increase in reactive oxygen species in the body. In this study, we tested the effect of antioxidant thiamine pyrophosphate (TPP) on propofol-induced liver damage. The eighteen rats were split into three groups: HG, healthy; PP, propofol-treated (50 mg/kg) and PT, treated with propofol (50 mg/kg) and TPP (25 mg/kg). Total glutathione (tGSH), total oxidant (TOS), and total antioxidant (TAS) levels were tested together with aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH) and malondialdehyde (MDA). Histopathological examination of the tissues was performed. We have found that levels of MDA, TOS, ALT, AST, and LDH were all higher in PP group than in HG and PT groups (p < 0.05). In PP group, the TAS and tGSH levels were statistically substantially lower. The PT for oxidants levels showed a statistically significant reduction. In PT group, the levels of antioxidants were found to be considerably higher. The epitheliums, glands, and vascular structures of the PTs were histologically close to normal. By boosting antioxidants, TPP may help to reduce propofolinduced liver damage.

Evaluation of Propofol in Inhibiting Proliferation of Cardiac Fibroblasts in Angiotensin II-Induced Mouse.

The present study was conducted to investigate the molecular mechanism of propofol in inhibiting the proliferation of mouse cardiac fibroblasts (CFs) induced by angiotensin II (Ag II). The ventricles of SPF mice from Kunming were cultured for the second to third generation of CFs under aseptic condition. On the basis of the different adding conditions, the mice were divided into five groups: (1) control group: no drug were added; (2) Ag II group: 100 nmol/L Ag II were added; (3) 10 µmol/L propofol + 100 nmol/L Ag II group; (4) 30 µmol/L propofol + 100 nmol/L Ag II group; (5) 50 µmol/L propofol + 100 nmol/L Ag II group. The effects of propofol on the proliferation of CFs induced by Ag II, the expression of CFs ET-1, the activity of NADPH oxidase and the formation of ROS were analyzed. In addition, our study also explored the potential role of Akt-eNOS-nitric oxide pathway regarding the inhibition of proliferation of Ag II induced CFs by propofol. We found that the proliferation of CFs, the secretion of ET-1, the activity of NADPH oxidase and the level of intracellular ROS and fibronectin expression were significantly increased after CFs exposure to Ag II for 24 h. The abovementioned indexes decreased significantly in CFs after treated with propofol for 24 h (10, 30, or 50 µmol/L) with significant statistical difference (P < 0.05). Akt and eNOS siRNA transfection significantly decreased the levels of Akt and eNOS protein, respectively. Blocking pathway of Akt-eNOS-nitric oxide decreased the inhibitory effect of propofol on Ag II-induced cell proliferation of CFs. Propofol exerts effect in inhibiting ET-1 and fibronectin expression and the formation of ROS induced by Ag II. Moreover, Akt-eNOS-nitric oxide signaling pathway may be involved in the effect of propofol on the proliferation of CFs induced by Ag II.

Exhaled Propofol Concentrations Correlate With Plasma and Brain Tissue Concentrations in Rats.

BACKGROUND: Propofol can be measured in exhaled gas. Exhaled and plasma propofol concentrations correlate well, but the relationship with tissue concentrations remains unknown. We thus evaluated the relationship between exhaled, plasma, and various tissue propofol concentrations. Because the drug acts in the brain, we focused on the relationship between exhaled and brain tissue propofol concentrations. METHODS: Thirty-six male Sprague-Dawley rats were anesthetized with propofol, ketamine, and rocuronium for 6 hours. Animals were randomly assigned to propofol infusions at 20, 40, or 60 mg·kg·h (n = 12 per group). Exhaled propofol concentrations were measured at 15-minute intervals by multicapillary column-ion mobility spectrometry. Arterial blood samples, 110 µL each, were collected 15, 30, and 45 minutes, and 1, 2, 4, and 6 hours after the propofol infusion started. Propofol concentrations were measured in brain, lung, liver, kidney, muscle, and fat tissue after 6 hours. The last exhaled and plasma concentrations were used for linear regression analyses with tissue concentrations. RESULTS: The correlation of exhaled versus plasma concentrations (R = 0.71) was comparable to the correlation of exhaled versus brain tissue concentrations (R = 0.75) at the end of the study. In contrast, correlations between plasma and lung and between lung and exhaled propofol concentrations were poor. Less than a part-per-thousand of propofol was exhaled over 6 hours. CONCLUSIONS: Exhaled propofol concentrations correlate reasonably well with brain tissue and plasma concentrations in rats, and may thus be useful to estimate anesthetic drug effect. The equilibration between plasma propofol and exhaled gas is apparently independent of lung tissue concentration. Only a tiny fraction of administered propofol is eliminated via the lungs, and exhaled quantities thus have negligible influence on plasma concentrations.

Circ_MUC16 attenuates the effects of Propofol to promote the aggressive behaviors of ovarian cancer by mediating the miR-1182/S100B signaling pathway.

BACKGROUND: Propofol is commonly used for anesthesia during surgery and has been demonstrated to inhibit cancer development, which is shown to be associated with deregulation of non-coding RNAs (ncRNAs). The objective of this study was to explore the role of circular RNA mucin 16 (circ_MUC16) in Propofol-mediated inhibition of ovarian cancer. METHODS: The expression of circ_MUC16, microRNA-1182 (miR-1182) and S100 calcium-binding protein B (S100B) mRNA was measured by quantitative real-time polymerase chain reaction (qPCR). The expression of S100B protein was checked by western blot. Cell proliferation was assessed by 3-(4, 5-di methyl thiazol-2-yl)-2, 5-di phenyl tetrazolium bromide (MTT) assay and colony formation assay. Glycolysis metabolism was assessed by glucose consumption, lactate production and ATP level. Cell migration and cell invasion were assessed by transwell assay. Cell migration was also assessed by wound healing assay. Animal study was conducted in nude mice to determine the role of circ_MUC16 in vivo. The relationship between miR-1182 and circ_MUC16 or S100B was validated by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. RESULTS: Propofol inhibited ovarian cancer cell proliferation, glycolysis metabolism, migration and invasion, which were partly recovered by circ_MUC16 overexpression. Circ_MUC16 was downregulated in Propofol-treated ovarian cancer cells. Besides, circ_MUC16 knockdown enhanced the effects of Propofol to further inhibit tumor growth in vivo. MiR-1182 was a target of circ_MUC16, and circ_MUC16 knockdown-inhibited cell proliferation, glycolysis metabolism, migration and invasion were partly restored by miR-1182 inhibition. In addition, S100B was a target of miR-1182, and miR-1182-suppressed cell proliferation, glycolysis metabolism, migration and invasion were partly restored by S100B overexpression. CONCLUSION: Circ_MUC16 overexpression alleviated the effects of Propofol to promote the aggressive behaviors of ovarian cancer by targeting the miR-1182/S100B network.

The role of propofol hydroxyl group in 5-lipoxygenase recognition.

Propofol is a clinically important intravenous anesthetic. We previously reported that it directly inhibited 5-lipoxygenase (5-LOX), a key enzyme for leukotriene biosynthesis. Because the hydroxyl group in propofol (propofol 1-hydroxyl) is critical for its anesthetic effect, we examined if its presence would be inevitable for 5-lipoxygenase recognition. Fropofol is developed by substituting the hydroxy group in propofol with fluorine. We found that propofol 1-hydroxyl was important for 5-lipoxygenase recognition, but it was not absolutely necessary. Azi-fropofol bound to 5-LOX at one of the two propofol binding sites of 5-LOX (pocket around Phe-187), suggesting that propofol 1-hydroxyl is important for 5-LOX inhibition at the other propofol binding site (pocket around Val-431). Interestingly, 5-hydroperoxyeicosatetraenoic acid (5-HpETE) production was significantly increased by stimulation with calcium ionophore A23187 in HEK293 cells expressing 5-LOX, suggesting that the fropofol binding site is important for the conversion from 5-HpETE to leukotriene A4. We also indicated that propofol 1-hydroxyl might have contributed to interaction with wider targets among our body.

Propofol Inhibits Cell Proliferation, Migration, and Invasion via mir-410-3p/Transforming Growth Factor-ß Receptor Type 2 (TGFBR2) Axis in Glioma.

BACKGROUND Propofol is a common intravenous anesthetic used to induce and maintain anesthesia. Numerous studies have reported that propofol plays an anti-tumor role in diverse human cancers, including glioma. In this research, we explored the roles of propofol and its related molecular mechanisms in glioma. MATERIAL AND METHODS U251 and A172 cells were exposed to different doses of propofol for 24 h. Cell proliferation, migration, and invasion in glioma were evaluated using MTT assay and Transwell assay, respectively. The levels of microRNA-410-3p (miR-410-3p) and transforming growth factor-ß receptor type 2 (TGFBR2) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) assay and Western blot assay, respectively. The association between miR-410-3p and TGFBR2 was predicted by TargetScan and confirmed by dual-luciferase reporter assay. RESULTS Propofol inhibited the proliferation, migration, and invasion of glioma cells in a concentration-dependent way. miR-410-3p was induced and TGFBR2 was inhibited by different concentrations of propofol treatment. Moreover, TGFBR2 was confirmed to be a target gene of miR-410-3p and TGFBR2 was inversely modulated by miR-410-3p in glioma cells. Depletion of miR-410-3p reversed the inhibition of propofol treatment on U251 and A172 cell growth and metastasis, but the effects were further abolished by knocking down the expression of TGFBR2. CONCLUSIONS Propofol can suppress cell growth and metastasis by regulating the miR-410-3p/TGFBR2 axis in glioma.

The Binding Mechanisms and Inhibitory Effect of Intravenous Anesthetics on AChE In Vitro and In Vivo: Kinetic Analysis and Molecular Docking.

Inhibitors of acetylcholinesterase (AChE), which have an important role in the prevention of excessive AChE activity and ß-amyloid (Aß) formation are widely used in the symptomatic treatment of Alzheimer's disease (AD). The inhibitory effect of anesthetic agents on AChE was determined by several approaches, including binding mechanisms, molecular docking and kinetic analysis. Inhibitory effect of intravenous anesthetics on AChE as in vitro and in vivo have been discovered. The midazolam, propofol and thiopental have shown competitive inhibition type (midazolam > propofol > thiopental) and Ki values were found to be 3.96.0 ± 0.1, 5.75 ± 0.12 and 29.65 ± 2.04 µM, respectively. The thiopental and midazolam showed inhibition effect on AChE in vitro, whereas they showed activation effect in vivo when they are combined together. The order of binding of the drugs to the active site of the 4M0E receptor was found to be midazolam > propofol > thiopental. This study on anesthetic agents that are now widely used in surgical applications, have provided a molecular basis for investigating the drug-enzyme interactions mechanism. In addition, the study is important in understanding the molecular mechanism of inhibitors that are effective in the treatment of AD.

Marmoset cytochrome P450 2B6, a propofol hydroxylase expressed in liver.

The common marmoset (Callithrix jacchus) is a useful experimental animal to evaluate the pharmacokinetics of drug candidates. Cytochrome P450 (P450) 2B enzyme in marmoset livers has been identified; however, only limited information on the enzymatic properties and distribution has been available. Marmoset P450 2B6 amino acids showed high sequence identities (>86%) with those of primates including humans and cynomolgus monkeys. Phylogenetic analysis using amino acid sequences indicated that marmoset P450 2B6 was closer to human and cynomolgus monkey P450 2B6 than to P450 2B orthologs of other species, including pigs, dogs, rabbits and rodents. Quantitative polymerase chain reaction analysis using specific primers showed P450 2B6 mRNA predominantly expressed in livers among the five marmoset tissues, similar to those of humans and cynomolgus monkeys. Marmoset P450 2B6 heterologously expressed in Escherichia coli membranes oxidized 7-ethoxycoumarin, pentoxyresorufin, propofol and testosterone, at roughly similar rates to those of humans and/or cynomolgus monkeys. A high capacity of marmoset P450 2B6 with propofol 4-hydroxylation (at low ionic strength conditions) with a low Km value was relatively comparable to that for marmoset livers. These results collectively indicated a high propofol 4-hydroxylation activity of P450 2B6 expressed in marmoset livers.

High Constitutive Activity Accounts for the Combination of Enhanced Direct Activation and Reduced Potentiation in Mutated GABAA Receptors.

GABAA receptors activated by the transmitter GABA are potentiated by several allosterically acting drugs, including the intravenous anesthetic propofol. Propofol can also directly activate the receptor, albeit at higher concentrations. Previous functional studies have identified amino acid residues whose substitution reduces potentiation of GABA-activated receptors by propofol while enhancing the ability of propofol to directly activate the receptor. One interpretation of such observations is that the mutation has specific effects on the sites or processes involved in potentiation or activation. We show here that divergent effects on potentiation and direct activation can be mediated by increased constitutive open probability in the mutant receptor without any specific effect on the interactions between the allosteric drug and the receptor. By simulating GABAA receptor activity using the concerted transition model, we demonstrate that the predicted degree of potentiation is reduced as the level of constitutive activity increases. The model further predicts that a potentiating effect of an allosteric modulator is a computable value that depends on the level of constitutive activity, the amplitude of the response to the agonist, and the amplitude of the direct activating response to the modulator. Specific predictions were confirmed by electrophysiological data from the binary α1ß3 and concatemeric ternary ß2α1γ2L+ß2α1 GABAA receptors. The corollaries of reduced potentiation due to increased constitutive activity are isobolograms that conform to simple additivity and a loss of separation between the concentration-response relationships for direct activation and potentiation.

Propofol Is an Allosteric Agonist with Multiple Binding Sites on Concatemeric Ternary GABAA Receptors.

GABAA receptors can be directly activated and potentiated by the intravenous anesthetic propofol. Previous photolabeling, modeling, and functional data have identified two binding domains through which propofol acts on the GABAA receptor. These domains are defined by the ß(M286) residue at the ß"+"-α"-" interface in the transmembrane region and the ß(Y143) residue near the ß"-" surface in the junction between the extracellular and transmembrane domains. In the ternary receptor, there are predicted to be two copies of each class of sites, for a total of four sites per receptor. We used ß2α1γ2L and ß2α1 concatemeric constructs to determine the functional effects of the ß(Y143W) and ß(M286W) mutations to gain insight into the number of functional binding sites for propofol and the energetic contributions stemming from propofol binding to the individual sites. A mutation of each of the four sites affected the response to propofol, indicating that each of the four sites is functional in the wild-type receptor. The mutations mainly impaired stabilization of the open state by propofol, i.e., reduced gating efficacy. The effects were similar for mutations at either site and were largely additive and independent of the presence of other Y143W or M286W mutations in the receptor. The two classes of sites appeared to differ in affinity for propofol, with the site affected by M286W having about a 2-fold higher affinity. Our analysis indicates there may be one or two additional functionally equivalent binding sites for propofol, other than those modified by substitutions at ß(Y143) and ß(M286).

Interactions of Propofol With Human Voltage-gated Kv1.5 Channel Determined by Docking Simulation and Mutagenesis Analyses.

Propofol blocks the voltage-gated human Kv1.5 (hKv1.5) channel by preferentially affecting in its open state. A previous mutational study suggested that several amino acids within the pore region of the hKv1.5 channel are involved in mediating the blocking action of propofol. The present investigation was undertaken to elucidate the predicted binding modes of propofol within the pore cavity of the open-state hKv1.5 channel, using computational docking and mutagenesis approaches. The docking simulation using a homology model of the hKv1.5 channel, constructed based on the crystal structure of the Kv1.2 channel, predicted that propofol was positioned at the base of the pore cavity of hKv1.5 channel, adjacent to 4 amino acids Thr479, Thr480, Val505, and Ile508, and formed arene-H interactions with Val505. The patch-clamp experiments on wild-type and mutant hKv1.5 channels constructed by site-directed mutagenesis revealed that the blocking potency of propofol was significantly reduced in T480A, V505A, and I508A but not in T479A mutants compared with wild-type hKv1.5 channel. These computational docking and experimental mutational analyses suggest that propofol is positioned at the base of the pore cavity and forms functional contact with Thr480, Val505, and Ile508 to directly block the hKv1.5 channel.

Propofol Promotes Activity and Tumor-Killing Ability of Natural Killer Cells in Peripheral Blood of Patients with Colon Cancer.

BACKGROUND We investigated the effect of propofol on activities and tumor-killing ability of natural killer (NK) cells in patients with colon cancer. MATERIAL AND METHODS Twenty colon cancer patients and 20 healthy subjects were included. Peripheral blood (5 ml) was collected from all patients and healthy subjects. NK cells in peripheral blood were separated by negative screening using immunomagnetic beads. Flow cytometry was used to determine expression of activated receptors, inhibitory receptors, killing effector molecules, and proliferation-associated markers on NK cell surfaces. After in vitro treatment with propofol for 24 h, expression of activated receptors, inhibitory receptors, killing effector molecules, and proliferation-associated markers on NK cell surfaces was examined again. In addition, the tumor-killing effect of NK cells was studied by co-culture with K562 cells or colon cancer SW620 cells at a ratio of 1: 1. RESULTS The number of NK cells in peripheral blood from colon cancer patients was increased compared with healthy subjects, but activities and proliferation ability of the NK cells were decreased. The tumor-killing effect of NK cells isolated from colon cancer patients was decreased. Of note, propofol promoted activation of NK cells from colon cancer patients. In addition, propofol increased expression of tumor-killing effector molecules by NK cells and the proliferation ability of NK cells. Propofol also enhanced the killing effect of NK cells on colon cancer cells. CONCLUSIONS The present study demonstrates that propofol promotes the activity and tumor-killing ability of NK cells in peripheral blood of patients with colon cancer.

Propofol Inhibits HeLa Cells by Impairing Autophagic Flux via AMP-Activated Protein Kinase (AMPK) Activation and Endoplasmic Reticulum Stress Regulated by Calcium.

BACKGROUND Propofol has antitumor effects against various cancers. However, the mechanism of action of propofol in HeLa human cervical cancer cells has not been elucidated. MATERIAL AND METHODS We treated HeLa human cervical cancer cells with different concentrations of propofol. Cell viability was evaluated with Cell Counting Kit-8 and apoptosis was analyzed by annexin V-fluorescein isothiocyanate and propidium iodide staining and flow cytometry. Autophagosome formation was evaluated based on microtubule-associated protein light chain (LC)3 conversion and light chain 3 puncta formation. Autophagosome clearance was assessed according to p62 protein level and autolysosome generation. RESULTS We found that propofol decreased cell viability and increased autophagosome generation in HeLa cells. Autophagosome formation was evaluated based on LC3 conversion and LC3 puncta formation. Autophagosome clearance was assessed according to p62 protein level. The AMPK/mTOR signaling pathway was found to be activated in propofol-induced autophagosome accumulation. Fluorescence analysis using LysoTracker dye revealed that propofol blocked autophagosome-lysosome fusion. Administration of rapamycin increased autophagosome clearance in propofol-treated HeLa cells. Additionally, propofol induced endoplasmic reticulum (ER) stress and disrupted intracellular Ca2+ balance, thereby enhancing autophagosome accumulation. Suppressing ER stress by treatment with tauroursodeoxycholic acid (TUDCA) enhanced these effects, suggesting that the cytotoxicity of propofol is related to induction of ER stress. CONCLUSIONS This study is the first to provide evidence that propofol-mediated autophagy regulation is an underlying part of the mechanism by which propofol regulates HeLa cells progression.

The Actions of Drug Combinations on the GABAA Receptor Manifest as Curvilinear Isoboles of Additivity.

Drug interactions are often analyzed in terms of isobolograms. In the isobologram, the line connecting the axial points corresponding to the concentrations of two different drugs that produce an effect of the same magnitude is termed an isobole of additivity. Although the isobole of additivity can be a straight line in some special cases, previous work has proposed that it is curvilinear when the two drugs differ in their maximal effects or Hill slopes. Modulators of transmitter-gated ion channels have a wide range of maximal effects as well as Hill slopes, suggesting that the isoboles for drug actions on ion channel function are not linear. In this study, we have conducted an analysis of direct activation and potentiation of the human α1ß2γ2L GABAA receptor to demonstrate that: 1) curvilinear isoboles of additivity are predicted by a concerted transition model where the binding of each GABAergic drug additively and independently reduces the free energy of the open receptor compared with the closed receptor; and 2) experimental data for receptor activation using the agonist pair of GABA and propofol or potentiation of responses to a low concentration of GABA by the drug pair of alfaxalone and propofol agree very well with predictions. The approach assuming independent energetic contributions from GABAergic drugs enables, at least for the drug combinations tested, a straightforward method to accurately predict functional responses to any combination of concentrations.

X-ray structures of general anaesthetics bound to a pentameric ligand-gated ion channel.

General anaesthetics have enjoyed long and widespread use but their molecular mechanism of action remains poorly understood. There is good evidence that their principal targets are pentameric ligand-gated ion channels (pLGICs) such as inhibitory GABA(A) (γ-aminobutyric acid) receptors and excitatory nicotinic acetylcholine receptors, which are respectively potentiated and inhibited by general anaesthetics. The bacterial homologue from Gloeobacter violaceus (GLIC), whose X-ray structure was recently solved, is also sensitive to clinical concentrations of general anaesthetics. Here we describe the crystal structures of the complexes propofol/GLIC and desflurane/GLIC. These reveal a common general-anaesthetic binding site, which pre-exists in the apo-structure in the upper part of the transmembrane domain of each protomer. Both molecules establish van der Waals interactions with the protein; propofol binds at the entrance of the cavity whereas the smaller, more flexible, desflurane binds deeper inside. Mutations of some amino acids lining the binding site profoundly alter the ionic response of GLIC to protons, and affect its general-anaesthetic pharmacology. Molecular dynamics simulations, performed on the wild type (WT) and two GLIC mutants, highlight differences in mobility of propofol in its binding site and help to explain these effects. These data provide a novel structural framework for the design of general anaesthetics and of allosteric modulators of brain pLGICs.