Propofol and thiopental attenuate adenosine triphosphate-sensitive potassium channel relaxation in pulmonary veins

Pulmonary veins (PV) make a significant contribution to total pulmonary vascular resistance. We investigated the cellular mechanisms by which the intravenous anesthetics propofol and thiopental alter adenosine triphosphate-sensitive potassium (KATP+) channel relaxation in canine PV. The effects of KATP+ channel inhibition (glybenclamide), cyclooxygenase inhibition (indomethacin), nitric oxide synthase inhibition (L-NAME), and L-type voltage-gated Ca2+ channel inhibition (nifedipine) on vasorelaxation responses to levcromakalim (KATP+ channel activator) alone and in combination with the anesthetics were assessed. The maximal relaxation response to levcromakalim was attenuated by removing the endothelium and by L-NAME, but not by indomethacin. Propofol (10(-5), 3x10(-5), and 10(-4) M) and thiopental (10(-4) and 3x10(-4) M) each attenuated levcromakalim relaxation in endothelium-intact (E+) rings, whereas propofol (3x10(-5) and 10(-4) M) and thiopental (3x10(-4) M) attenuated levcromakalim relaxation in endothelium-denuded (E-) rings. In E+ rings, the anesthesia-induced attenuation of levcromakalim relaxation was decreased after pretreatment with L-NAME but not with indomethacin. In E-strips, propofol (10(-4) M) and thiopental (3x10(-4) M) inhibited decreases in tension and intracellular Ca2+ concentration ([Ca2+]i) in response to levcromakalim, and these changes were abolished by nifedipine. These findings indicate that propofol and thiopental attenuate the endothelium-dependent component of KATP+ channel-induced PV vasorelaxation via an inhibitory effect on the nitric oxide pathway. Both anesthetics also attenuate the PV smooth muscle component of KATP+ channel-induced relaxation by reducing the levcromakalim-induced decrease in [Ca2+]i via an inhibitory effect on L-type voltage-gated Ca2+ channels.     

Effect of thiopental, propofol, and etomidate on vincristine toxicity in PC12 cells

Neurotoxicity is the dose-limiting side-effect of vincristine in cancer therapy. Using the nerve growth factor (NGF)-dependent neurite outgrowth and cell proliferation of the PC12 pheochromocytoma cell line as an in vitroassay, the protective effect of different intravenous anesthetics was assessed. Vincristine (1 nmol/L) significantly decreased the percentage of neurite-forming cells from 68%±9% to 27%±7% within a 3-day incubation period. The longer neurites (>2× cell body) in particular proved to be extremely sensitive to vincristine (from 17%±4% to 0% of total neurite-expressing cells). Flow cytometry results revealed an S-phase percentage of 15.85%±3.25% after NGF induction, with vincristine reducing this percentage to 0.68%±0.38%. Reversal of the inhibitory effect of vincristine was noted in the cells treated with thiopental or propofol but not etomidate. Bicuculline partially antagonized the protective effect of thiopental and propofol in both studies. We conclude that thiopental and propofol, but not etomidate, have a protective effect in vincristine-induced neurotoxicity. The protective effect produced by thiopental and propofol is probably secondary to activation of GABA_Areceptors. This revised version was published online in August 2006 with corrections to the Cover Date.     

Abeta peptide interactions with isoflurane, propofol, thiopental and combined thiopental with halothane: a NMR study

Abeta peptide is the major component of senile plaques (SP) which accumulates in AD (Alzheimer's disease) brain. Reports from different laboratories indicate that anesthetics interact with Abeta peptide and induce Abeta oligomerization. The molecular mechanism of Abeta peptide interactions with these anesthetics was not determined. We report molecular details for the interactions of uniformly (15)N labeled Abeta40 with different anesthetics using 2D nuclear magnetic resonance (NMR) experiments. At high concentrations both isoflurane and propofol perturb critical amino acid residues (G29, A30 and I31) of Abeta peptide located in the hinge region leading to Abeta oligomerization. In contrast, these three specific residues do not interact with thiopental and subsequently no Abeta oligomerization was observed. However, studies with combined anesthetics (thiopental and halothane), showed perturbation of these residues (G29, A30 and I31) and subsequently Abeta oligomerization was found. Perturbation of these specific Abeta residues (G29, A30 and I31) by different anesthetics could play an important role to induce Abeta oligomerization.     

Aβ peptide interactions with isoflurane, propofol, thiopental and combined thiopental with halothane: A NMR study

Aβ peptide is the major component of senile plaques (SP) which accumulates in AD (Alzheimer's disease) brain. Reports from different laboratories indicate that anesthetics interact with Aβ peptide and induce Aβ oligomerization. The molecular mechanism of Aβ peptide interactions with these anesthetics was not determined. We report molecular details for the interactions of uniformly ¹⁵N labeled Aβ40 with different anesthetics using 2D nuclear magnetic resonance (NMR) experiments. At high concentrations both isoflurane and propofol perturb critical amino acid residues (G29, A30 and I31) of Aβ peptide located in the hinge region leading to Aβ oligomerization. In contrast, these three specific residues do not interact with thiopental and subsequently no Aβ oligomerization was observed. However, studies with combined anesthetics (thiopental and halothane), showed perturbation of these residues (G29, A30 and I31) and subsequently Aβ oligomerization was found. Perturbation of these specific Aβ residues (G29, A30 and I31) by different anesthetics could play an important role to induce Aβ oligomerization.     

Tourniquet-induced ischemia-reperfusion injuries during extremity surgery at children's age: impact of anesthetic chemical structure

Abstract

Objectives

The aim of this study was to determine the relationship between the antioxidant profile of anesthetics and its relation to total antioxidant capacity (TAC) of plasma in children who underwent tourniquet-induced ischemia-reperfusion (IR) injury during extremity operations.

Methods

Children were randomized into three groups: general inhalational anesthesia with sevoflurane (group S), total intravenous anesthesia (TIVA) with propofol (group T), and regional anesthesia (group R). Venous blood samples were obtained before peripheral nerve block and induction of general anesthesia (baseline), 1 minute before tourniquet release (BTR), and 5 and 20 minutes after tourniquet release (ATR). Plasma TAC as well as antioxidant potential of propofol, thiopental, and bupivacaine were measured using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay.

Results

Plasma TAC in group T was increased significantly at 20 minutes ATR in comparison with basal and BTR values, and also was significantly higher in comparison with plasma TAC in groups S and R measured at the same time point. The radical scavenging activity of anesthetics in vitro indicated that only propofol possessed a significant antioxidative activity in the reaction with DPPH radical in comparison with thiopental and bupivacaine.

Discussion

These data confirm that TIVA with propofol attenuates oxidative stress related to tourniquet-induced ischaemia-reperfusion injury in children.     

Differential effects of intravenous anesthetics on capacitative calcium entry in human pulmonary artery smooth muscle cells

We assessed the roles of the protein kinase C (PKC) and the tyrosine kinase (TK) signaling pathways in regulating capacitative calcium entry (CCE) in human pulmonary artery smooth muscle cells (PASMCs) and investigated the effects of intravenous anesthetics (midazolam, propofol, thiopental, ketamine, etomidate, morphine, and fentanyl) on CCE in human PASMCs. Fura-2-loaded human PASMCs were placed in a dish (37 degrees C) on an inverted fluorescence microscope. Intracellular Ca2+ concentration ([Ca2+]i) was measured as the 340/380 fluorescence ratio in individual PASMCs. Thapsigargin, a sarcoplasmic reticulum Ca2+-adenosine triphosphatase inhibitor, was used to deplete intracellular Ca2+ stores after removing extracellular Ca2+. CCE was then activated by restoring extracellular Ca2+ (2.2 mM). The effects of PKC activation and inhibition, TK inhibition, and the intravenous anesthetics on CCE were assessed. Thapsigargin caused a transient increase in [Ca2+]i. Restoring extracellular Ca2+ caused a rapid peak increase in [Ca2+]i, followed by a sustained increase in [Ca2+]i; i.e., CCE was stimulated in human PASMCs. PKC activation attenuated (P < 0.05), whereas PKC inhibition potentiated (P < 0.05), both peak and sustained CCE. TK inhibition attenuated (P < 0.05) both peak and sustained CCE. Midazolam, propofol, and thiopental each attenuated (P < 0.05) both peak and sustained CCE, whereas ketamine, etomidate, morphine, and fentanyl had no effect on CCE. Our results suggest that CCE in human PASMCs is influenced by both the TK and PKC signaling pathways. Midazolam, propofol, and thiopental each attenuated CCE, whereas ketamine, etomidate, morphine, and fentanyl had no effect on CCE.     

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 β-amyloid (Aβ) 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.

     

The comparative effects of propofol, thiopental, and diazepam, administered intravenously, on pentylenetetrazol seizure threshold in the rabbit.

The anticonvulsant effects of propofol, thiopental, and diazepam, administered intravenously, on pentylenetetrazol (PTZ) seizure threshold were studied and compared in the rabbit. The PTZ seizure threshold determined in various rabbit groups during the control phase of conducted experiments, was found to be in the range of 10.1 +/- 2.0 to 13.5 +/- 3.7 mg/kg. Intravenous administration of comparable doses of propofol, thiopental, and diazepam resulted in marked and significant increases in PTZ seizure threshold. At all administered doses (1.25-10.0 mg/kg), propofol was found to be more effective than thiopental in increasing the PTZ threshold dose. However, the anticonvulsant effects of diazepam were more marked than those of propofol, except at a dose of 10 mg/kg where both agents exhibited equipotent activities. These data demonstrate that propofol enjoys a considerable degree of anticonvulsant activity in the rabbit. This anticonvulsant action is greater than that of thiopental at doses ranging from 2.5 to 10 mg/kg and equipotent with diazepam at the 10 mg/kg dose.     

Increased Expression of β-Amyloid Protein Precursor and Microtubule-Associated Protein τ During the Differentiation of Murine Embryonal Carcinoma Cells

Expression of the genes encoding the beta/A4 amyloid protein precursor (APP) and microtubule-associated protein tau was studied in an embryonal carcinoma cell line (P19) that differentiates in vitro into cholinergic neurons after treatment with retinoic acid. Expression of APP increased 34- (mRNA) and 50-fold (protein) during neuronal differentiation; APP-695 accounted for most of this increase. These remarkable increases in APP expression coincided with a proliferation of neuronal processes and with an increase in content of tau mRNA. Moreover, subsequent decreases in the levels of APP and tau mRNA coincided with the onset of the degeneration of the neuronal processes. Immunocytochemical staining suggested that greater than 85% of the P19-derived neurons are cholinergic and that APP is present in the neuronal processes and cell bodies. These results suggest that APP may play an important role in construction of neuronal networks and neuronal differentiation and also indicate that this embryonal carcinoma cell line provides an ideal model system to investigate biological functions of APP and the roles of APP and tau protein in development of Alzheimer's disease in cholinergic neurons.     

Comparison of different anaesthetic methodologies for sedation during in vitro fertilization procedures: effects on patient physiology and oocyte competence

The main goal of the present retrospective study is to compare four analgesic methodologies (EMLA cream, propofol, thiopental sodium, sevoflurane) for in vitro fertilization (IVF) oocyte retrieval. We found that most anaesthetic parameters were not significantly different among all treatments. In contrast, significant differences were revealed in all groups for total number of oocytes retrieved per patient, rate of mature oocytes at metaphase II stage (MII) and percentage of fertilization and embryo development. In the EMLA cream and thiopental sodium groups we observed the highest percentage of MII oocytes (P < 0.001). Fertilization rate in the EMLA and sevoflurane groups were similar but significantly higher than the propofol and thiopental sodium groups (P < 0.001). The highest rate of anomalous fertilization was observed in the propofol group. Rate of embryo development was similar in all groups but sevoflurane group had a lower percentage of good embryos. In conclusion, by comparing different anaesthetic techniques with different mechanisms of action and administration, potential negative effects of these drugs on the initial stages of human IVF procedure were revealed. Therefore, a local anaesthetic cream is proposed as an acceptable alternative option for anaesthesia during transvaginal oocyte retrieval.     

Amyloid precursor protein knockdown by siRNA impairs spontaneous alternation in adult mice

The cleavage-product of amyloid precursor protein (APP) constitutes the core component of plaques found in the brains of Alzheimer's disease (AD) patients. APP is ubiquitously expressed and its precise physiological functions remain unclear. This protein has been proposed to regulate synaptic function and processes underlying learning and memory. While APP knockout mice show behavioral impairments, these may occur due to early changes during development and/or due to abolition of APP function in adult. To investigate the acute effects of APP knockdown without involving developmental processes, APP expression was reduced using RNA interference in adult mouse brain. Small interfering RNAs (siRNAs) that down-regulated mouse APP protein levels (APP-siRNA) were identified using an APP plasmid-siRNA co-transfection assay in mouse NIH/3T3 fibroblast cells. Infusion of APP-siRNAs into the ventricular system for 2 weeks also down-regulated APP mRNA in mouse brain. Highest knockdown of APP mRNA levels was found in the CA2-CA3 regions of the hippocampus. Mice treated with the most active APP-siRNA showed a significant reduction in spontaneous alternation rate in the Y-maze, without effects on forelimb grip strength or locomotor activity. These data suggest that acute knockdown of APP in adult mouse brain impairs hippocampus-dependent spatial working memory.     

Recognition of anesthetic barbiturates by a protein binding site: a high resolution structural analysis

Barbiturates potentiate GABA actions at the GABA_A receptor and act as central nervous system depressants that can induce effects ranging from sedation to general anesthesia. No structural information has been available about how barbiturates are recognized by their protein targets. For this reason, we tested whether these drugs were able to bind specifically to horse spleen apoferritin, a model protein that has previously been shown to bind many anesthetic agents with affinities that are closely correlated with anesthetic potency. Thiopental, pentobarbital, and phenobarbital were all found to bind to apoferritin with affinities ranging from 10–500 µM, approximately matching the concentrations required to produce anesthetic and GABAergic responses. X-ray crystal structures were determined for the complexes of apoferritin with thiopental and pentobarbital at resolutions of 1.9 and 2.0 Å, respectively. These structures reveal that the barbiturates bind to a cavity in the apoferritin shell that also binds haloalkanes, halogenated ethers, and propofol. Unlike these other general anesthetics, however, which rely entirely upon van der Waals interactions and the hydrophobic effect for recognition, the barbiturates are recognized in the apoferritin site using a mixture of both polar and nonpolar interactions. These results suggest that any protein binding site that is able to recognize and respond to the chemically and structurally diverse set of compounds used as general anesthetics is likely to include a versatile mixture of both polar and hydrophobic elements.     

A dinucleotide deletion in amyloid precursor protein (APP) mRNA associated with sporadic Alzheimer's disease results in efficient secretion of truncated APP isoforms from neuroblastoma cell cultures

Recently, two dinucleotide deletions were detected in the mRNA of the amyloid precursor protein (APP) from cerebral cortex neurons of patients with sporadic Alzheimer's disease (AD) or Down's syndrome. These deletions resulted in truncation of APP, producing an APP isoform with a 38-kDa N-terminus and a novel carboxyl terminus (APP+1). We investigated the subcellular localization and the processing of APP+1 in the neuroblastoma cell line B103. cDNA constructs were generated encoding fusion proteins of APP+1 or full-length APP with the enhanced green fluorescent protein (eGFP). In transient transfection experiments using B103 cells, the APP+1-eGFP fusion protein showed a reticular localization with intense staining in the Golgi complex. Unlike full-length APP fused to eGFP, the APP+1-eGFP fusion protein did not localize to the perinuclear area or to the plasma membrane. Western blot analysis of cell extracts confirmed the translation of the expected fusion proteins. Analysis of the supernatant by western blot indicated that the APP+1-eGFP fusion protein was efficiently secreted from B103 cells, whereas the secreted form of full-length APP fusion protein (APPs) was hardly detectable. Thus, both dinucleotide deletions in the APP mRNA result in truncated APP+1 that is not membrane associated and is readily secreted from neurons.     

Amyloid precursor protein promotes endoplasmic reticulum stress-induced cell death via C/EBP homologous protein-mediated pathway

The accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) is known to activate the ER, which is termed ER stress. Here, we demonstrated that amyloid precursor protein (APP) is a novel mediator of ER stress-induced apoptosis through the C/EBP homologous protein (CHOP) pathway. Expression of APP mRNA was elevated by tunicamycin- or dithiothreitol-induced ER stress. The levels of C83 and APP intracellular domain (AICD) fragments, which are cleaved from APP, were significantly increased under ER stress, although the protein level of full-length APP was decreased. Cellular viability was reduced in APP-over-expressing cells, which was attenuated by treatment with a γ-secretase inhibitor, N -[ N -(3,5-difluorophenacetyl)-L-alanyl]- S -phenylglycine t -butyl ester (DAPT). Cellular viability was also reduced in AICD-FLAG-over-expressing cells. The mRNA and protein levels of CHOP, an ER stress-responsive gene, were remarkably increased by APP over-expression, which was attenuated by treatment with DAPT. CHOP mRNA induction was also found in AICD-FLAG-over-expressing cells. Cell death and CHOP up-regulation by ER stress were attenuated by APP knockdown. Data obtained with a luciferase assay and chromatin immunoprecipitation assay indicated that AICD associates with the promoter region of the CHOP gene. In conclusion, ER stress-induced APP undergoes α- and γ-secretase cleavage and subsequently induces CHOP-mediated cell death.     

A high-throughput drug screen targeted to the 5'untranslated region of Alzheimer amyloid precursor protein mRNA

The authors employed a novel approach to identify therapeutics effective in Alzheimer disease (AD). The 5'untranslated region (5'UTR) of the mRNA of AD amyloid precursor protein (APP) is a significant regulator of the levels of the APP holoprotein and amyloid beta (Abeta) peptide in the central nervous system. The authors generated stable neuroblastoma SH-SY5Y transfectants that express luciferase under the translational control of the 146-nucleotide APP mRNA 5'UTR and green fluorescent protein (GFP) driven by a viral internal ribosomal entry site. Using a high-throughput screen (HTS), they screened for the effect of 110,000 compounds obtained from the library of the Laboratory for Drug Discovery on Neurodegeneration (LDDN) on the APP mRNA 5'UTR-controlled translation of the luciferase reporter. This screening yielded several nontoxic specific inhibitors of APP mRNA 5'UTR-driven luciferase that had no effect on the GFP expression in the stable SH-SY5Y transfectants. Moreover, these compounds either did not inhibit or inhibited to a much lower extent the expression of the luciferase reporter regulated by a prion protein (PrP) mRNA 5'UTR, used as an alternative mRNA structure to counterscreen APP mRNA 5'UTR in stably transfected SH-SY5Y cell lines. The hits obtained from this robust, specific, and highly quantitative HTS will be characterized to identify agents that may be developed into useful future therapeutic agents to limit APP translation and Abeta production for AD.     

General anesthetics inhibit erythropoietin induction under hypoxic conditions in the mouse brain

Background

Erythropoietin (EPO), originally identified as a hematopoietic growth factor produced in the kidney and fetal liver, is also endogenously expressed in the central nervous system (CNS). EPO in the CNS, mainly produced in astrocytes, is induced under hypoxic conditions in a hypoxia-inducible factor (HIF)-dependent manner and plays a dominant role in neuroprotection and neurogenesis. We investigated the effect of general anesthetics on EPO expression in the mouse brain and primary cultured astrocytes.

Methodology/Principal Findings

BALB/c mice were exposed to 10% oxygen with isoflurane at various concentrations (0.10–1.0%). Expression of EPO mRNA in the brain was studied, and the effects of sevoflurane, halothane, nitrous oxide, pentobarbital, ketamine, and propofol were investigated. In addition, expression of HIF-2α protein was studied by immunoblotting. Hypoxia-induced EPO mRNA expression in the brain was significantly suppressed by isoflurane in a concentration-dependent manner. A similar effect was confirmed for all other general anesthetics. Hypoxia-inducible expression of HIF-2α protein was also significantly suppressed with isoflurane. In the experiments using primary cultured astrocytes, isoflurane, pentobarbital, and ketamine suppressed hypoxia-inducible expression of HIF-2α protein and EPO mRNA.

Conclusions/Significance

Taken together, our results indicate that general anesthetics suppress activation of HIF-2 and inhibit hypoxia-induced EPO upregulation in the mouse brain through a direct effect on astrocytes.     

Translation of the alzheimer amyloid precursor protein mRNA is up-regulated by interleukin-1 through 5'-untranslated region sequences

The amyloid precursor protein (APP) has been associated with Alzheimer's disease (AD) because APP is processed into the beta-peptide that accumulates in amyloid plaques, and APP gene mutations can cause early onset AD. Inflammation is also associated with AD as exemplified by increased expression of interleukin-1 (IL-1) in microglia in affected areas of the AD brain. Here we demonstrate that IL-1alpha and IL-1beta increase APP synthesis by up to 6-fold in primary human astrocytes and by 15-fold in human astrocytoma cells without changing the steady-state levels of APP mRNA. A 90-nucleotide sequence in the APP gene 5'-untranslated region (5'-UTR) conferred translational regulation by IL-1alpha and IL-1beta to a chloramphenicol acetyltransferase (CAT) reporter gene. Steady-state levels of transfected APP(5'-UTR)/CAT mRNAs were unchanged, whereas both base-line and IL-1-dependent CAT protein synthesis were increased. This APP mRNA translational enhancer maps from +55 to +144 nucleotides from the 5'-cap site and is homologous to related translational control elements in the 5'-UTR of the light and and heavy ferritin genes. Enhanced translation of APP mRNA provides a mechanism by which IL-1 influences the pathogenesis of AD.