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1.  Fluorescence polarization studies of rat intestinal microvillus membranes. 
Journal of Clinical Investigation  1977;59(3):536-548.
Rat intestinal microvillus membranes and lipid extracts prepared from them have been studied by fluorescence polarization with three lipid-soluble fluorophores: diphenylhexatriene, retinol, and anthroyl-stearate. The degree of fluorescence polarization of diphenylhexatriene, which provides an index of the "microviscosity" of the lipid regions of the membrane, is exceptionally high in microvillus membranes, the highest yet reported in normal biological membranes. Both the membrane proteins and lipids were found to contribute to the high values. With each of the three probes the polarization values are higher in ileal microvillus membranes as compared to membranes from proximal intestinal segments. Temperature-dependence studies of the fluorescence polarization of diphenylhexatriene and anthroylstearate demonstrate a phase transition in microvillus membranes and in liposomes prepared from their lipid extracts at approximately 26+/-2 degrees C. Ambient pH influences markedly the diphenylhexatriene fluorescence polarization in microvillus membranes but has little effect on that of human erythrocyte ghost membranes. The "microviscosity" of jejunal microvillus membranes is maximal at pH 6.5-7.0 and decreases as much as 50% at pH 3.0, an effect which depends largely upon the membrane proteins. Addition of calcium ions to suspensions of microvillus membranes increases the fluorescence polarization of retinol and anthroyl-stearate, but not that of diphenyl-hexatriene. This confirms the localization of the last compound to the hydrophobic interior of the membrane, relatively distant from the hydrophilic head groups of the polar lipids. Microvillus membrane proteins solubilized with Triton X-100 give relatively high fluorescence polarization and intensity values with retinol, suggesting the presence of binding proteins which could play a role in the normal absorptive mechanism for the vitamin.
PMCID: PMC333391  PMID: 14174
2.  Molecular Details of Membrane Fluidity Changes during Apoptosis and Relationship to Phospholipase A2 Activity 
Biochimica et biophysica acta  2012;1828(2):887-895.
Summary
Secretory phospholipase A2 exhibits much greater activity toward apoptotic versus healthy cells. Various plasma membrane changes responsible for this phenomenon have been proposed, including biophysical alterations described as “membrane fluidity” and “order.” Understanding of these membrane perturbations was refined by applying studies with model membranes to fluorescence measurements during thapsigargin-induced apoptosis of S49 cells using probes specific for the plasma membrane: Patman and trimethylammonium-diphenylhexatriene. Alterations in emission properties of these probes corresponded with enhanced susceptibility of the cells to hydrolysis by secretory phospholipase A2. By applying a quantitative model, additional information was extracted from the kinetics of Patman equilibration with the membrane. Taken together, these data suggested that the phospholipids of apoptotic membranes display greater spacing between adjacent headgroups, reduced interactions between neighboring lipid tails, and increased penetration of water among the heads. The phase transition of artificial bilayers was used to calibrate quantitatively the relationship between probe fluorescence and the energy of interlipid interactions. This analysis was applied to results from apoptotic cells to estimate the frequency with which phospholipids protrude sufficiently at the membrane surface to enter the enzyme’s active site. The data suggested that this frequency increases 50–100-fold as membranes become susceptible to hydrolysis during apoptosis.
doi:10.1016/j.bbamem.2012.08.024
PMCID: PMC3529823  PMID: 22967861
two-photon microscopy; merocyanine 540; Laurdan; hydrolysis kinetics; Patman; diphenylhexatriene
3.  The Phenyltetraene Lysophospholipid Analog PTE-ET-18-OMe as a Fluorescent Anisotropy Probe of Liquid Ordered Membrane Domains (Lipid Rafts) and Ceramide-Rich Membrane Domains 
Biochimica et biophysica acta  2007;1768(9):2213-2221.
The conjugated phenyltetraene PTE-ET-18-OMe (all-(E)-1-O-(15’-Phenylpentadeca-8’,10’,12’,14’-tetraenyl)-2-O-methyl-rac-glycero-3-phosphocholine), is a recently developed fluorescent lysophospholipid analog of edelfosine, (Quesada et al. (2004) J. Med. Chem. 47, 5333–5335). We investigated the use of this analog as a probe of membrane structure. PTE-ET-18-OMe was found to have several properties that are favorable for fluorescence anisotropy (polarization) experiments in membranes, including low fluorescence in water and moderately strong association with lipid bilayers. PTE-ET-18-OMe has absorbance and fluorescence properties similar to those of diphenylhexatriene (DPH) probes, with about as large a difference between its fluorescence anisotropy in liquid disordered (Ld) and ordered states (gel and Lo) as observed for DPH. Also like DPH, PTE-ET-18-OMe has a moderate affinity for both gel state ordered domains and Lo state ordered domains (rafts). However, unlike fluorescent sterols or DPH (Megha and London (2004) J. Biol. Chem. 279, 9997–10004), PTE-ET-18-OMe is not displaced from ordered domains by ceramide. Also unlike DPH, PTE-ET-18-OMe shows only slow exchange between the inner and outer leaflets of membrane bilayers, and can thus be used to examine anisotropy of an individual leaflet of a lipid bilayer. Since PTE-ET-18-OMe is a zwitterionic molecule, it should not be as influenced by electrostatic interactions as are other probes that do not cross the lipid bilayer but have a net charge. We conclude that PTE-ET-18-OMe has some unique properties that should make it a useful fluorescence probe of membrane structure.
doi:10.1016/j.bbamem.2007.05.008
PMCID: PMC2034443  PMID: 17573036
4.  Acyl-Chain Mismatch Driven Superlattice Arrangements in DPPC/DLPC/Cholesterol Bilayers 
The journal of physical chemistry. B  2010;114(31):10105-10113.
Fluorescence and infrared spectroscopy and cholesterol oxidase activity were employed to investigate the effect of phosphatidylcholine (PC) acyl chain length mismatch on the lateral organizations of lipids in liquid-ordered dipalmitoyl-PC/dilauroyl-PC/cholesterol (DPPC/DLPC/CHOL) bilayers. Plots of steady-state fluorescence emission anisotropy of diphenylhexatriene (DPH) labeled PC (DPH-PC) embedded in the DPPC/DLPC/CHOL bilayers revealed significant peaks at several DPPC mole fractions (YDPPC) when the cholesterol mole fraction (XCHOL) was fixed to particular values. Analogously, the DPH-PC anisotropy peaked at several critical XCHOL’s when YDPPC was fixed. Acyl chain C–H and C=O vibrational peak frequencies of native PC as well as the activity of cholesterol oxidase also revealed dips and peaks at similar YDPPC’s. Importantly, most of the observed peaks/dips coincide with the critical mole fractions predicted by the Superlattice (SL) model. A three-dimensional map of DPH-PC anisotropy versus composition in the range 0.32 ≤ XCHOL ≤ 0.50; 0.54 ≤ YDPPC ≤ 0.72 revealed a prominent peak at (XCHOL, YDPPC) ≈ (0.42, 0.64). This suggests a simultaneous presence of two different types of superlattices, one where cholesterol is the quest molecule in a PC host lattice and another where DPPC is the guest in the DLPC host lattice. Time-resolved measurements of DPH-PC fluorescence indicated the existence of an ordered, rotationally hindered environment of acyl chains at that “critical” composition consistent with the existence of SL arrangements. We propose that beside CHOL/PC superlattices, DPPC, and DLPC as well tend to adopt regular SL-like lateral distributions relative to each other, presumably because the less hydrophobic DLPC molecule is slightly displaced toward the aqueous phase, thus allowing more room and mobility for the head groups of both DPPC and DLPC as well as for the acyl chain tails of DPPC. The parallel presence of two kinds of superlattices, that is, CHOL/PC-SL and DPPC/DLPC-SL as demonstrated here, has intriguing implications regarding lipid homeostasis of eukaryote membranes.
doi:10.1021/jp105104f
PMCID: PMC2924151  PMID: 20684633
5.  Interaction of 3β-amino-5-cholestene with phospholipids in binary and ternary bilayer membranes 
Langmuir  2011;28(1):648-655.
3β-Amino-5-cholestene (aminocholesterol) is a synthetic sterol whose properties in bilayer membranes have been examined. In fluid palmitoyl sphingomyelin (PSM) bilayers, aminocholesterol and cholesterol were equally effective in increasing acyl chain order, based on changes in diphenylhexatriene (DPH) anisotropy. In fluid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers, aminocholesterol ordered acyl chains, but slightly less efficiently than cholesterol. Aminocholesterol eliminated the PSM and DPPC gel-to-liquid crystalline phase transition enthalpy linearly with concentration, and the enthalpy approached zero at 30 mol% sterol. Whereas cholesterol was able to increase the thermostability of ordered PSM domains in a fluid bilayer, aminocholesterol under equal conditions failed to do this, suggesting that its interaction with PSM was not as favorable as cholesterol’s. In ternary mixed bilayers, containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), PSM or DPPC, and cholesterol at proportions to contain a liquid-ordered phase (60:40 by mol of POPC and PSM or DPPC, and 30 mol% cholesterol), the average life-time of trans parinaric acid (tPA) was close to 20 ns. When cholesterol was replaced with aminocholesterol in such mixed bilayers, the average life-time of tPA was only marginally shorter (about 18 ns). This observation, together with acyl chain ordering data, clearly shows that aminocholesterol was able to form a liquid-ordered phase with saturated PSM or DPPC. We conclude that aminocholesterol should be a good sterol replacement in model membrane systems for which a partial positive charge is deemed beneficial.
doi:10.1021/la203589u
PMCID: PMC3265605  PMID: 22128897
membrane structure; differential scanning calorimetry; cholestatrienol; trans parinaric acid
6.  Cholesterol modulates rat renal brush border membrane phosphate transport. 
Journal of Clinical Investigation  1990;85(1):231-237.
In dietary phosphate (Pi) deprivation and in aging there is an inverse correlation between renal proximal tubular brush border membrane (BBM) cholesterol (Chol) content, BBM fluidity, and BBM sodium gradient-dependent Pi transport activity (Na-Pi cotransport). The purpose of this study was to determine whether in vitro enrichment of renal BBM with Chol has a direct modulating effect on Na-Pi cotransport. 12 and 24 mol % increases in Chol content caused dose-dependent decreases in Na-Pi cotransport activity, 2,000 in control, vs. 1,450 in Chol (+12%), vs. 900 pmol/5 s/mg BBM protein in Chol (+24%), all P less than 0.01, which was paralleled by dose-dependent increases in the fluorescence anisotropy of diphenylhexatriene, rDPH, i.e., decrease in BBM fluidity, 0.203 in control, vs. 0.210 in Chol (+12%), vs. 0.219 in Chol (+24%), all P less than 0.01. We found that increasing ambient temperature, which increases BBM fluidity independent of changes in Chol content, increased Na-Pi cotransport. When Na-Pi cotransport was analyzed as a function of BBM fluidity, 1/rDPH, we found that at an equivalent BBM fluidity BBM Chol enrichment still resulted in a dose-dependent decrease in Na-Pi cotransport. Finally, in BBM isolated from rats fed a low Pi diet in vitro enrichment with Chol completely reversed the adaptive increases in Na-Pi cotransport and fluidity. Our study therefore, indicates that Chol is a direct modulator of renal BBM Na-Pi cotransport activity, and that in vivo alterations in BBM Chol content most likely plays an important role in the regulation of renal tubular Pi transport.
PMCID: PMC296410  PMID: 1967258
7.  Role of Leaflet Asymmetry in the Permeability of Model Biological Membranes to Protons, Solutes, and Gases 
The Journal of General Physiology  1999;114(3):405-414.
Bilayer asymmetry in the apical membrane may be important to the barrier function exhibited by epithelia in the stomach, kidney, and bladder. Previously, we showed that reduced fluidity of a single bilayer leaflet reduced water permeability of the bilayer, and in this study we examine the effect of bilayer asymmetry on permeation of nonelectrolytes, gases, and protons. Bilayer asymmetry was induced in dipalmitoylphosphatidylcholine liposomes by rigidifying the outer leaflet with the rare earth metal, praseodymium (Pr3+). Rigidification was demonstrated by fluorescence anisotropy over a range of temperatures from 24 to 50°C. Pr3+-treatment reduced membrane fluidity at temperatures above 40°C (the phase-transition temperature). Increased fluidity exhibited by dipalmitoylphosphatidylcholine liposomes at 40°C occurred at temperatures 1–3°C higher in Pr3+-treated liposomes, and for both control and Pr3+-treated liposomes permeability coefficients were approximately two orders of magnitude higher at 48° than at 24°C. Reduced fluidity of one leaflet correlated with significantly reduced permeabilities to urea, glycerol, formamide, acetamide, and NH3. Proton permeability of dipalmitoylphosphatidylcholine liposomes was only fourfold higher at 48° than at 24°C, indicating a weak dependence on membrane fluidity, and this increase was abolished by Pr3+. CO2 permeability was unaffected by temperature. We conclude: (a) that decreasing membrane fluidity in a single leaflet is sufficient to reduce overall membrane permeability to solutes and NH3, suggesting that leaflets in a bilayer offer independent resistances to permeation, (b) bilayer asymmetry is a mechanism by which barrier epithelia can reduce permeability, and (c) CO2 permeation through membranes occurs by a mechanism that is not dependent on fluidity.
PMCID: PMC2229456  PMID: 10469730
barrier function; epithelia; membrane fluidity; CO2; NH3
8.  Isoflurane preconditioning increases B-cell lymphoma-2 expression and reduces cytochrome c release from the mitochondria in the ischemic penumbra of rat brain 
European journal of pharmacology  2008;586(1-3):106-113.
We and others have shown that prior exposure to the volatile anesthetic isoflurane induces ischemic tolerance in the brain. Our results also suggest that isoflurane preconditioning reduces cell apoptosis in the penumbral region of rat brain. We designed this study to determine whether isoflurane preconditioning decreased mitochondria-dependent cell apoptosis. Adult male Sprague-Dawley rats were exposed to or not exposed to 2% isoflurane for 30 min at 24 h before the permanent middle cerebral arterial occlusion. Western blotting was used to quantify protein expression in the cytosolic and mitochondrial fractions of non-ischemic brain cortex and brain cortex in the ischemic core and penumbra. Isoflurane preconditioning significantly decreased the infarct volume of cerebral cortex and improved neurological outcome. Isoflurane increased the expression of the antiapoptotic B-cell lymphoma-2 (Bcl-2) proteins in the cerebral cortex of rats without brain ischemia. Rats preconditioned with isoflurane before brain ischemia had increased Bcl-2 expression in the penumbra. Isoflurane preconditioning reduced the release of cytochrome c from the mitochondria and the activation of caspase 3 in the penumbra. However, isoflurane preconditioning did not alter the translocation of Bid and Bax from the cytosol to the mitochondria, identified mechanisms for Bcl-2 to block the release of cytochrome c from the mitochondria. Our results suggest that isoflurane preconditioning increases Bcl-2 expression to block the release of cytochrome c from the mitochondria to decrease the cell apoptosis in the penumbra.
doi:10.1016/j.ejphar.2008.02.073
PMCID: PMC2429852  PMID: 18355806
isoflurane; preconditioning; neuroprotection; cytochrome c; Bcl-2; mitochondria
9.  MODEL AND CELL MEMBRANE PARTITIONING OF PERFLUOROOCTANESULFONATE IS INDEPENDENT OF THE LIPID CHAIN LENGTH 
Perfluorooctanesulfonic acid (PFOS) is a persistent environmental pollutant that may cause adverse health effects in humans and animals by interacting with and disturbing of the normal properties of biological lipid assemblies. To gain further insights into these interactions, we investigated the effect of PFOS potassium salt on dimyristoyl- (DMPC), dipalmitoyl- (DPPC) and distearoylphosphatidylcholine (DSPC) model membranes using fluorescence anisotropy measurements and differential scanning calorimetry (DSC) and on the cell membrane of HL-60 human leukemia cells and freshly isolated rat alveolar macrophages using fluorescence anisotropy measurements. PFOS caused a concentration-dependent decrease of the main phase transition temperature (Tm) and an increased peak width (ΔTw) in both the fluorescence anisotropy and the DSC experiments, with a rank order DMPC > DPPC > DSPC. PFOS caused a fluidization of the gel phase of all phosphatidylcholines investigated, but had the opposite effect on the liquid crystalline phase. The apparent partition coefficients of PFOS between the phosphatidylcholine bilayer and the bulk aqueous phase were largely independent of the phosphatidylcholine chain length and ranged from 4.4 × 104 to 8.8 × 104. PFOS also significantly increased the fluidity of membranes of cells. These findings suggest that PFOS readily partitions into lipid assemblies, independent of their composition, and may cause adverse biological effects by altering their fluidity in a manner that depends on the membrane cooperativity and state (e.g., gel versus liquid crystalline phase) of the lipid assembly.
doi:10.1016/j.colsurfb.2009.10.025
PMCID: PMC2818369  PMID: 19932010
Fluorescence anisotropy; differential scanning calorimetry; cooperativity; phosphatidylcholines; DPH; TMA-DPH; HL-60 human leukemia cells; alveolar macrophages
10.  Complex I and ATP synthase mediate membrane depolarization and matrix acidification by isoflurane in mitochondria 
European journal of pharmacology  2012;690(0):149-157.
Short application of the volatile anesthetic isoflurane at reperfusion after ischemia exerts strong protection of the heart against injury. Mild depolarization and acidification of the mitochondrial matrix are involved in the protective mechanisms of isoflurane, but the molecular basis for these changes is not clear. In this study, mitochondrial respiration, membrane potential, matrix pH, matrix swelling, ATP synthesis and -hydrolysis, and H2O2 release were assessed in isolated mitochondria. We hypothesized that isoflurane induces mitochondrial depolarization and matrix acidification through direct action on both complex I and ATP synthase. With complex I-linked substrates, isoflurane (0.5 mM) inhibited mitochondrial respiration by 28±10%, and slightly, but significantly depolarized membrane potential and decreased matrix pH. With complex II- and complex IV-linked substrates, respiration was not changed, but isoflurane still decreased matrix pH and depolarized mitochondrial membrane potential. Depolarization and matrix acidification were attenuated by inhibition of ATP synthase with oligomycin, but not by inhibition of mitochondrial ATP- and Ca2+-sensitive K+ channels or uncoupling proteins. Isoflurane did not induce matrix swelling and did not affect ATP synthesis and hydrolysis, but decreased H2O2 release in the presence of succinate in an oligomycin- and matrix pH-sensitive manner. Isoflurane modulated H+ flux through ATP synthase in an oligomycin-sensitive manner. Our results indicate that isoflurane-induced mitochondrial depolarization and acidification occur due to inhibition of the electron transport chain at the site of complex I and increased proton flux through ATP synthase. K+ channels and uncoupling proteins appear not to be involved in the direct effects of isoflurane on mitochondria.
doi:10.1016/j.ejphar.2012.07.003
PMCID: PMC3653412  PMID: 22796646
isoflurane; mitochondria; mitochondrial potassium channels; uncoupling proteins; ATP-synthase
11.  Isoflurane modulates neuronal excitability of the nucleus reticularis thalami in vitro 
The thalamus has a key function in processing sensory information, sleep, and cognition. We examined the effects of a common volatile anesthetic, isoflurane, on modulation of neuronal excitability in reticular thalamic nucleus (nRT) in intact brain slices from immature rats. In current-clamp recordings, isoflurane (300–600 µM) consistently depolarized membrane potential, decreased input resistance and inhibited both rebound burst firing and tonic spike firing modes of nRT neurons. The isoflurane-induced depolarization persisted not only in the presence of tetrodotoxin, but after replacement of Ca2+ with Ba2+ ions in external solution; it was abolished by partial replacement of extracellular Na+ ions with N-methyl-D-glucamine. In voltage-clamp recordings, we found that isoflurane slowed recovery from inactivation of T-type Ca2+ current. Thus, at clinically relevant concentrations, isoflurane inhibits neuronal excitability of nRT neurons in developing brain via multiple ion channels. Inhibition of the neuronal excitability of thalamic cells may contribute to impairment of sensory information transfer in the thalamocortical network by general anesthetics. The findings may be important for understanding cellular mechanisms of anesthesia such as loss of consciousness and potentially damaging consequences of general anesthetics on developing mammalian brains.
doi:10.1111/j.1749-6632.2009.05172.x
PMCID: PMC2906252  PMID: 20633107
thalamus; low-threshold-activated; isoflurane; calcium; sodium; reticular thalamic nucleus
12.  Paired Assessment of Volatile Anesthetic Concentrations with Synaptic Actions Recorded In Vitro 
PLoS ONE  2008;3(10):e3372.
The volatile anesthetic isoflurane poses a number of experimental challenges in the laboratory. Due to its rapid evaporation, the open conditions of most in vitro electrophysiological recording systems make the determination of actual isoflurane concentrations a challenge. Since the absolute anesthetic concentration in solution is directly related to efficacy, concentration measurements are important to allow comparisons between laboratory and clinical studies. In this study we quantify the sources of isoflurane loss during experimentation and describe a method for the measurement of isoflurane concentrations using gas chromatography and mass spectrometry simultaneous to in vitro electrophysiological measurements. Serial samples of perfused bath solution allowed correlation of isoflurane concentrations with ongoing biological effects. Saturated physiological solutions contained 13.4±0.2 mM isoflurane and were diluted to desired “nominal” concentrations for experiments. The perfusion system established stable isoflurane concentrations within the bath by 2 minutes. However, bath isoflurane concentrations varied substantially and unpredictably between experiments. The magnitudes of such discrepancies in isoflurane concentrations spanned clinically important levels. Our studies suggest that, despite countermeasures, solution handling significantly impacted the isoflurane content in the tissue bath. The magnitude of these discrepancies appears to necessitate systematic direct measurement of bath isoflurane concentrations during most in vitro conditions.
doi:10.1371/journal.pone.0003372
PMCID: PMC2556393  PMID: 18841202
13.  Volatile Anesthetic Effects on Midbrain-elicited Locomotion Suggest that the Locomotor Network in the Ventral Spinal Cord Is the Primary Site for Immobility 
Anesthesiology  2008;108(6):1016-1024.
Background
Volatile anesthetics produce immobility primarily by action in the spinal cord; however, anesthetic effects among different neuronal classes located in different spinal regions, and how they relate to immobility, are not understood.
Methods
In decerebrated rats, effects of isoflurane and halothane on movement elicited by electrical microstimulation of the mesencephalic locomotor region (MLR) were assessed in relation to minimum alveolar concentration (MAC). Anesthetic effects on step frequency and isometric limb force were measured. The authors also examined effects of MLR stimulation on responses of nociceptive dorsal horn neurons and limb force responses to tail clamp.
Results
Mean isoflurane requirements to block MLR-elicited stepping were slightly but significantly greater than MAC by 10%. Mean halothane requirements to block MLR-elicited stepping were greater than those for isoflurane and exceeded MAC by 20%. From 0.4 to 1.3 MAC (but not 0.0 to 0.4 MAC), there was a dose-dependent reduction in the frequency and force of hind limb movements elicited by MLR stimulation during both anesthetics. MLR stimulation inhibited noxious stimulus evoked responses of dorsal horn neurons by approximately 80%. Aptly, MLR stimulation produced analgesia that outlasted the midbrain stimulus by at least 15 s, as indicated by an 81% reduction in hind limb force elicited noxious tail clamp.
Conclusions
Because electrical stimulation of the MLR elicits movement independent of dorsal horn activation, the results suggest that the immobilizing properties of isoflurane and halothane are largely independent of action in the dorsal horn. The results suggest that volatile anesthetics produce immobility mainly by action on ventral spinal locomotor networks.
doi:10.1097/ALN.0b013e3181730297
PMCID: PMC2713759  PMID: 18497602
14.  The volatile anesthetic isoflurane induces ecto-5′-nucleotidase (CD73) to protect against renal ischemia and reperfusion injury 
Kidney international  2013;84(1):90-103.
The volatile anesthetic isoflurane protects against renal ischemia and reperfusion injury by releasing renal tubular TGF-β1. Since adenosine is a powerful cytoprotective molecule, we tested whether TGF-β1 generated by isoflurane induces renal tubular ecto-5′-nucleotidase (CD73) and adenosine to protect against renal ischemia and reperfusion injury. Isoflurane induced new CD73 synthesis and increased adenosine generation in cultured kidney proximal tubule cells and in mouse kidney. Moreover, a TGF-β1 neutralizing antibody prevented isoflurane-mediated induction of CD73 activity. Mice anesthetized with isoflurane after renal ischemia and reperfusion had significantly reduced plasma creatinine and decreased renal tubular necrosis, neutrophil infiltration and apoptosis compared to pentobarbital-anesthetized mice. Isoflurane failed to protect against renal ischemia and reperfusion injury in CD73 deficient mice, in mice pretreated with a selective CD73 inhibitor or mice treated with an adenosine receptor antagonist. The TGF-β1 neutralizing antibody or the CD73 inhibitor attenuated isoflurane-mediated protection against HK-2 cell apoptosis. Thus, isoflurane causes TGF-β1-dependent induction of renal tubular CD73 and adenosine generation to protect against renal ischemia and reperfusion injury. Modulation of this pathway may have important therapeutic implications to reduce morbidity and mortality arising from ischemic acute kidney injury.
doi:10.1038/ki.2013.43
PMCID: PMC3676468  PMID: 23423261
acute kidney injury; adenosine; apoptosis; inflammation; necrosis; transforming growth factor-β1
15.  Validation and Insights of Anesthetic Action in an Early Vertebrate Network: The Isolated Lamprey Spinal Cord 
Anesthesia and analgesia  2011;113(5):1033-1042.
Background
The lamprey spinal cord is a well-characterized vertebrate network that could facilitate our understanding of anesthetic action. We tested several hypotheses concerning the lamprey’s clinical application to anesthesia, and the sites/mechanisms of anesthetic action.
Methods
In isolated lamprey spinal cords, minimum immobilizing concentrations (MIC) were determined for halothane, isoflurane, sevoflurane, desflurane, propofol, or the nonimmobilizer F6 (1,2-dichlorohexafluorocyclobutane)- applied during D-glutamate-induced fictive swimming or noxious tail stimulation. Isoflurane and propofol effects on fictive swimming were tested in the presence and absence of strychnine and/or picrotoxin.
Results
Volatile anesthetic MICs were clinically comparable. Isoflurane MIC for fictive swimming and noxious stimulus-evoked movement were the same. F6 did not produce immobility, but decreased the amplitude and phase lag of fictive swimming. Isoflurane decreased fictive swimming cycle frequency, amplitude, autocorrelation, rostrocaudal phase lag, and coherence. Strychnine and picrotoxin elicited only disorganized motor activity under isoflurane and caused small increases in MIC. Propofol’s effects differed from isoflurane for all locomotor rhythm variables except amplitude. The propofol MIC was much larger in lampreys compared to mammals. However, picrotoxin reversed propfol-induced immobility by reinitiating coordinated locomotor activity and increasing MIC >8-fold.
Conclusions
The lamprey spinal cord is a relevant and tractable vertebrate network model for anesthetic action. Isoflurane disrupts interneuronal locomotor networks. Gamma-aminobutyric acid A and glycine receptors play marginal roles in isoflurane-induced immobility in lampreys. Propofol’s selective gamma-aminobutyric acid AA-receptor-mediated immobilizing mechanism is conserved in lampreys. The differential immobilizing mechanisms of isoflurane versus propofol reflect those in mammals, and further suggest different network modes of immobilizing action.
doi:10.1213/ANE.0b013e3182273c34
PMCID: PMC3200493  PMID: 21788314
16.  Anesthetic-Like Modulation of Receptor Function by Surfactants: A Test of the Interfacial Theory of Anesthesia 
Anesthesia and analgesia  2008;107(3):868-874.
Introduction
Inhaled anesthetics are interfacially active, concentrating at interfaces such as the protein/water or bilayer/water interfaces. We tested the hypothesis that interfacial activity was a sufficient condition for anesthetic-like modulation of receptor function by applying surfactants to γ-aminobutyric acid type A (GABAA), glycine, and N-methyl-D-aspartate (NMDA) receptors. We defined anesthetic-like modulation as an increase in currents through native channels that isoflurane and ethanol increased currents through, and a decrease in currents through channels that isoflurane and ethanol decreased currents through. We also tested the null hypothesis that there would be no difference in modulation of channel currents by surfactants in receptors with point mutations that diminished their response to isoflurane and ethanol compared to the native version of these receptors.
Methods
The effect of seven surfactants with different head group charges (anionic, cationic, zwitterionic, and uncharged) and tail lengths (eight carbons and twelve carbons) on homomeric wild type α1 and mutant α1 (S267I) glycine receptors, wild type α1β2γ2s and mutant α1(S270I)β2γ2s GABAA receptors, and wild type NR1/NR2A and mutant NR1(F639A)/NR2A NMDA receptors was studied. Receptors were expressed in Xenopus laevis oocytes and studied using two-electrode voltage clamping.
Results
All seven surfactants, isoflurane, and ethanol potentiated GABAA receptor function. Six of seven surfactants, isoflurane, and ethanol potentiated glycine receptor function. Six of seven surfactants, isoflurane, and ethanol inhibited NMDA receptor function. For the mutant receptors, five of seven surfactants increased currents through GABAA receptors, while six of seven surfactants increased currents through glycine receptors. Six of seven surfactants decreased currents through the NMDA receptor. In contrast to isoflurane and ethanol, surfactants as a group did not diminish modulation of mutant compared to wild type receptors.
Conclusion
These findings identify another large class of compounds (surfactants) that modulate the function of GABAA, glycine, and NMDA receptors in a manner that is qualitatively similar to inhaled anesthetics. We cannot reject the hypothesis that interfacial activity is a sufficient condition for anesthetic-like modulation of these receptors. Mutations that diminish the modulatory effect of isoflurane and ethanol did not diminish the modulatory effect of the surfactants.
doi:10.1213/ane.0b013e31817ee500
PMCID: PMC2668573  PMID: 18713898
Anesthesia; GABAA receptor; Glycine receptor; NMDA receptor; detergent; surface-active agents; interfacial activity
17.  Isoflurane reduces hypoxia/reoxygenation-induced apoptosis and mitochondrial permeability transition in rat primary cultured cardiocytes 
BMC Anesthesiology  2014;14:17.
Background
The volatile anesthetic isoflurane protects the heart from hypoxia/reperfusion (H/R) injury. However, it is still incompletely understood whether isoflurane exerts its protective role through preventing mitochondrial permeability transition pore (MPTP) opening.
Methods
Primary cultured cardiocytes were exposed to H/R in the absence or presence of isoflurane. Cell cytotoxicity and apoptosis were detected by MTT assay and TUNEL staining, respectively. MPTP function was monitored by confocal imaging after reoxygenation. ROS production and activation of caspase-3 were determined by fluorescent reader and western blot, respectively.
Results
As compared to the control group, H/R led to significant cell cytotoxicity and apoptosis, while application of isoflurane markedly reversed the effects. Furthermore, isoflurane significantly inhibits the formation of H/R-induced excess ROS production. Finally, isoflurane attenuated the onset of mitochondrial permeability transition pore (MPTP) occurred during hypoxia/reoxygenation, and in turn inhibited activation of caspase-3.
Conclusions
These data indicate that isoflurane has a protective effect on cardiocytes exposed to H/R by reducing excess ROS production, blocking open of MPTP and further reducing apoptosis.
doi:10.1186/1471-2253-14-17
PMCID: PMC3975578  PMID: 24612850
Isoflurane; Hypoxia/reoxygenation; Cardiocyte; Apoptosis; Mitochondrial permeability transition; ROS
18.  Mechanism of Amphotericin B Resistance in Clinical Isolates of Leishmania donovani 
The clinical value of amphotericin B, the mainstay therapy for visceral leishmaniasis in sodium antimony gluconate-nonresponsive zones of Bihar, India, is now threatened by the emergence of acquired drug resistance, and a comprehensive understanding of the underlying mechanisms is the need of the hour. We have selected an amphotericin B-resistant clinical isolate which demonstrated 8-fold-higher 50% lethal doses (LD50) than an amphotericin B-sensitive strain to explore the mechanism of amphotericin B resistance. Fluorimetric analysis demonstrated lower anisotropy in the motion of the diphenylhexatriene fluorescent probe in the resistant strain, which indicated a higher fluidity of the membrane for the resistant strain than for the sensitive strain. The expression patterns of the two transcripts of S-adenosyl-l-methionine:C-24-Δ-sterol methyltransferase and the absence of ergosterol, replaced by cholesta-5,7,24-trien-3β-ol in the membrane of the resistant parasite, indicate a decreased amphotericin B affinity, which is evidenced by decreased amphotericin B uptake. The expression level of MDR1 is found to be higher in the resistant strain, suggesting a higher rate of efflux of amphotericin B. The resistant parasite also possesses an upregulated tryparedoxin cascade and a more-reduced intracellular thiol level, which helps in better scavenging of reactive oxygen species produced by amphotericin B. The resistance to amphotericin B was partially reverted by the thiol metabolic pathway and ABC transporter inhibitors. Thus, it can be concluded that altered membrane composition, ATP-binding cassette transporters, and an upregulated thiol metabolic pathway have a role in conferring amphotericin B resistance in clinical isolates of Leishmania donovani.
doi:10.1128/AAC.00030-11
PMCID: PMC3264217  PMID: 22123699
19.  A study of the Interaction Between Cetirizine and Plasma Membrane of Eosinophils, Neutrophils, Platelets and Lymphocytes using A fluorescence Technique 
Mediators of Inflammation  1994;3(3):229-234.
The effect of cetirizine on plasma membrane fluidity and heterogeneity of human eosinophils, neutrophils, platelets and lymphocytes was investigated using a fluorescence technique. Membrane fluidity and heterogeneity were studied by measuring the steady-state fluorescence anisotropy and fluorescence decay of 1-(4- trimethylammonium-phenyl)-6-phenyl-1, 3, 5-hexatriene (TMA-DPH) incorporated in the membrane. The results demonstrate that cetirizine (1 μg/ml) induced a significant increase in the Hpid order in the exterior part of the membrane and a decrease in membrane heterogeneity in eosinophils, neutrophils and platelets. Moreover, cetirizine blocked the PAF induced changes in membrane fluidity in these cells. Cetirizine did not influence significantly the plasma membrane of lymphocytes. These data may partially explain the effect ofcetirizine on inflammatory cell activities.
doi:10.1155/S0962935194000335
PMCID: PMC2367040  PMID: 18472948
20.  Combined use of steady-state fluorescence emission and anisotropy of merocyanine 540 to distinguish crystalline, gel, ripple, and liquid crystalline phases in dipalmitoylphosphatidylcholine bilayers 
PMC Biophysics  2010;3:14.
The various lamellar phases of dipalmitoylphosphadtidylcholine bilayers with and without cholesterol were used to assess the versatility of the fluorescent probe merocyanine 540 through simultaneous measurements of emission intensity, spectral shape, and steady-state anisotropy. Induction of the crystalline phase (Lc') by pre-incubation at 4°C produced a wavelength dependence of anisotropy which was strong at 15 and 25°C, weak at 38°C, and minimal above the main transition (>~41.5°C) or after returning the temperature from 46 to 25°C. The profile of anisotropy values across this temperature range revealed the ability of the probe to detect crystalline, gel (Lβ'), and liquid crystalline (Lα) phases. The temperature dependence of fluorescence intensity was additionally able to distinguish between the ripple (Pβ') and gel phases. In contrast, the shape of the emission spectrum, quantified as the ratio of merocyanine monomer and dimer peaks (585 and 621 nm), was primarily sensitive to the crystalline and gel phases because dimer fluorescence requires a highly-ordered environment. This requirement also explained the diminution of anisotropy wavelength dependence above 25°C. Repetition of experiments with vesicles containing cholesterol allowed creation of a phase map. Superimposition of data from the three simultaneous measurements provided details about the various phase regions in the map not discernible from any one of the three alone. The results were applied to assessment of calcium-induced membrane changes in living cells.
PACS Codes: 87.16.dt
doi:10.1186/1757-5036-3-14
PMCID: PMC2993649  PMID: 21054864
21.  Mixing of Perfluorooctanesulfonic Acid (PFOS) Potassium Salt with Dipalmitoyl Phosphatidylcholine (DPPC) 
Perfluorooctane-1-sulfonic acid (PFOS) is emerging as an important persistent environmental pollutant. To gain insight into the interaction of PFOS with biological systems, the mixing behavior of dipalmitoylphosphatidylcholine (DPPC) with PFOS was studied using differential scanning calorimetry (DSC) and fluorescence anisotropy measurements. In the DSC experiments the onset temperature of the DPPC pretransition (Tp) decreased with increasing PFOS concentration, disappearing at XDPPC ≤ 0.97. The main DPPC phase transition temperature showed a depression and peak broadening with increasing mole fraction of PFOS in both the DSC and the fluorescence anisotropy studies. From the melting point depression in the fluorescence anisotropy studies, which was observed at a concentration as low as 10 mg/L, an apparent partition coefficient of K = 5.7 × 104 (mole fraction basis) was calculated. These results suggest that PFOS has a high tendency to partition into lipid bilayers. These direct PFOS-DPPC interactions are one possible mechanism by which PFOS may contribute to adverse effects, for example neonatal mortality, in laboratory studies and possibly in humans.
doi:10.1016/j.colsurfb.2006.05.013
PMCID: PMC2593940  PMID: 16814996
Perfluorinated surfactants; DPPC; DSC; liposome; membrane fluidity
22.  Inhibition of T-type calcium current in rat thalamocortical neurons by isoflurane 
Neuropharmacology  2012;63(2):266-273.
Thalamocortical (TC) neurons provide the major sensory input to the mammalian somatosensory cortex. Decreased activity of these cells may be pivotal in the ability of general anesthetics to induce loss of consciousness and promote sleep (hypnosis). T-type voltage-gated calcium currents (T-currents) have a key function regulating the cellular excitability of TC neurons and previous studies have indicated that volatile general anesthetics may alter the excitability of these neurons.
Using a patch-clamp technique, we investigated the mechanisms whereby isoflurane, a common volatile anesthetic, modulates isolated T-currents and T-current-dependent excitability of native TC neurons in acute brain slices of the rat.
In voltage-clamp experiments, we found that isoflurane strongly inhibited peak amplitude of T-current, yielding an IC50 of 1.1% at physiological membrane potentials. Ensuing biophysical studies demonstrated that inhibition was more prominent at depolarized membrane potentials as evidenced by hyperpolarizing shifts in channel availability curves. In current-clamp experiments we found that isoflurane decreased the rate of depolarization of low-threshold-calcium spikes (LTCSs) and consequently increased the latency of rebound spike firing at the same concentrations that inhibited isolated T-currents. This effect was mimicked by a novel selective T-channel blocker 3,5-dichloro-N-[1-(2,2-dimethyl-tetrahydro-pyran-4-ylmethyl)-4-fluoro-piperidin-4-ylmethyl]-benzamide (TTA-P2). In contrast, isoflurane and TTA-P2 had minimal effect on resting membrane potential and cell input resistance. We propose that depression of thalamic T-currents may contribute to some clinical properties of isoflurane.
doi:10.1016/j.neuropharm.2012.03.018
PMCID: PMC3372673  PMID: 22491022
T-type calcium current; rodent thalamocortical neurons; isoflurane; anesthetics; burst firing; low-voltage-activated
23.  Fatty acyl chain composition in the determination of renal membrane order. 
Journal of Clinical Investigation  1986;77(3):768-773.
The relative roles of phospholipid fatty acyl chain length and phospholipid fatty acyl chain unsaturation in the determination of rat renal brush border membrane order were examined using multilamellar liposomes. Exposure of brush border membranes to sphingomyelinase resulted in a time- and concentration-dependent decrement in sphingomyelin content. Liposomes prepared from lipid extracts of these membranes were reconstituted to defined phosphatidylcholine (PC)/sphingomyelin (SPH) ratios with pure synthetic PCs of defined chain length and degrees of unsaturation. Mixed-acid PCs from bovine liver, egg, and the rat renal brush border membrane were also examined. The steady state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) at 37 degrees C was used to reflect acyl chain packing. The steady state anisotropy of DPH in liposomes isolated from the rat renal brush border membrane averaged 0.205 +/- 0.001, n = 8. When liposomes were reconstituted to PC/SPH ratios of 1.1, 1.6, and 2.4 with saturated PCs of acyl chain length 16 to 22, differences in anisotropy between groups were not observed. However, when PCs containing unsaturated or mixed-acid fatty acyl chains were introduced, anisotropy decreased in a concentration dependent fashion. These data suggest that phospholipid fatty acyl chain unsaturation, but not acyl chain length, has a powerful influence on renal brush border membrane order and the PC/SPH ratio is an important determinant of renal membrane order by virtue of the unsaturated fatty acids normally present with these phospholipids.
PMCID: PMC423462  PMID: 3949976
24.  A Comparison of Post-Operative Recovery Between Isoflurane and Enflurane for Pediatric Dental Outpatient Anesthesia 
Anesthesia Progress  1985;32(6):229-231.
Isoflurane is a volatile liquid anesthetic agent reported to have recovery times shorter than many anesthetic agents in current use. This study compared postoperative recovery times in pediatric patients receiving dental treatment under isoflurane to those receiving enflurane anesthesia. The study consisted of a retrospective review of anesthesia records for pediatric patients receiving isoflurane anesthesia. These patients were then matched to patients receiving enflurane anesthesia for age, weight, sex, race, and treatment time. A total of nine matched pairs were available for review. The average recovery time (as measured from extubation to discharge) for isoflurane was 53 minutes and for enflurane, 46 minutes. When the matched cases were analyzed by paired t-text, no statistically significant difference in recovery times was demonstrated at the 0.05 level. These findings suggest that there is no significant difference in postoperative recovery times between isofluane and enflurane in pediatric dental outpatients undergoing general anesthesia.
PMCID: PMC2175417  PMID: 3868305
25.  Effect of Nedocromil Sodium on Polymorphonuclear Leukocyte Plasma Membrane 
Mediators of Inflammation  1994;3(7):S21-S24.
The effect of nedocromil sodium on the plasma membrane fluidity of polymorphonuclear leukocytes (PMNs) was investigated by measuring steady-state fluorescence anisotropy of 1-[4-trimethylammonium-phenyl]-6-phenyl- 1,3,5-hexatriene (TMA-DPH) incorporated in the membrane. Our results show that nedocromil sodium 300 μM significantly decreased membrane fluidity of PMNs. The decrease in membrane fluidity of PMNs induced by fMLP was abolished in the presence of nedocromil sodium. These data suggest that nedocromil sodium interferes with the plasma membranes of PMNs and modulates their activities.
doi:10.1155/S0962935194000700
PMCID: PMC2365593  PMID: 18475598

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