Effect of the local anesthetic dibucaine on the surface membrane in sterol-manipulated Tetrahymena pyriformis studied by freeze-fracture electron microscopy

The effect of the local anesthetic dibucaine on the membrane ultrastructure of sterol-manipulated Tetrahymena pyriformis (NT-1 strain) was studied by freeze-fracture electron microscopy. Dibucaine-treated, ergosterol-replaced Tetrahymena cells had marked alterations in their plasma membranes. IMP-free small depressions (exoplasmic fracture face) and protrusions (protoplasmic fracture face) were formed on the plasma membranes which was in contact with the outer alveolar membrane. In addition, large IMP-free surface "blebs" covered with hexagonally-arranged depressions and protrusions appeared on both the plasma and outer alveolar membranes. These "blebs" were pinched off when the membranes were severely affected. Our previous study (28) demonstrated that the plasma membrane of dibucaine-treated native Tetrahymena cells that contain tetrahymanol showed vertical displacement of its intramembranous particles and that subsequently a smooth, flat surface appeared. Therefore, the structural changes in ergosterol-replaced membranes produced by dibucaine differ strikingly from changes in the native membranes. The remarkable difference in the ultrastructural deformation of the plasma membrane probably is due to a difference in the membrane lipid composition induced by sterol-manipulation.     

Catabolism of Exogenous and Endogenous Sphingomyelin and Phosphatidylcholine by Homogenates and Subcellular Fractions of Cultured Neuroblastoma Cells. Effects of Anesthetics

Cultured murine neuroblastoma cells contain a neutral, Mg2+-stimulated sphingomyelinase and an alkaline phosphatidylcholine-hydrolyzing activity that are enriched in the plasma membrane fraction. The reaction products of sphingomyelin catabolism are phosphocholine and ceramide and those of phosphatidylcholine, glycerophosphocholine and fatty acid. These reactions were studied with endogenous as well as exogenous liposomal substrates. With both exogenous and endogenous substrates, the sphingomyelinase activity was stimulated two- to threefold by Mg2+ and a further three- to fourfold by volatile anesthetic agents. Stimulation was concentration-dependent and corresponded to anesthetic potency: methoxyflurane greater than halothane greater than enflurane. Greater than 80% of the plasma membrane sphingomyelin was hydrolyzed within 2 h in the presence of Mg2+ and anesthetic. In contrast, the activity with exogenous and endogenous phosphatidylcholine was unaffected by Mg2+ or Ca2+ and was markedly inhibited (50-80%) by anesthetic agents. The degree of inhibition was concentration-dependent and corresponded to anesthetic potency. The quantitative importance of choline-containing lipids in cell membranes, the relatively exclusive localization of the neutral Mg2+-stimulated sphingomyelinase in cells of neural origin, the totally different type of hydrolytic attack on phosphatidylcholine, and the reciprocal effects of anesthetics on the hydrolysis of these two lipids strongly suggest important roles for these activities in cell membranes in general and in the neuron in particular.     

Biochemical and ultrastructural changes in rat liver plasma membranes after temporary ischemia

In this study we have attempted to correlate reversible and irreversible cell damage induced by in vivo or in vitro ischemia with characteristics of the plasma membranes of liver parenchymal cells, as detected biochemically and ultrastructurally. The effects of in vivo or in vitro ischemia appeared to be similar. It was virtually impossible to isolate a substantial membrane fraction from ischemic livers, probably because of changes in the physical properties of the membranes by ischemia. The isolated membranes of ischemic liver cells show ultrastructural changes including the occurrence of many vesicular profiles and alterations in junctional complexes expressed by extended and smudged electron densities along the lateral surfaces. The microvilli of the bile canaliculi disappeared after only 15 min ischemia and cytoplasmic densities associated with junctional complexes also appeared extended and smudged. These changes correspond with the alterations observed in ischemic isolated membranes. After 30 min in vivo ischemia the activity of 5'-mononucleotidase used as a marker enzyme for plasma membranes, decreased by 75%, whereas the activity of thymidine 5'-phosphodiesterase was reduced only slightly. The changes in these enzyme activities were more prominent after in vitro ischemia than after in vivo. The morphological and biochemical changes observed in rat hepatocyte plasma membrane during the early stage of injury have no value in predicting the occurrence of necrosis in a later phase of the process since profound changes occur in plasma membrane properties after even short periods of ischemia (i.e. during the reversible stage).     

Plasma membrane coating with cationic silica particles and osmotic shock alters the morphology of bovine aortic endothelial cells

We have used a published method of membrane preparation based on the precoating of the apical membrane of aortic endothelial cells with cationic silica microbeads (with or without polyacrylic acid) in combination with an osmotic shock and mechanical shearing to isolate the apical from the basal plasma membranes of these cells, in vitro. After labeling of the plasma membrane of adherent endothelial cells with a fluorescent derivative of phosphatidylcholine and by using laser confocal fluorescence scanning microscopy, we found that this method of membrane isolation rapidly induced invaginations of the basal plasma membrane to an extent which makes this method unsuitable for further membrane lipid analysis. Morphological analysis of the cells and fluorescence recovery after photobleaching experiments on the plasma membranes were performed at each step of the purification procedure and showed that only hypotonic shock and mechanical shearing of the cells enabled the basal plasma membranes to be purified without significant morphological changes.     

Modulation of prolactin binding sites in vitro by membrane fluidizers. Effects on male prostatic and female hepatic membranes in alcohol-fed rats

The objectives of this study were (i) to determine if in vivo administration of ethanol to rats produced changes in apparent lipid fluidity and prolactin binding capacity of male prostatic and female hepatic membranes and (ii) to compare the effects of membrane fluidizers (aliphatic alcohols) in vitro on prolactin binding of prostatic and hepatic membranes in control and alcohol-fed animals. In vitro ethanol has been shown by us previously to increase prolactin receptor levels presumably by unmasking cryptic prolactin receptors. The degree of fluidization was monitored by a fluorescence polarization method using 1,6-diphenylhexatriene. Adult male and female rats were given either water or 4% ethanol as the sole source of drinking fluid for a period of 6 weeks. No significant changes in plasma prolactin were observed between control and ethanol-treated groups of either sex. However, the microviscosity parameter, inversely related to lipid fluidity, was increased approx. 34% and 40%, respectively, in male prostatic and female rat hepatic membranes after ethanol feeding. Furthermore, 125I-prolactin binding capacity was decreased approx. 30% and 26%, respectively, in prostatic and hepatic membranes of alcohol fed animals. In vitro treatment with aliphatic alcohols had no effect on either microviscosity or prolactin binding in hepatic or prostatic membranes from ethanol-fed rats, but both fluidized and increased prolactin binding in the same membrane preparations from control rats. Our observations are consistent with the direct relationship between membrane fluidity and prolactin receptor levels. The changes in prostatic and hepatic membranes after alcohol feeding, namely decreased prolactin receptor levels, decreased fluidity and increased resistance to the fluidizing effects of in vitro aliphatic alcohols may reflect a fundamental membrane defect.     

Lack of effect of flurothyl, a non-anesthetic fluorinated ether, on rat brain synaptic plasma membrane calcium-ATPase

Plasma membrane Ca2+-ATPase (PMCA), a regulator of intracellular calcium, is inhibited by volatile anesthetics and by xenon and nitrous oxide. Response of a cellular system to anesthetics, particularly to volatile agents, raises the question of non-specific, even toxic, side effects unrelated to anesthetic action. Compounds with chemical and physical properties similar to halogenated anesthetics, but which lack anesthetic effect, have been used to address this question. We have compared the effects of halothane and flurothyl, a non-anesthetic fluorinated ether, on PMCA Ca2+ transport across isolated brain synaptic plasma membranes (SPM). Flurothyl, at concentrations predicted by the Meyer-Overton curve to range from 0.4 to 2.6 MAC (minimum alveolar concentration), had no significant on PMCA activity. In contrast halothane, 1.3 MAC, reduced Ca2+ transport 30 to 40%. These findings provide further evidence for a specific effect of inhalation anesthetics on neuronal plasma membrane Ca2+-ATPase.     

Effect of hyperthermia on the plasma membrane structure of Chinese hamster V79 fibroblasts: a quantitative freeze-fracture study

Hyperthermia in the range 41-45 degrees C can induce wide biochemical, physiological, and morphological changes in mammalian cells both in vivo and in vitro. In general, its effects on membranes, particularly on the plasma membrane, are still poorly understood. To investigate the effects of heat on this cell structure, Chinese hamster V79 fibroblasts were exposed to 43 degrees C hyperthermia for 1 h, immediately fixed with glutaraldehyde after treatment, and freeze-fractured for electron microscopic examination. Particular attention was given to the density and size of intramembranous particles (IMPs) on both protoplasmic (PF) and external (EF) fracture faces of the plasma membrane. The quantitative study performed by an interactive image analyzer on the IMPs, generally reported as plasma membrane proteins, showed in heat-treated cells a statistically significant increase in their density and size on both fracture faces. The differences observed demonstrate that in our experimental conditions, hyperthermia in plasma membranes produces structural changes whose biological significance has to be clarified. Moreover, our findings seem to support recent data indicating an involvement of membrane proteins in the cell response to hyperthermia.     

Activities of enzymes from canine kidney plasma membranes under effects of polyene antibiotics in vivo and in vitro]

The effects of amphotericin B drug containing sodium deoxycholate (DOC) and those of DOC and nistatin on the activities of Na+, K+-ATPase and 5'-nucleotidase of canine kidney plasma membranes were studied. It was found that the activities of Na+, K+-ATPase and 5'-nucleotidase were markedly inhibited only after intravenous injection of amphotericin B, whereas the other agents tested caused no changes in the enzyme activities. Similar results were obtained in vitro. In the presence of amphotericin B the activity of Na+, K+-ATPase was noticeably inhibited already at the antibiotic concentration of 0,1 mkg per mg of membrane protein. It was found that the injection of amphotericin B, DOC and nistatin did not qualitatively or quantitatively affect the phospholipid composition of the plasma membranes. This is indicative of the lack of correlation between the enzyme activities and changes in the phospholipid composition of the plasma membranes under effects of amphotericin B. The pyrimidine derivative--amygluracyl--markedly removes the inhibiting effect of amphotericin B on the enzyme activity of plasma membranes.     

The nitroxides pirolin and pirolid protect the plasma membranes of rat cardiomyocytes against damage induced by anthracyclines

This study was performed to evaluate the protective effects of pyrroline and pyrrolidine nitroxides Pirolin, PL, and Pirolid, PD, on the plasma membranes of rat cardiomyocytes treated in vitro with anthracycline drugs aclarubicin (ACL) and doxorubicin (DOX). The influence of two concentrations of drugs (10 and 20 microM) and nitroxides (0.1 and 1 mM) as well as their combinations (a drug and a nitroxide) on membrane fluidity was investigated. The plasma membranes of cardiomyocytes were labelled with a hydrophobic fluorescence probe 12-AS and membrane fluidity was estimated on the basis of the fluorescence anisotropy of the probe. We found that aclarubicin and doxorubicin induced a significant dose-dependent decrease in membrane fluidity, whereas the nitroxides (PL and PD) caused its increase. Preincubation of cardiomyocytes with Pirolin entirely protected plasma membranes of these cells against damage caused by DOX. In the same conditions no protective effect of Pirolid was observed. What is more, Pirolid in combination with DOX caused fluidisation of the plasma membranes of cardiomyocytes. Both nitroxides at low concentration (0.1 mM) protected plasma membranes against rigidification induced by aclarubicin, while high concentration (1 mM) was ineffective and caused fluidisation of the plasma membranes of cardiomyocytes.     

Reconstitution of cyc- S49 membranes by in vitro translated Gs alpha. Membrane anchorage and functional implications

After ADP-ribosylation by cholera toxin which promotes dissociation of the subunits, the alpha-subunit of Gs (Gs alpha) remained strongly associated with plasma membranes of wild-type S49 cells, since its interaction with the membrane was insensitive to 1 M KCl. Its association with the membrane was partially disrupted by 6 M urea and totally abolished by treatment with alkali at pH greater than or equal to 11.5. In vitro translated Gs alpha could interact with plasma membranes from the cyc- mutant of S49 cells as revealed by its cosedimentation with the membrane fraction and incubation of reconstituted membranes with GTP gamma S did not alter anchorage of Gs alpha. The characteristics of the association of in vitro translated Gs alpha with cyc- membranes after GTP gamma S treatment, i.e. sensitivity to 1 M KCl, 6 M urea and alkali treatment, were very similar to those described for the ADP-ribosylated form in wild-type membranes. Restoration of the coupling between the adrenergic receptor and adenylate cyclase further confirmed the vectorial reconstitution of cyc- membranes by in vitro translated alpha-subunit of Gs.     

Effects of 2-hydroxyoleic acid on the structural properties of biological and model plasma membranes

Genetic hypertension is associated with alterations in lipid metabolism, membrane lipid composition and membrane-protein function. 2-Hydroxyoleic acid (2OHOA) is a new antihypertensive molecule that regulates the structure of model membranes and their interaction with certain peripheral signalling proteins in vitro. While the effect of 2OHOA on elevated blood pressure is thought to arise through its influence on signalling proteins, its effects on membrane lipid composition remain to be assessed. 2OHOA administration altered the lipid membrane composition of hypertensive and normotensive rat plasma membranes, and increased the fluidity of reconstituted liver membranes from hypertensive rats. In spontaneously hypertensive rats (SHR), treatment with 2OHOA increased the cholesterol and sphingomyelin content while decreasing that of phosphatidylserine-phosphatidylinositol lipids. In addition, monounsaturated fatty acid levels increased as well as the propensity of reconstituted membranes to form HII-phases. These data suggest that 2OHOA regulates lipid metabolism that is altered in hypertensive animals, and that it affects the structural properties of liver plasma membranes in SHR. These changes in the structural properties of the plasma membrane may modulate the activity of signalling proteins that associate with the cell membrane such as the Galphaq/11 protein and hence, signal transduction.     

Modulation of prolactin binding sites in vitro by membrane fluidizers. IV. Differential effects on plasma membrane and Golgi fractions of male prostate and female liver in the rat

In vitro treatment of crude particulate fractions of male rat ventral prostate and female rat liver with membrane fluidizers (aliphatic alcohols) has been previously reported by us to increase prolactin (PRL) receptor levels, presumably by unmasking cryptic prolactin receptors. The objective of this study was to determine if similar in vitro treatment of purified plasma membrane- and Golgi-rich fractions of male rat prostate and female rat liver with ethanol produced differential effects on prolactin binding in these two subcellular fractions. The degree of fluidization was monitored by a fluorescence polarization method using 1,6-diphenylhexatriene. 125I-PRL specific binding to Golgi-rich fractions of male ventral prostate and female liver was approximately 4-fold higher than that observed in plasma membrane-rich fractions. The microviscosity parameter, inversely related to lipid fluidity, was consistently lower in Golgi-rich fractions than that in plasma membrane-rich fractions in both prostate and liver. In vitro ethanol treatment of prostatic and hepatic plasma membrane fractions produced a dose-related increase and then decline in prolactin binding and a maximal (60-75%) increase in prolactin binding was observed at 4.8% and 2.0% ethanol in prostatic and hepatic membranes, respectively. This in vitro treatment also produced a significant increase in apparent lipid fluidity of plasma membrane-rich fractions of prostate gland and liver. However, similar in vitro ethanol treatment of Golgi fractions of both prostate gland and liver exhibited little increase in prolactin binding without changing microviscosity. Our observations are consistent with the direct relationship between membrane fluidity and prolactin receptor levels. The changes in prostatic and hepatic plasma membrane fractions following in vitro ethanol treatment suggest that prolactin receptors located on the plasma membranes may be modulated (via membrane lipid microviscosity changes) in vivo to a greater extent by various physiological agents than those located within the Golgi fraction.     

Alcohol and the heart: theoretical considerations

It is now well established that consumption of ethyl alcohol, both acute and chronic, exerts deleterious effects on the heart. Evidence is presented that the initial event that precipitates both acute and chronic changes reflects the physical effects of alcohol on membrane phospholipids and perhaps proteins. The presence of alcohol increases membrane fluidity, a condition that leads to an adaptive change in the phospholipid composition of the membranes, with resultant greater rigidity of the membranes. The effects of alcohol on the lipid bilayer of the plasma membrane, when combined with other nonspecific insults, may lead to a drastic increase in calcium permeability; the resulting calcium influx may cause cell necrosis and initiate irreversibly cardiomyopathy. It is likely that changes in membrane fluidity also exert profound effects on enzyme and transport activities of membrane-bound proteins. In addition, alcohol may interact directly with the hydrophobic regions of proteins. Such interactions may play an important role not only in membrane-bound proteins, but also in alcohol-induced changes in contractile proteins of the heart. It is suggested that, in general, the effects of alcohol are similar to those of other anesthetic agents, and that the elucidation of the pathogenesis of alcoholic cardiomyopathy may require a deeper understanding of the physical interaction among alcohol, phospholipids, and proteins.     

Uptake of dibucaine into large unilamellar vesicles in response to a membrane potential

Local amine anesthetics appear to exert their effects in the charged (protonated) form on the cytoplasmic side of excitable membranes. Two features of interest are the mechanism whereby these drugs move across the membrane to the inner monolayer and the actual membrane concentrations achieved. In this work, we have investigated the influence of a K+ diffusion potential, delta psi, on the transmembrane distribution and concentration of the local anesthetic dibucaine employing large unilamellar vesicle systems. It is demonstrated that egg phosphatidylcholine large unilamellar vesicles exhibiting a delta psi (interior negative) actively accumulate dibucaine to achieve high interior concentrations. 31P and 13C nuclear magnetic resonance studies show that the internalized drug is localized to the vesicle inner monolayer, and suggest that the protonated form of the anesthetic is the species that is actively transported. The inner monolayer anesthetic concentrations thus achieved can be an order of magnitude or more larger than predicted on the basis of anesthetic lipid-water partition coefficients. It is suggested that these effects may be related to the mechanisms whereby local anesthetics are localized and concentrated at their sites of action in nerve membranes.     

'Piggy-back' transport of Xenopus hyaluronan synthase (XHAS1) via the secretory pathway to the plasma membrane

Hyaluronan is the sole glycosaminoglycan whose biosynthesis takes place directly at the plasma membrane. The mechanism by which hyaluronan synthase (HAS) becomes inserted there, as well as the question of how the enzyme discriminates between particular membrane species in polarized cells, are largely unknown. In vitro translation of HAS suggested that the nascent protein becomes stabilized in the presence of microsomal membranes, but would not insert spontaneously into membranes after being translated in the absence of those. We therefore monitored the membrane attachment of enzymatically active fusion proteins consisting of Xenopus HAS1 and green fluorescent protein shortly after de novo synthesis in Vero cells. Our data strongly suggest that HAS proteins are directly translated on the ER membrane without exhibiting an N-terminal signal sequence. From there the inactive protein is transferred to the plasma membrane via the secretory pathway. For unknown reasons, HAS inserted into membranes other than the plasma membrane remains inactive.     

Change in membrane lipid fluidity induced by phospholipase A activation: a mechanism of endotoxic shock

The effects of endotoxin administration on the fluidity of dog liver plasma membranes and their relationship with changes in phospholipase A2 activity were studied. Endotoxin administration decreased the fluidity of liver plasma membranes and this decrease was reversible by phosphatidylcholine. The endotoxin-induced decrease in membrane fluidity could be mimicked by digesting control liver membranes with exogenous phospholipase A2. Endotoxin administration also increased the endogenous phospholipase A2 activity. Endotoxin in vitro had no phospholipase A2-like activity but it activated the hydrolytic activity of exogenous phospholipase A2. Based on these data, it is concluded that endotoxin administration decreased the fluidity of canine liver plasma membranes by acting through activation of phospholipase A2. The decrease in membrane lipid fluidity induced by endotoxin administration may play a significant role in the development of the pathophysiology of endotoxic shock at the cellular level.     

Depression of phase-transition temperature by anesthetics: nonzero solid membrane binding

The anesthetic-induced depression of the main phase-transition temperature of phospholipid membranes is often analyzed according to the van't Hoff model on the freezing point depression. In this procedure, zero interaction between anesthetics and solid-gel membranes is assumed. Nevertheless, anesthetics bind to solid-gel membranes to a significant degree. It is necessary to analyze the difference in the anesthetic binding between the liquid-crystal and solid-gel membranes to probe the anesthetic action on the lipid membranes. This article describes a theory to estimate the anesthetic binding to each state at the phase-transition temperature. The equations derived here reveal the relation between the partition coefficients of anesthetics and the anesthetic effects on the transition characters: the change in the transition temperature, and the broadening of transition. The theory revealed that the width of transition temperature is determined primarily by the membrane/buffer partition coefficients of anesthetics. Our previous data on the local anesthetic action on the transition temperature of the dipalmitoylphosphatidylcholine vesicle membrane (Ueda, I., Tashiro, C. and Arakawa, K. (1977) Anesthesiology 46, 327-332) are analyzed by this method. The numerical values for the partition of local anesthetics into the liquid-crystal and solid-gel dipalmitoyl-phosphatidylcholine vesicle membranes at the phase-transition temperature are: procaine 8.0 x 10(3) and 4.7 x 10(3), lidocaine, 3.7 x 10(3) and 2.3 x 10(3), bupivacaine 4.1 x 10(4), and 2.6 x 10(4), and tetracaine 7.3 x 10(4) and 4.7 x 10(4), respectively.     

Propofol, a general anesthetic, promotes the formation of fluid phase domains in model membranes

The molecular site of anesthetic action remains an area of intense research interest. It is not clear whether general anesthetics act through direct binding to proteins or by perturbing the membrane properties of excitable tissues. Several studies indicate that anesthetics affect the properties of either membrane lipids or proteins. However, gaps remain in our understanding of the molecular mechanism of anesthetic action. Recent developments in membrane biology have led to the concept of small-scale domain structures in lipid and lipid--protein coupled systems. The role of such domain structures in anesthetic action has not been studied in detail. In the present study, we investigated the effect of anesthetics on lipid domain structures in model membranes using the fluorescent spectral properties of Laurdan (6-dodecanoyl-2-dimethylamino naphthalene). Propofol, a general anesthetic, promoted the formation of fluid domains in model membranes of dipalmitoyl phosphatidyl choline (DPPC) or mixtures of lipids of varying acyl chains (DPPC:DMPC dimyristoyl phosphatidyl choline 1:1). The estimated size of these domains is 20--50 A. Based on these studies, we speculate that the mechanism of anesthetic action may involve effects on protein--lipid coupled systems through alterations in small-scale lipid domain structures.     

Lipid dynamics in the plasma membrane of ram and bull spermatozoa after washing and exposure to macromolecules BSA and PVP.

Seminal plasma proteins and macromolecules in the external medium have a major influence on the functionality of sperm plasma membranes. In this investigation we have examined their effects on lipid diffusion in the surface membrane of ram and bull spermatozoa as measured by fluorescence recovery after photobleaching (FRAP). Results show that progressive removal of seminal plasma from ram spermatozoa by repeated centrifugation and resuspension in media +/- 4% bovine serum albumin (BSA) or 0.4% polyvinlypyrrolidone (PVP) causes a reduction in lipid diffusion in all regions of the membrane. By contrast, bull sperm membranes respond with an increase in diffusion in all regions. Repeated washing of bull spermatozoa whose membranes were previously immobile (i.e., showed no recovery after FRAP) restored lipid diffusion suggesting an inhibitory effect of seminal plasma proteins. Further analysis by atomic force microscopy revealed a close association between BSA and the plasma membrane. It is concluded that diffusion of lipids in the plasma membrane of ejaculated ram and bull spermatozoa is influenced by seminal plasma proteins and the composition of the suspending medium. Mol. Reprod. Dev. 59:306-313, 2001.     

Increased cholesterol content of erythrocyte and brain membranes in ethanol-tolerant mice

Mice were treated with ethanol for eight or nine days, using a liquid diet regimen known to produce physical dependence. In previous experiments, synaptosomal plasma membranes and erythrocyte ghosts from such ethanoltreated animals were found to be resistant to the fluidizing effects of ethanol in vitro, as measured by electron paramagnetic resonance. In the present experiments, corresponding membranes were analysed for phospholipid and cholesterol. The ratio of cholesterol to phospholipid was found to be significantly increased in both types of membrane after chronic ethanol treatment. The changed ratio was produced by an increase in cholesterol. There was little or no change in phospholipid content of the membranes. Increased cholesterol may explain the previously observed alteration of physical properties of the membranes.