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Zeitschrift Fur Naturforschung. C,... 2001The degree and time-course of expansion of palmitoyloleoylphosphatidylcholine (PC) and bovine brain phosphatidylserine (PS)/PC (75:25, mol/mol) monolayers at 32 mN/m...
The degree and time-course of expansion of palmitoyloleoylphosphatidylcholine (PC) and bovine brain phosphatidylserine (PS)/PC (75:25, mol/mol) monolayers at 32 mN/m caused by differently charged amphiphiles (detergents) added to the sub-phase buffer (pH 7.4, 22 degrees C) were followed. Amphiphiles were added to the sub-phase at a concentration/monolayer area corresponding to the concentration/erythrocytes surface area where sphero-echinocytic or sphero-stomatocytic shapes are induced (0.46-14.6 microM). Nonionic, cationic and anionic amphiphiles expanded the PS/PC monolayer significantly more (1.7-4.2 times) than the PC monolayer. A zwitterionic amphiphile expanded both monolayers to a similar extent. The initial rate of monolayer-expansion was higher for all amphiphiles (1.7-20.4 times) in the PS/PC monolayer than in the PC monolayer. It is suggested that hydrophobic interactions govern the intercalation of amphiphiles into monolayers, and that monolayer packing, modulated by phospholipid head group interactions and alkyl chain saturation, strongly influence amphiphile intercalation. A possible relation between the monolayer-expanding effect of amphiphiles and their effect on erythrocyte shape is discussed.
Topics: Animals; Brain Chemistry; Buffers; Cattle; Chlorpromazine; Dibucaine; Fatty Alcohols; Kinetics; Liposomes; Phosphatidylcholines; Phosphatidylserines; Surface-Active Agents
PubMed: 11724390
DOI: 10.1515/znc-2001-9-1024 -
Zeitschrift Fur Naturforschung. C,... 2001The interaction of the local anesthetic dibucaine with the isolated toad skin and membrane models is described. The latter consisted of human erythrocytes, isolated...
The interaction of the local anesthetic dibucaine with the isolated toad skin and membrane models is described. The latter consisted of human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), large unilamellar vesicles (LUV) of dimyristoylphosphatidylcholine (DMPC) and phospholipid multilayers built-up of DMPC and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Results indicate a significant decrease in the potential difference (PD) and in the short-circuit current (Isc) after the application of dibucaine in toad skin, which may be interpreted as reflecting inhibition of the active transport of ions. This finding might be explained on the basis of the results obtained from fluorescence spectroscopy and X-ray diffraction studies on membrane models. In fact, dibucaine induced structural perturbations in IUM, DMPC LUV and phospholipid multilayers. Scanning electron microscopy revealed that dibucaine induced erythrocyte stomatocytosis. According to the bilayer couple hypothesis an echinocytic type of shape change would have been expected given the preferential interaction of dibucaine with DMPC. Although it is still premature to define the molecular mechanism of action of dibucaine, the experimental results confirm the important role played by the phospholipid bilayers in the association of the anesthetic with cell membranes.
Topics: Animals; Anura; Biological Transport; Dibucaine; Dimyristoylphosphatidylcholine; Erythrocyte Membrane; Female; Humans; In Vitro Techniques; Lipid Bilayers; Liposomes; Male; Microscopy, Electron, Scanning; Models, Biological; Phosphatidylethanolamines; Skin; Sodium; X-Ray Diffraction
PubMed: 11531098
DOI: 10.1515/znc-2001-7-822 -
Biochimica Et Biophysica Acta Apr 2001Macrophages recognize oxidatively damaged autologous erythrocytes, and cell surface fibronectin of macrophages enhances the recognition (Beppu et al., FEBS Lett. 295...
Macrophages recognize oxidatively damaged autologous erythrocytes, and cell surface fibronectin of macrophages enhances the recognition (Beppu et al., FEBS Lett. 295 (1991) 135-140). In the present study, mechanisms of enhanced macrophage recognition of oxidatively damaged erythrocytes by fibronectin were investigated. Monolayers of thioglycollate-induced mouse peritoneal macrophages with cell surface fibronectin recognized autologous erythrocytes oxidized with an iron catalyst ADP/Fe(3+). The macrophage recognition of the oxidized erythrocytes was inhibited partially by pretreatment of the macrophage monolayers with a Ca(2+) channel blocker (diltiazem), calmodulin inhibitors (W-7, trifluoperazine, chlorpromazine and dibucaine), an inhibitor of myosin light chain kinase (ML-9), a microfilament formation inhibitor (cytochalasin B), phospholipase A(2) inhibitors (4-bromophenacyl bromide, mepacrine) and cyclooxygenase inhibitors (indomethacin and aspirin). Monolayers of macrophages depleted of fibronectin by trypsinization lost the ability of recognizing oxidized erythrocytes, but acquired the ability when stimulated with a fibronectin-coated coverslip. The recognition of fibronectin-stimulated trypsinized macrophages was also inhibited by the above inhibitors. On treatment with Ca ionophore A23187, trypsinized macrophages acquired the ability to recognize oxidized erythrocytes. The recognition of Ca ionophore-stimulated trypsinized macrophages was inhibited by the above inhibitors except the Ca(2+) channel blocker. These results indicate that the Ca(2+) signaling including Ca(2+) influx, calmodulin activation and myosin light chain phosphorylation are involved in the fibronectin stimulation of the recognition of macrophages for oxidized erythrocytes. Involvement of microfilament formation and arachidonate cascade in the fibronectin stimulation was also suggested.
Topics: Animals; Calcium; Cell Adhesion; Cells, Cultured; Enzyme Inhibitors; Erythrocytes; Fibronectins; Macrophages, Peritoneal; Male; Mice; Oxidative Stress; Receptors, Cell Surface; Signal Transduction
PubMed: 11336783
DOI: 10.1016/s0167-4889(00)00106-3 -
British Journal of Pharmacology Apr 2001AM-36 is a novel neuroprotective agent incorporating both antioxidant and Na(+) channel blocking actions. In cerebral ischaemia, loss of cellular ion homeostasis due to...
AM-36 is a novel neuroprotective agent incorporating both antioxidant and Na(+) channel blocking actions. In cerebral ischaemia, loss of cellular ion homeostasis due to Na(+) channel activation, together with increased reactive oxygen species (ROS) production, are thought to contribute to neuronal death. Since neuronal death in the penumbra of the ischaemic lesion is suggested to occur by apoptosis, we investigated the ability of AM-36, antioxidants and Na(+) channel antagonists to inhibit toxicity induced by the neurotoxin, veratridine in cultured cerebellar granule cells (CGC's). Veratridine (10 - 300 microM) concentration-dependently reduced cell viability of cultured CGC's. Under the experimental conditions employed, cell death induced by veratridine (100 microM) possessed the characteristics of apoptosis as assessed by morphology, TUNEL staining and DNA laddering on agarose gels. Neurotoxicity and apoptosis induced by veratridine (100 microM) were inhibited to a maximum of 50% by the antioxidants, U74500A (0.1 - 10 microM) and U83836E (0.03 - 10 microM), and to a maximum of 30% by the Na(+) channel blocker, dibucaine (0.1 - 100 microM). In contrast, AM-36 (0.01 - 10 microM) completely inhibited veratridine-induced toxicity ( IC(50) 1.7 (1.5 - 1.9) microM, 95% confidence intervals (CI) in parentheses) and concentration-dependently inhibited apoptosis. These findings suggest veratridine-induced toxicity and apoptosis are partially mediated by generation of ROS. AM-36, which combines both Na(+) channel blocking and antioxidant activity, provided superior neuroprotection compared with agents possessing only one of these actions. This bifunctional profile of activity may underlie the potent neuroprotective effects of AM-36 recently found in a stroke model in conscious rats.
Topics: Animals; Apoptosis; Cell Survival; Cells, Cultured; DNA Fragmentation; Depression, Chemical; Dibucaine; Electrophoresis, Agar Gel; Free Radical Scavengers; Immunohistochemistry; In Situ Nick-End Labeling; Mice; Neurons; Neuroprotective Agents; Piperazines; Sodium Channel Agonists; Sodium Channel Blockers; Tetrodotoxin; Veratridine
PubMed: 11309240
DOI: 10.1038/sj.bjp.0704018 -
Biochimica Et Biophysica Acta Mar 2001Interaction of the local anesthetic dibucaine with small unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) and dioleoyl phosphatidylcholine (DOPC) containing...
Interaction of the local anesthetic dibucaine with small unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) and dioleoyl phosphatidylcholine (DOPC) containing different mol percents of cholesterol has been studied by fluorescence spectroscopy. Fluorescence measurements on dibucaine in presence of phospholipid vesicles containing various amounts of cholesterol yielded a pattern of variation of wavelength at emission maximum and steady-state anisotropy which indicated that the microenvironment of dibucaine is more polar and flexible in membranes that contain cholesterol than in membranes without cholesterol. Experiments on quenching of fluorescence from membrane-associated dibucaine by potassium iodide showed a marked increase in quenching efficiency as the cholesterol content of the vesicles was increased, demonstrating increased accessibility of the iodide quenchers to dibucaine in the presence of cholesterol, when compared to that in its absence. Total emission intensity decay profiles of dibucaine yielded two lifetime components of approximately 1 ns and approximately 2.8--3.1 ns with mean relative contributions of approximately 25 and approximately 75%, respectively. The mean lifetime in vesicles was 20--30% smaller than in the aqueous medium and showed a moderate variation with cholesterol content. Fluorescence measurements at two different temperatures in DMPC SUVs, one at 33 degrees C, above the phase transition temperature and another at 25 degrees C, around the main phase transition, indicated two different mode of dibucaine localization. At 25 degrees C dibucaine partitioned differentially in presence and absence of cholesterol. However, at 33 degrees C the apparent partition coefficients remained unaltered indicating differences in the microenvironment of dibucaine in presence and absence of cholesterol in the phospholipid membranes.
Topics: Anesthetics, Local; Anisotropy; Cholesterol; Dibucaine; Dimyristoylphosphatidylcholine; Drug Interactions; Lipid Bilayers; Phosphatidylcholines; Phospholipids; Spectrometry, Fluorescence; Temperature
PubMed: 11248213
DOI: 10.1016/s0005-2736(01)00268-1 -
Biophysical Journal Mar 2001We investigated the voltage dependence of membrane capacitance of pituitary nerve terminals in the whole-terminal patch-clamp configuration using a lock-in amplifier....
We investigated the voltage dependence of membrane capacitance of pituitary nerve terminals in the whole-terminal patch-clamp configuration using a lock-in amplifier. Under conditions where secretion was abolished and voltage-gated channels were blocked or completely inactivated, changes in membrane potential still produced capacitance changes. In terminals with significant sodium currents, the membrane capacitance showed a bell-shaped dependence on membrane potential with a peak at approximately -40 mV as expected for sodium channel gating currents. The voltage-dependent part of the capacitance showed a strong correlation with the amplitude of voltage-gated Na+ currents and was markedly reduced by dibucaine, which blocks sodium channel current and gating charge movement. The frequency dependence of the voltage-dependent capacitance was consistent with sodium channel kinetics. This is the first demonstration of sodium channel gating currents in single pituitary nerve terminals. The gating currents lead to a voltage- and frequency-dependent capacitance, which can be well resolved by measurements with a lock-in amplifier. The properties of the gating currents are in excellent agreement with the properties of ionic Na+ currents of pituitary nerve terminals.
Topics: Animals; Kinetics; Membrane Potentials; Nerve Endings; Patch-Clamp Techniques; Pituitary Gland, Posterior; Rats; Rats, Sprague-Dawley; Sodium Channels
PubMed: 11222286
DOI: 10.1016/S0006-3495(01)76098-5 -
Anaesthesia Jun 2000We studied 100 men who were scheduled for urological surgery (Group 1) and another 50 men for orthopaedic surgery (Group 2). We attempted to anaesthetise both sides of... (Comparative Study)
Comparative Study
We studied 100 men who were scheduled for urological surgery (Group 1) and another 50 men for orthopaedic surgery (Group 2). We attempted to anaesthetise both sides of the lower body in Group 1 and to anaesthetise one leg in Group 2 by injecting 0.3% hyperbaric dibucaine intrathecally. The presence or absence of the cremasteric reflex and loss of sensation to pinprick higher than the first lumbar dermatome were examined by two researchers who were blind to each other's results. In Group 1, both the reflex and the pinprick sensation were always absent bilaterally 5 min after intrathecal injection. In Group 2, in 23 of 50 patients the reflex had become absent bilaterally; in all these patients, bilateral sensory loss was detected. In the remaining 27 patients, both the reflex and the pinprick sensation were absent on the operation side, whereas both were present on the nonoperation side. Sensitivity, specificity and positive or negative predictive value for the cremasteric reflex were all 100%. Disappearance of the cremasteric reflex is a simple objective indicator of spinal anaesthesia at the first lumbar dermatome. This test may be useful in patients who cannot give reliable answers to conventional tests, such as the pinprick test.
Topics: Adult; Aged; Aged, 80 and over; Anesthesia, Spinal; Anesthetics, Local; Dibucaine; Humans; Male; Middle Aged; Monitoring, Intraoperative; Muscle, Skeletal; Orthopedic Procedures; Reflex; Urologic Surgical Procedures, Male
PubMed: 10866724
DOI: 10.1046/j.1365-2044.2000.01424.x -
International Journal of Hyperthermia :... 2000Local anaesthetics, in addition to anaesthesia, induce the synthesis of heat shock proteins (HSPs), sensitize cells to hyperthermia, and increase the aggregation of...
Local anaesthetics, in addition to anaesthesia, induce the synthesis of heat shock proteins (HSPs), sensitize cells to hyperthermia, and increase the aggregation of nuclear proteins during heat shock. Anaesthetics are membrane active agents, and anaesthesia appears to be due to altered ion channel activity; however, the direct effect of heat shock is protein denaturation. These observations suggest that local anaesthetics may sensitize cells to hyperthermia by interacting with and destabilizing membrane proteins such that protein denaturation is increased. It is shown, using differential scanning calorimetry (DSC), that the local anaesthetics procaine, lidocaine, tetracaine and dibucaine destabilize the transmembrane domains of the Ca2+ -ATPase of sarcoplasmic reticulum and the band III anion transporter of red blood cells. The transmembrane domain of the Ca2+ -ATPase has a transition temperature (Tm) of denaturation of 61 degrees C which is decreased, for example, to 53 degrees C by 15 mM lidocaine. The degree of destabilization (deltaTm) by each anaesthetic is proportional to the lipid to water partition coefficient, and the increased sensitization by anaesthetics with larger partition coefficients and at higher pH suggests that the uncharged forms of the anaesthetics are responsible for destabilization. A Hill analysis of deltaTm for the Ca2+ -ATPase as a function of the concentration of anaesthetic in water gives dissociation constants (Kd) on the order of 10(-4) M, if binding occurs directly from the aqueous phase. This demonstrates moderate affinity binding. However, dissociation constants of 1-3 M are obtained, if binding occurs through the lipid phase, which demonstrates low affinity binding. Thus, the interaction of local anaesthetics with the Ca2+ -ATPase may be moderately specific or non-specific depending on the mechanism of interaction. The observation that local anaesthetics also destabilize the transmembrane domain of the band III protein of erythrocytes suggests that destabilization of transmembrane proteins is a general property of anaesthetics, which is at least in part a mechanism of sensitization to hyperthermia.
Topics: Anesthesia, Local; Anesthetics, Local; Animals; Anion Exchange Protein 1, Erythrocyte; Calcium-Transporting ATPases; Dibucaine; Erythrocytes; Fever; Heat-Shock Proteins; Lidocaine; Procaine; Rabbits; Tetracaine
PubMed: 10669313
DOI: 10.1080/026567300285385 -
Anesthesiology Dec 1999An elevation of the intracellular calcium level, which is mediated by N-methyl-D-aspartate receptors and L-type Ca2+ channels both, activates the mitogen-activated...
BACKGROUND
An elevation of the intracellular calcium level, which is mediated by N-methyl-D-aspartate receptors and L-type Ca2+ channels both, activates the mitogen-activated protein (MAP) kinase signaling pathway involved in synaptic modification. It has recently been suggested that MAP kinase plays a role in coupling the synaptic excitation to gene expression in the nucleus of postsynaptic neurons. Because the effects of local anesthetics on cellular signal transduction in neuronal cells are not well-known, the authors investigated whether they affect the MAP kinase signaling pathway using PC12 cells.
METHODS
The cells were stimulated with either 50 mM KCl or 1 microM ionomycin, and activated MAP kinase was thus immunoprecipitated. The immunocomplexes were then subjected to an Elk1 phosphorylation assay. Both the phosphorylation of MAP kinase and the induction of c-Fos were detected by immunoblotting.
RESULTS
Pretreatment of the cells with 1 mM (ethylenedioxy)-diethyl-enedinitrilotetraacetic acid or 5 micron nifedipine blocked the MAP kinase activation induced by 50 mM KCl, whereas pretreatment with 2 microM omega-conotoxin GIVA did not. The expression of c-Fos induced by potassium chloride was also suppressed by dibucaine, tetracaine (concentrations that inhibited 50% of the activity of positive control [IC50s] were 16.2+/-0.2 and 73.2+/-0.7 microM, respectively), and PD 98059, a mitogen-activated/extracellular receptor-regulated kinase inhibitor. Higher concentrations of dibucaine and tetracaine were needed to suppress the activation of MAP kinase induced by ionomycin (the IC50 values of dibucaine and tetracaine were 62.5+/-2.2 and 330.5+/-32.8 microM, respectively) compared with potassium chloride (the IC50 values of dibucaine and tetracaine were 17.7+/-1.0 and 70.2+/-1.2 microM, respectively). Although probable targets of these local anesthetics might be L-type Ca2+ channels or components between Ca2+ and Ras in MAP kinase pathway, the possibility that they directly affect MAP kinase still remains.
CONCLUSIONS
Dibucaine and tetracaine at clinical concentrations were found to inhibit the activation of MAP kinase and the expression of c-Fos mediated by L-type Ca2+ channels in PC12 cells. The suppression of MAP kinase pathway may thus be a potential target site for the actions of dibucaine and tetracaine, including the modification of the synaptic functions.
Topics: Anesthetics, Local; Animals; Blotting, Western; Calcium Channels, L-Type; Dibucaine; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Ionomycin; Mitogen-Activated Protein Kinases; PC12 Cells; Phosphorylation; Potassium Chloride; Proto-Oncogene Proteins c-fos; Rats; Tetracaine
PubMed: 10598624
DOI: 10.1097/00000542-199912000-00034 -
Kidney International Oct 1999Oxalate, a common constituent of kidney stones, is cytotoxic for renal epithelial cells. Although the exact mechanism of oxalate-induced cell death remains unclear,...
BACKGROUND
Oxalate, a common constituent of kidney stones, is cytotoxic for renal epithelial cells. Although the exact mechanism of oxalate-induced cell death remains unclear, studies in various cell types, including renal epithelial cells, have implicated phospholipase A2 (PLA2) as a prominent mediator of cellular injury. Thus, these studies examined the role of PLA2 in the cytotoxic effects of oxalate.
METHODS
The release of [3H]-arachidonic acid (AA) or [3H]-oleic acid (OA) from prelabeled Madin-Darby canine kidney (MDCK) cells was measured as an index for PLA2 activity. The cell viability was assessed by the exclusion of ethidium homodimer-1.
RESULTS
Oxalate exposure (175 to 550 microM free) increased the release of [3H]-AA in MDCK cells but had no effect on the release of [3H]-OA. Oxalate-induced [3H]-AA release was abolished by arachidonyl trifluoromethyl ketone (AACOCF3), a selective inhibitor of cytosolic PLA2 (cPLA2), but was not affected by selective inhibitors of secretory PLA2 and calcium-independent PLA2. The [3H]-AA release could be demonstrated within 15 minutes after exposure to oxalate, which is considerably earlier than the observed changes in cell viability. Furthermore, AACOCF3 significantly reduced oxalate toxicity in MDCK cells.
CONCLUSIONS
Oxalate increases AA release from MDCK cells by a process involving cPLA2. In addition, based on the evidence obtained using a selective inhibitor of this isoform, it would appear that the activity of this enzyme is responsible, at least in part, for the cytotoxic effects of oxalate. The finding that oxalate can trigger a known lipid-signaling pathway may provide new insight into the initial events in the pathogenesis of nephrolithiasis.
Topics: Anesthetics, Local; Animals; Arachidonic Acid; Arachidonic Acids; Biological Transport; Cell Line; Cyclohexanones; Dibucaine; Diglycerides; Dogs; Enzyme Inhibitors; Epithelial Cells; Free Radicals; Kidney Tubules, Distal; Oleic Acid; Oxalates; Phospholipases A; Phospholipases A2; Protease Inhibitors; Quinacrine; Tritium
PubMed: 10504495
DOI: 10.1046/j.1523-1755.1999.00683.x