Adenosine, a cytoprotective autocoid: effects of adenosine on neutrophil plasma membrane viscosity and chemoattractant receptor display

At inflammatory sites neutrophils are stimulated to produce a variety of toxic agents, yet rarely harm the endothelium across which they migrate. We have recently found that endothelium releases adenosine which, acting via receptors on the surface of human neutrophils, inhibits generation of toxic metabolites by stimulated neutrophils but, paradoxically, promotes chemotaxis. Agents which diminish plasma membrane viscosity affect neutrophil function similarly, possibly by modulating chemoattractant receptor number or affinity. We therefore determined whether adenosine receptor agonists modulate neutrophil function by decreasing membrane viscosity and/or chaning the affinity of chemoattractant (N-fMet-Leu-Phe, FMLP) receptors. Surprisingly, 5′-(N-ethylcar☐amido)adenosine (NECA, 10 μM), the most potent agonist at neutrophil adenosine receptors, increased plasma membrane viscosity, as measured by fluorescence anisotropy of the plasma membrane specific probe 1-(4-trimethylaminophenyl)-6-diphenyl-1,3,5-hexatriene (TMA-DPH), in unstimulated neutrophils from a mean microviscosity of 1.67 ± 0.02 (S.E.) to 1.80 ± 0.02 (p < 0.001) while inosine (10 μM), a poor adenosine receptor agonist, had no effect (1.73 ± 0.04, p =n.s. vs. control, p < 0.01 vs. NECA). Adenosine receptor agonists increased plasma membrane viscosity in neutrophils with the same order of potency previously seen for inhibition of superoxide anion generation and enhancement of chemotaxis (NECA > adenosine = N⁶-phenylisopropyladenosine). The adenosine receptor antagonist 8-(p-sulfophenyl)theophylline reversed the effect of NECA on plasma membrane viscosity. Unlike other agents which modulate plasma membrane viscosity, NECA (10 μM) did not significantly change the number or affinity of [³H]FMLP binding sites on neutrophils. In contrast to the hypothesis of Yuli et al. these results indicate that occupancy of adenosine receptors on neutrophils increases plasma membrane viscosity without affecting chemoattractant receptor display.     

Alterations in cardiac membrane Ca2+ transport during oxidative stress

Although cardiac dysfunction due to ischemia-reperfusion injury is considered to involve oxygen free radicals, the exact manner by which this oxidative stress affects the myocardium is not clear. As the occurrence of intracellular Ca²⁺ overload has been shown to play a critical role in the genesis of cellular damage due to ischemia-reperfusion, this study was undertaken to examine whether oxygen free radicals are involved in altering the sarcolemmal Ca²⁺-transport activities due to reperfusion injury. When isolated rat hearts were made globally ischemic for 30 min and then reperfused for 5 min, the Ca²⁺ -pump and Na⁺-Ca²⁺ exchange activities were depressed in the purified sarcolemmal fraction; these alterations were prevented when a free radical scavenger enzymes (superoxide dismutase plus catalase) were added to the reperfusion medium. Both the Ca²⁺- pump and Na⁺- Ca²⁺ exchange activities in control heart sarcolemmal preparations were depressed by activated oxygen-generating systems containing xanthine plus xanthine oxidase and H₂O₂; these changes were prevented by the inclusion of superoxide dismutase and catalase in the incubation medium. These results support the view that oxidative stress during ischemia-reperfusion may contribute towards the occurrence of intracellular Ca²⁺ overload and subsequent cell damage by depressing the sarcolemmal mechanisms governing the efflux of Ca²⁺ from the cardiac cell.     

Increase in hepatic microsomal lipid peroxidation mediated by α-adrenergic system under cold stress and noradrenaline treatment

A membrane protein recognized by monoclonal antibody SQM1 was identified in human squamous carcinomas, including those originating in the head and neck (SqCHN), lung and cervix. Cell lines derived from SqCHN of previously untreated patients expressed high amounts of this protein. In contrast, many cell lines established from SqCHN of patients previously treated with chemotherapy and/or radiation showed diminished amounts of this SQM1 protein. The expression of SQM1 antigen was determined in several SgCHN cell lines made resistant by exposure to methotrexate (MTX) in vitro. The parent cell lines all exhibited strong binding to SQM1 antibody. The MTX-resistant sublines showed much lower membrane binding of SQM1. The lowest SQM1 reactivity was found in cell lines with high resistance to MTX and with diminished rate of MTX transport. Some highly MTX-resistant cell lines which had high levels of dihydrofolate reductase, but which retained a high rate of MTX transport, also retained high levels of SQM1 binding. Reduced SQM1 protein was also found in SgCHN cells which developed resistance to the alkylating drug cis-platinum (CDDP) and which showed reduced membrane transport of CDDP. Cell growth kinetics and non-specific antigenic shifts were not responsible for the differences in SQM1 binding between the parent cell lines and their drug-resistant sublines. The finding of a novel protein which is reduced in cells resistant to MTX and CDDP could contribute to our understanding of the basic mechanisms of drug resistance. By detecting SQM1 protein in clinical specimens, it may be possible to monitor the development of drug resistance in tumors.Abbreviations SqCHN Squamous Carcinoma of the Head and Neck - MTX Methotrexate - CDDP Cis-Platinum - DHFR Dihydrofolate Reductase     

Fatty acid-binding to erythrocyte ghost membranes and transmembrane movement

Summary Palmitate binding to human erythrocyte ghost membranes has been investigated with ghost preparations suspended in 0.2% albumin solutions. Free unbound palmitate in the extracellular water phase was measured in equilibrium studies using albumin-filled acid loaded ghosts as small semipermeable bags. The apparent dissociation constant of binding to the membrane is 13.5 nM and the binding capacity 19 nmoles per 7.2 × 109 cells.The 0°C exchange efflux kinetics of palmitate from albumin-filled ghosts is described by a model, which provides estimates of the rate constant of membrane transfer, k₃ = 0.024 s^–1, independent of the molar ratio of palmitate to albumin () and of a mean dissociation rate constant of the palmitate-albumin complex, k₁ = 0.0015 s^–1 at 0.2, allowing for a heterogeneity of the palmitate binding to albumin.The values of a third kinetically determined dependent model constant, Q, the ratio of palmitate bound to the membrane inner surface to palmitate on intracellular albumin, are not different from the Q values obtained by equilibrium experiments.The temperature dependences of k₁ and k₃ in the interval 0°C to 15°C give activation energies of 96 and 103 kJ/mole, respectively. The 0°C exchange efflux increases about 2 fold in response to a rise of pH from 6 to 9. The results suggest a carrier mediated palmitate flux at low with a V_max about 2 pmoles min^–1 cm^–2 at 0°C pH 7.3.     

Impact of salt adaptation on esterified fatty acids and cytochrome oxidase in plasma and thylakoid membranes from the cyanobacterium Anacystis nidulans

During adaptation of photoautotrophically growing fresh water cyanobacterium Anacystis nidulans to high salinity the cells showed a pronounced increase of proton-sodium antiporter activity, and of cytochrome c oxidase in isolated and purified plasma membrane. At the same time the concentrations of plasma membrane-bound EDTA-resistant copper and iron (determined by inductively coupled plasma atomic emission spectrometry) rose proportionately, accompanied by an increase in whole cell respiration. In plasma membranes from salt adapted cells lipid/protein ratios were markedly higher than in control cells, levels of esterified saturated and long-chain fatty acids being significantly higher than the respective levels of unsaturated and short-chain fatty acids which explains the higher lipid-phase transition temperatures derived from Arrhenius plots. Immunoblotting of the membrane proteins with antisera raised against the cytochrome c oxidases from Paracoccus denitrificans and A. nidulans gave two cross-reacting bands with apparent molecular weights around 50000 and 30000 (subunits I and II, respectively) which were more pronounced in plasma membranes from salt adapted cells when compared to control cells. The protein pattern of plasma membranes from salt adapted cells also showed the appearance of bands at apparent molecular weights of 44000–48000 and 54000–56000 which might stem from the proton/sodium-antiporter in this membrane.Abbreviations CM cytoplasmic or plasma membrane - ICM intracytoplasmic or thylakoid membrane - cyt cytochrome - DCCD N,N-dicyclohexylcarbodiimide - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonate - ICP-AES inductively coupled plasma atomic emission spectrometry - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - EPR electron paramagnetic resonance spectrometry     

Analysis and separation of synaptosomal membrane proteins

Synaptosomal membrane proteins solubilized with 8% CHAPS-8 M urea were analyzed with twodimensional electrophoresis (2DE). The membrane proteins were resolved up to 250 spots on a 2DE map, ranging in isoelectric points (pI) from 3.5 to 10.0 and molecular weights (MW) from 10 kDa to 200 kDa. Comparison of the mapped proteins of synaptosomal membranes with those of myelin and mitochondorial membranes revealed that synaptosomal membrane proteins were characteristic in the area of pI from 4.0 to 7.5 and MW from 20 kDa to 130 kDa, and that at least 30 spots were synaptosomal membrane-specific proteins. Most of these 30 proteins have not been previously described, named, and characterized Serial numbers (from SY1 to SY30) were assigned to the proteins on the map in order to investigate them systematically. A preliminary attempt to separate synaptosomal membrane proteins was carried out using a reversed-phase HPLC system. Several proteins could either be isolated or enriched. SY10 (pI 4.6; MW 56 kDa) was one of these proteins, and was of particular interest for its unusual behavior on the reversed-phase column, and for its binding to an immobilized protein A-gel.     

Influence of isolation media on synaptosomal properties: Intracellular pH,pCa, and Ca2+ uptake

Preparations of synaptosomes isolated in sucrose or in Na⁺-rich media were compared with respect to internal pH (pH₁), internal Ca²⁺ concentration ([Ca²⁺]_i), membrane potential and⁴⁵Ca²⁺ uptake due to K⁺ depolarization and Na⁺/Ca²⁺ exchange. We found that synaptosomes isolated in sucrose media have a pH_i of 6.77±0.04 and a [Ca²⁺]_i of about 260 nM, whereas synaptosomes isolated in Na⁺-rich ionic media have a pH_i of 6.96±0.07 and a [Ca²⁺]_i of 463 nM, but both types of preparations have similar membrane potentials of about –50 mV when placed in choline media. The sucrose preparation takes up Ca²⁺ only by voltage sensitive calcium channels (VSCC'S) when K⁺-depolarized, while the Na⁺-rich synaptosomes take up⁴⁵Ca²⁺ both by VSCC'S and by Na⁺/Ca²⁺ exchange. The amiloride derivative 2, 4 dimethylbenzamil (DMB), at 30 M, inhibits both mechanisms of Ca²⁺ influx, but 5-(N-4-chlorobenzyl)-2, 4 dimethylbenzamil (CBZ-DMB), at 30 M, inhibits the Ca²⁺ uptake by VSCC'S, but not by Na⁺/Ca²⁺ exchange. Thus, DMB and CBZ-DMB permit distinguishing between Ca²⁺ flux through channels and through Na⁺/Ca²⁺ exchange. We point out that the different properties of the two types of synaptosomes studied account for some of the discrepancies in results reported in the literature for studies of Ca²⁺ fluxes and neurotransmitter release by different types of preparations of synaptosomes.Abbreviations used BCECF 2,7-Biscarboxyethyl-5(6)-carboxyfluorescein - BCECF/AM acetoxymethyl ester of BCECF - [Ca²⁺]_i Internal free calcium ion concentration - CBZ-DMB 5-(N-4-chlorobenzyl)-2,4-dimethylbenzamil - DMB 2, 4-dimethylbenzamil - DMSO dimethyl sulfoxide - Indo-1/AM acetoxymethyl ester of Indo-1 - MES 2-|N-Morpholino|ethanesulfonic acid - NMG N-methyl-D-glucamine - pH_i internal pH - TPP⁺ tetraphenylphosphonium - _p plasma membrane potential     

消炎痛对大鼠肝线粒体微粒体^45Ca摄取及膜流动性的影响

我们曾报道消炎痛预处理在大鼠能引起明显的肝保护作用,为了进一步探讨消炎痛肝保护作用的机制,本工作观察了它对大鼠肝线粒体、微粒体钙调节作用及膜流动性的影响。结果表明,经消炎痛整体预处理的大鼠肝线粒体和微粒体的钙摄取及膜流动性均明显增加,但是,将消炎痛直接加入由正常大鼠分离的线粒体或微粒体中,则反而使膜的流动性降低。这些变化可能与消炎痛的肝保护作用有关。     

EMA,an epithelial membrane-associated antigen during early development and morphogenesis ofXenopus laevis

Summary We raised monoclonal antibodies against a membrane fraction ofXenopus neurulae in order to detect tissue-specific cell-surface markers. Here we describe a monoclonal antibody that recognizes an epithelial membrane-associated antigen (EMA) in immunohistological stainings. The tissue-specific and membrane-associated antigen detected in immunohistological stainings could serve as useful marker in epithelium differentiation and membrane organization of the early embryo. In tadpoles and adults EMA was found in specific epithelial tissues derived from different germ layers such as kidney, skin, gut, pancreas, epiphysis and choroid plexus. In the cleaving embryo this antibody stained newly formed membranes between blastomeres from the two-cell stage onwards. Cytoplasmic staining in large oocytes and early embryos was also observed. The possibility that the cytoplasmic signal represents a maternal store of membrane material is discussed.     

植物单盐毒害和离子拮抗机理研究Ⅰ.单盐和混合盐对植物原生质体膜某些特性、超氧物歧化酶和过氧化氢酶活性的影响

单盐(KCl, CaCl_2或MgCl_2)和混合盐(KC_1+CaCl_2或KCl+MgCl_2)对植物原生质体完整率、存活率和膜透性等均有明显影响。K~+、Ca~(2+)或Mg~(2+)等单种阳离子明显降低原生质体膜完整率和存活率而增加其物质渗漏量,其中以单价阳离子K~+的影响为甚。上述单种阳离子还明显降低小麦幼叶超氧物歧化酶(SOD)和过氧化氢酶活性。只有由单价和二价阳离子组成的平衡混合盐才能使原生质体维持较高的完整率、存活率和较正常的膜透性.并能使细胞维持较高的SOD和过氧化氢酶活性。 认为单盐毒害机理可能是首先引起细胞膜发生不正常的膜相变或细胞累积较多的有害氧自由基,引起膜脂发生过氧化或脱酯化而破坏膜结构。在离子平衡混合盐作用下,膜系才能维持正常液晶相,具有较高活性的SOD和过氧化氢酶等生物保护性酶系是离子拮抗作用之原因。