共查询到20条相似文献,搜索用时 15 毫秒
1.
Human medulloblastoma gangliosides 总被引:2,自引:0,他引:2
To establish a model system for the study of ganglioside metabolismof the human brain tumor, medulloblastoma, we have chemicallycharacterized the gangliosides of the Daoy cell line. Thesecells contain a high concentration of gangliosides (143 ±13 nmol LBSA/108 cells). The major species have been structurallyconfirmed to be GM2 (65.9%), GM3 (13.0%), and GDla (10.3%).Isolation of individual gangliosides homogeneous in both carbohydrateand ceramide moieties by reversed-phase HPLC and analysis bynegative-ion fast atom bombardment collisionally activated dissociationtandem mass spectrometry have allowed us to unequivocally characterizeceramide structures. In the case of GM2, 10 major ceramide subspecieswere identified: d18:1-hC16:0, d18:1-C16:0, d18:0-C16:0, d18:1-C18:0,d18:1-C20:0, d18:1-C22:0, d18:2-C24:1, d18: 1-C23:1, d18:1-C24:1,and d18:1-C24:0. Taken together with previous studies, thesefindings in human medullo-blastoma cells support the view thathigh expression and marked heterogeneity of ceramide structureare general characteristics of tumor gangliosides, moleculeswhich are shed by the tumor cells and which are biologicallyactive in vivo. medulloblastoma gangliosides ceramide structure HPLC mass spectrometry 相似文献
2.
D. Cerretani L. Micheli A.I. Fiaschi G. Giorgi 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1996,678(2):365
A simple high-performance liquid chromatographic (HPLC) method for the determination of flufenamic acid in rat plasma is described. After liquid-liquid extraction, the drug is separated by HPLC on a 5-μm octadecylsilica column (Nucleosil C18) with ultraviolet detection at 280 nm. Linear calibration graphs for flufenamic acid were constructed from 0.5 to 15 μg/ml. The method has been applied to a pharmacokinetic study in animals. 相似文献
3.
Yamaguchi Isomaro; Nakazawa Hideyuki; Nakagawa Ryusuke; Suzuki Yoshihito; Kurogochi Shin; Murofushi Noboru; Takahashi Nobutaka; Weiler Elmar W. 《Plant & cell physiology》1990,31(8):1063-1069
Radioimmunoassays and enzyme-linked immunosorbent assays formethyl esters of gibberellins A1, A3, A4, and A7 were establishedusing an antiserum specific for GA1-Me. The antiserum was characterizedby high titer and specificity for such C19-GAs with 3ß-hydroxylgroup as GA1, GA3, GA4 and GA7. Combination of this antiserumand HPLC enabled us to identify and quantify GA, and GA4 fromthe pollen of Zea mays with a high degree of reliability. Similarly,identification and quantification of GA9 and GA20 were alsomade possible by use of an antiserum specific for GA20-Me. Combineduse of immunoassays and GC/MS enabled us to identify nine GAsfrom the pollen and four from the anthers of Zea mays. The identificationof non-13-hydroxylated GAs, such as GA4 and GA9, in additionto 13-hydroxylated GAs from the pollen and the anthers suggeststhat the early-non-hydroxylation pathway, as well as the early-13-hydrox-ylationpathway, operates in the male reproductive organs of Zea mays,and that the organ-specific biosynthesis and/or localizationof GAs in Zea mays is similar to that in Oryza saliva. (Received May 7, 1990; Accepted August 20, 1990) 相似文献
4.
《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1999,735(1):103-109
A rapid and sensitive method using HPLC has been developed for the quantification of nicorandil (SG-75) in human plasma samples for routine bioequivalence studies. The sample preparation needs two liquid–liquid extractions, first with CH3Cl and HClO4 as denaturation reagent and second with addition of ethyl acetate and Na2CO3(aq). Detection wavelength was 256 nm. The obtained correlation coefficient for weighted linear curve in the range from 5.0 to 300 ng/ml was higher than 0.9950. The limit of quantitation (LOQ) was established at 5.0 ng/ml. The HPLC separation was accomplished on Nucleosil Phenyl (5 μm) stainless steel column within 7 min. The mixture of 0.01 M ammonium acetate buffer (pH 6.2) and acetonitrile 10:3 (v/v) was used as the mobile phase. The same separation method was examined on HPLC–MS system. Using this system, the LOQ was established at 1.0 ng/ml and the linearity was obtained in the range from 1.0 to 150 ng/ml. 相似文献
5.
Whi-Gun Chae 《Biotechnology and Bioprocess Engineering》2000,5(3):191-195
Quantitation ofN7-methyldeoxyguanosine (N7-MedG) produced in thein vitro N-methyl-N-introsourea (NMU) action on calf thymus DNA has been achieved by enzymatic degradation, liquid chromatographic separation
and desorption chemical ionization tandem mass spectrometry. In conjunction with the resolving power of HPLC in the separation
of isomers, desorption chemical ionization tandem mass spectrometry has been utilized in determining modified nucleosides
at low levels using a stable-isotope labeled compound as an established by an independent HPLC analysis of methylated calf
thymus DNA. A sensitive and specific methodology for the quantitation ofN7-MedG at the picomole level using HPLC combined with tandem mass spectrometry without radioisotope labeling process is presented.
The potential of the liquid chromatographic tandem mass spectrometric analysis shows the detection ofN7-MedG as a possible marker for human exposure to methylating agentsin vitro. 相似文献
6.
Rood, S. B. and Larsen, K. M. 1988. Gibberellins, amylase, andthe onset of heterosis in maize seedlings.J. exp. Bot.39: 223233. The possible involvement of gibberellins and amylase in heterosisof maize seedlings was investigated in two parental inbreds,CM7 and CM49, and their single cross F1 hybrid, CM7xCM49. Germinationof all three genotypes was complete within 36 h after the onsetof imbibition. By 48 h, heterosis (hybrid vigour) for increasedshoot and root length was consistently observed. The endogenousconcentration of gibberellin A1 (GA1) was measured in 48 h seedlingsby gas chromatography-mass spectrometry with selected ion monitoring(GC-SIM) using [2H2]-GA1 as an internal standard. The GA1 concentrationwas highest in the hybrid (59 ng g1 dry wt.), intermediatein CM49 (9.0 ng g1), and lowest in CM7 (<5.0 ng g1).Amylase activities in all three genotypes were very low at 24h, but increased during the next 24 h, after which time amylaseactivity in the hybrid was significantly higher than that ofeither parental inbred. Inhibitors of gibberellin (GA) biosynthesis,AMO-1618 or CCC, inhibited germination, shoot and root growth,and amylase activity in all three genotypes. Conversely, exogenousgibberellic acid (GA3) increased amylase activity, particularlyin the inbred CM7. Amylase isozymes were separated through polyacrylamidegel electrophoresis and generally similar profiles of starchdegrading enzymes were observed in the three genotypes. SinceGA is known to control a-amylase biosynthesis in some cereals,these results are consistent with the hypothesis that GAs areinvolved in the regulation of heterosis in maize. A higher endogenousGA1 concentration in the hybrid could result in increased amylaseactivity in the hybrid seedlings and consequently, more rapidstarch hydrolysis which fuels heterosis for early growth. Key words: Amylase, germination, gibberellic acid, Gibberellin A1, heterosis, hybrid vigour, Zea mays 相似文献
7.
A Modified Micro-Drop Bioassay Using Dwarf Rice for Detection of Femtomol Quantities of Gibberellins 总被引:1,自引:0,他引:1
The sensitivity of the micro-drop assay with dwarf rice (Oryzasativa L., cv. Tan-ginbozu and cv. Waito-Q to gibberellins (GAs)was increased conspicuously by the use of assay plants thathas been treated with uniconazole (S-3307), an inhibitor ofthe biosynthesis of GAs. The Tan-ginbozu plants treated withS-3307 responded to 10 fmol/plant of GA3 (ca. 3.5 pg/plant)and to 30 fmol/plant of gibberellins A1, A4, A7, A19 and A20.Waito-C plants treated with S-3307 responded to 10 fmol of GA3and to 30 fmol/plant of gibberellins A1, A4 and A7. GibberellinsA9, A19 and A20 had much less of an effect on the treated Waito-Cplants than did gibberellins A1, A3, A4 and A7. Furthermore,treatment with S-3307 counteracted the inhibition of growthof both cultivars by abscisic acid. Thus, the modified micro-dropassay should prove very useful for the detection of minute amountsof GAs in plant extracts. (Received October 3, 1988; Accepted March 29, 1989) 相似文献
8.
Klein Andre; Diaz Sandra; Ferreira Isabel; Lamblin Genevieve; Roussel Philippe; Manzi Adriana E. 《Glycobiology》1997,7(3):421-432
Sialic acids are a family of 9-carbon carboxylated sugars, wheredifferent substitutions of the backbone define over 30 members.Biological roles of these substitutions have been missed untilrecently because of their low abundance and lability to conventionalisolation/purification methods. This new approach characterizessialic acids using electrospray ionization-mass spectrometry(ESI-MS) to monitor the HPLC separation of their DMB (1,2-diamino-4,5-methylenedioxy-benzene)derivatives (quinoxalinones). A combination of retention timesand spectra characteristics allows definition of the type andposition of the various substituents. This approach requiresno previous purification, involving a simple derivatizationreaction followed by direct injection on the microbore HPLCcolumn. A complete spectrum, including molecular ions and CADfragments of a sialic acid qainoxalinone, is obtained by injecting1020 pmol of the compound. Individual quinoxalinonescan be purified by regular RP-HPLC and analyzed by direct-injectionESI-MS or LSIMS. Using this approach, we identified 28 differentsialic acids, including the following new species: Neu5Gc9Lt(BSM), anhydro derivatives of Neu5Ac other than the 4,8-anhydro(horse serum hydrolyzates), KDNS(7)Ac and KDN5(7),9Ac2 (amphibianPleurodeles waltl), four isomers of Neu5Gc8MexAc and three anhydroderivatives of Neu5Gc8MS (glycolipids of the starfish Pisasterbrevirpinus), and Neu5Ac8S (in addition to Neu5Gc8S, in theglycolipids of the sea urchin Lovenia cordiformis). Resultsshow the usefulness of LC-ESIMS to study sialic acid diversity,and identification of small amounts of unexpected sialic acidsor new members of their family. sialic acid purification electrospray ionizationmass spectrometry quinoxalinone 相似文献
9.
Microheterogeneity of the oligosaccharides carried by the recombinant bovine lactoferrin expressed in Mamestra brassicae cells 总被引:1,自引:0,他引:1
Lopez Michel; Coddeville Bernadette; Langridge James; Plancke Yves; Sautiere Pierre; Chaabihi Hassan; Chirat Frederic; Harduin-Lepers Anne; Cerutti Martine; Verbert Andre; Delannoy Philippe 《Glycobiology》1997,7(5):635-651
The development of therapeutic glycoprotein production usingthe baculovirus expression system depends on the ability ofinsect cell lines to reproduce site specific mammalian-likeN-glycans. A combination of 1H-NMR and mass spectrometry techniques(MALD-MS, ES-MS, and CID-MS-MS) allowed us to elucidate theN-linked oligosaccharides microheterogeneity on three differentN-glycosylation sites, Asn233, Asn476, and Asn545, of a baculovirus-expressedrecombinant bovine lactoferrin produced in Mamestra brassicae.Two families of N-glycan structures have been found: first,oligomannosidic glycans (Man95GlcNAC2) and secondly, short truncatedpartially fucosylated glycans (Man32[Fuc01]GlcNAc2).These results indicate that Mamestra brassicae cell line isnot able to synthesize complex N-glycans, even if an 相似文献
10.
Yamaguchi Isomaro; Nakagawa Ryusuke; Kurogochi Shin; Murofushi Noboru; Takahashi Nobutaka; Weiler Elmar W. 《Plant & cell physiology》1987,28(5):815-824
Polyclonal anti-GA5-antiseruin and anti-GA20-antiserum wereprepared by immunizing rabbits with conjugates of N-GA5-ß-alanylBSA and N-GA20-ß-alanyl BSA. By radioimmunoassay usingthese antisera, GA5 and GA20 in developing fruits of Pharbitisnil Chois were analyzed after several purification steps andthe results were compared with those obtained by GC-MS to examinethe reliability of the analysis by radioimmunoassay. The analyticalresults by radioimmunoassay did not agree with those by GC-MSuntil two-step purifications by HPLC, indicating that samplesmust be suitably purified prior to radioimmunoassay to obtainreliable results. (Received November 13, 1986; Accepted April 17, 1987) 相似文献
11.
Mass spectrometry has been used to investigate the transportof CO2 in the freshwater diatom Navicula pelliculosa. The timecourseof CO2 formation in the dark after addition of 100 mmol m3dissolved inorganic carbon (DIC) to cell suspensions showedthat no external carbonic anhydrase (CA) was present in thesecells. Upon illumination, cells pre-incubated at pH 75 with100 mmol m3 DIC, removed almost all free CO2 from themedium at an initial rate of 285 µmol CO2 mg1Chl h1. Equilibrium between HCO3 and CO2 in themedium occurred rapidly upon addition of bovine CA, showingthat CO2 depletion resulted from a selective uptake of CO2 ratherthan an uptake of all inorganic carbon species. However, photosyntheticO2 evolution rate remained constant after CO2 had been depletedfrom the medium indicating that photosynthesis is sustainedprimarily by active HCO3 uptake. Treatment of cells with2-iodoacetamide (83 mol m3) completely inhibited CO2fixation but had little effect on CO2 transport since initialrates of CO2 depletion were about 81% that of untreated cells.Transfer of iodoacetamide-treated cells to the dark caused arapid increase in the CO2 concentration in the medium largelydue to the efflux of the unfixed intracellular DIC pool whichwas found to be about 194 times the concentration of that inthe external medium. These results indicate that Navicula pelliculosaactively takes up molecular CO2 against a concentration gradientby a process distinct from HCO3 transport. Key words: Dissolved inorganic carbon, carbonic anhydrase, bicarbonate transport, CO2 transport, mass spectrometry 相似文献
12.
Flowering (number of flowers) of a short-day duckweed, Lemnapaucicostata 6746, in continuous darkness at 26?C was affectedby a red light pulse in various ways depending on the time ofapplication. A conspicuous inhibition and a slight promotionwere respectively caused by the pulse given at the 7th and 19thhours of the dark period. Of the recently introduced floral parameters (4), a (vegetativegrowth rate) and (flowering ratio) were almost unchanged bythe pulse given at any time. P1 (pre-flower induction period)was extended by one day when the pulse was given at about the7th hour of the dark period. The pulse greatly extended P2 (flowerinduction period) when given at about the 7th hour of the darkperiod. A pulse given earlier or later was increasingly ineffectiveon P2. P4 (flower production period) changed rhythmically (i.e.,was extended or shortened) with the time of the red light pulse,the maximum extension and shortening being induced by the pulsegiven at about the 7th and 19th hours, respectively. Differenttiming mechanisms were suggested as controlling the sensitivitiesto the red light pulse of P1 and P2 or P4. The floral response (number of flowers) vs. the red light pulseapplication time curve was explained in terms of the sum ofthe responses of P2 and P4 to the pulse. Floral parameters P1and P2 were defined more clearly. (Received September 4, 1978; ) 相似文献
13.
Maitotoxin and P2Z/P2X7 purinergic receptor stimulation activate a common cytolytic pore 总被引:5,自引:0,他引:5
Schilling William P.; Wasylyna Tanya; Dubyak George R.; Humphreys Benjamin D.; Sinkins William G. 《American journal of physiology. Cell physiology》1999,277(4):C766
The effects ofmaitotoxin (MTX) on plasmalemma permeability are similar to thosecaused by stimulation of P2Z/P2X7ionotropic receptors, suggesting that1) MTX directly activatesP2Z/P2X7 receptors or2) MTX andP2Z/P2X7 receptor stimulationactivate a common cytolytic pore. To distinguish between these twopossibilities, the effect of MTX was examined in1) THP-1 monocytic cells before andafter treatment with lipopolysaccharide and interferon-, a maneuverknown to upregulate P2Z/P2X7receptor, 2) wild-type HEK cells andHEK cells stably expressing theP2Z/P2X7 receptor, and3) BW5147.3 lymphoma cells, a cellline that expresses functional P2Z/P2X7 channels that are poorlylinked to pore formation. In control THP-1 monocytes, addition of MTXproduced a biphasic increase in the cytosolic freeCa2+ concentration([Ca2+]i);the initial increase reflects MTX-inducedCa2+ influx, whereas the secondphase correlates in time with the appearance of large pores and theuptake of ethidium. MTX produced comparable increases in[Ca2+]iand ethidium uptake in THP-1 monocytes overexpressing theP2Z/P2X7 receptor. In bothwild-type HEK and HEK cells stably expressing theP2Z/P2X7 receptor, MTX-inducedincreases in[Ca2+]iand ethidium uptake were virtually identical. The response of BW5147.3cells to concentrations of MTX that produced large increases in[Ca2+]ihad no effect on ethidium uptake. In both THP-1 and HEK cells, MTX- andBz-ATP-induced pores activate with similar kinetics and exhibit similarsize exclusion. Last, MTX-induced pore formation, but not channelactivation, is greatly attenuated by reducing the temperature to22°C, a characteristic shared by theP2Z/P2X7-induced pore. Together,the results demonstrate that, although MTX activates channels that aredistinct from those activated byP2Z/P2X7 receptor stimulation, thecytolytic/oncotic pores activated by MTX- and Bz-ATP are indistinguishable. 相似文献
14.
Yang Young-Yell; Yamaguchi Isomaro; Takeno-Wada Kiyotoshi; Suzuki Yoshihito; Murofushi Noboru 《Plant & cell physiology》1995,36(2):221-227
Gibberellin A1, (GA1), GA19, and GA20 in phloem exudates andcotyledons of seedlings of Pharbitis nil cv. Violet, grown underdifferent photoperiodic conditions, were qualitatively and semi-quantitativelyanalyzed by a combination of high performance-liquid chromatography(HPLC) and radioimmunoassays (RIA). The levels of GA19 and GA20were higher in cotyledons from plants grown under dark treatment(DT) conditons of 16 h-light/8 h-dark for 6 days followed by8 h-light/16 h-dark for 3 days than in those grown under continuouslight (CL) for 9 days. This relationship was also observed forthe GAs in phloem exudates, although the levels were much lowerthan in the cotyledons. When GAs were applied to the cotyledons,elongation of the epicotyl was promoted more by GA20 than byGA1 or GA19, especially under the CL treatment. The relativeeffect of GA1 and GA20 on the epicotyl elongation was reversedwhen these GAs were applied to epicotyls pre-treated with prohexadione,an inhibitor of 2-oxoglutarate-dependent dioxygenases.
3Present address: Frontier Research Program, The Institute ofPhysical and Chemical Research (RIKEN), 2-1 Hirosawa, Wakoshi,Saitama, 351-01 Japan
4Present address: Laboratory of Horticulture, Faculty of Agriculture,Nagoya University, Nagoya, 464-01 Japan 相似文献
15.
Kobayashi Masatomo; Kamiya Yuji; Sakurai Akira; Saka Hitoshi; Takahashi Nobutaka 《Plant & cell physiology》1990,31(2):289-293
Cell-free extracts were prepared from anthers of normal anddwarf rice (Oryza sativa L.), and the metabolism of radioisotope-labeledgibberellins in the extracts was analyzed by HPLC and gas chromatography-massspectrometry (GC/MS). GA12 was converted to GA15 and GA34 inthe extracts. GA20 was converted to GA1, GA8 and GA29, but GA9was converted only to GA34. The extracts of the dwarf cultivar,Waito-C (dy mutant), showed the same 3ß-hydroxylationactivity as did those of the normal cultivar, Nihonbare, indicatingthat the dy gene is not expressed in the anthers. These resultssuggest that the regulation of the biosynthesis of gibberellinsin rice is organ-specific. (Received November 9, 1989; Accepted January 10, 1990) 相似文献
16.
Relative optical cross sections for flash-induced O2- and N2-productionwith water and 1 mM NH2OH as electron donors to PSII, respectively,as well as that for PSI-mediated O2-uptake have been measuredin tobacco chloroplasts by mass spectrometry. In the wild typetobacco these three reactions are driven by three populationsof photosynthetic units different with respect to the antennasize. The antenna size of O2-evolving units is twice as largecompared to the N2-evolving one, but both of these have thesame spectral characteristics in the far red region. In contrastto the wild type, the antenna sizes of O2- and N2-evolving unitsin the chlorophyll b-deficient tobacco mutant Su/su var. Aureaare the same as the sizes of the N2-evolying units in the wildtype chloroplasts. Taking into account the data of Thielen andVan Gorkom [Biochim. Biophys. Acta (1981) 635:111] on a strongdifference in the relative amounts of PSIIß and PSIIßin the mutant compared with the wild type (the ratio about 1: 3 and 3 : 1 , respectively) it is concluded that mainly thestroma-exposed PSIIß units are competent to N2-evolution. (Received November 11, 1993; Accepted April 25, 1994) 相似文献
17.
The kinetic properties of phosphoenolpyruvate (PEP) carboxylasehave been studied among several Flaveria species: the C3 speciesF. cronquistii, the C3C4 species F. pubescens and F.linearis, and the C4 species F. trinervia. At either pH 7 or8, the maximum activities (in µmol.mg Chl1.h1)for F. pubescens and linearis (187513) were intermediateto those of the C3 species (1219) and the C4 species(2,1822,627). The response curves of velocity versusPEP concentration were hyperbolic for the C3 and C3C4species at either pH 7 or 8 while they were sigmoidal for theC4 species at pH 7 and hyperbolic at pH 8. The Km values forPEP determined from reciprocal plots were lowest in the C3 species,and of intermediate value in the C3C4 species comparedto the K' values of the C4 species determined from Hill plotsat either pH 7 or 8. Glucose-6-phosphate (G6P) decreased theKm values for PEP at both pH 7 and 8 in the C3 and C3C4species. In the C4 species, G6P decreased the K' values at pH8 but increased the K' values at pH 7. In all cases, G6P hadits effect by influencing the activity at limiting PEP concentrationswith little or no effect on the maximum activity. At pH 8 andlimiting concentrations of PEP the degree of stimulation ofthe activity by G6P was greatest in the C4 species, intermediatein F. linearis, a C3C4 species, and lowest in the C3species. In several respects, the PEP carboxylases of the C3C4Flaveria species have properties intermediate to those of theC3 and C4 species. (Received April 30, 1983; Accepted August 22, 1983) 相似文献
18.
Self-incompatibility is a mechanism developed by many plantsto prevent inbreeding. The products of the selfincompatibility(S)-locus in the styles of solanaceous plants are a series ofglycoproteins with ribonuclease activity. In this study, wereport on the N-glycans from the stylar selfincompatibilityS3- and S6-ribonucleases of Nicotiana alata, which were enzymicallyreleased and fractionated by high-pH anion-exchange HPLC. Atotal of 14 N-glycans were identified and characterized by acombination of electrospray-ionization mass-spectrometry, 1H-NMRspectroscopy, chemical degradation, and methylation analyses.This pattern of N-glycosylation is much more complex than thatpreviously found on the N.alata S1- and S2-RNases each of whichcontained only four N-glycans. N-glycan Nicotiana alata ribonuclease selfincompatibility 相似文献
19.
Khoo Kay-Hooi; Dell Anne; Morris Howard R.; Breman Patrick J.; Chatterjee Delphi 《Glycobiology》1995,5(1):117-127
Based on chemical analysis, we have previously concluded thatthe biologically important lipoarabinomannan (LAM) and lipomannan(LM) from Mycobacterium are multiglycosylated forms of the phosphatidylinositolmannosides (PIMs), the characteristic cell envelope mannophosphoinositidesof mycobacteria Using definitive analytical techniques, we havenow re-examined the reported multiacylated nature of PIMs inorder to gain a better insight into their possible roles asbiosynthethic precursors of LM and LAM. High-sensitivity fastatom bombardment-mass spectrometry analyses of the perdeuteroacetyland permethyl derivatives of PIMs from Mycobacterium tuberculosisand Mycobacterium leprae enabled us to define the exact fattyacyl compositions of the multiacylated, heterogeneous PIM families,notably the dimannoside (PIM2) and the hexamannoside (PIM6).Specifically, in conjunction with other chemical and gas chromatography-massspectrometry (GCMS) analyses, the additional C16 fatty acylsubstituent on PIM2 and its lyso form were defined as attachedto the C6 position of mannose. We also present evidence fortriacylated mannophosphoinositide as a common lipid anchor forboth LM and LAM, and further postulate that acylation of PIM2may constitute a key regulatory step in their biosynthesis. FAB-MS lipoarabinomannan lipomannan Mycobacterium tuberculosis phosphatidylinositol mannosides 相似文献
20.
Faria RX Defarias FP Alves LA 《American journal of physiology. Cell physiology》2005,288(2):C260-C271
Stimulation of the P2X7 receptor by ATP induces cell membrane depolarization, increase in intracellular Ca2+ concentration, and, in most cases, permeabilization of the cell membrane to molecules up to 900 Da. After the activation of P2X7, at least two phenomena occur: the opening of low-conductance (8 pS) cationic channels and pore formation. At least two conflicting hypotheses have been postulated to reconcile these findings: 1) the P2X7 pore is formed as a result of gradual permeability increase (dilation) of cationic channels, and 2) the P2X7 pore represents a distinct channel, possibly activated by a second messenger and not directly by extracellular nucleotides. In this study, we investigated whether second messengers are necessary to open the pore associated with the P2X7 receptor in cells that expressed the pore activity by using the patch-clamp technique in whole cell and cell-attached configurations in conjunction with fluorescent imaging. In peritoneal macrophages and 2BH4 cells, we detected permeabilization and single-channel currents in the cell-attached configuration when ATP was applied outside the membrane patch in a condition in which oxidized ATP and Lucifer yellow were maintained within the pipette. Our data support Ca2+ as a second messenger associated with pore formation because the permeabilization depended on the presence of intracellular Ca2+ and was blocked by BAPTA-AM. In addition, MAPK inhibitors (SB-203580 and PD-98059) blocked the permeabilization and single-channel currents in these cells. Together our data indicate that the P2X7 pore depends on second messengers such as Ca2+ and MAP kinases. electrophysiology; pore formation 相似文献