共查询到20条相似文献,搜索用时 15 毫秒
1.
Rosado JA Redondo PC Salido GM Sage SO Pariente JA 《American journal of physiology. Cell physiology》2005,288(1):C214-C221
We recently reported that store-operated Ca2+ entry (SOCE) in nonexcitable cells is likely to be mediated by a reversible interaction between Ca2+ channels in the plasma membrane and the endoplasmic reticulum, a mechanism known as "secretion-like coupling." As for secretion, in this model the actin cytoskeleton plays a key regulatory role. In the present study we have explored the involvement of the secretory proteins synaptosome-associated protein (SNAP-25) and vesicle-associated membrane protein (VAMP) in SOCE in pancreatic acinar cells. Cleavage of SNAP-25 and VAMPs by treatment with botulinum toxin A (BoNT A) and tetanus toxin (TeTx), respectively, effectively inhibited amylase secretion stimulated by the physiological agonist CCK-8. BoNT A significantly reduced Ca2+ entry induced by store depletion using thapsigargin or CCK-8. In addition, treatment with BoNT A once SOCE had been activated reduced Ca2+ influx, indicating that SNAP-25 is needed for both the activation and maintenance of SOCE in pancreatic acinar cells. VAMP-2 and VAMP-3 are expressed in mouse pancreatic acinar cells. Both proteins associate with the cytoskeleton upon Ca2+ store depletion, although only VAMP-2 seems to be sensitive to TeTx. Treatment of pancreatic acinar cells with TeTx reduced the activation of SOCE without affecting its maintenance. These findings support a role for SNAP-25 and VAMP-2 in the activation of SOCE in pancreatic acinar cells and show parallels between this process and secretion in a specialized secretory cell type. synaptosome-associated protein; vesicle-associated membrane protein; pancreatic acinar cells; cytoskeleton; calcium entry 相似文献
2.
Pancreatitis is an inflammatory disease of pancreatic acinar cells whereby intracellular calcium concentration ([Ca2+]i) signaling and enzyme secretion are impaired. Increased oxidative stress has been suggested to mediate the associated cell injury. The present study tested the effects of the oxidant, hydrogen peroxide, on [Ca2+]i signaling in rat pancreatic acinar cells by simultaneously imaging fura-2, to measure [Ca2+]i, and dichlorofluorescein, to measure oxidative stress. Millimolar concentrations of hydrogen peroxide increased cellular oxidative stress and irreversibly increased [Ca2+]i, which was sensitive to antioxidants and removal of external Ca2+, and ultimately led to cell lysis. Responses were also abolished by pretreatment with (sarco)endoplasmic reticulum Ca2+-ATPase inhibitors, unless cells were prestimulated with cholecystokinin to promote mitochondrial Ca2+ uptake. This suggests that hydrogen peroxide promotes Ca2+ release from the endoplasmic reticulum and the mitochondria and that it promotes Ca2+ influx. Lower concentrations of hydrogen peroxide (10–100 µM) increased [Ca2+]i and altered cholecystokinin-evoked [Ca2+]i oscillations with marked heterogeneity, the severity of which was directly related to oxidative stress, suggesting differences in cellular antioxidant capacity. These changes in [Ca2+]i also upregulated the activity of the plasma membrane Ca2+-ATPase in a Ca2+-dependent manner, whereas higher concentrations (0.1–1 mM) inactivated the plasma membrane Ca2+-ATPase. This may be important in facilitating "Ca2+ overload," resulting in cell injury associated with pancreatitis. oxidant stress; pancreatitis; calcium pump 相似文献
3.
Characterization of ATPases Associated with Various Cellular Membranes Isolated from Etiolated Hypocotyls of Vigna radiata (L.) Wilczek 总被引:1,自引:0,他引:1
Cellular membrane fractions, including endoplasmic reticum (ER),Golgi-enriched membrane, plasma membrane and tonoplasts, wereisolated from Vigna radiata seedlings. Each of these membranefractions was associated with specific ATPases which were highlydependent on Mg2+. ATPases of ER, Golgi-enriched membrane andplasma membrane were sensitive to vanadate but the tonoplastATPase was not. ATPases were mostly dependent on Cl1, but aslight stimulation by K+ was observed in the case of ATPasesof Golgi-enriched membrane and plasma membrane. KNO3 inhibitedtonoplast ATPase but stimulated the other ATPases. ER ATPasecan be distinguished from other ATPases by the following characteristics:specific inhibition by KNO2 and Triton X-100, stimulation bylow concentrations of diethylstilbestrol and 4,4'-diisothiocyanostilbene-2,2'-disulfonicacid, and high sensitivity to heat. The ATPases showed typicalMichaelis-Menten kinetics and had Km values of 0.5 to 0.6 ITIMMg2+-ATP for ER, Golgienriched-membrane and tonoplast ATPases,and 2.27 msi Mg2+-ATP for plasma membrane ATPase. ATPases ofGolgi-enriched membranes and plasma membranes had similar properties,but they were still distinguishable by the differences in theirKm values and their responses to Triton X-100. Based on theseresults, it is postulated that each cellular membrane is associatedwith a specific ATPase in cells of V. radiata.
1Contribution No. 3171 from the Institute of Low TemperatureScience. (Received April 22, 1988; Accepted September 28, 1988) 相似文献
4.
ATPase activity in the cell membrane of a salt-stressed cyanobacterium,Nostoc muscorum M-14, was examined cytochemically by three differentstaining protocols. Application of Hulstaert's method resultedin distinct precipitation of the reaction products of ATPaseinside the cell membrane exclusively. No reaction products wereformed when ATP was replaced by GTP or when dicyclohexylcarbodiimideor N-ethylmaleimide was present in the reaction mixture. Bycontrast, low levels were detectable after the reaction in thepresence of ouabain. Bafilomycin did not affect the formationof products. Mayahara's method, which is considered to demonstratethe reaction of Na+,K+-ATPase activity, revealed the presenceof a ouabain-sensitive Na+,K+-ATPase in the cell membrane, whileWachstein-Meisel's method revealed the presence of an ATPaseactivity that was resistant to ouabain. It appears, therefore,that cell membranes of Nostoc muscorum contain both ouabain-sensitiveATPase and ouabain-insensitive ATPase. Comparison of the stainingprofiles of salt-stressed cells with those of control cellssuggested that a high-salt environment activates the ouabain-sensitiveNa+,K+-ATPase, which seems likely to be involved in the effluxof Na+ ions. (Received February 7, 1995; Accepted August 9, 1995) 相似文献
5.
Caloxin: a novel plasma membrane Ca2+ pump inhibitor 总被引:1,自引:0,他引:1
Chaudhary J Walia M Matharu J Escher E Grover AK 《American journal of physiology. Cell physiology》2001,280(4):C1027-C1030
Plasma membrane (PM) Ca2+ pump is aCa2+-Mg2+-ATPase that expels Ca2+from cells to help them maintain low concentrations of cytosolic Ca2+. There are no known extracellularly acting PMCa2+ pump inhibitors, as digoxin and ouabain are forNa+ pump. In analogy with digoxin, we define caloxins asextracellular PM Ca2+ pump inhibitors and describe caloxin2A1. Caloxin 2A1 is a peptide obtained by screening a random peptidephage display library for binding to the second extracellular domain(residues 401-413) sequence of PM Ca2+ pump isoform1b. Caloxin 2A1 inhibits Ca2+-Mg2+-ATPase inhuman erythrocyte leaky ghosts, but it does not affect basalMg2+-ATPase or Na+-K+-ATPase in theghosts or Ca2+-Mg2+-ATPase in the skeletalmuscle sarcoplasmic reticulum. Caloxin 2A1 also inhibitsCa2+-dependent formation of the 140-kDa acid-stableacylphosphate, which is a partial reaction of this enzyme. Consistentwith inhibition of the PM Ca2+ pump in vascularendothelium, caloxin 2A1 produces an endothelium-dependent relaxationthat is reversed byNG-nitro-L-arginine methyl ester.Thus caloxin 2A1 is a novel PM Ca2+ pump inhibitor selectedfor binding to an extracellular domain. 相似文献
6.
Two membrane fractions were obtained from 16%/26% and 34%/40%interfaces following discontinuous sucrose density gradientcentrifugation of a 10,00080,000xg pellet from mung bean(Phaseolus mungo L.) roots. The ATPases in the fractions differedfrom each other in their sensitivity toward various inhibitors,activation with salts, dependence of activity on pH, and Kmfor ATP.Mg2+. Judging from their sensitivity toward inhibitors,the ATPases in the low and high density membranes are consideredmainly of tonoplast and plasma membrane origin, respectively.Both ATPases were activated by gramicidin D and nigericin. ATP-inducedquenching of quinacrine fluorescence in both fractions requiredMg2+ and permeant anions such as Cl and quenching wascollapsed by carbonylcyanide p-trifluoromethoxyphenyl hydrazone.The sensitivities of quenching to the inhibitors were essentiallythe same as those of ATPase activity in the membranes. Thesefindings suggest the involvement of ATPases in H+-pumping acrossa plasma membrane and tonoplast. (Received April 12, 1985; Accepted October 11, 1985) 相似文献
7.
Phototropins (phot1 and phot2) are blue light (BL) receptorsthat mediate responses including phototropism, chloroplast movementand stomatal opening, and increased cytosolic Ca2+. BL absorbedby phototropins activates plasma membrane H+-ATPase in guardcells, resulting in membrane hyperpolarization, and drives K+uptake and stomatal opening. However, it is unclear whetherthe phototropin-mediated Ca2+ increase activates the H+-ATPase.Here, we determined cytosolic Ca2+ concentrations in guard cellprotoplasts (GCPs) from Arabidopsis transformed with aequorin.Cytosolic Ca2+ increased rapidly in response to BL in GCPs fromboth the wild type and phot1 phot2 double mutants, but was mostlysuppressed by an inhibitor of photosynthetic electron flow (DCMU).With depleted external K+, we observed another slower Ca2+ increase,which was phototropin- dependent. Fusicoccin, a H+-ATPase activator,mimicked the effect of BL. The slow Ca2+ increase thus appearsto result from membrane hyperpolarization. The slow Ca2+ increasewas suppressed by external K+ and was restored by blockers ofinward-rectifying K+ channels, CsCl and tetraethylammonium,suggesting the preferential uptake of K+ over Ca2+. Such efficientK+ uptake in response to BL was not found in mesophyll cells.Both the fast and the slow Ca2+ increases were inhibited byCa2+ channel blockers (CoCl2 and LaCl3) and a chelating agent(EGTA). These results indicate that the phototropin-mediatedCa2+ increase was not observed prior to H+-ATPase activationin guard cells and that Ca2+ entered guard cells via Ca2+ channelsthrough photosynthesis and phototropin-mediated membrane hyperpolarization. 相似文献
8.
Ichimiya Masato; Chang Seung H.; Liu Hui; Berezesky Irene K.; Trump Benjamin F.; Amstad Paul A. 《American journal of physiology. Cell physiology》1998,275(3):C832
The mechanism by which Bcl-2 inhibits cell death is unknown. Ithas been suggested that Bcl-2 functions as an antioxidant. BecauseBcl-2 is localized mainly to the membranes of the endoplasmic reticulum(ER) and the mitochondria, which represent the main intracellularstorage sites for Ca2+, wehypothesized that Bcl-2 might protect cells against oxidant injury byaltering intracellular Ca2+homeostasis. To test this hypothesis, we examined the effect of oxidanttreatment on viability in normal rat kidney (NRK) cells and in NRKcells stably transfected with Bcl-2 in the presence or absence ofintracellular Ca2+, and wecompared the effect of Bcl-2 expression on oxidant-induced intracellular Ca2+ mobilizationand on ER and mitochondrial Ca2+pools. NRK cells transfected with Bcl-2 (NRK-Bcl-2) were significantly more resistant toH2O2-inducedcytotoxicity than control cells. EGTA-AM, an intracellularCa2+ chelator, as well as theabsence of Ca2+ in the medium,reducedH2O2-inducedcytotoxicity in both cell lines. Compared with controls, cellsoverexpressing Bcl-2 showed a delayed rise in intracellularCa2+ concentration([Ca2+]i)afterH2O2treatment. After treatment with theCa2+ ionophore ionomycin,Bcl-2-transfected cells showed a much quicker decrease after themaximal rise than control cells, suggesting stronger intracellularCa2+ buffering, whereas treatmentwith thapsigargin, an inhibitor of the ERCa2+-ATPases, transientlyincreased[Ca2+]iin control and in Bcl-2-transfected cells. Estimates of mitochondrial Ca2+ stores using an uncoupler ofoxidative phosphorylation show that NRK-Bcl-2 cells have a highercapacity for mitochondrial Ca2+storage than control cells. In conclusion, Bcl-2 may prevent oxidant-induced cell death, in part, by increasing the capacity ofmitochondria to store Ca2+. 相似文献
9.
Faraday Christopher D.; Spanswick Roger M.; Bisson Mary A. 《Journal of experimental botany》1996,47(4):589-594
Plasma membranes were isolated using the aqueous polymer two-phasepartition method from the algae Chara corallina and Chara longifolia,algae which differ in their ability to grow in saline environments.Enrichment of plasma membrane and depletion of tonoplast relativeto the microsomal fraction was monitored using phosphohydrolaseassays and crossreactions to antibodies raised against higherplant transporters. Antibodies to the vacuolar ATPase and pyrophosphatasecross-reacted with epitopes in the microsomal fraction, butshowed little affinity for the plasma membrane fraction. Pyrophosphataseactivity also declined in the plasma membrane fraction relativeto the microsomal fraction. The V-type H+ -ATPase activity,sensitive to nitrate or bafilomycin, was low in both fractions,though the cross-reaction to the antibody was reduced in theplasma membrane fraction. By contrast, the antibody recognitionof a P-type H+-ATPase amino acid sequence from Arabidopsis didnot occur strongly in the anticipated 90100 kDa range.While there was enhanced recognition of a polypeptide at around140 kDa in the plasma membrane fraction, salt treatment of Charalongifolia resulted in plasma membrane fractions with reducedamounts of this epitope, but no change in vanadate-sensitiveATPase activity, suggesting that it does not represent the onlyP-type ATPase. Microsomal membranes from saltadapted C. longifoliahave higher reactivity with the antibody to the tonoplast ATPase. Key words: Chara, plasma membrane, salt tolerance, ATPase 相似文献
10.
Optical measurement of stimulus-evoked membrane dynamics in single pancreatic acinar cells 总被引:6,自引:0,他引:6
Giovannucci David R.; Yule David I.; Stuenkel Edward L. 《American journal of physiology. Cell physiology》1998,275(3):C732
Stimulation ofpancreatic acinar cells induces the release of digestive enzymes viathe exocytotic fusion of zymogen granules and activates postfusiongranule membrane retrieval and receptor cycling. In the present study,changes in membrane surface area of rat single pancreatic acinar cellswere monitored by cell membrane capacitance(Cm)measurements and by the membrane fluorescent dye FM1-43. When measuredwith the Cmmethod, agonist treatment evoked a graded, transient increase in acinarcell surface area averaging 3.5%. In contrast, a 13% increase insurface area was estimated using FM1-43, corresponding to the fusion of48 zymogen granules at a rate of 0.5 s1. After removal of FM1-43from the surface-accessible membrane, a residual fluorescence signalwas shown by confocal microscopy to be localized in endosome-likestructures and confined to the apical regions of acinar cells. Thedevelopment of an optical method for monitoring the membrane turnoverof single acinar cells, in combination with measurements ofCm changes,reveals coincidence of exocytotic and endocytotic activity in acinarcells after hormonal stimulation. 相似文献
11.
Nakajima Nobuyoshi; Saji Hikaru; Aono Mitsuko; Kondo Noriaki 《Plant & cell physiology》1995,36(5):919-924
cDNA encoding the plasma membrane H+-ATPase of guard cells ofVicia faba L. was isolated. The clone encoded a 105-kDa polypeptide(956 amino acids) that was 7985% identical in terms ofamino acid sequence to other plant H+-ATPases. High levels ofmRNA explain the high H+-ATPase activity of these plasma membranes. (Received December 24, 1994; Accepted April 12, 1995) 相似文献
12.
Xu Chang Hong; Nejidat Ali; Belkin Shimshon; Boussiba Sammy 《Plant & cell physiology》1994,35(5):737-741
Partition in aqueous dextran-polyethylene glycol two-phase systemwas used to isolate the plasma membranes from the alkalophiliccyanobacterium Spirulina platensis. The upper phase containeda colorless membranes obtained in relatively short time, 34h. This fraction had a different protein profile than that ofthe thylakoid fraction obtained in the lower phase. It did notcontain cytochrome c-oxidase activity, but retained characteristicMg2+-ATPase activity that is sensitive to vanadate, stimulatedby K+, and has a pH optimum near 8.5. These data support ourassumption that the upper phase of the gradient consist of theplasma membrane of S. platensis. (Received November 25, 1993; Accepted April 12, 1994) 相似文献
13.
Amlal Hassane; Wang Zhaohui; Soleimani Manoocher 《American journal of physiology. Cell physiology》1997,273(4):C1194
The response ofH+-ATPase to lethal acid stress isunknown. A mutant strain (called NHE2d) was derived from cultured inner medullary collecting duct cells (mIMCD-3 cells) following three cyclesof lethal acid stress. Cells were grown to confluence on coverslips,loaded with2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, andmonitored for intracellular pH(pHi) recovery from an acid load. The rate of Na+-independentpHi recovery from an acid load inmutant cells was approximately fourfold higher than in parent cells(P < 0.001). TheNa+-independentH+ extrusion was ATP dependent and K+ independent and wascompletely inhibited in the presence of diethylstilbestrol, N, N'-dicyclohexylcarbodiimide,or N-ethylmaleimide. Theseresults indicate that theNa+-independentH+ extrusion in cultured medullarycells is mediated via H+-ATPaseand is upregulated in lethal acidosis. Northern hybridization experiments demonstrated that mRNA levels for the 16- and 31-kDa subunits of H+-ATPase remainedunchanged in mutant cells compared with parent cells. We propose thatlethal acid stress results in increased H+-ATPase activity in innermedullary collecting duct cells. Upregulation ofH+-ATPase could play a protectiverole against cell death in severe intracellular acidosis. 相似文献
14.
Partial inhibition of SERCA is responsible for extracellular Ca2+ dependence of AlF-4-induced [Ca2+]i oscillations in rat pancreatic 总被引:1,自引:0,他引:1
Chong SA Hong SY Moon SJ Park JW Hong JH An JM Lee SI Shin DM Seo JT 《American journal of physiology. Cell physiology》2003,285(5):C1142-C1149
AlF4-is known to generate oscillations in intracellular Ca2+ concentration ([Ca2+]i) by activating G proteins in many cell types. However, in rat pancreatic acinar cells, AlF4--evoked [Ca2+]i oscillations were reported to be dependent on extracellular Ca2+, which contrasts with the [Ca2+]i oscillations induced by cholecystokinin (CCK). Therefore, we investigated the mechanisms by which AlF4- generates extracellular Ca2+-dependent [Ca2+]i oscillations in rat pancreatic acinar cells. AlF4--induced [Ca2+]i oscillations were stopped rapidly by the removal of extracellular Ca2+ and were abolished on the addition of 20 mM caffeine and 2 µM thapsigargin, indicating that Ca2+ influx plays a crucial role in maintenance of the oscillations and that an inositol 1,4,5-trisphosphate-sensitive Ca2+ store is also required. The amount of Ca2+ in the intracellular Ca2+ store was decreased as the AlF4--induced [Ca2+]i oscillations continued. Measurement of 45Ca2+ influx into isolated microsomes revealed that AlF4-directly inhibited sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). The activity of plasma membrane Ca2+-ATPase during AlF4- stimulation was not significantly different from that during CCK stimulation. After partial inhibition of SERCA with 1 nM thapsigargin, 20 pM CCK-evoked [Ca2+]i oscillations were dependent on extracellular Ca2+. This study shows that AlF4- induces [Ca2+]i oscillations, probably by inositol 1,4,5-trisphosphate production via G protein activation but that these oscillations are strongly dependent on extracellular Ca2+ as a result of the partial inhibition of SERCA. cholecystokinin; plasma membrane adenosine 5'-triphosphatase; G proteins; caffeine 相似文献
15.
Munc-18-2 regulates exocytosis of H(+)-ATPase in rat inner medullary collecting duct cells 总被引:2,自引:0,他引:2
Nicoletta JA Ross JJ Li G Cheng Q Schwartz J Alexander EA Schwartz JH 《American journal of physiology. Cell physiology》2004,287(5):C1366-C1374
Exocytic insertion of H+-ATPase into the apical membrane of inner medullary collecting duct (IMCD) cells is dependent on a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein target receptor (SNARE) complex. In this study we determined the role of Munc-18 in regulation of IMCD cell exocytosis of H+-ATPase. We compared the effect of acute cell acidification (the stimulus for IMCD exocytosis) on the interaction of syntaxin 1A with Munc-18-2 and the 31-kDa subunit of H+-ATPase. Immunoprecipitation revealed that cell acidification decreased green fluorescent protein (GFP)-syntaxin 1A and Munc-18-2 interaction by 49 ± 7% and increased the interaction between GFP-syntaxin 1A and H+-ATPase by 170 ± 23%. Apical membrane Munc-18-2 decreased by 27.5 ± 4.6% and H+-ATPase increased by 246 ± 22%, whereas GP-135, an apical membrane marker, did not increase. Pretreatment of IMCD cells with a PKC inhibitor (GO-6983) diminished the previously described changes in Munc-18-2-syntaxin 1A interaction and redistribution of H+-ATPase. In a pull-down assay of H+-ATPase by glutathione S-transferase (GST)-syntaxin 1A bound to beads, preincubation of beads with an approximately twofold excess of His-Munc-18-2 decreased H+-ATPase pulled down by 64 ± 16%. IMCD cells that overexpress Munc-18-2 had a reduced rate of proton transport compared with control cells. We conclude that Munc-18-2 must dissociate from the syntaxin 1A protein for the exocytosis of H+-ATPase to occur. This dissociation leads to a conformational change in syntaxin 1A, allowing it to interact with H+-ATPase, synaptosome-associated protein (SNAP)-23, and vesicle-associated membrane protein (VAMP), forming the SNARE complex that leads to the docking and fusion of H+-ATPase vesicles. soluble N-ethylmaleimide-sensitive factor attachment protein target receptor; cell pH; acid secretion 相似文献
16.
Wada Masato; Satoh Shinobu; Kasamo Kunihiro; Fujii Tadashi 《Plant & cell physiology》1989,30(6):923-928
Highly purified plasma membranes were isolated from Heterosigmaakashiwo cells, a marine raphidophycean unicellular biflagellate,by the silica microbead method, and the ATPase activity of themembranes was characterized. The ionic requirements and spectrumof effective inhibitors enable us to identify a novel Na+-activatedATPase in the plasma membrane of this organism. Furthermore,we detected two phosphorylated intermediate forms of ATPases,with molecular weights of 150 kDa and 95 kDa as judged by acidSDS-polyacrylamide gel electrophoresis of extracts of isolatedplasma membrane. The 150 kDa intermediate was phosphorylated in the presenceof both Mg2+ and Na+, while the 95 kDa intermediate was phosphorylatedin the presence of Mg2+ alone. Both were dephosphorylated inthe presence of monovalent cations. These results indicate thatthe former intermediate was a Na+-activated ATPase, similarto Na+,K+-ATPases from animals, and the latter was similar toH+,K+-ATPases from higher plants. The physiological significanceof the two kinds of ATPase in the plasma membrane of marinealgae. (Received March 15, 1989; Accepted June 23, 1989) 相似文献
17.
Salinity Effects on the Activity of Plasma Membrane H+and Ca2+Transporters in Bean Leaf Mesophyll: Masking Role of the Cell Wall 总被引:1,自引:0,他引:1
Net fluxes of H+and Ca2+were measured in the mesophyll tissueof broad bean (Vicia faba L.) leaves and in protoplasts derivedfrom these cells. NaCl at 90 m M enhanced H+extrusion in bothprotoplasts and tissue, but in different ways. Proton extrusionwas inhibited by vanadate, suggesting the involvement of theplasma membrane H+-ATPase in cell responses to salinity. Therewas virtually no effect of NaCl on the net Ca2+flux in protoplasts,while in the tissue a large transient Ca2+efflux followed thesalt treatment. Salt-induced Ca2+efflux was essentially independentof external Ca2+concentrations in the range 0.1 to 10 m M. Also,Ca2+flux responses were saturated above 50 m MNaCl. It is suggested that almost all the measured Ca2+fluxoriginates from Na+/Ca2+and H+/Ca2+ion exchange in the cellwall. This conclusion was supported by the results of modellingcation exchange in the cell wall. Copyright 2000 Annals of BotanyCompany Salinity, membrane transporters, wall ion exchange, proton, calcium, Vicia faba 相似文献
18.
Cell suspension cultures of Corydalis sempervirens have provenideal for the study of fusicoccin action [Schulz et al. (1990)Planta 183: 83] and express the fusicoccin-binding protein aswell as a plasma membrane H+-ATPase which is activated by thefungal toxin. Microsomal vesicles prepared from these cellsaccumulate Ca2+ in the presence of Mg-ATP. The protonophorecar-bonylcyanide m-chlorophenylhydrazone did not inhibit theMg-ATP dependent Ca2+-transport into the vesicles. This processis thus due to the activity of at least one primary active,ATP-driven, Ca2+-pump. The enzyme was characterized in detail.It has a pH optimum of 7.2, an apparent Km of 0.3 mu (ATP),12pm (Ca2+), accepts ATP>ITP GTP>CTP UTP, and is strongly(Ki, app 0.75 µmM) inhibited by erythrosine B but lessso (Ki, app 95 µM) by or-thovanadate. These characteristicsare typical for the plasma membrane Ca2+-ATPase characterizedfrom differentiated tissues [Graf and Weiler (1990) Physiol.Plant. 75: 634]. Fusicoccin activates the erythrosine-sensitiveCa2+-pump by lowering its Km for ATP, when added to living cellsprior to tissue homogenization. Thus, fusicoccin appears toactivate at least two ion-translocating ATPases in one and thesame tissue, suggesting that the toxin's mechanism of actionis complex and not restricted to activation of the H+-ATPase.FC has no effect when administered to microsomes. The microsomalenzyme was solubilized and reconstituted into asolec-tin liposomesin functional form. The reconstituted, erythrosine sensitiveCa2+-ATPase was insensitive to fusicoccin. Thus, componentsessential for toxin action are either lost or inactivated duringsubcellular fractionation. It is likely that FC action requiressoluble components. (Received April 22, 1991; Accepted July 24, 1991) 相似文献
19.
Role of plasma membrane Ca2+-ATPase in contraction-relaxation processes of the bladder: evidence from PMCA gene-ablated mice 总被引:1,自引:0,他引:1
Liu L Ishida Y Okunade G Shull GE Paul RJ 《American journal of physiology. Cell physiology》2006,290(4):C1239-C1247
We investigated the roles and relationships of plasma membrane Ca2+-ATPase (PMCA), sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2, and Na+/Ca2+ exchanger (NCX) in bladder smooth muscle contractility in Pmca-ablated mice: Pmca4-null mutant (Pmca4/) and heterozygous Pmca1 and homozygous Pmca4 double gene-targeted (Pmca1+/Pmca4/) mice. Gene manipulation did not alter the amounts of PMCA1, SERCA2, and NCX. To study the role of each Ca2+ transport system, contraction of circular ring preparations was elicited with KCl (80 mM) plus atropine, and then the muscle was relaxed with Ca2+-free physiological salt solution containing EGTA. We measured the contributions of Ca2+ clearance components by inhibiting SERCA2 (with 10 µM cyclopiazonic acid) and/or NCX (by replacing NaCl with N-methyl-D-glucamine/HCl plus 10 µM KB-R7943). Contraction half-time (time to 50% of maximum tension) was prolonged in the gene-targeted muscles but marginally shortened when SERCA2 or NCX was inhibited. The inhibition of NCX significantly inhibited this prolongation, suggesting that NCX activity might be augmented to compensate for PMCA4 function in the gene-targeted muscles under nonstimulated conditions. Inhibition of SERCA2 and NCX as well as gene targeting all prolonged the relaxation half-time. The contribution of PMCA to relaxation was calculated to be 2530%, with that of SERCA2 being 20% and that of NCX being 70%. PMCA and SERCA2 appeared to function additively, but the function of NCX might overlap with those of other components. In summary, gene manipulation of PMCA indicates that PMCA, in addition to SERCA2 and NCX, plays a significant role in both excitation-contraction coupling and the Ca2+ extrusion-relaxation relationship, i.e., Ca2+ homeostasis, of bladder smooth muscle. ATP2B; sarco(endo)plasmic reticulum Ca2+-ATPase 2; Na+/Ca2+ exchanger; homeostasis 相似文献
20.
When microsomal membranes from maize (Zea mays L. cv. Clipper)coleoptiles were separated by isopyc-nic centrifugation on acontinuous 1045% sucrose gradient, bafilomycin A1-inhibitedATPase activity co-localized with the activities of the tonoplastmarker-enzymes, nitrate-Inhibited ATPase and K+-dependent pyrophosphatase.Thus, bafilomycin A1 is a specific inhibitor of the vacuolarH+-ATPase of maize coleoptiles. Inhibition of the vacuolar H+-ATPaseby bafilomycin A1 was strictly dependent upon the concentrationof the enzyme present in the assay medium, suggesting a stoichiometricassociation between bafilomycin A1 and the vacuolar H+-ATPase.In tonoplast-enriched preparations, half-maximal inhibitionwas obtained at 43 pmol bafilomycin A1 mg1 protein. BafilomycinA1 inhibited the vacuolar H+-ATPase in a simple non-competitivemanner: increasing bafilomycin A1 concentrations reduced theVmax, of the H+ -ATPase, but had no effect on its Km towardsATP. Key words: Bafilomycin A1, coleoptile, H+-ATPase (vacuolar), maize, Zea mays L 相似文献