Cleavage of SNAP-25 and VAMP-2 impairs store-operated Ca2+ entry in mouse pancreatic acinar cells

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     

Oxidant-impaired intracellular Ca2+ signaling in pancreatic acinar cells: role of the plasma membrane Ca2+-ATPase

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     

Characterization of ATPases Associated with Various Cellular Membranes Isolated from Etiolated Hypocotyls of Vigna radiata (L.) Wilczek

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 K_m 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 theirK_m 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)     

Selective Effect of Salt Stress on the Activity of Two ATPases in the Cell Membrane of Nostoc muscorum

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)     

Caloxin: a novel plasma membrane Ca2+ pump inhibitor

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 byN^G-nitro-L-arginine methyl ester.Thus caloxin 2A1 is a novel PM Ca²⁺ pump inhibitor selectedfor binding to an extracellular domain.

     

Comparison of Plasma Membrane and Tonoplast H+-Translocating ATPases in Phaseolus mungo L. Roots

Two membrane fractions were obtained from 16%/26% and 34%/40%interfaces following discontinuous sucrose density gradientcentrifugation of a 10,000–80,000xg 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 K_mfor 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)     

Measurement of Changes in Cytosolic Ca2+ in Arabidopsis Guard Cells and Mesophyll Cells in Response to Blue Light

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.     

Effect of Bcl-2 on oxidant-induced cell death and intracellular Ca2+ mobilization

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⁺.

     

Plasma membrane isolation from freshwater and salt-tolerant species of Chara: antibody cross-reactions and phosphohydrolase activities

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 90–100 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     

Optical measurement of stimulus-evoked membrane dynamics in single pancreatic acinar cells

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(C_m)measurements and by the membrane fluorescent dye FM1-43. When measuredwith the C_mmethod, 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 ofC_m changes,reveals coincidence of exocytotic and endocytotic activity in acinarcells after hormonal stimulation.

     

Isolation of cDNA for a Plasma Membrane H+-ATPase from Guard Cells of Vicia faba L.

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 79–85% 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)     

Isolation and Characterization of the Plasma Membrane by Two-Phase Partitioning from the Alkalophilic Cyanobacterium Spirulina platensis

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, 3–4h. 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)     

Functional upregulation of H+-ATPase by lethal acid stress in cultured inner medullary collecting duct cells

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(pH_i) recovery from an acid load. The rate of Na⁺-independentpH_i 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.

     

Partial inhibition of SERCA is responsible for extracellular Ca2+ dependence of AlF-4-induced [Ca2+]i oscillations in rat pancreatic

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     

Munc-18-2 regulates exocytosis of H(+)-ATPase in rat inner medullary collecting duct cells

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     

Presence of a Na+-activated ATPase in the Plasma Membrane of the Marine Raphidophycean Heterosigma akashiwo

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)     

Salinity Effects on the Activity of Plasma Membrane H+and Ca2+Transporters in Bean Leaf Mesophyll: Masking Role of the Cell Wall

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     

Characterization, Functional Reconstitution and Activation by Fusicoccin of a Ca2+-ATPase from Corydalis sempervirens Pers. Cell Suspension Cultures

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 K_m 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 K_m 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)     

Role of plasma membrane Ca2+-ATPase in contraction-relaxation processes of the bladder: evidence from PMCA gene-ablated mice

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 25–30%, 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     

Bafilomycin A1 is a non-competitive inhibitor of the tonoplast H+-ATPase of maize coleoptiles

When microsomal membranes from maize (Zea mays L. cv. Clipper)coleoptiles were separated by isopyc-nic centrifugation on acontinuous 10–45% 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 mg^–1 protein. BafilomycinA1 inhibited the vacuolar H⁺-ATPase in a simple non-competitivemanner: increasing bafilomycin A1 concentrations reduced theV_max, of the H+ -ATPase, but had no effect on its K_m towardsATP. Key words: Bafilomycin A1, coleoptile, H⁺-ATPase (vacuolar), maize, Zea mays L