Glycosylphosphatidylinositol-anchored proteins and actin cytoskeleton modulate chloride transport by channels formed by the Helicobacter pylori vacuolating cytotoxin VacA in HeLa cells

The vacuolating cytotoxin VacA is an important virulence factor of Helicobacter pylori. Removing glycosylphosphatidylinositol-anchored proteins (GPI-Ps) from the cell surface by phosphatidylinositol-phospholipase C or disrupting the cell actin cytoskeleton by cytochalasin D reduced VacA-induced vacuolation of cells. Using the fluorescent dye 6-methoxy-N-ethylquinolinium chloride, an indicator for cytosolic chloride, we have investigated the role of either GPI-Ps or actin cytoskeleton in the activity of the selective anionic channel formed by VacA at the plasma membrane level. Removal of GPI-Ps from HeLa cell surfaces did not impair VacA localization into lipid rafts but strongly reduced VacA channel-mediated cell influx and efflux of chloride. Disruption of the actin cytoskeleton of HeLa cells by cytochalasin D did not affect VacA localization in lipid rafts but blocked VacA cell internalization and inhibited cell vacuolation while increasing the overall chloride transport by the toxin channel at the cell surface. Specific enlargement of Rab7-positive compartments induced by VacA could be mimicked by the weak base chloroquine alone, and the vacuolating activities of either chloroquine alone or VacA were blocked with the same potency by the anion channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid shown to inhibit VacA channel activity. We suggest that formation of functional VacA channels at the cell surface required GPI-Ps and that endocytosis of these channels by an actin-dependent process increases the chloride content of late endosomes that accumulate weak bases, provoking their enlargement by osmotic swelling.     

Inhibition of the vacuolating and anion channel activities of the VacA toxin of Helicobacter pylori.

VacA, the vacuolating cytotoxin secreted by Helicobacter pylori, is believed to be a major causative factor in the development of gastroduodenal ulcers. This toxin causes vacuolation of cultured cells and it has recently been found to form anion-selective channels upon insertion into planar bilayers as well as in the plasma membrane of HeLa cells. Here, we identify a series of inhibitors of VacA channels and we compare their effectiveness as channel blockers and as inhibitors of VacA-induced vacuolation, confirming that the two phenomena are linked. This characterization opens the way to studies in other experimental systems and to the search for a specific inhibitor of VacA action.     

Sphingomyelin functions as a novel receptor for Helicobacter pylori VacA

The vacuolating cytotoxin (VacA) of the gastric pathogen Helicobacter pylori binds and enters epithelial cells, ultimately resulting in cellular vacuolation. Several host factors have been reported to be important for VacA function, but none of these have been demonstrated to be essential for toxin binding to the plasma membrane. Thus, the identity of cell surface receptors critical for both toxin binding and function has remained elusive. Here, we identify VacA as the first bacterial virulence factor that exploits the important plasma membrane sphingolipid, sphingomyelin (SM), as a cellular receptor. Depletion of plasma membrane SM with sphingomyelinase inhibited VacA-mediated vacuolation and significantly reduced the sensitivity of HeLa cells, as well as several other cell lines, to VacA. Further analysis revealed that SM is critical for VacA interactions with the plasma membrane. Restoring plasma membrane SM in cells previously depleted of SM was sufficient to rescue both toxin vacuolation activity and plasma membrane binding. VacA association with detergent-resistant membranes was inhibited in cells pretreated with SMase C, indicating the importance of SM for VacA association with lipid raft microdomains. Finally, VacA bound to SM in an in vitro ELISA assay in a manner competitively inhibited by lysenin, a known SM-binding protein. Our results suggest a model where VacA may exploit the capacity of SM to preferentially partition into lipid rafts in order to access the raft-associated cellular machinery previously shown to be required for toxin entry into host cells.     

A dominant negative mutant of Helicobacter pylori vacuolating toxin (VacA) inhibits VacA-induced cell vacuolation

Most Helicobacter pylori strains secrete a toxin (VacA) that causes structural and functional alterations in epithelial cells and is thought to play an important role in the pathogenesis of H. pylori-associated gastroduodenal diseases. The amino acid sequence, ultrastructural morphology, and cellular effects of VacA are unrelated to those of any other known bacterial protein toxin, and the VacA mechanism of action remains poorly understood. To analyze the functional role of a unique strongly hydrophobic region near the VacA amino terminus, we constructed an H. pylori strain that produced a mutant VacA protein (VacA-(Delta6-27)) in which this hydrophobic segment was deleted. VacA-(Delta6-27) was secreted by H. pylori, oligomerized properly, and formed two-dimensional lipid-bound crystals with structural features that were indistinguishable from those of wild-type VacA. However, VacA-(Delta6-27) formed ion-conductive channels in planar lipid bilayers significantly more slowly than did wild-type VacA, and the mutant channels were less anion-selective. Mixtures of wild-type VacA and VacA-(Delta6-27) formed membrane channels with properties intermediate between those formed by either isolated species. VacA-(Delta6-27) did not exhibit any detectable defects in binding or uptake by HeLa cells, but this mutant toxin failed to induce cell vacuolation. Moreover, when an equimolar mixture of purified VacA-(Delta6-27) and purified wild-type VacA were added simultaneously to HeLa cells, the mutant toxin exhibited a dominant negative effect, completely inhibiting the vacuolating activity of wild-type VacA. A dominant negative effect also was observed when HeLa cells were co-transfected with plasmids encoding wild-type and mutant toxins. We propose a model in which the dominant negative effects of VacA-(Delta6-27) result from protein-protein interactions between the mutant and wild-type VacA proteins, thereby resulting in the formation of mixed oligomers with defective functional activity.     

Helicobacter pylori vacuolating toxin forms anion-selective channels in planar lipid bilayers: possible implications for the mechanism of cellular vacuolation

The Helicobacter pylori VacA toxin plays a major role in the gastric pathologies associated with this bacterium. When added to cultured cells, VacA induces vacuolation, an effect potentiated by preexposure of the toxin to low pH. Its mechanism of action is unknown. We report here that VacA forms anion-selective, voltage-dependent pores in artificial membranes. Channel formation was greatly potentiated by acidic conditions or by pretreatment of VacA at low pH. No requirement for particular lipid(s) was identified. Selectivity studies showed that anion selectivity was maintained over the pH range 4.8-12, with the following permeability sequence: Cl- approximately HCO3- > pyruvate > gluconate > K+ approximately Li+ approximately Ba2+ > NH4+. Membrane permeabilization was due to the incorporation of channels with a voltage-dependent conductance in the 10-30 pS range (2 M KCl), displaying a voltage-independent high open probability. Deletion of the NH2 terminus domain (p37) or chemical modification of VacA by diethylpyrocarbonate inhibited both channel activity and vacuolation of HeLa cells without affecting toxin internalization by the cells. Collectively, these observations strongly suggest that VacA channel formation is needed to induce cellular vacuolation, possibly by inducing an osmotic imbalance of intracellular acidic compartments.     

Association of Helicobacter pylori vacuolating toxin (VacA) with lipid rafts

A variety of extracellular ligands and pathogens interact with raft domains in the plasma membrane of eukaryotic cells. In this study, we examined the role of lipid rafts and raft-associated glycosylphosphatidylinositol (GPI)-anchored proteins in the process by which Helicobacter pylori vacuolating toxin (VacA) intoxicates cells. We first investigated whether GPI-anchored proteins are required for VacA toxicity by analyzing wild-type Chinese hamster ovary (CHO) cells and CHO-LA1 mutant cells that are defective in production of GPI-anchored proteins. Whereas wild-type and mutant cells differed markedly in susceptibility to aerolysin (a bacterial toxin that binds to GPI-anchored proteins), they were equally susceptible to VacA. We next determined whether VacA physically associates with lipid rafts. CHO or HeLa cells were incubated with VacA, and Triton-insoluble membranes then were separated by sucrose density gradient centrifugation. Immunoblot analysis revealed that a substantial proportion of cell-associated toxin was associated with detergent-resistant membranes (DRMs). DRM association required acid activation of the purified toxin prior to contact with cells, and acid activation also was required for VacA cytotoxicity. Treatment of cells with methyl-beta-cyclodextrin (a cholesterol-depleting agent) did not inhibit VacA-induced depolarization of the plasma membrane, but interfered with the internalization or intracellular localization of VacA and inhibited the capacity of the toxin to induce cell vacuolation. Treatment of cells with nystatin also inhibited VacA-induced cell vacuolation. These data indicate that VacA associates with lipid raft microdomains in the absence of GPI-anchored proteins and suggest that association of the toxin with lipid rafts is important for VacA cytotoxicity.     

Vacuolation induced by VacA toxin of Helicobacter pylori requires the intracellular accumulation of membrane permeant bases, Cl(-) and water.

The protein vacuolating toxin A (VacA) of Helicobacter pylori converts late endosomes into large vacuoles in the presence of permeant bases. Here it is shown that this phenomenon corresponds to an accumulation of permeant bases and Cl(-) in HeLa cells and requires the presence of extracellular Cl(-). The net influx of Cl(-) is due to electroneutral, Na(+), K(+), 2Cl(-) cotransporter-mediated transport. Cell vacuolation leads to cell volume increase, consistent with water flux into the cell, while hyper-osmotic media decreased vacuole formation. These data represent the first evidence that VacA-treated cells undergo an osmotic unbalance, reinforcing the hypothesis that the VacA chloride channel is responsible for cell vacuolation.     

Identification and modulation of a voltage-dependent anion channel in the plasma membrane of guard cells by high-affinity ligands.

Guard cell anion channels (GCAC1) catalyze the release of anions across the plasma membrane during regulated volume decrease and also seem to be involved in the targeting of the plant growth hormones auxins. We have analyzed the modulation and inhibition of these voltage-dependent anion channels by different anion channel blockers. Ethacrynic acid, a structural correlate of an auxin, caused a shift in activation potential and simultaneously a transient increase in the peak current amplitude, whereas other blockers shifted and blocked the voltage-dependent activity of the channel. Comparison of dose-response curves for shift and block imposed by the inhibitor, indicate two different sites within the channel which interact with the ligand. The capability to inhibit GCAC1 increases in a dose-dependent manner in the sequence: probenecid less than A-9-C less than ethacrynic acid less than niflumic acid less than IAA-94 less than NPPB. All inhibitors reversibly blocked the anion channel from the extracellular side. Channel block on the level of single anion channels is characterized by a reduction of long open transitions into flickering bursts, indicating an interaction with the open mouth of the channel. IAA-23, a structural analog of IAA-94, was used to enrich ligand-binding polypeptides from the plasma membrane of guard cells by IAA-23 affinity chromatography. From this protein fraction a 60 kDa polypeptide crossreacted specifically with polyclonal antibodies raised against anion channels isolated from kidney membranes. In contrast to guard cells, mesophyll plasma membranes were deficient in voltage-dependent anion channels and lacked crossreactivity with the antibody.     

Single-channel properties of a rat brain endoplasmic reticulum anion channel.

Many intracellular membranes contain ion channels, although their physiological roles are often poorly understood. In this study we incorporated single anion channels colocalized with rat brain endoplasmic reticulum (ER) ryanodine-sensitive Ca(2+)-release channels into planar lipid bilayers. The channels opened in bursts, with more activity at negative (cytoplasm-ER lumen) membrane potentials, and they occupied four open conductance levels with frequencies well described by the binomial equation. The probability of a protomer being open decreased from approximately 0.7 at -40 mV to approximately 0.2 at +40 mV, and the channels selected between different anions in the order PSCN > PNO3 > PBr > PCl > PF. They were also permeant to cations, including the large cation Tris+ (PTris/PCl = 0.16). Their conductance saturated at 170 pS in choline Cl. The channels were inactivated by 15 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and blocked with low affinity (KD of 1-100 microM) by anthracene-9-carboxylic acid, ethacrynic acid, frusemide (furosemide), HEPES, the indanyloxyacetic acid derivative IAA-94, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), and Zn2+. Unlike protein translocation pores, the channels were unaffected by high salt concentrations or puromycin. They may regulate ER Ca2+ release, or be channel components en route to their final cellular destinations. Alternatively, they may contribute to the fusion machinery involved in intracellular membrane trafficking.     

Prion channel proteins and their role in vacuolation and neurodegenerative diseases

The prion encephalopathies, which are characterized by neuropathological changes that include vacuolation, astrocytosis, the development of amyloid plaques and neuronal loss, are associated with the conversion of a normal cellular isoform of prion protein (PrP(c)) to an abnormal pathologic scrapie isoform (PrP(Sc)). The use of PrP[106-126] and its isoforms in studies of channels in lipid bilayers has revealed that it forms heterogeneous channels reflecting modifications in the peptide's structure and differences in the properties of the formed oligomeric aggregates and their intermediates. We propose that the accumulation of pathological isoforms of prion are linked to membrane abnormalities and vacuolation in prion diseases. The interlinked changes in membrane fluidity and endogenous channels induced by prion isoforms can occur independently and concurrently with channel formation, i.e. they are not mutually exclusive. We suggest that vacuolation is a cellular response triggered in order to immobilize pathological prion isoforms having the ability to form channels that compromise cellular membranes. This mechanism is similar to that of other channel-forming proteins that induce vacuolation, e.g. the well-established VacA of Helicobacter pylori, Vero cells and aerolysin, as well as melittin-induced micellization and membrane fusion. We conclude that channel formation is part of the molecular mechanisms responsible for the vacuolation associated with prion diseases. The initial vacuolation could be an adaptive cellular response to compartmentalize the increase in pathogenic prion isoforms, while an excessive accumulation of pathologic prion isoforms in later stages represents the inability of the cell to continue to compartmentalize these misfolded proteins in vacuoles.     

How the loop and middle regions influence the properties of Helicobacter pylori VacA channels.

VacA is a pore-forming cytotoxin produced by Helicobacter pylori in several strain-specific isoforms, which have been classified in two main families, m1 and m2, according to the sequence of a variable "midregion." Both forms are associated with gastric pathologies and can induce vacuolation of cultured cells. The comparison of two representative toxins, m1 17874 and m2 9554, has indicated that the m2 form is less powerful in vacuolation assays and that its effects are more strongly cell type dependent. To rationalize these differences and to investigate structure-function relationships in this toxin, we have compared the properties of the channels formed by these two variants and by a construct derived from 17874 by deleting a loop that connects the two toxin domains, which is shorter in 9554 than in 17874. Although the channels formed by all three proteins are similar, m2 9554 channels have, on average, a lower conductance and are less anion-selective and more voltage-dependent than the m1 pores. Furthermore, the rate of incorporation of 9554 VacA into planar bilayers depends on lipid composition much more strongly than that of 17874. The comparison with the behavior of the loop deletion mutant indicates that this latter property, as well as a portion of the conductance decrease, may be attributed to the reduction in loop length. The differences in pore properties are proposed to account in part for the different cytotoxicity exhibited by the two toxin isoforms. We furthermore present evidence suggesting that the conformation of the membrane-embedded toxin may be influenced by the lipid composition of the membrane itself.     

Identification of High-Affinity Slow Anion Channel Blockers and Evidence for Stomatal Regulation by Slow Anion Channels in Guard Cells

Slow anion channels in the plasma membrane of guard cells have been suggested to constitute an important control mechanism for long-term ion efflux, which produces stomatal closing. Identification of pharmacological blockers of these slow anion channels is instrumental for understanding plant anion channel function and structure. Patch clamp studies were performed on guard cell protoplasts to identify specific extracellular inhibitors of slow anion channels. Extracellular application of the anion channel blockers NPPB and IAA-94 produced a strong inhibition of slow anion channels in the physiological voltage range with half inhibition constants (K1/2) of 7 and 10 [mu]M, respectively. Single slow anion channels that had a high open probability at depolarized potentials were identified. Anion channels had a main conductance state of 33 [plus or minus] 8 pS and were inhibited by IAA-94. DIDS, which has been shown to be a potent blocker of rapid anion channels in guard cells (K1/2 = 0.2 [mu]M), blocked less than 20% of peak slow anion currents at extracellular or cytosolic concentrations of 100 [mu]M. The pharmacological properties of slow anion channels described here differ from those recently described for rapid anion channels in guard cells, fortifying the finding that two highly distinct types or modes of voltage- and second messenger-dependent anion channel currents coexist in the guard cell plasma membrane. Bioassays using anion channel blockers provide evidence that slow anion channel currents play a substantial role in the regulation of stomatal closing. Interestingly, slow anion channels may also function as a negative regulator during stomatal opening under the experimental conditions applied here. The identification of specific blockers of slow anion channels reported here permits detailed studies of cell biological functions, modulation, and structural components of slow anion channels in guard cells and other higher plant cells.     

A 12-Amino-Acid Segment, Present in Type s2 but Not Type s1 Helicobacter pylori VacA Proteins, Abolishes Cytotoxin Activity and Alters Membrane Channel Formation

Helicobacter pylori, a gram-negative bacterium associated with gastritis, peptic ulceration, and gastric adenocarcinoma in humans, secretes a protein toxin, VacA, that causes vacuolar degeneration of epithelial cells. Several different families of H. pylori vacA alleles can be distinguished based on sequence diversity in the "middle" region (i.e., m1 and m2) and in the 5' end of the gene (i.e., s1 and s2). Type s2 VacA toxins contain a 12-amino-acid amino-terminal hydrophilic segment, which is absent from type s1 toxins. To examine the functional properties of VacA toxins containing this 12-amino-acid segment, we analyzed a wild-type s1/m1 VacA and a chimeric s2/m1 VacA protein. Purified s1/m1 VacA from H. pylori strain 60190 induced vacuolation in HeLa and Vero cells, whereas the chimeric s2/m1 toxin (in which the s1 sequence of VacA from strain 60190 was replaced with the s2 sequence from strain Tx30a) lacked detectable cytotoxic activity. Type s1/m1 VacA from strain 60190 formed membrane channels in a planar lipid bilayer assay at a significantly higher rate than did s2/m1 VacA. However, membrane channels formed by type s1 VacA and type s2 VacA proteins exhibited similar anion selectivities (permeability ratio, P(Cl)/P(Na) = 5). When an equimolar mixture of the chimeric s2/m1 toxin and the wild-type s1/m1 toxin was added to HeLa cells, the chimeric toxin completely inhibited the activity of the s1/m1 toxin. Thus, the s2/m1 toxin exhibited a dominant-negative phenotype similar to that of a previously described mutant toxin, VacA-(Delta6-27). Immunoprecipitation experiments indicated that both s2/m1 VacA and VacA-(Delta6-27) could physically interact with a c-myc epitope-tagged s1/m1 VacA, which suggests that the dominant-negative phenotype results from the formation of heterooligomeric VacA complexes with defective functional activity. Despite detectable differences in the channel-forming activities and cytotoxic properties of type s1 and type s2 VacA proteins, the conservation of type s2 sequences in many H. pylori isolates suggests that type s2 VacA proteins retain an important biological activity.     

Structure and interaction of VacA of Helicobacter pylori with a lipid membrane.

In its mature form, the VacA toxin of Helicobacter pylori is a 95-kDa protein which is released from the bacteria as a low-activity complex. This complex can be activated by low-pH treatment that parallels the activity of the toxin on target cells. VacA has been previously shown to insert itself into lipid membranes and to induce anion-selective channels in planar lipid bilayers. Binding of VacA to lipid vesicles and its ability to induce calcein release from these vesicles were systematically compared as a function of pH. These two phenomena show a different pH-dependence, suggesting that the association with the lipid membrane may be a two-step mechanism. The secondary and tertiary structure of VacA as a function of pH and the presence of lipid vesicles were investigated by Fourier-transform infrared spectroscopy. The secondary structure of VacA is identical whatever the pH and the presence of a lipid membrane, but the tertiary structure in the presence of a lipid membrane is dependent on pH, as evidenced by H/D exchange.     

Essential role of a GXXXG motif for membrane channel formation by Helicobacter pylori vacuolating toxin

Helicobacter pylori secretes a toxin, VacA, that can form anion-selective membrane channels. Within a unique amino-terminal hydrophobic region of VacA, there are three tandem GXXXG motifs (defined by glycines at positions 14, 18, 22, and 26), which are characteristic of transmembrane dimerization sequences. The goals of the current study were to investigate whether these GXXXG motifs are required for membrane channel formation and cytotoxicity and to clarify the role of membrane channel formation in the biological activity of VacA. Six different alanine substitution mutations (P9A, G13A, G14A, G18A, G22A, and G26A) were introduced into the unique hydrophobic region located near the amino terminus of VacA. The effects of these mutations were first analyzed using the TOXCAT system, which permits the study of transmembrane oligomerization of proteins in a natural membrane environment. None of the mutations altered the capacity of ToxR-VacA-maltose-binding protein fusion proteins to insert into a membrane, but G14A and G18A mutations markedly diminished the capacity of the fusion proteins to oligomerize. We then introduced the six alanine substitution mutations into the vacA chromosomal gene of H. pylori and analyzed the properties of purified mutant VacA proteins. VacA-G13A, VacA-G22A, and VacA-G26A induced vacuolation of HeLa cells, whereas VacA-P9A, VacA-G14A, and VacA-G18A did not. Subsequent experiments examined the capacity of each mutant toxin to form membrane channels. In a planar lipid bilayer assay, VacA proteins containing G13A, G22A, and G26A mutations formed anion-selective membrane channels, whereas VacA proteins containing P9A, G14A, and G18A mutations did not. Similarly, VacA-G13A, VacA-G22A, and VacA-G26A induced depolarization of HeLa cells, whereas VacA-P9A, VacA-G14A, and VacA-G18A did not. These data indicate that an intact proline residue and an intact G(14)XXXG(18) motif within the amino-terminal hydrophobic region of VacA are essential for membrane channel formation, and they also provide strong evidence that membrane channel formation is essential for VacA cytotoxicity.     

High cell sensitivity to Helicobacter pylori VacA toxin depends on a GPI-anchored protein and is not blocked by inhibition of the clathrin-mediated pathway of endocytosis

Helicobacter pylori vacuolating toxin (VacA) causes vacuolation in a variety of cultured cell lines, sensitivity to VacA differing greatly, however, among the different cell types. We found that the high sensitivity of HEp-2 cells to VacA was impaired by treating the cells with phosphatidylinositol-specific phospholipase C (PI-PLC) which removes glycosylphosphatidylinositol (GPI)-anchored proteins from the cell surface. Incubation of cells with a cholesterol-sequestering agent, that impairs both structure and function of sphingolipid-cholesterol-rich membrane microdomains ("lipid rafts"), also impaired VacA-induced cell vacuolation. Overexpression into HEp-2 cells of proteins inhibiting clathrin-dependent endocytosis (i.e., a dominant-negative mutant of Eps15, the five tandem Src-homology-3 domains of intersectin, and the K44A dominant-negative mutant of dynamin II) did not affect vacuolation induced by VacA. Nevertheless, F-actin depolymerization, known to block the different types of endocytic mechanisms, strongly impaired VacA vacuolating activity. Taken together, our data suggest that the high cell sensitivity to VacA depends on the presence of one or several GPI-anchored protein(s), intact membrane lipid rafts, and an uptake mechanism via a clathrin-independent endocytic pathway.     

The Phytotoxic Lipodepsipeptide Syringopeptin 25A from Pseudomonas syringae pv syringae Forms Ion Channels in Sugar Beet Vacuoles

Syringopeptin 25A (SP(25)A) belongs to a family of cyclic lipodepsipeptides (LDPs) produced by the gram-negative bacterium Pseudomonas syringae, a phytopathogenic organism that affects several plants of agronomic interest. LDPs increase the permeability of plasma and, possibly, intracellular membranes in plant cells. Consistently, SP(25)A forms ion channels in planar lipid bilayers and other model membranes. Here we used sugar beet tonoplasts as a new biological model system to study toxin action. When applied to the vacuoles by a fast perfusion procedure, SP(25)A increases membrane permeability by forming discrete ion channels even at low applied potentials. The SP(25)A channel displays anion selectivity (with a Cl-/K+ permeability ratio of 6.7 +/- 1.3) and has intrinsic rectification properties that derive from a different channel conductance at negative and positive voltages, presumably owing to an asymmetric distribution of fixed charges on the pore. Substitution of chloride with different anions reveals the following selectivity sequence NO3- approximately Cl-> F- > gluconate-, suggesting that the permeation pore is filled with water. The properties of the SP(25)A channels in vacuolar membranes are similar to those observed in planar lipid membranes prepared with asolectin. This work provides a direct demonstration of toxin effects on a native plant membrane, extending to a biological system previous results obtained on artificial planar lipid membranes.     

钙激活氯离子通道对大鼠肺动脉张力的调节作用

目的：研究钙激活氯离子通道及其通道阻断剂尼氟灭酸（niflumic acid,NFA）、indaryloxyacetic acid（IAA-94）在苯福林（phenylephrine,PE）引起的肺动脉收缩中的作用。方法：常规离体血管灌流法检测肺动脉环张力;采用钙荧光探针（Fura-2／AM）负载急性酶分离法（胶原酶Ⅰ型和木瓜蛋白酶）获得的大鼠肺动脉平滑肌细胞（PASMCs）,观察NFA和IAA-94对PE诱导的PASMCs胞浆游离钙离子浓度（[Ca^2＋]i）的影响,用荧光分光光度计法检测[Ca^2＋]i。结果：钙激活氯离子通道阻断剂NFA和IAA-94可以舒张PE引起的肺动脉环收缩;NFA和IAA-94对KCl引起的血管收缩无影响;PE可以引起[Ca^2＋]i升高,NFA和IAA-94对PE诱导[Ca^2＋]i升高无影响。结论：钙激活氯离子通道在生理状态下与血管活性药（PE）引起的肺动脉张力变化有关,这为研究其在低氧肺血管收缩中的作用提供了新的线索。     

Functional properties of the p33 and p55 domains of the Helicobacter pylori vacuolating cytotoxin

Helicobacter pylori secretes an 88-kDa vacuolating cytotoxin (VacA) that may contribute to the pathogenesis of peptic ulcer disease and gastric cancer. VacA cytotoxic activity requires assembly of VacA monomers into oligomeric structures, formation of anion-selective membrane channels, and entry of VacA into host cells. In this study, we analyzed the functional properties of recombinant VacA fragments corresponding to two putative VacA domains (designated p33 and p55). Immunoprecipitation experiments indicated that these two domains can interact with each other to form protein complexes. In comparison to the individual VacA domains, a mixture of the p33 and p55 proteins exhibited markedly enhanced binding to the plasma membrane of mammalian cells. Furthermore, internalization of the VacA domains was detected when cells were incubated with the p33/p55 mixture but not when the p33 and p55 proteins were tested individually. Incubation of cells with the p33/p55 mixture resulted in cell vacuolation, whereas the individual domains lacked detectable cytotoxic activity. Interestingly, sequential addition of p55 followed by p33 resulted in VacA internalization and cell vacuolation, whereas sequential addition in the reverse order was ineffective. These results indicate that both the p33 and p55 domains contribute to the binding and internalization of VacA and that both domains are required for vacuolating cytotoxic activity. Reconstitution of toxin activity from two separate domains, as described here for VacA, has rarely been described for pore-forming bacterial toxins, which suggests that VacA is a pore-forming toxin with unique structural properties.     

Clustering and redistribution of late endocytic compartments in response to Helicobacter pylori vacuolating toxin

Helicobacter pylori VacA is a secreted protein toxin that may contribute to the pathogenesis of peptic ulcer disease and gastric adenocarcinoma. When added to cultured mammalian cells in the presence of weak bases (e.g., ammonium chloride), VacA induces the formation of large cytoplasmic vacuoles. Here, we report a previously unrecognized capacity of VacA to induce clustering and perinuclear redistribution of late endocytic compartments. In contrast to VacA-induced cell vacuolation, VacA-induced clustering and redistribution of late endocytic compartments are not dependent on the presence of weak bases and are not inhibited by bafilomycin A1. VacA mutant toxins defective in the capacity to form anion-selective membrane channels fail to cause clustering and redistribution. VacA-induced clusters of late endocytic compartments undergo transformation into vacuoles after the addition of ammonium chloride. VacA-induced clustering and redistribution of late endocytic compartments occur in cells expressing wild-type or constitutively active Rab7, but not in cells expressing dominant-negative mutant Rab7. In VacA-treated cells containing clustered late endocytic compartments, overexpression of dominant-negative Rab7 causes reversion to a nonclustered distribution. Redistribution of late endocytic compartments to the perinuclear region requires a functional microtubule cytoskeleton, whereas clustering of these compartments and vacuole formation do not. These data provide evidence that clustering of late endocytic compartments is a critical mechanistic step in the process of VacA-induced cell vacuolation. We speculate that VacA-induced alterations in late endocytic membrane traffic contribute to the capacity of H. pylori to persistently colonize the human gastric mucosa.