1 Dipartimento di Fisiologia Generale e Ambientale, Università di Bari, 70126 Bari; 2 Dipartimento di Scienze Biomediche e Oncologia Umana, Sezione di Patologia Generale, Università di Bari, 70124 Bari; and 3 Centro CNR Biomembrane e Dipartimento di Scienze Biomediche, Università di Padova, 35121 Padova, Italy
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Human infection by the
bacterium Helicobacter pylori (Hp) may lead to severe
gastric diseases by an ill-understood process involving several
virulence factors. Among these, the cytotoxin VacA is associated with
higher tissue damage. In this study, the isolated frog stomach model
was used to characterize the acute effects of VacA on the gastric
epithelium. Our results show that VacA partially inhibits gastric acid
output by increasing HCO-selective
microelectrodes on surface epithelial gastric cells (SECs) and single
gastric glands show that VacA does not impair the activity of the
oxyntic cells but renders the apical membrane of SECs more permeable to
HCO
. Inhibition of this
permeation by 5-nitro-2-(3-phenylpropylamino) benzoic acid indicates
that this may be due to the formation of anion-selective pores by the
toxin. We suggest that VacA-dependent HCO
gastric secretion; anion channel
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
INFECTION BY THE GRAM-NEGATIVE bacterium Helicobacter pylori (Hp) is very common in humans (2). Hp colonizes the stomach and establishes a life-long chronic inflammation, which can evolve into severe gastritis, ulcer, and gastric adenocarcinoma (9).
Studies conducted in recent years clearly show that a plethora of
different adaptations and virulence factors are needed to guarantee Hp
early colonization and persistence within and below the mucus layer
covering surface epithelial gastric cells (SECs) (39). A
cytosolic bacterial urease, which generates NH3 and HCO
A set of ~30 genes grouped in the so-called pathogenicity island cag (PAI cag) encodes for different factors essential in gastric colonization (9). Among these are the protein cag A and a type IV secretory apparatus that is responsible for its injection into host epithelial cells (49, 50).
Two other factors encoded outside PAI cag have been identified and intensively studied: the Hp-neutrophil-activating protein, which stimulates polymorphonucleates and monocytes in proinflammatory terms (48), and the vacuolating toxin (VacA) (38).
This latter factor is a 95-kDa protein, encoded by the polymorphic gene vaca (1), which can be isolated from the cell-free medium as an inactive oligomer. Biological activation of purified VacA follows exposure to acidic or alkaline conditions (15, 59) and is believed to be accompanied by an increased cell binding (40), membrane insertion (36, 37, 41), and internalization by clathrin-independent endocytosis (35, 46).
Topical addition of VacA to different cell lines (MDCK, T84, epH4, HeLa) has revealed different cytotoxic effects ranging from cell vacuolation, due to enlargement of late endosomes, to the selective permeabilization of polarized epithelia by decreasing the resistance of the paracellular pathway (42, 43). Recently, VacA has been shown to form low-conducting, anion-selective channels in model and plasma membranes, an activity that appears essential for the main characterized biological action of VacA membranes (14, 52, 55).
One of the main goals of these studies was to define the molecular and cellular action of VacA to extrapolate its functional and pathological role in vivo. For example, epithelial and plasma membrane permeabilization have been proposed to serve to a higher nutrient supply from the gastric tissue to infecting Hp bound to the surface of gastric cells (43, 44, 52, 55). Therefore, experimental approaches exploiting an intact gastric tissue are needed not only to test hypotheses generated in simpler models but also to identify the consequences of a given molecular action on the complex functional performance of a whole tissue. Indeed, very little is known about the acute functional alterations induced by the toxin on the actual target of the bacterium: the gastric epithelium.
To gather novel insight into the role played by VacA in the Hp
parasitic cycle, we have challenged the intact gastric mucosa from
Rana esculenta and analyzed one of its major physiological functions: the acid and alkaline secretion balance. Our data show that,
despite the phylogenetic distance, this amphibian gastric model can be
very useful in the study of Hp virulence factors. Moreover, we
discovered that an early effect of VacA consists of a modification of
the permeability of the apical plasma membrane of SECs to
HCO
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissue preparation. Experiments on gastric mucosa of Rana esculenta were performed in accordance with the Italian guidelines for animal experimentation. The frogs were kept in an aquarium at room temperature and killed by decapitation and destruction of the spinal cord and brain. The stomach was removed, and the fundic mucosa was separated from the muscle layer by blunt dissection. The isolated stomach preparation was mounted in one of two different types of Lucite chambers for measuring either acid/alkaline secretion or transepithelial and cell membrane electrical parameters.
Acid and alkaline secretion measurements. Tissues were mounted vertically between two halves of a Lucite chamber having an exposed area of 0.64 cm2. Each half-chamber consisted of a circular fluid canal of 2.5 ml total volume filled with modified Ringer solution that was constantly recirculated by means of a bubble lift.
The control Ringer solution on the serosal side contained (in mM): 102.4 Na+, 4.0 K+, 1.8 Ca2+, 0.8 Mg2+, 91.4 ClIntracellular measurements.
The dissected gastric mucosa was mounted horizontally as a flat sheet
between two halves of an open-top Lucite chamber (aperture 0.2 cm2). Both the serosal and mucosal surfaces of the tissue
were continuously superfused with Ringer solution at room temperature
(20-24°C) containing (in mM): 102.4 Na+, 4.0 K+, 1.8 Ca2+, 0.8 Mg2+, 91.4 Cl, 17.8 HCO
when this
ion was reduced to 2 mM; in this solution, raising Ca2+ to
5 mM through addition of calcium gluconate kept the Ca2+
activity constant.
Microelectrodes.
Double-barrelled pH microelectrodes were constructed as previously
described (16). Briefly, two molten pieces of
filament-containing aluminum silicate glass tubing of different
diameters (Hilgenberg, Malsfeld, Germany) were twisted together. The
capillaries were then pulled (tip length 20 mm) in a PE2
vertical puller (Narishige, Tokyo, Japan). The thick channel was
silanized in dimethyldichlorosilane vapor (Serva, Heidelberg, Germany).
The tip was back-filled with H+ ligand (Hydrogen Ionophore
II, Cocktail A/Cocktail B; Fluka, Buchs, Switzerland), and the shaft
was then filled with a buffer solution containing (in mM): 500 KCl,
64.7 NaH2PO4, and 85.3 Na2HPO4, pH 7.0. The reference channel
contained 500 mM KCl. Average slope and resistance of the electrodes
were 55.6 ± 0.4 mV/pH unit (n = 23), 292 ± 29 G
(selective channel), and 187 ± 19 M
(reference channel). All microelectrodes were calibrated in the upper half of the
chamber before and after each puncture by flushing the chamber with
NaCl solutions containing a mixture of KH2PO4
and Na2HPO4 to yield pH values between 6.8 and
7.8 (osmolarity: 230 mosM).
Toxin. VacA was extracted from the extracellular medium from cultures of the Hp strain CCUG 17874 as previously described (34) and purified by fractionized precipitation, ion exchange, and HPLC chromatography and stored at 4°C in PBS at concentrations of 0.1-0.2 mg/ml. Activation was achieved by pretreatment at pH 2.0 for 5 min at 37°C (15). The toxin concentration was chosen according to the results of pilot experiments, in which the range of 10-60 nM was tested, with the aim of achieving maximal effects with the lowest toxin concentration.
Data analysis and statistics. All measurements are expressed as mean values ± SE of m individual transepithelial experiments or n individual micropuncture recordings on m tissues from which data were analyzed. The significance of the observation was evaluated by Student's t-test for paired or unpaired data as appropriate and a P value <0.05 denoted a statistical difference.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Studies in the whole intact epithelium.
Hp resides within the mucus layer or is anchored to the luminal surface
by means of the adhesines (3, 18, 27, 28). Hence, VacA
toxin is presumably released by the bacterium directly onto the apical
cell membrane. To mimic these in vivo conditions, before VacA addition,
the mucus layer of stomach preparations was removed either by gentle
suction or by pretreatment with a mucolytic solution (see
Measurements in the gastric gland lumen). The
gastric fundus mucosa was bathed in HCO
Effect of VacA on acid secretion.
Stimulation of control gastric tissue with histamine (500 µM)
elicited an average rate of acid secretion of 3.86 ± 0.22 µeq · cm2 · h
1, whereas
the Vt in these conditions was
23.0 ± 1.5 mV (lumen negative; m = 25). Tissue exposition to a
placebo control solution (consisting of 60 µl PBS and 6 µl 0.1 N
HCl, a cocktail that represents the VacA solvent) did not modify acid
secretion (from 3.91 ± 0.43 to 3.66 ± 0.35 µeq · cm
2 · h
1;
m = 9). Whereas similar results were obtained when the
epithelia were exposed to na-VacA, exposure to 40 nM a-VacA
significantly reduced HCl secretion by ~25% within 3 h (from
3.82 ± 0.37 to 2.66 ± 0.38 µeq · cm
2 · h
1;
m = 9; P < 0.001; Fig.
1A). During this period,
Vt depolarized (
Vt = 4.5 ± 1.5 mV;
P < 0.01) in control conditions.
Vt changes either in a-VacA or na-VacA were not
significant.
|
Effect of VacA on alkaline secretion.
ASR of the whole tissue was measured after inhibition of spontaneous
acid secretion by the H2 receptor antagonist cimetidine (100 µM in
the serosal bath). Under control conditions, i.e., in the presence of
the placebo cocktail (see Effect of VacA on acid secretion),
ASR decreased significantly over a period of 3 h, from 1.15 ± 0.1 to 0.65 ± 0.09 µeq · cm2 · h
1
(n = 13; P < 0.01). After correction
for the control data, ASR values recorded following addition of toxin
indicated that na-VacA does not induce significant changes in ASR. On
the contrary, a-VacA elicited a significant increase of ~50% in the
alkali output (Fig. 1B).
Measurements in the gastric gland lumen.
VacA-induced reduction of acid secretion and relative increase of ASR
may depend on the targeting of either the SECs, most directly exposed
to the toxin, and/or of acid-secreting oxyntopeptic cells (OCs) located
deeply in the glands. To discriminate between the two possibilities,
the action of VacA on the OC secretory activity was followed directly
by monitoring HCl secretion in the lumen of single gastric glands with
a method recently developed in our laboratory (16). Unique
observations of the glandular pH (pHgl) were made in situ
using double-barrelled proton-sensitive microelectrodes. The insertion
of the microelectrode in the gland lumen was achieved by first impaling
an oxyntopeptic cell and then gradually advancing the electrode until
the tip broke into the gland lumen (Fig.
2A). The correct positioning
of the microelectrode tip was established by the following criteria:
1) the near identity of the glandular potential
(Vgl), the voltage recorded via the nonselective
channel of the microelectrode, with the Vt;
2) the near identity of the electrical resistance recorded
between the microelectrode reference channel and serosal bath
macroelectrode with Rt (the resistance recorded
between serosal and mucosal bath macroelectrodes); 3) the
strong acidification of the gland lumen in response to stimulation with
histamine; and 4) the eventual, but not immediate, response
to pH changes in the luminal bath. The latter was also employed to
evaluate whether mucosally applied toxin might penetrate the gastric
pits and contact the OCs directly. The time course of the change in
pHgl shows that the gland content may be effectively
replaced by the bathing solution in ~5 min. Because protons are much
more diffusible in aqueous solution than other molecules, we
repeated the same type of experiment with Ca2+-sensitive
microelectrodes. By exposing the luminal side of the epithelium to
solutions with different Ca2+ concentrations, it was found
that a change in Ca2+ concentration was also recorded
within 5 min after the change of solutions (Caroppo R, Debellis L, and
Curci S, unpublished results). It is therefore likely that the
apical membrane of the OC can be slowly reached by the cytotoxin.
|
|
Measurements in surface epithelial cells. Given that the OCs appeared to be functionally intact following treatment with the toxin, we next investigated SECs as the targets of VacA. Double-barrelled proton-sensitive microelectrodes were used to measure SEC apical membrane potential (Va) and intracellular pH (pHi) in resting tissues. In these experiments, the dissected mucosa was mounted horizontally, with the mucosal side facing up. Micropunctures were preceded by removal of the surface mucus layer by gentle suction to improve the VacA efficacy, as explained above. The intracellular pH measured in six cells averaged 7.35 ± 0.06 pH units and remained unchanged ~90 min after exposure to 40 nM a-VacA.
The effect of the toxin was hence evaluated by measuring the pHi response to sudden changes in luminal HCO
|
|
Effect of NPPB on ionic permeability induced by VacA.
The data presented above indicate that exposure to VacA increases the
anionic permeability of the SEC apical membrane, an effect observed
previously in artificial planar lipid bilayers (55) and in
HeLa cell plasma membranes (52, 56). In the latter
studies, VacA was shown to form anion-selective channels, inhibited by
the Cl-channel blocker NPPB but insensitive to IAA-94.
VacA-induced increase of anion permeability in the SEC apical membrane
was also blocked by NPPB. As shown in the experiment of Fig.
6 and in analogy with the previous
observations on other cell types, exposure to 50 µM NPPB decreased
the pHi response to luminal [HCO
0.04 ± 0.03 pH units
(n = 3; P < 0.01). At variance, IAA-94
did not modify the VacA effect (not shown).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Detailed studies of the action of Hp virulence factors on the physiology of the gastric mucosa have been limited by the lack of suitable experimental systems for measurements at the cellular level. The amphibian model used here affords the possibility to study directly, with electrophysiological techniques applied to OCs and SECs, the effects of acute topical addition of the cytotoxin VacA to the luminal surface of the isolated gastric mucosa perfused in vitro. In the same preparation, we also developed an approach that allowed us to monitor pH changes in the lumen of single gastric glands in situ. The present study tried to mimic as closely as possible the in vivo situation to ensure a rapid contact of the toxin with the cell surface. In fact, our data demonstrate that VacA is capable of interacting efficiently with amphibian gastric cells.
The most important implication of our observations is that one of the
early events in the Hp infection appears to be the membrane insertion
of VacA with the formation of anion-selective channels that can
increase efflux of HCO
A similar result was also obtained in a recent study performed on the rat duodenum perfused in vivo, where an increase in alkaline secretion was described in response to treatment with Hp water extracts (23).
Our data clearly indicate that, at least in the early stage of
intoxication, VacA gives rise to an increased alkali efflux in the
amphibian gastric mucosa. This conclusion is based on the following
lines of evidence: 1) the significant reduction in HCl secretion during stimulation with histamine (Fig. 1A);
2) the functional integrity of the OCs as tested in the
experiments with pH microelectrodes in the gland lumen (Fig. 3);
3) the increase in ASR monitored in response to the
cytotoxin (Fig. 1B); and 4) the observation that
the transepithelial response to luminal Cl reduction
(Fig. 5) or to HCO
The fact that the decrease in acid secretion is not quantitatively
comparable with the increase in alkali output is not against this
conclusion, because direct quantitative comparison between these two
phenomena cannot be accomplished. In fact, as discussed in a previous
work (11), ASR is most probably underestimated due to
CO2 diffusion from the
HCO
Furthermore, according to previous studies, alkaline secretion seems to originate not only from the SECs (22, 54) by mechanisms that are not fully understood (6) but also from the OCs (11, 17). The fact that pHgl, either at rest or after stimulation with histamine, did not change in response to the toxin clearly precludes any short-term involvement of these cells in the change in ASR. Therefore, because amphibian gastric mucosa is composed of only SECs and OCs, with the exception of a few neck cells (see Fig. 2A), the SECs are the most likely cell type to be involved in the change in ASR.
The experiments using micropuncture of the SECs with pH microelectrodes
confirmed that these cells are responsible for the change in alkaline
output and that this effect has to be attributed to a single virulence
factor: VacA. As shown here and also in previous studies from our
laboratories, the anionic permeability of the SEC apical membrane is
negligible under control conditions. Reducing luminal
[Cl] does not significantly alter SEC intracellular
Cl
activity (12, 13) (see also Fig. 5), and
reduction of luminal [HCO
reduction. This implies that exposure to the
cytotoxin resulted in a modification of the SEC apical membrane anion permeability.
An additional point of evidence in support of this conclusion is
provided by the changes observed in the Va and
in the ratio of the SEC apical/basolateral membrane resistance,
expressed as VDR. After mucosal perfusion with low or high
HCO
On the other hand, the observation that the responses of
Vt and of SEC Va to
luminal low Cl were not influenced by the toxin confirms
that the OCs were not affected by VacA. Such responses originate from
the OC apical membrane (6, 12, 31) rather than from the
SECs, which scarcely contribute to the tissue conductance
(31).
Furthermore, the observation that Vt and
Va responses to luminal Cl
reduction were larger than the responses to HCO
conductance of the OC apical membrane is larger than the conductance to
other anions, such as the HCO
In view of the finding that the specific target cell of the toxin is the SEC, it is not surprising that VacA does not elicit significant changes in transepithelial electrical parameters such as Vt and Rt. In fact, SECs represent a low-conductance pathway across the gastric mucosa, which operates in parallel with the prevalent OC conductance, and therefore the amount of SEC contribution does not exceed 10% of total transepithelial conductance (31). In such a situation, any electrical signal that originates at the apical membrane of SECs will be strongly attenuated.
Moreover, in a study on confluent monolayers of different cell lines
(MDCK, T84, epH4, Caco-2), the paracellular permeability increased and
the Rt decreased (43) only when the
monolayer Rt values were >1
k · cm2. Therefore, the electrical resistance of
the amphibian stomach having, as observed, a value of ~600
· cm2 is expected not to be modified by VacA.
The most straightforward interpretation of our results is that
anion-selective channels made by VacA directly modify the apical plasma
membrane permeability of SECs. In fact, the VacA channel conducts
Cl and HCO
An important aspect of these findings is their possible significance
for clinical observations. The reduction in HCl secretion during
stimulation with histamine observed in our experiments appears to fit
with previous clinical studies on humans (20, 24, 26, 30)
and in animals in vivo (4, 29). Nevertheless, it is also
evident that the amount of alkali that may leave the SECs through VacA
channels in our model (1 µeq · cm2 · h
1) is too
modest to explain the significant pH changes observed in the gastric
lumens of Hp-infected patients. In these studies, such a decrease was
attributed to either a dysfunction of secreting cells or to a marked
reduction of their number after Hp infection. In our model, on the
contrary, the decrease in acid secretion observed on the whole tissue
was not the result of an altered secretory activity of the OCs.
However, we document that HCO
Therefore it seems unlikely that, at least in the short term, VacA is responsible for the impairment of the secretory function of OCs observed in chronic infections (8, 21, 25, 32, 51).
A relevant implication of the alkali exit from SECs, to which bacterial
cells are intimately bound, can be envisaged anyway inasmuch as an
increased HCO
![]() |
ACKNOWLEDGEMENTS |
---|
We thank K. Padovano, M. Paradiso, B. Pesetti A. Gerbino, S. Ciraci, and A. Colasuonno for participation in some of the measurements and Dr. A. Hofer (Harvard Medical School), Prof. M. Zoratti, and Dr. I. Szabò (University of Padova) for useful discussions and suggestions.
![]() |
FOOTNOTES |
---|
This work was supported by Consiglio Nazionale delle Ricerca (Grants 97.01168.PF49 and 99.02443.C), by Grants MURST 40% on 1) "molecular and cellular mechanisms of cellular and tissue damage and response induced by Helicobacter pylori virulence" and 2) "inflammation."
Address for reprint requests and other correspondence: L. Debellis, Dipartimento di Fisiologia Generale e Ambientale, Università di Bari, via Amendola 165/A, 70126 Bari, Italy (E-mail: debellis{at}biologia.uniba.it).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 5 February 2001; accepted in final form 16 August 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Atherton, JC,
Cao P,
Peek RMJ,
Tummuru MK,
Blaser MJ,
and
Cover TL.
Mosaicism in vacuolating cytotoxin alleles of Helicobacter pylori. Association of specific VacA types with cytotoxin production and peptic ulceration.
J Biol Chem
270:
17771-17777,
1995
2.
Blaser, MJ.
Helicobacter pylori: microbiology of a "slow" bacterial infection.
Trends Microbiol
1:
255-259,
1993[Medline].
3.
Boren, T,
Falk P,
Roth KA,
Larson G,
and
Normark S.
Attachment of Helicobacter pylori to human gastric epithelium mediated by blood group antigens.
Science
262:
1892-1895,
1993[ISI][Medline].
4.
Brzozowski, T,
Konturek PC,
Konturek SJ,
Bobrzynski A,
Pajdo R,
Stachura J,
Ghiara P,
and
Hahn EG.
Gastric secretion and ulcer healing in mouse stomach infected with cytotoxin expressing strain of Helicobacter pylori.
J Physiol Pharmacol
49:
387-403,
1998[ISI][Medline].
5.
Burns, BP,
Hazell SL,
and
Mendez GL.
Acetyl-CoA carboxilase activity in Helicobacter pylori and the requirement of increased CO2 for growth.
Microbiology
141:
3113-3118,
1995[Abstract].
6.
Caroppo, R,
Debellis L,
Valenti G,
Alper S,
Frömter E,
and
Curci S.
Is resting state HCO-HCO
7.
Chu, S,
Tanaka S,
Kaunitz J,
and
Montrose M.
Dynamic regulation of gastric surface pH by luminal pH.
J Clin Invest
103:
605-612,
1999
8.
Claeys, D,
Faller G,
Appelmelk BJ,
Negrini R,
and
Kirchner T.
The gastric H+,K+-ATPase is a major autoantigen in chronic Helicobacter pylori gastritis with body mucosa atrophy.
Gastroenterology
115:
340-347,
1998[ISI][Medline].
9.
Covacci, A,
Telford JL,
Del Giudice G,
Parsonnet J,
and
Rappuoli R.
Helicobacter pylori virulence and genetic geography.
Science
284:
1328-1333,
1999
10.
Cuppoletti, J,
Baker AM,
and
Malinowska DH.
Cl channels of the gastric parietal cell that are active at low pH.
Am J Physiol Cell Physiol
264:
C1609-C1618,
1993
11.
Curci, S,
Debellis L,
Caroppo R,
and
Frömter E.
Model of bicarbonate secretion by resting frog stomach fundus mucosa. I. Transepithelial measurements.
Pflügers Arch
428:
648-654,
1994[ISI][Medline].
12.
Curci, S,
and
Schettino T.
Effect of external sodium on intracellular chloride activity in the surface cells of frog gastric mucosa.
Pflügers Arch
401:
152-159,
1984[ISI][Medline].
13.
Curci, S,
Schettino T,
and
Frömter E.
Hystamine reduces Cl activity in surface ephitelial cells of frog gastric mucosa.
Pflügers Arch
406:
204-211,
1986[ISI][Medline].
14.
Czajkowsky, DM,
Iwamoto H,
Cover TL,
and
Shao Z.
The vacuolating toxin from Helicobacter pylori forms hexameric pores in lipid bilayers at low pH.
Proc Natl Acad Sci USA
96:
2001-2006,
1999
15.
De Bernard, M,
Papini E,
de Filippis V,
Gottardi E,
Telford J,
Manetti R,
Fontana A,
Rappuoli R,
and
Montecucco C.
Low pH activates the vacuolating toxin of Helicobacter pylori, which becomes acid and pepsin resistant.
J Biol Chem
270:
23937-23940,
1995
16.
Debellis, L,
Caroppo R,
Frömter E,
and
Curci S.
Alkaline secretion by frog gastric gland measured with pH microelectrodes in the gland lumen.
J Physiol (Lond)
513:
235-241,
1998
17.
Debellis, L,
Iacovelli C,
Frömter E,
and
Curci S.
Model of bicarbonate secretion by resting frog stomach fundus mucosa. II. Role of the oxyntopeptic cells.
Pflügers Arch
428:
655-663,
1994[ISI][Medline].
18.
Doig, P,
Austin JW,
Kostrzynska M,
and
Trust TJ.
Production of a conserved adhesin by the human gastroduodenal pathogen Helicobacter pylori.
J Bacteriol
174:
2539-2547,
1992[Abstract].
19.
Eaton, KA,
Brooks CL,
Morgan DR,
and
Krakowka S.
Essential role of urease in pathogenesis of gastritis induced by Helicobacter pylori in gnotobiotic piglets.
Infect Immun
59:
2470-2475,
1991[ISI][Medline].
20.
El-Omar, EM,
Oien K,
El-Nujumi A,
Gillen D,
Wirz A,
Dahill S,
Williams C,
Ardill JE,
and
McColl KE.
Helicobacter pylori infection and chronic gastric acid hyposecretion.
Gastroenterology
113:
15-24,
1997[ISI][Medline].
21.
Figura, N,
Vindigni C,
Covacci A,
Presenti L,
Burroni D,
Vernillo R,
Banducci T,
Roviello F,
Marrelli D,
Biscontri M,
Kristodhullu S,
Gennari C,
and
Vaira D.
cag A positive and negative Helicobacter pylori strains are simultaneously present in the stomach of most patients with non-ulcer dyspepsia: relevance to histological damage.
Gut
42:
772-778,
1998
22.
Flemström, G.
Cl dependence of HCO
23.
Flemström, G,
Hällgren A,
Nylander O,
Engstrand L,
Wilander E,
and
Allen A.
Adherent surface mucus gel restricts diffusion of macromolecules in rat duodenum in vivo.
Am J Physiol Gastrointest Liver Physiol
277:
G375-G382,
1999
24.
Furuta, T,
Baba S,
Takashima M,
Futami H,
Arai H,
Kajimura M,
Hanai H,
and
Kaneko E.
Effect of Helicobacter pylori infection on gastric juice pH.
Scand J Gastroenterol
33:
357-363,
1998[ISI][Medline].
25.
Gööz, M,
Hammond CE,
Larsen K,
Mukin YV,
and
Smolka AJ.
Inhibition of human gastric H+(K+)-ATPase alpha-subunit gene expression by Helicobacter pylori.
Am J Physiol Cell Physiol
278:
C981-C991,
2000.
26.
Iijima, K,
Ohara S,
Sekine H,
Koike T,
Kato K,
Asaki S,
Shimosegawa T,
and
Toyota T.
Changes in gastric acid secretion assayed by endoscopic gastrin test before and after Helicobacter pylori eradication.
Gut
46:
20-26,
2000
27.
Ilver, D,
Arnqvist A,
Ogren J,
Frick IM,
Kersulyte D,
Incecik ET,
Berg DE,
Covacci A,
Engstrand L,
and
Boren T.
Helicobacter pylori adhesin binding fucosylated histo-blood group antigens revealed by retagging.
Science
279:
373-377,
1998
28.
Kobayashi, Y,
Okazaki K,
and
Murakami K.
Adhesion of Helicobacter pylori to gastric epithelial cells in primary cultures obtained from stomach of various animals.
Infect Immun
61:
4058-4063,
1993[Abstract].
29.
Konturek, PC,
Brzozowski T,
Konturek SJ,
Stachura J,
Karczewska E,
Pajdo R,
Ghiara P,
and
Hahn EG.
Mouse model of Helicobacter pylori infection: studies of gastric function and ulcer healing.
Aliment Pharmacol Ther
13:
333-346,
1999[ISI][Medline].
30.
Konturek, PC,
Konturek SJ,
Bobrzynski A,
Kwiecien N,
Obtulowicz W,
Stachura J,
Hahn EG,
and
Rembiarz K.
Helicobacter pylori and impaired gastric secretory functions associated with duodenal ulcer and atrophic gastritis.
J Physiol Pharmacol
48:
365-367,
1997[ISI][Medline].
31.
Kottra, G,
Iacovelli C,
Caroppo R,
Curci S,
Bakos P,
and
Frömter E.
Contribution of surface epithelial cells to total conductance of Necturus gastric fundus mucosa.
Am J Physiol Gastrointest Liver Physiol
270:
G902-G908,
1996
32.
Kuipers, EJ,
Uyterlinde AM,
Pena AS,
Roosendaal R,
Pals G,
Nelis GF,
Festen HP,
and
Meuwissen SG.
Long-term sequelae of Helicobacter pylori gastritis.
Lancet
345:
1525-1528,
1995[ISI][Medline].
33.
Labigne, A,
Cussac V,
and
Courcoux P.
Shuttle cloning and nucleotide sequences of Helicobacter pylori genes responsible for urease activity.
J Bacteriol
173:
1920-1931,
1991[ISI][Medline].
34.
Manetti, R,
Massari P,
Burroni D,
de Bernard M,
Marchini A,
Olivieri R,
Papini E,
Montecucco C,
Rappuoli R,
and
Telford JL.
Helicobacter pylori cytotoxin: importance of native conformation for induction of neutralizing antibodies.
Infect Immun
63:
4476-4480,
1995[Abstract].
35.
McClain, MS,
Schraw W,
Ricci V,
Boquet P,
and
Cover TL.
Acid activation of Helicobacter pylori vacuolating cytotoxin (VacA) results in toxin internalization by eukaryotic cells.
Mol Microbiol
37:
433-442,
2000[ISI][Medline].
36.
Molinari, M,
Galli C,
de Bernard M,
Norais N,
Ruysschaert JM,
Rappuoli R,
and
Montecucco C.
The acid activation of Helicobacter pylori toxin VacA: structural and membrane binding studies.
Biochem Biophys Res Commun
248:
334-340,
1998[ISI][Medline].
37.
Moll, G,
Papini E,
Colonna R,
Burroni D,
Telford JL,
Rappuoli R,
and
Montecucco C.
Lipid interaction of the 37-kDa and 58-kDa fragments of the Helicobacter pylori citotoxin.
Eur J Biochem
234:
947-952,
1995[Abstract].
38.
Montecucco, C,
Papini E,
de Bernard M,
Telford JL,
and
Rappuoli R.
Helicobacter pylori vacuolating cytotoxin and associated pathogenic factors.
In: The Comprehensive Sourcebook of Bacterial Protein Toxin, edited by Alouf JE,
and Freer JH.. San Diego: Academic, 1999, p. 264-283.
39.
Montecucco, C,
Papini E,
de Bernard M,
and
Zoratti M.
Molecular and cellular activities of Helicobacter pylori pathogenic factors.
FEBS Lett
452:
16-21,
1999[ISI][Medline].
40.
Padilla, PI,
Wada A,
Yahiro K,
Kimura M,
Niidome T,
Aoyagi H,
Kumatori A,
Anami M,
Hayashi T,
Fujisawa J,
Saito H,
Moss J,
and
Hirayama T.
Morphologic differentiation of HL-60 cells is associated with appearance of RPTPbeta and induction of Helicobacter pylori VacA sensitivity.
J Biol Chem
275:
15200-15206,
2000
41.
Pagliaccia, C,
Wang XM,
Tardy F,
Telford JL,
Ruysschaert JM,
and
Cabiauw V.
Structure and interaction of VacA of Helicobacter pylori with a lipid membrane.
Eur J Biochem
267:
104-109,
2000
42.
Papini, E,
De Bernard M,
Milia E,
Bugnoli M,
Zerial M,
Rappuoli R,
and
Montecucco C.
Cellular vacuoles induced by Helicobacter pylori originate from late endosomal compartments.
Proc Natl Acad Sci USA
91:
9720-9724,
1994
43.
Papini, E,
Satin B,
Norais N,
de Bernard M,
Telford JL,
Rappuoli R,
and
Montecucco C.
Selective increase of the permeability of polarized epithelial cell monolayers by Helicobacter pylori vacuolating toxin.
J Clin Invest
102:
813-820,
1998
44.
Pelicic, V,
Reyrat JM,
Sartori L,
Pagliaccia C,
Rappuoli R,
Telford JL,
Montecucco C,
and
Papini E.
Helicobacter pylori VacA cytotoxin associated with the bacteria increases epithelial permeability independently of its vacuolating activity.
Microbiology
145:
2043-2050,
1999[Abstract].
45.
Quigley, EM,
and
Turnberg LA.
pH of the microclimate lining human gastric and duodenal mucosa in vivo. Studies in control subjects and in duodenal ulcer patients.
Gastroenterology
92:
1876-1884,
1987[ISI][Medline].
46.
Ricci, V,
Galmiche A,
Doye A,
Necchi V,
Solcia E,
and
Boquet P.
High cell sensitivity to Helicobacter VacA toxin depends on GPI-anchored protein and is not blocked by inhibition of the clathrin-mediated pathway of endocytosis.
Mol Biol Cell
11:
3897-3909,
2000
47.
Saccomani, G,
Psarras CG,
Shmith PR,
Kirk KL,
and
Shoemaker LR.
Histamine-induced chloride channels in apical membrane of isolated rabbit parietal cells.
Am J Physiol Cell Physiol
260:
C1000-C1011,
1991
48.
Satin, B,
Del Giudice G,
Della Bianca V,
Dusi S,
Laudanna C,
Tonello F,
Kelleher D,
Rappuoli R,
Montecucco C,
and
Rossi F.
The neutrophil-activating protein (HP-NAP) of Helicobacter pylori is a protective antigen and a major virulence factor.
J Exp Med
191:
1467-1476,
2000
49.
Segal, ED,
Cha J,
Lo J,
Falkow S,
and
Tompkins LS.
Altered states: involvement of phosphorylated cag A in the induction of host cellular growth changes by Helicobacter pylori.
Proc Natl Acad Sci USA
96:
14559-14564,
1999
50.
Stein, M,
Rappuoli R,
and
Covacci A.
Tyrosine phosphorylation of the Helicobacter pylori cag A antigen after cag-driven host cell translocation.
Proc Natl Acad Sci USA
97:
1263-1268,
2000
51.
Steininger, H,
Faller G,
Dewald E,
Brabletz T,
Jung A,
and
Kirchner T.
Apoptosis in chronic gastritis and its correlation with antigastric autoantibodies.
Virchows Arch
433:
13-18,
1998[ISI][Medline].
52.
Szabò, I,
Brutsche S,
Tombola F,
Moschioni M,
Satin B,
Telford JL,
Rappuoli R,
Montecucco C,
Papini E,
and
Zoratti M.
Formation of anion-selective channels in the cell plasma membrane by the toxin VacA of Helicobacter pylori is required for its biological activity.
EMBO J
18:
5517-5527,
1999
53.
Takeuchi, K,
Magee D,
Critchlow J,
Matthews J,
and
Silen W.
Studies of the pH gradient and thickness of frog gastric mucus gel.
Gastroenterology
84:
331-340,
1983[ISI][Medline].
54.
Takeuchi, K,
Merhav A,
and
Silen W.
Mechanism of luminal alkalinization by bullfrog fundic mucosa.
Am J Physiol Gastrointest Liver Physiol
243:
G377-G388,
1982
55.
Tombola, F,
Carlesso C,
Szabò I,
De Bernard M,
Reyrat JM,
Telford JL,
Rappuoli R,
Montecucco C,
Papini E,
and
Zoratti M.
Helicobacter pylori vacuolating toxin forms anion-selective channels in planar lipid bilayer: possible implications for the mechanism of cellular vacuolation.
Biophys J
76:
1401-1409,
1999
56.
Tombola, F,
Oregna F,
Brutsche S,
Szabò I,
Del Giudice G,
Rappuoli R,
Montecucco C,
Papini E,
and
Zoratti M.
Inhibition of the vacuolating and anion channels activities of the VacA toxin of Helicobacter pylori.
FEBS Lett
460:
221-225,
1999[ISI][Medline].
57.
Tsuda, M,
Karita M,
Morshed MG,
Okita K,
and
Nakazawa T.
A urease-negative mutant of Helicobacter pylori constructed by allelic exchange mutagenesis lacks the ability to colonize the nude mouse stomach.
Infect Immun
62:
3586-3589,
1994[Abstract].
58.
Weeks, DL,
Eskandari S,
Scott DR,
and
Sachs G.
A H+-gated urea channel: the link between Helicobacter pylori urease and gastric colonization.
Science
287:
482-485,
2000
59.
Yahiro, K,
Niidome T,
Kimura M,
Hatakeyama T,
Aoyagi H,
Kurazono H,
Imagawa K,
Wada A,
Moss J,
and
Hirayama T.
Activation of Helicobacter pylori VacA toxin by alkaline or acid conditions increases its binding to a 250-kDa receptor protein-tyrosine phosphatase beta.
J Biol Chem
274:
36693-36699,
1999