Documente Academic
Documente Profesional
Documente Cultură
Background & Aims: The pathophysiology behind Heli- with peptic ulcer is the ability of the duodenal mucosa
cobacter pyloriinduced gastroduodenal dysfunction is to increase its alkaline secretion in response to acid expo-
incompletely understood. The aim of this study was to sure.4 The physiological mediation of acid-induced duo-
investigate if a water extract of H. pylori distorts acid- denal mucosal alkaline secretion involves several regula-
induced duodenal mucosal alkaline secretion. Meth- tory principles, e.g., local synthesis of prostaglandins and
ods: Chloralose-anesthetized rats were prepared for du- neurohumoral control.5 Furthermore, recent studies
odenal luminal perfusion and in situ pH-stat titration of
showed that the acid-induced response involves also the
mucosal alkaline secretion. Results: Mucosal bicarbon- 6,7
L-arginine/nitric oxide pathway. The question was
ate secretion increased approximately 55%60% after
a 5-minute exposure to 10 mmol/L HCl. This response raised if luminal factors, released from the H. pylori infec-
was absent when water extracts of three strains of H. tion in patients with duodenal ulcer, disturb the mucosal
pylori (protein content, 0.220 mg/mL) had been responsiveness to luminal acid, via interference with the
added to the perfusate. Presence of 3 mmol/L L-argi- L-arginine/NO axis. Theoretically, this could be related
nine, but not the stereoisomer D-arginine, in the luminal to the microbe itself by excreted toxins disturbing NO
perfusate reversed the H. pylori extract blockade of formation or to indirect effects related to the inflamma-
acid-induced mucosal alkaline secretion. High-perfor- tory reaction. We decided to test the former possibility
mance liquid chromatographybased analyses showed in an in vivo rat model by briefly exposing the duodenal
that the endogenous nitric oxide synthase inhibitor mucosa to a water extract of H. pylori. Presence of H.
asymmetric dimethyl arginine (ADMA) increased four- pylori extracts (Hpx) markedly inhibited the mucosal re-
fold in duodenal perfusate and fivefold in duodenal tis-
sponsiveness to acid because of local interference with
sue after H. pylori extract exposure. In vitro proteolysis
of H. pylori extract also resulted in a substantial accu-
NO synthase (NOS). Some of these data have been pre-
mulation of ADMA. Exogenously administered ADMA, sented in abstract form previously.8
giving similar tissue concentrations, inhibited the mu- Materials and Methods
cosal alkaline response to acid exposure. Conclusions:
Water extracts of H. pylori inhibit acid-induced mucosal General
alkaline secretion via interference with mucosal NO The experiments were approved by the Animal Ethics
synthase. Committee of Goteborg University. Male SpragueDawley
rats (Mollegard, Denmark) were anesthetized with methohexi-
tal injected intraperitoneally (75 mg/kg body wt) and a-chlor-
H elicobacter pylori is now recognized as a key factor in
the pathogenesis of chronic gastroduodenitis and
peptic ulcer disease. A number of H. pylorirelated
alose injected intravenously (bolus, 50 mg/kg plus 25
mgrkg01rh01). The body temperature was maintained at
387C with a heating pad and lamp. A catheter was inserted
pathogenic mechanisms causing mucosal injury have into the trachea to ensure free airways. A femoral artery and
been suggested, e.g., the excretion of cytotoxic substances one vein were catheterized with polyethylene 50 tubes for
and indirect effects such as neutrophilic production of subsequent blood pressure measurements and drug infusions,
oxygen radicals with effects on mucosal viability. In addi- respectively. Blood pressure was recorded using a Statham
tion to direct damage of the gastroduodenal mucosa, H. P23Dc transducer (Statham, Hato Rey, Puerto Rico). To avoid
pylori infection is associated with functional disturbances,
Abbreviations used in this paper: ADMA, asymmetric dimethyl argi-
e.g., defects in the feedback regulation of gastrin release
nine; Hpx, Helicobacter pylori extract; NOS, nitric oxide synthase.
and gastric acid secretion.1 3 Another function reported q 1997 by the American Gastroenterological Association
to be disturbed by the presence of H. pylori in patients 0016-5085/97/$3.00
acidosis and dehydration caused by surgical trauma and the was centrifugated at 23,000g for 20 minutes (47C), and the
long period of general anesthesia, a 2% glucose solution con- supernatant was stored at 0707C until analysis.
taining 0.04 mol/L NaHCO3 was given intravenously through-
out the experiments at a rate of 1 mL/h. Drugs
For anesthesia, methohexital (Brietal; Lilly Inc., India-
Secretion napolis, IN) and a-chloralose (Sigma Chemical Co., St. Louis,
Duodenal mucosal alkaline (HCO30) secretion was MO) were used. L- and D-arginine hydrochloride (Sigma)
measured as described previously.9 Briefly, after a midline lapa- and proteinase K (Boerhinger Mannheim Scandinavia AB,
rotomy, a 1.6-cm segment of the duodenum with intact vascu- Bromma, Sweden) were freshly dissolved as stock solutions.
lar supply was isolated between two glass tubes connected to
a water-jacketed (387C) reservoir containing 150 mmol/L Experimental Protocol
NaCl. The saline was circulated through the segment using a The animals were left undisturbed for 1 hour after
gas-lift (pure air). The common bile duct was catheterized to surgery. Acid exposure was performed after a 4560-minute
avoid contamination of the segment by bile and pancreatic control period, and the circulating saline in the titration-cham-
juice. Alkaline secretion to the perfusate was continuously ber was then changed to body tempered 10 mmol/L HCl (made
titrated with HCl by pH-stat equipment. isotonic with the addition of NaCl) during 5 minutes. In a
separate series, without acid exposure, the acute effects of lumi-
Bacterial Water Extracts
nal administration of, e.g., bacterial extracts and L-arginine,
H. pylori strains E50 (provided by Dr. J.-P. Butzler, were studied. The animals were divided into different groups
WHO Collaborating Centre for Enteric Campylobacter, Brus- (each 6 animals) depending on treatment. The experiments
sels, Belgium); C21, a vacuolating cytotoxin negative strain were terminated by induction of cardiac arrest with intravenous
(provided by Dr. N. Figura, Siena, Italy); and Hel73, a vac- KCl.
uolating cytotoxin-positive strain (our clinical isolate from a
patient with duodenal ulcer), were used. The strains were kept Statistics
at 0707C until use and were then grown on Columbia agar Analysis of variance (ANOVA) for repeated measure-
plates with 8% horse blood in microaerophilic atmosphere of ments and Bonferronis post hoc test were used to evaluate
10% CO2 and 5% O2 for 4872 hours. Bacteria from agar significance of changes within groups. Secretory data obtained
plates were inoculated into Brucella broth supplemented with during the control period immediately before any interference
1% dimethyl-b-cyclodextrin (a gift from Teijin Ltd., Tokyo, (e.g., acid exposure) were used as basal conditions. Responses
Japan), vancomycin, trimethoprim, and polymyxin. The liquid to stimulations were analyzed by using the induced net change
bacterial cultures were grown for 72 hours under microaero- in secretion (prestimulatory value minus the value most differ-
philic conditions without shaking. Escherichia coli K12 strain ent from the prestimulatory value within 20 minutes after
C 600 was grown in LuriaBertani broth at 377C overnight. the stimulation period). Net changes were compared between
H. pylori from 1 L of liquid culture or 25 mL of E. coli culture groups by using one-way ANOVA and, when appropriate, a
was pelleted by centrifugation at 6000g and resuspended in t test. A P value of 0.05 was considered significant.
5 mL of distilled water. The bacterial suspensions were kept
at room temperature for 20 minutes and shaken occasionally. Results
Proteins extracted by this procedure were separated from bacte-
Effects of Bacterial Extracts on Basal
ria by centrifugation at 20,000g. The protein contents were
estimated by subtracting the absorbance value at 310 nm from
Secretion
the value at 280 nm, and protein concentration was expressed Basal mucosal alkaline secretion by the duodenal
in milligrams per milliliter. The water extracts were stored at segment in untreated controls ranged between 10 and
0707C until use. 17 mmolrcm01rh01. Similar steady-state secretory levels
Analyses of Arginine Analogues were observed in the groups with bacterial extracts added
to the perfusate resulting in a protein concentration of
Occurrence of monomethyl arginine, asymmetric di- 20 mg/mL (Figure 1). The acute effect of adding E50
methyl arginine (ADMA), and symmetric dimethyl arginine Hpx was studied in greater detail. A transient increase
in luminal perfusates and in the tissue of the duodenal segment
(P 0.05) in secretion with a magnitude and duration
under study was analyzed using a modified high-performance
liquid chromatographic method with fluorescence detection.10
of approximately 70% and 20 minutes, respectively, was
The luminal perfusates were frozen at 0707C until analysis. recorded immediately after addition of E50-Hpx to the
The duodenal segment, arranged for secretion study as de- perfusate (not shown in figure). This transient increase
scribed above, was dissected and immediately homogenized in in secretory values could not be attributed to either the
4 mL of 150 mmol/L NaCl (47C) using a Polytron PT homoge- titration technique, because the acid buffering capacity
nizer (Kinematic AG, Lucerne, Switzerland). The homogenate was similar in the control solutions, or to any significant
Effects of Arginine
Short-term addition L-arginine (3 mmol/L) to the
duodenal perfusate transiently lowered the alkaline secre-
tion by approximately 50%. The secretion returned to
baseline values within 20 minutes (n 6; data not
shown). Presence of L-arginine did not influence the re- Figure 3. Dose-response relationship between the presence of strain
sponse to 10 mmol/L HCl (n 6; data not shown). E50 Hpx at various concentrations and the increase in duodenal muco-
When 3 mmol/L L-arginine had been added to the Hpx- sal alkaline secretion elicited by a 5-minute exposure to 10 mmol/L
containing (2 mg/mL) luminal perfusate, acid exposure HCl. The effect of acid exposure was tested in 6 animals for each
Hpx concentration. A plotted value represents mean { SEM from each
increased the duodenal mucosal alkaline secretion to a group. *P 0.05, *** P 0.001; significan difference from control
level similar to one observed in untreated controls (Figure (equals zero concentration).
patients with duodenal ulcer.4 Furthermore, NO is a driks L. A water extract of Helicobacter pylori inhibits acid induced
increase in duodenal mucosal bicarbonate secretion in the rat
regulatory factor of several principal gastrointestinal (abstr). Gastroenterology 1996; 110:A136.
functions (e.g., local blood perfusion, mucosa-protective 9. Flemstrom G, Garner A, Nylander O, Hurst BC, Heylings JR. Sur-
functions, epithelial permeability, motility), and NO is face epithelial HCO 30 transport by mammalian duodenum in vivo.
Am J Physiol 1982; 243:G348 G358.
used as a cytotoxic agent in inflammatory reactions.14 17 10. Halawa I, Baig S, Qureshi GA. Use of high-performance liquid
Consequently, it may be of interest to consider inhibition chromatography in definin the abnormalities in the free amino
of the mucosal L-arginine/NO system as a virulence factor acid patterns in cerebrospinal flui of patients with aseptic men-
for gastrointestinal pathogens other than H. pylori. ingitis. Biomed Chromatograph 1991; 5:216 220.
11. Takeuchi K, Ohuchi T, Miyake H, Okabe S. Stimulation by nitric
In summary, the present results suggest that the water oxide synthase inhibitors of gastric and duodenal HCO 30 secre-
extract of H. pylori contains certain peptides that, when tion in rats. J Pharmacol Exp Ther 1993; 266:1512 1519.
in contact with the duodenal lumen, probably by means 12. Hallgren A, Flemstrom G, Sababi M, Nylander O. Effects of nitric
oxide inhibition on duodenal function in rats: involvement of neu-
of the inherent digestive proteolytic systems, become ral mechanisms. Am J Physiol 1995; 269:G246 G254.
degraded into free amino acid residues, accumulating 13. Hamlet A, Jonson C, Fandriks L. The mediation of increased
within the mucosa. Some of these amino acid residues, duodenal alkaline secretion in response to 10 mM HCl in the
anaesthetized rat. Support for the involvement of capsaicin-sen-
e.g., the presently analyzed ADMA, are potent inhibitors sitive nerve elements. Acta Physiol Scand 1992; 146:519 525.
of NOS that interfere with the mediation of the mucosal 14. Stark ME, Szurszewski JH. Role of nitric oxide in gastrointestinal
alkaline secretory response to luminal acid. and hepatic function and disease (review). Gastroenterology
1992; 103:1928 1949.
References 15. Whittle BJR. Nitric oxide in gastrointestinal physiology and pathol-
ogy. In: Johnson LR, ed. Physiology of the gastrointestinal tract.
1. El-Omar EM, Penman ID, Ardill JES, Chittajallu RS, Howie C, New York: Raven, 1994:267 294.
McColl KEL. Helicobacter pylori infection and abnormalities of 16. Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology, patho-
acid secretion in patients with duodenal ulcer disease. Gastroen- physiology, and pharmacology (review). Pharmacol Rev 1991; 43:
terology 1995; 109:681 691. 109 142.
2. McGowan CC, Cover TL, Blaser MJ. Helicobacter pylori and gas- 17. Anggard E. Nitric oxide: mediator, murderer, and medicine. Lan-
tric acid: biological and therapeutic implications. Gastroenterol- cet 1994; 343:1199 1206.
ogy 1996; 110:926 938. 18. Vallance P, Leone A, Calver A, Collier J, Moncada S. Accumulation
3. Olbe L, Hamlet A, Dalenback J, Fandriks L. A mechanism by which of an endogenous inhibitor of nitric oxide synthesis in chronic
Helicobacter pylori infection of the antrum contributes to the renal failure. Lancet 1992; 339:572 575.
development of duodenal ulcer. Gastroenterology 1996; 110: 19. Ueno S, Sano A, Kotani K, Kondoh K, Kakimoto Y. Distribution
1386 1394. of free methylarginines in rat tissues and the bovine brain. J
4. Hogan DL, Rapier RC, Dreilinger A, Koss MA, Basuk PM, Neurochem 1992; 59:2012 2016.
Weinstein WM, Nyberg LM, Isenberg JI. Duodenal bicarbonate 20. Webb KJ. Intestinal absorption of protein hydrolysis products: a
secretion: eradication of Helicobacter pylori and duodenal struc- review. J Anim Sci 1990; 68:3011 3022.
ture and function in humans. Gastroenterology 1996; 110:705
716.
5. Allen A, Flemstrom G, Garner A, Kivilaakso E. Gastroduodenal Received December 30, 1996. Accepted July 18, 1997.
mucosal protection. Physiol Rev 1993; 73:823 857. Address requests for reprints to: Lars Fandriks, M.D., Centre for
6. Bilski J, Konturek SJ. Role of nitric oxide in gastroduodenal alka- Gastroenterological Research, Goteborg University, P.O. Box 75032,
line secretion. J Physiol Pharmacol 1994; 45:541 553. S-400 36 Goteborg, Sweden. Fax: (46) 31-82-1866.
7. Johansson B, Bothmer vC, Holm M, Pettersson A, Jonson C, Supported by grant no. 8663 the Swedish Medical Research Coun-
Fandriks L. Acid induced increase in duodenal mucosal bicarbon- cil, the Bank of Sweden Tercentenary Foundation, the Gothenburg
ate secretion in the rat involves the L-Arg/NO pathway (abstr). Medical Association, and the Knut and Alice Wallenberg Foundation.
Gastroenterology 1996; 110:A144. The authors thank Anna Casselbrant and Soren Lundberg for excel-
8. Holm M, Bothmer vC, Johansson B, Bolin I, Pettersson A, Fan- lent technical assistance.