Journal of Gastroenterology and Hepatology (2001) 16, 755–762

gesting a dominant functional, rather than morphol-ogical alteration.10 In addition, it has been observed thatPHG is associated with a decrease in chief cell11 andparietal cell12 mass. A diminution of gastric acid secre-tion in partial portal vein ligated Wistar rats has beenattributed to a low energy state in the congested gastricmucosa in PHG.13

Various secretagogues that act on parietal cells andstimulate acid secretion are histamine, acetylcholineand gastrin. Histamine acts mainly through the secondmessenger cyclic adenosine monophosphate (cAMP),14

although reports also suggest that it can increase intra-cellular calcium [(Ca2+)i].15

Carbachol, which is a synthetic analog of acetyl-choline, stimulates acid secretion via an increase in

INTRODUCTION

Portal hypertensive gastropathy (PHG) is a well estab-lished clinical entity comprising of gastric vascularectasia1,2 at one end and fundic gland atrophy3 at theother end of the spectrum. The portal hypertensive(PHT) gastric mucosa has unique functional and mor-phological abnormalities that correspond to its in-creased sensitivity to damage by noxious agents such asalcohol4 and bile acids,5 which may be caused byimpaired mucosal oxygenation.6 The morphologic al-terations are very well studied.7,8 Functionally, it hasbeen observed that pentagastrin-stimulated acid secre-tion in rats is blunted.9 Hypochlorhydria, in the absenceof atrophy, has been reported in cirrhotic patients, sug-

GASTRITIS, GASTRIC ULCER, GASTRIC METAPLASIA:CLINICAL AND EXPERIMENTAL STUDIES

Modulation of acid secretion in common bile duct ligation-relatedgastropathy in Wistar rats

SIMRAN KAUR,* UPJEET KAUR,* NAVNEET AGNIHOTRI,* CHANDAN D TANDON† ANDSIDDHARTHA MAJUMDAR*

*Department Of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education andResearch and †Department of Biochemistry, Panjab University, Chandigarh, India

AbstractBackground: Portal hypertensive gastropathy is associated with fundic gland atrophy, resulting in adecrease in chief and parietal cells, and diminished acid secretion.Methods: Acid secretion by isolated parietal cells was measured (acridine orange retention), along withthe levels of various second messengers (intracellular Ca2+, cyclic adenosine monophosphate and proteinkinase C) in the common bile duct, ligated portal hypertensive rats and compared with sham-operated controls.Results: There was a significant decrease in the response of isolated parietal cells to the secretagogueshistamine and carbachol. This resulted in the blunted acid secretion in the common bile duct ligatedgroup. In addition, all the second messengers studied were significantly decreased as compared with thesham-operated controls.Conclusion: These results suggest that the blunted acid secretory response in the portal hypertensiverat is caused by an alteration in the intracellular signal transduction mechanism.© 2001 Blackwell Science Asia Pty Ltd

Key words: calcium, carbachol, gastropathy, histamine, portal hypertension, second messengers.

Correspondence: Dr U Kaur, Additional Professor, Department of Experimental Medicine and Biotechnology, PostgraduateInstitute of Medical Education & Research, Chandigarh 160012, India. Email: medinst@pgi.chd.nic.in

Accepted for publication 9 March 2001.

intracellular calcium, which is more marked as com-pared to histamine.16 Carbachol induces a biphasicresponse in intracellular calcium, with an initial spikebelieved to be caused by the release of calcium fromintracellular stores followed by a sustained plateaubecause of an increased influx of calcium from theextracellular medium.17

Gastrin has both a stimulatory and inhibitory effecton the parietal cells. The presence of gastrin receptorson parietal cells in the rat is controversial, and mostprobably gastrin (in the rat) acts by releasing histaminefrom the enterochromaffin-like cells18.Therefore, in thisstudy, the role of both histamine and carbachol only onisolated parietal cells was studied.

Gastropathy in partial portal vein ligated rats is asso-ciated with altered calcium homeostasis and decreasedlevels of cAMP and adenosine triphosphate (ATP) inisolated parietal cells.13

The status of various second messengers such ascalcium, cAMP, cyclic guanosine monophosphate(cGMP) and protein kinase C (PKC) in parietal cellsin intrahepatic portal hypertensive gastropathy has notbeen reported. Therefore, in this study, alterations inthese second messengers in relation to acid secretionwere studied.

METHODS

Chemicals

Histamine, carbachol, bovine serum albumin, pronase,collagenase, EGTA, percoll, acridine orange, Fura-2/AM, histone IIIS, dithiothreitol (DTT), diolein andphosphatidyl serine were obtained from the SigmaChemical Company (Sigma, St Louis, MO, USA). Allother chemicals were of a reagent grade.

Animals

Male Wistar rats weighing between 120 and 150 g wereobtained from the Central Animal House, PostgraduateInstitute of Medical Education and Research, Chandi-garh, and given humane care. The study formed a partof a PhD thesis and was approved by the Institute’sEthics Committee.

Study design

The animals were divided into two groups; the experi-mental group underwent a common bile duct ligation(CBDL) by using the method of Shibayama andNakata.19 In the sham-operated group (SO), the bileduct was exposed but not ligated.The intrasplenic pulppressure (ISPP) was measured 18 days after the respec-tive surgeries in all the animals to estimate the portalpressure. The animals were killed immediately afterISPP measurement.The parietal cells were isolated andbiochemical estimations performed.

756 S Kaur et al.

Common bile duct ligation

Rats fasted overnight, but with free access to water,were anesthetized intraperitoneally with ketamine (100 mg/kg bodyweight) after induction with ether.The abdomen was opened by a 2-cm long midline incision and the duodenum was reflected.The bile ductwas divided between ligatures and the abdomen wasclosed in layers.

Secretagogues/drugs used

The stomach was removed and the animals were killed by exsanguination. The isolated parietal cells from both groups (CBDL and SO) were divided into three aliquots or subgroups: (i) subgroup I, un-stimulated parietal cells; (ii) subgroup II, parietal cells stimulated with 10-3 mol/L histamine; and (iii)subgroup III, parietal cells stimulated with 10-3 mol/Lcarbachol.

Intrasplenic pulp pressure

The ISPP was measured by using a modification of themethod of Atkinson and Sherlock,20 and Agnihotri etal.21 The abdomen was opened by a left paramedianincision, and the stomach and spleen were brought tothe exterior through the incision.The spleen was punc-tured with a 20 gauge scalp vein needle attached to asaline manometer. The whole assembly was perfusedwith heparinized normal saline prior to the commence-ment of the procedure. A small amount (0.5 mL) ofheparinized saline was injected into the splenic pulp toflush the needle. The column of saline was allowed tofall until a steady level was attained.The vertical heightof the column above the heart was measured in cen-timeters. A mean of three such readings was taken to bethe ISPP.

Isolation of parietal cells

The parietal cells were isolated by using the modifiedmethod of Mardh et al.22 The stomach was removed,washed with oxygenated Hanks’ balanced salt solution(HBSS; pH 7.4), and incubated with pronase (1 mg/mL) for 30 min at 37°C. After washing with HBSS containing 0.1% bovine serum albumin (BSA) and 2 mmol/L EGTA (pH 7.4), the mucosa was scraped andincubated with collagenase (25 units/mL) for 45 min at37°C in HBSS containing 1% BSA and 0.5 mmol/LCaCl2 with constant gentle manual shaking. The sus-pension was filtered by using nylon filters (MilliporeCorporation, Bedford, MA, USA), centrifuged, washedand resuspended in HBSS containing 1% BSA. Thiscrude preparation was purified on a linear percolldensity (40–60%) gradient. The purified cell prepara-tion was collected and washed free of percoll (SigmaChemical Company, St Louis, MO, USA). The viablecells were counted on a hemocytometer (Neubauer

improved, 0.0025 mm2, depth 0.100 mm; Poly-OptikGmbH, Germany; viability was ascertained by 0.1%trypan blue exclusion).

Acid secretion

Acid secretion was measured by using acridine orange retention, and by using the method adaptedfrom Berglindh et al.23 and modified by Agnihotri et al.13

Acridine orange is a pH sensitive naturally fluores-cent probe. Being a weak base, it is uncharged at cyto-plasmic pH and this form (uncharged acridine orange,AO) is freely permeable across the membrane. In anacidic environment, however, it becomes charged(AOH+), losing its permeability and accumulates in the acidic compartment in accordance with the pH.This results in the quenching of its fluorescence, andthe degree of quenching is an indirect measure of thepH.

To a suspension of isolated parietal cells in HBSS containing 1% BSA with and without 1 mmol/LCaCl2, 10-3 mol/L histamine or 10-3 mol/L carbacholwas added and incubated for 30 min at 37°C. Acridineorange (50 mmol/L) was added and incubated for 5 min at 37∞C. The cell suspension was centrifuged,washed and the pellet resuspended in HBSS with 1% BSA. Fluorescence was measured (Kontron spectrofluorometer model SFM 25; Kontron instru-ments, Zurich, Switzerland) at 493/530 nm (excita-tion/emission). The percentage quenching of acridineorange fluorescence with secretagogues was calculatedand taken as a measure of acid secretion. The percent-age quenching of AO fluorescence was calculated asfollows:

Intracellular free calcium [(Ca2+)i]

The intracellular free calcium level was measured byusing the modified method of Mardh et al.15 The iso-lated purified parietal cells were incubated for 30 minat 37∞C in HBSS containing 1% BSA, 1 mmol/LCaCl2, and Fura-2 acetoxy methyl ester (Fura-2/AM;4 mmol/L), with and without 10-3 mol/L histamine orcarbachol. Following centrifugation at 2500 r.p.m. for15 min, the pellet was resuspended in HBSS contain-ing 1% BSA, but without CaCl2. Fluorescence wasmeasured (Kontron spectrofluorometer) at 340/510 nm(excitation/emission) wavelength by using a Kd of 224for Fura-2 AM.

Protein kinase C

The protein kinase C activity in the cytoplasm andmembrane was assayed by measuring the incorporation

F unstimulated F stimulatedF unstimulated

( ) - ( ) ¥( )

100

Modulation of acid secretion in PHG 757

of 32P from (g 32P)-ATP into lysine-rich histone by themodified method of Ostrowski and Bomsztyk.24

The isolated, purified parietal cells were resuspendedin 1 mL cold lysing buffer containing 20 mmol/L Tris-HCl, 10 mmol/L EGTA, 2.0 mmol/L EDTA,2.0 mmol/L DTT (pH 7.5) and sonicated at 4°C for 3 min. The preparation was then centrifuged at 100 000 g (Sorvall, OTD Combii; Dupont Company,Newton, CT, USA) for 60 min at 4°C, and the super-natant (representing the cytosolic fraction) was savedfor PKC assay. The membrane pellet was resuspendedin 1 mL 0.1% Triton-X 100 in lysis buffer, and thesamples slowly rotated for 1 h at 4°C. After centrifuga-tion (100 000 g) for 60 min at 4°C, the supernatant(representing the membrane fraction) was used for thedetermination of PKC activity.

The reaction for PKC estimation was as follows:15 mL cytoplasm or membrane sample was added to the PKC assay mixture to yield a final reaction volumeof 50 mL. The assay mixture contained 20 mmol/L Tris-HCI, 5.6 mmol/L DTT, 10 mmol/L MgCl2,0.6 mmol/L EDTA, 4.5 mmol/L CaCl2, 3.0 mmol/LEGTA (pH 7.5) and 1 mg/mL histone III-S, 50 mmol/L(g 32P)-ATP (18.5 kBq, specific activity 111 TBq),6 mg/mL diolein, 60 mg/mL phosphatidyl serine. Theassay was terminated after 5 min of incubation at 30°Cby the addition of 200 mL cold 25% trichloroacetic acid(TCA).The acid precipitable material was collected onMillipore membrane filters (Millipore Corporation,Bedford, MA, USA; pore size: 0.45 mm) and washedfivefold with 5% TCA (8 mL each).The bound 32P wasdetermined by using a liquid scintillation counter(model LKB 1214 Rackbeta; Wallace Oy, Turku,Finland). The enzyme activity was determined by sub-tracting bound 32P in the absence of Ca2+, phosphatidylserine and diolein from that in the presence of Ca2+,phosphatidyl serine and diolein.

Cyclic adenosine monophosphate

Cyclic AMP was measured by using standard radioim-munoassay kits from Amersham International,Aylesbury, Buckinghamshire, UK. The isolated parietalcells were collected as a pellet and resuspended in acidicethanol (1 mL 1N HCl/mL ethanol) and allowed tostand for 5 min. After centrifugation at 12 000 g, thesupernatant was collected. The precipitate was washedwith 1 mL ethanol/water (2 : 1, v/v) and centrifuged.The supernatants were combined and evaporated todryness at 60°C under a stream of nitrogen.The resultsare expressed as pmol of cAMP/105 cells.

Statistical analysis

Statistical analysis was conducted by using two-wayANOVA and Neuman–Keuls’ post-hoc test if a signifi-cant F statistic was obtained. A P value less than 0.05 was considered significant. Data were analyzed byusing EXCEL-MS OFFICE 97 (Microsoft Corpora-tion, Seattle, WA, USA).

RESULTS

Intrasplenic pulp pressure measurements

In the common bile duct ligated group, the ISPP was20.60 ± 0.65 cm of normal saline (n = 11) and in thesham-operated group it was 11.67 ± 0.33 cm of normalsaline (n = 12; P < 0.001). After an initial rise, the ISPPstabilized approximately at 18–20 days, indicating thedevelopment of collaterals at this stage. There was nolinear relationship between the ISPP and duration of portal hypertension (Fig. 1). Subsequently, all ISPPmeasurements were made on the 18th post opera-tive day. In the animals used for PKC estimation, ISPPmeasurement was not performed because five to sixanimals had to be killed simultaneously in order to poolthe harvested cells.

Parietal cell count

The viable parietal cell count in the CBDL group was0.55 ¥ 106 ± 0.02 ¥ 106 cells/mL (n = 20), while in thesham-operated group, the value was 0.7 ¥ 106 ± 0.03 ¥106 cells/mL (n = 10). There was a significant decrease(P < 0.001) after bile duct ligation. This diminution inthe parietal cell count did not bear a linear relationshipwith the duration of portal hypertension. However,the parietal cell count correlated significantly with thepresence of an elevated ISPP (results not shown).The viability of the parietal cell preparations rangedbetween 95 and 98% throughout all experiments.

Acid secretion

Mean ± SEM intrasplenic pulp pressure (in cm ofnormal saline) was 20.58 ± 0.65 and 11.46 ± 0.53 cmsaline in CBDL and SO groups, respectively, in thepresence of calcium and 19.50 ± 0.24 and 10.70 ± 0.29in CBDL and SO groups, respectively, in the absence

758 S Kaur et al.

of calcium. The acid secretion by isolated parietal cellswas measured by the quenching of acridine orange flu-orescence. In the CBDL group, both histamine (P <0.001) and carbachol (P < 0.01) resulted in significantquenching in AO fluorescence in comparison to thesham-operated controls in the presence of calcium(Table 1). The removal of CaCl2 from the incubationmedium did not make a significant difference to thequenching of fluorescence (Fig. 2).

Intracellular free calcium ([Ca2+]i)

The mean ± SEM intrasplenic pulp pressure (in cm ofnormal saline) was 21.50 ± 0.46 in the CBDL groupand 10.92 ± 0.40 in SO group. In the CBDL group, themean resting level of [Ca2+]i was 64.80 ± 6.04 nmol/L (n = 6); histamine stimulation caused the [Ca2+]i level to rise to 90.05 ± 5.25 nmol/L (n = 6), while carbacholelevated the [Ca2+]i level to 144.0 ± 15.6 nmol/L (n = 6).In the sham-operated group, the resting level of [Ca2+]i

was 128.00 ± 7.72 nmol/L (n = 6). Both histamine andcarbachol elevated the level of [Ca2+]i to 158.03 ±6.72 nmol/L and 243.90 ± 13.16 nmol/L, respectively.The intracellular free calcium levels in the CBDL groupwere significantly lower with histamine (P < 0.001) andcarbachol (P < 0.001) as compared to their sham-operated controls (Fig. 3).

Cyclic AMP

The results of cAMP are given in Table 2. In the CBDLrats, the value of cAMP in the resting parietal cells was 0.01 ± 0.01 pmol/105 cells. Histamine increasedcAMP level to 0.03 ± 0.03 pmol/105 cells, while carba-chol increased the level of cAMP to 0.35 ± 0.04 pmol/

40

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25

20

15

10

5

08 9 10 21 26 27 28 30 35 36

Days postoperative

ISP

P (

cm o

f nor

mal

sal

ine)

Figure 1 Scatter diagram showing intrasplenic pulp pres-sure (ISPP) measurements in common bile duct ligated(CBDL) and sham-operated (SO) rats. Subsequently, allanimals were killed on the 18th postoperative day immediatelyafter ISPP measurement for further biochemical estimations.(�) Common bile duct ligation, (�) SO.

Table 1 Effect of secretagogues on acid secretion in isolatedparietal cells in common bile duct ligated and sham-operatedrats in the presence of extracellular calcium

% Quenching with acridine orangeCommon bile duct ligated Sham-operated

(CBDL) (SO)Histamine Carbachol Histamine Carbachol

26.78 ± 3.20* 49.75 ± 3.50† 51.40 ± 2.69 63.36 ± 1.01

Mean ± SEM intrasplenic pulp pressure (in cm of normalsaline) was 20.58 ± 0.65 in the CBDL group, and 11.46 ± 0.53in the SO group. Values are expressed as mean ± SEM of sixexperiments. Each experiment was performed by obtainingmaterial from one rat in the SO group and pooling materialfrom two rats in the CBDL group. The individual valuesobtained were calculated by assuming the value without histamine/carbachol to be 100%. The results were analyzed by using a two-way ANOVA. *P < 0.001 as compared to its SO control; †P < 0.01 as compared to its SO control; F valuefor the CBDL group is P < 0.01; F value for the SO group isP < 0.01.

105 cells. In the sham-operated group, the unstimulatedcAMP levels were 0.54 ± 0.28 pmol/105 cells, which wassignificantly (P < 0.01) higher as compared to the corre-sponding CBDL group. Histamine increased cAMPlevel to 11.61 ± 5.83 pmol/105 cells, while carbacholdecreased the level of cAMP to 0.22 ± 0.03 pmol/105 cells in the SO group.

Protein kinase C

In the SO rats, cytosolic PKC was significantlydecreased with secretagogues as compared to the basal

Modulation of acid secretion in PHG 759

value, although the PKC activity in the membrane frac-tion increased by 4.4, 0.8 and 2.52 pmol phosphate/minper 106 cells in the basal, histamine and carbachol sub-groups, the translocation of PKC activity in the basalstate was exceeded only by the increased translocationin the carbachol subgroup. Likewise, in the CBDL rats,the cytosolic PKC decreased significantly with secreta-gogues as compared to their basal value. Although thePKC activity in the membrane fraction increased by2.0, 1.0 and 1.1 pmol phosphate/min per 106 cells in thebasal, histamine and carbachol subgroups, the translo-cation of PKC activity in the basal state was exceededonly by the increased translocation in the histaminesubgroup.

Basal cytosolic and membrane PKC and carbachol-stimulated membrane PKC were significantly de-creased in the CBDL rats as compared with the SO rats.In contrast, there was no significant difference in thePKC activity of cytosolic or membrane fractions withhistamine stimulation between CBDL and SO rats.

70

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30

20

10

0

% A

ge q

uenc

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of a

crid

ine

oran

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With histamine With carbachol

* †

§‡

Figure 2 Effect of secretagogues on acid secretion incommon bile duct ligated (CBDL) and sham-operated (SO)rats in the presence and absence of extracellular calcium.Values are expressed as mean ± SEM of six and five experi-ments in the presence and absence of calcium, respectively,and in the CBDL group and six experiments in each SOgroup. Each experiment was performed by obtaining materialfrom one rat in the SO groups and pooling material from tworats in the CBDL group, except the fifth experiment in theabsence of calcium, which was performed with one rat. Thevalues are expressed as percentage quenching of acridineorange by assuming the value without histamine/carbacholwas 100%. The results were analyzed by using the Student’sunpaired t-test and a two-way ANOVA.( ) CBDL group +Ca2+, ( ) CBDL group - Ca2+, ( ) SO + Ca2+, ( ) SO - Ca2+.*P < 0.001; †P < 0.01, ‡P < 0.01, §P < 0.01 as compared torespective SO controls (the F value for the CBDL group wasP < 0.01; §F-value for the SO group was P < 0.01).

300

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50

0

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acel

lula

r ca

lciu

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Basal Histamine Carbachol

*

†

‡

Figure 3 Effect of secretagogues on intracellular calciumlevels in common bile duct ligated (CBDL) and SO rats. Theresults are expressed as mean ± SEM of six experiments. Eachexperiment was performed by obtaining material from one ratin the SO group and pooling material from two rats in theCBDL group, except one experiment, which was performedwith one rat from the CBDL group.The results were analyzedby using the Student’s unpaired t-test and a two-way ANOVA.(�) CBDL, ( ) SO. *P < 0.001, †P < 0.001, ‡P < 0.001 as com-pared to respective SO controls (F value for the CBDL groupwas P < 0.001; F value for the SO group was P < 0.001).

Table 2 Effect of secretagogues on cAMP levels in isolated parietal cells in common bile duct ligated and sham-operated rats

cAMP levels (pmol/105 cells)Common bile duct ligated (CBDL) Sham operated (SO)

Basal Histamine Carbachol Basal Histamine Carbachol

0.01 ± 0.010* 0.03 ± 0.03† 0.35 ± 0.040 0.54 ± 0.28 11.61 ± 5.83 0.22 ± 0.03

Mean ± SEM intrasplenic pulp pressure (in cm of normal saline) was 22.36 ± 0.37 in the CBDL group, and 10.83 ± 0.33 inthe SO group. Values are expressed as mean ± SEM of three experiments. Each experiment was performed by pooling materialfrom two rats in the SO group and pooling material from three rats in the CBDL group. The results were analyzed by using aStudent’s paired t-test and a two-way ANOVA. *P < 0.01 as compared to its SO control; †P < 0.01 as compared to basal level. AnF value of the sham-operated group is P < 0.05.

As is evident from Table 3, there was a significantincrease in the PKC activity in the membrane fractioncompared to the cytosolic fraction of CBDL and SOrats with both secretagogues, inspite of the overalldecreased activity as compared with their respectivebasal values.

DISCUSSION

Before accepting an animal model, it is mandatory todocument structural as well as functional similarities tothe condition sought to be reproduced.

Our results showed that there was a significant ele-vation in the ISPP in the CBDL group compared withthe sham-operated controls. In addition, there werestructural alterations in the liver resulting in bile duc-tular hyperplasia, which resulted in the elevation ofintrasplenic pulp pressure. Portal hypertension-inducedchanges were also seen in the spleen and stomach.Therefore, the presence of portal hypertension was confirmed by ISPP measurement and histopathologicalalterations (results not shown).

760 S Kaur et al.

Acid secretion

We found that in the CBDL group, there was adecreased response of acid secretion in parietal cells fol-lowing stimulation with secretagogues, and the responsewas statistically significant as compared with the sham-operated group.The addition of extracellular calcium tothe medium did not make a significant alteration in acidsecretion, thus suggesting that extracellular calciumdoes not play a significant role in histamine or carba-chol-stimulated acid secretion in the isolated parietalcell preparation.

One of the possible explanations for the blunting instimulated acid response could be that the parietal cellsin the CBDL animals had already been exposed to ahigh concentration of these secretagogues prior to beingkilled. Indeed, there is a report of increased activity ofthe cholinergic nervous system in the stomachs of CCl4-treated portal hypertensive rats.25 In addition,hypergastrinemia has been observed in both cirrhoticpatients26 and in patients with extrahepatic portalvenous obstruction.27 These effects might explain the decreased further stimulation of acid secretion bycarbachol and histamine in the portal hypertensivegroup. A similar observation of blunted stimulated acidsecretion has been made in extrahepatic portal hyper-tension following partial portal vein ligation in rats.13

As we observed a decreased acid stimulatoryresponse of parietal cells to secretagogues, the secondmessengers regulating acid secretion were studied to look for any alteration in the regulation of acid secretion.

Intracellular free calcium

Calcium is a well-studied second messenger in the regulation of acid secretion. Its basal level in normalparietal cells is known to be affected by the acid secretagogues, histamine and carbachol.28,29

Our results in the CBDL group showed that the basal[Ca2+]i levels were significantly lower as compared withthose in the sham-operated group. In addition, the ele-vation in the level of [Ca2+]i by histamine and carbacholwas lower in the CBDL group as compared with thesham-operated group.

Gastric mucosal hypoxia,6 decreased transmucosalpotential difference30 and decreased ATP levels havebeen observed in PHG.31 Altered plasma membranepermeability and decreased ATP levels have also beendemonstrated in parietal cells obtained from extra-hepatic portal hypertensive gastric mucosa.13 The neteffect of these aberrations could be a substantial disruption of the regulatory mechanisms resulting insuboptimal [Ca2+]i levels.

Hypoxia is known to cause damage to many celltypes, especially the highly specialized and oxygen-dependent oxynticopeptic cells.32 Therefore, all thesefactors may lead to a greater dysfunction of the parietalcells in the portal hypertensive gastric mucosa, which in turn may lead to a gross disruption of the regula-tory pathway of acid secretion. This situation may beoperational in our animal model also.

Table 3 Effect of secretagogues on protein kinase C levels inisolated parietal cells in common bile duct ligated (CBDL)and sham-operated (SO) rats

PKC activity (pmol phosphate/min per 106 cells)

Common bile duct ligated Sham-operated

BasalCytosol 1.90 ± 0.26* 3.20 ± 0.42Membrane 3.90 ± 0.26† 7.60 ± 0.12Fold translocation 2.04 ± 0.23 2.52 ± 0.32

HistamineCytosol 0.50 ± 0.06‡ 0.70 ± 0.13‡‡

Membrane 1.50 ± 0.17§ 1.50 ± 0.20§§

Fold translocation 2.90 ± 0.26 2.46 ± 0.63Carbachol

Cytosol 1.20 ± 0.21¶ 0.98 ± 0.25¶¶

Membrane 2.30 ± 0.28**,†† 3.50 ± 0.50***Fold translocation 2.04 ± 0.32 3.98 ± 0.54

Values are expressed as mean ± SEM of four experiments.All experiments were performed by pooling material from sixrats in the CBDL group and five rats in the SO group. Theresults were analyzed by using a two-way ANOVA and a pairedt-test. *P < 0.01 as compared to SO (basal), †P < 0.01 as com-pared to SO (basal), ‡P < 0.05 as compared to its basal level;§P < 0.01 as compared to its cytosolic level; ¶P < 0.001 as com-pared to its basal level; **P < 0.05 as compared to SO (carba-chol); ††P < 0.05 as compared to its cytosolic level; ‡‡P < 0.01as compared to its basal level; §§P < 0.05 as compared to its cytosolic level; ¶¶P < 0.01 as compared to its basal level;***P < 0.01 as compared to its cytosolic level. An F value forthe CBDL groups is P < 0.01; F value for the SO groups is P < 0.01.

The reduced intracellular calcium reported in theportal hypertensive group hence could be explained on the basis of hypoxic stress and decreased ATP production.

Cyclic AMP

There have been many studies on the role of cAMP inthe regulation of gastric acid secretion.31,32 The stimu-latory effect of histamine on acid secretion is viacAMP.34 In addition, finite levels of cAMP are requiredfor both gastrin and carbachol-mediated aminopyrineaccumulation in rat parietal cells.35

Some authors, however, believe that gastrin and acetylcholine have no effect on the cellular cAMP level. Our results revealed that although histamineincreased cAMP levels, it was to a lesser extent in thecommon bile duct-ligated animals. Carbachol showed a similar trend, which did not, however, achieve significance.

Cyclic AMP is formed from ATP by the action of theenzyme adenylate cyclase. Various authors have foundATP levels to be low because of hypoxic stress in theportal hypertensive gastric mucosa.13,31 This couldexplain low cAMP levels in the CBDL group. Thereduced stimulatory effect of histamine on cAMP levelsmay explain the blunting of acid secretory response asseen by acridine orange quenching in the parietal cellsfrom the CBDL group. Low cAMP levels are consistentwith a low metabolic state of parietal cells in the PHTgastric mucosa.

Cyclic GMP levels were also measured by radio-immunoassay (results not shown). Our observationshowed that both histamine and carbachol did not altercGMP levels to any significant extent in the CBDL andsham-operated groups. In addition, there was no significant alteration in the histamine or carbachol stimulated cGMP levels between the CBDL and sham-operated groups. Thus, it appears that cGMP does nothave a significant role to play in the regulation of acidsecretion of PHG.

Protein kinase C

In isolated parietal cells from CBDL-related PHG,there is: (i) a decrease in cytosolic and membrane PKCactivity in unstimulated cells; (ii) a further decrease incytosolic PKC with secretagogues accompanied by asignificant increase in PKC activity of the correspond-ing membrane fraction; and (iii) the translocating abilityof PKC is not lost, and although the efficiency oftranslocation is preserved with histamine, it appears tobe reduced to basal levels with carbachol. This obser-vation suggests that some intracellular factor responsiveto, or regulated by, histamine may be modulating PKCactivity in CBDL-related PHG.Thus, it appears that inCBDL-related PHG, some as yet unidentified intra-cellular mechanism of secondary importance but, re-sponsive to histamine assumes a dominant role in theregulation of PKC translocation, whereas under normal

Modulation of acid secretion in PHG 761

circumstances, a primary intracellular mechanism moreresponsive to carbachol is operative.

The better preserved PKC levels with carbachol incomparison to histamine, along with the comparativelyhigher levels of intracellular cytosolic free Ca2+ couldexplain the relatively better acid stimulatory effect ofcarbachol compared to histamine in this model.

Finally, to summarize the results, the parietal cells’response to histamine and carbachol is blunted inCBDL rats because of reduced levels of second messengers like calcium, cyclic AMP and protein kinaseC.

Therefore, to conclude, stimulated acid secretion inPHG is decreased because of an inefficiency of theintracellular modulatory mechanism that is unable torespond to stimulation with secretagogues.

REFERENCES

1 McCormack TT, Sims J, Eyre-Brook I et al. Gastric lesionsin the CBDL rats and portal hypertension: Inflammatorygastritis or congestive gastropathy. Gut 1985; 26: 1226–32.

2 Papazian A, Braillon A, Dupas JL, Sevinet F, Capron JP.Portal hypertensive gastric mucosa: An endoscopic study.Gut 1986; 27: 1199–1203.

3 Nishida H, Kodama T, Satoh T, Fuse Y, Yamashita S.Changes in gastric microcirculation and mucosal lesion inportal hypertensive rats. J. Gastroenterol. Hepatol. 1989; 4(Suppl.): 88–90.

4 Sarfeh IJ, Malki A, Tarnawski A, Ivey KJ, Mason GR.Portal hypertension and gastric mucosal injury in rats.Effect of alcohol. Gastroenterology 1983; 84: 987–93.

5 Maeda R, Guilmette E, Tarnawski A, Sarfeh IJ. Bile acidinduced gastric mucosal injury. Significance of portalhypertension and capillary mucosal permeability. J. Surg.Res. 1984; 36: 312–14.

6 Sarfeh IJ, Soliman H,Waxman K et al. Impaired oxygena-tion of gastric mucosa in portal hypertension. The basisfor increased susceptibility to injury. Dig. Dis. Sci. 1989;34: 225–8.

7 Smart HL, Triger DR. Clinical features, pathophysiologyand relevance of portal hypertensive gastropathy.Endoscopy 1991; 23: 224–8.

8 Tarnawski AS, Sarfeh IJ, Stachura J et al. Microvascularabnormalities of the portal hypertensive gastric mucosa.Hepatology 1988; 8: 1488–94 .

9 Pique JM, Leung FW, Kitahore T, Sarfeh IJ,Tarnawski A,Guth PH. Gastric mucosal blood flow and acid secretionin portal hypertensive rats. Gastroenterology 1988; 95:727–33.

10 Perez-Ayuso RM, Pique JM, Saperas EI et al. Gastric vas-cular ectasia in cirrhosis: Association with hypoacidity notrelated to gastric atrophy. Scand. J. Gastroenterol. 1989; 24:1073–9.

11 Mitsunaga A, Yokoyama S, Hashimoto H et al. Clinicalstudy of the aggressive factors of the gastric mucosa inliver cirrhosis. J. Gastroenterol. Hepatol. 1989; 4 (Suppl. 1):266–7.

12 Agnihotri N, Kaur S, Dilawari JB, Bhusnurmath SR, KaurU. Dimunition in parietal cell number in experimentalportal hypertensive gastropathy. Dig. Dis. Sci. 1997; 42:431–9.

13 Agnihotri N, Kaur U, Dhawan V, Dilawari JB. Extra-hepatic portal hypertensive gastropathy in Wistar rats.Modulation of acid secretion in isolated parietal cells. Dig.Dis. Sci. 1998; 43: 56–66.

14 Thompson WJ, Chang LX, Rosenfeld GC. Histamine regulation of adenyl cyclase of enriched rat gastric parietal cells. Am. J. Physiol. 1981; 240: G76–84.

15 Mardh S, Song YH, Carlsson C, Bjorkman T. Mechanismsof stimulation of acid production in parietal cells isolatedfrom the pig gastric mucosa. Acta Physiol. Scand. 1987;131: 589–98.

16 Muallem S, Sachs G. Ca2+ metabolism during cholinergicstimulation of acid secretion. Am. J. Physiol. 1985; 248:G216–28.

17 Wilkes JM, Scott DR, Hersey SJ, Sachs G. Second mes-sengers in the gastric gland. A focus on calcium. Scand. J.Gastroenterol. 1991; 26 (Suppl. 180): 70–84.

18 Waldum HL, Sandvik AK, Brenne E, Petersen H. Gastrinhistamine sequence in the regulation of gastric acid secre-tion. Gut 1991; 32: 698–701.

19 Shibayama Y, Nakata Y. Haemodynamic alterations andtheir morphological basis in biliary obstruction. Liver1992; 12: 175–8.

20 Atkinson M, Sherlock S. Intrasplenic pulp pressure as anindex of portal venous pressure. Lancet 1954; 1: 1325–7.

21 Agnihotri N, Bhusnurmath SR, Narsimhan KL, DilawariJB, Majumdar S, Kaur U. Experimental extrahepaticobstruction of portal vein: Documentation of histopatho-logical alterations in the liver and extrahepatic tissues. J.Gastroenterol. Hepatol. 1996; 11: 971–7.

22 Mardh S, Norberg L, Ljungstrom M, Humble L, Borg T,Carlsson C. Preparation of cells from pig gastric mucosa.Isolation of parietal cells by isopyknic centrifugation onlinear density gradients of percoll. Acta Physiol. Scand.1984; 122: 607–13.

23 Berglindh T, Dibona D, Ito S, Sachs G. Probes of parietalcell function. Am. J. Physiol. 1980; 238: G165–76.

24 Ostrowski J, Bomsztyk K. Interaction of signal transduc-tion pathways in mediating acid secretion by rat parietalcells. Am. J. Physiol. 1989; 256: G873–9.

25 Nakamura M, Oda M, Nishizaki Y, Kaneko K, TsuchiyaM. Histochemical characterizations of gastric erosionformed in chronic CCL4-treated rats. J. Gastroenterol.Hepatol. 1989; 4 (Suppl. 1): 123–5.

26 Quintero E, Pique JM, Bondi JA et al. Gastric mucosalvascular ectasias causing bleeding in cirrhosis: A dis-tinct entity associated with hypergastrinemia and low

762 S Kaur et al.

serum levels of pepsinogen 1. J. Gastroenterol. 1987; 93:1054–61.

27 Maruyama T, Kinoshita E, Futagawa S. Study of themucosal damage of gastropathy in portal hypertension. J.Gastroenterol. Hepatol. 1989; 4 (Suppl. 1): 151–3.

28 Chew CS. Cholecystokinin, carbachol, gastrin, histamineand forskolin increased [Ca2+]i in gastric gland. Am. J.Physiol. 1986; 250: G814–23.

29 Negulescu PA, Reenstram W, Machen TF. IntracellularCa2+ requirement for stimulus and secretion coupling inparietal cells. Am. J. Physiol. 1989; 257: G241–51.

30 Sarfeh IJ, Tarnawski A. Functional histologic and ultra-structural characteristics of the portal hypertensive gastricmucosa. J. Gastroenterol. 1987; 93: 1054–61.

31 Kawano S, Tanimuru H, Tsuji S et al. Impaired gastricmucosal energy metabolism in congestive gastropathy incirrhotic patients. J. Gastroenterol. 1994; 29: 245–9.

32 Yanaka A, Ito S, Carter KJ, Goddard PJ, Silen W. Effectsof hypoxia on function and morphology of in vitro froggastric mucosa. Am. J. Physiol. 1992; 262: G403–19.

33 Kimberg DV. Cyclic nucleotides and their role in gas-trointestinal secretion. Gastroenterology 1974; 67:1023–64.

34 Dousa TP, Dozois RR. Interrelationship between hista-mine, prostaglandin and cyclic AMP in gastric secretion:A hypothesis. Gastroenterology 1977; 73: 904–12.

35 Li Z-Q, Cabero JL, Mardh S. Gastrin and carbacholrequire cAMP to elicit aminopyrine accumulation in iso-lated pig and rat parietal cells. Am. J. Physiol. 1995; 258:G82–9.

36 Brown MR, Chew CS. Carbachol induced protein phos-phorylation in parietal cells. Regulation by [Ca2+]i. Am. J.Physiol. 1989; 257: G499–11.

37 Nishizuka Y. Studies and perspectives of protein kinase C.Science 1986; 233: 305–12.

38 Anderson NY, Hanson PJ. Involvement of calcium sensi-tive phospholipid dependent protein kinase in control ofacid secretion by isolated rat parietal cells. Biochem. J.1985; 232: 609–11.

39 McKenna JP, Hanson P. Inhibition by RO31–8220 of acidsecretory activity induced by carbachol indicates a stimu-latory role for protein kinase C in the action of muscarinicagonists on isolated rat parietal cells. Biochem. Pharmacol.1993; 46: 583–88.

40 Beil W, Mannschede W, Sewing KF. Protein kinase C andparietal cell function. Biochem. Biophys. Res. Commun.1987; 149: 720–8.