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Pharmacological Research 64 (2011) 249– 257

Contents lists available at ScienceDirect

Pharmacological Research

jo ur n al hom epage: www.elsev ier .com/ locate /yphrs

elatonin independent protective role of l-tryptophan in experimental refluxsophagitis in rats�

ratibha Singha, Neetu Singha, Ausaf Ahmada,b, Omprakash Singha, Gautam Palit a,∗

Division of Pharmacology, Central Drug Research Institute, CSIR, Lucknow 226001, Uttar Pradesh, IndiaAmity Institute of Biotechnology, Lucknow campus, Amity University, Lucknow 226016, India

r t i c l e i n f o

rticle history:eceived 1 November 2010eceived in revised form 6 March 2011ccepted 11 April 2011

eywords:-Tryptophanelatoninastroesophageal reflux disorderGE2

rylalkylamine-N-acetyltransferase

a b s t r a c t

Melatonin is implicated in sustaining the esophageal integrity in gastro-esophageal reflux disease. How-ever, the role of its synthetic precursor l-tryptophan is not clear in this pathology. The present study wasdesigned to explore the effects of l-tryptophan on esophageal damage following reflux esophagitis (RE)-establishment and concurrent alterations in factors possibly influencing esophageal integrity such asesophageal melatonin level, luminal acidity, H+K+-ATPase activity, mucin and gastric PGE2 levels. RE wasestablished in rats by simultaneous ligation of pylorus region and fore-stomach. RE significantly decreasedthe esophageal-melatonin level and the expression of its synthesizing enzymes: arylalkylamine-N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT). Administration ofl-tryptophan significantly decreased the RE-induced esophageal mucosal damage, without altering thelevels of melatonin. l-Tryptophan pretreatment also normalized the esophageal mucosal damage causedby melatonin receptor antagonist-luzindole. Simultaneously, l-tryptophan significantly increased theRE-decreased expression of AA-NAT with insignificant effect on HIOMT gene expression. In contrast, l-tryptophan per se caused a significant elevation in the esophageal melatonin level, with no significanteffect on the expression of AA-NAT and HIOMT enzymes. Further, l-tryptophan significantly normalizedthe RE-induced changes in the gastric juice volume, acidity and pH. However, it did not significantly inhibitthe H+K+-ATPase activity in vitro. Also, l-tryptophan significantly increased the RE-reduced mucin level,COX-2 activity and thereby PGE2 levels. Interestingly, indomethacin (PGE2 synthesis blocker), aggravatedthe RE-induced tissue injury with simultaneous changes in the gastric volume, acidity, pH and mucin

content, which l-tryptophan failed to reverse, suggesting that the attenuating effect of l-tryptophan ongastric secretions could be PGE2 driven. Thus the current study provide evidences that protective func-tions of l-tryptophan against RE is independent of its conversion into melatonin, and possibly involvemobilization of factors such as COX-2 derived PGE2 and mucin that counterbalance the detrimentaleffect of gastric acid on esophageal mucosa, signifying the therapeutic efficacy of l-tryptophan againstthe esophageal pathologies.

. Introduction

Gastroesophageal reflux disorder (GERD) is classified as a func-ional gastrointestinal (GI) disorder, in which gastric acid refluxesnto the esophagus, causing irritations and inflammation of its

ucosa. The prevalence of GERD is ranging from less than 5% in Asiao 10–20% in the Western world [1]. It is also reported that chronic

Abbreviations: RE, reflux esophagitis; AA-NAT, arylalkylamine-N-acetyl-ransferase; HIOMT, hydroxyindole-O-methyltransferase; PGE2, prostaglandinE2;ERD, gastroesophageal reflux disorder; GI, gastrointestinal; GIT, gastrointestinal

ract.� CDRI communication no. 8050.∗ Corresponding author. Tel.: +91 522 2612411 418x4303;

ax: +91 522 2623405/2623938.E-mail addresses: pratibha02in@gmail.com (P. Singh),

palitcdri@gmail.com (G. Palit).

043-6618/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.phrs.2011.04.004

© 2011 Elsevier Ltd. All rights reserved.

heart burn-symptoms may lead to the development of oesophagealadenocarcinomas [2].

The present mainstay therapy for GERD is based on gastricacid-suppression, and healing of injured oesophageal mucosa toprevent further complications [3]. Although these therapies areeffective in producing acute symptom relief and mucosal healing,there is still a need of alternative treatment strategies becauseof their limitations such as daily administration, failure to pro-vide complete symptom relief and frequent relapses following drugwithdrawal [3].

The major breakthrough in the search for alternative approachesfor GERD treatment is the discovery of the protective influences of

melatonin in GERD patients [4,5]. Supplementation of melatonin,alone or in combination with acid suppressant is found to bringrapid regression of GERD associated symptoms [6]. Moreover, theesophageal erosions in GERD are associated with declined levels

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50 P. Singh et al. / Pharmacolog

f circulating melatonin, signifying its role in the protection ofsophageal integrity [7].

Melatonin has been isolated primarily from the pineal gland8], but following studies have shown its widespread occurrencen other organs also such as GIT, suprarenal gland, thyroid gland,hymus, placenta, etc. [9,10]. Besides the presence of melatoninn enterochromaffin (EC) cells [11], its receptors and biosyn-hetic enzymes arylalkylamine-N-acetyltransferase (AA-NAT; EC.3.1.37) and hydroxyindole-O-methyltransferase (HIOMT; EC.1.1.4) were also detected in GIT [12,13]. The clinical applicabilityf melatonin in the treatment of GERD patient and the evidencesf its possible role in the maintenance of esophageal integrityntrigued us to evaluate the role of its precursor, l-tryptophan inhe esophageal pathology. Also in GERD subjects, a supplemen-ary formulation containing l-tryptophan were administered andound extremely effective in reducing the GERD associated symp-oms [4]. The author’s rationale of using it was to alleviate theain by enhancing the availability or efficacy of neuro-inhibitororadrenalin and serotonin. Several studies indicate that GIT load-

ng of l-tryptophan, increases the levels of circulating melatoninn the day time [14,15]. It was also hypothesized that the pro-ective influences of l-tryptophan may depend on its conversiono melatonin [16,17]. Thus, it is essential to understand the intri-ate relationship between l-tryptophan and melatonin systemn the protection of esophageal integrity and to elucidate their

ode of function by deciphering their synergism or indepen-ent action. In the current study we also focused an important

ssue that the effect of l-tryptophan on the melatonin levels inhe esophagus is subjected to change under pathological con-itions such as RE when the esophagus mucosal tissue become

njured.In this regard, our finding address following two issues. Our

rst concern was to evaluate the effect of oral administration of-tryptophan on the esophageal melatonin and expression lev-ls of AA-NAT and HIOMT. It is well perceived that RE is not aimple consequence of acid accumulation, and a number of fac-ors possibly governing through l-tryptophan are involved. Thus,ur second issue was to evaluate that whether the changes in l-ryptophan and/or melatonin system would lead to the protectiongainst RE associated mucosal injury, and if so, these systems workynergistically or independently. Hence, parallel perturbations inactors influencing esophageal integrity such as luminal acidity,

+K+-ATPase activity, mucin and gastric mucosal PGE2 level werexamined in all the groups.

. Materials and methods

.1. Materials

All the chemicals used in the study were obtained from M/s.igma Chemicals, St Louis, MO, USA unless otherwise mentioned.

.2. Animals

Experimental protocols were approved by the Institutionalthical and Usage Committee of Central Drug Research InstituteCDRI), Lucknow, following the guidelines of the Committee forhe Purpose of Control and Supervision of Experiments on Ani-

als (CPCSEA). Adult Sprague Dawley rats, weighing 180–220 grocured from National Laboratory Animal Centre, CDRI, were used

n the study. Rats were housed three to four per cage, in a room withemperature regulated at 22 ± 2 ◦C, with a 12 h/12 h light/dark cyclelights on 07:00 h, lights off 19:00 h). Standard chow pellets andater were given ad libitum, except during the period when foodeprivation was applied.

esearch 64 (2011) 249– 257

2.3. Induction of reflux esophagitis

Animals were deprived of food for 18 h before induction ofesophagitis but water was provided ad libitum. RE was inducedin rats according to the method described earlier [18] with minormodifications. Briefly, under pentobarbital anesthesia (30 mg/kg,i.p.) the abdomen was incised along the midline and then both thepyloric end of the stomach and limiting ridge (transitional regionbetween the fore stomach and corpus) were simultaneously ligatedtightly, resulting in the reflux of the gastric juice into the esophagus.After 5 h of ligation animals were killed, esophagus was removedand incised lengthwise. Haemorrhagic lesions were observed underTrinocular zoom microscope (SZ-CTV Olympus) and area of lesions(sq.mm) developed was measured using Biovis image analyzer soft-ware (Expert Vision Lab Private Ltd., Mumbai, India).

2.4. Experimental procedure

After 1 week of acclimatization, the rats were randomly dividedinto various groups, each consisting of 6 animals.

Normal control: Rats underwent sham operation and were notgiven any treatment.

Reflux esophagitis control: RE was induced in rats as describedabove (RE) and given same vehicle solutions as used for the disso-lution of respective drugs and used for the purpose of comparisonaccordingly.l-tryptophan per se group: Normal rats were pretreated with l-

tryptophan (100–400 mg/kg) alone to evaluate drug per se effect(l-100–400 per se).

Treatment groups: Rats were treated with graded dosesof l-tryptophan (100–400 mg/kg; l-100–400 + RE), omeprazole(10 mg/kg; Omz + RE) and indomethacin (5 mg/kg; IND + RE),45 min prior to the induction of RE. The dose of omeprazole andindomethacin was selected on the basis of our pilot studies andavailable literature [19,20].

In a separate experimental group, rats were pretreated withindomethacin (5 mg/kg) followed by l-tryptophan (200 mg/kg)45 min prior to the induction of RE (IND + l-200 + RE).

Additionally, rats were divided into two more groups: in onegroup rats were treated with melatonin receptor antagonist-luzindole (2 mg/kg) 45 min prior to the induction of (LUZ + RE). Inanother group rats were pretreated with luzindole followed bygraded doses of l-tryptophan (100–400 mg/kg) 45 min prior to RE-induction (LUZ + l-100–400 + RE).l-Tryptophan was dissolved in normal saline with a drop of 0.1

HCl. Omeprazole was suspended in aqueous solution containing0.5% carboxymethyl cellulose (CMC). l-Tryptophan and omepra-zole were administered orally using a ball ended feeding needle.Both l-tryptophan and omeprazole were administered to animalsat a volume of 1 ml/200 g body weight. Indomethacin was dis-solved in the minimal amount of DMSO and diluted in water.Indomethacin was administered subcutaneously. Luzindole wasdissolved in DMSO, diluted in water and given intraperitoneally(i.p.).

2.5. Estimation of melatonin and l-tryptophan level inesophageal mucosa

Immediately after 5 h of RE-induction, rats were killed byinhalation of anesthetic ether, and the esophageal tissues wereimmediately removed. The esophageal tissues were first weighedand 200 ng/ml of internal standard HPA (4-hydroxyphenylacetic

acid) was added to each sample. Homogenization was performedin 0.1 M perchloric acid with an Ultra-Turrax homogenizer (ModelT25, IKA-Laborthechnik, Germany). Homogenates were then cen-trifuged at 13 200 × g (Sigma centrifuge, model 3K30, USA) at 4 ◦C,

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P. Singh et al. / Pharmacolog

upernatant was passed through a 0.22 �m membrane filter andsed for the analysis. The endogenous levels of l-tryptophan andelatonin were determined by reverse phase HPLC with electro-

hemical detection [21]. The sample (20 �l) was injected via a HPLCump (Model 1525, Binary Gradient Pump, Waters, Milford, MA,SA) into a column (Spherisorb, RP C18, 5 �m particle size, 4.6 mm

.d. × 250 mm at 30 ◦C) connected to an electrochemical detectorModel 2465, Waters, Milford, MA, USA). Oxidation potential wasxed at 0.80 V using a glass carbon working electrode versus ang/AgCl reference electrode. The mobile phase consisted of 0.1 Mitric acid, 0.1 M sodium acetate, 0.03 mM Na2EDTA and 30% (v/v)ethanol. pH of the mobile phase was adjusted to 4.2. Separa-

ion was carried out at a flow rate of 1.0 ml/min. The l-tryptophannd melatonin levels were quantified using Breeze version 3.2oftware purchased from Waters HPLC systems (Hietzinger 145,1130 Wien, Austria). The levels were expressed in nanograms of-tryptophan or melatonin per gram of wet weight of esophagealissue. Quantification was made by comparing peak heights of theamples to the corresponding standard curve.

.6. Semi-quantitative RT-PCR (reverse transcriptase polymerasehain reaction) analysis of AA-NAT and HIOMT gene expression

Total RNA was extracted from esophageal samples using TRIzoleagent (InvitrogenTM, Carlsbad, CA, USA). cDNA was generated

rom 5 �g of total RNA using ReverAidTM H Minus First strandDNA synthesis kit (Fermentas life sciences, Burlington, Canada)ollowing manufacturer’s instructions. Genes for AA-NAT, HIOMTnd �-actin were amplified with specific primer sets as previ-usly described [22]. cDNA samples were run on Bioer XP Cyclernd amplified for 28 cycles with the following cycling conditions:enaturation at 94 ◦C (5 min), 94 ◦C for 1 min, respective annealingemperature for AA-NAT-58 ◦C, HIOMT-62 ◦C and �-actin-55 ◦C for

min and extension at 72 ◦C for 1 min followed by a final extensiont 72 ◦C for 10 min. PCR products were electrophoresed on a 1.0%garose gel using O’RangeRulerTM 100-bp DNA Ladder (Fermen-as life sciences, Burlington, Canada) and intensity was measuredsing Biovis gel documentation software. Results were expresseds relative intensity of PCR-product/�-actin ratio.

.7. Analysis of gastric secretions

Samples of gastric juice obtained from rats of different stud-ed groups were measured for volume and an aliquot was usedor determination of acid concentration with a Radiometer Auto-itration instrument. Samples were titrated against 0.1 N NaOH ton endpoint of pH 7.0. The pH of collected gastric juice was alsoeasured using pH meter (MFRS Toshniwal, instrument manu-

acturing limited, Ajmer, India). Mucin level in gastric juice wasuantified with a fluorometric assay and expressed as �g ofucin/ml of gastric juice [23].

.8. Preparation of H+-K+-ATPase-enriched subfraction oficrosomes and in vitro assay of H+K+-ATPase activity

H+-K+-ATPase-containing microsomes were prepared fromasted rat stomach as previously reported with minor modifications24]. Briefly, the stomach of sacrificed rats was removed and the

ucosa from the fundus and body region was scraped, minced, andomogenized in a hypotonic solution containing ice-cold 125 mMannitol, 40 mM sucrose, 1 mM EDTA, and 5 mM piperazine-N,N′-

is(2-ethanesulfonic acid) (PIPES), pH 6.7. The homogenate was

entrifuged at 14 500 × g for 10 min at 4 ◦C. The supernatant wasurther centrifuged at 100 000 × g for 1 h. The resulting pellet wasesuspended in a suspending medium (300 mM sucrose, 5 mM Tris,nd 0.2 mM EDTA, pH 7.4). The crude microsomal suspension was

esearch 64 (2011) 249– 257 251

overlaid on the gradients of 33%, 27% and 21% sucrose (wt/vol)in 5 mM Tris and 0.2 mM EDTA, pH 7.4, and centrifuged for 2 h at100 000 × g using a Beckman SW27 rotor. H+-K+-ATPase-enrichedrat gastric microsomes were collected as the flotation layer at aninterface between 27% and 21% layers and stored in aliquots at−20 ◦C until use.

For the enzyme assay, gastric microsomes incubated withor without different concentrations of l-tryptophan as well asstandard drug omeprazole for 10 min at 37 ◦C. Buffer containing150 mM KCl, 10 mM PIPES, 1 mM MgSO4, 5 mM Mg ATP, 1 mM EGTAand 0.1 mM ouabain, at pH 7.2 and 10 �g/ml valinomycin, 2.5 �g/mloligomycin was added. The reaction was carried out at 37 ◦C for20 min and was stopped by adding 10% ice-cold trichloroaceticacid. After centrifugation at 2000 × g for 1 min, inorganic phosphate(Pi) released in the supernatant was determined spectrophotomet-rically at 310 nm wavelength [25] and expressed as �M/(h mg)protein.

2.9. Estimation of gastric prostaglandin PGE2 level

PGE2 assay was performed as described by manufacturerinstructions (Amersham Pharmacia Biotech, USA). Briefly, the gas-tric mucosa of different groups was excised and homogenized inan ice-cold Tris/HCl buffer containing 50 mM Tris/HCl (pH 7.4),100 mM sodium chloride, 1 mM calcium chloride, 1 mg/ml d-glucose and 28 �M indomethacin. PGE2 recovery and purificationwas conducted according to protocols provided with the PGE2 EIAkit. Purified PGE2 samples were stored at −80 ◦C until assayed. Sam-ples were dissolved in 0.5–1.0 ml of sample buffer and assayed in96-well plates. The quantities of PGE2 were determined by PGE2standards ranging from 2.5 to 320 pg/ml. Total protein concentra-tion was determined using Lowry protein assay [26]. Results wereexpressed as pg of PGE2/mg of protein.

2.10. Determination of activities of cyclooxygenase (COX) 1 and 2

Gastric mucosal scrapings from experimental groups wereimmediately taken after sacrifice and homogenized on ice in 100mM Tris–HCl, pH 7.5, containing leupeptin (0.5 �g/ml) and phenyl-methylsulfonyl fluoride (PMSF) (1 mM). The homogenates weresubjected to centrifugation at 10 000 × g for 15 min at 4 ◦C. Thesupernatant was used for assays of COX activity, using a commer-cially available kit (COX Fluorescent Activity Assay Kit, 700200,Cayman Chemical Company, USA) and a fluorescent microplatereader (Varian fluorescence spectrophotometer, Netherlands) fol-lowing the instructions provided by the manufacturer.

2.11. Statistical analysis

Data were evaluated by one-way analysis of variance (ANOVA)followed by Newman–Keuls post hoc test. Significance was ascer-tained at p < 0.05. All the data are presented as means ± SEM(standard error of the means).

3. Results

3.1. Effect of l-tryptophan on RE-induced esophageal damage

In order to assess the effect of l-tryptophan on RE model,we performed pilot studies using graded doses of l-tryptophan.As represented in Fig. 1, RE-control rats exhibited formation of

macroscopically evident lesions along the entire esophagus dur-ing the 5 h duration. Pre-treatment with l-tryptophan (100, 200and 400 mg/kg), dose dependently reduced the area of lesions. l-Tryptophan significantly (P < 0.001) reduced the area of lesions at

252 P. Singh et al. / Pharmacological R

Fig. 1. (A) Pictomicrograph representative of the gross appearance of whole esoph-agus in different studied groups. (B) Bar diagram representing the changes in thearea of haemorrhagic lesions in reflux esophagitis (RE) control and various drugtreated groups. Results were expressed as means ± SEM, with six rats (n = 6) in eachg

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roup. *P < 0.05 and ***P < 0.001, respectively (df = 6,35 and F value = 19.87).

00 and 400 mg/kg when compared to RE-control group. Omepra-ole also significantly (P < 0.001) protected the esophagus damages compared to RE-control group.

Administration of indomethacin (a PGE2 synthesis blocker)aused a significant (P < 0.05) increase of RE-induced oesophagealnjury. l-Tryptophan (200 mg/kg) pre-treatment produced no sig-ificant alteration in the indomethacin exacerbated esophagealamage in RE-rats.

As shown in Fig. 7, LUZ + RE group exhibited a non-significantncrease in the area of haemorrhagic lesions compared to RE-ontrol group. Administration of l-tryptophan dose dependentlyeduced the area of lesion showing significant reduction at00 mg/kg (P < 0.01) and 400 mg/kg (P < 0.01) doses compared toUZ + RE group.

.2. Effect of l-tryptophan loading on the esophageal melatoninnd l-tryptophan level

Estimation of esophageal melatonin revealed that induction ofE in rats significantly (P < 0.001) lowered the melatonin contentf esophagus in comparison to normal control group, as shownn Fig. 2A. l-Tryptophan failed to reverse this RE-induced deficit

f melatonin (Fig. 2A) while its own endogenous concentrationncreased significantly in a dose-dependent approach (Fig. 2B). In l-00 + RE and l-400 + RE groups, the esophageal l-tryptophan level

ncreased significantly (P < 0.05 and P < 0.001, respectively) in com-

esearch 64 (2011) 249– 257

parison to RE control group, however, at 100 mg/kg the increasewas insignificant. Contrastingly, l-200 and l-400 per se groupshowed significantly (P < 0.01 and P < 0.001, respectively) elevatedlevels of esophageal melatonin compared to normal control group(Fig. 3A) while exhibit no alterations in esophageal l-tryptophancontents over normal control values (Fig. 3B, respectively).

3.3. Effect of l-tryptophan on gene expression of AA-NAT andHIOMT

As represented in Fig. 4, infliction of RE significantly (P < 0.05 andP < 0.001, respectively) repressed the gene expression of both mela-tonin synthesizing enzymes, AA-NAT and HIOMT in comparison tonormal control rats. l-100–400 + RE group exhibited significantly(P < 0.05) increased expression of AA-NAT compared to RE-rats.Further, l-100–400 + RE groups showed no significant reversal ofRE-suppressed expression of HIOMT gene. Further as representedin Fig. 5, l-100–400 per se group exhibited insignificant alterationin the expression of both AA-NAT and HIOMT genes compared tonormal control group.

3.4. Effect of l-tryptophan on gastric secretions

As summarized in Table 1, the gastric secretion study showedthat l-tryptophan pre-treatment significantly reduced the accumu-lated volume of gastric juice and total acidity in a dose dependentmanner with respect to RE-control group. l-200 + RE and l-400 + REgroup showed significantly lowered gastric volume (P < 0.001) andacidity (P < 0.05) compared to RE-group, whereas l-100 + RE groupfailed to produce significant suppression of RE-induced increasein gastric volume and acidity. Omz + RE group displayed the sig-nificant reduction in the gastric juice volume (P < 0.001) and totalacidity (P < 0.05) over RE-group. Also, gastric juice pH increasedsignificantly in the l-200 + RE and l-400 + RE group (P < 0.01 andP < 0.001, respectively) compared to RE-control group. Moreover,l-200 + RE and l-400 + RE group showed significantly (P < 0.05 andP < 0.001, respectively) elevated the mucin level compared to RE-control rats.

Further, as shown in Table 2, IND + RE group exhibited signifi-cantly reduced gastric juice volume (P < 0.05), acidity (P < 0.01), pH(P < 0.05) and mucin (P < 0.05) content over RE-control values. Theseparameters did not change significantly in l-200 + IND + RE groupcompared to IND + RE group.

3.5. Effect of l-trptophan on H+K+ATPase activity

The effect of l-tryptophan on the in vitro H+ K+-ATPase activitywas examined. As shown in Fig. 6, l-tryptophan in the dose range(10–100 �g/ml) failed to inhibit the gastric H+ K+-ATPase activitycompared to control values. In contrast, omeprazole (10–50 �g/ml),used as a positive control, significantly reduced the H+ K+-ATPaseactivity with an IC50 value of 30.24 �g/ml (Fig. 6).

3.6. Effect of l-tryptophan on gastric PGE2 level

As represented in Table 2, significant reduction in the gastricPGE2 (P < 0.05) level was observed in RE-group compared to nor-mal control rats. l-200 + RE group exhibited significantly (P < 0.001)elevated gastric PGE2 level compared to RE-control rats. IND + RE

group showed significantly (P < 0.05) reduced gastric PGE2 levelcompared to RE-control groups. The gastric PGE2 level did notchange significantly in l-200 + IND + RE group compared to IND + REgroup.

P. Singh et al. / Pharmacological Research 64 (2011) 249– 257 253

Fig. 2. (A) Alterations in the esophageal melatonin levels in normal Control, Reflux esophagitis (RE) control, and l-tryptophan pretreated RE rats (l-100 + RE, l-200 + RE andl-400 + RE). Results were represented as means ± SEM, with six rats (n = 6) in each group. ***P < 0.001 (df = 4,25 and F value = 8.16). (B) Changes in the esophageal l-tryptophancontent in normal control rats and l-tryptophan pretreated RE rats (l-100 + RE, l-200 + RE and l-400 + RE). Results were represented in means ± SEM, with six rats (n = 6) ineach group. *P < 0.05 and ***P < 0.001, respectively (df = 4,25 and F value = 9.26).

Fig. 3. (A) Alterations in the esophageal melatonin levels in normal control and l-tryptophan pretreated normal rats (l-100 per se, l-200 per se and l-400 per se, respectively).Results were represented as means ± SEM, with six rats (n = 6) in each group. **P < 0.01 and ***P < 0.001, respectively (df = 3,20 and F value = 23.18). (B) Changes in theesophageal l-tryptophan content in normal control rats and l-tryptophan pretreated normal rats (l-100 per se, l-200 per se and l-400 per se, respectively). Results wererepresented in means ± SEM, with six rats (n = 6) in each group (df = 3,20 and F value = 0.3880).

Table 1Alterations in the total gastric volume, acidity, pH and mucin contents in control RE group (RE control), RE groups pretreated with l-tryptophan at the doses of 100–400 mg/kg(l-100 + RE, l-200 + RE, l-400 + RE, respectively) and omeprazole (Omz + RE) at the dose of (10 mg/kg).

Group Gastric volume (ml) Total acidity (�Eq/ml) pH Mucin content (�g/ml of gastric juice)

RE control 4.41 ± 0.50 157.20 ± 15.85 2.14 ± 0.23 548.10 ± 58.87l-100 + RE 3.76 ± 0.45 133.30 ± 29.94 1.91 ± 0.27 656.00 ± 75.14l-200 + RE 2.12 ± 0.38*** 73.67 ± 5.20* 3.37 ± 0.26** 894.6 ± 77.66*

l-400 + RE 1.27 ± 0.18*** 58.79 ± 5.46* 4.53 ± 0.42*** 1455.00 ± 135.40***

Omz + RE 0.80 ± 0.12*** 54.78 ± 6.09* 5.20 ± 0.31*** 919.60 ± 83.65*

Results were expressed as means ± SEM with n = 6 rats in each group.* P < 0.05 in comparison to control.

** P < 0.01 in comparison to control.*** P < 0.001 in comparison to control.

254 P. Singh et al. / Pharmacological Research 64 (2011) 249– 257

Fig. 4. Histogram representing the changes in the gene expression of AA-NAT andHIOMT in normal control rats, reflux esophagitis (RE) control group and RE grouppretreated with l-tryptophan at graded doses (l-100 + RE, l-200 + RE and l-400 + RE,respectively). Results were expressed as means ± SEM with three rats (n = 3) ineach group. The gel image provided was representative of experiments performediv

32

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Fig. 5. Histogram representing the changes in the gene expression of AA-NAT andHIOMT in normal control rats, normal control rats pretreated with l-tryptophanat graded doses (l-100 per se, l-200 per se and l-400 per se, respectively). Resultswere expressed as means ± SEM with three rats (n = 3) in each group. The gel

logical functions. GIT synthesized its own melatonin from precursor

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n triplicates. *P < 0.05 and ***P < 0.001, respectively (for AA-NAT, df = 4,10 and Falue = 4.54 and for HIOMT, df = 4,10 and F value = 17.55).

.7. Effect of l-tryptophan on gastric cycloxygenase (COX) 1 and activities

As shown in Fig. 8, significant reduction in the COX-2 activityP < 0.05) was observed in RE-group compared to normal controlats with no significant change in the activity of COX-1 isoformn these groups. l-200 + RE group showed significantly (P < 0.01)levated COX-2 activity compared to RE-control rats; however,

OX-1 activity remained unaltered. IND + RE significantly (P < 0.05)educed the activity of both COX-1 and COX-2 enzymes as com-ared to RE-control groups. However, in l-200 + IND + RE group

able 2hanges in the total gastric volume, acidity, pH, mucin contents and PGE2 level in con

ndomethacine (IND + RE) and indomethacin + l-200 simultaneously (IND + l-200 + RE).

Group Gastric volume (ml) Total acidity (�Eq/ml) pH

Normal control – – –

RE control 4.41 ± 0.50 157.20 ± 15.85 2.14 ± 0.23

l-200 + RE 2.12 ± 0.38*** 73.67 ± 5.20* 3.37 ± 0.26**

IND + RE 5.93 ± 0.35# 260.60 ± 42.13## 1.00 ± 0.07#

IND + l-200 + RE 4.96 ± 0.42 238.90 ± 28.56 1.84 ± 0.35

esults were expressed as means ± SEM with n = 6 rats in each group.* P < 0.05 in comparison to RE control.

** P < 0.01 in comparison to RE control.*** P < 0.001 in comparison to RE control.∧ P < 0.05, with respect to normal control group.# P < 0.05 in comparison to RE control group.

## P < 0.01 in comparison to RE control group.

image provided was representative of the three separate experiments. *P < 0.05and ***P < 0.001, respectively (for AA-NAT, df = 3,8 and F value = 1.70 and for HIOMT,df = 3,8 and F value = 0.3454).

there was no significant change in the activities of both COX-1 andCOX-2 when compared with IND + RE group.

4. Discussion

Discovery of GIT as the major extra-pineal source of melatoninhas added new dimensions to the understanding of its diverse bio-

amino acid l-tryptophan, independent of pineal gland [27]. Variousstudies show that l-tryptophan administration can improve the cir-culating melatonin levels produced largely from GIT [14,15,28,29].

trol RE group (RE control), RE group pretreated with l-tryptophan (l-200 + RE),

Mucin content (�g/ml of gastric juice) Gastric PGE2 level (pg/mg protein)

– 4487 ± 662.30548.10 ± 58.87 2569 ± 459.40∧

894.6 ± 77.66* 9981 ± 764.00***

253.00 ± 31.92# 1229 ± 194.90#

428.5 ± 93.52 1715 ± 286.50

P. Singh et al. / Pharmacological Research 64 (2011) 249– 257 255

Fip

Iwcm

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Fig. 8. Changes in the activities of cycloxygenase (COX) 1 and 2 in normalcontrol group, RE group (RE control), RE group pretreated with l-tryptophan(l-200 + RE), indomethacine (IND + RE) and indomethacin + l-200 simultaneously(IND + l-200 + RE). Results were represented as means ± SEM, with six rats (n = 6)

ig. 6. Effect of l-tryptophan and standard drug omeprazole on H+ K+-ATPase activ-ty in the rat gastric microsomes. Dots and lines are means ± SEM of experimentserformed in triplicates.

t is also shown that l-tryptophan supplementation in combinationith melatonin and vitamins significantly reduced the GERD asso-

iated symptoms [4] in patients observed separately to have poorelatonin levels [7]. However, the study deciphering the exact role

ig. 7. (A) Pictomicrograph representative of the gross appearance of whole esopha-us in RE-control group and various drug treated group. (B) Bar diagram representinghe changes in the area of haemorrhagic lesions in reflux esophagitis (RE) controlnd various drug treated groups. Results were expressed as means ± SEM, with sixats (n = 6) in each group. **P < 0.01 (df = 4,25 and F value = 6.78).

in each group. *P < 0.05 and **P < 0.01 (df = 4,25 and F value = 0.8588 for COX-1 anddf = 4,25 and F value = 17.10 for COX-2, respectively).

of l-tryptophan in reflux esophagitis is largely unknown, we there-fore aim to investigate the role of l-tryptophan in experimental REand its contribution in the esophageal level of melatonin.

Present study supported the previous notion that establishmentof RE caused a deficit in local production of melatonin [7]. Ourhypothesis that l-tryptophan treatment would improve this loss ofesophageal melatonin level accompanying RE-induction failed, asvarious doses of l-tryptophan were not being able to overcome thisdeficit. l-Tryptophan is a substrate of two important biosyntheticpathways, one leading to the synthesis of serotonin and mela-tonin [30] and other to the production of niacin [31]. It is reportedthat chronic immune activation pushes l-tryptophan toward niacinproduction [32]. Furthermore, previous report demonstrates thatesophagitis imposed esophageal mucosa to massive inflammatoryand oxidative load [33]. Thus here it can be hypothesized that l-tryptophan entering into GIT could be converted into niacin ratherthan melatonin, causing a decreased levels of melatonin duringRE. Paradoxically, increasing the doses of l-tryptophan in RE-ratsresulted into significant enhancement of its own endogenous lev-els which were not seen in l-tryptophan per se group. The possibleexplanation for these discrepancies could be that during normalcondition l-tryptophan that reaches the GIT may get metabolizedinto the melatonin, and because of the large size of GIT it mayget consumed rapidly to convert into the melatonin. While duringesophagitis since l-tryptophan may not enter the pathway to pro-duce melatonin, it could be less efficiently used in GIT, consequentlyits concentration may build up with the increasing doses.

Earlier immuno-histochemical studies revealed the presence ofmelatonin in practically all parts of rat GIT [34]. Further detec-tion of the melatonin synthesizing enzymes HIOMT and AANATin GIT confirmed the occurrence of its synthesis rather thanjust passive accumulation [12]. In concur to this our result alsoshowed the occurrence of both these enzymes in the esophagusof normal rat sustaining the possibility of melatonin synthesis inesophagus. Establishment of RE caused significant decline in thegene expression of both enzymes which further supported our

interpretation that the process of esophageal melatonin synthe-sis may get blocked in RE-rats. l-Tryptophan administration inRE rats though significantly upregulated the expression of AA-

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AT enzyme, it was not being able to counteract the RE-repressedevel of HIOMT enzymes. In rats, biosynthesis of melatonin is pos-tively regulated through sympathetic neurons projecting into theland. The norepinephrine (NE) released from these nerve end-ngs at night stimulates serotonin N-acetyltransferase (AA-NAT),esulting in increased melatonin levels [13,35,36]. It is also shownhat the proteolytic degradation of AA-NAT resulting from ter-

ination of NE-induced stimulation of cAMP is responsible fornding melatonin synthesis [37]. Esophagitis mucosa also showedepressed cholinoreceptor mediated responses [38]. Thus possiblyhe insufficient cholinergic stimulus due to the esophagus injury

ay cause proteosomal degradation of AA-NAT, and despite ofhe l-tryptophan mediated increase in its expression we observedecreased melatonin synthesis in RE-rats. Altered melatonin syn-hesis in RE model can also be attributed to the decline in thexpression of HIOMT enzyme, which l-tryptophan failed to reverse.urther examination of enzyme activity is essential to test theseypotheses.

Apart from these results, our observation of l-tryptophan pere effect showed that in normal control rats the melatonin levelnhanced manifold with increasing l-tryptophan doses reflectinghat it possibly participates in the mechanism of esophageal mela-onin synthesis. These results are in agreement to a study showinghe role of l-tryptophan in GIT synthesis of melatonin [14]. How-ver, in RE this pathway may get hampered due to the injureducosa, which can be associated with reduced cholinoreceptor

ignals during esophageal inflammation which regulate the activ-ty of melatonin synthesizing enzyme [39]. Moreover the intakef l-tryptophan in normal control rats did not change the normalxpression of AA-NAT and HIOMT gene, showed that esophagusave the ability to produce melatonin under normal condition, thisotion is in agreement with other studies also [14,28,29].

Our direct examination of melatonin level following l-ryptophan administration also reflected that the ameliorativeffects of l-tryptophan on esophageal damage ensuing RE-nduction may involve other complex mechanism and is notependent on its conversion into melatonin. To further confirmhat protective action of l-tryptophan against RE is independent of

elatonin, we blocked melatonin actions using its antagonist luzin-ole. l-Tryptophan pretreatment protected the esophagus fromamage even in the presence of luzindole further supporting ourypothesis that l-tryptophan mediated protection in RE is inde-endent of melatonin.

In an attempt to find the possible mechanism of l-tryptophanxerted protection against RE, we noted that it offered great reduc-ion in the total gastric output with corresponding decline in totalcidity. l-Tryptophan also greatly elevated the pH and mucin con-ent of accumulated gastric juice. The dose-related reduction ofhe volume of gastric juice indicates that this inhibition may note due to a non-specific effect such as a dilution effect result-

ng from a break in the mucosal barrier. Our results thus inferredhat l-tryptophan might protect against esophageal damage viaegulating the gastric secretions and follow the mechanism thatontrolled the flow of gastric juice into lumen and potentiated theucin secretions. Since the scant secretion ensures the adequate

ime for the mucus or alkaline mucosa to act on it [40,41], thebserved increase in pH after l-tryptophan might be due to neu-ralizing effect of mucin on accumulated gastric juice. In support ofhis Pavlov also stated that abundant secretion of the gastric glandave higher acidity then scant secretions due to the fact that rapidowing of juice offer less opportunity to mucus or alkaline mucosao neutralize it [42].

Several endogenous mediators are involved in the secretion ofastric acid, which initiate unique series of events that finally con-erge into activation of the enzyme H+ K+-ATPase. Thus, we nextxamined the effect of l-tryptophan on the H+K+-ATPase activity in

esearch 64 (2011) 249– 257

gastric microsomal preparation. It was observed that l-tryptophanfailed to inhibit the H+ K+-ATPase activity under in vitro condi-tion compared to positive control omeprazole. However, underin vivo experiments, the acid suppressing effect of l-tryptophanwas essentially similar to the omeprazole. The differential responseof l-tryptophan on acid secretion under in vivo and in vitro circum-stances signified that l-tryptophan did not directly influence theH+K+-ATPase activity like omeprazole [43]. However, unlike pro-ton pump inhibitors, its antisecretory functions seemed to be moredependent on the actions of gastric acid secretagogue. The similaracid attenuating effect of l-tryptophan has been studied in stressinduced gastric ulcer model [16], where it offered gastro-protectionvia stimulating the function of acid secretagogue such as gastrinand PGE2. In view of these literatures, we next examined the roleof l-tryptophan on gastric PGE2 concentration.

We found that l-tryptophan treatment markedly augmentedthe gastric PGE2 content which agrees with previous studies[16,22]. The participation of PGE2 in the anti-secretory functionsin our model was confirmed by blocking it via indomethacin.Indomethacin not only increased the gastric volume and acidity, italso lowered the pH and mucin content of gastric juice in the RE rats.Similar kind of association has also been noted in gastroduodenalmucosa treated with indomethacin [44] suggesting the regulatoryrole played by PGE2 in gastric secretions. However, l-tryptophanfailed to overcome the changes produced by indomethacin inRE-group. This result suggested that l-tryptophan mediated aug-mentation of gastric PGE2 might be the possible explanation for itsantisecretory functions, a view shared by others [45–47].

In order to examine the source of the increased PGE2 fol-lowing l-tryptophan treatment we examined the changes in theactivities of cycloxegenase (COX) 1 and 2. It was observed thatl-tryptophan significantly increased the RE-lowered activity ofCOX-2 enzyme. However, it showed no effect on COX-1 activity.Further l-trtyptophan was unable to increase the COX-2 activitywhen blocked by indomethacin co-administration. These resultsclearly showed that the increased PGE2 obtained after l-tryptophantreatment in RE-rats was COX-2 derived. Hence the l-tryptophanimparted protection against RE seemed to be resultant of its attenu-ating effect on gastric acid secretions and subsequent mobilizationof mucosal defensive factors such as mucin and COX-2 derived PGE2that counteracted the potential detrimental effects of gastric acid.

In conclusion, we for the first time demonstrated that the onsetof RE caused impairment of melatonin synthesizing pathway viarepressing the expression of rate limiting enzymes involve in itssynthesis. In addition, we showed that l-tryptophan prevented theonset of RE via a mechanism that is independent of its conversioninto melatonin. l-Tryptophan apparently ameliorates RE-inducedesophageal damage via inhibiting the gastric acid secretion as wellas activating the factors such as gastric mucin and COX-2 derivedPGE2 that counterbalance its detrimental effects on esophagealmucosa.

Acknowledgements

We sincerely thank Indian council of medical research (ICMR,New Delhi, India) for providing financial support and Mrs. ShibaniSengupta for her technical help.

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