Melatonin, (N- acetyl-5-metoksytryptamine) discovered in 1958 by Aaron Lerner as a main product of pineal gland is produced from amino acid L-tryptophan in a four step reaction (1, 2). Beside its production in the central nervous system (CSN) melatonin is synthesized in retina, cilliary bodies, kidney, respiratory tract, but the main extrapineal source of this indoloamine remains the gastrointestinal tract (3).

The presence of melatonin was confirmed in many organs of humans digestive system such as pancreas, liver, esophagus, stomach, duodenum, ileum, colon or rectum (4, 5). Melatonin is produced in the gastrointestinal tract (GIT) in the enteroendocrine cells (EE cells) of its mucosa. Synthesis of this indole significantly increased following oral administration of L-tryptophan (6-9), but the physiological role of this indoloamine in digestive system is still unknown.

The effects of melatonin are mediated via specyfic receptors which are localized in CNS as well as in various peripheral tissues such as heart, thyroid gland and also in the gut and in the pancreas (10-12). It has been proved that melatonin is able to influence both endo or exocrine pancreatic functions, but the mechanisms of above melatonin effects have not been clarified. Melatonin has been reported to reduce glucose-induced secretion of insulin leading to hyperglycemia (13). On the other hand it has been shown that this indole could stimulate insulin secretion from isolated pancreatic beta cells (14). Exogenous melatonin, as well as this synthesized from L-tryptophan, has been demonstrated to protect pancreatic tissue from the damage caused by caerulein overstimulation or ischemia/reperfusion, leading to the spectacular reduction in proinflammatory cytokines production and to the marked decrease of lipid peroxidation (15, 16).

In our recently published study we have reported that melatonin, as well as its precursor, L-tryptophan when given introperitoneally to the rats produced dose-dependent stimulation of pancreatic enzyme secretion, however above substances failed to affect in vitro amylase release from pancreatic acini (17). Melatonin also has been demonstrated to stimulate production and phosphorylation of HSP27 in human pancreatic carcinoma cells (18).

Pancreatic enzyme secretion is controlled by neural and hormonal mechanisms, involving the sensory fibers, vagal nerves, central nervous system with DVC (dorsal vagal complex), enteric nervous system (ENS) and hormones like CCK, serotonin and many others (19-21). According to Lee & Owyang, CCK which is released from I cells in the duodenal mucosa is able to stimulate pancreatic exocrine secretion through the activation of specific CCK1 receptors on vagal afferents which trigger vago-vagal enteropancreatic reflex (22).

The purpose of this study was to investigate the influence of melatonin or its precursor; L-tryptophan administered directly into duodenal lumen, on pancreatic protein secretion in the anaesthetized rats with pancreato-biliary fistulas, and to assess the involvement of vagal nerves and CCK in above pancreatic exocrine secretory effects of melatonin or L-tryptophan.


MATERIALS AND METHODS
Materials

Following items were purchased: melatonin, L-tryptophan and specific CCK1 receptor antagonist; lorglumide, were from Sigma Co (St. Louis, MO, USA); CCK radioimmunoassay commercial kit was from DRG International Inc. (Mountainside, WI, USA). Vetbutal (Pentobarbitalum) was from BIOWET (Pu³awy, Poland). Silicone tubes were purchased from Rainchelt Chemietechnik, GmBH, (Helderberg, Germany)

The experimental protocol was approved by the Jagiellonian University Ethical Committee for Animal Experimentation and run in accordance to the statements of European Union regarding handling of experimental animals.

Animal preparation.

The study was performed on male Wistar rats weighting about 350 g. Animals were housed in cages under standard conditions at 24 hours light/dark cycle at constant temperature with free access to standard laboratory chow and water. Rats were deprived from food for 24h before the experiment with continued access to water. The surgery was performed under pentobarbiturate anesthesia (Vetbutal), given intraperitoneally (i.p) at a dose of 15 mg/300g body weight. Supplemental doses were used every 2 h as needed to maintain adequate anesthesia.

Through an upper midline laparotomy, the duodenum was identified and the bile-pancreatic duct was isolated as its entrance to the duodenum. A silicone tube diameter 0,5 mm was inserted into the common bile-pancreatic duct for bile and pancreatic juice collection and was secured with a fine suture. A second silicone cannula diameter 0,7 mm was placed into the duodenum and its tip fixed proximal to the ampulla for reinfusion of previously harvested bile-pancreatic juice (after dilution with saline 1:2) and for administration the investigated substances (melatonin, L-tryptophan or lorglumide). The abdominal incision was sutured with a double layer suture and the animals were kept under the heating lamps to maintain the right body temperature (37° C). At the end of experiment, the abdominal vena cava was exposed and the blood was withdrawn into EDTA containing tubes for determination of CCK by radioimmunoassay (RIA).

During the experiment the animals were placed in individual Bollmann cages. The bile-pancreatic juice (BPJ) samples were collected in small preweighted vials in 15 min aliquots to measure the volume of each sample and protein concentration. Basal secretion of pancreatic juice was measured by collecting of BPJ for 60 min to allow for stabilization of flow affected by surgical manipulation. Protein concentration of each sample was measured by spectrophotometric method, as described previously (23). The results were expressed as total protein (mg per 15 min) outputs. During all experiments, except those with diversion of BPJ to the exterior (DPBJ), previously collected pancreato-biliary juice was re-infused via duodenal cannula into duodeum at the rate of 1 ml/h (24).

The study consisted of three series of experiments. In series I the effects of melatonin or its precursor L-tryptophan given alone on pancreatic exocrine secretion under basal conditions or following the stimulation of this secretion with diversion of pancreatic juice to the exterior (DPBJ) were studied. Series II included group of animals with bilaterally transected vagal nerves. In series III, CCK1 receptor antagonist; lorglumide, was used to determine the involvement of CCK in the secretory effects of melatonin or L-tryptophan on exocrine pancreas. For each part of the experiment the animals groups of 5-6 rats were used. Melatonin was dissolved in a drop of DMSO (dimethyl sulfoxide) and then diluted in physiologic saline to obtain an appropriate concentration. L-tryptophan was dissolved in physiologic saline containing a drop of 0.1N HCl. Melatonin or its precursor L-tryptophan were given in a volume of 0,5 ml to the rats intraduodenally (i.d.) as a bolus injection.

Series I

The following experiments, with pancreato-biliary juice (PBJ) returned to the duodenum throughout the experiment were performed: 1. Control (i.d. bolus injection of vehicle saline); 2. Melatonin at doses of 1; 5; 25 mg/kg or L-tryptophan at doses of 10; 50; 250 mg/kg were administered i.d to the rats. Each dose of melatonin or L-tryptophan was given to the separate group of the animals.

In this part of the study diversion of pancreato-biliary juice to the exterior throughout the experiment was performed to stimulate pancreatic secretion. The following study groups, each consisting of 6-8 animals, were employed to investigate the postdiversional pancreatic stimulation including: 1. Control (DPBJ alone); 2. DPBJ combined with melatonin or L-tryptophan given i.d to the animals at doses of 1; 5, 25 mg/kg or 10; 50; 250 mg/kg, respectively. Each dose of melatonin or its precursor L-tryptophan was administered to the separate group of rats.

Series II

This series was designed to evaluate the role of vagal nerves in the pancreatic secretory effects of melatonin or its precursor L- tryptophan in the rats with bilateral subdiaphragmatic vagotomy. The animals were anesthetized with pentobarbiturate and the esophagus was exposed through a midline laparotomy and subdiaphragmatic vagal trunks were isolated at the level of gastric cardia. Both anterior and posterior trunks of vagal nerves were transsected, and the abdomen was sutured. Rats were allowed to recovery for 10 days before the start of experiments. Pancreato-biliary juice was returned into the duodenum and selected dose of melatonin (5 mg/kg i.d.) or L-tryptophan (50 mg/kg i.d.), were given to the animals. The control group of the animals received the intraduodenal bolus of the vehicle saline instead of melatonin or L-tryptophan.

Series III

This study was used to determine the effects of intraduodenal infusion of CCK1 receptor antagonist, lorglumide, on pancreatic enzymatic secretory response to melatonin or its precursor, L-tryptophan. Lorglumide was given i.d as a bolus injection 15 min prior the administration of melatonin or L-tryptophan at selected doses in a volume of 0.5 ml.

Several study groups, (each group consist of 6-8 rats) were employed including: 1) Vehicle (0.5 ml of saline) injected i.d. as a bolus, 2) Lorglumide (1 mg/kg) given i.d.; followed 15 min later by i.d. injection of vehicle saline; 3) Lorglumide (1 mg/kg i.d.) followed 15 min later by melatonin (at dose of 5 mg/kg i.d.) or L-tryptophan (at dose of 50 mg/kg i.d).

Determination of CCK plasma level:

Plasma CCK concentrations was measured by radioimmunoassay (RIA) using rat CCK radioimmunoassay commercial kit, according to the manufacturer's protocol.

Statistical analysis

Comparison of the differences between the mean values of various groups of experiments was made by analysis of variance and the Student's t test for unpaired data. Differences with a p value of < 0.05 were considered statistically significant. Results are expressed as means ± SEM.


RESULTS
During reinfusion of pancreato-biliary juice (PBJ) into the duodenum in anesthetized rats with acute pancreatic fistulas the basal protein output was well sustained and averaged 4,09 ± 0,39 mg/15 min (Figs. 1 and 2, Table 1). Melatonin at doses of 1, 5 or 25 mg/kg increased dose-dependently protein output, reaching about 12,4 ± 1,6 mg/15 min, at dose of 25 mg/kg i.d. of this indoloamine (Fig. 1).

Fig. 1. Time course of protein outputs of pancreato-biliary juice following the bolus of intraduodenal (i.d.) administration of melatonin at doses of 1, 5 or 25 mg/kg. Control = protein output in rats treated with vehicle instead of melatonin. The results are means ± SEM of separate experiments, each performed on group of 6-8 animals. Asterisk (*) indicates significant (p< 0.05) increase above the control value.

Administration of L-tryptophan, melatonin precursor also resulted in significant and dose-dependent enlargement of protein output, that reached about 9,45 ± 1,1 mg/15 min while the dose of 250 mg/15 min of this amino acid was used (Fig. 2).

Fig. 2. Time course of protein secretions of pancreato-biliary juice following the i.d. administration of melatonin precursor, L-tryptophan at doses of 10, 50 or 250 mg/kg. Control = protein output in rats treated with vehicle instead of L-tryptophan. The results are means ± SEM of separate experiments, each performed on of 6-8 rats. Asterisk (*) indicates significant (p< 0.05) increase above the control value.

In the experimental groups with diversion of pancreato-biliary juice to the exterior (DPBJ) the protein secretion increased above the basal level, reaching a highest value at 135 min and then remaining elevated for the rest of the experiment. DPBJ caused a significant increase in protein secretion to 7,52 ± 0,9 mg/15 min (by about 46 %), as compared to the control values (4,09 ± 0,39 mg/15 min) obtained in the animals with the pancreato-biliary juice returned to the duodenum (Figs 3 and 4). Application of melatonin at a dose of 5 or 25 mg/kg significantly and dose-dependently stimulated this protein output reaching the highest value that was 13,9 ± 1,6 mg/15min at dose of 25 mg/kg. Intraduodenal administration of melatonin at dose of 1mg/kg failed to affect significantly stimulated with DPBJ pancreatic secretion (Fig. 3).

Fig. 3. Effect of intraduodenal (i.d.) administration of melatonin (1, 5 or 25 mg/kg) on pancreatic protein secretions stimulated by diversion of pancreato-biliary juice (DPBJ). Control = protein output in rats treated with vehicle instead of melatonin. The results are means ± SEM of separate experiments, each performed on group of 6-8 animals. Asterisk (*) indicates significant (p< 0.05) increase above the value obtained with DPBJ alone.

Administration of melatonin precursor, L-tryptophan at doses of 50 or 250 mg/kg resulted in significant and dose dependent rises of protein secretion, while the highest used dose of this amino acid caused the extreme augmentation of protein output (11,2±1,4 mg/15 min). However, this increase was less pronounced than the stimulatory effect of melatonin (Figs. 3 and 4). The lowest dose of L-tryptophan (10 mg/kg i.d.) has no effect on the pancreatic protein secretion stimulated with DPBJ (Fig. 4).

Fig. 4. Effect of i.d. administration of L-tryptophan at doses of 10, 50 or 250 mg/kg on pancreatic protein secretion stimulated by diversion of pancreato-biliary juice (DPBJ). Control = protein output in rats treated with vehicle instead of melatonin precursor. The results are means ± SEM of separate experiments, each performed on of 6-8 rats. Asterisk (*) indicates significant (p< 0.05) increase above the value obtained with DPBJ alone.

Bilateral, subdiaphragmatic vagotomy failed to affect significantly basal pancreatic protein secretion but completely abolished the increases of protein output in response to melatonin (5 mg/kg ) or L-tryptophan (50 mg/kg), administered directly into the duodenal lumen (Figs 5 and 6).

Fig. 5. Protein output in response to application of melatonin (5 mg/kg i.d.) in the rats with intact to vagal nerves and in the animals subjected to bilateral vagotomy. Control = protein secretion in animals injected with saline instead of melatonin. The results are the means ± SEM of 4 separate experiments, each performed on 6-8 rats. Asterisks (*) indicates significant (p< 0.05) increase above the control value. Cross (+) indicates significant (p< 0.05) decrease below the value obtained in animals with melatonin alone.

Fig. 6. Protein output in response to administration of L-tryptophan (50 mg/kg i.d.) in the rats with intact vagal nerves and in the animals subjected to bilateral supradiaphragmatic vagotomy. Control = protein secretion in animals injected with vehicle instead of investigated substance. The results are the means ± SEM of 4 separate experiments, each performed on 6-8 rats. Asterisks (*) indicates significant (p< 0.05) increase above the control value. Cross (+) indicates significant (p< 0.05) decrease below the value obtained in animals with L-tryptophan alone.

Intraduodenal application of lorglumide, CCK1 receptor antagonist, at dose of 1 mg/kg did not elicit any significant alterations in pancreatic protein secretion under basal conditions. The pretreatment of rats with pancreato-biliary fistulas with this CCK1 receptor blocker, resulted in complete reversion of rises of protein outputs caused by melatonin (5 mg/kg i.d.) or L-tryptophan (50 mg/kg i.d.) (Table 1).

Table 1. Protein outputs (mg/15 min) in rats under basal conditions in response to melatonin (5 mg/kg i.d.), or L-tryptophan (50 mg/kg i.d.) alone or after pretreatment of the rats with lorglumide, CCK1, receptor blocker (1 mg/kg i.d.). Control = protein secretion in animals injected with vehicle instead of investigated substances. The results are means ± SEM from the separate experiments, each performed on 6-8 animals. Asterisk (*) indicates significant (p< 0.05) increases above the control value. Cross (+) indicates significant (p< 0.05) decrease below the value obtained in animals with melatonin or L-tryptophan alone.

Under basal conditions plasma level of CCK averaged about 15,4 ± 1,8 pg/ml. Pretreatment of the rats with increasing doses of melatonin (1, 5, or 25 mg/kg id) resulted in the significant and dose-dependent enhancements in plasma CCK immunoreactivities above the level detected in control group, reaching 49 ± 3,5 pg/ml at dose of 25 mg/kg of melatonin (Fig. 7). L-tryptophan, administered intraduodenally at doses of 10, 50 or 250 mg/kg also increased in dose-dependent manner plasma CCK concentrations, reaching 41,2 ± 4.0 pg/ml, achieved following administration of highest dose of melatonin precursor (250 mg/kg) (Fig. 7).

Fig. 7. Effect of increasing concentration of melatonin (1, 5 or 25 mg/kg) or L-tryptophan (10, 50 or 250 mg/kg, respectively) given intraduodenally (i.d.) under basal conditions on CCK plasma level (pg/ml). The results are means ± SEM from the separate experiments, each performed on 6-8 rats. Asterisk (*) indicates significant (p< 0.05) increase above the control value. Control = CCK plasma level in the group of animals treated with injection of 0,9% NaCl.

In the DPBJ statement plasma CCK immunoreactivity gained to 48,7 ± 5,1 pg/ml.

Intraduodenal application of melatonin (1, 5 or 25 mg/kg) or its precursor, L-tryptophan (10, 50 or 250 mg/kg ) resulted in dose-dependent augmentation of CCK plasma level, averaging 119 ± 10,8 pg/ml or 98 ± 10,1 pg/ml, respectively after pretreatment of the rats subjected to DBPJ with the highest doses of examined substances (Fig. 8).

Fig. 8. Effect of i.d. administration of various doses of melatonin (1, 5 or 25 mg/kg) or L-tryptophan (10, 50 or 250 mg/kg) following the stimulation of pancreatic secretion with DPBJ on CCK plasma level (pg/ml). The results are means ± SEM from the separate experiments, each performed on 6-8 rats. Asterisk (*) indicates significant (p< 0.05) increase above the value obtained with DPBJ alone. Control = CCK plasma level in the group of animals treated with injection of saline.

DISCUSSION
The present study demonstrates that; 1) intraduodenal administration of melatonin or its precursor; L-tryptophan increases pancreatic protein secretion under basal conditions or following the stimulation of this secretion with diversion of pancreato-biliary juice (DBPJ), 2) bilateral vagotomy, as well as CCK1 receptor blockade with lorglumide completely repealed the effects caused by melatonin or L-tryptophan on pancreatic exocrine function, 3) intraduodenal application of examined substances results in a dose-dependent rises of plasma CCK level.

The presence of melatonin in gastrointestinal tract was evidenced about 40 years ago (6). The main source of melatonin presented in the gut lumen appears the enteroendocrine cells, as well as the food, bile and intestinal microorganisms (25). It has been reported that melatonin could act as a modulator of many gastrointestinal functions to synchronize digestive processes (5). This substance causes smooth muscle relaxation (26, 27), decreases food transit time (28), modulates the electrolytes secretion in the intestinal lumen (27) and also via MT1 receptors reduces the neurotransmission in the gastrointestinal tract (30).

Melatonin is widely known as the gastroprotective agent. It is well evidenced that melatonin, as well as its precursor L-tryptophan is implicated in the prevention of gastric mucosal lesions induced by ethanol, stress, aspirin or ischaemia-reperfusion (32, 33). The mechanism of above beneficial action of melatonin has been related to the ability of this indole to attenuate lipid membrane peroxidation, to the influence on cyclooxygenase-prostaglandin (COX-PG) and nitric oxide (NO) systems as well as to the activation of brain-gut axis (34, 35).

The presence of specific melatonin receptors in the pancreatic tissue proclaims that melatonin is able to control the endocrine as well as the exocrine pancreatic functions. Several observations suggest that melatonin could inhibit insulin secretion (13). To the contrary some authors have reported the stimulatory effects of this indoloamine on the endocrine pancreatic function (14). Melatonin has been shown to exert the beneficent effect on the course of experimental acute pancreatitis suggesting the implication of this indoloamine in the pancreatic defense against inflammation (36, 37).

In our previous study we have demonstrated that exogenous melatonin as well as its precursor L-tryptophan when administered intraperitoneally increased pancreatic amylase secretion. The pancreatostimulatory effects of examined substances were indirect because neither melatonin nor L-tryptophan significantly affected amylase release from isolated pancreatic acini (17, 38). We have also revealed that the release of CCK is strictly attributed to above stimulatory effects of melatonin or its precursor (17).

According to the hypothesis of Li and Owyang CCK at low, physiological doses stimulates pancreatic enzyme secretion through indirect mechanism depending on the activation of specific CCK1 receptors localized on vagal afferent fibers in the duodenal mucosa and involving the vago-vagal enteropancreatic reflex (20, 21). Previous study has documented that CCK-8 is able to stimulate pancreatic secretion in the calf exerting its effect from duodenal lumen via cholinergic pathway (39). As we have shown recently intraluminal administration of exogenous melatonin or its precursor, L-tryptophan resulted in the significant and dose-dependent rises of CCK plasma immunoreactivity in the rats with pancreato-biliary fistulas (40). Herein we demonstrate, for the first time, that above increases of plasma CCK in response to melatonin or to its precursor, have been observed not only under basal conditions but also following the stimulation of pancreatic secretion by DPBJ.

The involvement of CCK in the stimulatory effects of intraduodenally administrated examined substances has been confirmed by the results showing that the blockade of CCK1 receptor by lorglumide reversed the increases of protein outputs obtained by i.d. application of melatonin or L-tryptophan. Our results are in agreement with the previous data published by our team already (40), however instead of lorglumide, another CCK1 antagonist, tarazepide was employed that time. Above results are also in agreement with the study published by Niebergall at al. (41, 42). They have reported in dogs that intrajejunal infusion of L-tryptophan increased significantly the carbohydrates output and that above effect has been abolished by preatreatment of the animals with CCK1 receptor blocker L-364,718 or telenzepine (muscarinic receptor antagonist).

Sensory nerves are implicated in the regulation of pancreatic exocrine secretion through the activation of vago-vagal enteropancreatic reflexes. Deactivation of sensory fibers by capsaicin decreased the postprandial secretory response of pancreatic gland (23). Brzozowski at al. have documented that sensory nerves are involved in the protective effects of melatonin on gastric acute lesions and in the healing of gastric ulcerations (43). In our previous study capsaicin deactivation of sensory fibers completely reversed the stimulatory effects of melatonin or L-tryptophan on pancreatic amylase secretion (40). The present data for the first time, strongly and meaningfully demonstrates that bilateral subdiaphragmatic vagotomy completely reversed the stimulatory effects caused by intraduodenal application of melatonin or L-tryptophan. This observation suggests that vagal nerves are implicated in the pancreatostimulatory action of examined substances on exocrine pancreatic function.

In conclusion: our study demonstrates that in anesthetized rats exogenous melatonin or its precursor L-tryptophan when administered directly into the duodenal lumen significantly and dose-dependently stimulates pancreatic protein secretion. The mechanism of this effect could be related to the CCK release and to the activation of neural pathway; sensory nerves and vago-vagal enteropancreatic reflex.


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