Previous studies revealed that melatonin and its precursor, L-tryptophan, when applied exogenously, are highly effective in prevention of the formation of acute gastric lesions induced by ethanol, stress, aspirin and ischemia-reperfusion (1-3). The mechanism of melatonin’s beneficial action has been attributed to the ability of this indole to stabilize lipid cell membranes and to the antioxidizing activity of this compound, particularly due to its high lipophilic properties allowing for the rapid entrance into the cells to protect their subcellular compartments (4). Moreover, this hormone exhibits immunomodulatory action by influencing the activity of nitric oxide synthase (NOS) and cytokine production during inflammatory and carcinogenic processes (5-8). Our recent studies (9-11) fully confirmed previous observations that both, melatonin and its precursor, L-tryptophan, dose-dependently reduced the number of acute gastric lesions, attenuated lipid peroxidation and enhanced activity of antioxidant enzymes in gastric mucosa in rats exposed to 3.5 h of water immersion and restraint (WRS), representing typical oxidative stress-induced gastric disorder. These protective effects were accompanied by gradual increase in plasma melatonin levels suggesting that topical melatonin exerts a local protective action on gastric mucosa, acting via
circulation following its absorption form the gut. Furthermore, L-tryptophan as highly hydrophobic substance, easily penetrates the gastrointestinal barrier to be quickly transformed into melatonin within gastrointestinal mucosa, showing the same activity as oral melatonin to achieve the same plasma indole levels. Our and others previous studies demonstrated that liver causes marked inactivation of melatonin on its way to pass from the gut lumen into the circulation (10).
The mechanism of the gastroprotection afforded by melatonin and its precursor, L-tryptophan, has been attributed to the stimulation of cyclooxygenase (COX)/PG system and the scavenge of free radicals as described before (1-3, 9, 11-15). These beneficial effects of melatonin released from the gastrointestinal (GIT) mucosa were supported by the finding that pinealectomy, that is to deplete the major source of melatonin in the body, reduced plasma levels of this indole but failed to affect melatonin contents in the GIT (16). Also, it was shown that pinealectomy in rats (17) which resulted in strong attenuation of diurnal melatonin level and suppressed the night surge of this hormone reversed, in part, the stress-induced gastric lesions, suggesting that normally the nocturnal melatonin limits to some extent of stress-induced gastric injury. However, during the day, pinealectomized rats were more vulnerable to stress-induced gastric lesions and the supplementation of these rats with melatonin or its precursor L-tryptophan reversed the stress-induced gastric ulcerogenicity in these pinealectomized animals (16, 17). If the GIT-originated melatonin is indeed involved in the local mucosal protection, it is expected that exogenous melatonin and its precursor, L-tryptophan should be protective against the mucosal lesions even after pinealectomized rats. Indeed, pinealectomy significantly reduced the basal plasma levels of melatonin and enhanced gastric ulcerogenicity of stress but failed to prevent the gastroprotective activity of exogenous melatonin and its precursor, L-tryptophan (9, 10). The question remains whether the presence of pineal gland and its hormonal activity are really relevant to the circadian rhythm of the formation of stress-induced gastric lesions and for the reduction in stress-induced lesions observed at the dark phase. The circadian rhythm of the formation of gastric lesions has so far been studied mainly in rats with intact pineal gland and little attempts were made to check whether this circadian rhythm of stress-induced gastric is affected in pinealectomized animals.
MATERIAL AND METHODS
This study was approved by the Committee of Animal Research of Jagiellonian University and run in accordance to Helsinki Declaration.
Induction of WRS lesions in rats with intact pineal gland and after pinealectomy
Male Sprague-Dawley rats weighing 220-250 g were maintained on Purina rat chow
and tap water at libitum
and were placed in room with an automatically
regulated lighting cycle with a 12 h light/12 h dark cycle (light on at 08.00
h). The animals were fasted for 24 h prior to experiments but allowed free access
to water preceding the beginning of each experimentation. The studies were performed
at 3 h after lights on and off. For the diurnal studies, light intensity in
experimental room was 600 lux, sufficient to inhibit endogenous pineal melatonin
synthesis. Nocturnal experiments were carried out using a photo safe dim red
light source while the animals were handled.
In separate group of 60 rats, the pineal gland was removed (pinealectomy) according
to the procedure described previously by Kato et al
. (18). Briefly, under
the pentobarbital anesthesia, rats were placed in a prone position and mounted
in a head holder. A sagittal incision was made, and the parietal and interparietal
bones were drilled with a dental burr, allowing visualization of the sagittal
and transverse sinus confluence. The pineal gland was removed by aspiration
a specially constructed 22-G needle, which reached the pineal gland
through the sagittal sinus. Sham-operation consisted of insertion and withdrawal
of needle without applying suction. Rats were used in experiments after 1 week
recovery from the pinealectomy. Complete recovery was confirmed based on locomotor’s
activity compared to that of normal rats. In addition, the brain specimens were
collected at the day of rat autopsies to confirm the successful removal of the
pineal gland by histology determination. Acute gastric stress lesions were induced
by exposure of rats with their intact pineal gland or in those with pineal gland
surgically removed to water immersion restraint stress (WRS) method as described
before (9). Briefly, the animals were placed in restraint metal cages and immersed
vertically to the level of the xyphoid process into a water bath of 23 °C for
3.5 h. The rats were mounted in individual restraint cages for 3.5 h at two
points in the light/dark cycle (from 10.00 a.m. to 2 p.m. and from 10.00 p.m.
to 2.00 a.m.). After the end of 3.5 h of WRS, the rats were lightly anesthetized
with ether, the abdomen was opened and the stomach was exposed. GBF was measured
in the oxyntic gland area of stomach by means of local H2
clearance method using an electrolytic regional blood flow meter (Biomedical
Science, Model RBF-2, Osaka, Japan) as described before (19). The measurements
were made in the three areas of the mucosa and the mean values of the measurements
were calculated and expressed as percent changes of those recorded in the rats
not exposed to WRS (control group) saline-treated animals. The stomach was then
removed, opened along the greater curvature and placed flat to count the number
of gastric lesions by two investigators, unaware of the treatment given as described
in our previous studies (1-3). The stress lesions were defined as round or linear
mucosal defects of at least 0.1 mm in diameter (Fig. 1
). Blood samples
of about 1.5 mL were withdrawn from the jugular vein under ether anesthesia
3.5 h after the initiation of WRS and at the corresponding time before an after
stress exposure during the light and dark phases for measurement of plasma melatonin
in the non-stressed group samples serving as control values for the measurement
of blood plasma immunoreactive melatonin. After counting the number of lesions
in each stomach, the corpus mucosa samples (about 100 mg) were excised from
each stomach for the assessment of the generation of PGE2
Measurement of PGE2 generation in the gastric
mucosa exposed to WRS at the day and night
Corpus mucosa samples were obtained separately during the light and dark phases
from control groups and those subjected to WRS. Mucosal samples were obtained
immediately after removal of the stomach, the corpus mucosa being stripped,
weighed and processed for ex vivo
measurement as previously described (2). Briefly, the tissue was minced with
scissors for 1 min in microfuge plastic tubes containing 1 mL of phosphate buffer
(pH 7.4) and centrifuged in a fixes-speed bench centrifuge at 15 000 r.p.m.
for 30 s. After the supernatant was discarded, the tissue was suspended in 1
mL of phosphate buffer and mixed by vortex for 1 min at room temperature. Then,
10 µL of indomethacin was added to each sample to inhibit further formation
and release of PGs. The samples were centrifuged for 1 min and the supernatant
stored at -20 oC until assay using radioimmunoassay kit (PGE2
NEN Life Science Products Inc. Boston, MA, USA). The assay medium was 0.0255
ml/L phosphate buffered medium azide (0.05%), pH 6.8. Generative capacity was
expressed as nanograms of PGE2
per gram wet
tissue weight (ng/g). Sensitivity of the assay was 1 pg/mL. Recovery as determined
by adding 25 and 50 pg of PGE2
to the samples
was about 50 ± 7% (mean ± S.EM, n = 10).
Determination of plasma and gastric luminal concentration of melatonin
At the termination of experiments with exogenously administered melatonin at
the day and night in animals with or without pinealectomy, the blood samples
(about 3 ml) were taken from the vena cava
(into tubes containing 2500
U Trasylol, Bayer, FRG and 0.5 mg/ml of EDTA) and plasma was separated. The
plasma samples were stored at -20°C until radioimmunoassay (RIA) of melatonin
using commercially available kit purchased from DRG Instruments GmbH (Marburg,
Germany) and described in detail previously (9).
Results are expressed as means ± SEM. Statistical analysis was performed using Mann-Whitney and Friedman two-way analysis of variance. Differences with p<0.05 were considered as significant.
Effect of various time durations of WRS on the WRS lesions and changes in the GBF in rats without or with pinealectomy
As presented in Fig. 1A
, removal of pineal glend by pinealectomy
aggravated the number of WRS-induced gastric lesions.
Representative photomicrograph of gastric mucosa of rat with intact pineal gland exposed to 3.5 h of WRS (A) and that from the pinealectomized animal also exposed to 3.5 h of WRS (B). Note that the number of WRS-induced erosions is enhanced in the rat subjected to pinealectomy as compared to that observed in rat with intact pineal gland.
(upper and lower panel) shows the effect of various time exposures
to WRS on the number of WRS lesions, alterations in the GBF in rats with intact
pineal glands and in those subjected to pinealectomy and plasma levels and luminal
concentrations of melatonin. Exposure to 1.5 h of WRS resulted in the formation
of gastric lesions and produced a significant decrease in the GBF as compared
to non-stressed controls (Fig. 2
, upper panel). When the WRS was extended
up to 3 h and 6 h, the number of gastric erosions was significantly increased
and this effect was accompanied by the greater fall in the GBF as compared to
the respective values obtained in animals exposed to WRS at 1.5 h. Pinealectomy
augmented the WRS-induced gastric erosions and significantly decreased the GBF
as compared to the values achieved in non-pinealectomized animals (Fig. 2
upper panel). Plasma and luminal concentration of melatonin in pinealectomized
rats were markedly lower than those in animals with intact pineal gland, but
these values exhibited a small, though significant increase in response to each
time extension of WRS exposure (Fig. 2
, lower panel).
The number of water immersion and restraint stress (WRS) lesions and alterations
in the gastric blood flow (GBF) (upper panel) and mucosal and luminal
concentrations of melatonin (lower panel) in rats exposed to various time
durations of WRS ranging from 1.5 h up 6 h. Asterisk indicates significant
change as compared to the value obtained at 1.5 h of WRS. Cross indicates
significant change as compared to the respective values obtained in animals
with intact pineal gland.
As shown in Fig. 3
, the number of WRS lesions in rats with an intact
pineal gland during light phase, averaged about 28 ± 3 per stomach and this
was significantly higher than the number of stress lesions (20 ± 2) during the
night. In these animals, the GBF and mucosal generation of PGE2
were significantly reduced (Fig. 3, Table 1
), while plasma melatonin
concentrations showed a significant (by about 30%) rise in response to WRS as
compared to non-stressed animals (data not shown). Rats subjected to WRS at
night, showed the lesion number about 30% lower and the mucosal PGE2
generation and GBF were also significantly reduced as compared with those in
animals with intact pineal gland (Fig. 3, Table 1
). Plasma melatonin
levels reached significantly higher values than those obtained in rats subjected
to WRS during daily hours.
Gastric mucosal PGE2 generation in intact
rats and in those without or with pinealectomy exposed to 3.5 h of WRS
at the day and night. Results are mean ± SEM of 6-8 rats. Asterisk indicates
a significant change as compared to the values obtained in intact animals.
Cross indicates a significant change as compared to the values obtained
in non-pinealectomized rats subjected to WRS during the day. Asterisk
and cross indicate a significant change as compared to the values obtained
in non-pinealectomized rats subjected to WRS during the light phase.
The number of WRS-induced gastric lesions, plasma melatonin levels and
gastric blood flow (GBF) in rats with or without pinealectomy exposed
to 3.5 h of WRS during the day or at night. Asterisk indicates a significant
change as compared to the values obtained in animals without pinealectomy.
Cross indicates a significant change as compared to the values obtained
in rats subjected to WRS during the light phase. Asterisk and cross indicate
a significant change as compared to the values obtained in rats with pinealectomy
subjected to WRS during the light phase.
Following pinealectomy, the number of gastric lesions in response to WRS during
the light phase was significantly higher than that in rats with an intact pineal
gland (Fig. 3
, lower panel), but these animals also developed less gastric
lesions at night hours. The same tendency in GBF and PGE2
was observed in animals subjected to WRS at night hours, though the GBF and
generation were significantly higher as
compared with respective values recorded at daily hours.
This study shows that stress ulcerogenesis involves diurnal/nocturnal rhythm
in formation of gastric lesions in rats with intact pineal glands because these
stress-induced gastric lesions were much more pronounced at the day than in
the night suggesting that nocturnal melatonin could contribute to the gastric
mucosal defense especially at dark phase, resulting in limitation of stress-induced
gastric bleeding erosions. This confirms and extends previous finding of Kato
. (3) and Otsuka et al
. (13), who described this phenomenon
in rats and suggested that such a fluctuation in stress ulcerogenesis could
be attributable to nocturnal melatonin. We found that various time-related exposures
to WRS caused a time-dependent increase in the gastric lesion incidence and
significantly decreased GBF and gastric mucosal generation PGE2
Moreover, the removal of pineal gland by pinealectomy, augmented WRS-induced
gastric lesions at each time duration of WRS determined and caused a more profound
fall in the GBF than that observed in animals with intact pineal gland. This
suggests that pineal melatonin and the interaction of this indole with COX/PGE2
system could be of importance in the stress ulcerogenesis.
However, Bubenik et al
. (17) demonstrated that 4-week administration of melatonin in the diet significantly reduced the incidence of spontaneous (chronic) gastric ulcers in young pigs. It is of interest that the pigs with such ulcers exhibited lower contents of melatonin in the gastric mucosa and in the blood suggesting that these spontaneous ulcers originate from the local deficiency of indole. They also demonstrated that coarsely ground diet, in contrast to finely ground diet, exerted stronger protective effects on the gastric mucosa by stimulating more extensively the production of endogenous melatonin from the gastric mucosa (20). Otsuka et al
. (21) reported, however, that acute stress-induced gastric lesions in rats are accompanied by increased plasma melatonin. The proposed explanation for the rise in melatonin is that its production increases under stressful stimuli in both, experimental animals and human as suggested by Oxenkrug and McIntyre (22) and Karasek and Winczyk (23). Results of the present study remain, in part, in agreement with those studies regarding the lower gastric stress ulcerogenesis at night in animals with intact pineal gland or in those subjected to pinealectomy. Also in our hands, plasma melatonin levels accompanying a stressful procedure (WRS) showed a significant increase even in pinealectomized animals when the pre-WRS plasma levels of this indole exhibited much lower level. Our study is also corroborative with findings of Klupinska et al
(24) that melatonin secreted in great amounts at night attenuated dyspepsia-like- and gastric reflux episodes in patients with symptoms of GERD and NERD.
Recent evidence indicates that melatonin may exert a beneficial action against gastric injury due to the activation of the COX/PG system, heat shock proteins as well as the NOS/NO system (25-28). This notion agrees with previous reports from our laboratory showing that suppression of COX by a non-selective COX inhibitor, i.e. indomethacin, and selective COX-1 and COX-2 inhibitors attenuated the protective effects of melatonin against mucosal damage induced by stress and ischemia-reperfusion (1, 2, 12). Based on these observations, the hypothesis has been put forward that PG and NO play pivotal roles in the acceleration of ulcer healing by melatonin (24). The protective and ulcer-healing effects by melatonin in the stomach are considered to be receptor specific because melatonin-induced gastroprotection and acceleration of ulcer healing with an accompanying rise in the GBF in the ulcer area, were abolished by luzindole, a specific antagonist of the membrane melatonin M2 receptors (26, 28).
Another mediator of the action of melatonin on gastroprotection and ulcer healing may be the gut-brain axis. To examine this possibility, animals with functionally deactivated sensory nerves using neurotoxic dose of capsaicin should be used. Such capsaicin-denervated animals were previously employed to test the mechanisms of gastric mucosal defense, the mucosal repair from damage and acceleration of ulcer healing induced by strong irritants (29-32). It was shown that functional ablation of afferent nerves delayed healing of gastric ulcers at 1 and 2 weeks after their production (by acetic acid) and this delay was associated with a marked and persistent decrease in tissue CGRP-like immunoreactivity related to afferent nerve stimulation by melatonin (32, 33).
This beneficial action of melatonin against stress ulcerogenesis seems to involve
the activity of antioxidant enzymes and lipid peroxidation as documented in
and in vitro
conditions (12, 14, 34). It has been shown (10)
that gastric stress induced by the exposing of rats to water immersion and restraint,
suppressed the activity of SOD and GSH while increasing lipid peroxidation.
Suppression by WRS of SOD activity combined with enhanced lipid peroxidation
results in massive gastric damage most likely resulting from generation of reactive
oxygen metabolites such as hydrogen peroxide, the hydroxyl radical and peroxynitrite
anion. Moreover, a deficit in gastroprotective PGE2
combined with the reduction in antioxidative enzymes, enhanced lipid peroxidation
and accompanying reduction in mucosal microcirculation results in the formation
of multiple gastric erosions. Our present study revealed that this stress-mediated
ulcerogogenesis is, however, less pronounced in dark phase under stressful conditions
since, as shown in this report and in previous studies (12, 13, 21), there is
increased antioxidative enzyme activity, reduced lipid peroxidation and increased
gastric blood flow in the gastric mucosa subjected to stress at night. The crucial
question remains what is the common factor that limits stress-induced ulcerogenesis
at night compared with that during the day, especially in pinealectomized rats.
In our opinion, this factor is the small but significant rise in melatonin at
night, probably released from an extrapineal source e.g
Another mediator involved in the melatonin-induced attenuation of stress ulcerogenesis observed at dark phase may be the gut-brain axis. To examine this possibility, animals with functionally deactivated sensory nerves using a neurotoxic dose of capsaicin could be used. Our preliminary observation (data not included) seems to favor the concept that the gut derived melatonin in combination with sensory nerve activation are responsible for dark phase reduction in stress ulcerogenesis in both, non-pinealectomized as well as pinealectomized animals.
Among the other possible candidates responsible for the beneficial effect of melatonin against formation of WRS-induced gastric lesions might be a major gastric hormone, gastrin, which is known to posses gastroprotective and trophic action in the stomach. Melatonin was reported to elevate plasma gastrin levels, suggesting that this hormone could not only contribute to ulcer healing process but also to the acceleration of ulcer healing by melatonin (35-38). Further studies are needed to resolve the question of the contribution of these factors in diurnal variation of gastric stress-mediated ulcerogenesis.
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