1 Division of Physiology, Department of Neuroscience, Uppsala University, SE-751 23 Uppsala; and 2 Gastroenterology Unit, Department of Medical Sciences, Uppsala University Hospital, SE-751 85 Uppsala, Sweden
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ABSTRACT |
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The amount of melatonin present in
enterochromaffin cells in the alimentary tract is much higher than that
in the central nervous system, and melatonin acting at MT2
receptors mediates neural stimulation of mucosal HCO
enterochromaffin cells; enterocyte clusters; intracellular calcium; mucosal protection; syncytium
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INTRODUCTION |
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DUODENAL MUCOSAL
HCO 2.0) in the duodenal luminal bulk solution. The major
physiological stimulant of the HCO
1-adrenoceptor agonist phenylephrine (20,
34), and stimulation has also been observed after
intracerebroventricular administration of some neuropeptides (13,
21) and benzodiazepines (29). Vagal and sympathetic
nerves as well as
-endorphin released from the pituitary gland
mediate the centrally elicited influence on duodenal
HCO
Our recent studies have shown that melatonin, a product of the
intestinal enterochromaffin (EC) cells (4) as well as the pineal gland in the central nervous system, is a potent stimulant of
the duodenal mucosal HCO
Melatonin receptors are distributed throughout the gastrointestinal
tract. Effects of melatonin are mediated by specific high-affinity membrane-associated melatonin receptors that belong to the superfamily of G protein-coupled receptors (27, 38). On the basis of
pharmacological evidence, three subtypes are reported. The mammalian
MT1 and MT2 receptors, but not yet the
MT3 subtype, have been cloned (10, 27). The
signaling pathways downstream of the melatonin receptors are not fully
clarified. Both the MT1 and the MT2 receptor
share the ability to inhibit adenylyl cyclase activity via pertussis toxin (PTX)-sensitive Gi-proteins (2, 10, 26).
Furthermore, the -subunits of Gi-proteins are known
to be involved in the transient mobilization of intracellular free
calcium concentration ([Ca2+]i) via the
inositol-specific phospholipase C pathway. A recent study by Brydon et
al. (3) showed, in both primary cultures of ovine pars
tuberalis and HEK-293 cells stably expressing MT1 receptors, that melatonin induced cytosolic Ca2+
mobilization via PTX-insensitive G proteins. This increase in [Ca2+]i was mediated by Gq/11
(3).
The aim of the present study was to establish and elucidate the effects of melatonin on duodenal epithelium intracellular calcium signaling. No direct effects of melatonin on the small intestinal epithelium in humans have been reported before (5) and we therefore studied human as well as rat duodenal enterocytes. Freshly isolated clusters of human and rat duodenal enterocytes loaded with fura-2 AM were mounted in a perfusion chamber, and [Ca2+]i was measured by imaging. Clusters (10-50 interconnecting cells) increase the viability of the duodenal enterocytes and, furthermore, enable studies of cell-to-cell communication in the duodenal epithelium. It was found that melatonin increases [Ca2+]i in crypt-like enterocytes and, in addition, enhances the rise in [Ca2+]i in response to the duodenal secretagogues carbachol and cholecystokinin octapeptide (CCK-8). Our choice of preparation should exclude the possibility that the neurohormone acted via enteric neurons. This was further confirmed by the absence of an effect of the muscarinic antagonist atropine.
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MATERIALS AND METHODS |
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Chemicals and drugs. Membrane filter (polycarbonate, diameter 25 mm, 25-µm thickness, 3 × 106 pores/cm2, pore diameter 3 µm) were from Osmonics, Livemore, CA. Dispase II was purchased from Boehringer-Mannheim (Mannheim, Germany). Melatonin was obtained from Research Biochemicals International (Natick, MA). The melatonin receptor agonists 2-iodomelatonin and 2-iodo-N-butanoyl-5-methoxytryptamine (2-Ibmt) and the melatonin receptor antagonists luzindole and N-pentanoyl-2-benzyltryptamine (DH 97) were obtained from Tocris Cookson (Avonmouth, UK). CCK-8 and CCK 26-33 were from Peninsula Europe (Merseyside, UK). Carbachol (carbamylcholine chloride), DMEM/Ham's F-12 medium (DMEM/F-12), HEPES, TES, EGTA, atropine (sulfate), probenesid, and gentamicin were obtained from Sigma (St. Louis, MO), and fura-2 AM was from Calbiochem (La Jolla, CA). Ionophores 4-bromo A 23187 and ionomycin, and Pluronic F-127, the fura-2 calcium imaging calibration kit (F-6774) were obtained from Molecular Probes (Eugene, OR). Fetal calf serum (Svanocolone FBS Super) was from the National Veterinary Institute (Håtunaholm, Sweden). All agents were dissolved in isotonic saline at pH 7.4 on the day of use.
Tissue preparation. Experiments were approved by the Uppsala Ethics Committee for Experiments with Animals. Male F1-hybrids of Lewis and Dark Agouti rats (Animal Department, Biomedical Center, Uppsala, Sweden), weighing 230-300 g were raised in a conditioning unit under standardized temperature and light conditions (21-22°C, 12:12-h light-dark cycle). The rats were kept in cages in groups of two or more and had access to tap water and pelleted food (Ewos, Södertälje, Sweden) ad libitum.
Animals were deprived of food overnight before the experiments, but had free access to drinking water. Experiments were begun at 0830, and to avoid possible stimulatory effects of anesthetics on intestinal mucus release, rats were decapitated. A 3-cm segment of duodenum, starting 2-3 mm distal to the pylorus was promptly excised via an abdominal midline incision and freed from mesentery. The segment was opened along the antemesenterial axis and the luminal surface was rinsed with a normal respiratory medium (NRM) containing (in mM) 114.4 Na+, 5.4 K+, 1.0 Ca2+, 1.2 Mg2+, 121.8 ClHuman biopsies.
Human duodenal biopsies were obtained from patients undergoing upper
endoscopy at the Gastroenterology Unit, Uppsala University Hospital and
found to have endoscopically normal duodenal and gastric mucosae. The
project was approved by the Ethics Committee of the Medical Faculty at
Uppsala University, and all subjects provided written informed consent.
Biopsies were taken between 0900 and 1000 with radial jaw (large
capacity with needle) single-use biopsy forceps (Boston Scientific, MA)
and were immediately placed into 20 ml of sterile DMEM/F-12 medium
(plus 15 mM HEPES and L-glutamine, 1 mM
HCO
Data analyses. All statistical analyses were performed on an IBM-compatible computer using StatView 5.0 software (SAS Institute, Cary, NC). When appropriate, statistical significances were calculated by using Student's t-test. Nonlinear curve-fitting of the data was made by use of SigmaPlot for Windows 4.01 (Jandel Scientific, Corte Madera, CA).
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RESULTS |
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Samples were taken from the suspended final pellet up to 6 h
after the isolation procedure and placed in the perfusion chamber in
which the enterocytes were studied for up to 30 min. The preparations were composed of clusters of 10-50 interconnected enterocytes (Fig. 1, A and B),
as well as single enterocytes. Clusters provided the possibility to
study intestinal cell-to-cell communication and, in addition, pilot
experiments showed better viability of enterocytes in clusters than of
single enterocytes. As tested by trypan blue exclusion, enterocyte
viability was >95% immediately after the preparation procedure and
>85% after a 6-h period. There were no changes in excitation ratio in
enterocytes not responding to melatonin or receptor agonists.
Intracellular calcium concentration ([Ca2+]i)
remained stable at ~100 nM, further indicating good viability of
enterocytes in cluster preparation. The majority of clusters isolated
by the present procedure and chosen for study were composed of
enterocytes with secretory structures characteristic of crypt enterocytes (8, 37). Control experiments were performed by measuring [Ca2+]i in human and rat
preparations perfused with the electrolyte solution alone (no agonists
or antagonists added).
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Initial experiments demonstrated that HCO
Effects of melatonin on
[Ca2+]i in duodenal
enterocytes.
In a first experimental protocol (Fig.
2), melatonin was added to the
consecutively increasing concentrations of 0.1, 1.0, 5.0, and 100 nM
(199 cells in 13 clusters), No changes in
[Ca2+]i were observed in rat duodenal
enterocytes exposed to the lowest concentration of melatonin (0.1 nM).
At a concentration of 1.0 nM, melatonin increased
[Ca2+]i in a few enterocytes (4%), and 5.0 nM of the neurohormone increased [Ca2+]i in
11% of the enterocytes examined. The higher concentration of 100 nM
induced a robust rise in [Ca2+]i in ~35%
of the rat enterocytes.
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Melatonin receptor ligands.
In further experiments, we examined the effects of some melatonin
receptor ligands. The full melatonin receptor agonist 2-Ibmt (87-fold
more potent than melatonin in Xenopus laevis melanophores) (35) was added and present in the perfusate in increasing
concentrations (10, 100, and 500 nM or 10, 100, and 1,000 nM,
respectively) during consecutive 3-min periods. The agonist induced the
same pattern of enterocyte [Ca2+]i signaling
(Fig. 8) as did melatonin (102 cells in 6 clusters) as well as in human (41 cells in 3 clusters, not shown)
preparations. Significant increases in
[Ca2+]i, occurred in 15% of the human and
33% of the rat enterocytes exposed to 10 or 100 nM of the compound,
and the higher concentrations of 500 and 1,000 nM caused no further
effects.
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Nonresponding enterocytes.
As tested by trypan blue exclusion, enterocyte viability was >95%
immediately after preparations and >85% when tested after 6 h.
However, as described above, the percentage of examined cells responding to melatonin receptor ligands with a rise in
[Ca2+]i was always below 40%. To verify
intracellular deesterization of fura-2 AM and further establish the
viability of enterocytes in clusters, the ionophores 4-bromo A 23187 (10 µM) and ionomycin (10 µM) were added at the end of experiments
(Fig. 10). These ionophores increase
[Ca2+]i by direct actions on cell membranes,
i.e., absence of melatonin receptors should not affect the response. As
illustrated in Fig. 10, the majority (92%) of rat duodenal enterocytes
responded to Ca2+ ionophores (163 cells in 11 clusters).
With clusters from human duodenum, 91% of the enterocytes responded to
ionophores (79 cells in 4 clusters, not shown). A response to
ionophores thus occurred despite the absence of a response to
melatonin. As a further test, Ca2+ in the perfusate was
removed and reintroduced at the end of some experiments (6 clusters
from rat and 3 from human duodenum, not shown). This caused transient
changes in [Ca2+]i in >90% of all
enterocytes tested. The [Ca2+]i response in
cells that responded to melatonin was very similar to that in those
apparently insensitive to the compound.
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Melatonin-induced oscillations.
Another interesting type of response was observed in ~21% of all rat
as well as human enterocytes responding to melatonin. The initial
transient increase in [Ca2+]i was followed by
slow, rhythmic oscillations of high amplitude (Fig.
11). Oscillations continued throughout
the perfusate period (20-25 min), despite cessation of the
exposure to melatonin and the frequency was one period in ~5 to 6 min. Oscillations were never observed in the absence of
Ca2+ in the perfusate and, furthermore, oscillations did
not occur in the presence of the melatonin antagonist luzindole. There
was a time delay between individual cells (Fig. 11) with respect to the
start of each start of rise in [Ca2+]i,
suggesting spread of this pattern of signaling by cell-to-cell communication within the cluster.
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Potentiation of the response to other secretagogues.
Enterocytes in primary culture respond to carbachol and CCK-8 with an
transient increase in [Ca2+]i
(8). Similar responses to these intestinal secretagogues were observed in the present preparation of freshly isolated clusters of duodenal enterocytes. Melatonin may, like other EC-cell products, exert (paracrine) regulatory actions on epithelial function. This made
it of interest to study effects of melatonin on the
[Ca2+]i response to other stimuli. Cell
clusters were perfused with melatonin (10, 100, and 500 nM for 3 min
each concentration) before exposure to 100 µM of carbachol and 100 nM
of CCK-8 (93 cells in 8 human clusters and 146 cells in 17 rat clusters
were examined). As illustrated in Fig.
12, there was a marked enhancement of
the enterocyte [Ca2+]i response to the latter
secretagogues.
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DISCUSSION |
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The total amount of melatonin in the alimentary tract is much
higher (~400-fold) than that in the central nervous system (4, 5), but the role of melatonin in gastrointestinal function has
been largely unknown. In the intestinal mucosa, the hormone is produced
by EC cells that are in close contact with fibers from the autonomic
nervous system. The present findings that melatonin increases
[Ca2+]i in duodenal enterocytes demonstrate
for the first time that melatonin has a direct action on intestinal
epithelium and, furthermore, that melatonin affects intestinal mucosa
also in humans. Low concentrations of melatonin, EC50 being
17.0 ± 2.6 nM, and of agonists 2-iodomelatonin and 2-Ibmt,
increased enterocyte [Ca2+]i, and the
receptor antagonists (MT2>MT1) luzindole
and DH 97 abolished the responses to melatonin. The combined results
strongly suggest that melatonin acts at MT2-receptors in
the human and rat enterocyte membrane. Furthermore, the results support
and extend recent observations in rat duodenum in situ that local (intra-arterial or luminal) administration of melatonin stimulates mucosal HCO1 · h
1)
(34). One possible explanation is undeveloped expression
of receptors, a potential problem in evaluating actions of
secretagogues by use of transformed cell lines.
In the present study, we used isolated clusters of enterocytes, a
preparation that should be devoid of neural tissue. Pretreatment with
the muscarinic antagonist atropine did not affect basal
[Ca2+]i or the response to melatonin, further
excluding the possibility that melatonin might act at remaining enteric
nervous system rather than the enterocyte cell membrane. Only low
concentrations of proteases (collagenase and dispase) and brief times
of exposure were used for isolation of clusters. It would seem likely
that enterocytes are better viable after mild digestion than after use
of more extensive digestion procedures resulting in release of mainly
single cells. Small intestinal enterocytes in situ are programmed to a
very restricted life span (4-6 days in human intestine). In
addition, enterocytes in situ rapidly respond to irritating compounds
in the intestinal lumen by apoptosis and expulsion. Very
probably reflecting these physiological characteristics, small
intestinal enterocytes appear more difficult than, for instance, gastric parietal cells or pancreatic -cells to maintain viable after
isolation (9). As in other cells and tissues,
agonist-induced [Ca2+]i signaling is probably
of utmost importance in control of various aspects of enterocyte
function, but very few studies of [Ca2+]i
signaling in enterocytes have been reported. The present study demonstrates that clusters of freshly isolated enterocytes from the
proximal small intestine can be maintained viable and provide a
suitable model for studies of agonist-induced
[Ca2+]i signaling. The viability of
enterocytes in clusters as studied by trypan blue exclusion was good
(>85% after 6 h). It may be compared with the viability (10%
after 2-4 h) reported in studies of intracellular pH in acutely
isolated villus tips from rat duodenum (39).
However, the percentage of enterocytes in clusters responding to melatonin never exceeded 40%. The lack of a response in all enterocytes may reflect absence or loss of receptor expression in some cells and/or a decline of cell-to-cell communication in some preparations. Furthermore, freshly isolated clusters were used in the present study and the individual enterocytes were probably in different stages of differentiation (15). Melatonin receptor distribution within the intestinal mucosa has never been studied. It is thus not known whether all enterocytes express melatonin receptors. Nor is it known whether the procedure of overnight fast before study (used in humans as well as rats) affects intestinal melatonin receptor expression. Proper measurement of [Ca2+]i was strongly supported by the findings (cf. Fig. 10) that the Ca2+ ionophores 4-bromo A 23187 and ionomycin, as well as changes in external Ca2+ concentration, affected [Ca2+]i very similarly in melatonin-responding and nonresponding enterocytes. It should also be noted that the percentage of cells responding to ionophores (~90%) was very similar to that of cells excluding trypan blue (85-95%). It would thus seem possible that <40% of duodenal enterocytes express melatonin receptors.
In the main type of melatonin-induced signaling pattern,
[Ca2+]i spiked rapidly and then slowly
returned to basal or almost basal line values. In a smaller number of
cells, [Ca2+]i tended to remain at a plateau
level. The magnitude of initial rise in
[Ca2+]i was dependent on the perfusate
concentration of melatonin in some enterocytes (Figs. 2 and
4B). In other experiments (Figs. 4A and 5), there
was a rapid downregulation of the response, similar to the
desensitization observed with CCK-8 in duodenal enterocytes in primary
culture (8). Interestingly, there is a dose-dependent rise
in mucosal HCO1 · h
1) dose of
the compound. The similarity may support a role of
[Ca2+]i in mediating melatonin-induced
stimulation of the secretion.
Perfusion with calcium-free solutions (Fig. 6) abolished the plateau phase but not the initial increase in [Ca2+]i in rat duodenal enterocytes. A biphasic Ca2+ response to agonists is characteristic of many nonexcitable cell types and a substantial amount of evidence indicates that the initial spike in [Ca2+]i is the result of release of Ca2+ from an intracellular storage site(s), whereas the later sustained phase is due to the influx of Ca2+ across the cell membrane. In duodenal enterocytes in primary culture, carbachol (acting at muscarinic M3 receptors) induced biphasic [Ca2+]i responses (8), similar to those observed with melatonin in the present study. The sustained phase of the rise in [Ca2+]i was, as found here with melatonin, dependent of external Ca2+.
Another interesting type of [Ca2+]i response
to melatonin was observed in ~21% of the responding preparations.
The initial transient increase in [Ca2+]i was
followed by slow rhythmic oscillations in
[Ca2+]i of high amplitude, which spread
throughout the cluster of enterocytes (Fig. 11). Oscillations (and
spread of oscillations) were never observed in the absence of
Ca2+ in the perfusate in the present study, suggesting that
influx of Ca2+ contributes to the phenomenon. Presence of
extracellular Ca2+ may, however, also be important in
maintaining mucosal cell-to-cell communication. Agonists that provoke
inositol lipid hydrolysis have been shown to induce oscillations in
[Ca2+]i in some, but not all, nonexcitable
cell types, but the physiological significance of these oscillations is
not clearly established. For example, carbachol induces sustained
oscillations in [Ca2+]i in mouse pancreatic
-cells exposed to 20 mM of glucose (17). Subpopulations
of rat (but not of human) duodenocytes in primary culture, responded to
carbachol with an initial, transient rise in
[Ca2+]i followed by oscillations in
[Ca2+]i of low and declining amplitude
(8). The melatonin-induced oscillations observed in the
present study of clusters of rat as well as human duodenal enterocytes
occurred with about the same frequency (~1 period in 5 min) but seem
of a different nature. Thus there was no decline but a time-dependent
gain in amplitude and oscillations spread within the cell cluster (Fig.
11). At maximum, 40% of all enterocytes responded to melatonin
receptor ligands, and cell-to-cell spread of
[Ca2+]i signaling may thus be important in
mediating the epithelial response to melatonin.
Cellular responses depend on the pattern and magnitude of [Ca2+]i signaling (1). Simultaneous measurement of [Ca2+]i signaling and enterocyte acid/base transport seems a prerequisite for further understanding of enterocyte stimulus-secretion coupling and is an interesting subject of further studies. In mouse ileal crypts, carbachol increased [Ca2+]i in Paneth's cells at the bottom of the crypts (30). In contrast to the present study and previous studies (8) of duodenal preparations from humans and rats, neither carbachol nor CCK-8 affected enterocyte [Ca2+]i in the ileal preparation (30), and no propagation of a [Ca2+]i wave along the crypt could be observed. The absence of enterocyte [Ca2+]i responses and a wave in the ileum may reflect differences in preparation and/or differences between the two segments (ileum and duodenum) of the intestine. However, cell-to-cell communication of [Ca2+]i signaling has been demonstrated in colonic crypts isolated from rats (22). Acetylcholine, acting at M3 receptors, initiated a [Ca2+]i signal in the base of colonic crypts, which spread from this region to the remainder of the lower half of the crypt in a wave-like manner. The combined evidence suggests that enterocytes in duodenal (small intestinal) as well as in colonic epithelium interact and function as syncytia.
Interestingly, melatonin potentiated the rise in
[Ca2+]i in response to carbachol and CCK-8 in
the present study (Fig. 12). Furthermore, there was a long-lasting
sustained [Ca2+]i plateau in some enterocytes
exposed to melatonin alone (Fig. 7). These phenomena cannot, presently,
be fully explained but may reflect an action of melatonin on channels
for uptake of extracellular Ca2+ (1) and/or
the presence of cell-to-cell spread of signaling within clusters of
enterocytes. A synergistic action in Ca2+ dependent
stimulation of Cl secretion in T84 cells has been
reported for histamine acting at H1-receptors. The
duodenal secretagogues dopamine, VIP, and prostaglandin E2
increase intracellular cyclic AMP production (25, 28), and
intracellular cyclic GMP is involved in mediating the
HCO
Physiological role of intestinal melatonin. It would seem very likely that melatonin influencing the duodenal epithelium is of mainly intestinal (EC-cell) origin, in particular at daytime when release from the central nervous system (the pineal gland) is low. The amount of melatonin in the gastrointestinal tract is thus ~400 times higher than that in the pineal organ at any time, and the highest levels are found in the rectum, colon, and duodenum (4, 6). Furthermore, the half-life of melatonin, if injected into the general circulation, is relatively short (~20 min in rats and ~40 min in humans) (40). It has been suggested that melatonin released from the gastrointestinal tract appears in the general circulation and that this occurs particularly when increased amounts of tryptophan are added to the diet (cf. Ref. 5). It should, however, be noted that melatonin is absorbed from portal blood and metabolized (hydroxylation to 6-hydroxy-melatonin) by the liver. Paracrine modulation of the intestinal epithelium and, in addition, mediation of neural stimuli would seem a more likely main role of intestinal endogenous melatonin.
The present study thus demonstrates that melatonin, evoking [Ca2+]i responses in enterocytes, influences duodenal enterocytes from humans as well as that from rats. The potentiation of the response to carbachol and CCK-8 may suggest one additional important role of melatonin in regulating intestinal electrolyte transport and mucosal integrity. Whether other EC-cell products, e.g., guanylin and orexin A (14, 23, 24), exert similar effects is of considerable interest. ![]() |
ACKNOWLEDGEMENTS |
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The authors thank Gunilla Jedstedt for excellent technical assistance.
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FOOTNOTES |
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Support for this study was from Swedish Research Council Grant 3515, the Wallenberg Foundation, and the Swedish Society for Medical Research.
Address for reprint requests and other correspondence: G. Flemström, Division of Physiology, Dept. of Neuroscience, Uppsala University, Biomedical Center, P.O. Box 572, SE-751 23 Uppsala, Sweden (E-mail: Gunnar.Flemstrom{at}fysiologi.uu.se).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published February 12, 2003;10.1152/ajpgi.00500.2002
Received 25 November 2002; accepted in final form 10 February 2003.
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