Melatonin-induced calcium signaling in clusters of human and rat duodenal enterocytes

Markus Sjöblom1, Bengt Säfsten2, and Gunnar Flemström1

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion in duodenum in vivo. We have examined effects of melatonin and receptor ligands on intracellular free calcium concentration ([Ca2+]i) signaling in human and rat duodenal enterocytes. Clusters of interconnecting enterocytes (10-50 cells) were isolated by mild digestion (collagenase/dispase) of human duodenal biopsies or rat duodenal mucosa loaded with fura-2 AM and attached to the bottom of a temperature-controlled perfusion chamber. Clusters provided viable preparations and respond to stimuli as a syncytium. Melatonin and melatonin receptor agonists 2-iodo-N-butanoyl-5-methoxytryptamine and 2-iodomelatonin (1.0-100 nM) increased enterocyte [Ca2+]i, EC50 of melatonin being 17.0 ± 2.6 nM. The MT2 receptor antagonists luzindole and N-pentanoyl-2-benzyltryptamine abolished the [Ca2+]i responses. The muscarinic antagonist atropine (1.0 µM) was without effect on basal [Ca2+]i and did not affect the response to melatonin. In the main type of response, [Ca2+]i spiked rapidly and returned to basal values within 4-6 min. In another type, the initial rise in [Ca2+]i was followed by rhythmic oscillations of high amplitude. Melatonin-induced enterocyte [Ca2+]i signaling as well as mucosal cell-to-cell communication may be involved in stimulation of duodenal mucosal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion.

enterochromaffin cells; enterocyte clusters; intracellular calcium; mucosal protection; syncytium


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DUODENAL MUCOSAL HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> SECRETION has a key role in duodenal protection against the pulses of hydrochloric acid (and pepsin) intermittently discharged from the stomach. The duodenal enterocytes transport HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> into the continuous layer of viscoelastic mucus gel on top of the epithelial surface, maintaining pH in its cell-facing portion at neutrality despite high acidities (pH <=  2.0) in the duodenal luminal bulk solution. The major physiological stimulant of the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion is gastric acid expelled into the duodenal lumen, and the acid-induced HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> response is mediated by enteric nervous pathways, involving release of vasoactive intestinal polypeptide and acetylcholine (11, 12), as well as by E-type prostaglandins released from mucosal cells (36). In addition to the local intestinal control, the alkaline secretion is under central nervous influence. Up to fourfold increases in secretion occur after intracerebroventricular infusion of the alpha 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 beta -endorphin released from the pituitary gland mediate the centrally elicited influence on duodenal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion.

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<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion in the rat in vivo (33, 34). Furthermore, the melatonin MT2-receptor antagonist luzindole abolished the rise in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion induced by central nervous intracerebroventricular administration of phenylephrine. The response was also abolished by ligation of all nerves around the carotid arteries but was unaffected by removal of either the pineal gland or pituitary gland (34). The results thus strongly suggest intestinal (EC-cell) melatonin as one mediator of neurally mediated intestinal secretion.

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 beta gamma -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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Cl-, 1.2 SO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, 6.0 phosphate, 15.0 HEPES, 1.0 pyruvate, and 10 glucose plus 10 mg/l phenol red, 0.1 mg/ml gentamicin, and 2.0% fetal calf serum (8). The pH was adjusted to 7.40 immediately before use, and temperature was maintained at 37°C. The sheet of duodenal wall was then put on a precleaned glass slide (lumen side up) and the mucosa was gently scraped off. The depth of mucosal tissue removed by the scraping procedure (and used for experiments) was tested by morphological examination of the remaining tissue (fixed in 10% neutral buffered formalin and stained with hematoxylin-eosin). The duodenal remnant contained some crypt bases and all submucosa containing Brunner's glands. Cells originating from the latter glands were thus excluded from the studied preparations.

The scraped-off mucosa was then cut into 0.3- to 0.8-mm diameter pieces that were dispersed and briefly shaken in NRM solution also containing 0.5 mM DTT. After sedimentation for 2-3 min, the supernatant was removed, and the tissue fragments (in the sediment) were washed three times in NRM solution (not containing DTT). After brief gassing with 100% O2, the tissue fragments (15-20 µl) were then exposed to mild digestion for 3 min by inoculation in 10 ml NRM solution containing 0.1 mg/ml collagenase type H (Sigma) and 0.1 mg/ml dispase II (Boehringer-Mannheim). Digestion was performed at 37°C in a horizontal shaking water bath, and it was stopped by adding DTT (to a concentration of 0.3 mM) followed by centrifugation of the solution (3 min at 1,000 g). The pellet was washed three times by suspension in 10 ml DMEM/F-12 (with 15 mM HEPES and 2.5 mM glutamine) followed by centrifugation (3 min at 1,000 g). HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (1 mM), gentamicin (0.01 mg/ml), and fetal calf serum (2.0%) were always added to the DMEM/F-12, and pH was adjusted to 7.40. The preparation procedure yielded clusters of interconnected duodenal enterocytes as well as smaller amounts of single cells, and clusters of 10-50 interconnected cells were selected for imaging analysis. Submucosal tissue was not included and no (enteric) neurons were detectable under light microscopy or during microscopy of fura-2 AM loaded preparations. The clusters were composed predominately of cells with morphologies characteristic of crypt cells (8, 37). The viability after preparation was tested by trypan blue exclusion (>95% after the isolation procedure). The final pellet was suspended in ~1 ml of DMEM/F-12 (with the same additives) solution and immediately put on ice, a procedure found to increase the viability of the enterocyte clusters compared with keeping the cells at 37°C.

For measurement of [Ca2+]i, 70 µl of the cell cluster suspension was loaded at 37°C with fura-2 AM (2 µM) for 20-30 min in an electrolyte solution (in mM): 141.2 Na+, 5.4 K+, 1.0 Ca2+, 1.2 Mg2+, 146.4 Cl-, 0.4 phosphate, 20.0 TES, and 10 glucose, pH 7.40 found appropriate for studies of cell aggregates from other tissues (32). Probenesid (1 mM), pluronic F-127 (0.02%) and fetal calf serum (2.0%) were present during the loading procedure. The fura-2 AM-loaded cell aggregates were spun down and placed on an uncoated and precleaned circular glass coverslip (diameter 25 mm; Knittel, Braunschweig, Germany) at the bottom of a temperature-controlled (37°C) perfusion chamber and fixed on top of the coverslip by a uniformly sized pore polycarbonate membrane filter (32). The covering filter and the cell preparation were perfused (1 ml/min) with the electrolyte solution and receptor ligands to be tested by inclusion in the perfusate.

Calibration of the fluorescence data was accomplished in vitro according to the method used by Grynkiewicz et al., (16). The fluorescence ratio calibration curve was made by use of a commercially available fura-2 calcium imaging calibration kit (F-6774; Molecular Probes). Maximum fluorescence was obtained by applying 10 µM ionomycin and minimum by adding 10 mM EGTA. Changes in [Ca2+]i in the fura-2 AM-loaded cells were measured 30 times per minute by the dual-wavelength excitation ratio technique by exposure to alternating 340 and 380 nm light with the use of a filter changer under the control of an InCytIM-2 system (Intracellular Imaging, Cincinnati, OH) and a dichroic mirror (DM430; Nikon, Tokyo, Japan). Emission was measured through a 510-nm barrier filter with an integrating charge-coupled device camera (Cohu, San Diego, CA).

The average [Ca2+]i peak response in all duodenal enterocytes (responding as well as nonresponding) in the examined cluster preparations was calculated in some experiments. Cluster-containing suspension (70 µl) was loaded with fura-2 AM and placed in the perfusion chamber as described above. Melatonin was added to different concentrations (0.1, 1.0, 5.0, 10, 100, 500, or 1,000 nM) in different experimental setups. Changes in enterocyte [Ca2+]i were measured and expressed as percentage of the maximal peak response in each enterocyte (by using comparison with the response to 10 µM ionomycin).

Human 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<UP><SUB>3</SUB><SUP>−</SUP></UP>) containing 2.0% fetal calf serum and 0.01 mg/ml gentamicin, and were then transported to the laboratory, rinsed, and minced at room temperature within 20-40 min after removal. Clusters of duodenal enterocytes were then prepared and the viability was approved as described above for rat duodenum.

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).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Clusters of duodenal enterocytes (10-50 cells) were obtained by mild digestion (dispase/collagenase) of human duodenal biopsies or gently scraped-off rat duodenal mucosa. A: 3 human clusters with ~20 cells and 1 single cell are shown. B: clusters obtained from rat mucosa. Enterocytes had secretory structures characteristic of crypt cells. Bar = 25 µm.

Initial experiments demonstrated that HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> at a concentration of 1 mM in the DMEM/F-12 solution improved duodenal enterocyte viability, compared with the absence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> or presence of 3 or 5 mM of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Adverse effects of absence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> may reflect its involvement in regulation of duodenal enterocyte acid/base transport and pHi (12, 18, 19), and adverse effects of 3 with 5 mM of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> may reflect the continuous rise of pH in those solutions (reflecting loss of CO2 with transfer of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> to CO<UP><SUB>3</SUB><SUP>2−</SUP></UP>). When measured over an 180-min period, there was an increase in pH from 7.44 to 7.60 ± 0.01 (n = 4) in DMEM/F-12 containing 3 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and an increase from 7.49 to 7.94 ± 0.01 (n = 4) with 5 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. With 1 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, pH remained at 7.40. Gassing with low concentrations of CO2 was tested but caused shifting values of pH in the present open circuit (32) perfusion system.

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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Fig. 2.   Rat duodenal enterocytes (clusters) were loaded with fura-2 AM, and changes in intracellular free calcium concentration ([Ca2+]i) were determined by ratio imaging of fura-2 fluorescence and converted to actual levels of calcium (nM). Melatonin at perfusate concentrations >= 1.0 nM caused a rapid spike in [Ca2+]i, which then slowly returned to basal or close to basal values.

The average [Ca2+]i peak response in all caged duodenal enterocytes (responding as well as nonresponding) in the examined rat clusters was measured in some experiments (>= 214 cells in >= 14 clusters with each melatonin concentration). Clusters were exposed to 0.1, 1.0, 5.0, 10, 100, 500, or 1,000 nM of melatonin for 3 min, and changes in enterocyte [Ca2+]i were compared with the maximal peak response during exposure to 10 µM ionomycin in each individual enterocyte. The concentration-response relation is shown in Fig. 3 and the calculated EC50 value of melatonin was 17.0 ± 2.63 nM. The maximal observed response to melatonin was ~14% of that to 10 µM ionomycin.


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Fig. 3.   Concentration-response relation for melatonin in freshly isolated duodenal enterocytes in clusters from rats. The average [Ca2+]i peak response to melatonin (3-min exposures) was compared with the (maximal) average peak response to 10 µM ionomycin in the same enterocytes. All caged enterocytes, responding as well as nonresponding, were examined. The maximal stimulation obtained with 1,000 nM melatonin was ~14% of that obtained with 10 µM of ionomycin. The average [Ca2+]i response to each concentration of melatonin is shown as %maximal stimulation by melatonin (1,000 nM). Means ± SE are shown (>= 214 cells in >= 14 clusters with each melatonin concentration).

Effects of melatonin in clusters from human duodenal biopsies and rat duodenal mucosa were compared in other experimental protocols. Melatonin added to the consecutively increasing concentrations of 10, 100, and 500 nM increased [Ca2+]i in human (88 cells in 8 clusters) as well as in rat (629 cells in 43 clusters) duodenal enterocytes. A significant rise in [Ca2+]i occurred in ~18% of all human and 36% of all rat enterocytes tested. Already the lowest concentration of melatonin (10 nM) induced a clear response in about one-half of the responding human and rat enterocytes (in 7 and 17%, respectively, of all enterocytes examined). These findings demonstrate that melatonin acts on duodenal epithelium of human (as well as rat) origin.

Melatonin, like other neurohormones, may influence intestinal secretion via direct action at enterocyte membrane receptors. Another possibility is that agents act primarily at receptors within the enteric nervous system, and responses secondarily mediated to the epithelium by other transmitters. However, only preparations microscopically devoid of enteric neurons were used in the present experiments. To further exclude the absence of enteric neurons in the cluster preparations and the possibility that endogenous acetylcholine mediated or modulated the response to melatonin, the muscarinic antagonist atropine was added in some experiments. Pretreatment with atropine (1.0 µM) did not affect basal [Ca2+]i or the magnitude or pattern of the [Ca2+]i response induced by 10 and 100 nM melatonin (181 enterocytes in 10 clusters from rat duodenum, not shown).

Three main types of signaling patterns were observed in duodenal enterocytes exposed to melatonin. In the major type of response, [Ca2+]i spiked rapidly and then slowly returned to basal or almost baseline values within 4-6 min after start of melatonin exposure (Fig. 4, A and B, human enterocytes; and Figs. 2 and 5, rat enterocytes). There was a desensitization of the response, in particular with higher concentrations of the compound (Figs. 4A and 5). Use of higher concentrations of melatonin (1,000, 1,500 and 2,000 nM, 79 cells in 7 rat clusters, not shown) did not cause greater rises in [Ca2+]i. Nor did these higher concentrations increase the percentage of (tested) enterocytes responding to melatonin.


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Fig. 4.   Human duodenal enterocytes (clusters of ~20 cells) were loaded with fura-2 AM, and changes in [Ca2+]i were determined by ratio imaging of fura-2 fluorescence and converted to actual levels of calcium (nM). A: response to perfusion with 10 and 100 nM melatonin. The smaller increase in [Ca2+]i at the higher dose (100 nM) of melatonin, suggests desensitization of melatonin receptors. B: desensitization is not evident at 100 nM but neither preparation (A nor B) showed a clear response to 500 nM of the agonist.



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Fig. 5.   Rat duodenal enterocytes in a cluster of ~20 cells responded with a large increase in [Ca2+]i to 10 nM of melatonin. As in the human duodenal enterocytes (Fig. 4A), [Ca2+]i responses at higher concentrations of melatonin suggest desensitization of the melatonin receptors.

Melatonin increased [Ca2+]i also in the absence of Ca2+ in the perfusate and preparation solutions (94 cells in 8 clusters tested, rat preparations). However, as illustrated in Fig. 6, only the initial, transient increase in [Ca2+]i and no (prolonged, slowly declining) plateau phase occurred in the absence of extracellular Ca2+. In contrast to effects in the presence of extracellular Ca2+, a low concentration of melatonin (10 nM) did not affect [Ca2+]i in absence of extracellular Ca2+. However, 23% of all enterocytes tested responded to 100 nM of melatonin and an additional 3% responded to 500 nM of the neurohormone. Melatonin thus appears to induce release of Ca2+ from intracellular stores as well as influx of extracellular Ca2+.


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Fig. 6.   Effects of melatonin (100 nM) in rat duodenal enterocytes from clusters prepared and perfused with calcium-free solutions. Melatonin increased [Ca2+]i also in the absence of extracellular Ca2+. However, only initial transient rises in [Ca2+]i and no prolonged, slowly declining plateau phase, were observed.

A second type of signaling pattern was observed in a small number (<5%) of responding cells. There was a rapid and profound increase in [Ca2+]i followed by a sustained plateau at ~75-100% of the maximal [Ca2+]i response (Fig. 7).


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Fig. 7.   Another type of signaling pattern was observed, in a small number (<5%) of responding rat duodenal enterocytes. The rapid and profound increase in [Ca2+]i was followed by a sustained plateau at ~75% of the maximal [Ca2+]i response, which remained throughout the experimental period (<= 20 min).

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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Fig. 8.   The full melatonin receptor agonist 2-iodo-N-butanoyl-5-methoxytryptamine (2-Ibmt) was present in increasing concentration in the perfusate during consecutive 3-min periods. The agonist induced the same pattern of enterocyte [Ca2+]i signaling as did melatonin. A: desensitization of the response. B: concentration-dependent increase in [Ca2+]i.

The pattern of [Ca2+]i response to another potent agonist, 2-iodomelatonin (Fig. 9), was the same as that to melatonin and 2-Ibmt. 2-Iodomelatonin 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. Enterocytes exposed to 10 and 100 nM of the agonist increased [Ca2+]i and 500 or 1,000 nM induced no further effects. Responses were observed in 19% of the human (93 cells in 8 clusters, not shown) and in 39% of the rat (107 cells in 7 clusters, not shown) enterocytes examined. A response to the lowest concentration of 2-iodomelatonin tested (10 nM) occurred in ~11% of human and 19% of rat enterocytes.


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Fig. 9.   Exposure of rat duodenal enterocytes to the melatonin receptor agonist 2-iodomelatonin induced [Ca2+]i responses very similar to those observed with melatonin.

The MT1/MT2 selective (MT2>MT1) melatonin receptor antagonist luzindole was tested at concentrations of 10, 100, 500, 1,000, 1,500, and 2,000 nM (3 min at each concentration) in clusters of human (136 cells in 8 clusters, not shown) and rat (186 cells in 10 clusters, not shown) duodenal enterocytes. No effects on [Ca2+]i were observed after perfusion with luzindole alone, excluding the fact that presence of endogenous melatonin affecting [Ca2+]i is present in the preparations. Luzindole at a concentration of 500 nM was then selected to test whether the antagonist prevents the response to melatonin. Clusters were preperfused with luzindole for 3 min, and the antagonist was then present throughout experiments. Luzindole abolished the rise in enterocyte [Ca2+]i in response to melatonin (500 nM, 3 min exposure) in human (22 cells in 2 clusters) as well as in rat (140 cells in 8 clusters) duodenal preparations, suggesting that melatonin increases [Ca2+]i via action on enterocyte membrane MT2 receptors.

DH 97, another and more selective MT2 receptor antagonist, was tested in further experiments. The concentration (500 nM) and experiment protocol were the same as described for luzindole. Similar to luzindole, DH 97 abolished the rise in enterocyte [Ca2+]i in response to melatonin (500 nM) in rat duodenal clusters (69 cells in 5 clusters, not shown).

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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Fig. 10.   The Ca2+ ionophores 4-bromo A 23187 (10 µM for 3 min) followed by ionomycin (10 µM for 3 min) were added to the perfusate at the end of experiments. Ionophores increased [Ca2+]i in cells showing marked responses to a low concentration of melatonin as well as in cells not responding to melatonin. Nonresponding as well as responding enterocytes showed (stable) values of [Ca2+]i (~100 nM). Four enterocytes in a rat duodenal cluster are shown.

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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Fig. 11.   Example of rhythmic oscillations in [Ca2+]i induced by 10-100 nM melatonin. The initial transient increase in [Ca2+]i was followed by slow rhythmic oscillations of high amplitude and these oscillations in [Ca2+]i continued throughout the experimental period (>= 20 min). The frequency of these oscillations [Ca2+]i was 1 in ~5 to 6 min. A time delay between individual cells with respect to the start of each rise in [Ca2+]i indicates cell-to-cell communication within the cluster. Approximately 21% of all rat, as well as human, duodenal enterocytes responding to melatonin showed this pattern of rhythmic oscillation pattern. Four cells in a cluster of 23 cells from rat duodenum are shown.

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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Fig. 12.   The marked enhancement of the [Ca2+]i response to secretagogues carbachol and cholecystokinin octapeptide (CCK-8) in a cluster of rat duodenal enterocytes pretreated with melatonin. Inserted in the figure (dashed-line box) is a representative example of the magnitude of [Ca2+]i responses to carbachol and CCK-8 alone.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion (33, 34). These in vivo experiments, in addition, presented strong evidence that mucosal melatonin mediates centrally elicited neural stimulation of the duodenal secretion. A previous study (7) provided evidence that melatonin increases anion (chloride) transport in monolayers of colonic T84 cells. The concentrations of melatonin required for an effect (EC50 100 µM) were, however, much higher than that found effective in the present study of [Ca2+]i in isolated duodenal enterocytes (EC50 17 nM) and of duodenal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion in vivo (20 nmol · kg-1 · 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 beta -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 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion as well as apparent desensitization of the response when melatonin is administered to rat duodenum in situ (34). The latter occurs during infusion of a relatively high (2 µmol · kg-1 · 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 beta -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<UP><SUB>3</SUB><SUP>−</SUP></UP> secretory responses to guanylin and heat-stable enterotoxin (STa) (31). Interactions between these pathways and enterocyte [Ca2+]i signaling would seem likely.

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

The authors thank Gunilla Jedstedt for excellent technical assistance.


    FOOTNOTES

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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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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