Abstract
In bile duct-ligated (BDL) rats, large cholangiocytes proliferate by activation of cAMP-dependent signaling. Melatonin, which is secreted from pineal gland as well as extrapineal tissues, regulates cell mitosis by interacting with melatonin receptors (MT1 and MT2) modulating cAMP and clock genes. In the liver, melatonin suppresses oxidative damage and ameliorates fibrosis. No information exists regarding the role of melatonin in the regulation of biliary hyperplasia. We evaluated the mechanisms of action by which melatonin regulates the growth of cholangiocytes. In normal and BDL rats, we determined the hepatic distribution of MT1, MT2, and the clock genes, CLOCK, BMAL1, CRY1, and PER1. Normal and BDL (immediately after BDL) rats were treated in vivo with melatonin before evaluating 1) serum levels of melatonin, bilirubin, and transaminases; 2) intrahepatic bile duct mass (IBDM) in liver sections; and 3) the expression of MT1 and MT2, clock genes, and PKA phosphorylation. In vitro, large cholangiocytes were stimulated with melatonin in the absence/presence of luzindole (MT1/MT2 antagonist) and 4-phenyl-2-propionamidotetralin (MT2 antagonist) before evaluating cell proliferation, cAMP levels, and PKA phosphorylation. Cholangiocytes express MT1 and MT2, CLOCK, BMAL1, CRY1, and PER1 that were all upregulated following BDL. Administration of melatonin to BDL rats decreased IBDM, serum bilirubin and transaminases levels, the expression of all clock genes, cAMP levels, and PKA phosphorylation in cholangiocytes. In vitro, melatonin decreased the proliferation, cAMP levels, and PKA phosphorylation, decreases that were blocked by luzindole. Melatonin may be important in the management of biliary hyperplasia in human cholangiopathies.
Keywords: cAMP, cholestasis, mitosis, PKA, secretin
cholangiocytes are the target cells in human cholangiopathies, including primary biliary cirrhosis and primary sclerosing cholangitis (5), and animal models of cholestasis such as bile duct ligation (BDL) and acute administration of carbon tetrachloride (CCl4) (4, 40). These pathologies are characterized by cholangiocyte hyperplasia/damage (5) that is restricted to bile ducts of certain sizes (3, 40). In BDL rats only large cholangiocytes (lining large ducts) (2, 6, 23) proliferate (leading to enhanced large intrahepatic bile ductal mass, IBDM) (3, 40) by the activation of cAMP→PKA signaling (3, 24, 40). Studies (4, 24, 40, 42) have demonstrated the key role of secretin and its receptor (SR, only expressed by cholangiocytes in rodent liver) (2, 8, 24) in the regulation and functional evaluation of biliary hyperplasia/damage. Following BDL the enhanced biliary hyperplasia is associated with increased SR expression and secretin-stimulated cAMP levels and bile secretion (3, 4, 7, 8, 40, 42). Conversely, during damage of cholangiocytes, there is reduced SR expression and functional response to secretin (40, 41). A number of neuroendocrine factors and neuropeptides such as secretin, sex hormones, vascular endothelial growth factor, α-calcitonin gene-related peptide, serotonin, cholinergic and adrenergic receptor agonists, and biogenic amines have been shown to exert inhibitory and/or stimulatory effects on biliary growth in normal and cholestatic conditions (3, 5, 9, 20–22, 24, 39, 42).
Melatonin is an indole formed enzymatically from l-tryptophan by the activity of the enzymes serotonin N-acetyltransferase (AANAT), and hydroxyindole-0-methyltransferase (32, 37) and is produced predominantly by the pineal gland (53). Extrapineal sites (e.g., the gastrointestinal tract) of melatonin production have been demonstrated (13). Melatonin exerts its effects by interacting with G protein-coupled membrane receptors, such as melatonin 1A receptor (MT1), MT2, and MT3 (found only in nonmammals) (37, 55), modulating intracellular messengers such as cAMP, a key molecule regulating large cholangiocyte functions (40), and [Ca2+]i, an important signaling molecule regulating the function of small cholangiocytes (21). Indeed, melatonin-activation of G protein-coupled receptors inhibits cAMP levels in a number of cells including rat pancreatic β-cells (50).
Melatonin receptors are distributed in the central nervous system (46) as well as in peripheral tissues including small intestine and hepatocytes (45, 51). In the liver, melatonin suppresses oxidative damage, attenuates proliferation, and stimulates apoptosis of hepatocytes in rats subjected to partial hepatectomy (35). Melatonin improves liver fibrosis in rats with BDL (62) and ameliorates BDL-induced systemic oxidative stress in cholestatic rats (19). No information exists regarding the role and mechanisms of action by which melatonin modulates biliary hyperplasia in cholestatic rats. Melatonin regulates cell mitosis by modulation of the circadian rhythm, which is under the control of a core set of clock genes: Period 1, 2, and 3 (PER1–3); Cryptochrome 1 and 2 (CRY1 and CRY2); CLOCK; and BMAL1 and BMAL2 (33). For example, melatonin exerts antiproliferative effects in breast cancer cells by resynchronization of deregulated core clock circuitry (27). The aims of the study were to demonstrate in the BDL rats that 1) melatonin decreases the proliferation of large cholangiocytes and liver damage and 2) melatonin inhibition of large biliary hyperplasia is associated with downregulation of cAMP-dependent phosphorylation of PKA and modulation of clock genes.
METHODS AND MATERIALS
Materials.
Reagents were purchased from Sigma Chemical (St. Louis, MO) unless otherwise indicated. The following antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA): 1) mouse monoclonal antibody against rat proliferating cell nuclear antigen (PCNA); 2) MT1, an affinity-purified goat polyclonal antibody raised against a peptide mapping near the COOH terminus of MT1 of rat origin; 3) MT2, an affinity-purified goat polyclonal antibody raised against a peptide mapping within an internal region of MT2 of mouse origin; and 4) the rabbit polyclonal antibodies to the CLOCK transcription factor, the goat polyclonal antibody to BMAL1 transcription factor, CRY1, and PER1 proteins. The mouse anti-cytokeratin-19 (CK-19) antibody was purchased from Caltag Laboratories (Burlingame, CA). The phospho-PKA catalytic subunit antibody was purchased from Cell Signaling (Boston, MA). The RNeasy Mini Kit to purify RNA was purchased from Qiagen (Valencia, CA). The radioimmunoassay (RIA) kits for the determination of cAMP levels were purchased from GE Healthcare (Arlington Heights, IL). 4-Phenyl-2-propionamidotetralin (4-P-PDOT, a specific MT2 antagonist, >300-fold selective for the MT2 vs. the MT1 subtype) (61) and the MT1/MT2 antagonist, luzindole (18), were purchased from Tocris Bioscience, Ellisville, MO.
Animal models.
Male 344 Fischer rats (150–175 g) were purchased from Charles River (Wilmington, MA) and kept in a temperature-controlled environment (22°C) with 12-h:12-h light/dark cycles. Animals were fed ad libitum and had free access to food and drinking water. The studies were performed in normal rats and in rats that, immediately after BDL (4) or bile duct incannulation (BDI, for bile collection) (4), had ad libitum access to water or water containing melatonin (20 mg/l corresponding to a melatonin intake of 2 mg/g body wt per day) (10) for 1 wk (Table 1). Melatonin (20 mg) was dissolved in 2.5 ml of ethanol and then diluted at 1 l with water. Control animals received water containing the same amount of ethanol. This estimated intake of melatonin is based on the fact that rats drink ∼15 ml of water per day mostly throughout the night (57) when melatonin secretion from the pineal gland is higher (37). The facts that 1) circulating melatonin is mostly metabolized (4–6 h in rats) (56) and excreted with the bile to small bowel and returns to the liver through enterohepatic circulation (with a minimal quantity excreted through the urine) (37, 65) and that 2) melatonin serum levels increased in our model (see Table 1) support the validity of our route of melatonin administration. The administration of melatonin by drinking water has been previously used in rodents (10). Before each surgical procedure, animals were anesthetized with pentobarbital sodium (50 mg/kg body wt ip). All animal experiments were performed in accordance with a protocol approved by the Scott and White and Texas A&M HSC IACUC Committee. All animals were used for the harvest of tissues and purification of liver cells at 8:00 AM. In all animals, we measured wet liver weight, body weight, and wet liver weight-to-body weight ratio, an index of cell growth (4).
Table 1.
Liver and body weight, liver-to-body weight ratio, and serum levels of melatonin, transaminases, and bilirubin in selected animal groups
| Groups | Liver Weight, g | Body Weight, g | Liver-to-Body Weight Ratio | Melatonin serum, pg/ml | SGPT, U/l | SGOT, U/l | Total Bilirubin, mg/l |
|---|---|---|---|---|---|---|---|
| Normal rats + tap water | 7.98 ± 0.7 (n = 5) | 190.4 ± 3.2 (n = 5) | 4.2 ± 0.4 (n = 5) | 45.5 ± 12.7 (n = 7) | 83.2 ± 14.8 (n = 7) | 194.5 ± 42.0 (n = 6) | <0.1 (n = 8) |
| Normal rats + tap water containing melatonin | 9.4 ± 0.4 (n = 5) | 206.6 ± 3.9 (n = 5) | 4.5 ± 0.2 (n = 5) | 140.2 ± 12.7 (n = 7) | 69.4 ± 45.9 (n = 7) | 143.0 ± 36.0 (n = 6) | <0.1 (n = 8) |
| BDL rats + tap water | 8.3 ± 0.2 (n = 15) | 145.6 ± 3.9 (n = 15) | 5.7 ± 0.1 (n = 15) | 117.6 ± 38.5 (n = 7) | 390.7 ± 107.0 (n = 6) | 1415.0 ± 325.8 (n = 6) | 12.4 ± 1.5 (n = 8) |
| BDL rats + tap water containing melatonin | 6.7 ± 0.1 (n = 15) | 150.1 ± 7.9 (n = 15) | 4.5 ± 0.1 (n = 15) | 164.7 ± 66.8 (n = 7) | 210.8 ± 45.9 (n = 6) | 585.0 ± 162.4 (n = 6) | 7.3 ± 1.9 (n = 8) |
Values are means ± SE.
BDL, bile duct ligation; SGOT, serum glutamic oxaloacetic transaminases; SGPT, serum glutamate pyruvate transaminases.
Expression of MT1 and MT2, CLOCK, BMAL1, CRY1, and PER1 in liver sections and purified cholangiocytes.
We evaluated the expression of MT1 and MT2 and CLOCK, BMAL1, CRY1, and PER1 in liver sections (4–5 μm thick) from normal and BDL rats by immunohistochemistry (42) and in total RNA (0.5 μg) by real-time PCR (21) from purified cholangiocytes.
Immunohistochemical observations were taken in a coded fashion by BX-51 light microscopy (Olympus, Tokyo, Japan) with a Videocam (Spot Insight; Diagnostic Instrument, Sterling Heights, MI) and analyzed with an Image Analysis System (Delta Sistemi, Rome, Italy). For all immunoreactions, negative controls (with normal serum from the same species substituted for the primary antibody) were included. To evaluate the expression of the messages for MT1 and MT2, CLOCK, BMAL1, CRY1, and PER1, we used the RT2 Real-Time assay from SABiosciences (Frederick, MD) (21). A ΔΔCT analysis was performed (21) using normal cholangiocytes as control. Data were expressed as relative mRNA levels ± SE of the selected gene-to-GAPDH ratio. The primers for melatonin receptors 1A and 1B, CLOCK, BMAL1, CRY1, and PER1 (SABiosciences) were designed according to the NCBI GenBank Accession numbers: XM_341441 (MT1), NM_053330 (MT2), NM_021856 (CLOCK gene), NM_024362 (BMAL1 gene), NM_198750 (CRY1), and NM_001034125 (PER1).
We evaluated by fluorescence-activated cell sorting (FACS) analysis (49) the expression of MT1 and MT2, CLOCK, BMAL1, CRY1, and PER1 in purified large cholangiocytes. FACS analysis was performed using a C6 flow cytometer and analyzed by CFlow Software (Accuri Cytometers, Ann Arbor, MI) (49). The expression of the selected protein was identified and gated on FL1-A/Count plots. The relative quantity of the selected protein (mean selected protein fluorescence) was expressed as mean FL1-A (samples)/mean FL-1A (secondary antibodies only).
Isolated and immortalized large cholangiocytes.
The isolation of cholangiocytes started each morning at 8:00 AM. Pure (100% by γ-glutamyltransferase histochemistry) (58) cholangiocytes were isolated by immunoaffinity separation (7) using a monoclonal antibody (from Dr. R. Faris, Brown University, Providence, RI). The rationale for performing these studies in large cholangiocytes is based on the fact that, following BDL, only these cells undergo mitosis (3, 23, 40). The in vitro studies were performed in immortalized large cholangiocytes (from large bile ducts) (63) displaying phenotypes similar to that of freshly isolated cholangiocytes (3, 21, 24, 63).
Evaluation of serum levels of melatonin, transaminases, and bilirubin and cholangiocyte proliferation and apoptosis.
The serum levels of the transaminases, glutamate pyruvate transaminases, and glutamic oxaloacetic transaminase and total bilirubin were evaluated using a Dimension RxL Max Integrated Chemistry system (Dade Behring, Deerfield, IL) by the Chemistry Department, Scott & White. Serum levels of melatonin were measured by commercially available ELISA kits (Genway, San Diego, CA).
We evaluated in liver sections (4–5 μm thick) 1) the percentage of cholangiocyte proliferation by semiquantitative immunolocalization for PCNA (41), 2) IBDM of cholangiocytes (41), and 3) the percentage of cholangiocyte apoptosis by semiquantitative terminal deoxynucleotidyltransferase biotin-dUTP nick-end labeling (TUNEL) kit (Apoptag; Chemicon International, Temecula, CA) (24). We evaluated by hematoxylin and eosin of sections whether melatonin administration induces the damage of kidney, heart, stomach, spleen, and small and large intestine. Sections were evaluated in a blinded fashion by a BX-51 light microscope (Olympus, Tokyo, Japan).
Measurement of PCNA expression and phosphorylation of PKA.
We evaluated by immunoblots (21) PCNA protein expression (21) and the phosphorylation of PKA in protein (10 μg) from spleen (positive) and large cholangiocytes from melatonin- or vehicle-treated BDL rats. The intensity of the bands was determined by scanning video densitometry using the phospho-imager Storm 860 and the ImageQuant TL software version 2003.02 (GE Healthcare).
Measurement of secretin-stimulated cAMP levels and bile and bicarbonate secretion.
We evaluated the effect of secretin on cAMP levels in large cholangiocytes and bile and bicarbonate secretion in bile fistula rats. Following isolation, cholangiocytes (1 × 105 cells) were incubated for 1 h at 37°C (31) before stimulation with 0.2% bovine serum albumin or secretin (100 nM) for 5 min at room temperature before evaluation of cAMP levels by RIA (3, 40). After anesthesia, rats were surgically prepared for bile collection (4). When steady-state bile flow was reached (60–70 min from the intravenous infusion of Krebs-Ringer-Henseleit solution, KRH), the animals were infused with secretin (100 nM) (39, 40) for 30 min followed by intravenous infusion of KRH for 30 min. Bicarbonate levels in bile were determined by a COBAS Mira Plus automated clinical chemistry analyzer (Bohemia, NY) (47).
In vitro effect of melatonin on the proliferation of large cholangiocytes.
We evaluated by immunofluorescence (21) and immunoblots (20) the expression of MT1 and MT2 receptors in immortalized large cholangiocytes. Images were visualized using an Olympus IX-71 confocal microscope. For all immunoreactions, negative controls were included. The intensity of the bands was determined by scanning video densitometry (see above).
We evaluated by RIA kits (3, 34, 40) cAMP levels in cholangiocytes treated with vehicle (basal) or melatonin (10−11 M for 5 min) in the absence/presence of preincubation with 4-P-PDOT or luzindole (both 10 μM) (18, 61). The rationale for using this dose (10−11 M) for melatonin is based on the finding that serum levels of melatonin in rodents and humans are on the picomolar to nanomolar ranges (12, 66). After trypsinization, cholangiocytes were treated at 37°C for 48 h with vehicle (DMSO diluted with 1× PBS), melatonin (10−11 M) in the absence/presence of preincubation with 4-P-PDOT or luzindole (10 μM) (18, 61), 4-P-PDOT or luzindole alone (10 μM) before evaluating by immunoblots (21) the expression of PCNA and PKA phosphorylation. Melatonin was first dissolved in DMSO and then diluted with 1× PBS at the stock solution of 2.5 × 10−2 M before being diluted to the desired working solution with 1× PBS.
Statistical analysis.
All data are expressed as means ± SE. Differences between groups were analyzed by Student's unpaired t-test when two groups were analyzed and ANOVA when more than two groups were analyzed, followed by an appropriate post hoc test.
RESULTS
Cholangiocytes express MT1 and MT2, CLOCK, BMAL1, CRY1, and PER1.
By immunohistochemistry in liver sections, normal bile ducts were weakly positive for MT1 (but not MT2) but showed strong immunoreactivity for MT1 and MT2 following BDL (Fig. 1A). The expression of CLOCK and BMAL1 was absent in normal bile ducts; however, immunoreactivity was observed in BDL bile ducts (Fig. 1B). Normal bile ducts stained positively for PER1 and CRY1, whose expression slightly increased in bile ducts from BDL rats (Fig. 1B). By real-time PCR and FACS analysis (Fig. 1, C and D), the expression of PER1, BMAL1, CRY1, and CLOCK increased in BDL compared with normal cholangiocytes but decreased in cholangiocytes from normal and BDL rats treated in vivo with melatonin compared with control cholangiocytes. The mRNA expression of MT1 and MT2 increased in BDL compared with normal cholangiocytes (Fig. 2A). By real-time PCR and FACS analysis, the expression of MT1 and MT2 decreased in cholangiocytes from BDL rats treated with melatonin in vivo compared with cholangiocytes from control BDL (Fig. 2, A and B).
Fig. 1.
A: by immunohistochemistry in liver sections, bile ducts (yellow arrow) from normal rats (NR) were weakly positive for melatonin 1A receptor (MT1) and did not stain for MT2; bile ducts (yellow arrows) from bile duct-ligated (BDL) rats showed immunoreactivity for both MT1 and MT2. Original magnification ×20. B: expression of CLOCK and BMAL1 was virtually absent in normal bile ducts; immunoreactivity was observed in BDL bile ducts (yellow arrows). Normal bile ducts stained positively for PER1 and CRY1, whose expression increased in bile ducts from BDL rats. Original magnification ×20. C: by real-time PCR, the mRNA expression of PER1, BMAL1, CRY1, and CLOCK increased in BDL compared with normal cholangiocytes but decreased in cholangiocytes from normal and BDL rats treated with melatonin in vivo compared with control cholangiocytes. D: by fluorescence-activated cell sorting (FACS) analysis, the protein expression of PER1, BMAL1, CRY1, and CLOCK decreased in cholangiocytes from BDL rats treated with melatonin in vivo compared with cholangiocytes from BDL rats treated with vehicle. Data are means ± SE of 3 evaluations. *P < 0.05 vs. the values of normal cholangiocytes. #P < 0.05 vs. the values of normal and BDL cholangiocytes from rats treated with regular tap water.
Fig. 2.
A: by real-time PCR, the mRNA expression of MT1 and MT2 increased in BDL compared with normal cholangiocytes but decreased in purified cholangiocytes from BDL rats treated with melatonin in vivo compared with cholangiocytes from control BDL rats. Data are means ± SE of 3 evaluations. *P < 0.05 vs. the values of normal cholangiocytes. #P < 0.05 vs. the values of BDL cholangiocytes from rats treated with regular tap water. B: by FACS analysis the protein expression of MT1 and MT2 decreased in cholangiocytes from BDL rats treated with melatonin in vivo compared with cholangiocytes from BDL rats treated with vehicle. Data are means ± SE of 3 evaluations. #P < 0.05 vs. the values of BDL cholangiocytes from rats treated with regular tap water.
Evaluation of serum levels of melatonin and transaminases, and bilirubin levels, cholangiocyte proliferation, and apoptosis.
There was a 15–20% decrease of body weight in BDL compared with normal rats (Table 1). No difference was observed in body weight between normal and BDL rats treated with melatonin compared with controls (Table 1). There was a decrease in liver-to-body weight ratio in BDL rats treated with melatonin compared with BDL controls (Table 1). The serum levels of melatonin of normal rats were similar to that of previous studies and increased following BDL (64) and after the administration of melatonin to normal and BDL rats (Table 1). The serum levels of transaminases increased in BDL rats compared with normal rats and were decreased in both normal and BDL rats by the administration of melatonin (Table 1). The administration of melatonin to BDL rats decreased large BDM (Fig. 3 and Table 2) and the percentage of PCNA-positive cholangiocytes (Table 2) compared with control rats. Melatonin inhibition of biliary hyperplasia in BDL rats was associated with enhanced cholangiocyte apoptosis (Table 2). Melatonin had no effects in normal rats (Table 2). No morphological changes of kidney, heart, stomach, spleen, and small and large intestine were observed in rats treated with melatonin (not shown).
Fig. 3.
Effect of melatonin on large intrahepatic bile duct mass (IBDM) of BDL rats, immunoreactivity for CK19. The administration of melatonin to BDL rats decreased IBDM compared with their corresponding controls rats (for semiquantitative data see Table 2) (yellow arrows: bile ducts) Original magnification, ×20.
Table 2.
Percentage of PCNA- and TUNEL-positive cholangiocytes and IBDM
| Groups | PCNA-Positive Cholangiocytes, % | IBDM, % | Cholangiocytes Positive by TUNEL, % |
|---|---|---|---|
| Normal rats + tap water | 7.919 ± 0.338 | 0.28 ± 0.03 | Negative |
| Normal rats + tap water containing melatonin | 6.740 ± 0.303 | 0.24 ± 0.03 | Negative |
| BDL rats + tap water | 47.436 ± 1.227 | 3.657 ± 0.302 | 5.514 ± 0.142 |
| BDL rats + tap water containing melatonin | 41.501 ± 0.973* | 2.036 ± 0.100* | 7.146 ± 0.163* |
Data are mean ± SE.
P < 0.01 vs. the corresponding values of BDL rats.
IBDM, intrahepatic bile duct mass; PCNA, proliferating cell nuclear antigen; TUNEL, terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling.
Effect of secretin on cAMP levels in cholangiocytes and bile and bicarbonate secretion in bile fistula rats.
In BDL rats treated with melatonin, basal levels of cAMP and basal bile and bicarbonate secretion were lower than those of the corresponding values of BDL control rats (Fig. 4A and Table 3). As expected (7), secretin increased cAMP levels of large cholangiocytes from BDL rats but did not enhance the levels of cAMP of large cholangiocytes from BDL rats treated with melatonin for 1 wk (Fig. 4A). Secretin increased bile and bicarbonate secretion of BDL controls but not of melatonin-treated BDL rats (Table 3).
Fig. 4.
A: effect of secretin on cAMP levels in large cholangiocytes from BDL rats treated with regular tap water or melatonin in drinking water for 1 wk. Secretin increased the intracellular cAMP levels of large cholangiocytes from BDL rats. Secretin did not enhance the intracellular levels of cAMP of large cholangiocytes from melatonin-treated rats. Data are means ± SE of 6 evaluations from cumulative preparations of cholangiocytes. *P < 0.05 vs. the corresponding basal values of large cholangiocytes from BDL controls. B–C: effect of melatonin on the expression of proliferating cell nuclear antigen (PCNA) (B) and the phosphorylation of PKA (C) in purified large cholangiocytes. By immunoblots, there was decreased PCNA expression and PKA phosphorylation in large cholangiocytes from melatonin-treated BDL rats compared with large cholangiocytes from BDL controls. Data are means ± SE of 4 blots from cumulative preparations of cholangiocytes. *P < 0.05 vs. the corresponding values of large cholangiocytes from BDL controls.
Table 3.
Measurement of basal and secretin-stimulated bile flow and bicarbonate secretion in rats
| Bile Flow |
Bicarbonate Secretion |
|||
|---|---|---|---|---|
| Treatment | Basal, μl·min−1·kg body wt−1 | Secretin, μl·min−1·kg body wt−1 | Basal, μl·min−1·kg body wt−1 | Secretin, μl·min−1·kg body wt−1 |
| BDI rats + tap water (n = 4) | 124.6 ± 11.9 | 226.7 ± 28.2† | 4.0 ± 0.3 | 11.0 ± 1.4† |
| BDI rats + tap water containing melatonin (n = 4) | 90.1 ± 11.9* | 94.3 ± 11.6‡ | 2.5 ± 0.4* | 3.4 ± 0.4‡ |
Values are means ± SE. Differences between groups were analyzed by the Student's unpaired t-test when 2 groups were analyzed and ANOVA when more than 2 groups were analyzed.
P < 0.05 vs. basal values of bile flow, bicarbonate secretion of bile duct incannulation (BDI) treated with vehicle for 1 wk.
P < 0.05 vs. corresponding basal value of bile flow or bicarbonate secretion of BDI control rats.
Nonsignificant vs. corresponding basal value of bile flow, bicarbonate concentration, or bicarbonate secretion of BDI rats that had ad libitum access to regular tap water for 1 wk.
Effect of melatonin on the expression of PCNA and phosphorylation of PKA in large cholangiocytes.
There was decreased PCNA expression in large cholangiocytes from BDL rats treated with melatonin compared with cholangiocytes from BDL control rats (Fig. 4B). We found decreased phosphorylation of PKA in large cholangiocytes from melatonin-treated BDL rats compared with large cholangiocytes from BDL controls (Fig. 4C).
Effect of melatonin on the proliferation of large cholangiocytes.
By immunofluorescence and immunoblots, large cholangiocytes express both MT1 and MT2 (Fig. 5, left and right). Melatonin decreased cAMP levels, a decrease that was prevented by luzindole (a MT1/MT2 antagonist) (18) but not 4-P-PDOT (a specific MT2 antagonist) (61) (Fig. 6A), and MT1 and MT2 (Fig. 7B) compared with their corresponding basal value.
Fig. 5.
Evaluation of MT1 and MT2 expression by immunofluorescence in cell smears (left) and immunoblots in protein of large cholangiocytes (right). We demonstrated that large cholangiocytes express both MT1 and MT2. Bar = 50 μm. Specific receptor immunoreactivity is depicted in red, whereas cells were counterstained with DAPI (blue).
Fig. 6.
Effect of melatonin on cAMP levels (A), expression of mRNA PCNA (B), PCNA protein expression (C), and PKA phosphorylation (D) of large cholangiocyte lines. Melatonin decreased cAMP levels, a decrease that was prevented by luzindole but not 4-phenyl-2-propionamidotetralin (4-P-PDOT). Melatonin decreased PCNA protein expression and the phosphorylation of PKA, decreases that were prevented by luzindole but not 4-P-PDOT. Data are means ± SE of 6 evaluations from cumulative preparations of cholangiocytes. *P < 0.05 vs. the corresponding basal values of large cholangiocyte lines.
Fig. 7.
A: by FACS analysis the protein expression of PER1, BMAL1, CRY1, and CLOCK decreased in large mouse cholangiocytes treated with melatonin compared with basal large mouse cholangiocyte cell lines. Data are means ± SE of 3 evaluations. *P < 0.05 vs. the corresponding basal values of large cholangiocyte lines. B: by FACS analysis the protein expression of MT1 and MT2 decreased in large mouse cholangiocyte lines after melatonin treatment compared with basal large mouse cholangiocyte lines. Data are means ± SE of 3 evaluations. *P < 0.05 vs. the corresponding basal values of large cholangiocyte lines.
DISCUSSION
Our study demonstrated 1) that freshly isolated and lines of large cholangiocytes express MT1 and MT2, CLOCK, BMAL1, CRY1, and PER1 and 2) that in vivo administration of melatonin to BDL rats reduces the serum levels of transaminases and bilirubin and inhibits cholangiocyte proliferation and IBDM typical of BDL (4). The antiproliferative effects of melatonin on biliary growth were associated with decreased basal cAMP levels and spontaneous bile and bicarbonate secretion as well as with loss of responsiveness to secretin and enhanced biliary apoptosis. The biliary expression of MT1 and MT2 and clock genes increased in BDL compared with normal cholangiocytes and decreased in cholangiocytes from normal and BDL rats treated with melatonin. There was decreased phosphorylation of PKA in cholangiocytes. In vitro melatonin decreased proliferation, cAMP levels, and PKA phosphorylation in large cholangiocytes, decreases that were mediated by MT1 receptors.
As it mimics typical features of human cholangiopathies (5), the BDL model is commonly used for evaluating the mechanisms of biliary growth/damage (3, 4, 24). Cholangiopathies share common pathological characteristics such as the damage of cholangiocytes and the proliferation of residual ducts (as a mechanism of compensatory repair to maintain the homeostasis of the biliary tree) (9), but they evolve toward ductopenia that represents the terminal stage of these diseases (9).
We provided the first evidence for the presence of functional melatonin receptors in the biliary epithelium. A recent study showing that melatonin attenuates the damage caused by scolicidal agents on bile ducts alluded to the presence of melatonin receptors on cholangiocytes (60). MT1s are expressed by human gallbladder epithelia (11). The reason why these inhibitory melatonin receptors are upregulated in proliferating BDL cholangiocytes may be due to a compensatory mechanism. The decrease in the expression of MT1 and MT2 by melatonin is likely due to desensitization of these receptors as suggested by other studies (36). Another explanation may be due to the increased expression of AANAT (the enzyme regulating melatonin secretion by cholangiocytes, G. Alpini, unpublished observations), an increase that may lead to decreased expression of melatonin receptors. Studies are being undertaken in our laboratory to demonstrate the presence and role of this autocrine loop (AANAT→MT1/MT2) in the autocrine regulation of biliary growth.
The validity of our model was supported by the fact that, following chronic administration of melatonin, the serum levels of this hormone increased compared with BDL control rats. The serum levels of melatonin in normal rats were similar to that of previous studies (30) and, in agreement with previous findings (e.g., in liver cirrhosis) (14), increased following BDL. The finding that administration of melatonin to normal and BDL rats increases its circulating levels is supported by studies in rats (52) and humans (17). A number of studies support the inhibitory effect of melatonin on cell mitosis. For example, melatonin inhibits the hyperplastic growth of gastric mucosal in rats (1). The finding that the in vivo administration of melatonin decreases the serum levels of transaminases and bilirubin (observed in BDL rats) is supported by previous studies (48). This finding suggests that melatonin protects the biliary epithelium from cholestatic injury as supported by the fact that melatonin ameliorates oxidative stress in cholestatic rats (19).
We demonstrated that melatonin inhibition of biliary hyperplasia is associated with downregulation of basal and secretin-stimulated cAMP levels and bile secretion and phosphorylation of PKA, regulators of large cholangiocyte proliferation (3, 40–42). Indeed, activation of cAMP-dependent signaling has been shown to stimulate large cholangiocyte hyperplasia (22, 24). Conversely, downregulation of the cAMP-dependent transduction pathway inhibits BDL-induced biliary hyperplasia (20, 40).
We performed in vitro experiments in large cholangiocytes aimed to demonstrate that melatonin exerts its effects by direct interaction with specific melatonin receptors (MT1) by downregulating cAMP signaling and selected clock genes. The concept that MT1 (but not MT2) is the predominant receptor modulating the inhibitory effects of melatonin on biliary hyperplasia is supported by studies in other cells (43, 50). These findings raise the potential important concept that drug targeting of MT1 may be important in the management of cholangiopathies.
Recent studies have demonstrated the role of circadian rhythm (26) and the key circadian hormone, melatonin, in the pathogenesis of disease states and carcinogenesis (54, 59). Several studies have implicated melatonin in the pathogenesis of liver disease and damage. Melatonin synthesis, release, and resulting circadian rhythms are dysregulated in a number of liver diseases. Abnormal melatonin circadian rhythms are found in patients with hepatic cirrhosis and correlated with the severity of liver insufficiency (64). This melatonin arrhythmia is corrected after liver transplantation (15). In addition, melatonin has been shown to protect against liver damage by attenuating oxidative stress and apoptosis in animal models of hepatic cirrhosis and fibrosis (16, 28, 29). Melatonin regulates the expression of circadian genes, which include Per1 and Per2, Cry1 and Cry2, BMAL1, and CLOCK. Circadian genes are linked with downregulation of cell proliferation and dysregulation of cell-cycle control during carcinogenesis (38). Mice lacking the circadian genes Per1 and Per2 and Cry1 and Cry2 are deficient in cell-cycle regulation, and Per2 mutant mice are cancer prone (38). Studies have shown that clock genes regulate cell mitosis and that melatonin regulates cell proliferation by changes in clock gene expression (27). For example, the circadian clock component BMAL1 is a critical regulator of p21WAF1/CIP1 expression and hepatocyte proliferation (25). The loss of CLOCK activity in Cry1−/−/Cry2−/− double mutant mice results in delayed liver regeneration (44). Supporting these studies, we have demonstrated that melatonin inhibition of biliary hyperplasia is associated with downregulation of PER1, CLOCK, BMAL1, and CRY1 expression. These previous findings suggest that circadian genes play a key role in the mechanisms regulating liver cell proliferation, and our current findings suggest that clock genes may play an important role in liver pathogenesis. Future studies are necessary to fully elucidate the individual roles of each circadian gene in the proliferative signaling cascade by which melatonin regulates biliary proliferation. Studies aimed to evaluate the expression and function of clock genes in cholangiocytes in vivo over at least one full 24-h cycle are undergoing because melatonin secretion from the pineal gland (37) and the biliary epithelium (G. Alpini unpublished observations) is higher during the night. Our findings have important clinical implications because melatonin (an over-the-counter drug used for curing sleep disorders) may be an important therapeutic tool for managing the cholangiocyte hyperplasia in biliary disorders.
GRANTS
This work was supported partly by the Dr. Nicholas C. Hightower Centennial Chair of Gastroenterology from Scott & White, the VA Research Scholar Award, a VA Merit Award, and the NIH grant DK58411 and DK76898 to G. Alpini, a NIH grant DK081442 to S. Glaser, the NIH K01 grant award (DK078532), an NIH R01 grant award (DK082435) to S. DeMorrow, by University funds to P. Onori, and Federate Athenaeum funds from University of Rome “La Sapienza” to E. Gaudio, and by a grant from Health and Labor Sciences Research Grants for the Research on Measures for Intractable Diseases (from the Ministry of Health, Labor and Welfare of Japan) and from Grant-in Aid for-Scientific Research C (16590573) from JSPS. The study related to the measurement of bicarbonate levels in bile were performed at Yale Medical School and supported by the Yale-MMPC grant, U24 DK-76169.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the Texas A&M Health Science Center Integrated Microscopy and Imaging Laboratory, Temple, TX for assistance with the confocal microscopy and Bryan Moss (Medical Illustration, Scott & White, Graphic Services Department) for assistance in the preparation of the figures.
REFERENCES
- 1. Akbulut KG, Gonul B, Akbulut H. The role of melatonin on gastric mucosal cell proliferation and telomerase activity in ageing. J Pineal Res 47: 308–312, 2009 [DOI] [PubMed] [Google Scholar]
- 2. Alpini G, Glaser S, Robertson W, Rodgers RE, Phinizy JL, Lasater J, LeSage G. Large but not small intrahepatic bile ducts are involved in secretin-regulated ductal bile secretion. Am J Physiol Gastrointest Liver Physiol 272: G1064–G1074, 1997 [DOI] [PubMed] [Google Scholar]
- 3. Alpini G, Glaser S, Ueno Y, Pham L, Podila PV, Caligiuri A, LeSage G, LaRusso NF. Heterogeneity of the proliferative capacity of rat cholangiocytes after bile duct ligation. Am J Physiol Gastrointest Liver Physiol 274: G767–G775, 1998 [DOI] [PubMed] [Google Scholar]
- 4. Alpini G, Lenzi R, Sarkozi L, Tavoloni N. Biliary physiology in rats with bile ductular cell hyperplasia. Evidence for a secretory function of proliferated bile ductules. J Clin Invest 81: 569–578, 1988 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Alpini G, Prall RT, LaRusso NF. The pathobiology of biliary epithelia. In: The Liver: Biology & Pathobiology, edited by Arias IM, Boyer JL, Chisari FV, Fausto N, Jakoby W, Schachter D, Shafritz DA. Philadelphia, PA: Lippincott Williams & Wilkins, 2001, p. 421–435 [Google Scholar]
- 6. Alpini G, Roberts S, Kuntz SM, Ueno Y, Gubba S, Podila PV, LeSage G, LaRusso NF. Morphological, molecular, and functional heterogeneity of cholangiocytes from normal rat liver. Gastroenterology 110: 1636–1643, 1996 [DOI] [PubMed] [Google Scholar]
- 7. Alpini G, Ulrich C, Roberts S, Phillips JO, Ueno Y, Podila PV, Colegio O, LeSage G, Miller LJ, LaRusso NF. Molecular and functional heterogeneity of cholangiocytes from rat liver after bile duct ligation. Am J Physiol Gastrointest Liver Physiol 272: G289–G297, 1997 [DOI] [PubMed] [Google Scholar]
- 8. Alpini G, Ulrich CD, 2nd, Phillips JO, Pham LD, Miller LJ, LaRusso NF. Upregulation of secretin receptor gene expression in rat cholangiocytes after bile duct ligation. Am J Physiol Gastrointest Liver Physiol 266: G922–G928, 1994 [DOI] [PubMed] [Google Scholar]
- 9. Alvaro D, Mancino MG, Glaser S, Gaudio E, Marzioni M, Francis H, Alpini G. Proliferating cholangiocytes: a neuroendocrine compartment in the diseased liver. Gastroenterology 132: 415–431, 2007 [DOI] [PubMed] [Google Scholar]
- 10. Anisimov VN, Alimova IN, Baturin DA, Popovich IG, Zabezhinski MA, Manton KG, Semenchenko AV, Yashin AI. The effect of melatonin treatment regimen on mammary adenocarcinoma development in HER-2/neu transgenic mice. Int J Cancer 103: 300–305, 2003 [DOI] [PubMed] [Google Scholar]
- 11. Aust S, Thalhammer T, Humpeler S, Jager W, Klimpfinger M, Tucek G, Obrist P, Marktl W, Penner E, Ekmekcioglu C. The melatonin receptor subtype MT1 is expressed in human gallbladder epithelia. J Pineal Res 36: 43–48, 2004 [DOI] [PubMed] [Google Scholar]
- 12. Brzezinski A. Melatonin in humans. N Engl J Med 336: 186–195, 1997 [DOI] [PubMed] [Google Scholar]
- 13. Bubenik GA. Gastrointestinal melatonin: localization, function, and clinical relevance. Dig Dis Sci 47: 2336–2348, 2002 [DOI] [PubMed] [Google Scholar]
- 14. Celinski K, Konturek PC, Slomka M, Cichoz-Lach H, Gonciarz M, Bielanski W, Reiter RJ, Konturek SJ. Altered basal and postprandial plasma melatonin, gastrin, ghrelin, leptin and insulin in patients with liver cirrhosis and portal hypertension without and with oral administration of melatonin or tryptophan. J Pineal Res 46: 408–414, 2009 [DOI] [PubMed] [Google Scholar]
- 15. Cordoba J, Steindl P, Blei AT. Melatonin arrhythmia is corrected after liver transplantation. Am J Gastroenterol 104: 1862–1863, 2009 [DOI] [PubMed] [Google Scholar]
- 16. Cruz A, Padillo FJ, Torres E, Navarrete CM, Munoz-Castaneda JR, Caballero FJ, Briceno J, Marchal T, Tunez I, Montilla P, Pera C, Muntane J. Melatonin prevents experimental liver cirrhosis induced by thioacetamide in rats. J Pineal Res 39: 143–150, 2005 [DOI] [PubMed] [Google Scholar]
- 17. Dollins AB, Zhdanova IV, Wurtman RJ, Lynch HJ, Deng MH. Effect of inducing nocturnal serum melatonin concentrations in daytime on sleep, mood, body temperature, and performance. Proc Natl Acad Sci USA 91: 1824–1828, 1994 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18. Drazen DL, Bilu D, Bilbo SD, Nelson RJ. Melatonin enhancement of splenocyte proliferation is attenuated by luzindole, a melatonin receptor antagonist. Am J Physiol Regul Integr Comp Physiol 280: R1476–R1482, 2001 [DOI] [PubMed] [Google Scholar]
- 19. Esrefoglu M, Gul M, Emre MH, Polat A, Selimoglu MA. Protective effect of low dose of melatonin against cholestatic oxidative stress after common bile duct ligation in rats. World J Gastroenterol 11: 1951–1956, 2005 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20. Francis H, Franchitto A, Ueno Y, Glaser S, DeMorrow S, Venter J, Gaudio E, Alvaro D, Fava G, Marzioni M, Vaculin B, Alpini G. H3 histamine receptor agonist inhibits biliary growth of BDL rats by downregulation of the cAMP-dependent PKA/ERK1/2/ELK-1 pathway. Lab Invest 87: 473–487, 2007 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21. Francis H, Glaser S, DeMorrow S, Gaudio E, Ueno Y, Venter J, Dostal D, Onori P, Franchitto A, Marzioni M, Vaculin S, Vaculin B, Katki K, Stutes M, Savage J, Alpini G. Small mouse cholangiocytes proliferate in response to H1 histamine receptor stimulation by activation of the IP3/CaMK I/CREB pathway. Am J Physiol Cell Physiol 295: C499–C513, 2008 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22. Francis H, Glaser S, Ueno Y, LeSage G, Marucci L, Benedetti A, Taffetani S, Marzioni M, Alvaro D, Venter J, Reichenbach R, Fava G, Phinizy JL, Alpini G. cAMP stimulates the secretory and proliferative capacity of the rat intrahepatic biliary epithelium through changes in the PKA/Src/MEK/ERK1/2 pathway. J Hepatol 41: 528–537, 2004 [DOI] [PubMed] [Google Scholar]
- 23. Glaser S, Gaudio E, Rao A, Pierce LM, Onori P, Franchitto A, Francis HL, Dostal DE, Venter JK, DeMorrow S, Mancinelli R, Carpino G, Alvaro D, Kopriva SE, Savage JM, Alpini G. Morphological and functional heterogeneity of the mouse intrahepatic biliary epithelium. Lab Invest 89: 456–469, 2009 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24. Glaser S, Lam IP, Franchitto A, Gaudio E, Onori P, Chow BK, Wise C, Kopriva S, Venter J, White M, Ueno Y, Dostal D, Carpino G, Mancinelli R, Butler W, Chiasson V, DeMorrow S, Francis H, Alpini G. Knockout of secretin receptor reduces large cholangiocyte hyperplasia in mice with extrahepatic cholestasis induced by bile duct ligation. Hepatology 52: 204–214, 2010 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25. Grechez-Cassiau A, Rayet B, Guillaumond F, Teboul M, Delaunay F. The circadian clock component BMAL1 is a critical regulator of p21WAF1/CIP1 expression and hepatocyte proliferation. J Biol Chem 283: 4535–4542, 2008 [DOI] [PubMed] [Google Scholar]
- 26. Hardeland R. Melatonin, hormone of darkness and more: occurrence, control mechanisms, actions and bioactive metabolites. Cell Mol Life Sci 65: 2001–2018, 2008 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27. Hill SM, Frasch T, Xiang S, Yuan L, Duplessis T, Mao L. Molecular mechanisms of melatonin anticancer effects. Integr Cancer Ther 8: 337–346, 2009 [DOI] [PubMed] [Google Scholar]
- 28. Hong RT, Xu JM, Mei Q. Melatonin ameliorates experimental hepatic fibrosis induced by carbon tetrachloride in rats. World J Gastroenterol 15: 1452–1458, 2009 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29. Hu S, Yin S, Jiang X, Huang D, Shen G. Melatonin protects against alcoholic liver injury by attenuating oxidative stress, inflammatory response, and apoptosis. Eur J Pharmacol 616: 287–292, 2009 [DOI] [PubMed] [Google Scholar]
- 30. Huang LT, Tiao MM, Tain YL, Chen CC, Hsieh CS. Melatonin ameliorates bile duct ligation-induced systemic oxidative stress and spatial memory deficits in developing rats. Pediatr Res 65: 176–180, 2009 [DOI] [PubMed] [Google Scholar]
- 31. Ishii M, Vroman B, LaRusso NF. Isolation and morphologic characterization of bile duct epithelial cells from normal rat liver. Gastroenterology 97: 1236–1247, 1989 [DOI] [PubMed] [Google Scholar]
- 32. Iuvone PM, Tosini G, Pozdeyev N, Haque R, Klein DC, Chaurasia SS. Circadian clocks, clock networks, arylalkylamine N-acetyltransferase, and melatonin in the retina. Prog Retin Eye Res 24: 433–456, 2005 [DOI] [PubMed] [Google Scholar]
- 33. Jung-Hynes B, Huang W, Reiter RJ, Ahmad N. Melatonin resynchronizes dysregulated circadian rhythm circuitry in human prostate cancer cells. J Pineal Res 49: 60–68, 2010 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34. Kato A, Gores GJ, LaRusso NF. Secretin stimulates exocytosis in isolated bile duct epithelial cells by a cyclic AMP-mediated mechanism. J Biol Chem 267: 15523–15529, 1992 [PubMed] [Google Scholar]
- 35. Kirimlioglu H, Ecevit A, Yilmaz S, Kirimlioglu V, Karabulut AB. Effect of resveratrol and melatonin on oxidative stress enzymes, regeneration, and hepatocyte ultrastructure in rats subjected to 70% partial hepatectomy. Transplant Proc 40: 285–289, 2008 [DOI] [PubMed] [Google Scholar]
- 36. Kokkola T, Vaittinen M, Laitinen JT. Inverse agonist exposure enhances ligand binding and G protein activation of the human MT1 melatonin receptor, but leads to receptor down-regulation. J Pineal Res 43: 255–262, 2007 [DOI] [PubMed] [Google Scholar]
- 37. Konturek SJ, Konturek PC, Brzozowska I, Pawlik M, Sliwowski Z, Czesnikiewicz-Guzik M, Kwiecien S, Brzozowski T, Bubenik GA, Pawlik WW. Localization and biological activities of melatonin in intact and diseased gastrointestinal tract (GIT). J Physiol Pharmacol 58: 381–405, 2007 [PubMed] [Google Scholar]
- 38. Lee S, Donehower LA, Herron AJ, Moore DD, Fu L. Disrupting circadian homeostasis of sympathetic signaling promotes tumor development in mice. PLos One 5: e10995, 2010 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39. LeSage G, Alvaro D, Glaser S, Francis H, Marucci L, Roskams T, Phinizy JL, Marzioni M, Benedetti A, Taffetani S, Barbaro B, Fava G, Ueno Y, Alpini G. Alpha-1 adrenergic receptor agonists modulate ductal secretion of BDL rats via Ca(2+)- and PKC-dependent stimulation of cAMP. Hepatology 40: 1116–1127, 2004 [DOI] [PubMed] [Google Scholar]
- 40. LeSage G, Glaser S, Marucci L, Benedetti A, Phinizy JL, Rodgers R, Caligiuri A, Papa E, Tretjak Z, Jezequel AM, Holcomb LA, Alpini G. Acute carbon tetrachloride feeding induces damage of large but not small cholangiocytes from BDL rat liver. Am J Physiol Gastrointest Liver Physiol 276: G1289–G1301, 1999 [DOI] [PubMed] [Google Scholar]
- 41. Mancinelli R, Franchitto A, Gaudio E, Onori P, Glaser S, Francis H, Venter J, DeMorrow S, Carpino G, Kopriva S, White M, Fava G, Alvaro D, Alpini G. After damage of large bile ducts by gamma-aminobutyric acid, small ducts replenish the biliary tree by amplification of calcium-dependent signaling and de novo acquisition of large cholangiocyte phenotypes. Am J Pathol 176: 1790–1800, 2010 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42. Mancinelli R, Onori P, Gaudio E, DeMorrow S, Franchitto A, Francis H, Glaser S, Carpino G, Venter J, Alvaro D, Kopriva S, White M, Kossie A, Savage J, Alpini G. Follicle-stimulating hormone increases cholangiocyte proliferation by an autocrine mechanism via cAMP-dependent phosphorylation of ERK1/2 and Elk-1. Am J Physiol Gastrointest Liver Physiol 297: G11–G26, 2009 [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
- 43. Mao L, Yuan L, Slakey LM, Jones FE, Burow ME, Hill SM. Inhibition of breast cancer cell invasion by melatonin is mediated through regulation of the p38 mitogen-activated protein kinase signaling pathway (Abstract). Breast Cancer Res 12: R107, 2010 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44. Matsuo T, Yamaguchi S, Mitsui S, Emi A, Shimoda F, Okamura H. Control mechanism of the circadian clock for timing of cell division in vivo. Science 302: 255–259, 2003 [DOI] [PubMed] [Google Scholar]
- 45. Naji L, Carrillo-Vico A, Guerrero JM, Calvo JR. Expression of membrane and nuclear melatonin receptors in mouse peripheral organs. Life Sci 74: 2227–2236, 2004 [DOI] [PubMed] [Google Scholar]
- 46. Nakahara K, Murakami N, Nasu T, Kuroda H, Murakami T. Individual pineal cells in chick possess photoreceptive, circadian clock and melatonin-synthesizing capacities in vitro. Brain Res 774: 242–245, 1997 [DOI] [PubMed] [Google Scholar]
- 47. Norris KA, Atkinson AR, Smith WG. TBA (Abstract). Clin Chem 21: 1093, 1975 [PubMed] [Google Scholar]
- 48. Ohta Y, Kongo M, Kishikawa T. Melatonin exerts a therapeutic effect on cholestatic liver injury in rats with bile duct ligation. J Pineal Res 34: 119–126, 2003 [DOI] [PubMed] [Google Scholar]
- 49. Onori P, Wise C, Gaudio E, Franchitto A, Francis H, Carpino G, Lee V, Lam I, Miller T, Dostal DE, Glaser S. Secretin inhibits cholangiocarcinoma growth via dysregulation of the cAMP-dependent signaling mechanisms of secretin receptor. Int J Cancer 127: 43–54, 2010 [DOI] [PubMed] [Google Scholar]
- 50. Peschke E, Muhlbauer E, Musshoff U, Csernus VJ, Chankiewitz E, Peschke D. Receptor (MT(1)) mediated influence of melatonin on cAMP concentration and insulin secretion of rat insulinoma cells INS-1. J Pineal Res 33: 63–71, 2002 [DOI] [PubMed] [Google Scholar]
- 51. Poon AM, Choy EH, Pang SF. Modulation of blood glucose by melatonin: a direct action on melatonin receptors in mouse hepatocytes. Biol Signals Recept 10: 367–379, 2001 [DOI] [PubMed] [Google Scholar]
- 52. Porkka-Heiskanen T, Laakso ML, Stenberg D, Alila A, Johansson G. Increase in testosterone sensitivity induced by constant light in relation to melatonin injections in rats. J Reprod Fertil 96: 331–336, 1992 [DOI] [PubMed] [Google Scholar]
- 53. Reiter RJ. Pineal melatonin: cell biology of its synthesis and of its physiological interactions. Endocr Rev 12: 151–180, 1991 [DOI] [PubMed] [Google Scholar]
- 54. Reiter RJ, Tan DX, Korkmaz A. The circadian melatonin rhythm and its modulation: possible impact on hypertension. J Hypertens Suppl 27: S17–S20, 2009 [DOI] [PubMed] [Google Scholar]
- 55. Reppert SM, Godson C, Mahle CD, Weaver DR, Slaugenhaupt SA, Gusella JF. Molecular characterization of a second melatonin receptor expressed in human retina and brain: the Mel1b melatonin receptor. Proc Natl Acad Sci USA 92: 8734–8738, 1995 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 56. Rowe SA, Kennaway DJ. Melatonin in rat milk and the likelihood of its role in postnatal maternal entrainment of rhythms. Am J Physiol Regul Integr Comp Physiol 282: R797–R804, 2002 [DOI] [PubMed] [Google Scholar]
- 57. Rowland NE, Bellush LL, Fregly MJ. Nycthemeral rhythms and sodium chloride appetite in rats. Am J Physiol Regul Integr Comp Physiol 249: R375–R378, 1985 [DOI] [PubMed] [Google Scholar]
- 58. Rutenburg AM, Kim H, Fischbein JW, Hanker JS, Wasserkrug HL, Seligman AM. Histochemical and ultrastructural demonstration of gamma-glutamyl transpeptidase activity. J Histochem Cytochem 17: 517–526, 1969 [DOI] [PubMed] [Google Scholar]
- 59. Sahar S, Sassone-Corsi P. Metabolism and cancer: the circadian clock connection. Nat Rev Cancer 9: 886–896, 2009 [DOI] [PubMed] [Google Scholar]
- 60. Sezer A, Hatipoglu AR, Usta U, Altun G, Sut N. Effects of intraperitoneal melatonin on caustic sclerosing cholangitis due to scolicidal solution in a rat model. Curr Ther Res 71: 118–128, 2010 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 61. Shiu SY, Pang B, Tam CW, Yao KM. Signal transduction of receptor-mediated antiproliferative action of melatonin on human prostate epithelial cells involves dual activation of Galpha and Galpha proteins. J Pineal Res 49: 301–311, 2010 [DOI] [PubMed] [Google Scholar]
- 62. Tahan G, Akin H, Aydogan F, Ramadan SS, Yapicier O, Tarcin O, Uzun H, Tahan V, Zengin K. Melatonin ameliorates liver fibrosis induced by bile-duct ligation in rats. Can J Surg 53: 313–318, 2010 [PMC free article] [PubMed] [Google Scholar]
- 63. Ueno Y, Alpini G, Yahagi K, Kanno N, Moritoki Y, Fukushima K, Glaser S, LeSage G, Shimosegawa T. Evaluation of differential gene expression by microarray analysis in small and large cholangiocytes isolated from normal mice. Liver Int 23: 449–459, 2003 [DOI] [PubMed] [Google Scholar]
- 64. Velissaris D, Karanikolas M, Kalogeropoulos A, Solomou E, Polychronopoulos P, Thomopoulos K, Labropoulou-Karatza C. Pituitary hormone circadian rhythm alterations in cirrhosis patients with subclinical hepatic encephalopathy. World J Gastroenterol 14: 4190–4195, 2008 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 65. Waldhauser F, Waldhauser M, Lieberman HR, Deng MH, Lynch HJ, Wurtman RJ. Bioavailability of oral melatonin in humans. Neuroendocrinology 39: 307–313, 1984 [DOI] [PubMed] [Google Scholar]
- 66. Yie SM, Johansson E, Brown GM. Competitive solid-phase enzyme immunoassay for melatonin in human and rat serum and rat pineal gland. Clin Chem 39: 2322–2325, 1993 [PubMed] [Google Scholar]