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The expression of MT1 and MT2 melatonin receptor mRNA in several rat tissues
Life Sciences 76 (2005) 1123 – 1134 www.elsevier.com/locate/lifescie
The expression of MT1 and MT2 melatonin receptor mRNA in several rat tissues Pirkko Sallinena,*, Seppo Saarelaa, Mika Ilvesb, Olli Vakkurib, Juhani Lepp7luotob a
Department of Biology, P.O. Box 3000, 90014 University of Oulu, Finland b Department of Physiology, University of Oulu, Oulu, Finland Received 14 May 2004; accepted 16 August 2004
Abstract The mechanisms that mediate the various effects of melatonin in mammalian tissues are not always known. Therefore, the aim of this study was to investigate whether MT1 and MT2 melatonin receptors are expressed in certain tissues of the rat. The expression of MT1 and MT2 melatonin receptor mRNA was determined using a realtime quantitative RT-PCR method. In addition, we examined whether mRNA for either subtype of receptor shows any difference in the expression between midnight and noon, similar to the changes in melatonin concentrations in plasma and tissue samples. MT1 and MT2 melatonin receptor mRNAs were found in the rat hypothalamus, retina and small intestine. We also showed a low expression of MT2 mRNA in the rat liver and heart SA node. In the heart apex and the Harderian gland, no appearance of either of the receptor mRNAs was detectable. A significant difference in the expression of MT1 mRNA between day and night was found in the hypothalamus. In conclusion, our findings suggest that at least some effects of melatonin are mediated through membrane MT1 and MT2 receptors in the hypothalamus, the retina and the small intestine. Down-regulation of receptors might be one reason for the difference in the hypothalamic MT1 melatonin receptor mRNA expression between midnight and noon. In the liver and the heart SA node, the physiological significance of possible MT2 receptors remains unclear. According to our negative midnight and noon results in the Harderian gland and heart apex melatonin may exert its effect on these tissues by a non-receptor mechanism. D 2004 Elsevier Inc. All rights reserved. Keywords: Melatonin receptor; MT1 mRNA; MT2 mRNA; Real-Time Quantitative RT-PCR; Melatonin; Rat
* Corresponding author. Tel.: +358 8 5531240; fax: +358 8 5531061. E-mail address: [email protected] (P. Sallinen). 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.08.016
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Introduction Melatonin, an indole-derived neurosecretory product, is synthesized in the pineal gland and secreted into the blood in a circadian manner. However, melatonin is also synthesized in the retina, the Harderian gland and the gastrointestinal tract (Reiter, 1991). This indoleamine is known to be an important regulator of daily and seasonal behavior and physiology as well as a highly efficient antioxidant scavenger of, for example, the hydroxyl radical (Tan et al., 1993). Melatonin is also involved in many physiological functions depending on the circadian rhythm, such as the immune and the cardiovascular systems. The lipophilicity of melatonin permits its transfer through biological membranes, and therefore the indoleamine is found throughout the organism in all cell and cellular organelles including the nucleus (Menendez-Pelaez and Reiter, 1993). It has also been reported that melatonin has an impact on the function of many different tissues. For example, in the retina, it is suggested that melatonin acts as a paracrine signal of darkness and enhances the sensitivity of horizontal cells (Wiechmann et al., 1988). Antolin et al. (1996) have observed that in the Harderian gland melatonin prevents cell damage induced by free radicals generated by porphyrin metabolism. Melatonin also has an effect on the circadian rhythms of the neuronal firing rate in the suprachiasmatic nucleus (SCN) (Hunt et al., 2001). In addition, several reports have recently demonstrated that in the heart this indoleamine has beneficial effects on postischemic myocardial dysfunction (Kaneko et al., 2000; Lagneux et al., 2000; Lee et al., 2002; Sahna et al., 2002a,b; Szarszoi et al., 2001; Tan et al., 1998). Furthermore, it has been shown that melatonin affects the function of the liver and the gastrointestinal tract. Melatonin elevates the plasma glucose level probably by acting directly on the liver (Poon et al., 2001). Ohta et al. (2000) have found that melatonin exerts a therapeutic effect on carbon tetrachlorideinduced acute liver injury in rats. Because melatonin is also synthesized in the gastrointestinal tract, it has been suggested that melatonin is probably a paracrine agent that protects the epithelium of the gastrointestinal tract and stimulates the synthesis of other gastroprotective hormones (Motilva et al., 2001). It has also been shown that melatonin is involved in the motility changes of the small intestine especially at night (Merle et al., 2000), it may also synchronize the sequential digestive processes (Bubenik, 2001), and melatonin is involved in the control of bicarbonate secretion by the duodenal epithelium (Sjo¨blom and Flemstro¨m, 2003). Even though many studies indicate that melatonin has an impact on the function of many different tissues, it is not always clear how the actions of melatonin are mediated. It is possible that melatonin owes its influence to its antioxidant action, and its effects may also be mediated via cell membrane receptors which belong to the family of G-protein coupled receptors (MT1 and MT2) and the quinone reductase enzymes (MT3). Two of these receptor subtypes (MT1 and MT2) have been found in mammals (Reppert et al., 1996). Traditionally, melatonin receptors have been visualized using radioactive 2-[125I]-iodomelatonin (125I-Mel), and melatonin receptor messenger RNA levels have been quantified by Northern blot analysis. However, a more sensitive method is to use a real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) (Bustin, 2000), but because it is a fairly new method, the melatonin receptors’ mRNA levels of rat tissues have not previously been measured quantitatively using this method. In some tissues of rat melatonin receptor mRNAs have been detected using traditional RT-PCR, which is merely a semi-quantitative method related to a real-time quantitative RT-PCR-method. The MT1 melatonin receptor mRNA has been identified, for example, in the SCN (Sugden et al., 1999), and in other regions of the brain as well as in some peripheral tissues (Aust et al., 2004; Chucharoen et al., 2003; Fujieda et al., 1999; Li et al., 1998; Peschke et al., 2000; Poirel et al.,
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2003; Ting et al., 1999; Woo et al., 2001; Zhao et al., 2000) using a classical RT-PCR method. The MT2 melatonin receptor mRNA has been measured in the rat SCN (Sugden et al., 1999), in the human ovary (Woo et al., 2001), in the corpus epididymis (Li et al., 1998) and in the caudal artery of the rat (Masana et al., 2002). The above-mentioned findings raise the question about the mechanism which mediates the action of melatonin in certain tissues. Therefore, the aim of the present study was to investigate the expression of MT1- and MT2-receptors in rat hypothalamus, the retina, the Harderian gland, the small intestine, the heart and the liver. The real-time quantitative RT-PCR method was used to determine the expression of MT1 and MT2 melatonin receptor mRNA. The mRNA for the MT1 or MT2 subtype of melatonin receptor was examined for signs of a difference in the expression between midnight and noon. In addition, radioimmunoassay (RIA) was used to measure the concentration of melatonin at midnight and noon in plasma and in the studied tissues.
Materials and methods Animals and tissue preparations Sixteen adult male and female Sprague-Dawley rats (Rattus norvegicus), obtained from the Laboratory Animal Centre, University of Oulu, were used in the experiment. The body mass of the rats ranged from 372 to 437 g. The animals received food and water ad libitum and were maintained at constant temperature (+21F18C) and under a 12:12 h light:dark cycle (lights on at 08:00 h a.m.). The experimental protocol was approved by the Animal Experimentation Committee of the University of Oulu (Licence Nr 074/01). Half of the rats were killed at midnight (24.00 h, n=8) and the others were killed at noon (12.00 h, n=8). Animals were anaesthetized with carbon dioxide and blood samples were taken with heparinized syringes from the heart. After this, the animals were decapitated and tissue samples from the heart (apex and sinoatrial node), hypothalamus (including the SCN), Harderian gland, the eye (including the retina), small intestine and liver were collected. Tissue samples were immediately frozen in liquid nitrogen, and stored at -708C until RNA isolation was performed. Blood samples were centrifuged (3,000 g for 10 min) and plasma samples were stored at -708C until assayed for melatonin. For the rats killed at midnight, the samples were collected in the dark under dim red lighting (Philips PF 712E, Holland). Total RNA extraction and reverse transcription Total RNA was extracted from the tissue samples using an RNA isolation kit (QuickPrep Total RNA Extraction Kit, Amersham Pharmacia Biotech, USA) according to the manufacturer’s instructions. The amount of RNA was determined spectrophotometrically at 260 nm. Total RNA was converted into cDNA using a First Strand cDNA Synthesis Kit (#K1612, MBI Fermentas, Lithuania). Deionized, nuclear free water was added to the total RNA (0.5 Ag) to get a volume of 5.0 Al. After this random hexamer primer (0.1 Ag) was added and the mixture was heated to 708C for 5 min, then cooled immediately on ice. A ribonuclease inhibitor (10U), 5X reaction buffer (2 Al), 1.0 Al of 10 mM dNTP mix (dATP, dGTP, dCTP anp dTTP) and a MuLV-reverse transcriptase (20U) were added to make the final volume 10 Al. The reaction mixture was preincubated for 10 min at room temperature (+228C)
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before cDNA synthesis. Reverse transcription (RT) reactions were carried out for 60 min at +378C and then samples were heated to +708C for 10 min to terminate the RT reaction. Polymerase chain reaction Real time quantitative PCR was performed using a TaqManR 1000 Rxn Gold/Buffer Pack reagent Kit (Applied Biosystems, Foster City, CA, USA) and an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). TaqManR probes and primers were designed with the aid of the Primer Express program (Applied Biosystems, Foster City, CA, USA) from the previously reported partial MT1 (Poirel et al., 1999a) and MT2 (Poirel et al., 1999b) melatonin receptor sequences of rats. Probes and primers were obtained from Applied Biosystems UK (Cheshire). For the MT1 receptor the synthesized amplicon was 66 bp and the sequence of the probe and primers were as follows: probe, 5VTCCTGTACCTTCACCCAGTCCGTCAGC-3V (bases 516-542); forward primer, 5V-CAGTACGACCCCCGGATCTA-3V (bases 495-514); reverse primer 5V-GGCAATCGTGTACGCCG-3V (bases 544-560). For the MT2 receptor the synthesized amplicon was 65 bp and the sequence of the probe and primers were as follows: probe, 5V-CGCCATATGCTGGGCCCCC-3V(bases 366-384); forward primer 5VATGTTCGCAGTGTTTGTGGTTT-3V (bases 343-364); reverse primer 5V-ACTGCAAGGCCAATACAGTTGA-3V(bases 386-407). Ribosomal protein gene 18S rRNA was used as an endogenous control and the sequences of the probe and primers for the 18S rRNA were as follows: probe 5VCCTGGTGGTGCCCTTCCGTCA-3V; forward primer 5V-TGGTTGCAAAGCTGAAACTTAAAG-3V; reverse primer 5V-AGTCAAATTAAGCCGCAGGC-3V. Each reaction contained 5 Al RT reaction product as template DNA and 20 Al reaction mixture, which consisted of milli-Q water, MgCl2 (25.0 mM), reaction buffer (10X TaqManR Buffer A), dNTP mix (2.5 mM each), primers (3.0 AM), probe (5.0 AM) and AmpliTag Gold polymerase (5U/Al). The template was initially denaturated for 10 min at +958C followed by a 40-cycle program with 15 s at +958C, 1 min annealing and extension at +608C. The TaqMan probe consists of an oligonucleotide with a 5V-fluorescent reporter dye and a 3V-quencher dye. When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence. During PCR the reporter dye and quencher dye are separated, resulting in increased fluorescence of the reporter. The ABI Prism 7700 instrument reads each well every few seconds and measures the increase in fluorescence, the increase in fluorescence of the reporter being a direct consequence of the target amplification during PCR. The real-time results are reported as threshold cycle (Ct) values, the cycle at which fluorescence readings exceed the mean baseline readings by 10 standard deviations. The fewer cycles it takes to reach a detectable level of fluorescence, the greater the initial copy number (Bustin, 2000). Radioimmunoassay Plasma and tissue melatonin was measured radioimmunologically (Vakkuri et al., 1984). Tissue samples were homogenized in a Tricine-buffer (0.1 M, pH 7.8). Plasma samples of 0.4-1.0 ml and tissue samples of 1.0 ml were extracted with 4.0 ml of chloroform. The chloroform phase was washed with 2.0 ml of milli-Q water, dried overnight under vacuum and diluted into a sodium phosphate buffer (pH 6.0) for the MT-RIA. In brief, duplicate samples of 100 Al were incubated with melatonin antiserum and 2[125I]-iodomelatonin at +48C overnight and the immunocomplex was precipitated with 2.5 M ammoniumsulfate.
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The melatonin RIA was originally validated for cross-reactivity, sensitivity, intra- and interassay coefficients in the melatonin RIA paper (Vakkuri et al., 1984). Further validity of the present melatonin measurements are based on previous parallelism and HPLC studies carried out in connection of corresponding tissue extractions and melatonin assays (Vakkuri et al., 1985). Statistical analysis Student’s t-test was used to analyze the results statistically.
Results The messenger RNA expression of the MT1 melatonin receptor Fig. 1 shows the relative appearance of the MT1 melatonin receptor mRNA in different tissues at noon and midnight. If the baseline fluorescence was not exceeded during the 40-cycle program, the expression was regarded as being below the detection limit (in the figure a ratio value below 0.5). Of the rat tissues studied by the real-time quantitative RT-PCR method, retina, hypothalamus and small intestine showed notable MT1 receptor mRNA expressions. During the PCR-program the fluorescence of the heart, Harderian gland and liver tissues did not exceed the baseline significantly. Interestingly, the hypothalamic tissue showed a significant difference in the expression of the MT1 receptor mRNA between day and night (pb0.05), the expression being absent during the night. In retina and small intestine there were no changes in the expressions between day and night.
Fig. 1. The mean relative values of the MT1 melatonin receptor mRNA in studied tissues at noon (white bars) and at midnight (black bars). Each mean column and error bar (FS.E.) represents 6 to 8 rats. Signals that did not differ from the background were defined as not detected and were given the value 0. Values that were more than 2*SD away from the mean were left out. One retina sample, collected at midnight, was used as an internal control and it was given the ratio value 1.0. Tissues with no detectable MT1 mRNA expression were defined as those with a ratio value below 0.5. *pb0.05.
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Fig. 2. The mean relative values of the MT2 melatonin receptor mRNA in studied tissues at noon (white bars) and midnight (black bars). Each mean column and error bar (FS.E.) represents 5 to 8 rats. Signals that did not differ from the background were defined as not detected and were given the value 0. Values that were more than 2*SD away from the mean were left out. One retina sample, collected at midnight, was used as an internal control and it was given the ratio value 1.0. Tissues with no detectable MT2 mRNA expression were defined as those with a ratio value below 0.2.
The messenger RNA expression of the MT2 melatonin receptor The relative appearance of the MT2 melatonin receptor mRNA in different tissues at noon and midnight is shown in Fig. 2. Signals which did not exceed the baseline fluorescence during the PCRprogram were defined as not detected (in the figure a ratio value below 0.2). A clearly detectable MT2 melatonin receptor mRNA expression was observed in the retina, the hypothalamus and the small intestine using the real-time quantitative RT-PCR method. In the heart SA node and the liver the values of the relative appearance were hardly detectable, so the expression of possible MT2 melatonin receptors was very low. During the PCR-program the fluorescence of the heart apex and the Harderian gland tissues did not exceed the baseline significantly. There was only slight variation in the expression of the MT2 melatonin receptor mRNA between day and night samples in the studied tissues. The differences were not statistically significant in any of the tissues. Because of the difference between pools, the Table 1 The concentration of melatonin (mean F S.E.) at noon and midnight in different tissues of rat Tissue
n (noon) Concentration of melatonin at noon (pg/g) n (night) Concentration of melatonin at night (pg/g)
Heart (apex) Heart (SA node) Retina Hypothalamus Harderian gland Liver Small intestine Plasma
5 8 4 3 3 4 5 8
1362 4493 2381 1854 2476 4323 2570 20
* Relative to noon, pb0.05. ** Relative to noon, pb0.01.
F F F F F F F F
236 484 655 489 1433 1546 458 3 pg/ml
7 8 5 6 4 5 6 8
3941 5051 5151 2168 2803 5482 8719 78
F 1039* F 342 F 1076 F 949 F 829 F 1680 F 1701* F 15 pg/ml**
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relative values of the MT1 and MT2 melatonin receptor mRNA appearances are not directly comparable to each other. Concentration of melatonin in studied tissues Table 1 shows the concentration of melatonin in different tissues of rat at noon and midnight. In the heart apex and the small intestine the concentration of melatonin was significantly (pb0.05) higher at night than at noon. In other tissues, the melatonin level also tended to be higher at midnight than at noon, but no significant differences were observed. The plasma melatonin concentration at midnight was almost 4 times greater than at noon (pb0.01). The highest melatonin concentration (8 719 F 1 701 pg/g) of all tissues was found in the small intestine at night.
Discussion In this study, for the first time, we show the expression of both MT1 and MT2 melatonin receptor mRNA in the rat hypothalamus, retina and small intestine using a real-time quantitative RT-PCR method. In addition, we show a low expression of MT2 melatonin receptor mRNA in the rat liver and heart SA node. These findings suggest that at least some effects of melatonin could be mediated through the membrane MT1 and MT2 melatonin receptors in these tissues. As usual for melatonin receptors, the values of the relative appearance of mRNA were low. In the heart apex and Harderian gland the appearance of both receptor mRNAs was under the detection limit, so melatonin probably affects these tissues by means of a mechanism that does not include receptors. We also observed the difference between midnight and noon in the expression of the MT1 melatonin receptor mRNA in the hypothalamus. The melatonin concentration of studied tissues and plasma was higher at midnight than at noon, as expected. Tissue melatonin concentrations were clearly higher compared to melatonin concentration in plasma. This is not uncommon when measuring melatonin levels (Reiter and Tan, 2003). It could be assumed that lipophilic melatonin remains longer in tissues because they contain more lipids than plasma. The local synthesis of melatonin in some tissues and the rapidity of its use as a free radical scavenger in the cells also contribute to the concentration of melatonin. Of the studied tissues, the MT1 melatonin receptor mRNA was expressed most abundantly in the hypothalamus. This was expected because previous reports show that the suprachiasmatic nucleus (SCN) of the hypothalamus is a major site of 2-[125I]-iodomelatonin binding in the rodent brain (McArthur et al., 1997). Our result is also consistent with the findings of Sugden et al. (1999) and Poirel et al. (2003) who detected the MT1 melatonin receptor mRNA using a traditional RT-PCR method. We observed that the hypothalamic MT1 melatonin receptor mRNA expression was significantly higher at noon than at midnight (pb0.05). One reason for this may be down-regulation of receptors because the concentration of melatonin in the hypothalamus tended to be higher at midnight than at noon. This hypothesis supports previous findings that melatonin seems to affect the density of its own receptors (Gauer et al., 1993a,b). In addition Neu and Niles (1997) found a marked diurnal rhythm of melatonin MT1 receptor mRNA expression in the SCN; levels remained low during dark period but rose and then fell during 6 hour period in the middle of the light period. On the other hand, Sugden et al. (1999) did not find a significant rhythm in the expression of the MT1 receptor mRNA in the rat SCN. Due to these
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conflicting results further studies are needed to find out whether melatonin really regulates the expression of its own receptor mRNA or not. It has been also suggested that the transcription of the MT1 receptor gene may be regulated by the photoperiod and by the circadian clock through clock gene proteins (Witt-Enderby et al., 2003). Our results and the earlier report of Sugden et al. (1999) show that the MT2 receptor mRNA is also expressed in the rat hypothalamus. The SCN controls the synthesis of melatonin and according to Hunt et al. (2001) melatonin feeds back on the SCN to mediate phase advances via activation of MT2 melatonin receptor signaling. Probably some daily and seasonal effects of melatonin are also mediated through MT1 receptors given that the Siberian hamster (Phodopus sungorus) shows seasonal and circadian responses to melatonin even though its MT2 receptor gene cannot encode a functional receptor (Weaver et al., 1996). We showed that both MT1 and MT2 melatonin receptor mRNAs were expressed in the retina. Even though no earlier findings of the MT2 melatonin receptor mRNA in the rat retina have been reported, the result was expected because MT2 receptors are mainly found in the retina of other mammals (Reppert et al., 1995). In our study the MT2 melatonin receptor was also expressed most in the retina. The physiological role of melatonin in the retina is not fully understood, but probably the indoleamine mediates different effects of light and dark through its receptors (Fujieda et al., 1999). The Harderian gland is known to synthesize and release melatonin. Possibly melatonin has a local role in the tissue, because it has been shown that Harderectomy in the Syrian hamster does not modify daytime melatonin levels in blood (Djeridane et al., 1998). These findings give reason to expect that melatonin receptors are found in the Harderian gland. In our study we did not observe any significant expression of either the MT1 or MT2 melatonin receptor mRNA, although Poirel et al. (2003) detected the MT1 melatonin receptor mRNA in the Harderian gland. However, they used a different RT-PCR method compared to us and this might be the reason for the differences in the results. Also the difference in the time when samples where taken may affect the results. Even though the expression of either melatonin receptor mRNA was not significant at midnight or noon, these mRNA levels may reach a significant expression some other time during the 24 hour period. Lopez-Gonzalez et al. (1991) characterized melatonin-binding sites in the Harderian gland using 2-[125I]-iodomelatonin. Nevertheless, it has been reported that not all 2-[125I]-iodomelatonin binding sites are necessarily actual melatonin receptors. Whether those 2-[125I]-iodomelatonin binding sites represent melatonin receptors or not remains unsolved. It is also possible that melatonin functions in the Harderian gland without receptors. Moreover, a lipophilicity of this indoleamine permits its transfer through biological membranes, and the effects of melatonin have also been found to be mediated through its antioxidant action in the Harderian gland (Djeridane and Touitou, 2001). Furthermore, it has been suggested that melatonin, synthesized in the Harderian gland, could be a precursor of other biologically active compounds, for example, 5methoxytryptophol (Djeridane et al., 1998). In the heart the relative appearance of the MT1 receptor mRNA was under the detection limit at midnight and noon, so there is no proof that MT1 melatonin receptors are synthesized there. The expression of the MT2 melatonin receptor mRNA was hardly detectable, so we can assume that the synthesis of possible membrane MT2 receptors in the heart is very low at midnight and noon. This suggests that the known effects of melatonin in the heart are mainly mediated by a different mechanism, for example, through its antioxidant action. This is consistent with previously reported results which show that melatonin protects the heart sarcolemmal membrane function probably by means of its antioxidant capacity (Chen et al., 1994). Our heart results are inconsistent with those of Poirel et al. (2003) who recently reported that the heart expressed very high levels of MT1 mRNA. The reasons for
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the differences in the results published by Poirel et al. (2003) and the results of this present investigation might be attributable to methodological differences in the RT-PCR technique. There could also have been differences in the preparation of the heart samples and in the times when samples were taken. It is possible that MT1 and MT2 melatonin receptor mRNA expression reaches the detection limit sometimes during the 24 hour period and in this case melatonin may act also through its membrane receptors in the heart. Possible MT2 melatonin receptors of the small intestine have not been investigated until now with molecular biological methods. Poon et al. (1997) found 2-[125I]-iodomelatonin binding sites in the gastrointestinal tract of many mammals and birds. Poirel et al. (2003) reported an intestinal MT1 mRNA expression. We also showed the expression of MT1 receptor mRNA and in addition to this we found MT2 melatonin receptor mRNA, so it is probable that previously found 2-[125I]-iodomelatonin binding sites represented MT1 and MT2 melatonin receptors. It has been suggested that through the receptors melatonin protects the epithelium of the gastrointestinal tract, possibly stimulates the synthesis of other gastroprotective hormones and also enhances the submucosal blood flow (Motilva et al., 2001). In addition to receptor-mediated actions, it has been shown that melatonin can protect gastrointestinal mucosa from ulceration due to its antioxidant action (Bubenik, 2001). We did not observe any significant difference in the expression of either melatonin receptor subtype mRNA in the small intestine between midnight and noon. This result might indicate that instead of the photoperiod, the expression of melatonin receptors in the small intestine may be controlled by some other factors. It has been reported that the level of gastrointestinal melatonin seems to be elevated after food intake and also after long-term food deprivation (Bubenik, 2001). It would be interesting to investigate if these factors also influence the expression of melatonin receptors. Our results show that the concentration of melatonin in the small intestine was significantly (pb0.05) higher at midnight than at noon. This is in accordance with findings which demonstrate that in the gastrointestinal tract of birds (Saarela et al., 1999) a nd rodents there appears to be a diurnal rhythm of melatonin, with high levels in the dark period (Lee and Pang, 1993). These differences are probably caused by fluctuation of melatonin synthesis in the gastrointestinal tract, because according to Bubenik and Brown (1997) pinealectomy does not influence melatonin levels in the gastrointestinal tract of rats. It would be interesting to discover why melatonin synthesis in the intestine seems to respond to the photoperiod and what the physiological significance of this is. We found that there is a low level of MT2 melatonin receptor mRNA expression in the rat liver, but no evident expression of MT1 receptor mRNA. This differs from the results of Poirel et al. (2003) who reported the MT1 melatonin receptor mRNA expression in the rat liver. The reason for this might be the methodological differences in the RT-PCR technique. However, our results are consistent with the findings of Poon et al. (2001) who reported that MT2 receptors are found in the mouse liver. They suggested that melatonin may act directly on the liver to elevate the plasma glucose level, and in turn the changes in the plasma glucose level may affect hepatic melatonin binding. Melatonin has also been shown to influence the plasma insulin levels (Rodriguez et al., 1989). Probably the abovementioned effects of melatonin are mediated through MT2 melatonin receptors. We did not observe any significant difference in the expression of the liver MT2 melatonin receptor mRNA between noon and midnight. This indicates that the photoperiod does not control the expression of melatonin receptors in the rat liver and probably the fluctuation of the plasma melatonin level does not control it either. It would be interesting to investigate if nourishment has any effect on the expression of MT2 receptors in the liver. In addition to receptors, melatonin probably mediates some of its effects in the liver through its antioxidant action. For example, Ohta et al. (2000) found that melatonin protects the
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liver in the rat against carbon tetrachloride-induced acute liver injury possibly through its antioxidant action. In our study we used a real-time quantitative RT-PCR technique. This method significantly simplifies and accelerates the process of quantification of mRNAs. Also real-time RT-PCR assays are readily standardized, so it is easier and more reliable to compare results of different laboratories (Bustin, 2000). For example, Wang and Brown (1999) and Winer et al. (1999) have shown this method to be accurate and reliable.
Conclusion We found the expression of the MT1 melatonin receptor mRNA in hypothalamus, retina and small intestine using a real-time quantitative RT-PCR. We also showed the expression of the MT2 melatonin receptor mRNA in hypothalamus, retina, small intestine, heart SA node and liver. These findings suggest the expression of the membrane MT1 and MT2 melatonin receptor proteins in these tissues. Therefore at least some effects of melatonin are probably mediated through the membrane MT1 and MT2 melatonin receptors in these tissues. We could not find any detectable expression of the melatonin receptor mRNA in the heart apex and the Harderian gland at midnight and noon, so according to this study there is no proof that the melatonin receptors are synthesized in these tissues.
Acknowledgements We are grateful to Ms. Marja-Liisa Martimo, Ms. Tuula Taskinen and Ms. Helka Koisti for their technical assistance, and to lecturer Ian Morris-Wilson for revising the English of this paper. This study was supported by the Academy of Finland (Grant Nr. 102 286). References Antolin, I., Rodriguez, C., Sainz, R.M., Mayo, J.C., Uria, H., Kotler, M.L., Rodriguez-Colunga, M.J., Tolivia, D., MenendezPelaez, A., 1996. Neurohormone melatonin prevents cell damage: effect on gene expression for antioxidant enzymes. Faseb Journal 10 (8), 882 – 890. Aust, S., Thalhammer, T., Humpeler, S., Jager, W., Klimpfinger, M., Tucek, G., Obrist, P., Marktl, W., Penner, E., Ekmekcioglu, C., 2004. The melatonin receptor subtype MT1 is expressed in human gallbladder epithelia. Journal of Pineal Research 36 (1), 43 – 48. Bubenik, G.A., 2001. Localization, physiological significance and possible clinical implication of gastrointestinal melatonin. Biological Signals and Receptors 10 (6), 350 – 366. Bubenik, G.A., Brown, G.M., 1997. Pinealectomy reduces melatonin levels in the serum but not in the gastrointestinal tract of rats. Biological Signals 6 (1), 40 – 44. Bustin, S.A., 2000. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. Journal of Molecular Endocrinology 25 (2), 169 – 193. Chen, L.D., Kumar, P., Reiter, R.J., Tan, D.X., Manchester, L.C., Chambers, J.P., Poeggeler, B., Saarela, S., 1994. Melatonin prevents the suppression of cardiac Ca2+-stimulated ATPase activity induced by alloxan. American Journal of Physiology 267 (1), E57 – E62. Chucharoen, P., Chetsawang, B., Srikiatkhachorn, A., Govitrapong, P., 2003. Melatonin receptor expression in rat cerebral artery. Neuroscience Letters 341 (3), 259 – 261.
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Poon, A.M.S., Choy, E.H.Y., Pang, S.F., 2001. Modulation of blood glucose by melatonin: A direct action on melatonin receptors in mouse hepatocytes. Biological Signals and Receptors 10 (6), 367 – 379. Reiter, R.J., 1991. Melatonin: That ubiquitously acting pineal hormone. News in Physiological Sciences 6 (5), 223 – 227. Reiter, R.J., Tan, D.X., 2003. What constitutes a physiological concentration of melatonin? Journal of Pineal Research 34 (1), 79 – 80. Reppert, S.M., Godson, C., Mahle, C.D., Weaver, D.R., Slaugenhaupt, S.A., Gusella, J.F., 1995. Molecular characterization of a second melatonin receptor expressed in human retina and brain: The Mel1b melatonin receptor. Proceedings of the National Academy of Sciences 92 (19), 8734 – 8738. Reppert, S.M., Weaver, D.R., Godson, C., 1996. Melatonin receptors step into the light: cloning and classification of subtypes. Trends in Pharmacological Sciences 17 (3), 100 – 102. Rodriguez, V., Mellado, C., Alvarez, E., De Diego, J.G., Blazquez, E., 1989. Effect of pinealectomy on liver insulin and glucagons receptor concentrations in the rat. Journal of Pineal Research 6 (1), 77 – 88. Saarela, S., Vuori, M., Eloranta, E., Vakkuri, O., 1999. Melatonin, a candidate signaling molecule for energy sparing. Ornis Fennica 76, 231 – 235. Sahna, E., Acet, A., Ozer, M.K., Olmez, E., 2002a. Myocardial ischemia-reperfusion in rats: reduction of infarct size by either supplemental physiological or pharmacological doses of melatonin. Journal of Pineal Research 33 (4), 234 – 238. Sahna, E., Olmez, E., Acet, A., 2002b. Effects of physiological and pharmacological concentrations of melatonin on ischemiareperfusion arrhythmias in rats: can the incidence of sudden cardiac death be reduced? Journal of Pineal Research 32 (3), 194 – 198. Sjfblom, M., Flemstrfm, G., 2003. Melatonin in the duodenal lumen is a potent stimulant of mucosal bicarbonate secretion. Journal of Pineal Research 34 (4), 288 – 293. Sugden, D., McArthur, A.J., Ajpru, S., Duniec, K., Piggins, H.D., 1999. Expression of mt1 melatonin receptor subtype mRNA in the entrained rat suprachiasmatic nucleus: a quantitative RT-PCR study across the diurnal cycle. Molecular Brain Research 72 (2), 176 – 182. Szarszoi, O., Asemu, G., Vanecek, J., Ostadal, B., Kolar, F., 2001. Effects of melatonin on ischemia and reperfusion injury of the rat heart. Cardiovascular Drugs and Therapy 15 (3), 251 – 257. Tan, D.X., Chen, L.D., Poeggeler, B., Manchester, L.C., Reiter, R.J., 1993. Melatonin: A potent, endogenous hydroxyl scavenger. Endocrine Journal 1, 57 – 60. Tan, D.X., Manchester, L.C., Reiter, R.J., Qi, W., Kim, S.J., El-Sokkary, G.H., 1998. Ischemia/reperfusion-induced arrhythmias in the isolated rat heart: prevention by melatonin. Journal of Pineal Research 25, 184 – 191. Ting, K.N., Blaylock, N.A., Sugden, D., Delagrange, P., Scalbert, E., Wilson, V.G., 1999. Molecular and pharmacological evidence for MT1 melatonin receptor subtype in the tail artery of juvenile Wistar rats. British Journal of Pharmacology 127 (4), 987 – 995. Vakkuri, O., Lepp7luoto, J., Vuolteenaho, O., 1984. Development and validation of a melatonin radioimmunoassay using radioiodinated melatonin as tracer. Acta Endocrinology 106, 152 – 157. Vakkuri, O., Rintam7ki, H., Lepp7luoto, J., 1985. Presence of immunoreactive melatonin in different tissues of the pigeon (Columba livia). General and Comparative Endocrinology 58, 69 – 75. Wang, T., Brown, M.J., 1999. mRNA quantification by real time TaqMan polymerase chain reaction: validation and comparison with RNase protection. Analytical Biochemistry 269 (1), 198 – 201. Weaver, D.R., Liu, C., Reppert, S.M., 1996. Nature’s knockout: the Mel1b receptor is not necessary for reproductive and circadian responses to melatonin in Siberian hamsters. Molecular Endocrinology 10 (11), 1478 – 1487. Wiechmann, A.F., Yang, X.L., Wu, S.M., Hollyfield, J.G., 1988. Melatonin enhances horizontal cell sensitivity in salamander retina. Brain Research 453 (1-2), 377 – 380. Winer, J., Jung, C.K.S., Shackel, I., Williams, P.M., 1999. Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Analytical Biochemistry 270 (1), 41 – 49. Witt-Enderby, P.A., Bennett, J., Jarzynka, M.J., Firestine, S., Melan, M.A., 2003. Melatonin receptors and their regulation: biochemical and structural mechanisms. Life Sciences 72 (20), 2183 – 2198. Woo, M.M.M., Tai, C.J., Kang, S.K., Nathwani, P.S., Pang, S.F., Leung, P.C.K., 2001. Direct action of melatonin in human granulosa-luteal cells. Journal of Clinical Endocrinology and Metabolism 86 (10), 4789 – 4797. Zhao, H., Poon, A.M.S., Pang, S.F., 2000. Pharmacological characterization, molecular subtyping, and autoradiographic localization of putative melatonin receptors in uterine endometrium of estrous rats. Life Sciences 66 (17), 1581 – 1591.
The expression of MT1 and MT2 melatonin receptor mRNA in several rat tissues Pirkko Sallinena,*, Seppo Saarelaa, Mika Ilvesb, Olli Vakkurib, Juhani Lepp7luotob a
Department of Biology, P.O. Box 3000, 90014 University of Oulu, Finland b Department of Physiology, University of Oulu, Oulu, Finland Received 14 May 2004; accepted 16 August 2004
Abstract The mechanisms that mediate the various effects of melatonin in mammalian tissues are not always known. Therefore, the aim of this study was to investigate whether MT1 and MT2 melatonin receptors are expressed in certain tissues of the rat. The expression of MT1 and MT2 melatonin receptor mRNA was determined using a realtime quantitative RT-PCR method. In addition, we examined whether mRNA for either subtype of receptor shows any difference in the expression between midnight and noon, similar to the changes in melatonin concentrations in plasma and tissue samples. MT1 and MT2 melatonin receptor mRNAs were found in the rat hypothalamus, retina and small intestine. We also showed a low expression of MT2 mRNA in the rat liver and heart SA node. In the heart apex and the Harderian gland, no appearance of either of the receptor mRNAs was detectable. A significant difference in the expression of MT1 mRNA between day and night was found in the hypothalamus. In conclusion, our findings suggest that at least some effects of melatonin are mediated through membrane MT1 and MT2 receptors in the hypothalamus, the retina and the small intestine. Down-regulation of receptors might be one reason for the difference in the hypothalamic MT1 melatonin receptor mRNA expression between midnight and noon. In the liver and the heart SA node, the physiological significance of possible MT2 receptors remains unclear. According to our negative midnight and noon results in the Harderian gland and heart apex melatonin may exert its effect on these tissues by a non-receptor mechanism. D 2004 Elsevier Inc. All rights reserved. Keywords: Melatonin receptor; MT1 mRNA; MT2 mRNA; Real-Time Quantitative RT-PCR; Melatonin; Rat
* Corresponding author. Tel.: +358 8 5531240; fax: +358 8 5531061. E-mail address: [email protected] (P. Sallinen). 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.08.016
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Introduction Melatonin, an indole-derived neurosecretory product, is synthesized in the pineal gland and secreted into the blood in a circadian manner. However, melatonin is also synthesized in the retina, the Harderian gland and the gastrointestinal tract (Reiter, 1991). This indoleamine is known to be an important regulator of daily and seasonal behavior and physiology as well as a highly efficient antioxidant scavenger of, for example, the hydroxyl radical (Tan et al., 1993). Melatonin is also involved in many physiological functions depending on the circadian rhythm, such as the immune and the cardiovascular systems. The lipophilicity of melatonin permits its transfer through biological membranes, and therefore the indoleamine is found throughout the organism in all cell and cellular organelles including the nucleus (Menendez-Pelaez and Reiter, 1993). It has also been reported that melatonin has an impact on the function of many different tissues. For example, in the retina, it is suggested that melatonin acts as a paracrine signal of darkness and enhances the sensitivity of horizontal cells (Wiechmann et al., 1988). Antolin et al. (1996) have observed that in the Harderian gland melatonin prevents cell damage induced by free radicals generated by porphyrin metabolism. Melatonin also has an effect on the circadian rhythms of the neuronal firing rate in the suprachiasmatic nucleus (SCN) (Hunt et al., 2001). In addition, several reports have recently demonstrated that in the heart this indoleamine has beneficial effects on postischemic myocardial dysfunction (Kaneko et al., 2000; Lagneux et al., 2000; Lee et al., 2002; Sahna et al., 2002a,b; Szarszoi et al., 2001; Tan et al., 1998). Furthermore, it has been shown that melatonin affects the function of the liver and the gastrointestinal tract. Melatonin elevates the plasma glucose level probably by acting directly on the liver (Poon et al., 2001). Ohta et al. (2000) have found that melatonin exerts a therapeutic effect on carbon tetrachlorideinduced acute liver injury in rats. Because melatonin is also synthesized in the gastrointestinal tract, it has been suggested that melatonin is probably a paracrine agent that protects the epithelium of the gastrointestinal tract and stimulates the synthesis of other gastroprotective hormones (Motilva et al., 2001). It has also been shown that melatonin is involved in the motility changes of the small intestine especially at night (Merle et al., 2000), it may also synchronize the sequential digestive processes (Bubenik, 2001), and melatonin is involved in the control of bicarbonate secretion by the duodenal epithelium (Sjo¨blom and Flemstro¨m, 2003). Even though many studies indicate that melatonin has an impact on the function of many different tissues, it is not always clear how the actions of melatonin are mediated. It is possible that melatonin owes its influence to its antioxidant action, and its effects may also be mediated via cell membrane receptors which belong to the family of G-protein coupled receptors (MT1 and MT2) and the quinone reductase enzymes (MT3). Two of these receptor subtypes (MT1 and MT2) have been found in mammals (Reppert et al., 1996). Traditionally, melatonin receptors have been visualized using radioactive 2-[125I]-iodomelatonin (125I-Mel), and melatonin receptor messenger RNA levels have been quantified by Northern blot analysis. However, a more sensitive method is to use a real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) (Bustin, 2000), but because it is a fairly new method, the melatonin receptors’ mRNA levels of rat tissues have not previously been measured quantitatively using this method. In some tissues of rat melatonin receptor mRNAs have been detected using traditional RT-PCR, which is merely a semi-quantitative method related to a real-time quantitative RT-PCR-method. The MT1 melatonin receptor mRNA has been identified, for example, in the SCN (Sugden et al., 1999), and in other regions of the brain as well as in some peripheral tissues (Aust et al., 2004; Chucharoen et al., 2003; Fujieda et al., 1999; Li et al., 1998; Peschke et al., 2000; Poirel et al.,
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2003; Ting et al., 1999; Woo et al., 2001; Zhao et al., 2000) using a classical RT-PCR method. The MT2 melatonin receptor mRNA has been measured in the rat SCN (Sugden et al., 1999), in the human ovary (Woo et al., 2001), in the corpus epididymis (Li et al., 1998) and in the caudal artery of the rat (Masana et al., 2002). The above-mentioned findings raise the question about the mechanism which mediates the action of melatonin in certain tissues. Therefore, the aim of the present study was to investigate the expression of MT1- and MT2-receptors in rat hypothalamus, the retina, the Harderian gland, the small intestine, the heart and the liver. The real-time quantitative RT-PCR method was used to determine the expression of MT1 and MT2 melatonin receptor mRNA. The mRNA for the MT1 or MT2 subtype of melatonin receptor was examined for signs of a difference in the expression between midnight and noon. In addition, radioimmunoassay (RIA) was used to measure the concentration of melatonin at midnight and noon in plasma and in the studied tissues.
Materials and methods Animals and tissue preparations Sixteen adult male and female Sprague-Dawley rats (Rattus norvegicus), obtained from the Laboratory Animal Centre, University of Oulu, were used in the experiment. The body mass of the rats ranged from 372 to 437 g. The animals received food and water ad libitum and were maintained at constant temperature (+21F18C) and under a 12:12 h light:dark cycle (lights on at 08:00 h a.m.). The experimental protocol was approved by the Animal Experimentation Committee of the University of Oulu (Licence Nr 074/01). Half of the rats were killed at midnight (24.00 h, n=8) and the others were killed at noon (12.00 h, n=8). Animals were anaesthetized with carbon dioxide and blood samples were taken with heparinized syringes from the heart. After this, the animals were decapitated and tissue samples from the heart (apex and sinoatrial node), hypothalamus (including the SCN), Harderian gland, the eye (including the retina), small intestine and liver were collected. Tissue samples were immediately frozen in liquid nitrogen, and stored at -708C until RNA isolation was performed. Blood samples were centrifuged (3,000 g for 10 min) and plasma samples were stored at -708C until assayed for melatonin. For the rats killed at midnight, the samples were collected in the dark under dim red lighting (Philips PF 712E, Holland). Total RNA extraction and reverse transcription Total RNA was extracted from the tissue samples using an RNA isolation kit (QuickPrep Total RNA Extraction Kit, Amersham Pharmacia Biotech, USA) according to the manufacturer’s instructions. The amount of RNA was determined spectrophotometrically at 260 nm. Total RNA was converted into cDNA using a First Strand cDNA Synthesis Kit (#K1612, MBI Fermentas, Lithuania). Deionized, nuclear free water was added to the total RNA (0.5 Ag) to get a volume of 5.0 Al. After this random hexamer primer (0.1 Ag) was added and the mixture was heated to 708C for 5 min, then cooled immediately on ice. A ribonuclease inhibitor (10U), 5X reaction buffer (2 Al), 1.0 Al of 10 mM dNTP mix (dATP, dGTP, dCTP anp dTTP) and a MuLV-reverse transcriptase (20U) were added to make the final volume 10 Al. The reaction mixture was preincubated for 10 min at room temperature (+228C)
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before cDNA synthesis. Reverse transcription (RT) reactions were carried out for 60 min at +378C and then samples were heated to +708C for 10 min to terminate the RT reaction. Polymerase chain reaction Real time quantitative PCR was performed using a TaqManR 1000 Rxn Gold/Buffer Pack reagent Kit (Applied Biosystems, Foster City, CA, USA) and an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). TaqManR probes and primers were designed with the aid of the Primer Express program (Applied Biosystems, Foster City, CA, USA) from the previously reported partial MT1 (Poirel et al., 1999a) and MT2 (Poirel et al., 1999b) melatonin receptor sequences of rats. Probes and primers were obtained from Applied Biosystems UK (Cheshire). For the MT1 receptor the synthesized amplicon was 66 bp and the sequence of the probe and primers were as follows: probe, 5VTCCTGTACCTTCACCCAGTCCGTCAGC-3V (bases 516-542); forward primer, 5V-CAGTACGACCCCCGGATCTA-3V (bases 495-514); reverse primer 5V-GGCAATCGTGTACGCCG-3V (bases 544-560). For the MT2 receptor the synthesized amplicon was 65 bp and the sequence of the probe and primers were as follows: probe, 5V-CGCCATATGCTGGGCCCCC-3V(bases 366-384); forward primer 5VATGTTCGCAGTGTTTGTGGTTT-3V (bases 343-364); reverse primer 5V-ACTGCAAGGCCAATACAGTTGA-3V(bases 386-407). Ribosomal protein gene 18S rRNA was used as an endogenous control and the sequences of the probe and primers for the 18S rRNA were as follows: probe 5VCCTGGTGGTGCCCTTCCGTCA-3V; forward primer 5V-TGGTTGCAAAGCTGAAACTTAAAG-3V; reverse primer 5V-AGTCAAATTAAGCCGCAGGC-3V. Each reaction contained 5 Al RT reaction product as template DNA and 20 Al reaction mixture, which consisted of milli-Q water, MgCl2 (25.0 mM), reaction buffer (10X TaqManR Buffer A), dNTP mix (2.5 mM each), primers (3.0 AM), probe (5.0 AM) and AmpliTag Gold polymerase (5U/Al). The template was initially denaturated for 10 min at +958C followed by a 40-cycle program with 15 s at +958C, 1 min annealing and extension at +608C. The TaqMan probe consists of an oligonucleotide with a 5V-fluorescent reporter dye and a 3V-quencher dye. When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence. During PCR the reporter dye and quencher dye are separated, resulting in increased fluorescence of the reporter. The ABI Prism 7700 instrument reads each well every few seconds and measures the increase in fluorescence, the increase in fluorescence of the reporter being a direct consequence of the target amplification during PCR. The real-time results are reported as threshold cycle (Ct) values, the cycle at which fluorescence readings exceed the mean baseline readings by 10 standard deviations. The fewer cycles it takes to reach a detectable level of fluorescence, the greater the initial copy number (Bustin, 2000). Radioimmunoassay Plasma and tissue melatonin was measured radioimmunologically (Vakkuri et al., 1984). Tissue samples were homogenized in a Tricine-buffer (0.1 M, pH 7.8). Plasma samples of 0.4-1.0 ml and tissue samples of 1.0 ml were extracted with 4.0 ml of chloroform. The chloroform phase was washed with 2.0 ml of milli-Q water, dried overnight under vacuum and diluted into a sodium phosphate buffer (pH 6.0) for the MT-RIA. In brief, duplicate samples of 100 Al were incubated with melatonin antiserum and 2[125I]-iodomelatonin at +48C overnight and the immunocomplex was precipitated with 2.5 M ammoniumsulfate.
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The melatonin RIA was originally validated for cross-reactivity, sensitivity, intra- and interassay coefficients in the melatonin RIA paper (Vakkuri et al., 1984). Further validity of the present melatonin measurements are based on previous parallelism and HPLC studies carried out in connection of corresponding tissue extractions and melatonin assays (Vakkuri et al., 1985). Statistical analysis Student’s t-test was used to analyze the results statistically.
Results The messenger RNA expression of the MT1 melatonin receptor Fig. 1 shows the relative appearance of the MT1 melatonin receptor mRNA in different tissues at noon and midnight. If the baseline fluorescence was not exceeded during the 40-cycle program, the expression was regarded as being below the detection limit (in the figure a ratio value below 0.5). Of the rat tissues studied by the real-time quantitative RT-PCR method, retina, hypothalamus and small intestine showed notable MT1 receptor mRNA expressions. During the PCR-program the fluorescence of the heart, Harderian gland and liver tissues did not exceed the baseline significantly. Interestingly, the hypothalamic tissue showed a significant difference in the expression of the MT1 receptor mRNA between day and night (pb0.05), the expression being absent during the night. In retina and small intestine there were no changes in the expressions between day and night.
Fig. 1. The mean relative values of the MT1 melatonin receptor mRNA in studied tissues at noon (white bars) and at midnight (black bars). Each mean column and error bar (FS.E.) represents 6 to 8 rats. Signals that did not differ from the background were defined as not detected and were given the value 0. Values that were more than 2*SD away from the mean were left out. One retina sample, collected at midnight, was used as an internal control and it was given the ratio value 1.0. Tissues with no detectable MT1 mRNA expression were defined as those with a ratio value below 0.5. *pb0.05.
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Fig. 2. The mean relative values of the MT2 melatonin receptor mRNA in studied tissues at noon (white bars) and midnight (black bars). Each mean column and error bar (FS.E.) represents 5 to 8 rats. Signals that did not differ from the background were defined as not detected and were given the value 0. Values that were more than 2*SD away from the mean were left out. One retina sample, collected at midnight, was used as an internal control and it was given the ratio value 1.0. Tissues with no detectable MT2 mRNA expression were defined as those with a ratio value below 0.2.
The messenger RNA expression of the MT2 melatonin receptor The relative appearance of the MT2 melatonin receptor mRNA in different tissues at noon and midnight is shown in Fig. 2. Signals which did not exceed the baseline fluorescence during the PCRprogram were defined as not detected (in the figure a ratio value below 0.2). A clearly detectable MT2 melatonin receptor mRNA expression was observed in the retina, the hypothalamus and the small intestine using the real-time quantitative RT-PCR method. In the heart SA node and the liver the values of the relative appearance were hardly detectable, so the expression of possible MT2 melatonin receptors was very low. During the PCR-program the fluorescence of the heart apex and the Harderian gland tissues did not exceed the baseline significantly. There was only slight variation in the expression of the MT2 melatonin receptor mRNA between day and night samples in the studied tissues. The differences were not statistically significant in any of the tissues. Because of the difference between pools, the Table 1 The concentration of melatonin (mean F S.E.) at noon and midnight in different tissues of rat Tissue
n (noon) Concentration of melatonin at noon (pg/g) n (night) Concentration of melatonin at night (pg/g)
Heart (apex) Heart (SA node) Retina Hypothalamus Harderian gland Liver Small intestine Plasma
5 8 4 3 3 4 5 8
1362 4493 2381 1854 2476 4323 2570 20
* Relative to noon, pb0.05. ** Relative to noon, pb0.01.
F F F F F F F F
236 484 655 489 1433 1546 458 3 pg/ml
7 8 5 6 4 5 6 8
3941 5051 5151 2168 2803 5482 8719 78
F 1039* F 342 F 1076 F 949 F 829 F 1680 F 1701* F 15 pg/ml**
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relative values of the MT1 and MT2 melatonin receptor mRNA appearances are not directly comparable to each other. Concentration of melatonin in studied tissues Table 1 shows the concentration of melatonin in different tissues of rat at noon and midnight. In the heart apex and the small intestine the concentration of melatonin was significantly (pb0.05) higher at night than at noon. In other tissues, the melatonin level also tended to be higher at midnight than at noon, but no significant differences were observed. The plasma melatonin concentration at midnight was almost 4 times greater than at noon (pb0.01). The highest melatonin concentration (8 719 F 1 701 pg/g) of all tissues was found in the small intestine at night.
Discussion In this study, for the first time, we show the expression of both MT1 and MT2 melatonin receptor mRNA in the rat hypothalamus, retina and small intestine using a real-time quantitative RT-PCR method. In addition, we show a low expression of MT2 melatonin receptor mRNA in the rat liver and heart SA node. These findings suggest that at least some effects of melatonin could be mediated through the membrane MT1 and MT2 melatonin receptors in these tissues. As usual for melatonin receptors, the values of the relative appearance of mRNA were low. In the heart apex and Harderian gland the appearance of both receptor mRNAs was under the detection limit, so melatonin probably affects these tissues by means of a mechanism that does not include receptors. We also observed the difference between midnight and noon in the expression of the MT1 melatonin receptor mRNA in the hypothalamus. The melatonin concentration of studied tissues and plasma was higher at midnight than at noon, as expected. Tissue melatonin concentrations were clearly higher compared to melatonin concentration in plasma. This is not uncommon when measuring melatonin levels (Reiter and Tan, 2003). It could be assumed that lipophilic melatonin remains longer in tissues because they contain more lipids than plasma. The local synthesis of melatonin in some tissues and the rapidity of its use as a free radical scavenger in the cells also contribute to the concentration of melatonin. Of the studied tissues, the MT1 melatonin receptor mRNA was expressed most abundantly in the hypothalamus. This was expected because previous reports show that the suprachiasmatic nucleus (SCN) of the hypothalamus is a major site of 2-[125I]-iodomelatonin binding in the rodent brain (McArthur et al., 1997). Our result is also consistent with the findings of Sugden et al. (1999) and Poirel et al. (2003) who detected the MT1 melatonin receptor mRNA using a traditional RT-PCR method. We observed that the hypothalamic MT1 melatonin receptor mRNA expression was significantly higher at noon than at midnight (pb0.05). One reason for this may be down-regulation of receptors because the concentration of melatonin in the hypothalamus tended to be higher at midnight than at noon. This hypothesis supports previous findings that melatonin seems to affect the density of its own receptors (Gauer et al., 1993a,b). In addition Neu and Niles (1997) found a marked diurnal rhythm of melatonin MT1 receptor mRNA expression in the SCN; levels remained low during dark period but rose and then fell during 6 hour period in the middle of the light period. On the other hand, Sugden et al. (1999) did not find a significant rhythm in the expression of the MT1 receptor mRNA in the rat SCN. Due to these
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conflicting results further studies are needed to find out whether melatonin really regulates the expression of its own receptor mRNA or not. It has been also suggested that the transcription of the MT1 receptor gene may be regulated by the photoperiod and by the circadian clock through clock gene proteins (Witt-Enderby et al., 2003). Our results and the earlier report of Sugden et al. (1999) show that the MT2 receptor mRNA is also expressed in the rat hypothalamus. The SCN controls the synthesis of melatonin and according to Hunt et al. (2001) melatonin feeds back on the SCN to mediate phase advances via activation of MT2 melatonin receptor signaling. Probably some daily and seasonal effects of melatonin are also mediated through MT1 receptors given that the Siberian hamster (Phodopus sungorus) shows seasonal and circadian responses to melatonin even though its MT2 receptor gene cannot encode a functional receptor (Weaver et al., 1996). We showed that both MT1 and MT2 melatonin receptor mRNAs were expressed in the retina. Even though no earlier findings of the MT2 melatonin receptor mRNA in the rat retina have been reported, the result was expected because MT2 receptors are mainly found in the retina of other mammals (Reppert et al., 1995). In our study the MT2 melatonin receptor was also expressed most in the retina. The physiological role of melatonin in the retina is not fully understood, but probably the indoleamine mediates different effects of light and dark through its receptors (Fujieda et al., 1999). The Harderian gland is known to synthesize and release melatonin. Possibly melatonin has a local role in the tissue, because it has been shown that Harderectomy in the Syrian hamster does not modify daytime melatonin levels in blood (Djeridane et al., 1998). These findings give reason to expect that melatonin receptors are found in the Harderian gland. In our study we did not observe any significant expression of either the MT1 or MT2 melatonin receptor mRNA, although Poirel et al. (2003) detected the MT1 melatonin receptor mRNA in the Harderian gland. However, they used a different RT-PCR method compared to us and this might be the reason for the differences in the results. Also the difference in the time when samples where taken may affect the results. Even though the expression of either melatonin receptor mRNA was not significant at midnight or noon, these mRNA levels may reach a significant expression some other time during the 24 hour period. Lopez-Gonzalez et al. (1991) characterized melatonin-binding sites in the Harderian gland using 2-[125I]-iodomelatonin. Nevertheless, it has been reported that not all 2-[125I]-iodomelatonin binding sites are necessarily actual melatonin receptors. Whether those 2-[125I]-iodomelatonin binding sites represent melatonin receptors or not remains unsolved. It is also possible that melatonin functions in the Harderian gland without receptors. Moreover, a lipophilicity of this indoleamine permits its transfer through biological membranes, and the effects of melatonin have also been found to be mediated through its antioxidant action in the Harderian gland (Djeridane and Touitou, 2001). Furthermore, it has been suggested that melatonin, synthesized in the Harderian gland, could be a precursor of other biologically active compounds, for example, 5methoxytryptophol (Djeridane et al., 1998). In the heart the relative appearance of the MT1 receptor mRNA was under the detection limit at midnight and noon, so there is no proof that MT1 melatonin receptors are synthesized there. The expression of the MT2 melatonin receptor mRNA was hardly detectable, so we can assume that the synthesis of possible membrane MT2 receptors in the heart is very low at midnight and noon. This suggests that the known effects of melatonin in the heart are mainly mediated by a different mechanism, for example, through its antioxidant action. This is consistent with previously reported results which show that melatonin protects the heart sarcolemmal membrane function probably by means of its antioxidant capacity (Chen et al., 1994). Our heart results are inconsistent with those of Poirel et al. (2003) who recently reported that the heart expressed very high levels of MT1 mRNA. The reasons for
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the differences in the results published by Poirel et al. (2003) and the results of this present investigation might be attributable to methodological differences in the RT-PCR technique. There could also have been differences in the preparation of the heart samples and in the times when samples were taken. It is possible that MT1 and MT2 melatonin receptor mRNA expression reaches the detection limit sometimes during the 24 hour period and in this case melatonin may act also through its membrane receptors in the heart. Possible MT2 melatonin receptors of the small intestine have not been investigated until now with molecular biological methods. Poon et al. (1997) found 2-[125I]-iodomelatonin binding sites in the gastrointestinal tract of many mammals and birds. Poirel et al. (2003) reported an intestinal MT1 mRNA expression. We also showed the expression of MT1 receptor mRNA and in addition to this we found MT2 melatonin receptor mRNA, so it is probable that previously found 2-[125I]-iodomelatonin binding sites represented MT1 and MT2 melatonin receptors. It has been suggested that through the receptors melatonin protects the epithelium of the gastrointestinal tract, possibly stimulates the synthesis of other gastroprotective hormones and also enhances the submucosal blood flow (Motilva et al., 2001). In addition to receptor-mediated actions, it has been shown that melatonin can protect gastrointestinal mucosa from ulceration due to its antioxidant action (Bubenik, 2001). We did not observe any significant difference in the expression of either melatonin receptor subtype mRNA in the small intestine between midnight and noon. This result might indicate that instead of the photoperiod, the expression of melatonin receptors in the small intestine may be controlled by some other factors. It has been reported that the level of gastrointestinal melatonin seems to be elevated after food intake and also after long-term food deprivation (Bubenik, 2001). It would be interesting to investigate if these factors also influence the expression of melatonin receptors. Our results show that the concentration of melatonin in the small intestine was significantly (pb0.05) higher at midnight than at noon. This is in accordance with findings which demonstrate that in the gastrointestinal tract of birds (Saarela et al., 1999) a nd rodents there appears to be a diurnal rhythm of melatonin, with high levels in the dark period (Lee and Pang, 1993). These differences are probably caused by fluctuation of melatonin synthesis in the gastrointestinal tract, because according to Bubenik and Brown (1997) pinealectomy does not influence melatonin levels in the gastrointestinal tract of rats. It would be interesting to discover why melatonin synthesis in the intestine seems to respond to the photoperiod and what the physiological significance of this is. We found that there is a low level of MT2 melatonin receptor mRNA expression in the rat liver, but no evident expression of MT1 receptor mRNA. This differs from the results of Poirel et al. (2003) who reported the MT1 melatonin receptor mRNA expression in the rat liver. The reason for this might be the methodological differences in the RT-PCR technique. However, our results are consistent with the findings of Poon et al. (2001) who reported that MT2 receptors are found in the mouse liver. They suggested that melatonin may act directly on the liver to elevate the plasma glucose level, and in turn the changes in the plasma glucose level may affect hepatic melatonin binding. Melatonin has also been shown to influence the plasma insulin levels (Rodriguez et al., 1989). Probably the abovementioned effects of melatonin are mediated through MT2 melatonin receptors. We did not observe any significant difference in the expression of the liver MT2 melatonin receptor mRNA between noon and midnight. This indicates that the photoperiod does not control the expression of melatonin receptors in the rat liver and probably the fluctuation of the plasma melatonin level does not control it either. It would be interesting to investigate if nourishment has any effect on the expression of MT2 receptors in the liver. In addition to receptors, melatonin probably mediates some of its effects in the liver through its antioxidant action. For example, Ohta et al. (2000) found that melatonin protects the
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liver in the rat against carbon tetrachloride-induced acute liver injury possibly through its antioxidant action. In our study we used a real-time quantitative RT-PCR technique. This method significantly simplifies and accelerates the process of quantification of mRNAs. Also real-time RT-PCR assays are readily standardized, so it is easier and more reliable to compare results of different laboratories (Bustin, 2000). For example, Wang and Brown (1999) and Winer et al. (1999) have shown this method to be accurate and reliable.
Conclusion We found the expression of the MT1 melatonin receptor mRNA in hypothalamus, retina and small intestine using a real-time quantitative RT-PCR. We also showed the expression of the MT2 melatonin receptor mRNA in hypothalamus, retina, small intestine, heart SA node and liver. These findings suggest the expression of the membrane MT1 and MT2 melatonin receptor proteins in these tissues. Therefore at least some effects of melatonin are probably mediated through the membrane MT1 and MT2 melatonin receptors in these tissues. We could not find any detectable expression of the melatonin receptor mRNA in the heart apex and the Harderian gland at midnight and noon, so according to this study there is no proof that the melatonin receptors are synthesized in these tissues.
Acknowledgements We are grateful to Ms. Marja-Liisa Martimo, Ms. Tuula Taskinen and Ms. Helka Koisti for their technical assistance, and to lecturer Ian Morris-Wilson for revising the English of this paper. This study was supported by the Academy of Finland (Grant Nr. 102 286). References Antolin, I., Rodriguez, C., Sainz, R.M., Mayo, J.C., Uria, H., Kotler, M.L., Rodriguez-Colunga, M.J., Tolivia, D., MenendezPelaez, A., 1996. Neurohormone melatonin prevents cell damage: effect on gene expression for antioxidant enzymes. Faseb Journal 10 (8), 882 – 890. Aust, S., Thalhammer, T., Humpeler, S., Jager, W., Klimpfinger, M., Tucek, G., Obrist, P., Marktl, W., Penner, E., Ekmekcioglu, C., 2004. The melatonin receptor subtype MT1 is expressed in human gallbladder epithelia. Journal of Pineal Research 36 (1), 43 – 48. Bubenik, G.A., 2001. Localization, physiological significance and possible clinical implication of gastrointestinal melatonin. Biological Signals and Receptors 10 (6), 350 – 366. Bubenik, G.A., Brown, G.M., 1997. Pinealectomy reduces melatonin levels in the serum but not in the gastrointestinal tract of rats. Biological Signals 6 (1), 40 – 44. Bustin, S.A., 2000. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. Journal of Molecular Endocrinology 25 (2), 169 – 193. Chen, L.D., Kumar, P., Reiter, R.J., Tan, D.X., Manchester, L.C., Chambers, J.P., Poeggeler, B., Saarela, S., 1994. Melatonin prevents the suppression of cardiac Ca2+-stimulated ATPase activity induced by alloxan. American Journal of Physiology 267 (1), E57 – E62. Chucharoen, P., Chetsawang, B., Srikiatkhachorn, A., Govitrapong, P., 2003. Melatonin receptor expression in rat cerebral artery. Neuroscience Letters 341 (3), 259 – 261.
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