The Human Myometrium as a Target for Melatonin - Oxford Journals

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The Journal of Clinical Endocrinology & Metabolism 88(2):908 –913 Copyright © 2003 by The Endocrine Society doi: 10.1210/jc.2002-020449

The Human Myometrium as a Target for Melatonin ¨ LLER, N. SCHLABRITZ-LOUTSEVITCH, N. HELLNER, R. MIDDENDORF, D. MU

AND

J. OLCESE

Institute for Hormone and Fertility Research, University of Hamburg (N.S.-L., N.H., D.M., J.O.), and Institute of Anatomy, University of Hamburg Medical School (R.M.), 22529 Hamburg, Germany The circadian timing of spontaneous human deliveries results in births occurring statistically more often during the nocturnal phase of the 24-h cycle. The neuroendocrine mechanisms underlying this physiological phenomenon are not understood. In an effort to test the hypothesis that melatonin may serve as an endocrine signal for coordinating myometrial events in the human, we determined the mRNA expression of both MT1 and MT2 melatonin receptor isoforms in pregnant as well as nonpregnant myometrial biopsies by means of RT-PCR and in situ hybridiza-

tion histochemistry. Additionally, we could demonstrate specific, high affinity iodomelatonin binding to myometrial tissues of both pregnant and nonpregnant women. Primary cultures of myocytes responded differentially from melatonin in terms of cAMP signaling depending on the reproductive state. These results imply that melatonin may have the potential to modulate myometrial function in the human, a finding that could open up new possibilities for the development of novel therapeutic agents. (J Clin Endocrinol Metab 88: 908 –913, 2003)

T

HE UTERUS IS the central organ for the perpetuation of human life. An important feature of the uterus is the well defined 24-h rhythm of contractility and electrical and endocrine activities in rodents, primates, and humans (1; for review, see Ref. 2). A classic example of such circadian activity is the 24-h rhythm of spontaneous birth in humans (3, 4), with maximal birth rate values during the night, at a time coinciding with maximal pineal melatonin (5-methoxy-Nacetyltryptamine) secretion (5). Melatonin is recognized to be an important endocrine signal of the circadian timing system for coordinating many rhythmic events (5). With respect to rhythmic parameters in nonhuman primates, Ducsay et al. (6) have described a circadian rhythm of progesterone, cortisol, and estradiol in pregnant rhesus monkeys with peak values at night, although it is debatable whether these hormones contribute to the circadian timing of birth. Harbert and coworkers (7) have also demonstrated an increased mean placental blood flow during the night. The fact that the light-dark cycle can regulate the daily timing of birth and uterine contractility has been shown for rhesus macaques (8, 9) and rats (10). It is known that maternal melatonin crosses the placenta (11), and plasma melatonin levels have been reported to undergo biphasic dynamics during pregnancy, rising during the first 20 wk of gestation, then falling during wk 20 –36 before rising again at wk 36 – 42 (12, 13). However, the data on a potential influence of melatonin on human myometrial function are scarce (14), and a clear understanding of the molecular mechanisms of its action in this tissue is completely lacking. Therefore, in the present studies we sought to obtain a better understanding of the molecular mechanisms of melatonin action on uterine function in the human.

the Hamburg medical ethics committee. Samples were obtained between 0800 –1000 h from noncycling nonpregnant (NP) patients undergoing hysterectomy or pregnant (P) patients undergoing cesarean section (at wk 38 – 40, before labor). The average age of the P patients was 31.8 ⫾ 5.59 yr, and that of the NP patients was 46.67 ⫾ 5.74 yr. Indication for cesarean section was noncephalic presentation, whereas for hysterectomy it was uterine fibromyoma. The samples were used for primary cell culture, or they were immediately frozen in liquid nitrogen and stored at – 80 C until further investigation.

Quantitative RT-PCR RNA isolation was performed with TRIzol reagent (Life Technologies, Inc., Karlsruhe, Germany). Deoxyribonuclease I digestion and RT were carried out following the manufacturer’s instructions from 1 ␮g RNA (deoxyribonuclease I, ribonuclease-free, and RT system, Promega Corp., Mannheim, Germany). Primers for detection of the MT1 receptor were S1 and AS1, and those for the MT2 receptor were S3 and AS3, as specified in Table 1. As a positive control, the cDNA from cell lines (SKUT, CHO) that had been stably transfected with either the MT1 or MT2 receptor constructs were used. After amplification the PCR products were electrophoresed on a 2% agarose gel, stained with ethidium bromide, and visualized under UV light. Quantitative PCRs (Fast Start SYBR green kit, Light Cycler, Roche, Mannheim, Germany) were performed with the same primer sets as that for the standard PCR. The reaction conditions for the Light Cycler (Roche) were as follows: denaturation step, 95 C, 20 sec; annealing, 70 C (MT1 and MT2) and 65 C (Gs␣ and oxytocin receptor), 20 sec; elongation, 72 C, 25 sec; and acquisition, 83 C, 15 sec. Up to 45 PCR cycles were used. Melting curve analysis was performed at the end of each run. Variations in cDNA concentrations were normalized against Gs␣ cDNA, as expression of this mRNA has been shown not to fluctuate significantly during pregnancy (15). Reaction composition was as follows: primer concentrations, 0.5 ␮m (for both forward and reverse primers); MgCl2, 3 mm; deoxy-NTPs, buffer, and polymerase concentration were provided by supplier. Receptor transcripts were validated by sequencing. For each assay internal standard curves were developed (data not shown).

Subjects and Methods Human subjects and tissues

In situ hybridization

Myometrial tissue was taken from the upper edge of the lower uterine segment with written informed consent of the patients and approval of

Templates (306 bp MT1 cDNA and 424 bp MT2 receptor cDNA, produced by RT-PCR with primer set S2, AS2, S4, and AS4 as listed in Table 1) after ligation into the pGEM-T easy vector (Promega Corp.) were used for cRNA synthesis. Riboprobes (sense and antisense) were synthesized using SP6 or T7 RNA polymerase and digoxigenin-labeling

Abbreviations: GTP␥S, Guanosine 5⬘-O-3-thiophosphate; NP, nonpregnant; P, pregnant; 4P-PDOT, 4-phenyl-2-propionamidotetralin.

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TABLE 1. Primers used for quantitative PCR and for the generation of sense and antisense probes for in situ hybridization Gene

Accession no.

MT-1-melatonin receptor

U14108

MT2-melatonin receptor

U23541

Stimulatory G-protein ␣ subunit (Gs␣)

AH002748

Oxytocin receptor

X80282

Primer sequences (5⬘-3⬘)

Forward: tcc tgg tca tcc tgt cgg tgt atc Reverse: ctg ctg tac agt ttg tcg tac ttg Forward: gta cga ccc gag gat cta ctc g Reverse: cct agg cac cat ggt ggc gg Forward: tgg gca acc tcc tgg tga tcc tc Reverse: agc cag atg agg cag atg tgc aga Forward: tag gga gga gga agt gga tga c Reverse: ccg gaa cgc agg taa ttt gtt c Forward: aga agc agc tgc aga agg ac Reverse: aca atg gtt tca atc gcc tc Forward: tgg cggagc agc aca gg Reverse: gtg tca gca agc gtc aag c

dNTP mix following the manufacturer’s instructions (DIG labeling kit, Roche Molecular Biochemicals). The specificity of the probes was validated with ribonuclease protection assay (data not shown). The riboprobes were specific for one receptor type only, i.e. there was no crosshybridization between MT1 and MT2 receptor riboprobes. The hybridization procedure was carried out with 10-␮m frozen tissue sections as follows. After rehydration, sections were denatured in 0.2 n HCl, heat-denatured in 2⫻ standard saline citrate (2⫻ SSC), then postfixed with 4% paraformaldehyde, acetylated with 0.25% acetic anhydride in 0.1 triethanolamine, dehydrated, and air-dried. Slides were hybridized at 55 C overnight, then washed in 2⫻ SSC (at room temperature) and hybridization buffer (at 65 C), before treatment with ribonuclease A (20 ␮g/ml) and sequential washing in 1⫻ SSC. Finally, slides were rinsed in 0.1⫻ SSC, then incubated with buffer 1 [0.1 m Tris-HCl (pH 7.5) and 0.15M NaCl] and buffer 2 [0.1 m Tris-HCl (pH 9.5), 0.1 m NaCl, and 50 mm MgCl2, blocked with 20% normal sheep serum]. The AntiDig Detection system (Roche) was used for detection of digoxigenin-labeled cRNA.

[125I]Melatonin binding assay Crude membranes were prepared on ice as previously described (15). The binding of [125I]melatonin (Amersham Pharmacia Biotech, Little Chalfont, UK; specific activity, 2000 Ci/mmol) was determined as described previously (16). Briefly, membranes (80 ␮g protein) were incubated in Tris-HCl (50 mm) and 0.02 mm MgCl2 at room temperature for 90 min in the absence (total binding) or presence (nonspecific binding) of 10 ␮m unlabeled iodomelatonin (Sigma-Aldrich, Taufkirchen, Germany). Saturation and displacement studies were conducted in triplicate samples. In experiments to test for G protein coupling of the melatonin receptor, membranes were incubated with 100 pm [125I]melatonin in a total assay volume of 200 ␮l. Concomitantly, the nonhydrolyzable guanine nucleotide guanosine 5⬘-O-3-thiophosphate (GTP␥S; Calbiochem, Bad Soden, Germany) was employed at doses ranging from 1–100 nm. Reactions were terminated by the addition of 4 ml ice-cold Tris-HCl, followed by rapid filtration over presoaked glass-fiber filters (Schleicher & Schuell, Inc., Dassel, Germany). Each filter was thereafter washed twice in 4 ml buffer to remove unbound melatonin, and the radioactivity of the filters was determined in a gamma-counter.

Autoradiographic studies Autoradiography was performed following the method described by Seltzer et al. (17), Briefly, frozen sections of P human myometrial tissues (12 ␮m) were mounted on gelatin-coated slides and incubated for 2 h at 4 C with 50 pm [2-125I]melatonin (2000 Ci/mmol) in 50 mm Tris HCl buffer, containing 5 mm MgCl2 in the absence (total binding) or presence (nonspecific binding) of 1 ␮m melatonin. After incubation, the slides were washed twice for 5 min each time in cold buffer with 5% BSA. Slides were apposed to Hyperfilm (Kodak, Stuttgart, Germany) for 1 wk.

Primer name

S1 AS1 S2 AS2 S3 AS3 S4 AS4 S5 AS5 S6 AS6

References

14 14

15 16 16 17 17

FIG. 1. PCR products with MT1 and MT2 receptor-specific primers. A, SKUT cells transfected with the MT1 receptor; B, CHO cells transfected with the MT2 receptor; C, NP human myometrium; D, water control; E, myometrial cells from primary culture.

minced thoroughly and digested in Ham’s F-12 medium (SigmaAldrich) with 10 mg/ml collagenase type 2 (Worthington LS), 1000 U/ml deoxyribonuclease I (Roche), 100 IU/ml penicillin, 100 ␮g/ml streptomycin, and 125 ␮g/ml fungizone (Life Technologies, Inc.) for 15 h at 37 C before plating. Myometrial smooth muscle cells were isolated and maintained in monolayer cultures (maximum of three passages) in Ham’s F-12/DMEM with 4.5 g/liter glucose (BioWhittaker, Inc. Europe Cambrex Co., Apen, Germany), 3 mm glutamine, 100 IU/ml penicillin, 100 ␮g/ml streptomycin, and 10% fetal calf serum (Life Technologies, Inc.). For the cAMP determinations cells were plated in 12-well multidishes (Nunc, Naperville, IL) in 1 ml culture medium/well. For RNA isolation, cells were harvested at confluence in T175 flasks (in 10 ml culture medium). Immunofluorescence with ␣-actin antibody (DAKO Corp., Hamburg, Germany) was used to verify that the cells were myocytes (data not shown).

cAMP assay For the determination of total cAMP accumulation, an ELISA was employed, which is based on a previously characterized RIA (19). Plated myometrial cells were preincubated for 15 min in the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (0.25 mm) before stimulation with 10 ␮m forskolin (Sigma-Aldrich) in the presence or absence of melatonin or iodomelatonin for 15–30 min. In experiments to show the pharmacological specificity of melatonin’s effect, the melatonin receptor antagonist 4-phenyl-2-propionamidotetralin (4P-PDOT; Tocris Cookson, UK) at a dose of 10 nm was also included during the stimulation period. To terminate cAMP accumulation 2 ml ice-cold ethanol was added to the wells (final volume, 2.5 ml), whereupon they were placed at –20 C to facilitate the extraction of intracellular cAMP. After centrifugation and evaporation, samples were redissolved in ELISA buffer, acetylated, and assayed. The sensitivity was 5 fmol/tube. Intraassay coefficients of variation were typically 6 –10%.

Data analysis and statistics Primary myometrial cell culture Human myometrial cells were prepared as described by Kobayashi al (18) to establish a primary cell culture. Myometrial tissue (0.5 g) was

Experiments were conducted in triplicate with a minimum of three independent tissue samples and were repeated at least three times. In all figures the data represent the mean ⫾ se. Statistical analyses were

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performed using an ANOVA, followed by the Bonferroni post hoc test with a significance criterion of P ⬍ 0.05.

Results

We found transcripts representing both melatonin receptor subtypes (MT-1 and MT-2) in human myocytes (i.e. myometrial samples and myometrial cells in primary culture; Fig.

FIG. 2. Relative expression of oxytocin (OT) and melatonin (MT1 and MT2) receptor mRNA transcripts in human NP (䡺) and P (f) myometrium. Variations in cDNA loading were normalized against Gs␣ cDNA using primers specified in Table 1. Asterisks indicate significant differences (P ⬍ 0.05) with respect to NP values as determined by ANOVA. Data represent the mean ⫾ SE (n ⫽ 5 and 4, respectively).

Schlabritz-Loutsevitch et al. • Melatonin Receptors in Human Myometrium

1). Quantitative real-time RT-PCR revealed differences in melatonin receptor expression levels when P myometrial samples were compared with NP samples (n ⫽ 4 –5; Fig. 2). Whereas oxytocin receptor expression was, as expected, significantly higher in P myometrial biopsies, the expression of both melatonin receptor isoforms tended to be much lower in P myometrial samples, although statistical significance was only reached for the MT2-R (P ⬍ 0.05). By means of in situ hybridization in human myometrial tissue, both transcripts were also detected in NP myometrial tissue (Fig. 3), although no apparent differences in transcript distribution were noted. In P myometrial samples, however, we were unable to reproducibly detect melatonin receptors by means of in situ hybridization. Receptor autoradiography as well as radioreceptor assay showed that specific high affinity melatonin-binding sites exist in both the P and NP human myometrium samples (Fig. 4). On the basis of nonlinear regression analysis (PRISM, GraphPad Software, Inc., San Diego, CA) of ligand binding to myometrial membranes, affinity constants (Kd) of 1.29 ⫾ 0.4 nm (NP) and 2.25 ⫾ 1.6 nm (P) were calculated (no significant difference). The calculated receptor densities (Bmax) for ligand binding were 28.2 ⫾ 5 fmol/mg for NP and 0.41 ⫾

FIG. 3. In situ hybridization of MT1 (upper row) and MT2 (lower row) melatonin receptor mRNA on NP myometrial tissue sections. The two photomicrographs to the left represent hybridization with antisense riboprobes, whereas the micrographs to the right represent hybridization with sense probes. All micrographs are ⫻40. The small vertical bar represents 10 ␮m. Three negative controls were also performed: hybridization with sense cRNA for the MT1 and MT2 receptors, hybridization without the antidigoxigenin antibody, and hybridization without riboprobe but with antibody.

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FIG. 4. Radioreceptor assays and receptor autoradiography. Binding assay showing specific melatonin binding to membranes of NP (A) and P (B) myometrial tissues. C, NP membranes were assayed in the presence or absence of 1 nM GTP␥S. D, Displaceable [125I]melatonin binding to frozen nonpregnant myometrial sections by means of receptor autoradiography. Data represent the mean ⫾ SE (n ⫽ 3).

FIG. 5. Accumulation of cAMP in cultured myometrial cells. A, The specific melatonin receptor antagonist 4PPDOT (Œ, dotted lines) prevented melatonin from significantly inhibiting forskolin-induced (F) cAMP accumulation (f, solid lines). C, Unstimulated controls. The effect of melatonin was only seen in NP myometrium. B, In contrast, melatonin slightly increased basal cAMP levels (P ⱕ 0.05 for the highest dose) only in P myometrial cells culture. The infinity symbol (⬁) indicates unstimulated controls. The data (mean ⫾ SE; n ⫽ 3) are representative of three replicate experiments. See Subjects and Methods for further details.

0.16 fmol/mg for P tissues (P ⬍ 0.05). In the presence of 1 nm GTP␥S, specific binding was reduced to 8% of normal control values (Fig. 4C), and at higher concentrations specific iodomelatonin binding was abolished (data not shown). Differences in the effects of melatonin on cAMP accumulation in primary cultures of P and NP myometrial cells were also noted. Myocytes from NP uteri showed no response to melatonin alone (data not shown), but did show the expected reductions in forskolin-stimulated cAMP accumulation after addition of nanomolar concentrations of melatonin (Fig. 5A). The inhibitory effect of melatonin was absent in the presence

of 10 nm of the melatonin receptor antagonist 4P-PDOT. In contrast, cAMP accumulation from P uteri myocytes was not inhibited by melatonin after forskolin treatment (data not shown), but did show a modest increase in basal cAMP production after application of a nonphysiological (1 ␮m) melatonin concentration (Fig. 5B). Discussion

At present two functional isoforms of melatonin receptors have been demonstrated in the human: MT1 and MT2 (20,

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21). These receptors have been localized to both central nervous (e.g. hypothalamic suprachiasmatic nucleus, hippocampus, cerebral cortex, retina, and neonatal pituitary) and peripheral structures (e.g. in lymphocytes, platelets, granulosa cells, fetal kidney, and human coronary arteries) (22, 23; for review, see Ref. 24). In the present study we demonstrate for the first time that transcripts for both melatonin receptor isoforms are expressed in human myometrial tissue and primary myometrial cell cultures. The real-time PCR quantification of melatonin receptor transcripts showed a decline in MT2-R expression in the face of clearly up-regulated oxytocin receptor mRNA expression in P tissue (Fig. 3). Such up-regulation of oxytocin receptor transcripts and binding sites has been described by Ivell et al. (25) and Fuchs et al. (26). The decline in MT2-R transcript expression is also mirrored by the marked reduction in receptor density as assessed by ligand binding assay (Fig. 4). Doolen et al. (27) described a melatonin effect on smooth muscle activity in the rat caudal artery in which MT1 receptor activation causes contraction, whereas MT2 receptors mediate relaxation. Generally speaking, melatonin receptor signal transduction mechanisms appear to rather site specific (28, 29). Previous investigations on direct actions of melatonin on uterine function have been performed mostly in the rat, where melatonin has been shown to block prostaglandin generation (30) and depress spontaneous as well as oxytocininduced uterine contractility (31, 32). On the other hand, Ma¨ rtensson et al. (14) found an augmentation of contractile force in human myometrial strips by melatonin after the administration of noradrenaline. These differences are likely to relate to differences in the phase relation between nocturnal melatonin secretion and maximal myometrial contractile activity (high at night in primates, high during the day in rodents). It is well known that melatonin can inhibit cAMP signaling via the coupling of its receptors (MT1 and MT2) to pertussis toxin-sensitive G proteins (Gi2 or Gi3). The fact that melatonin binding to human myometrial membranes is abolished by coincubation with GTP␥S (Fig. 4) is consistent with G protein coupling of the melatonin receptors. Melatonin has been shown to act via the Gq/11 protein (33), which is also known to be involved in the oxytocin receptor regulatory pathway. The ability of the melatonin receptor antagonist 4P-PDOT to abolish melatonin’s inhibitory action on cAMP in the human myometrium (Fig. 5) also points to this effect being mediated specifically via one or both melatonin receptors. However, the effects of melatonin on cAMP signaling reported in the present study do not appear to be related to the tocotrophic effects of the hormone on myometrial contractions in late pregnancy as reported by Martensson et al. (14), as we see an inhibitory effect of melatonin on cAMP levels only in NP tissues. Melatonin may of course participate through other signaling pathways in the nocturnal switching mechanism from contractures to contractility as described by Nathanielsz (34). For example, it is known that melatonin can also modulate both potassium and calcium channel activities in various tissues (35, 36), although this has yet to be examined in myometrium. The switching mechanism between an inhibitory effect on cAMP signaling in myocytes from NP women to a loss of

Schlabritz-Loutsevitch et al. • Melatonin Receptors in Human Myometrium

effect in myocytes from P women might be related to the expression of specific ␤␥-stimulated adenylyl cyclase isoforms exclusively during pregnancy, as described by Price et al. (37). However, we cannot exclude other mechanisms underlying such a switching phenomenon, for example, differential coupling of melatonin to the MT1 and MT2 receptors in P compared with NP myometrial tissues. An analogous switching phenomenon has recently been reported to occur in the P myometrium in terms of adrenaline and noradrenaline actions via ␤- and ␣2-adrenergic receptors (38). In summary, our present data demonstrate for the first time the functional expression of both melatonin receptor isoforms in the NP and P human myometrium as well as a direct influence of melatonin on cAMP on NP myometrial cells in vitro. Taken together these findings clearly demonstrate that the human myometrium is a target for melatonin and point to interesting new horizons for the potential use of this hormone or antagonists of the melatonin receptors in the treatment of uterine contractile disturbances. Acknowledgments We thank C. Martinsen for the excellent assistance with the preparation of the graphics, and A. Bednorz, S. Frederichs, J. Fahnenstich, and I. Schro¨ der for technical help. For many helpful discussions and advice we are thankful to Prof. B. Hueneker, Prof. H. J. Schro¨ der, and Dr. C. Rybakowski (Department of Obstetrics and Gynecology, University of Hamburg) as well as to Prof. V. Lehmann (General Hospital, Hamburg-Altona). For donation of the MT2R-expressing cell line, we are grateful to Dr. P. Witt-Enderby. Finally, we wish to acknowledge the many patients whose tissue donations made these studies possible. Received March 21, 2002. Accepted November 18, 2002. Address all correspondence and requests for reprints to: Dr. James Olcese, Institute for Hormone and Fertility Research, University of Hamburg, Grandweg 64, 22529 Hamburg, Germany. E-mail: [email protected] This work was supported by a grant from the Leidenberger Forschung GmbH. A portion of these findings were submitted in fulfillment of the M.D. degree by N.H. Present address for N.S.-L.: Department of Experimental Gynecology, Clinic of Obstetrics and Gynecology, University of Hamburg Medical School, 22529 Hamburg, Germany.

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