Molecular and cellular analyses of melatonin receptor-mediated cAMP signaling in rat corpus...

J Pineal Res 1998: 25:219-228 Printed in the United States of AmericcFilll rights reserved

CopyrrRhr 0 Munksgaard, I998 Journal of Pineal Research

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Molecular and cellular analyses of melatonin receptor-mediated CAMP signaling in rat corpus epididymis

Li L, Xu JN, Wong YH, Wong JTY, Pang SF, Shiu SYW. Molecular and cellular analyses of melatonin receptor-mediated cAMP signaling in rat corpus epididymis. J. Pineal Res. 1998; 25:219-228.0 Munksgaard, Copenhagen

Abstract: By using 2-[1251]iodomelatonin receptor binding studies, we have previously demonstrated high affinity melatonin receptors, the binding activities of which are regulated by testosterone, in the corpus epididymis of rats. In this report, some of the basic molecular and cellular characteristics of these high affinity melatonin receptors in rat corpus epididymis were analyzed. MELIA and MELIB receptor mRNAs were expressed by rat corpus epididymal epithelial cells as revealed by in situ hybridization. Functionally, these high affinity melatonin receptors are negatively coupled to adenylyl cyclase via pertussis toxin (PTX) sensitive G, protein and the inhibitory effects of melatonin on forskolin-stimulated CAMP accumulation were enhanced by Sa-dihydrotestosterone (5a-DHT). Interestingly, opposing interactions between melatonin and P-adrenergic receptor signaling in rat epididymal epithelial cells were observed with melatonin inhibiting norepinephrine- and isoproterenol-stimulated cAMP accumulation. In conclusion, our data support a modulatory action of melatonin, mediated via pertussis toxin-sensitive G,-coupled MELIA and MELIB receptors, in androgenic and adrenergic regulation of rat corpus epididymal epithelial cell functions.

Li Li,' Jian N. Xu,' Yung H. Wong? Joseph T.Y. Wong,' Shiu F. Pang,' and Stephen Y. W. Shiu' 'Department of Physiology, The University of Hong Kong, Hong Kong, China; 'Department of Biology, Hong Kong University of Science and Technology, Kowloon, Hong Kong, China

Key words: corpus epididymis - G protein - melatonin receptor - 5a- dihydrotestosterone - epithelial cells - norepinephrine - isoproterenol

Address reprint requests to Dr. Stephen Y W. Shiu, Department of Physiology, The University of Hong Kong, Li Shu Fan Building, 5 Sassoon Road, Hong Kong, China. E-mail: sywshiu@hkucc.hku.hk

Received January 28,1998; accepted May 21, 1998.

Introduction

Melatonin, the neurohormone secreted predomi- nantly by the pineal gland, plays an important role in the synchronization of diurnal body rhythms in mammals including humans [Armstrong, 19891. Al- terations in the daily melatonin secretion pattern, induced by changes in the photoperiod, are also re- sponsible for timing circannual changes of the re- productive responses of many seasonal breeders such as hamsters [Reiter, 19801. In these animals, long days induce gonadal recrudescence while short days induce gonadal regression, in parallel with pho- toperiod-induced shortening and prolongation of the daily period when melatonin is secreted [Reiter et al., 19761. In light of such interesting observations, melatonin has been suggested to be nature's contra- ceptive for these seasonally-breeding animals [Lin- coln and Short, 19801. Until recently, the target sites of the observed gonad-regulatory actions of mela-

tonin were believed to be located predominantly at the hypothalamus of the central nervous system or in the pituitary gland [Fraschini et al., 1968; Pevet et al., 1987; Williams and Morgan, 19881. With the availability of radiolabeled melatonin agonist, high affinity 2-[ '251]iodomelatonin binding sites, satisfy- ing the pharmacokinetic properties of G protein- coupled receptors, have been demonstrated in peripheral tissues [Pang et al., 19931, including re- productive organs [Ayre et al., 1992; Yie et al., 1995; Laudon et al., 1996; Gilad et al., 1996; Shiu et al., 1996). It is now evident that the regulatory effects of melatonin on reproductive functions can be exerted directly on the target organs, in addition to its reported actions on the hypothalamic-pituitary axis [Fraschini et al., 1968; Pevet et al., 1987; Wil- liams and Morgan, 19881.

Recent successful gene cloning of different sub- types (MELIA, MELIB, Mel,,) of G protein-coupled

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melatonin receptor [Reppert et al., 1994, 1995a,b; Liu et al., 19951 has led to major advances in our understanding of the diverse signaling pathways that can be coupled to these cloned receptors expressed in transfected cells [Liu et al., 1995; Yung et al., 1995; Witt-Enderby and Dubocovich, 1996; Godson and Reppert, 19971. Nevertheless, there is generally a paucity of molecular information on G protein- coupled melatonin receptor signaling in primary cul- tures of native peripheral tissues “ash and Osbome, 1995; Barrett et al., 19961. Early radioreceptor stud- ies in our laboratories have identified a single class of high affinity receptor, which shows saturable, re- versible, stable, and specific binding to 2-[1251]- iodomelatonin, in the epididymis of rats [Yu et al., 19941. Subsequently, autoradiogra hic analysis has shown that these high affinity 2-[ I]iodomelatonin binding sites, which are subject to testosterone regu- lation, are localized in the corpus region of rat epi- didymis [Shiu et a]., 19961. Moreover, these epididymal melatonin receptors are likely to be members of the G protein-coupled receptor family, since MELIB receptor sequence has recently been identified by reverse transcription-polymerase chain reaction (RT-PCR) in the tissue [Shiu et al., 19971. As the signaling mechanisms of melatonin in the regulation of mammalian reproductive functions are largely undefined, it is fundamental to delineate, ini- tially, some of the basic molecular and cellular char- acteristics of these G protein-coupled receptors in melatonin signal transduction at the target reproduc- tive tissues. To this end, the present investigation was undertaken to determine the cellular localiza- tion of G protein-coupled melatonin receptor sub- types in the rat epididymis, as well as the functional properties of these receptors in relation to signal- ing via adenosine 3’,5’-cyclic monophosphate (CAMP), which is one of the well-studied second messengers known to mediate melatonin action [Carlson et al., 19891 in target tissues.

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Materials and methods

Chemicals and animals

Chemicals for cell culture were purchased from GIBCO BRL Chemical Co. (Grand Island, NY), while other reagents were obtained from Sigma Chemical Co. (St. Louis, MO), unless otherwise in- dicated. Male Sprague-Dawley (SD) rats (6-week- old) were obtained from the Laboratory Animal Unit of The University of Hong Kong and kept under a 12 hr light, 12 hr dark cycle (light on from 0300- 1500 hr) for 1 week. Light was provided by ceil- ing-mounted fluorescent lamps with an intensity of about 150-200 lux at the top of the cage. Tempera-

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ture was kept constant at 23 k 2°C. Standard food and water were provided ad libitum. Animals were decapitated at mid-light (0900 hr). Epididymides of the animals were collected and treated as described below.

Synthesis and labeling of rat MELIA and MELIB cDNAs

Poly (A)’ RNA was extracted from the corpus epi- didymides of SD rats by using the messenger RNA isolation kit (Stratagene, La Jolla, CA). Synthesis of rat MELIB cDNA by reverse transcription-poly- merase chain reaction (RT-PCR) has been reported previously [Shiu et al., 19971. By using a similar approach, first strand rat MELlA receptor cDNA was synthesized with antisense amplimer 5’-GTTaa- gcttGTCCTGCGGCTTCAGTTTGG-3’, designed from published partial MELlA receptor sequence of rats [Reppert et al., 19941, MELlA receptor cDNA was then subjected to nested PCR amplification with 200 nM each of antisense amplimer 5’-GTTaagctt- GTCCTGCGGCTTCAGTTTGG-3’ and sense amp 1 i mer 5’- TTT g g t ac c G TC T C A A G TATG A - TAGGATA-3’ for the first PCR and sense amplimer 5’-TCAggtaccTCATGCCCAACCTGCAAACC-3’ for the second PCR. The 30 cycles of PCR arnpli- fication was preceded by a heat denaturing step at 94°C for 2.5 min. Each cycle of PCR (50 pl) con- sisted of denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec, and extension at 72°C for 90 sec, in the presence of 2.5 U Tuq DNA polymerase (GIBCO BRL, Grand Island, NY.). The PCR was terminated with a prolonged extension step at 72°C for 10 min. PCR product of the correct predicted size for MELlA receptor cDNA was then gel-puri- fied and sequenced. Both rat MELIB receptor cDNA (264 bp) [Shiu et al., 19971 and MELlA receptor cDNA (242 bp) thus obtained were labeled with digoxigenin (DIG) using DIG DNA Labeling and Detection kit (Boehringer Mannheim, Mannheim, Germany) for subsequent detection of MELIA and MELIB receptor transcripts in epididymal tissue and cells by in situ hybridization studies.

In situ hybridization studies on tissue and cell samples

After the rat was decapitated, the epididymis was dissected and cut into two parts (proximal and dis- tal segments) before being frozen in liquid nitrogen. Frozen tissues were cut into 8-12 pm sections and fixed in 4% paraformaldehyde for 20 min. The slides were dehydrated with different concentrations of ethanol (30%, 40%, 60%, and 70%), before im- mersion in 0.2 N HC1 for 20 min at room tempera- ture and in 2.5 pg/ml of proteinase K solution at 37°C for 20 min. The sections were then post-fixed

Melatonin receptor-mediated cAMP signaling in rat epididymis

(4 mM), 5a-dihydrotestosterone (1 nM), 10% fetal bovine serum (FBS), penicillin (100 IU/ml), and streptomycin (100 pg/ml). The cell suspension was incubated in a flask at 32°C in a humidified atmo- sphere of 5 % COZ for 8 hr. During this period, non- epithelial cells such as fibroblasts and smooth muscle cells attach rapidly to the plastic surface of the flask, whereas epithelial cells remain suspended in the medium. The resulting cell suspension of the supernatant was thus free from non-epithelial cells. The epithelial cells in the supernatant were counted in a haemocytometer and cultured in a flask at a cell concentration of 1 x lo5 cells/ml at 32°C. The epi- didymal epithelial cell monolayers became con- fluent after 3 days of culture.

with 4% paraformaldehyde for 20 min. Slides were rinsed with 0.1 M triethylamine/0.25% acetic anhy- dride for 10 min and dehydrated in 70%, 85%, 90%, and 100% ethanol. Prehybridization was performed for 2 hr at 42°C. The prehybridization buffer con- sisted of 50% deionized formamide, 1 x Denhardt’s solution, 0.1 mg/ml sheared salmon sperm DNA, 0.25 mg/ml yeast transfer RNA, 5% dextran sulfate, and 4 x SSC. For hybridization, 20 ng of each DIG- labeled cDNA probe in 50 p1 prehybridization buffer was added to each section and incubated for 18 hr at 42°C. After incubation, the slides were washed in 2 x SSC, 1 x SSC, and 0.5 x SSC. For detection of DIG-labeled probe, sections were equilibrated in buffer I (0.1 M maleic acid buffer, pH 7.5; 2% lamb serum; and 0.3% Tween-20) for 30 min. Alkaline phosphatase-conjugated anti-DIG antibody ( I :500) in 200 p1 buffer I was added on to the section and incubated for 2 hr. The slides were washed in buffer I and then equilibrated in detection buffer (0.1 M Tris-HC1; 0.1 M NaC1; 50 mM MgC12, pH 9.5) for 2 min. The sections were incubated overnight in the dark in 100 p1 expression solution (45 pl of 75 mg/ ml nitroblue tetrazolium, 35 pl of 50 mg/ml 5- bromo-4-chloro-3-indolyl-phosphate, and 2.4 mg levamisol in 10 ml detection buffer). The reactions were terminated by incubating the sections with TE- buffer (10 mM Tris-HC1, 1 mM EDTA, pH 8.0) for 5 min. Controls for specificity of hybridization were carried out by pretreatment with RNase A before hybridization with DIG-labeled probes. For in situ hybridization on cells, rat corpus epididymal epithe- lial cells, cultured as described below, were grown on cover glass, fixed in 4% paraformaldehyde and dehydrated in 70% ethanol. The cells were hybrid- ized with 500 p1 hybridization buffer containing 40 ng DIG-labeled rat MELIA or MELIB receptor cDNA probe.

Primary culture of rat epididymal epithelial cells

Culture of rat epididymal epithelial cells was adapted from procedures previously described by Wong and co- workers [Cuthbert and Wong, 1986; Wong, 19881. Rat corpus epididymides were cut into small pieces and placed in sterile Hank’s balanced salt solution (HBSS) containing 0.25% trypsin. Tissues were incubated for 30 rnin at 32°C with vigorous shaking and separated by low-speed centrifugation (800g, 5 min). The super- natant was discarded. The pellet was then resus- pended in Eagle’s minimum essential medium (EMEM), containing collagenase IA (1 mg/ml) for 2 hr at 32°C with shaking. Cells were separated by centrifugation at 800g for 10 min. The pellet was then resuspended in EMEM containing nonessential amino acids, sodium pyruvate (1 mM), glutamine

Assay for intracellular cAMP accumulation

Epididymal epithelial cells, after two rounds of sub- culture, were seeded onto 24-well plates. At the time of the experiment, cells were washed twice with FBS-free EMEM and preincubated in FBS-free EMEM with 1 mM theophylline at 32°C for 2 hr. After preincubation, culture medium was quickly aspirated and the cells were treated with the follow- ing in 0.5 ml HBSS containing 1 mM theophylline: vehicle, melatonin (1 pM to 0.1 pM), forskolin (30 pM), norepinephrine (0.1 mM), isoproterenol (10 pM), or both forskolin and melatonin, norepineph- rine and melatonin, as well as isoproterenol and melatonin. After 30 rnin incubation, reactions were terminated by rapid aspiration of culture medium and replaced with 0.5 ml of 5 mM cold acetic acid solution. The culture plates were immediately placed in dry ice and then thawed in a 68°C water bath. This cycle was repeated thrice in order to disrupt the cell membranes and release intracellular cAMP into the acetic acid solution. The supernatant was collected from each well and centrifuged at 2,000g for 10 min, before transfer to a fresh eppendorf tube. The supernatant was frozen at -70°C prior to the measurement of CAMP. The remaining cell pellet and debris attached to the bottom of the well were pooled together for determination of its protein con- centration by Lowry’s method [Lowry et al., 19511.

For pertussis toxin (PTX) experiments, cells were preincubated for 12 hr at 32°C in EMEM with or without 100 ng/ml PTX. At the end of preincuba- tion, cells were washed thrice with FBS-free EMEM, preincubated in FBS-free EMEM contain- ing 1 mM theophylline for 2 hr, before incubation for 30 min in 0.5 ml HBSS containing 1 mM theo- phylline, added with either vehicle or 30 pM forskolin with and without 0.1 pM melatonin. For 5a-dihydrotestosterone (5a-DHT) experiments, cells were preincubated for 24 hr at 32°C in EMEM

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with 1 nM Sa-DHT, 10 nM Sa-DHT or without any Sa-DHT. At the end of the preincubation, cells were washed thrice with FBS-free EMEM, preincubated in FBS-free EMEM containing 1 mM theophylline for 2 hr, before incubation for 30 min in 0.5 ml HBSS containing 1 mM theophylline, added with either vehicle or 30 pM forskolin with and without melatonin (1 fM to 10 pM). The reactions were ter- minated as described above.

Acetic acid solution containing intracellular cAMP was used to measure cAMP level by radioimmunoas- say. Standards and samples were acetylated by the ad- dition of 5 p1 acetic anhydride-triethylamine (1 : 2), 50 pl iodinated tracer (10,OOO cpm), and 50 p1 CAMP-spe- cific antibody (1:2,500). The CAMP-specific antiserum was kindly supplied by Dr. D.C. Klein (NIH, Bethesda, MA). The reaction was carried out at 4°C overnight, and 1 ml ethanol was then added. The bound fraction was separated by precipitation. All determinations were made in triplicates and data shown are representative results from one of three independent experiments that yielded similar results. The cAMP levels were normal- ized for total protein per culture well.

Results

RT-PCR

In the present study, a specific PCR product of 242 bp was generated by nested PCR using MELlA re- ceptor sequence-specific primers after reverse tran- scription of mRNA purified from rat corpus epididymis (Fig. 1A). For RT-PCR using MELIB re- ceptor sequence-specific primers (Shiu et al., 1997),

M 1 2 3

B

a specific PCR product of 264 bp was generated (Fig. 1B). Nucleotide sequencing confirmed that the 242 bp and 264 bp cDNA fragments, with the ex- clusion of amplimer bases, encode sequences which are identical to nucleotides 118-301 of published rat MELIA receptor DNA sequence (Genbank accession no: U14409) [Reppert et al., 19941 and nucleotides 52-255 of published rat MELIB receptor DNA se- quence (Genbank accession no: U28218) [Reppert et al., 1995a1, respectively.

In situ hybridization

MELlA receptor mRNAs were expressed in basal and principal cells, as shown by tissue in situ hy- bridization using 20 ng of the respective DIG-la- beled cDNA probe (Fig. 2A,B). For epididymal epithelial cells, in situ hybridization with 40 ng DIG- labeled rat MELIA receptor cDNA probe re- vealed expression of MELlA receptor transcripts in the peri-nuclear cytoplasm of the cells (Fig. 2C). Similar expression patterns were observed in tissue and cells by in situ hybridization using rat MELIB receptor cDNA probe (Fig. 2a-2c). Specific signals were not detected in RNAase A-treated cells (Fig. 2D,d) and tissue (Fig. 2E,e).

Effects of melatonin on forskolin-stimulated cAMP accumulation with and without PTX

Forskolin stimulated, in a concentration-dependent manner, cAMP accumulation in rat corpus epididy- ma1 epithelial cells (2.3 t- 0.2-, 3.8 k 0.3-, 10 f 0.95- fold increases in intracellular cAMP upon respective

M 4 5 6

F i g . I . RT-PCR analysis of the melatonin receptor mRNA expres- sion in rat corpus epididymis. DNA gel electrophoresis of ampli- fication products generated by RT- PCR on poly (A)’ RNA purified from rat corpus epididymis using MELIA-specific amplimers (A) and MELIB-specific amplirners (B). M, DNA molecular weight marker $X- 174-RF DNA-Hae 111); 1, 4, Negative control without reverse transcriptase; 2, 5, Reagent con- trol with water as template; 3, cDNA generated by RT-PCR us- ing MELIA-specific amplirners 5 ’ - GTTaag c t t GTCCTGCGG - CTTCAGTTTGG-3’ and 5’-TC- AggtaccTCATGCCCAACCTGC- AAACC-3’. 6, cDNA generated by RT-PCR using MELIB-specific

MEL amplirners 5’-CTTaagcttGGC- Z b F CTGGAGCACCAGTATCCA-3’

and 5’-GCGgetaccCACCGAG- CCTGCAGTCk3TGG-3’.

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treatment with 1, 10, and 30 pM forskolin; each set of data was derived from three separate experi- ments). Since a reproducible 10-fold increase in in- tracellular cAMP was recorded with 30 p M forskolin, this concentration of forskolin was used in subsequent experiments. When preincubated with 30 pM forskolin and increasing concentration of melatonin (0.1 pM to 1 pM), there was a concen- tration-dependent inhibition of cAMP levels by me- latonin, with an ECjO value of 0.43 _+ 0.22 nM (three separate experiments) and maximal inhibition (28%) at 0.1 pM (Fig. 3A). Melatonin did not affect the basal level of cAMP in these cells at any concen- tration tested. After preincubation with 100 ng/ml PTX for 12 hr, the effect of 0.1 pM melatonin on 30 pM forskolin-stimulated cAMP accumulation was blocked (Fig. 3B). PTX alone did not affect basal levels or forskolin-stimulated cAMP accumu- lation (data not shown).

Melatonin receptor-mediated cAMP signaling in rat epididymis

lated cAMP accumulation by 30% (Fig. 5A). Simi- larly, the P-adrenergic agonist isoproterenol pro- duced a 2-fold elevation in cAMP level at a concentration of 10 pM. This effect was also inhib- ited by melatonin in a concentration-dependent man- ner with an EC50 of 0.19 * 0.05 nM (three separate experiments). Maximal inhibition by melatonin was observed at a concentration of 0.1 pM with 26% re- duction of isoproterenol-stimulated cAMP accumu- lation (Fig. 5B).

Discussion

Effects of 5a-DHT on melatonin- and forskolin-mediated cAMP accumulation

The level of intracellular cAMP accumulation, af- ter 30 pM forskolin stimulation, varied depending on whether the cells were preincubated in medium with or without 1 nM or 10 nM 5a-DHT. Forskolin- stimulated cAMP levels were 442 _+ 15 fmol/pg pro- tein, 344 & 29 fmol/pg protein, and 248 _+ 15 fmol/ pg protein (three separate experiments) for medium without 5a-DHT, with 1 nM 5a-DHT, and with 10 nM 5a-DHT, respectively. In the absence of forskolin, the level of cAMP accumulation in these cells did not vary significantly when preincubated in medium with or without 5a-DHT (data not shown). The ECjos of inhibition by melatonin were 4.26 k 0.91 nM, 0.43 k 0.22 nM, and 0.06 _+ 0.02 nM (three sepa- rate experiments) for cells preincubated in medium without Sa-DHT, with 1 nM 5a-DHT, and 10 nM 5a- DHT, respectively (Fig. 4).

Effects of melatonin on norepinephrine- and isoproterenol-stimulated cAMP accumulation

Intracellular cAMP in cultured epididymal epithe- lial cells was stimulated by norepinephrine or iso- proterenol (Fig. 5). When the cells were incubated with 0.1 mM norepinephrine for 30 min, a 2-fold elevation in intracellular cAMP level was recorded. Concentration-dependent inhibition by melatonin on norepinephrine-stimulated CAMP accumulation was observed (ECS0 = 4.67 * 1.22 nM; three separate experiments) when the cells were incubated with 0.1 mM norepinephrine and 1 pM to 0.1 pM melato- nin. The maximum effect was mediated by 0.1 pM melatonin, which inhibited norepinephrine-stimu-

In line with our previous results on the localization of melatonin receptors and identification of MELIB receptor cDNA sequences in rat corpus epididymis [Shiu et al., 1996, 19971, evidence is now provided on the molecular identities and cellular distribution of the transcripts of these receptors. In addition to MELIB receptor mRNA, MELlA receptor transcript was detected by RT-PCR in rat epididymis in the present analysis (Fig. 1). Furthermore, in situ hy- bridization studies using RT-PCR derived rat MELlA and MELIB receptor cDNA probes showed that the predominant cell types expressing the transcripts of the two receptor subtypes are the basal and princi- pal cells of the epididymal epithelium (Fig. 2). To date, three subtypes of G protein-coupled melato- nin receptor (MELIA, MELIB, and Mell,) have been identified in different vertebrates, of which only MELIA and MELIB receptors are found in mammals [Reppert et al., 1994, 1995; Reppert and Weaver, 1995; Shiu et al., 19971. Different distribution of MELIA and MELIB receptor transcripts has been pre- viously reported in neural tissues. By in situ hybrid- ization, expression of MELlA receptor mRNA was detected in rodent suprachiasmatic nucleus and pars tuberalis, which are, respectively, the presumed sites of the circadian and some of the reproductive ac- tions of melatonin [Reppert et al., 19941. Con- versely, MELIB receptor transcript was expressed in the human retina as detected by RT-PCR and the re- ceptor subtype has been suggested to mediate the retinal actions of melatonin [Reppert et al., 1995a1. Interestingly, expression of both MELlA and MELIB receptor transcripts by rat corpus epididymal epithe- lial cells, as detected by in situ hybridization, was observed in our study. This may be explained by dif- ferences in the regulation of melatonin receptor gene expression in different tissues. Taking into account of previously reported functional similarities be- tween the two receptor subtypes in relation to inhi- bition of cAMP accumulation and potency for 2-['251]iodomelatonin binding [Reppert and Weaver, 19951, and the fact that definitive subtype-selective drugs for melatonin receptors are not yet available,

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Melatonin receptor-mediated cAMP signaling in rat epididymis

B 3 110 g 100

70

50

10

0

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W $? v

F+M F+M+PTX -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 C F

Log [Melatonin] (M)

Fig. 3. Effects of melatonin on forskolin-stimulated cAMP accumulation in rat corpus epididymal epithelial cells. A: Cells were incubated with melatonin as indicated in the presence of forskolin (30 pM) for 30 min. Results are expressed as a per- centage of mean maximum forskolin-stimulated cAMP accu- mulation (100%). Data shown are the mean k standard error of three separate experiments assayed in triplicates. B: Cells were preincubated with vehicle or pertussis toxin (100 ng/ml)

for 12 hr before stimulation with vehicle, forskolin (30 pM), or forskolin (30 pM) plus melatonin (0.1 pM) for 30 min. C, control; F, forskolin; F + M, forskolin + melatonin; F + M + PTX, forskolin + melatonin + pertussis toxin. Data shown are the mean f. standard error of five separate experiments assayed in triplicates. *P < 0.05 compared to the forskolin response (Student’s t test).

it may not be possible to pinpoint which receptor subtype is important for any functional effects of melatonin on the rat epididymis at this stage.

In relation to melatonin receptor-mediated cAMP signaling, forskolin-stimulated cAMP accumulation in epididymal epithelial cells was significantly in- hibited by melatonin. The ECS0 of melatonin was 0.43 k 0.22 nM, which is in the physiological range of melatonin concentration in rat serum [Pang and Ralph, 19751. The inhibitory effect of melatonin on forskolin-stimulated cAMP accumulation in epididy- ma1 epithelial cells was found to be blocked by PTX (Fig. 3B), which implicates negative coupling of rat epididymal melatonin receptors to adenylyl cyclase through PTX-sensitive Gi protein. This is in accord with results obtained for melatonin receptors in the brain [Carlson et al., 1989; Morgan et al., 19941, spinal cord [Wan et al., 19971, pars tuberalis [Mor- gan et al., 19901, retina [Nash and Osborne, 1995; Iuvone and Gan, 19951, dermal melanophores

Fig. 2. Localization of melatonin MELIA and MELIB re- ceptor transcripts in rat corpus epididymis by in situ hybrid- ization. MELIA (A,B) and MELIB (a,b) receptor mRNA expression in basal and principal cells of corpus epididy- ma1 tissues. MELIA (C) and MELIB (c) receptor mRNA ex- pression in the peri-nuclear cytoplasm of rat corpus epididymal epithelial cells. RNAase A-treated epididymal epithelial cells (D,d) and rat corpus epididymal tissues (E,e). B, basal cells; P, principal cells; Arrow, hybridization dots. C,D,c,d sections stained with hematoxylin. A,E,a,e, ~400; B,C,D,b,c,d, X1,OOO.

[White et al., 19871, kidney [Song et al., 19961, ar- teries forming the circle of Willis [Capsoni et al., 19941, and testicular Leydig cells [Valenti et al., 19971. Additionally, our data support an enhance-

G 1 O T ’, ~ ~ ~ : ~ : ~ ~ ’ -16 -15 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5

Log [Melatonin] (M)

Fig . 4 . Effects of melatonin on 30 pM forskolin-stimulated cAMP accumulation in rat corpus epididymal epithelial cells after 24 hr preincubation in medium containing various con- centrations of 5a-dihydrotestosterone (Sa-DHT). Cells were preincubated in medium with 1 nM Sa-DHT (open circle), 10 nM Sa-DHT (closed triangle), or without Sa-DHT (closed circle). Results are expressed as a percentage of mean maxi- mum forskolin-stimulated cAMP accumulation (100%). Data shown are the mean zk standard error of three separate experi- ments assayed in triplicates.

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+ + +

NE plus melatonin (1 pM to 0.1 PM)

Fig. 5 . Effects of melatonin on norepinephrine- or isoproter- enol-stimulated cAMP accumulation in rat corpus epididymal epithelial cells. Cells were incubated with melatonin as indi- cated in the presence of 0.1 mM norepinephrine (A) or 10 pM isoproterenol (B) for 30 min. NE, norepinephrine; ISO, isopro-

ment of Gi-coupled melatonin receptor-mediated CAMP signaling pathway in the epididymis by 5a- DHT, as evidenced by the decrease in ECS0 of me- latonin-mediated inhibition of forskolin-stimulated cAMP accumulation with an increase of androgen in the preincubation medium (Fig. 4). The observed effect of Sa-DHT on melatonin receptor-mediated CAMP signaling may be exerted, in part, via inhi- bition of the activity of adenylyl cyclase in response to forskolin, since the level of intracellular cAMP accumulation stimulated by forskolin decreased with increased Sa-DHT in the preincubation medium without melatonin. Reduction of adenylyl cyclase response to forskolin with testosterone treatment has indeed been reported in rat hepatic membranes [Shima, 19921. Nevertheless, the possibility that 5a- DHT may regulate the transcription of epididymal adenylyl cyclase is not excluded. It is also likely that melatonin receptors constitute other sites of action for Sa-DHT, as suggested by previous observations of testosterone-dependent regulation of 2-['25J]- iodomelatonin binding to melatonin receptors in rat epididymis [Shiu et al., 19961, suprachiasmatic nu- cleus and pars tuberalis [Zitouni et al., 19961. Al- though the mechanism of androgen effect on melatonin receptor signaling is yet unknown, andro- gen-induced changes in melatonin receptor activity is a distinct possibility, as reflected by decrease in

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L L -1 + E I 5

IS0 plus melatonin (1 pM to 0.1 pM)

terenol. Results are expressed as a percentage of mean maxi- mum cAMP accumulation stimulated by adrenergic agonists (100%). Data shown are the mean k standard error of three separate experiments assayed in triplicates.

Kd without any change in B,,, of 2-['251]iodo- melatonin binding sites in rat corpus epididymis af- ter testosterone treatment [Shiu et al., 19971.

Notwithstanding the hitherto enigmatic functional significance of melatonin receptor-mediated cAMP signaling in epididymal cell biology, some possible roles played by this pathway in the regulation of specific cell processes can still be inferred from the functions of the cell types expressing melatonin re- ceptors and our current knowledge on epididymal epithelial cell physiology. As mentioned above, MELIA and MELIB receptor transcripts are predomi- nantly expressed by the basal and principal cells of the epididymal epithelium (Fig. 2). While the prin- cipal cells are active in transport and secretion of small organic molecules, protein synthesis, as well as secretion and absorption of epididymal fluid [Robaire and Hermo, 19881, the functions of basal cells are yet to be determined. Since the activity of anion transport by epididymal epithelial cells is stimulated by P-adrenergic receptor-mediated cAMP signaling [Wong, 1988; Wong and Huang, 1990; Chan et al., 19941, it would therefore be useful to support a putative modulatory role of melatonin in the regulation of epididymal anion transport, by demonstrating interaction between melatonin and p- adrenergic receptor-mediated cAMP accumulation in epididymal epithelial cells, which have been

Melatonin receptor-mediated cAMP signaling in rat epididymis

shown to express P-adrenergic receptors [Chan et al., 19941. Intriguingly, opposing interactions be- tween melatonin and P-adrenergic receptor signal- ing in rat epididymal epithelial cells were observed in our study with melatonin inhibiting norepineph- rine- and isoproterenol-stimulated cAMP accumu- lation (Fig. 5). While melatonin receptors have been shown to potentiate contractile responses to adrener- gic nerve stimulation in rat caudal artery at the tissue level [Krause et al., 19951, our present observation is significant as it demonstrates modulation of p-adren- ergic signaling by melatonin in native cells. Similarly, potent inhibition of D 1 dopamine receptor-mediated cAMP accumulation by melatonin was observed in neuronal cells [Iuvone and Gan, 19951 and transfected HEK-293 cells transiently co-expressing dopamine and melatonin receptors [Liu et al., 19951. In the HEK-293 cells system, the interaction of melatonin and dopam- ine appeared to be specific as melatonin could not in- hibit the glucagon-like peptide, glucose-dependent insulinotropic peptide, and PTH type 1 receptor me- diated cAMP accumulation in HEK-293 cells [Chan et al., 19971. Taken together, these data highlight the important functional roles of melatonin as physiologi- cal modulators of both dopaminergic and adrenergic neurotransmission in specific tissues.

Figure 6 summarizes our current data on mela- tonin receptor-mediated CAMP signaling in epididy- ma1 epithelial cell. In conclusion, our results support a modulatory action of melatonin, mediated via per- tussis toxin-sensitive Gi-coupled MEL,, and MELIB receptors, in androgenic and adrenergic regulation

Melatonin

I & ATP CAMP

Fig . 6 . Schematic diagram of the molecular and cellular ba- sis of melatonin receptor-mediated cAMP signaling in rat cor- pus epididymal epithelial cell. AC, adenylyl cyclase; PTX, pertussis toxin; NE, norepinephrine; ISO, isoproterenol; 5a- DHT, 5a-dihydrotestosterone; T, testosterone; Dotted arrow, refers to probable multi-step action leading to regulation of re- ceptor activity; (+), activating effect; (-), inhibitory effect.

of rat corpus epididymal epithelial cell functions. In light of the significant impact of the epididymal mi- cro-environment on male fertility via its effects on sperm maturation and storage, it is our belief that future research efforts directed towards unraveling the tissue, cellular, and molecular mechanisms of melatonin signaling in the epididymis may provide us with fundamental insights on the physiological regulation of male reproduction by this pineal gland neurohormone .

Acknowledgments

This work was supported by CRCG research grants (335-034- 0071 and 335-034-0083), Elaine GCF Tso Memorial Fund, and Neuroendocrinology Research Fund of The University of Hong Kong. The authors acknowledge Dr. D.C. Klein for the antise- rum used in cAMP radioimmunoassay, Dr. C.S. Pang for criti- cal comments, and Mr. Y.T. Wong for technical assistance. Ms. L. Li is supported by a postgraduate studentship of The Uni- versity of Hong Kong and the data presented are derived from part of her PhD thesis work.

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