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Endocrinology Vol. 138, No. 1 397-404
Copyright © 1997 by The Endocrine Society

ARTICLES

The Mel1a Melatonin Receptor Is Coupled to Parallel Signal Transduction Pathways1

Catherine Godson and Steven M. Reppert

Laboratory of Developmental Chronobiology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Dr. Catherine Godson, Laboratory of Developmental Chronobiology, Jackson 1226, Massachusetts General Hospital, Boston, Massachusetts 02114. E-mail: godson{at}helix.mgh.harvard.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The recent cloning of a family of high affinity melatonin receptors has provided us with a unique opportunity to define the signal transduction pathways used by these receptors. We have studied signaling through the human Mel1a receptor subtype by stable expression of receptor complementary DNA in NIH 3T3 cells. Our data indicate that the human Mel1a receptor is coupled to inhibition of forskolin-stimulated cAMP accumulation by a pertussis toxin-sensitive G protein. Although melatonin alone is without effect on phosphoinositide hydrolysis, it potentiates the effects of PGF2{alpha} stimulation on phospholipase C activation. Melatonin potentiates arachidonate release stimulated by PGF2{alpha} and by ionomycin. The effects of melatonin on arachidonate release are sensitive to inhibition of protein kinase C. They are independent of the effects of melatonin on cAMP and do not appear to involve activation of mitogen-activated protein kinase. The effects of melatonin on both phosphoinositide hydrolysis and arachidonate release are sensitive to pertussis toxin treatment. Thus, we show that the melatonin signal is transduced by parallel pathways involving inhibition of adenylyl cyclase and potentiation of phospholipase activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MELATONIN is a hormone secreted rhythmically from the vertebrate pineal gland. This hormone elicits potent circadian, reproductive, and hypnotic effects in mammals (1). Using expression cloning, a high affinity melatonin receptor has been cloned from Xenopus laevis melanophores (2). Subsequent work using reverse transcriptase-PCR (RT-PCR) has indicated the existence of three receptor subtypes in vertebrates (Mel1a, Mel1b, and Mel1c) (3, 4, 5). The Mel1a and Mel1b subtypes are found in mammals. Using 2-[125I]melatonin as a ligand, these receptor subtypes are pharmacologically indistinguishable (4). In situ hybridization indicates Mel1a receptor expression in hypophyseal pars tuberalis and the hypothalamic suprachiasmatic nucleus (3), the presumed loci of the reproductive and circadian effects of the hormone. Expression of the Mel1b receptor is not detectable by in situ hybridization, but RT-PCR indicates expression of the gene in brain and retina (4). Sequence analysis of the cloned melatonin receptors indicates that they comprise a novel group within the large, functionally diverse, G protein-coupled receptor (GPCR) superfamily (1). Despite the potent physiological effects of the hormone and its therapeutic potential, relatively little is known about the signal transduction pathways activated by the melatonin receptor family. Stable expression of these recombinant receptors in mammalian cells provides an unprecedented opportunity to study transduction of the melatonin signal.

Studies of the endogenous receptor in vertebrates indicate high affinity melatonin receptors negatively coupled to adenylyl cyclase by a pertussis toxin (PTX)-sensitive G protein (1). Using NIH 3T3 cells expressing the human Mel1a receptor, we investigated whether the receptor is coupled to inhibition of adenylyl cyclase activity and/or phosphoinositide hydrolysis and arachidonate (AA) release. Our data indicate that the human Mel1a receptor is coupled to inhibition of forskolin-stimulated cAMP accumulation via a PTX-sensitive G protein. Although melatonin alone does not stimulate phosphoinositide phospholipid hydrolysis or AA release in the Mel1a-expressing cells, it markedly potentiates the effects of calcium-mobilizing agents on these phenomena. These effects of melatonin are sensitive to PTX. The potentiation of AA release by melatonin is 1) sensitive to the protein kinase C (PKC) inhibitor GF 102903X and to PKC down-regulation, 2) independent of the effects of melatonin on cAMP, and 3) does not appear to involve activation of mitogen-activated protein (MAP) kinase. We conclude that the melatonin signal may be transduced by activation of parallel pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
All reagents were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise indicated. Data were analyzed by Student’s t test using the Statview (Abacus Concepts, Berkeley, CA) computer program.

Stable transfection and culture of NIH 3T3 cells
Cloning of the human melatonin receptor by RT-PCR has previously been described (3). The complementary DNA encoding the Mel1a receptor was subcloned into pcDNA-Neo (Invitrogen, San Diego, CA) and transfected into NIH 3T3 cells using Lipofectin (Life Technologies, Gaithersburg, MD). Colonies of transfected cells were selected by culture in 1 mg/ml G418 (Geneticin (Life Technologies). Cell lines expressing high levels of receptor were selected on the basis of 2-[125I]melatonin binding. The cell line used in the studies described here specifically bound more than 500 fmol [125I]melatonin/mg protein, with a Kd of 30 pM.

NIH 3T3 cells were routinely cultured in DMEM supplemented with 10% FCS, penicillin (50 U/ml), and streptomycin (50 µg/ml) at 37 C in a humidified atmosphere of 5% CO2. Culture medium for transfected stocks was supplemented with 1 mg/ml G418.

cAMP assay
Cells were plated at 1 x 105/35-mm plate and harvested 48 h later. After washing twice with DMEM, cells were preincubated for 10 min with 250 µM isobutylmethyxanthine and then stimulated with vehicle, forskolin (10 µM), melatonin (1 µM unless otherwise indicated), or both forskolin and melatonin in isobutylmethyxanthine as described above. After 10 min at 37 C, the medium was aspirated, and the cells were lysed by boiling in 50 mM CH3COOH. The lysates were centrifuged at 13,500 x g for 15 min, and cAMP in the supernatant was determined by RIA (DuPont-New England Nuclear, Boston, MA). All determinations were made in triplicate. The effects of PTX on cAMP accumulation were measured after overnight treatment of the cells with 100 ng/ml PTX (List Biologicals, Campbell, CA).

Inositol phosphate (InsP) accumulation
Cells were plated at 1 x 105/35-mm plate. Twenty-four hours later the cells were washed twice with DMEM and labeled overnight with 2 µCi/ml [3H]myo-inositol (DuPont-New England Nuclear) in DMEM containing 0.1% BSA. The cultures were washed twice with DMEM and preincubated for 10 min in 10 mM LiCl DMEM before the addition of vehicle, PGF2{alpha} (final concentration, 1 µM), or melatonin (1 µM) in 10 mM LiCl. After 30 min at 37 C, medium were aspirated, and the cells were scraped into 500 µl MeOH-HCl (100:1) and kept on dry ice for 15 min before extraction with 2 ml CHCl3, 800 µl H2O, and 1 ml MeOH-HCl (100:1). The resulting aqueous phase was diluted before applying it to anion exchange columns (AG-1X8, formate form, Bio-Rad Laboratories, Richmond, CA). After washing with water and 0.1 M formic acid, InsP were eluted with 2 M ammonium formate and 0.1 M formic acid.. Aliquots of the eluate were taken for liquid scintillation counting. All measurements were made in triplicate (elution profiles from the columns were verified using purified [3H]InsP; DuPont-New England Nuclear). The effects of PTX were determined as described above.

[3H]AA release
Cells were plated at 0.5 x 105/35-mm plate. After 48 h in culture, cells were labeled with 0.2 µCi [3H]AA/ml 0.5% FCS-DMEM. Cells were washed twice with DMEM before stimulation with various reagents in DMEM-1 mg/ml fatty acid-free BSA,(6) as indicated. After incubation at 37 C for the indicated times, free AA release into the extracellular medium was quantitated by liquid scintillation counting. The effect of PTX was determined as described above. The effects of cAMP-modulating agents (8-Br-cAMP and Rp-cAMP; Calbiochem, La Jolla, CA) were determined in cells treated with these agents (1 µM) in the presence of PGF2{alpha} and/or melatonin as indicated.

The effect of PKC down-regulation was investigated by pretreating the cells for 18 h with 500 nM PMA before measuring AA release. This treatment did not compromise [3H]AA incorporation into the cells. In experiments using the specific PKC inhibitor GF 109203X, bisindolylmaleimide (7) (Calbiochem) cells were pretreated with this compound (10 µM) or vehicle (0.1% dimethylsulfoxide) for 15 min before stimulation with various agents as indicated.

MAP kinase immunoblotting
Subconfluent cell cultures were rendered quiescent by serum starvation for 24–36 h. After this period, cells were stimulated with various agents in DMEM for 10 min as indicated. After washing with PBS (4 C), lysates were harvested in RIPA buffer containing protease inhibitors (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS containing 2 mM PMSF, 20 µM NaVO3, 50 mM NaF, 20 µM leupeptin, and 10 µg/ml aprotinin). The lysates were clarified by centrifugation at 15,500 x g for 10 min. Aliquots of the supernatant were removed for protein assay (Bio-Rad), and the remainder was denatured by incubation for 5 min at 95 C in SDS-PAGE sample buffer. The cell lysates (25 µg protein/well) were resolved on a 12% SDS-PAGE gel before blotting onto nitrocellulose membranes. Nonspecific sites on the membranes were blocked by incubation in 3% nonfat milk TBS. The blots were incubated with primary antibody (anti-ERK-2, Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:500 in blocking buffer containing 0.05% Tween-20 overnight at 4 C. After several washes, incubation with an alkaline phosphate-conjugated secondary antibody, and subsequent washing, specific antibody binding was detected with a colorimetric substrate (4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phospate, Kirkegaard and Perry, Gaithersburg, MD).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The human Mel1a receptor is negatively coupled to adenylyl cyclase by Gi
Melatonin inhibited forskolin stimulated cAMP accumulation in cells expressing the human Mel 1a receptor. This inhibition was dose dependent, with an estimated IC50 value of 5 x 10-10 M (Fig. 1aGo). Melatonin was without effect on forskolin-stimulated cAMP accumulation in wild-type NIH 3T3 cells or in cells transfected with the pcDNA vector (data not shown). Melatonin has also been shown to inhibit forskolin-stimulated cAMP accumulation in cells expressing the recombinant Mel1a, Mel1b, and Mel1c receptors from different species with comparable IC50 values (3, 4, 5).



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  		Figure 1. Stimulation of the human high affinity melatonin receptor inhibits forskolin-stimulated cAMP accumulation by a PTX-sensitive mechanism. Using NIH 3T3 cells stably transfected with the human Mel1a receptor, the following investigations were conducted. A, The effect of increasing concentrations of melatonin on forskolin-stimulated cAMP accumulation was investigated. Cells were incubated with melatonin as indicated in the presence of forskolin (1 µM) for 10 min. Accumulated intracellular cAMP was determined by RIA. Results are expressed as a percentage of the maximum cAMP accumulation (i.e. that observed after stimulation with forskolin alone) and are the mean of three independent experiments assayed in triplicate. B, Cells were preincubated with either vehicle (-PTX) or PTX (+PTX; 100 ng/ml) for 18 h before stimulation with forskolin (1 µM), melatonin (1 µM), or forskolin and melatonin as indicated for 10 min. Results are expressed as a percentage of the mean forskolin-stimulated value (100%). Data shown are the mean ± SD of four independent experiments assayed in triplicate.

 
PTX treatment of the cells abolished the effects of melatonin on forskolin-stimulated cAMP accumulation (Fig. 1bGo). This suggests that the human Mel1a receptor is coupled through Gi, the only PTX substrate expressed in these cells (8). PTX sensitivity of the effects of melatonin on cAMP has also been demonstrated on expression of the other recombinant receptors and on the endogenous receptors of vertebrates (1, 9).

Melatonin potentiates phosphoinositide hydrolysis
Melatonin stimulation of NIH 3T3 cells expressing the Mel1a receptor did not alter phosphoinositide phospholipid hydrolysis. However, PGF2{alpha}-stimulated phosphoinositide phospholipid hydrolysis was potentiated by costimulation with melatonin (PGF2{alpha} alone, 2.5 ± 0.3-fold basal; PGF2{alpha} and melatonin, 7.4 ± 0.9-fold basal; P < 0.001; Fig. 2Go). Melatonin was without effect on PGF2{alpha}-stimulated phosphoinositide metabolism in wild-type NIH 3T3 cells (data not shown).



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  		Figure 2. Melatonin potentiates PGF2{alpha}-stimulated phosphoinositide hydrolysis. NIH 3T3 cells stably transfected with the human Mel1a receptor were labeled with 2 µCi/ml [3H]myo-inositol for 24 h and preincubated with either vehicle (-PTX) or PTX (+PTX; 100 ng/ml) for 18 h before 30-min stimulation with PGF2{alpha} (1 µM), melatonin (1 µM), or PGF2{alpha} and melatonin as indicated in the presence of 20 mMLiCl. 3H-Labeled inositides were extracted and resolved on ion exchange columns. Results are expressed as the fold basal amount of recovered label. Data are the mean ± SEM of four independent experiments. All measurements were made in triplicate.

 
This potentiation of phosphoinositide hydrolysis by melatonin was via Gi, as it was not observed in PTX-treated cells (Fig. 2Go). It was insensitive to inhibition of PKC with GF 109203X (data not shown). In PTX-treated cells, we consistently observed an increase in PG-stimulated inositide production. The reason for this is not clear, but it suggests possible tonic inhibition of Gq{alpha}-activated phospholipase Cß (PLCß) (10).

Melatonin potentiates AA release
Although melatonin alone was without effect on AA release from the Mel1a-transfected cells, melatonin potentiated the effects of PGF2{alpha} on AA release (PGF2{alpha}, 1.85 ± 0.07-fold basal; PGF2{alpha} and melatonin, 5.8 ± 0.2-fold basal; Fig. 3Go). The EC50 for melatonin potentiation of AA release was approximately 0.5 x 10-9 M (data not shown). Melatonin also potentiated ATP- and thrombin-stimulated AA release (data not shown). In cells pretreated with PTX, melatonin potentiation of AA release was not observed (Fig. 4AGo). PTX treatment enhanced the sensitivity of the cells to PGF2{alpha} stimulation of AA release, similar to its enhancement of PGF2{alpha}-stimulated InsP hydrolysis. (That we did not observe potentiation of AA release on costimulation with melatonin was not because PGF2{alpha} alone generated the maximal AA response, because in parallel experiments combined ionomycin and PMA stimulation generated greater fold basal AA release.)



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  		Figure 3. Melatonin potentiates PGF2{alpha}-stimulated AA release. NIH 3T3 cells stably transfected with the human Mel1a receptor were labeled with 0.2 µCi/ml [3H]arachidonic acid for 18 h before 30-min stimulation with PGF2{alpha} (1 µM), melatonin (1 µM), or PGF2{alpha} and melatonin as indicated. [3H]AA released into the culture medium was determined by liquid scintillation counting. Results shown are the mean ± SEM of five experiments.

 


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  		Figure 4. Melatonin potentiation of AA release is PTX sensitive, independent of changes in cAMP, and sensitive to PKC inhibition and down-regulation. Cells were labeled with arachidonic acid and the following treatments were given. A, Pretreatment with PTX, as previously described, before stimulation with hormones as described above. Results shown are the mean ± SEM of four independent experiments. All observations were made in triplicate. B, Treatment with hormone, as previously described, in the presence of vehicle, 8-Br-cAMP, or Rp-cAMP as indicated. The results shown are the mean ± SEM of three independent experiments. All observations were made in triplicate. C, Pretreatment for 18 h with 500 nM PMA or vehicle (0.1% ethanol) before stimulation as described above. The results shown are the mean ± SEM of three experiments. All observations were made in triplicate. D, Pretreated with 10 µM GFX109203X or vehicle (0.1% dimethylsulfoxide) for 15 min before stimulation with hormones as described above. Results shown are the mean ± SEM of 10 experiments. All observations were made in triplicate.

 
Melatonin also potentiated ionomycin-stimulated AA release, but was without effect on PMA-stimulated AA release [ionomycin alone, 4.3-fold basal; ionomycin and melatonin, 6.6-fold basal (P < 0.05); PMA alone, 3.8-fold basal; PMA and melatonin, 3.4-fold basal; Fig. 5Go). These data indicate that melatonin can potentiate AA release in combination with agents that elevate intracellular calcium.



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  		Figure 5. The effects of melatonin on ionomycin and PMA-stimulated AA release. Cells were labeled with [3H]arachidonic acid, and [3H]AA release was determined in response to 45-min stimulation with PMA (100 nM), ionomycin (500 nM), melatonin (1 µM), or the combinations indicated. Results are the mean ± SEM of four independent experiments (ionomycin) or five independent experiments (PMA). All observations were made in triplicate.

 
Melatonin potentiation of AA release is independent of changes in cAMP
To determine whether the effects of melatonin on AA release are related to the effects of the hormone on intracellular cAMP, we assayed AA release from cells treated with agents that should mimic (Rp-cAMP) or block (8-Br-cAMP) these effects of melatonin (11). Neither of these agents had any effect on basal or PGF2{alpha}-, melatonin-, or combined melatonin- and PGF2{alpha}-stimulated AA release (Fig. 4BGo).

Melatonin potentiation of AA release is PKC dependent
To investigate whether functional PKC was required for the observed effects of melatonin, we used two approaches: pretreatment with the PKC inhibitor GF 109203X and down-regulation of the kinase by overnight incubation with phorbol ester. Both of these manipulations resulted in an inhibition of the effect of melatonin on potentiation of AA release (Fig. 4Go, C and D). PGF2{alpha} and melatonin treatment of PKC-down-regulated cells resulted in 1.7 ± 0.1-fold basal AA release, whereas under control conditions, the fold basal AA release was 5.6 ± 0.2 (P < 0.001). GF 102903X pretreatment of cells reduced the fold basal AA release stimulated by PGF2{alpha} and melatonin from 4.9 ± 0.5 to 3 ± 0.27 (P < 0.05). The specificity of GF 109203X as a PKC inhibitor has previously been demonstrated in intact 3T3 cells (7). These data suggest that functional PKC is required for the observed effects of melatonin on potentiation of AA release.

To control for the possibility that the loss of responsiveness in the PKC-down-regulated cells was not attributable to a loss in receptor number, as has been shown for some other GPCR (12), we determined [125I]melatonin binding in phorbol ester-treated and control cells. [125I]Melatonin binding was unchanged by PKC down-regulation (data not shown).

Melatonin potentiation of AA release is independent of MAP kinase activation
Recent evidence suggests a role for MAP kinase in the activation of hormone-stimulated AA release. Several Gi-coupled receptors have been shown to activate MAP kinase (13, 14). To determine whether the potentiation of AA release by melatonin is related to a change in MAP kinase activation, we used immunoblotting with an anti-MAP kinase-specific antibody in an electrophoretic mobility gel shift assay (Fig. 6Go). Our data indicate that PGF2{alpha} stimulation is associated with a shift in the mobility of both p42 and p44 forms of MAP kinase, consistent with the modification of electrophoretic mobility of the activated phosphorylated form compared with the nonphosphorylated inactive form (15). In contrast, melatonin treatment did not alter the electrophoretic mobility of either the p42 or p44 form of the kinase. Furthermore, when cells were stimulated with both melatonin and PGF2{alpha}, we did not detect any potentiation of the effects observed with PGF2{alpha} alone. The observed effects on MAP kinase hyperphosphorylation were PTX insensitive.



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  		Figure 6. Melatonin does not alter MAP kinase phosphorylation stimulated by PGF2{alpha}. NIH 3T3 cells expressing the Mel1a receptor were serum starved for 36 h and pretreated for 18 h with or without PTX (100 ng/ml) as indicated before treatment with vehicle, melatonin (1 µM), PGF2{alpha} (1 µM), or both hormones as indicated. Cell lysates were resolved on 12% SDS-PAGE before transfer to nitrocellulose and immunoblotting with specific MAP kinase antisera. The antisera used preferentially recognized the p42 form of the kinase, but also recognized the p44 form. The positions of both proteins in their basic and hyperphosphorylated forms are indicated by the arrows. Qualitatively similar data were obtained in three other experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Until recently, it has been difficult to define the intracellular signaling pathways activated by melatonin. Expression of recombinant melatonin receptors in mammalian cells provides a useful model to this end. Our data indicate that activation of the Mel1a receptor expressed in NIH 3T3 fibroblasts modulates parallel signaling pathways via a PTX-sensitive mechanism: inhibition of adenylyl cyclase and potentiation of hormone-stimulated PLC activity and AA release. The effects of melatonin on potentiation of PLC activation and AA release reported here could not be mimicked or abolished by Rp-cAMP or 8-Br-cAMP, respectively, indicating that these are independent of the effects of melatonin on cAMP.

The physiological relevance of our findings is supported by several observations. First, the reported effects on cAMP and phospholipases can be achieved at physiological concentrations of melatonin. Data on the endogenous receptor obtained from vertebrate tissue consistently shows that the receptor is coupled to inhibition of adenylyl cyclase (1, 9). Second, there is evidence of melatonin potentiating a PLC-mediated phenomenon; studies from rat caudal artery indicate that {alpha}1-adrenergic receptor-stimulated vasoconstriction is potentiated by physiological concentrations of melatonin (16). Third, the circadian clock in the hypothalamic suprachiasmatic nucleus (SCN) has been identified as a major target of melatonin’s action (1). Interestingly, in the context of the data presented here, recent evidence suggests a role for PKC in modulating the effects of melatonin on phase shifting of electrical activity in the SCN (17). Furthermore, melatonin has been proposed to activate a K+ current in SCN neurons (18). AA modulation of several K+ currents has also been demonstrated previously (14, 19, 20).

GPCR activation of phospholipase C can be mediated through PTX-insensitive and sensitive G proteins (e.g. Gq and Gi/Go, respectively) (reviewed in Ref.21). Activation of phosphoinositide-specific PLC generates a bifurcating second messenger system: inositol 1,4,5-trisphosphate (InsP3), which releases Ca2+ from intracellular stores, and 1,2-diacylglcerol (DAG), which activates PKC. Hormone-sensitive AA release is modulated by the recently described cytosolic PLA2 (cPLA2) (22). This protein is a member of the Ca2+-dependent phospholipid binding domain family of pro-teins (23, 24). It requires elevated calcium to promote its translocation to the membrane, and phosphorylation is required for maximal catalytic activity (23, 25). cPLA2 activation may occur subsequent to PLC and protein kinase activation. Activation of the enzyme on phosphorylation by both MAP kinase and PKC has been shown (26, 27, 28). Direct coupling of the enzyme to a PTX-sensitive G protein has been suggested in some systems (29). AA release is the rate-determining step in the synthesis of PGs and leukotrienes. Recent evidence suggests an important role for the eicosanoids in ion channel regulation and synaptic transmission (reviewed in Refs. 19 and 20).

To date, evidence for a role of melatonin in modulation of phospholipid metabolism has been inconclusive. In neonatal rat gonadotrophs, melatonin is without effect on DAG and AA release, but can inhibit LHRH-dependent activation of these pathways (30). In ovine pars tuberalis melatonin is without effect on phospholipid metabolism (31, 32). In chick retina, dopaminergic increases in cAMP can be inhibited by melatonin, which is without effect on the D1 receptor-induced increase InsP production (33). The data presented here demonstrate that melatonin can potentiate PGF2{alpha} activation of PLC. This effect is PTX sensitive. Melatonin also potentiates PGF2{alpha}- and ionomycin-stimulated AA release. Melatonin potentiation of AA release is PTX sensitive and is dependent on functional PKC.

The potentiation of hormone-stimulated AA release in response to stimulation of several Gi-coupled receptors has previously been shown. D2 dopaminergic, {alpha}2-adrenergic, and M2 and M4 muscarinic receptors act synergistically with Ca-mobilizing agents to promote AA release. These phenomena are sensitive to PTX and can be mimicked by guanosine 5'-O-(3-thiotriphosphate) (11, 34), but are independent of effects on PLC activation (11, 35). In contrast, adenosine potentiation of muscarinic receptor-stimulated AA release appears to involve activation of PLC in some systems (36). Receptor-dependent potentiated AA release has also been shown after stimulation of endogenous receptors in neurons and astrocytes (37, 38).

The sensitivity of melatonin-facilitated AA release to PKC inhibition and down-regulation that we report here suggests that melatonin may activate PKC. This activation may be isoform selective (6, 39, 40), as melatonin alone does not stimulate AA release, whereas PMA stimulation does. Differential regulation of PLC and AA metabolism by PKC{alpha} and -{epsilon} has been demonstrated in NIH 3T3 cells (40, 41). The activation of conventional PKC isoforms is dependent on DAG. That melatonin alone does not stimulate phosphoinositide hydrolysis argues against the hormone acting as a classical PKC activator. However, DAG produced by hydrolysis of other phospholipids, e.g. phosphatidylcholine, has frequently been shown to activate PKC (42). Further evidence supporting possible PKC activation by melatonin is suggested by our observation that melatonin cannot potentiate AA release stimulated by the PKC activator PMA. In contrast, melatonin potentiates AA release stimulated by the calcium ionophore ionomycin. Thus, one might envisage that maximal AA release could be facilitated by ionomycin-induced increases in intracellular calcium, resulting in cPLA2 translocation in combination with melatonin-activated PKC and subsequent cPLA2 phosphorylation.

cPLA2 can be activated by MAP kinase. Activation of p42 and p44 MAP kinase by Gi-coupled receptors has previously been shown (13, 14). PGF2{alpha} activation of MAP kinase by a PTX-insensitive pathway has recently been demonstrated in NIH 3T3 cells. (43). This suggests that MAP kinase activation might be an upstream locus at which the PGF2{alpha} and melatonin signals converge under permissive conditions to activate cPLA2. However, the data presented here do not support this hypothesis: We have demonstrated PGF2{alpha} activation of both p42 and p44 MAP kinases, but we did not discern any effect of melatonin on either form of MAP kinase when it was added alone or in combination with PGF2{alpha}. Recent evidence from ovine pars tuberalis also indicates that melatonin does not activate either p42 or p44 MAP kinase (44). A further possibility is that melatonin might activate a p38 MAP kinase that has recently been described in thrombin-activated platelets. cPLA2 is a downstream target of this kinase (45).

That melatonin alone is without effect on AA release or PLC activation raises the question of what do PGF2{alpha} and ionomycin do to sensitize the system to the effects of the hormone? A hypothetical scenario is summarized in Fig. 7Go. The lack of effect of melatonin on AA release argues against direct coupling of the receptor to cPLA2. In NIH 3T3 cells, the PGF2{alpha} receptor is coupled through Gq to PLC activation (46). The effects of melatonin that we report here are coupled through the PTX-sensitive G protein, G. Gß{gamma} activation of PLCß has previously been shown (47, 48, 49). Activation of some forms of PLCß by ß{gamma} are calcium dependent (50). We hypothesize that the G protein ß{gamma}-subunits released after melatonin receptor stimulation can conditionally activate PLC subsequent to PGF2{alpha} stimulation. This may explain how melatonin potentiates both PLC and cPLA2 activation. Thus, the increase in intracellular Ca2+ produced subsequent to PGF2{alpha}-mediated Gq{alpha} activation of PLC and InsP3 generation facilitates ß{gamma} activation of PLCß. Activation of PLC, in turn, generates InsP3 and DAG. This may involve superactivation of the PLCß species initially activated by Gq{alpha} or activation of an alternative form of PLC (10). As PTX inhibits receptor-mediated dissociation of Gi/Go into its {alpha}- and ß{gamma}-subunits the sensitivity of PLC activation and AA release to PTX can be explained by inhibition of ß{gamma} production. The finding that the ß{gamma}-subunits produced from the Gi heterotrimer may activate PLC in preference to the ß{gamma}-subunits of the Gq complex might be explained by the relative abundance of the Gi complex, which is typically found in a 10-fold greater abundance than the Gq complex (10). Some preliminary data support this hypothesis. Melatonin potentiates intracellular calcium mobilization of PGF2{alpha}-stimulated cells loaded with fura-2. The melatonin-induced increase in Ca2+ is PTX sensitive and independent of extracellular calcium.2



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  		Figure 7. Hypothetical scheme by which melatonin may activate parallel signaling pathways: inhibition of adenylyl cyclase and potentiation of PLC and cPLA2 activation. Stimulation of the Mel1a receptor generates Gi{alpha} and Giß{gamma} subunits. The Gi{alpha} subunits are negatively coupled to adenylyl cyclase. The Giß{gamma} subunits may activate PLCß in the presence of elevated intracellular calcium (10, 47) subsequent to PGF2{alpha}-mediated Gq{alpha} activation of PLC and InsP3 generation. The resulting diacylglycerol may activate PKC (PKC*), which, in turn, can activate cPLA2 (cPLA2*). PTX inhibits dissociation of the Gi{alpha}ß{gamma} heterotrimer, thereby inhibiting the effects of melatonin on both adenylyl cyclase and phospholipid metabolism. GF 109203X and PKC down-regulation inhibit PKC activation of cPLA2 and subsequent potentiation of AA release.

 
Our data demonstrate the potential importance of melatonin in regulating cross-talk between different signals. The results presented here are on signaling through the Mel1a receptor. Preliminary studies on signaling through the Mel1b receptor subtype (4) have indicated similar potentiation of lipid metabolism.3 This suggests that potentiation of PLC activation and AA release may be a common mechanism by which melatonin exerts its effects through its receptor subtypes.


   Acknowledgments
 
We thank Dr. Paul Insel, Department of Pharmacology, University of California-San Diego, for helpful comments and discussion.


   Footnotes
 
1 This work was supported by Grant R37-HD-14427 and a Sponsored Research Agreement with Bristol Myers Squibb. Back

2 Vanecek, J., C. Godson, and S. M. Reppert, unpublished. Back

3 Godson, C., and S. M. Reppert, unpublished. Back

Received July 15, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Reppert SM, Weaver DR, Godson C 1996 Melatonin receptors step into the light. Trends Pharmacol Sci 17:100–102[CrossRef][Medline]
  2. Ebisawa T, Karne S, Lerner MR, Reppert SM 1994 Expression cloning of a high-affinity melatonin receptor from Xenopus dermal melanophores. Proc Natl Acad Sci USA 91:6133–6137[Abstract/Free Full Text]
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