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Endocrinology Vol. 138, No. 10 4255-4261
Copyright © 1997 by The Endocrine Society

ARTICLES

Inactivation of Melatonin Receptors by Protein Kinase C in Human Prostate Epithelial Cells

Eli Gilad, Haim Matzkin and Nava Zisapel

Department of Neurobiochemistry, George S. Wise Faculty of Life Sciences (E.G., N.Z.), and the Department of Urology (H.M.), Tel Aviv Medical Center and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel

Address all correspondence and requests for reprints to: Prof. Nava Zisapel, Ph.D. Department of Neurobiochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel. E-mail: navazis{at}ccsg.tau.ac.il


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The pineal hormone melatonin regulates seasonal reproduction and pubertal development in mammals. We recently found melatonin receptors in the human benign prostate tissue, primarily associated with the microsome-enriched fraction of the epithelial cells. In cultured benign prostate epithelial cells, melatonin, at physiological concentrations, suppressed [3H]thymidine incorporation and cGMP levels. The effects of melatonin were transient, suggesting inactivation of the receptors. In the present study, the possibility of inactivation of the prostate melatonin receptors by protein kinase C (PKC) was explored.

Treatment of the microsome-enriched fraction with crude rat brain PKC in the presence of phorbol 12-myristate 13-acetate (TPA) or CaCl2 abolished the specific [125I]melatonin binding. This effect was prevented by the PKC inhibitor bisindolylmaleimide (GF-109203). [125I]Melatonin binding could be reinstated by iodoacetamide treatment.

In benign prostate epithelial cells in culture, TPA pretreatment markedly reduced the apparent affinity of [125I]melatonin binding. In addition, TPA ablated the cells responses to melatonin, namely the suppression of [3H]thymidine incorporation and cGMP levels. Pretreatment with GF-109203 prevented the TPA effects on [125I]melatonin binding and responses. In addition, GF-109203 slowed down the inactivation of the melatonin-mediated inhibition of [3H]thymidine incorporation.

Taken together, these data show that melatonin receptors are desensitized by PKC and imply that the transient response to melatonin may be the outcome of a direct or indirect melatonin-mediated activation of endogenous PKC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE NOCTURNAL production of melatonin by the pineal gland of all vertebrates and its release into the circulation represent the night-dark cycles in the organism (1). The biological clock in the suprachiasmatic nucleus, mediobasal hypothalamus, and pars tuberalis of the pituitary are thought to be the major target sites for melatonin action (1). Two types of high affinity membrane-associated melatonin receptors have been recently cloned and found to be expressed in the human suprachiasmatic nuclei and retina and recently also in the cerebellum (2, 3). These transmembrane melatonin receptors of the Mel-1 family can apparently inhibit the forskolin-activated elevation in cAMP through a pertussis toxin-sensitive G protein (2). In addition, melatonin has recently been found to bind and activate at nanomolar concentrations an orphan of the nuclear receptor superfamily (4).

Accumulating evidence indicates the presence of melatonin receptors in peripheral organs (for review, see 5 . We recently found specific binding sites for 125I-labeled melatonin ([125I]melatonin) in human benign prostate tissue (6). Equilibrium binding and competition experiments revealed specific binding of [125I]melatonin to the microsome-enriched fraction of the prostate epithelial cells, whereas the plasma membrane-enriched fraction from the cells did not display such binding. The binding (apparent half-saturation at 120 pM [125I]melatonin) was inhibited by GTP analogs, suggesting G protein coupling (6). Cultured epithelial cells from the human benign prostate were also found to display high affinity melatonin binding (7). The whole cell sites resembled the microsomal sites in apparent affinity, specificity, and sensitivity to GTP analogs (7). Melatonin, at physiological concentrations, inhibited DNA and protein synthesis in these cells and reduced their viability (7). The effects of melatonin on [3H]thymidine and [3H]uridine incorporation in prostate epithelial cells were transient (7). The responses to melatonin were maximal within 1 h, and the incorporation had returned to near-basal values within 24 h of treatment (7), suggesting desensitization of the receptors or downstream signal transduction pathways.

No direct evidence exists for melatonin receptor desensitization. However, daily injections of melatonin in the morning have been shown in some species to prevent the antigonadal response to melatonin injected in the afternoon (8, 9). The apparent density of melatonin-binding sites in the rodent brain exhibits diurnal and seasonal variations that are out of phase of the melatonin rhythm (10, 11). In addition, melatonin administration apparently inhibits melatonin binding in the rat suprachiasmatic nuclei (12). These observations are compatible with agonist (melatonin)-induced desensitization of the melatonin receptors (12, 13).

Protein kinase C (PKC) has been shown to induce desensitization of several classes of neurotransmitter receptors, e.g. M2 muscarinic, angiotensin 2,5-hydroxytryptamine 2A (14, 15, 16). Some evidence points to a possible involvement of PKC in melatonin responses: PKC activators prevented and reversed the melatonin-induced pigment aggregation in Xenopus laevis melanophores (17), pretreatment with PKC activators prevented the melatonin-induced secretion of interleukin-1 in human monocytes (18), and subthreshold concentrations of melatonin and a PKC activator synergistically enhanced interleukin-1 secretion in human monocytes (18). Finally, the Mel-1a melatonin receptor contains seven consensus sites for PKC phosphorylation, six of which are apparently located at the intracellular domain of the receptor (2). Inhibition of PKC has recently been shown to partially inhibit melatonin responses in Mel-1a receptor-transfected NIH-3T3 cells (19). Modulation by PKC of melatonin receptors has not been demonstrated.

In the present study we explored the effects of activation of PKC on melatonin receptor binding and responses in human benign prostate epithelial cells. Human benign prostate hyperplastic (BPH) tissue has been reported to contain phospholipid-dependent, phorbol 12-myristate 13-acetate (TPA)-activated PKC isoforms (20). Thus, the possibility that PKC mediates the desensitization of melatonin responses in the cells was investigated.

Besides inhibition of thymidine incorporation (7), we recently observed a melatonin-mediated decrease in cGMP in human prostate epithelial cells. Hence, we used the melatonin-mediated inhibition of thymidine incorporation and cGMP levels as markers for melatonin responses in prostate epithelial cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Melatonin, polyethylenimine, BSA, phenylmethylsulfonylfluoride (PMSF), phosphatidylserine (PS), dihydrotestosterone (DHT), and TPA were obtained from Sigma Chemical Co. (St. Louis, MO). Bisindolylmaleimide-GF-109203X (GF) was obtained from Calbiochem (La Jolla, CA). [Methyl-3H]thymidine was obtained from Rotem Industries (Beer-Sheva, Israel). Na125I and the cGMP RIA kit were obtained from Amersham (Arlington Heights, IL). [125I]Melatonin was prepared as previously described (21). Culture media [RPMI 1640 and RPMI 1640 without phenol red supplemented with L-glutamine (RPMI-P)], sera [newborn calf serum (NBS) and charcoal-stripped NBS (NBSC)], insulin, transferrin, selenium, and antibiotics were purchased from Biological Industries (Beit Haemek, Israel).

Preparation of microsome-enriched fractions (MF)
Human benign prostate tissue samples were obtained from patients undergoing open transabdominal prostatectomies for benign prostate hyperplasia (BPH). All patients (aged 55–80 yr; n = 24) were otherwise healthy. Approval for the use of the tissue was obtained from the local ethical committee. After removal, BPH was confirmed using histopathology. The rest of the tissue was put immediately in cold RPMI 1640 medium and used for preparation of MF (6) or epithelial cell cultures. MF samples were stored at -70 C until used. Five such samples (or more) from specimens from different patients were pooled for each experiment.

Epithelial cell cultures
Epithelial cells were cultured as previously described (7) and grown at 37 C in growth medium (RPMI containing 10% NBS, 10 ng/ml epidermal growth factor, 5 ng/ml insulin, 5 ng/ml transferrin, 5 ng/ml selenium, 50 U/ml penicillin, 50 µg/ml streptomycin, 250 ng/ml amphotericin B, and 10 ng/ml DHT) in a humidified atmosphere with 5% CO2. Before each experiment, cells were harvested by trypsin and adjusted to a density of 106 cells/ml in culture medium (RPMI containing 10% NBSC, 10 ng/ml epidermal growth factor, 5 ng/ml insulin, 5 ng/ml transferrin, 5 ng/ml selenium, 50 U/ml penicillin, 50 µg/ml streptomycin, and 10 ng/ml DHT) and replated in 24-well multiplates. After 24 h, the medium was replaced with fresh culture medium containing 5% NBSC. Protein content was determined in each plate (22), and data for all experiments were normalized to protein content.

Treatment with PKC, activators, and inhibitors
Preparation of crude PKC.
Whole rat brain was suspended in 4 ml/g tissue of homogenizing buffer [20 mM Tris-HCl (pH 7.9), 5 mM EGTA, 2 mM EDTA, 200 µM PMSF, 1 mM iodoacetamide (IAA), 1 mM dithiothreitol (DTT), and 250 mM sucrose] and homogenized on ice (8 strokes) in a Teflon-glass homogenizer. The homogenate was spun at 10,000 x g for 10 min to remove cell debris and then spun at 100,000 x g for 1 h. The supernatants were collected and used as the crude PKC preparation. Samples were stored at -70 C until used (23).

Treatment of MF.
MF samples (0.6–0.8 mg protein/ml) were incubated in 50 mM Tris buffer, pH 7.4, containing 10 mM MgCl2 in the absence or presence of 100 µM ATP, 10 µg/ml PS, 5 mM CaCl2, 100 ng/ml TPA, and 500 nM GF, or their combinations. The reaction was initiated by the addition of a 1:6 dilution (vol/vol) of the crude PKC preparation. The samples were incubated for 30 min at 37 C and diluted with 5 vol/vol ice-cold Tris buffer (50 mM; pH 7.4), and MF was collected by centrifugation (100,000 x g, 2 h, 4 C).

Treatment of epithelial cells.
Cells attached to the plates were treated with TPA (100 ng/ml, 30 min or 18 h) or GF (500 nM, 30 min) or were preincubated with GF for 30 min before the addition of TPA, and incubation was resumed for an additional 30-min period. In some experiments, cells were preincubated with or without GF (500 nM; 30 min at 37 C). Buffer or melatonin was then added, and incubation was resumed for additional periods of 1, 4, 8, or 24 h.

[125I]Melatonin binding
In MF.
[125I]Melatonin binding was assessed as previously described (6). Briefly, MF aliquots were incubated with 100 or 500 pM [125I]melatonin in 50 mM Tris buffer, pH 7.4, containing 0.15% Triton X-100 in the absence (total binding) or presence (nonspecific binding) of 50 µM melatonin for 1 h at 37 C. The reaction was terminated by the addition of 4 ml ice-cold Tris buffer, and binding was determined on GF/F filters (Whatman, Clifton, NJ) preincubated in 0.3% polyethylenimine and counted in a {gamma}-counter.

In thiol-reduced and alkylated MF.
In specified experiments, MF samples were incubated with 4 mM DTT for 30 min. Excess DTT was removed by dialysis (50 mM Tris buffer, pH 7.4, contains 200 µM PMSF; three times, 8 h at 4 C).

In some experiments, the PKC- or DTT-treated MF were incubated with 10 mM IAA or vehicle for 30 min at 37 C. Excess IAA was removed by dialysis (50 mM Tris buffer, pH 7.4, contains 200 µM PMSF; three times, 8 h at 4 C).

In epithelial cells.
Equilibrium binding studies were carried out as previously described (7). Briefly, cells were incubated for 60 min at 37 C with 10 pM to 1 nM [125I]melatonin (2000 Ci/mmol) in RPMI-P medium in the absence (total binding) or presence (nonspecific binding) of 2 µM melatonin. The media were then removed, and cells were immediately washed (twice, 2 ml) with ice-cold PBS dissolved in 0.1 M NaOH. Aliquots were removed for determination of protein content. The amount of radioactivity associated with the cells was determined in a {gamma}-counter.

The equilibrium binding data at the concentration range used was compatible with a single type of binding site (Scatchard analysis). The equilibrium binding parameters were obtained by nonlinear regression analysis (SigmaPlot).

Thymidine incorporation
[3H]Thymidine incorporation assays were performed as previously described (7). Briefly, cells attached to the plates were incubated with buffer or melatonin for 1 h at 37 C. [3H]Thymidine (60 Ci/mmol; 1 µCi/well) was then added, and incubation was resumed for 1 h. Media were discarded, and the cells were washed (twice, 2 ml) with ice-cold PBS and harvested by trypsin. Aliquots were retained for protein determination. Trichloroacetic acid was added, and insoluble materials were collected by filtration on GF/C glass fiber filters. The amount of radioactivity was determined by scintillation spectrometry.

cGMP assay
Cells were washed twice with PBS, harvested by a rubber policeman in RPMI-P medium containing 200 µM PMSF, then gently suspended and incubated for 10 min at 37 C with 10-6 M of the phosphodiesterase inhibitor isobutylmethylxanthine. Melatonin (10-10–10-6M) or the same volume of vehicle was added for 10 min. The reaction mixture was boiled for 4 min, frozen, thawed, sonicated, and centrifuged for 10 min (10,000 x g). The supernatant was collected, and the cGMP content was determined by RIA (Amersham). Data were normalized to protein content.

Statistical analyses
Results were compared by ANOVA followed by Student-Newman-Keuls test for multiple comparisons; significance was set at P < 0.050 (24).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of PKC on [125I]melatonin binding
MF.
The effects of PKC on [125I]melatonin (500 pM) binding in MF are shown in Fig. 1aGo. Incubation of the MF with the crude rat brain PKC preparation in the presence of ATP and PS did not affect binding. However, when either CaCl2 (5 mM) or TPA (100 ng/ml) was present, [125I]melatonin binding to these preparations was completely abolished (Fig. 1aGo). TPA did not affect [125I]melatonin binding to MF in the absence of crude PKC. The PKC inhibitor GF prevented both Ca2+ and TPA-dependent, PKC-mediated inhibition of [125I]melatonin binding (Fig. 1bGo).



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  		Figure 1. Effects of PKC, GF, and IAA on specific [125I]melatonin binding to microsome-enriched fraction from human benign prostate tissue. a, MF samples were incubated with buffer (Cont.) or crude rat brain PKC in the absence (PKC) or presence of TPA (PKCT) or Ca2+ (PKCCa) for 30 min. b, MF samples were incubated for 30 min with GF and treated as described in a. c, MF samples were incubated with 4 mM DTT for 30 min or treated as described in a and then treated with 10 mM IAA for 30 min. A sample treated with DTT without subsequent IAA treatment is also presented. Specific binding (mean ± SEM; n = 5) of [125I]melatonin (500 pM) was determined at equilibrium and calculated from the difference between the amount of [125I]melatonin bound in the absence and presence of 50 µM melatonin. *, P < 0.01, within pairs.

 
The effects of thiol reduction with DTT and alkylation with IAA on [125I]melatonin (500 pM) binding to untreated and PKC- treated MF are shown in Fig. 1cGo. DTT treatment markedly reduced [125I]melatonin binding to MF (by ~50%; P < 0.05). IAA restored [125I]melatonin binding in the DTT-treated MF without affecting binding in the control untreated MF. Surprisingly, IAA completely reinstated [125I]melatonin binding in MF pretreated with PKC in the presence of TPA. In MF treated with PKC in the presence of Ca2+, IAA treatment restored only 66% of the binding (Fig. 1cGo). Similar results were obtained with lower (100 pM) [125I]melatonin concentrations (not shown).

Epithelial cells.
The effects of TPA treatment (30 min) on [125I]melatonin-binding sites in benign prostate epithelial cells in culture are shown in Fig. 2Go. In TPA-treated cells, the apparent affinity of [125I]melatonin-binding sites was significantly lower than that in untreated cells (Kd = 753 ± 35 vs. 80 ± 5 pM, respectively; Fig. 2Go, a and b, and Table 1Go). Yet, the apparent density of binding sites was not affected (Table 1Go). Incubation of the cells with GF (60 min) decreased the apparent affinity of epithelial [125I]melatonin binding sites, although the effect was less pronounced than that of TPA (Fig. 2cGo and Table 1Go). Here, too, the apparent density of the sites was unaffected. Preincubation of the cells with GF (30 min) prevented the TPA-induced decrease in apparent affinity of [125I]melatonin-binding sites, but the apparent density of the sites significantly decreased (Fig. 2dGo and Table 1Go). Prolonged incubation (18 h) of the cells with TPA also reduced the apparent affinity of [125I]melatonin-binding sites (Kd = 398 ± 76 pM; Table 1Go) without affecting the apparent binding site density (Fig. 2eGo and Table 1Go).



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  		Figure 2. Equilibrium [125I]melatonin binding to human BPH epithelial cells as a function of [125I]melatonin concentrations. Cells were incubated for 30 min with vehicle (a), TPA (b), or GF and then with GF and either buffer (c) or TPA (d) for 30 min. In e, cells were treated with TPA for 18 h. The concentration dependency of [125I]melatonin binding was determined at equilibrium in each preparation. Specific binding was calculated from the difference between the amount of [125I]melatonin bound in the absence and presence of 2 µM melatonin. Results are the mean ± SEM (n = 12). The solid lines are theoretical curves reconstructed from the mean Kd and binding capacity values calculated from the Scatchard plot of the specific [125I]melatonin binding data (inset).

 

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  		Table 1. Apparent Kd and binding capacity (Bmax) values calculated from the Scatchard analyses of specific [125I]melatonin binding data presented in Fig. 3Go

 
Effects of TPA on melatonin responses in the prostate epithelial cells
Thymidine incorporation.
The effects of TPA on melatonin-induced inhibition of [3H]thymidine incorporation by prostate epithelial cells are shown in Fig. 3Go. Incubation of the cells with melatonin (1 h) resulted in a dose-dependent inhibition of [3H]thymidine incorporation into the cells [half-maximal effect (IC50) at 0.5 nM; Fig. 3aGo]. Incubation of the cells with TPA (100 ng/ml; 30 min) resulted in a 40% decrease in [3H]thymidine incorporation into the cells (Fig. 3aGo). No further inhibition of [3H]thymidine incorporation by melatonin was evident in TPA-pretreated cells (Fig. 3aGo). Prolonged (18-h) incubation of cells with TPA inhibited [3H]thymidine incorporation almost completely, and no further effect of melatonin was observed (Fig. 3aGo).



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  		Figure 3. Effects of melatonin on [3H]thymidine incorporation in human prostate epithelial cells. a, Cell were incubated with vehicle ({blacktriangledown}) or TPA ({square}) for 30 min or were treated with TPA for 18 h ({blacksquare}). b, Cells were incubated with GF for 30 min, with GF and either buffer ({circ}) or TPA (•) for 30 min, and then with various melatonin concentrations or the same volume of vehicle for 1 h. The incorporation of [3H]thymidine was then assessed. Results are the mean ± SEM of three independent experiments performed in quintuplicate and are expressed as a percentage of the incorporation in control vehicle-treated cells.

 
Incubation of the cells with GF (500 nM; 60 min) did not affect [3H]thymidine incorporation by the cells, and the inhibitory effect of melatonin was retained (Fig. 3bGo). Preincubation of the cells with GF (30 min) before the addition of TPA (30 min in the presence of GF) prevented the TPA-mediated abolition of the melatonin response (Fig. 3bGo), and the inhibitory effect of melatonin on [3H]thymidine incorporation was maintained. Interestingly, GF alone or in combination with TPA reduced the apparent IC50 for melatonin to less than 0.1 nM (Fig. 3bGo).

cGMP.
The effects of melatonin on cGMP levels in prostate epithelial cells are shown in Fig. 4Go. In control cells, melatonin (1 nM) reduced the cellular cGMP content by up to 29 ± 3% in a dose-dependent manner (IC50 = ~5 nM). Incubation of the cells with TPA (30 min) also reduced cGMP in the cells (by 27%), but prevented the inhibitory effect of melatonin on cGMP (Fig. 4aGo). Prolonged incubation of the cells with TPA (18 h) did not affect the basal cGMP level, but prevented the melatonin-mediated decrease in cGMP (Fig. 4aGo).



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  		Figure 4. Effects of melatonin on cGMP concentrations in human benign prostate epithelial cells. a, Cells were incubated with vehicle ({blacktriangledown}) or TPA ({square}) for 30 min or treated with TPA for 18 h ({blacksquare}). b, Cells were incubated with GF for 30 min and with GF and either buffer ({circ}) or TPA (•) for 30 min and then harvested and treated with various melatonin concentrations or the same volume of vehicle for 10 min. Results are the mean ± SEM of three independent studies performed in triplicate and are expressed as a percentage of the cGMP content in control cells treated with vehicle.

 
Incubation of the cells with GF (30 min) significantly augmented cGMP levels, but the inhibitory effect of melatonin was retained (Fig. 4bGo). Preincubation of the cells with GF (30 min) before the addition of TPA (30 min in the presence of GF) prevented the TPA-mediated abolition of melatonin’s effects (Fig. 4bGo). GF, alone or in combination with TPA, reduced the apparent IC50 for the melatonin-mediated suppression of cGMP to less than 0.5 nM (Fig. 4bGo).

Inactivation of the [3H]thymidine incorporation response.
The effects of melatonin (1 nM), alone or in combination with GF (500 nM), on [3H]thymidine incorporation by the cells is shown in Fig. 5Go. In the absence of GF, maximal inhibition of [3H]thymidine incorporation was observed 1 h after the addition of melatonin, and the incorporation recovered to 84 ± 5% of the control value within 24 h of treatment. In the presence of GF, the inactivation of melatonin’s effect was significantly delayed, so that maximal inhibition of [3H]thymidine incorporation was maintained for over 4 h, and by 24 h of melatonin treatment, [3H]thymidine incorporation recovered to only 64 ± 6% of control values (Fig. 5Go).



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  		Figure 5. Effects of melatonin on [3H]thymidine incorporation into prostate epithelial cells. Cells were pretreated with buffer (open bars) or GF (solid bars) for 30 min and then incubated with melatonin (10-8 M) or the same volume of vehicle for the specified period of time, and the incorporation of [3H]thymidine was assessed. Results are the mean ± SEM of three independent studies performed in quintuplicate and are expressed as a percentage of the incorporation in control vehicle-treated cells. *, P < 0.05, within pairs.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The data presented here show for the first time that melatonin receptors can be desensitized by PKC and that this desensitization may be a major cause of the transient nature of melatonin responses in human prostate cells. The inactivation of [125I]melatonin-binding sites by PKC was observed for MF as well as intact cells, suggesting a similarity between the receptors present in the cultured cells and those in tissue-derived microsome-enriched fraction. Melatonin is able to cross the blood-brain barrier and cell membranes and enter cellular compartments (9). Nevertheless, melatonin might be acting through plasma membrane receptors whose density is too low to be measured by radioligand binding. It is presently unknown whether the prostate melatonin receptors belong to any of the hitherto cloned melatonin receptor families. Like the Mel-1 receptors, the microsome-associated receptors seem to be coupled to GTP-binding proteins (6, 7). Some G protein-coupled receptors have been shown to undergo recycling through intracellular endosomes as part of a ligand-induced desensitization mechanism. Although the existence of such mechanisms have not been studied in Mel-1 receptors, the possibility exists that the microsome-associated melatonin receptors in the prostate represent recycling of the cell surface melatonin receptors. Preliminary findings (Gilad, E., A. Rimler, G. M. Brown, H. Matzkin, and N. Zisapel, unpublished observations) suggest that the human prostate microsomal receptors contain epitopes that are recognized by specific anti Mel-1a receptor antibodies (25). Inactivation by PKC is compatible with the presence of multiple PKC phosphorylation sites in the intracellular domain of the Mel-1a receptors (2). However, in NIH-3H3 cells transfected with this clone, Mel-1a receptors apparently do not become desensitized by PKC (19). These results suggest nonidentity of the prostate epithelial and Mel-1a melatonin receptors. Direct evidence for phosphorylation of the prostate receptors awaits the development of specific antibodies for the melatonin receptor subtypes.

Activation of endogenous PKC in prostate cells by TPA induced a marked (~10-fold) decrease in the apparent affinity of the receptors without an apparent change in the density of sites. Such phenomena may be indicative of uncoupling of the receptors and associated G proteins, as shown for a number of G protein-coupled receptors (26, 27). Concomitantly with the decrease in [125I]melatonin binding, TPA desensitized prostate epithelial cells to melatonin, as evidenced by the loss of ability of melatonin to inhibit [3H]thymidine incorporation and suppress the cGMP content. These effects of TPA were all prevented by the PKC inhibitor GF. Together, these data are compatible with the idea that a PKC phosphorylation site is localized at or close to a site that participates in melatonin binding or coupling to G protein.

Interestingly, alkylation by IAA restored [125I]melatonin binding in PKC-treated as well as DTT-treated MF. These results imply that the PKC phosphorylation sites are in proximity to a cysteine residue(s) that is crucial for melatonin binding or for the interaction of melatonin receptors with the coupled G proteins. The suppression of agonist binding by DTT treatment may resemble that demonstrated in other G protein-coupled receptors, e.g. N-methyl-D-aspartate (28) and GH-releasing hormone receptors (29).

Complete reinstatement of the binding by IAA treatment was observed with TPA-activated PKC, whereas only partial reinstatement was observed with Ca2+-activated PKC. This implies differences in phosphorylation sites of the receptor protein between TPA-activated and Ca2+-activated PKC subtypes. The brain PKC preparation used by us is known to contain both subtypes, whereas human BPH tissue reportedly contains TPA-activated, but is devoid of Ca2+-activated, PKC isoforms (20). Hence, in prostate epithelial cells, the phosphorylation of melatonin receptors may be predominantly mediated by the TPA-activated PKC isoform(s).

Preincubations (>4 h) with TPA have been repeatedly shown to cause down-regulation of PKC (30). However, long term (18-h), like short term (30-min), treatment of epithelial cells with TPA effected a decrease in the apparent binding affinity of melatonin receptors. In addition, melatonin responses were not reinstated in the cells after long term treatment with TPA. Several possible explanations may be offered for the apparent lack of down-regulation of PKC, which inactivates melatonin receptors. First, the PKC present in prostate epithelial cells may belong to a nonregulating or slowly down-regulating isoform (30). Secondly, the inactivation of melatonin receptors may not be reversible, or reactivation may be slow. In such a case, reinstatement of active receptor may take longer than the 18 h of the experiment. Another possibility is that, as in the steroid-dependent prostate tumor cells LNCaP (31), long term treatment of benign prostate epithelial cells with TPA induced cell death (not shown). The diminished [3H]thymidine incorporation after 18 h of TPA treatment may be too low for further inhibition by melatonin to be detected.

The PKC-induced inactivation of melatonin receptors in the epithelial cells may underlie the transient nature of melatonin responses in these cells. This possibility is compatible with the ability of GF to retard the inactivation of melatonin-mediated suppression of [3H]thymidine incorporation. Moreover, in the presence of GF, the apparent IC50 for the melatonin-mediated suppression of [3H]thymidine incorporation and cGMP in the cells decreased about 10-fold, suggesting supersensitivity to melatonin. These data imply that receptor desensitization limits the action of melatonin to a period comparable to the duration of nocturnal melatonin production.

The involvement of PKC in agonist-induced desensitization of melatonin receptors implies that melatonin activates PKC either directly or indirectly in the course of its action. A melatonin-mediated activation of PKC is compatible with recent findings in human monocytes and in Mel-1a receptor-transfected NIH-3T3 cells (18, 19). Melatonin has been shown to decrease Ca2+ influx in several systems, including neonatal rat pituitary, rat hypothalamus, and muscles (32, 33, 34, 35). Hence, activation of PKC by melatonin may be indirect rather than via Ca2+-mediated activation of phospholipase C. The latter conclusion is compatible with the fact that melatonin responses in the epithelial cells (inhibition of thymidine incorporation and cGMP) were not inhibited by GF. Future studies will be dedicated to the elucidation of this possibility in prostate cells.

Received June 12, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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