- Email: [email protected]
Melatonin elicits protein kinase C-mediated calcium response in immortalized GT1–7 GnRH neurons
BR A IN RE S E A RCH 1 4 35 ( 20 1 2 ) 2 4 –28
Available online at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research Report
Melatonin elicits protein kinase C-mediated calcium response in immortalized GT1–7 GnRH neurons Haluk Kelestimura,⁎, Mete Ozcanb , Emine Kacara , Ergul Alcinc , Bayram Yılmazd , Ahmet Ayare a
Firat University, Medical School, Department of Physiology, Elazig, Turkey Firat University, Medical School, Department of Biophysics, Elazig, Turkey c Inonu University, Medical School, Department of Physiology, Malatya, Turkey d Yeditepe University, Medical School, Department of Physiology, Istanbul, Turkey e Karadeniz Technical University, Medical School, Department of Physiology, Trabzon, Turkey b
A R T I C LE I N FO
AB S T R A C T
Article history:
Melatonin is suggested to have effects on hypothalamic–pituitary–gonadal (HPG) axis. The
Accepted 18 November 2011
pulsatile pattern of GnRH release, which results in the intermittent release of
Available online 1 December 2011
gonadotropic hormones from the pituitary, has a critical importance for reproductive function but the factors responsible from this release pattern are not known. Calcium is a
Keywords:
second messenger involved in hormone release. Therefore, investigation of the effects of
Melatonin
melatonin on intracellular free calcium levels ([Ca2+]i) would provide critical information
Calcium signaling
on hormone release in immortalized GnRH neurons. The pattern of melatonin-induced in-
GT1–7 cell
tracellular calcium signaling was investigated by fluorescence calcium imaging using the immortalized GnRH-secreting GT1–7 hypothalamic neurons. Melatonin caused a significant increase in [Ca2+]i, which was greatly blocked by luzindole, a melatonin antagonist, or attenuated by pre-treatment with protein kinase C inhibitor. This study suggests that melatonin seems to have a direct effect on GnRH neurons. © 2011 Elsevier B.V. All rights reserved.
1.
Introduction
Melatonin is produced and secreted by the pineal gland with a circadian rhythm characterized by low levels during the day and peak values at night (Armstrong, 1989; Moller, 1992). Therefore, it is so-called darkness hormone. It has been known that there is a connection between the pineal gland and human reproductive function for a long time through clinical observation of the effects of pineal tumors on human sexual development (Binkley, 1988). It is suggested that the pineal gland exerts an inhibitory role on the pubertal maturation in humans because there is a causal relationship between puberty onset and a reduction in pineal melatonin
production at the period of pubertal development (Attanasio et al., 1985). Melatonin is also suggested to be involved in the precocious puberty process (de Holanda et al., 2010). Melatonin levels were found to be low in precocious puberty (Waldhauser et al., 1991) and high in women with stressinduced, exercise-induced or functional hypothalamic hypogonadism (Berga et al., 1988; Bergiannaki et al., 1995; Laughlin et al., 1991). It has been postulated that, before puberty, the decline of serum melatonin represents the activating signal for the hypothalamic pulsatile secretion of GnRH, which is necessary for the activation of gonadotropic axis, and thereafter the onset of pubertal changes (Silman, 1991). In rodents and in seasonal breeders, the pineal gland has been shown to
⁎ Corresponding author at: Firat University Medical School, Department of Physiology, 23119 Elazig, Turkey. Fax: + 90 424 2379138. E-mail address: [email protected] (H. Kelestimur). 0006-8993/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2011.11.040
BR A I N R ES E A RCH 1 4 35 ( 20 1 2 ) 2 4 –28
convey photoperiodic information through its nocturnal melatonin secretion, and to modulate reproductive activity (Arendt, 1995; Goldman and Elliott, 1988). In animals, it has been reported that the pineal exerts an inhibitory role on the pubertal maturation (Buchanan and Yellon, 1991; Sizonenko et al., 1985). In the present experiment, the effects of melatonin on intracellular cascade were investigated in immortalized GnRH neurons. The pulsatile release of GnRH resulting in the intermittent release of gonadotropic hormones from the pituitary (Terasawa, 1995) has a critical importance for reproductive function. Therefore, it is important that intracellular signaling cascade initiated by melatonin treatment in GnRH neurons is established. The mechanism by which melatonin exerts its effects on hypothalamus–pituitary–gonadal axis is controversial. Melatonin is suggested to have a direct (Roy and Belsham, 2002; Roy et al., 2001) or an indirect action (Glass and Knotts, 1987; Lincoln and Maeda, 1992; Malpaux et al., 1993; Revel et al., 2006) on GnRH neurons. In the present experiment, we looked at the effects of melatonin on intracellular calcium concentration, which is a critical component in the exocytosis, in GT1–7 cells to obtain additional evidence for the mechanism in the release of GnRH. Protein kinase C (PKC) inhibitor was used to explore whether phospholipase C (PLC)–PKC pathway is involved in effects of melatonin in GT1–7 cells. Immortalized GT1–7 cells (Mellon et al., 1990) are generally used as an in vitro model system of GnRH neurones because neurons responsible for producing GnRH are too rare and scattered to study in the hypothalamus regions.
2.
Results
Melatonin caused intracellular [Ca2+]i transients in GT1–7 cells in a dose-dependent manner. Effects of melatonin on the fura-2AM 340/380 nm fluorescence ratio due to changes in [Ca2+]i were tested for 1, 10 and 100 μM (Fig. 1). The increases in [Ca2+]i as % of preceding control (baseline) levels were 117.8 ± 4.9% (p < 0.01, n = 68) and 157.5 ± 7.2% (p < 0.001, n = 86) produced by 10 μM and 100 μM melatonin, respectively
Fig. 2 – Melatonin (100 μM) induces a transient Ca2+ elevation in a GT1–7 cell bathed in calcium free solution (n = 36) as seen in the graphs (A) (original trace) and (B) (bar graph). * p < 0.01 vs baseline level (unpaired t test). (Fig. 1). However 1 μM melatonin (n = 25) evoked only a small increase as 102.0 ± 4.7% in [Ca2+]i above pretest baseline levels but this tiny effect was not significant (Fig. 1). A significant elevation in [Ca2+]i response to melatonin also occurred in calcium-free conditions (p < 0.01, n = 36; Fig. 2). We used luzindole, the melatonin receptor antagonist, for pharmacological characterization of the melatonin-induced [Ca2+]i response. Peak [Ca2+]i response to melatonin (100 μM) was significantly reduced to 9.1 ± 3.7% by the application of 10 μM luzindole (Figs. 3A–C; P < 0.001, n = 54). One of the possibilities of the mechanism of the melatonin induced [Ca2+]i rise could be the activation of PKC. The ability of the PKC antagonist, chelerythrine chloride, to affect the [Ca2+]i response to melatonin was examined. When the GT1–7 cells were tested with melatonin in the presence of 1 μM PKC inhibitor, the peak [Ca 2+]i response to 100 μM melatonin was significantly reduced (p < 0.001, n = 62) to 15.8 ± 6.5% (Figs. 3B and C). Apparently PKC-mediated signaling pathways play an important role in the melatonin-induced [Ca2+]i response in immortalized GT1–7 cells.
3.
Fig. 1 – Melatonin provokes calcium elevation in GT1–7 cells in a dose-dependent manner. 1 μM (n = 25), 10 μM (n = 68) and 100 μM (n = 86) melatonin stimulated Ca2+ responses in different GT1–7 cells, in the presence of extracellular Ca2+. Bar graph compares effects of different concentrations of melatonin on percentage of total fluorescence ratio. * p < 0.01 and ** p < 0.001 vs baseline level (one-way analysis of variance followed by a post-hoc Tukey HSD test).
25
Dıscussıon
The results from this study indicate an increase in [Ca2+]i levels in GT1–7 cell lines treated with melatonin. This result may be considered as indirect evidence on the expression of melatonin receptors in GT1–7 cells. Inhibition of the effects of melatonin on [Ca2+]i levels by the melatonin receptor antagonist luzindole provides further evidence that melatonin action on intracellular calcium levels is mediated by melatonin receptors in the cell membrane. The melatonin-induced increases in [Ca2+]i levels were inhibited by protein kinase C inhibitor indicating involvement of PKC-mediated pathway.
26
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Fig. 3 – Summary of the effects of (A) melatonin receptor antagonist luzindole, (B) PKC inhibitor, chelerythrine chloride (ChCl) on melatonin fluorescence calcium responses in GT1–7 cells. (C) Cells were stimulated by melatonin (100 μM) and then [Ca2+]i response to the luzindole (10 μM, n = 54) and ChCl (10 μM, n = 62) were investigated in the presence of extracellular Ca2+. *p < 0.01, **p < 0.001 vs melatonin treatment (unpaired t test).
Considering the fact that melatonin inhibits GnRH secretion in GT1–7 cells (Roy and Belsham, 2002; Roy et al., 2001), the observed effect of melatonin-induced increase in [Ca2+]i in this study may seem controversial. However, the pattern of melatonin-induced increase in [Ca2+]i does not resemble kisspeptin-induced increase in [Ca2+]i, which is suggested to be responsible for the pulsatile nature of GnRH secretion
(Ozcan et al., 2011). Kisspeptins are the most potent activators of hypothalamic–pituitary–gonadal (HPG) axis reported to date. Kisspeptin-10 caused a triphasic change characterized by an initial small increase followed by a significant decrease and increase in intracellular free calcium concentrations. Thus, it can be interpreted that the increase in [Ca2+] resulting from melatonin treatment may impair pulsatile pattern of GnRH release, which may cause inactivation in gonadotropic cells in the pituitary. Therefore, it can be considered that melatonin acting as GnRH analogue. Due to antigonadal effects, melatonin has been implicated to be used as contraceptive, and it should be further assessed that at high concentrations it may act as GnRH analogue. With regard to the expression of melatonin receptors in GT1–7 cells, some studies report expression of both two subtypes of melatonin receptors (Gillespie et al., 2003; Roy and Belsham, 2002; Roy et al., 2001). Inhibition of the autocrine– paracrine effects of GnRH on GT1–7 cells in the latest study by treatment with the GnRH antagonist cetrorelix suggested that the melatonin 1 (MT1) receptors are expressed in GT1–7 cell but downregulated by GnRH (Ishii et al., 2009). There is also evidence that melatonin indirectly affects GnRH neuronal activity through acting on its afferent neurons (Glass and Knotts, 1987; Lincoln and Maeda, 1992; Malpaux et al., 1993; Revel et al., 2006). However, melatonin suppresses GnRH gene expression in GT1–7 cells (Roy and Belsham, 2002; Roy et al., 2001) and can suppress GnRH secretion by about 45% in immortalized GnRH neurons, indicating direct actions of melatonin on GnRH neurons. Inhibitory actions of melatonin on [Ca2+]i levels in cultured rat sensory neurons (Ayar et al., 2001) indicate that melatonin actions may be mediated through different cellular pathways in different cells. In a previous study, melatonin has been found to cause about 1.6 times increases in the PKC activity compared to the basal activity level. By using the protein kinase C inhibitor bisindolylmaleimide, it has been shown that protein kinase C may be involved in the melatonin-mediated cyclical regulation of GnRH mRNA expression (Roy et al., 2001). Melatonin may have different effects on male and female GnRH neurons. For example, effect of melatonin on the GABAAR currents in male and female GnRH neurons is different, augmenting these currents in male neurons while reducing the currents in female neurons (Sato et al., 2008). Thus, MT1 and MT2 receptor expressions are different male and females. Melatonin also down-regulates rGnRH-1 mRNA expression in GT1–7 cells in a 24-h cyclical pattern (Lee et al., 2008). The results from this study clearly indicate that melatonin causes intracellular calcium signaling in GT1–7 cells. However, the physiological implication of this effect is not completely clear and there is a need for further comprehensive studies targeting mechanism of action.
4.
Experimental procedures
4.1.
Cell culture of GT1–7 cells and reagents
GT1–7 cells (a gift from Dr. Mellon, University of California, San Francisco, CA) were cultured in DMEM (Life Technologies, Inc., Gaithersburg, MD) with 10% fetal bovine serum (JRH, Lenexa, KS), 100 U/ml penicillin, and 100 μg/ml streptomycin (Life
BR A I N R ES E A RCH 1 4 35 ( 20 1 2 ) 2 4 –28
Technologies, Inc.) without phenol red. Cells were plated on poly-D-lysine-coated coverslips for intracellular Ca2+ imaging.
4.2.
unpaired Student's t-test. For all analyses, p < 0.05 was accepted as evidence of significance.
Measurement of [Ca 2+]i
Cells were loaded with the Ca2+-sensitive dye fura-2 acetoxymethyl ester (fura 2-AM, 1 μM, Invitrogen) for about 60 min at 37 °C in a 5% CO2 humidified incubator. The imaging bath solution contained (in mM): 130.0 NaCl, 3.0 KCl, 0.6 MgCl2, 2.0 CaCl2, 1.0 NaHCO3, 5.0 glucose, and 10.0 HEPES. The pH was adjusted with NaOH to 7.4 and the osmolarity was adjusted to 310–320 mOsm by sucrose. All imaging experiments were performed in the dark, at room temperature. Glass coverslips with fura-2-loaded cells were mounted in an imaging/perfusion chamber equipped with perfusion valve system (Warner Instruments, Hamden, CT, USA) which was mounted and viewed through an inverted microscope (Nikon TE2000S, Japan). The bath volume of the chamber was 600 μl. The fura-2 loaded GT1–7 cells were alternately illuminated with 340 nm and 380 nm wavelengths from a 175-W Xenon ozone-free lamp source (Sutter Instruments Co., Novato, CA, USA) optically coupled to the microscope with a liquid guide. A computercontrolled filter wheel (Lambda-10; Sutter Instruments, Novato, CA) was used to switch filters of 340 and 380 nm into the light path. Emitted light was passed through an S-flour 40× objective (oil: N.A. 1.30, W.D. 0.22 mm, Nikon) attached to a Nikon TE2000S inverted microscope, through a 510 nm band-pass emission filter (fura-2 filter set, Semrock Brightline, Invitrogen), and finally into a cooled CCD (charge-coupled device) camera (C4742-95; Hamamatsu Photonics, Japan). An estimate of [Ca2+]i was calculated from the ratio of 340/ 380 nm fluorescence intensity values (after correction by subtraction of background fluorescence) and expressed as a ratio (F340/F380). The maximum fura-2 emission ratio (340/380 nm) response to the application of 100 μM melatonin was used as 100% to normalize PKC inhibitor chelerythrine chloride (ChCl) for each cell. To avoid variations, control and drug treatment values were collected from the same cells, so that data from the same cells could be compared directly. Fura2AM was dissolved in dimethylsulfoxide (DMSO). The final concentration of DMSO in the bathing solution did not exceed 0.2% (v/v), which did not elicit any change in [Ca2+]i by itself in control experiments. Melatonin and luzindole (Sigma Chemical Co., St. Louis, MO, USA) were dissolved in DMSO, and aliquots were frozen. Chelerythrine chloride (Tocris Bioscience) was dissolved in the imaging bath solution, and aliquot was frozen. Each stock solution was diluted to the required concentration minutes before bath application. Melatonin (1 μM, 10 μM and 100 μM) was delivered in the imaging bath solution.
4.3.
27
Statistical analysis
All values illustrated are the mean ± SD. Differences between the baselines and the doses of 1 μM, 10 μM and 100 μM of melatonin on [Ca2+]i were calculated by means of one-way analysis of variance followed by a post-hoc Tukey HSD test. In the presence of melatonin receptor antagonist luzindole and PKC inhibitor chelerythrine chloride (ChCl), the changes in melatonin-induced [Ca2+]i transients and effects of melatonin on [Ca2+]i in the calcium free condition were evaluated using
Acknowledgments We thank Dr. Pamela L. Mellon, Department of Reproductive Medicine, University of California, San Diego, for kindly providing GT1–7 cells. This work was supported by a grant from TUBITAK (Project No. 107T825).
REFERENCES
Arendt, J., 1995. Melatonin and the Mammalian Pineal Gland. Chapman & Hall, London. Armstrong, S.M., 1989. Melatonin and circadian control in mammals. Experientia 45, 932–938. Attanasio, A., Borrelli, P., Gupta, D., 1985. Circadian rhythms in serum melatonin from infancy to adolescence. J. Clin. Endocrinol. Metab. 61, 388–390. Ayar, A., Martin, D.J., Ozcan, M., Kelestimur, H., 2001. Melatonin inhibits high voltage activated calcium currents in cultured rat dorsal root ganglion neurones. Neurosci. Lett. 313, 73–77. Berga, S.L., Mortola, J.F., Yen, S.S., 1988. Amplification of nocturnal melatonin secretion in women with functional hypothalamic amenorrhea. J. Clin. Endocrinol. Metab. 66, 242–244. Bergiannaki, J.D., Paparrigopoulos, T.J., Syrengela, M., Stefanis, C.N., 1995. Low and high melatonin excretors among healthy individuals. J. Pineal Res. 18, 159–164. Binkley, S., 1988. The Pineal: Endocrine and Nonendocrine Function. Prentice Hall, Englewood CliVs, NJ. Buchanan, K.L., Yellon, S.M., 1991. Delayed puberty in the male Djungarian hamster: effect of short photoperiod or melatonin treatment on the Gn-RH neuronal system. Neuroendocrinology 54, 96–102. de Holanda, F.S., Tufik, S., Bignotto, M., Maganhin, C.G., Vieira, L.H., Baracat, E.C., Soares, J.M., 2010. Evaluation of melatonin on the precocious puberty: a pilot study. Gynecol. Endocrinol. 20, 1–5. Gillespie, J.M., Chan, B.P., Roy, D., Cai, F., Belsham, D.D., 2003. Expression of circadian rhythm genes in gonadotropin-releasing hormone-secreting GT1–7 neurons. Endocrinology 144, 5285–5292. Glass, J.D., Knotts, L.K., 1987. A brain site for the antigonadal action of melatonin in the whitefooted mouse (Peromyscus leucopus): involvement of the immunoreactive GnRH neuronal system. Neuroendocrinology 46, 48–55. Goldman, B.D., Elliott, J.A., 1988. Photoperiodism and seasonality in hamsters: role of the pineal gland. In: Stetson, M.H. (Ed.), Processing of Environmental Information in Vertebrates. Springer Verlag, New York. Ishii, H., Sato, S., Yin, C., Sakuma, Y., Kato, M., 2009. Cetrorelix, a gonadotropin-releasing hormone antagonist, induces the expression of melatonin receptor 1a in the gonadotropin-releasing hormone neuronal cell line GT1–7. Neuroendocrinology 90, 251–259. Laughlin, G.A., Loucks, A.B., Yen, S.S., 1991. Marked augmentation of nocturnal melatonin secretion in amenorrheic athletes, but not in cycling athletes: unaltered by opioidergic or dopaminergic blockade. J. Clin. Endocrinol. Metab. 73, 1321–1326. Lee, V.H., Lee, L.T., Chow, B.K., 2008. Gonadotropin releasing hormone: regulation of the GnRH gene. FEBS J. 275, 5458–5478. Lincoln, G.A., Maeda, K.I., 1992. Reproductive effects of placing micro-implants of melatonin in the mediobasal hypothalamus and preoptic area in rams. J. Endocrinol. 132, 201–215.
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Malpaux, B., Daveau, A., Maurice, F., Gayrard, V., Thiery, J.C., 1993. Short-day effects of melatonin on luteinizing hormone secretion in the ewe: evidence for central sites of action in the mediobasal hypothalamus. Biol. Reprod. 48, 752–760. Mellon, P., Windle, J.J., Goldsmith, P.C., Padula, C.A., Roberts, J.L., Weiner, R.I., 1990. Immortalization of hypothalamic GnRH neurons by genetically targeted tumorigenesis. Neuron 5, 1–10. Moller, M., 1992. Fine structure of the pinealopetal innervation of the mammalian pineal gland. Microsc. Res. Tech. 21, 188–204. Ozcan, M., Alcin, E., Ayar, A., Yılmaz, B., Sandal, S., Kelestimur, H., 2011. Kisspeptin-10 elicits triphasic cytosolic calcium responses in immortalized GT1–7 GnRH neurones. Neurosci. Lett. 492, 55–58. Revel, F.G., Saboureau, M., Masson-Pevet, M., Pevet, P., Mikkelsen, J.D., Simonneaux, V., 2006. Kisspeptin mediates the photoperiodic control of reproduction in hamsters. Curr. Biol. 16, 1730–1735. Roy, D., Belsham, D.D., 2002. Melatonin receptor activation regulates GnRH gene expression and secretion in GT1–7 GnRH neurons. Signal transduction mechanisms. J. Biol. Chem. 277, 251–258.
Roy, D., Angelini, N.L., Fujieda, H., Brown, G.M., Belsham, D.D., 2001. Cyclical regulation of GnRH gene expression in GT1–7 GnRH-secreting neurons by melatonin. Endocrinology 142, 4711–4720. Sato, S., Yin, C., Teramoto, A., Sakuma, Y., Kato, M., 2008. Sexually dimorphic modulation of GABA A receptor currents by melatonin in rat gonadotropin-releasing hormone neurons. J. Physiol. Sci. 58, 317–322. Silman, R.E., 1991. Melatonin and the human gonadotrophin-releasing hormone pulse generator. J. Endocrinol. 128, 7–11. Sizonenko, P.C., Lang, U., Rivest, R.W., Aubert, M.L., 1985. Pineal and pubertal development. In: Evered, D., Clark, S. (Eds.), Photoperiodism, Melatonin and the Pineal. Pitman, 117. Ciba Foundation Symposium, London, pp. 208–230. Terasawa, E., 1995. Control of luteinizing hormone-releasing hormone pulse generation in nonhuman primates. Cell. Mol. Neurobiol. 15, 141–164. Waldhauser, F., Boepple, P.A., Schemper, M., Mansfield, M.J., Crowley Jr., W.F., 1991. Serum melatonin in central precocious puberty is lower than in age-matched prepubertal children. J. Clin. Endocrinol. Metab. 73, 793–796.
Available online at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research Report
Melatonin elicits protein kinase C-mediated calcium response in immortalized GT1–7 GnRH neurons Haluk Kelestimura,⁎, Mete Ozcanb , Emine Kacara , Ergul Alcinc , Bayram Yılmazd , Ahmet Ayare a
Firat University, Medical School, Department of Physiology, Elazig, Turkey Firat University, Medical School, Department of Biophysics, Elazig, Turkey c Inonu University, Medical School, Department of Physiology, Malatya, Turkey d Yeditepe University, Medical School, Department of Physiology, Istanbul, Turkey e Karadeniz Technical University, Medical School, Department of Physiology, Trabzon, Turkey b
A R T I C LE I N FO
AB S T R A C T
Article history:
Melatonin is suggested to have effects on hypothalamic–pituitary–gonadal (HPG) axis. The
Accepted 18 November 2011
pulsatile pattern of GnRH release, which results in the intermittent release of
Available online 1 December 2011
gonadotropic hormones from the pituitary, has a critical importance for reproductive function but the factors responsible from this release pattern are not known. Calcium is a
Keywords:
second messenger involved in hormone release. Therefore, investigation of the effects of
Melatonin
melatonin on intracellular free calcium levels ([Ca2+]i) would provide critical information
Calcium signaling
on hormone release in immortalized GnRH neurons. The pattern of melatonin-induced in-
GT1–7 cell
tracellular calcium signaling was investigated by fluorescence calcium imaging using the immortalized GnRH-secreting GT1–7 hypothalamic neurons. Melatonin caused a significant increase in [Ca2+]i, which was greatly blocked by luzindole, a melatonin antagonist, or attenuated by pre-treatment with protein kinase C inhibitor. This study suggests that melatonin seems to have a direct effect on GnRH neurons. © 2011 Elsevier B.V. All rights reserved.
1.
Introduction
Melatonin is produced and secreted by the pineal gland with a circadian rhythm characterized by low levels during the day and peak values at night (Armstrong, 1989; Moller, 1992). Therefore, it is so-called darkness hormone. It has been known that there is a connection between the pineal gland and human reproductive function for a long time through clinical observation of the effects of pineal tumors on human sexual development (Binkley, 1988). It is suggested that the pineal gland exerts an inhibitory role on the pubertal maturation in humans because there is a causal relationship between puberty onset and a reduction in pineal melatonin
production at the period of pubertal development (Attanasio et al., 1985). Melatonin is also suggested to be involved in the precocious puberty process (de Holanda et al., 2010). Melatonin levels were found to be low in precocious puberty (Waldhauser et al., 1991) and high in women with stressinduced, exercise-induced or functional hypothalamic hypogonadism (Berga et al., 1988; Bergiannaki et al., 1995; Laughlin et al., 1991). It has been postulated that, before puberty, the decline of serum melatonin represents the activating signal for the hypothalamic pulsatile secretion of GnRH, which is necessary for the activation of gonadotropic axis, and thereafter the onset of pubertal changes (Silman, 1991). In rodents and in seasonal breeders, the pineal gland has been shown to
⁎ Corresponding author at: Firat University Medical School, Department of Physiology, 23119 Elazig, Turkey. Fax: + 90 424 2379138. E-mail address: [email protected] (H. Kelestimur). 0006-8993/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2011.11.040
BR A I N R ES E A RCH 1 4 35 ( 20 1 2 ) 2 4 –28
convey photoperiodic information through its nocturnal melatonin secretion, and to modulate reproductive activity (Arendt, 1995; Goldman and Elliott, 1988). In animals, it has been reported that the pineal exerts an inhibitory role on the pubertal maturation (Buchanan and Yellon, 1991; Sizonenko et al., 1985). In the present experiment, the effects of melatonin on intracellular cascade were investigated in immortalized GnRH neurons. The pulsatile release of GnRH resulting in the intermittent release of gonadotropic hormones from the pituitary (Terasawa, 1995) has a critical importance for reproductive function. Therefore, it is important that intracellular signaling cascade initiated by melatonin treatment in GnRH neurons is established. The mechanism by which melatonin exerts its effects on hypothalamus–pituitary–gonadal axis is controversial. Melatonin is suggested to have a direct (Roy and Belsham, 2002; Roy et al., 2001) or an indirect action (Glass and Knotts, 1987; Lincoln and Maeda, 1992; Malpaux et al., 1993; Revel et al., 2006) on GnRH neurons. In the present experiment, we looked at the effects of melatonin on intracellular calcium concentration, which is a critical component in the exocytosis, in GT1–7 cells to obtain additional evidence for the mechanism in the release of GnRH. Protein kinase C (PKC) inhibitor was used to explore whether phospholipase C (PLC)–PKC pathway is involved in effects of melatonin in GT1–7 cells. Immortalized GT1–7 cells (Mellon et al., 1990) are generally used as an in vitro model system of GnRH neurones because neurons responsible for producing GnRH are too rare and scattered to study in the hypothalamus regions.
2.
Results
Melatonin caused intracellular [Ca2+]i transients in GT1–7 cells in a dose-dependent manner. Effects of melatonin on the fura-2AM 340/380 nm fluorescence ratio due to changes in [Ca2+]i were tested for 1, 10 and 100 μM (Fig. 1). The increases in [Ca2+]i as % of preceding control (baseline) levels were 117.8 ± 4.9% (p < 0.01, n = 68) and 157.5 ± 7.2% (p < 0.001, n = 86) produced by 10 μM and 100 μM melatonin, respectively
Fig. 2 – Melatonin (100 μM) induces a transient Ca2+ elevation in a GT1–7 cell bathed in calcium free solution (n = 36) as seen in the graphs (A) (original trace) and (B) (bar graph). * p < 0.01 vs baseline level (unpaired t test). (Fig. 1). However 1 μM melatonin (n = 25) evoked only a small increase as 102.0 ± 4.7% in [Ca2+]i above pretest baseline levels but this tiny effect was not significant (Fig. 1). A significant elevation in [Ca2+]i response to melatonin also occurred in calcium-free conditions (p < 0.01, n = 36; Fig. 2). We used luzindole, the melatonin receptor antagonist, for pharmacological characterization of the melatonin-induced [Ca2+]i response. Peak [Ca2+]i response to melatonin (100 μM) was significantly reduced to 9.1 ± 3.7% by the application of 10 μM luzindole (Figs. 3A–C; P < 0.001, n = 54). One of the possibilities of the mechanism of the melatonin induced [Ca2+]i rise could be the activation of PKC. The ability of the PKC antagonist, chelerythrine chloride, to affect the [Ca2+]i response to melatonin was examined. When the GT1–7 cells were tested with melatonin in the presence of 1 μM PKC inhibitor, the peak [Ca 2+]i response to 100 μM melatonin was significantly reduced (p < 0.001, n = 62) to 15.8 ± 6.5% (Figs. 3B and C). Apparently PKC-mediated signaling pathways play an important role in the melatonin-induced [Ca2+]i response in immortalized GT1–7 cells.
3.
Fig. 1 – Melatonin provokes calcium elevation in GT1–7 cells in a dose-dependent manner. 1 μM (n = 25), 10 μM (n = 68) and 100 μM (n = 86) melatonin stimulated Ca2+ responses in different GT1–7 cells, in the presence of extracellular Ca2+. Bar graph compares effects of different concentrations of melatonin on percentage of total fluorescence ratio. * p < 0.01 and ** p < 0.001 vs baseline level (one-way analysis of variance followed by a post-hoc Tukey HSD test).
25
Dıscussıon
The results from this study indicate an increase in [Ca2+]i levels in GT1–7 cell lines treated with melatonin. This result may be considered as indirect evidence on the expression of melatonin receptors in GT1–7 cells. Inhibition of the effects of melatonin on [Ca2+]i levels by the melatonin receptor antagonist luzindole provides further evidence that melatonin action on intracellular calcium levels is mediated by melatonin receptors in the cell membrane. The melatonin-induced increases in [Ca2+]i levels were inhibited by protein kinase C inhibitor indicating involvement of PKC-mediated pathway.
26
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Fig. 3 – Summary of the effects of (A) melatonin receptor antagonist luzindole, (B) PKC inhibitor, chelerythrine chloride (ChCl) on melatonin fluorescence calcium responses in GT1–7 cells. (C) Cells were stimulated by melatonin (100 μM) and then [Ca2+]i response to the luzindole (10 μM, n = 54) and ChCl (10 μM, n = 62) were investigated in the presence of extracellular Ca2+. *p < 0.01, **p < 0.001 vs melatonin treatment (unpaired t test).
Considering the fact that melatonin inhibits GnRH secretion in GT1–7 cells (Roy and Belsham, 2002; Roy et al., 2001), the observed effect of melatonin-induced increase in [Ca2+]i in this study may seem controversial. However, the pattern of melatonin-induced increase in [Ca2+]i does not resemble kisspeptin-induced increase in [Ca2+]i, which is suggested to be responsible for the pulsatile nature of GnRH secretion
(Ozcan et al., 2011). Kisspeptins are the most potent activators of hypothalamic–pituitary–gonadal (HPG) axis reported to date. Kisspeptin-10 caused a triphasic change characterized by an initial small increase followed by a significant decrease and increase in intracellular free calcium concentrations. Thus, it can be interpreted that the increase in [Ca2+] resulting from melatonin treatment may impair pulsatile pattern of GnRH release, which may cause inactivation in gonadotropic cells in the pituitary. Therefore, it can be considered that melatonin acting as GnRH analogue. Due to antigonadal effects, melatonin has been implicated to be used as contraceptive, and it should be further assessed that at high concentrations it may act as GnRH analogue. With regard to the expression of melatonin receptors in GT1–7 cells, some studies report expression of both two subtypes of melatonin receptors (Gillespie et al., 2003; Roy and Belsham, 2002; Roy et al., 2001). Inhibition of the autocrine– paracrine effects of GnRH on GT1–7 cells in the latest study by treatment with the GnRH antagonist cetrorelix suggested that the melatonin 1 (MT1) receptors are expressed in GT1–7 cell but downregulated by GnRH (Ishii et al., 2009). There is also evidence that melatonin indirectly affects GnRH neuronal activity through acting on its afferent neurons (Glass and Knotts, 1987; Lincoln and Maeda, 1992; Malpaux et al., 1993; Revel et al., 2006). However, melatonin suppresses GnRH gene expression in GT1–7 cells (Roy and Belsham, 2002; Roy et al., 2001) and can suppress GnRH secretion by about 45% in immortalized GnRH neurons, indicating direct actions of melatonin on GnRH neurons. Inhibitory actions of melatonin on [Ca2+]i levels in cultured rat sensory neurons (Ayar et al., 2001) indicate that melatonin actions may be mediated through different cellular pathways in different cells. In a previous study, melatonin has been found to cause about 1.6 times increases in the PKC activity compared to the basal activity level. By using the protein kinase C inhibitor bisindolylmaleimide, it has been shown that protein kinase C may be involved in the melatonin-mediated cyclical regulation of GnRH mRNA expression (Roy et al., 2001). Melatonin may have different effects on male and female GnRH neurons. For example, effect of melatonin on the GABAAR currents in male and female GnRH neurons is different, augmenting these currents in male neurons while reducing the currents in female neurons (Sato et al., 2008). Thus, MT1 and MT2 receptor expressions are different male and females. Melatonin also down-regulates rGnRH-1 mRNA expression in GT1–7 cells in a 24-h cyclical pattern (Lee et al., 2008). The results from this study clearly indicate that melatonin causes intracellular calcium signaling in GT1–7 cells. However, the physiological implication of this effect is not completely clear and there is a need for further comprehensive studies targeting mechanism of action.
4.
Experimental procedures
4.1.
Cell culture of GT1–7 cells and reagents
GT1–7 cells (a gift from Dr. Mellon, University of California, San Francisco, CA) were cultured in DMEM (Life Technologies, Inc., Gaithersburg, MD) with 10% fetal bovine serum (JRH, Lenexa, KS), 100 U/ml penicillin, and 100 μg/ml streptomycin (Life
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Technologies, Inc.) without phenol red. Cells were plated on poly-D-lysine-coated coverslips for intracellular Ca2+ imaging.
4.2.
unpaired Student's t-test. For all analyses, p < 0.05 was accepted as evidence of significance.
Measurement of [Ca 2+]i
Cells were loaded with the Ca2+-sensitive dye fura-2 acetoxymethyl ester (fura 2-AM, 1 μM, Invitrogen) for about 60 min at 37 °C in a 5% CO2 humidified incubator. The imaging bath solution contained (in mM): 130.0 NaCl, 3.0 KCl, 0.6 MgCl2, 2.0 CaCl2, 1.0 NaHCO3, 5.0 glucose, and 10.0 HEPES. The pH was adjusted with NaOH to 7.4 and the osmolarity was adjusted to 310–320 mOsm by sucrose. All imaging experiments were performed in the dark, at room temperature. Glass coverslips with fura-2-loaded cells were mounted in an imaging/perfusion chamber equipped with perfusion valve system (Warner Instruments, Hamden, CT, USA) which was mounted and viewed through an inverted microscope (Nikon TE2000S, Japan). The bath volume of the chamber was 600 μl. The fura-2 loaded GT1–7 cells were alternately illuminated with 340 nm and 380 nm wavelengths from a 175-W Xenon ozone-free lamp source (Sutter Instruments Co., Novato, CA, USA) optically coupled to the microscope with a liquid guide. A computercontrolled filter wheel (Lambda-10; Sutter Instruments, Novato, CA) was used to switch filters of 340 and 380 nm into the light path. Emitted light was passed through an S-flour 40× objective (oil: N.A. 1.30, W.D. 0.22 mm, Nikon) attached to a Nikon TE2000S inverted microscope, through a 510 nm band-pass emission filter (fura-2 filter set, Semrock Brightline, Invitrogen), and finally into a cooled CCD (charge-coupled device) camera (C4742-95; Hamamatsu Photonics, Japan). An estimate of [Ca2+]i was calculated from the ratio of 340/ 380 nm fluorescence intensity values (after correction by subtraction of background fluorescence) and expressed as a ratio (F340/F380). The maximum fura-2 emission ratio (340/380 nm) response to the application of 100 μM melatonin was used as 100% to normalize PKC inhibitor chelerythrine chloride (ChCl) for each cell. To avoid variations, control and drug treatment values were collected from the same cells, so that data from the same cells could be compared directly. Fura2AM was dissolved in dimethylsulfoxide (DMSO). The final concentration of DMSO in the bathing solution did not exceed 0.2% (v/v), which did not elicit any change in [Ca2+]i by itself in control experiments. Melatonin and luzindole (Sigma Chemical Co., St. Louis, MO, USA) were dissolved in DMSO, and aliquots were frozen. Chelerythrine chloride (Tocris Bioscience) was dissolved in the imaging bath solution, and aliquot was frozen. Each stock solution was diluted to the required concentration minutes before bath application. Melatonin (1 μM, 10 μM and 100 μM) was delivered in the imaging bath solution.
4.3.
27
Statistical analysis
All values illustrated are the mean ± SD. Differences between the baselines and the doses of 1 μM, 10 μM and 100 μM of melatonin on [Ca2+]i were calculated by means of one-way analysis of variance followed by a post-hoc Tukey HSD test. In the presence of melatonin receptor antagonist luzindole and PKC inhibitor chelerythrine chloride (ChCl), the changes in melatonin-induced [Ca2+]i transients and effects of melatonin on [Ca2+]i in the calcium free condition were evaluated using
Acknowledgments We thank Dr. Pamela L. Mellon, Department of Reproductive Medicine, University of California, San Diego, for kindly providing GT1–7 cells. This work was supported by a grant from TUBITAK (Project No. 107T825).
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