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Serotonin increases glial cell line-derived neurotrophic factor release in rat C6 glioblastoma cells
Brain Research 1002 (2004) 167 – 170 www.elsevier.com/locate/brainres
Short communication
Serotonin increases glial cell line-derived neurotrophic factor release in rat C6 glioblastoma cells Kazue Hisaoka a,b,*, Akira Nishida a, Minoru Takebayashi a, Tetsuzo Koda a, Shigeto Yamawaki c, Yoshihiro Nakata b a
Department of Psychiatry and Neuroscience, Institute of Clinical Research, National Kure Medical Center, 3-1 Aoyama, Kure 737-0023, Japan b Department of Pharmacology, Division of Clinical Pharmaceutical Sciences, Programs of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan c Department of Psychiatry and Neurosciences, Division of Frontier Medical Science, Programs for Biomedical Research, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan Accepted 2 January 2004
Abstract Antidepressants, which increase monoamine levels, induce glial cell line-derived neurotrophic factor (GDNF) release in C6 cells. Thus, we examined whether monoamines affect on GDNF release in C6 cells. We found that serotonin (5-HT) specifically increased GDNF mRNA expression and GDNF release in a dose- and time-dependent manner. The 5-HT-induced GDNF release was mediated through the MEK/ mitogen-activated protein kinase (MAPK) pathway and, at least, 5-HT2A receptors. The action of 5-HT on GDNF release may provide important insights into the mechanism of antidepressants. D 2004 Elsevier B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Serotonin Keywords: Dopamine; Glial cell line-derived neurotrophic factor; Mitogen-activated protein kinase; Noradrenaline; Serotonin
Glial cell line-derived neurotrophic factor (GDNF) is a member of the GDNF family, which is a distant member of the transforming growth factor-h superfamily. GDNF and its receptor components are broadly expressed in the normal rat brain. Thus, GDNF plays an important role in the development, differentiation, maintenance and survival of distinct and overlapping neuronal populations within the central nervous system (CNS) [1]. GDNF synthesis and release are regulated by many biological factors and multiple signal transduction systems. For example, growth factors (FGF-1, -2 and -9) and cytokines (IL-1h, -6, INF-g and TNF-a) significantly increase GDNF release [2,22]. Ca2 + ionophores and PKC activators also enhance GDNF release, whereas compounds that
* Corresponding author. Department of Psychiatry and Neuroscience, Institute of Clinical Research, National Kure Medical Center, 3-1 Aoyama, Kure 737-0023, Japan. Tel.: +81-823-22-3111; fax: +81-823-21-0478. E-mail address: [email protected] (K. Hisaoka). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.01.009
increase levels of cAMP repress GDNF release in rat C6 glioblastoma cells (C6 cells) [22]. Recent evidence suggests the possibility that monoamines also affect GDNF synthesis and release. We previously demonstrated that long-term treatment with antidepressant drugs, which increase monoamine levels, leads to an increase in GDNF release in C6 cells [9]. Furthermore, R-()-1(benzofuran-2-yl)-2-propylaminopentane (a novel catecholaminergic and serotoninergic activity enhancer), selegiline (a selective inhibitor of monoamine oxidase-B) and apomorphine (a D1/D2 dopamine (DA) agonist) increased GDNF synthesis in mouse astrocytes [13,15,16]. However, it is unclear whether monoamines directly affect GDNF release, so we examined GDNF release in C6 cells using enzymelinked immunosorbent assay (ELISA), following cell treatment with monoamines. C6 cells were grown in Dulbecco’s modified Eagle’s medium (Bio Whittaker, Walkersville, MD) supplemented with 2 mM L-glutamine and 5% fetal bovine serum (JRH Biosciences, Lenexa, KS) in a humidified 10% CO2 atmo-
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sphere. We used C6 cells at between 10 and 40 passages. As described previously [9], the C6 cells were seeded into the wells of 12-well plates (for ELISA) or into 10-cm culture dishes (for RNA preparation) at a density of 2.5 104 cells/ cm2 in growth medium and allowed to adhere for 24 h. The medium was replaced with serum-free Opti-MEMR (Invitrogen, Carlsbad, CA) containing 0.5% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO), and the cells were incubated for another 24 h. The medium was subsequently replaced with fresh Opti-MEMR plus 0.5% BSA (0.5 ml) and the monoamine of interest. GDNF protein levels in cell-conditioned media were determined using a GDNF EmaxR ImmunoAssay System (Promega, Madison, WI) following the manufacturer’s protocols. Total RNA was isolated after serotonin (5-HT) treatment using an ISOGEN kit according to the manufacturer’s instructions (Nippon Gene, Tokyo, Japan). Semiquantitative reverse transcription – polymerase chain reaction (RT – PCR) was performed as described previously [9] using RNA PCR kit (AMV) ver.2.1 according to the manufacturer’s instructions (Takara Biomedicals, Shiga, Japan). Data are presented as mean F S.E.M. values for a number (n) of separate experiments. Statistical analysis was performed using the Student’s t test, Tukey – Kramer or Fisher’s PLSD test followed by a one-way analysis of variance (ANOVA) using SPSS software (SPSS, Chicago, IL). p Values < 0.05 were considered significant. The EC50 value was calculated by Hill plots with linear regression. 5-HT (100 AM, 48 h treatment) increased GDNF release (93.7 F 23.9 pg/ml, n = 6), but noradrenaline (NA; 100 AM, 48 h treatment) and DA (100 AM, 48 h treatment) did not have effects on GDNF release (3.2 F 3.7 and 11.5 F 6.6 pg/ ml, respectively, n = 5). In addition, we examined the effects of NA and DA in other several doses (10 AM to 1 mM) and other time points (9, 24, 33 and 48 h), but we did not find any changes in the GDNF release (data not shown). Although C6 cells express the h1- and h2-adrenergic receptors, dopamine receptors and 5-HT2A receptors [6,8,26], only 5-HT had an effect on GDNF release. The consistency of serotonergic effects in previous studies [9,13,16] and our results suggest the specific involvement of 5-HT in GDNF release. Fig. 1. Effects of 5-HT on GDNF release and GDNF mRNA expression in C6 cells. (A) Cells were treated with 100 AM of 5-HT for 9, 24, 33 and 48 h. The results represent the mean F S.E.M. values of independent experiments, each performed in duplicate (n = 3). **p < 0.01 indicates that the level was significantly different from that in media alone (Tukey – Kramer ). (B) Cells were treated with the indicated concentrations of 5-HT for 48 h. The results represent the mean F S.E.M. values of independent experiments, each performed in duplicate (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 indicate that the level was significantly different from that in media alone (Fisher’s PLSD test). (C) The representative result of RT – PCR analysis of GDNF mRNA in C6 cells is shown. Cells were treated with 100 AM of 5-HT for 24 h. M, marker. The PCR products for GDNF and G3PDH mRNA were 700 and 983 bp, respectively. (D) GDNF mRNA expression is shown as the ratio of GDNF versus G3PDH. The results represent the mean F S.E.M. values of independent experiments (n = 3). *p < 0.05 indicates that the level was significantly different from that in media alone (Student’s t test).
Then, C6 cells were treated with 5-HT (100 AM), and the amount of GDNF released into the medium was assessed after 9, 24, 33 and 48 h. 5-HT increased GDNF release in a time-dependent manner. Furthermore, 5-HT-induced GDNF release was significantly increased after 48 h treatment (Fig. 1A). Subsequently, C6 cells were treated at a range of 5-HT concentrations and the amount of GDNF released into the medium was assessed after 48 h. A significant increase in
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GDNF release was detected following treatment with 100 AM of 5-HT. 5-HT-induced GDNF release reached a maximum at 200 AM, and EC50 was 47.5 AM (Fig. 1B). We also examined the effect of 5-HT on GDNF mRNA expression by RT –PCR. 5-HT (100 AM, 24 h treatment) significantly increased GDNF mRNA expression before significant increase of GDNF release (48 h) in C6 cells (Fig. 1C,D). These data suggest that a close relationship between GDNF mRNA levels and amounts of secreted protein exists in C6 cells, and 5-HT most likely increases GDNF release through elevation of GDNF mRNA levels. It is well known that C6 cells endogenously express 5HT2A receptors [6]. To clarify whether 5-HT2A receptor activation is related to GDNF release induced by 5-HT, C6 cells were pretreated with ketanserin (1 AM), a 5-HT2A receptor antagonist or cyproheptadine (1 AM), a 5-HT2 receptor antagonist for 10 min at 37 jC and subsequently treated with 5-HT (100 AM) for 48 h. Ketanserin and cyproheptadine partially, but significantly, blocked 5-HTinduced GDNF release (Fig. 2). These antagonists had no effect on basal GDNF release. Thus, these findings indicate that the effect of 5-HT on GDNF release is mediated, at least partly, via 5-HT2A receptor activation. In order to clarify the intracellular mechanisms by which 5-HT induces GDNF release, we examined the effects of H89 (a PKA inhibitor), calphostin C (a PKC inhibitor), herbimycin A (a tyrosine kinase inhibitor) and U0126 (a MEK1 inhibitor) on 5-HTinduced GDNF release. We referred to some articles to
Fig. 2. Effect of 5-HT receptor antagonists on 5-HT-induced GDNF release. Cells were pretreated with ketanserin (1 AM) or cyproheptadine (1 AM) for 10 min at 37 jC and subsequently treated with 5-HT (100 AM) for 48 h. The results are expressed as the percentage of the 5-HT treatment group in each experiment, and represent the mean F S.E.M. of independent experiments (n = 9). The value for 100% release of GDNF was 82.4 F 16.8 pg/ml. yyy p < 0.001 compared with the vehicle group and **p < 0.01, ***p < 0.001 compared with the 5-HT treatment group (Student’s t test).
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Table 1 Effect of protein kinase inhibitors on 5-HT-induced GDNF release Treatment group
GDNF released (% of 5-HT treatment)
Vehicle 5-HT (100 AM) 5-HT + H89 (1 AM) 5-HT + U0126 (10 AM) 5-HT + calphostin C (100 nM) 5-HT + herbimycin A (100 nM)
47.3 F 9.9 100.0 F 16.9y 90.3 F 16.6 36.1 F 6.3* 104.2 F 11.4 103.1 F 10.3
Cells were pretreated with one of the inhibitors for 10 min at 37 jC and subsequently treated with 5-HT (100 AM) for 48 h. The results are expressed as the percentage of the 5-HT treatment group, and represent the mean F S.E.M. of independent experiments. (n = 4). The value for 100% release of GDNF was 107.6 F 18.2 pg/ml. y p < 0.05 compared with the vehicle group. * p < 0.05 compared with the 5-HT treatment group (Student’s t-test).
select the concentration of inhibitors [5,7,20,25]. C6 cells were pretreated with an inhibitor for 10 min at 37 jC and subsequently treated with 5-HT (100 AM) for 48 h. Only U0126 (10 AM), but not H89 (1 AM), calphostin C (100 nM) or herbimycin A (100 nM), inhibited the increase of GDNF release induced by 5-HT (Table 1). These data suggest that 5-HT-induced GDNF release occurs via the MEK/mitogenactivated protein kinase (MAPK)-dependent pathway, but probably independent of the PKA, PKC or Src pathways. Although there is no report showing that 5-HT increases GDNF release through CREB activation via MEK/MAPK pathways in C6 cells, some other reports by using different cells suggest this possibility [3,4,18]. Further investigations are needed to clarify this possibility. C6 cells endogenously express 5-HT2A receptors; nevertheless, ketanserin and cyproheptadine did not completely block the effect of 5HT. Thus, 5-HT-induced GDNF release could be mediated by direct activation of 5-HT2A receptors, but the induction of GDNF observed in C6 cells seems to involve other 5-HT receptor subtypes. There is at present molecular and functional evidence for the existence of at least 16 different subtypes of 5-HT receptors. Among them, 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2B and 5-HT7 receptor subtypes have been shown to activate the MAPK cascade [19]. Although there is little information on the presence of these receptor subtypes in C6 cells, additional studies with selective 5HT receptor drugs may further characterize the role of these receptor subtypes in the regulation of GDNF release. Serotonergic neuronal networks are widely distributed in the brain. 5-HT contributes to many physiological and psychological functions such as endocrine regulation, circadian rhythms, food intake, sleep, reproductive activity, motor function, cognition, mood and anxiety [14]. In addition, 5-HT influences neurite outgrowth, neuronal differentiation, neurogenesis and synaptogenesis, and displays a trophic function in the CNS [10,11]. It is possible that 5HT acts like growth factors through activation of its receptors and second messengers such as cAMP, PLC-h, Ca2 + and hg subunits of heteromeric G-proteins. This activation
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may initiate intracellular cascades and intersect with signaling pathways utilized by growth factors [24]. We demonstrated that 5-HT increased GDNF release in this study. Recent evidence shows that 5-HT also increases brainderived neurotrophic factor mRNA expression in C6 cells [12] and in rats [21]. Furthermore, 5-HT increases IL-6 and TNF-a mRNA in rat astrocytes [17]. Although there is no report about expressions of these cytokines in C6 cells, it has been demonstrated that IL-6 and TNF-a have neuroprotective actions [23]. Therefore, another possibility is that 5-HT may exert its trophic action through modulation of neurotrophic factors and cytokines in the CNS. 5-HT has been implicated in many psychiatric disorders. Many medications that are currently used for these disorders act by affecting the serotonergic system. Actually, it has been suggested that antidepressant drugs display their clinical efficacy through protein expression following a change in monoamine levels. The action of 5-HT on expression of neurotrophic factors may yield important insights into the etiology of 5-HT-related psychiatric disorders and may ultimately lead to more selective and effective therapies.
[9]
[10] [11]
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[16]
Acknowledgements This work was supported by a grant from the Ministry of Health, Labor and Welfare of Japan.
[17]
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Short communication
Serotonin increases glial cell line-derived neurotrophic factor release in rat C6 glioblastoma cells Kazue Hisaoka a,b,*, Akira Nishida a, Minoru Takebayashi a, Tetsuzo Koda a, Shigeto Yamawaki c, Yoshihiro Nakata b a
Department of Psychiatry and Neuroscience, Institute of Clinical Research, National Kure Medical Center, 3-1 Aoyama, Kure 737-0023, Japan b Department of Pharmacology, Division of Clinical Pharmaceutical Sciences, Programs of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan c Department of Psychiatry and Neurosciences, Division of Frontier Medical Science, Programs for Biomedical Research, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan Accepted 2 January 2004
Abstract Antidepressants, which increase monoamine levels, induce glial cell line-derived neurotrophic factor (GDNF) release in C6 cells. Thus, we examined whether monoamines affect on GDNF release in C6 cells. We found that serotonin (5-HT) specifically increased GDNF mRNA expression and GDNF release in a dose- and time-dependent manner. The 5-HT-induced GDNF release was mediated through the MEK/ mitogen-activated protein kinase (MAPK) pathway and, at least, 5-HT2A receptors. The action of 5-HT on GDNF release may provide important insights into the mechanism of antidepressants. D 2004 Elsevier B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Serotonin Keywords: Dopamine; Glial cell line-derived neurotrophic factor; Mitogen-activated protein kinase; Noradrenaline; Serotonin
Glial cell line-derived neurotrophic factor (GDNF) is a member of the GDNF family, which is a distant member of the transforming growth factor-h superfamily. GDNF and its receptor components are broadly expressed in the normal rat brain. Thus, GDNF plays an important role in the development, differentiation, maintenance and survival of distinct and overlapping neuronal populations within the central nervous system (CNS) [1]. GDNF synthesis and release are regulated by many biological factors and multiple signal transduction systems. For example, growth factors (FGF-1, -2 and -9) and cytokines (IL-1h, -6, INF-g and TNF-a) significantly increase GDNF release [2,22]. Ca2 + ionophores and PKC activators also enhance GDNF release, whereas compounds that
* Corresponding author. Department of Psychiatry and Neuroscience, Institute of Clinical Research, National Kure Medical Center, 3-1 Aoyama, Kure 737-0023, Japan. Tel.: +81-823-22-3111; fax: +81-823-21-0478. E-mail address: [email protected] (K. Hisaoka). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.01.009
increase levels of cAMP repress GDNF release in rat C6 glioblastoma cells (C6 cells) [22]. Recent evidence suggests the possibility that monoamines also affect GDNF synthesis and release. We previously demonstrated that long-term treatment with antidepressant drugs, which increase monoamine levels, leads to an increase in GDNF release in C6 cells [9]. Furthermore, R-()-1(benzofuran-2-yl)-2-propylaminopentane (a novel catecholaminergic and serotoninergic activity enhancer), selegiline (a selective inhibitor of monoamine oxidase-B) and apomorphine (a D1/D2 dopamine (DA) agonist) increased GDNF synthesis in mouse astrocytes [13,15,16]. However, it is unclear whether monoamines directly affect GDNF release, so we examined GDNF release in C6 cells using enzymelinked immunosorbent assay (ELISA), following cell treatment with monoamines. C6 cells were grown in Dulbecco’s modified Eagle’s medium (Bio Whittaker, Walkersville, MD) supplemented with 2 mM L-glutamine and 5% fetal bovine serum (JRH Biosciences, Lenexa, KS) in a humidified 10% CO2 atmo-
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sphere. We used C6 cells at between 10 and 40 passages. As described previously [9], the C6 cells were seeded into the wells of 12-well plates (for ELISA) or into 10-cm culture dishes (for RNA preparation) at a density of 2.5 104 cells/ cm2 in growth medium and allowed to adhere for 24 h. The medium was replaced with serum-free Opti-MEMR (Invitrogen, Carlsbad, CA) containing 0.5% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO), and the cells were incubated for another 24 h. The medium was subsequently replaced with fresh Opti-MEMR plus 0.5% BSA (0.5 ml) and the monoamine of interest. GDNF protein levels in cell-conditioned media were determined using a GDNF EmaxR ImmunoAssay System (Promega, Madison, WI) following the manufacturer’s protocols. Total RNA was isolated after serotonin (5-HT) treatment using an ISOGEN kit according to the manufacturer’s instructions (Nippon Gene, Tokyo, Japan). Semiquantitative reverse transcription – polymerase chain reaction (RT – PCR) was performed as described previously [9] using RNA PCR kit (AMV) ver.2.1 according to the manufacturer’s instructions (Takara Biomedicals, Shiga, Japan). Data are presented as mean F S.E.M. values for a number (n) of separate experiments. Statistical analysis was performed using the Student’s t test, Tukey – Kramer or Fisher’s PLSD test followed by a one-way analysis of variance (ANOVA) using SPSS software (SPSS, Chicago, IL). p Values < 0.05 were considered significant. The EC50 value was calculated by Hill plots with linear regression. 5-HT (100 AM, 48 h treatment) increased GDNF release (93.7 F 23.9 pg/ml, n = 6), but noradrenaline (NA; 100 AM, 48 h treatment) and DA (100 AM, 48 h treatment) did not have effects on GDNF release (3.2 F 3.7 and 11.5 F 6.6 pg/ ml, respectively, n = 5). In addition, we examined the effects of NA and DA in other several doses (10 AM to 1 mM) and other time points (9, 24, 33 and 48 h), but we did not find any changes in the GDNF release (data not shown). Although C6 cells express the h1- and h2-adrenergic receptors, dopamine receptors and 5-HT2A receptors [6,8,26], only 5-HT had an effect on GDNF release. The consistency of serotonergic effects in previous studies [9,13,16] and our results suggest the specific involvement of 5-HT in GDNF release. Fig. 1. Effects of 5-HT on GDNF release and GDNF mRNA expression in C6 cells. (A) Cells were treated with 100 AM of 5-HT for 9, 24, 33 and 48 h. The results represent the mean F S.E.M. values of independent experiments, each performed in duplicate (n = 3). **p < 0.01 indicates that the level was significantly different from that in media alone (Tukey – Kramer ). (B) Cells were treated with the indicated concentrations of 5-HT for 48 h. The results represent the mean F S.E.M. values of independent experiments, each performed in duplicate (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 indicate that the level was significantly different from that in media alone (Fisher’s PLSD test). (C) The representative result of RT – PCR analysis of GDNF mRNA in C6 cells is shown. Cells were treated with 100 AM of 5-HT for 24 h. M, marker. The PCR products for GDNF and G3PDH mRNA were 700 and 983 bp, respectively. (D) GDNF mRNA expression is shown as the ratio of GDNF versus G3PDH. The results represent the mean F S.E.M. values of independent experiments (n = 3). *p < 0.05 indicates that the level was significantly different from that in media alone (Student’s t test).
Then, C6 cells were treated with 5-HT (100 AM), and the amount of GDNF released into the medium was assessed after 9, 24, 33 and 48 h. 5-HT increased GDNF release in a time-dependent manner. Furthermore, 5-HT-induced GDNF release was significantly increased after 48 h treatment (Fig. 1A). Subsequently, C6 cells were treated at a range of 5-HT concentrations and the amount of GDNF released into the medium was assessed after 48 h. A significant increase in
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GDNF release was detected following treatment with 100 AM of 5-HT. 5-HT-induced GDNF release reached a maximum at 200 AM, and EC50 was 47.5 AM (Fig. 1B). We also examined the effect of 5-HT on GDNF mRNA expression by RT –PCR. 5-HT (100 AM, 24 h treatment) significantly increased GDNF mRNA expression before significant increase of GDNF release (48 h) in C6 cells (Fig. 1C,D). These data suggest that a close relationship between GDNF mRNA levels and amounts of secreted protein exists in C6 cells, and 5-HT most likely increases GDNF release through elevation of GDNF mRNA levels. It is well known that C6 cells endogenously express 5HT2A receptors [6]. To clarify whether 5-HT2A receptor activation is related to GDNF release induced by 5-HT, C6 cells were pretreated with ketanserin (1 AM), a 5-HT2A receptor antagonist or cyproheptadine (1 AM), a 5-HT2 receptor antagonist for 10 min at 37 jC and subsequently treated with 5-HT (100 AM) for 48 h. Ketanserin and cyproheptadine partially, but significantly, blocked 5-HTinduced GDNF release (Fig. 2). These antagonists had no effect on basal GDNF release. Thus, these findings indicate that the effect of 5-HT on GDNF release is mediated, at least partly, via 5-HT2A receptor activation. In order to clarify the intracellular mechanisms by which 5-HT induces GDNF release, we examined the effects of H89 (a PKA inhibitor), calphostin C (a PKC inhibitor), herbimycin A (a tyrosine kinase inhibitor) and U0126 (a MEK1 inhibitor) on 5-HTinduced GDNF release. We referred to some articles to
Fig. 2. Effect of 5-HT receptor antagonists on 5-HT-induced GDNF release. Cells were pretreated with ketanserin (1 AM) or cyproheptadine (1 AM) for 10 min at 37 jC and subsequently treated with 5-HT (100 AM) for 48 h. The results are expressed as the percentage of the 5-HT treatment group in each experiment, and represent the mean F S.E.M. of independent experiments (n = 9). The value for 100% release of GDNF was 82.4 F 16.8 pg/ml. yyy p < 0.001 compared with the vehicle group and **p < 0.01, ***p < 0.001 compared with the 5-HT treatment group (Student’s t test).
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Table 1 Effect of protein kinase inhibitors on 5-HT-induced GDNF release Treatment group
GDNF released (% of 5-HT treatment)
Vehicle 5-HT (100 AM) 5-HT + H89 (1 AM) 5-HT + U0126 (10 AM) 5-HT + calphostin C (100 nM) 5-HT + herbimycin A (100 nM)
47.3 F 9.9 100.0 F 16.9y 90.3 F 16.6 36.1 F 6.3* 104.2 F 11.4 103.1 F 10.3
Cells were pretreated with one of the inhibitors for 10 min at 37 jC and subsequently treated with 5-HT (100 AM) for 48 h. The results are expressed as the percentage of the 5-HT treatment group, and represent the mean F S.E.M. of independent experiments. (n = 4). The value for 100% release of GDNF was 107.6 F 18.2 pg/ml. y p < 0.05 compared with the vehicle group. * p < 0.05 compared with the 5-HT treatment group (Student’s t-test).
select the concentration of inhibitors [5,7,20,25]. C6 cells were pretreated with an inhibitor for 10 min at 37 jC and subsequently treated with 5-HT (100 AM) for 48 h. Only U0126 (10 AM), but not H89 (1 AM), calphostin C (100 nM) or herbimycin A (100 nM), inhibited the increase of GDNF release induced by 5-HT (Table 1). These data suggest that 5-HT-induced GDNF release occurs via the MEK/mitogenactivated protein kinase (MAPK)-dependent pathway, but probably independent of the PKA, PKC or Src pathways. Although there is no report showing that 5-HT increases GDNF release through CREB activation via MEK/MAPK pathways in C6 cells, some other reports by using different cells suggest this possibility [3,4,18]. Further investigations are needed to clarify this possibility. C6 cells endogenously express 5-HT2A receptors; nevertheless, ketanserin and cyproheptadine did not completely block the effect of 5HT. Thus, 5-HT-induced GDNF release could be mediated by direct activation of 5-HT2A receptors, but the induction of GDNF observed in C6 cells seems to involve other 5-HT receptor subtypes. There is at present molecular and functional evidence for the existence of at least 16 different subtypes of 5-HT receptors. Among them, 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2B and 5-HT7 receptor subtypes have been shown to activate the MAPK cascade [19]. Although there is little information on the presence of these receptor subtypes in C6 cells, additional studies with selective 5HT receptor drugs may further characterize the role of these receptor subtypes in the regulation of GDNF release. Serotonergic neuronal networks are widely distributed in the brain. 5-HT contributes to many physiological and psychological functions such as endocrine regulation, circadian rhythms, food intake, sleep, reproductive activity, motor function, cognition, mood and anxiety [14]. In addition, 5-HT influences neurite outgrowth, neuronal differentiation, neurogenesis and synaptogenesis, and displays a trophic function in the CNS [10,11]. It is possible that 5HT acts like growth factors through activation of its receptors and second messengers such as cAMP, PLC-h, Ca2 + and hg subunits of heteromeric G-proteins. This activation
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may initiate intracellular cascades and intersect with signaling pathways utilized by growth factors [24]. We demonstrated that 5-HT increased GDNF release in this study. Recent evidence shows that 5-HT also increases brainderived neurotrophic factor mRNA expression in C6 cells [12] and in rats [21]. Furthermore, 5-HT increases IL-6 and TNF-a mRNA in rat astrocytes [17]. Although there is no report about expressions of these cytokines in C6 cells, it has been demonstrated that IL-6 and TNF-a have neuroprotective actions [23]. Therefore, another possibility is that 5-HT may exert its trophic action through modulation of neurotrophic factors and cytokines in the CNS. 5-HT has been implicated in many psychiatric disorders. Many medications that are currently used for these disorders act by affecting the serotonergic system. Actually, it has been suggested that antidepressant drugs display their clinical efficacy through protein expression following a change in monoamine levels. The action of 5-HT on expression of neurotrophic factors may yield important insights into the etiology of 5-HT-related psychiatric disorders and may ultimately lead to more selective and effective therapies.
[9]
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[16]
Acknowledgements This work was supported by a grant from the Ministry of Health, Labor and Welfare of Japan.
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