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Endocrinology Vol. 138, No. 12 5275-5281
Copyright © 1997 by The Endocrine Society

ARTICLES

Norepinephrine Stimulates Mitogen-Activated Protein Kinase Activity in GT1–1 Gonadotropin-Releasing Hormone Neuronal Cell Lines

Takeshi Sawada, Masahide Ohmichi, Koji Koike, Yuuki Kanda, Akiko Kimura, Kanji Masuhara, Hiromasa Ikegami, Masaki Inoue, Akira Miyake and Yuji Murata

Department of Obstetrics and Gynecology, Osaka University Medical School (T.S., M.O., Y.K., A.K., K.M., H.I., A.M., Y.M.), 2–2 Yamadaoka, Suita-shi, Osaka 565; and the Department of Obstetrics and Gynecology, Kanazawa University Medical School (K.K., M.I.), 13–1 Takaramachi Kanazawa-shi, Ishikawa 920, Japan; and the Division of Endocrinology, Children’s Hospital Medical Center, and Perinatal Research Institute, University of Cincinnati College of Medicine (Y.K.), Cincinnati, Ohio 45229

Address all correspondence and requests for reprints to: Dr. Masahide Ohmichi, Osaka University Medical School, 2–2 Yamadaoka, Suita, Osaka 565, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The GT1–1 GnRH neuronal cell lines exhibit highly differentiated properties of GnRH neurons. We have used GT1–1 cells to study the roles of norepinephrine (NE), membrane depolarization, calcium influx, and phorbol esters in the regulation of mitogen-activated protein (MAP) kinase. NE, which is known to stimulate the release of GnRH, induced MAP kinase activity, the tyrosine phosphorylation of MAP kinase, and MAP kinase kinase activity. Forskolin led to activation of MAP kinase comparable with that induced by NE, and a selective inhibitor of cAMP-dependent protein kinase, H8, attenuated the NE-induced activation of MAP kinase. On the other hand, elimination of extracellular calcium by EGTA completely blocked NE-induced tyrosine phosphorylation of MAP kinase, and a selective inhibitor of calcium/calmodulin-dependent protein kinase, KN-62, attenuated the NE-induced activation of MAP kinase. Furthermore, depolarization of GT1–1 cells with 75 mM KCl, 10 µM BayK 8644, or 1 µM calcium ionophore (A23187) induced rapid tyrosine phosphorylation of MAP kinase. The omission of calcium from the extracellular medium completely abolished these effects of tyrosine phosphorylation of MAP kinase. Phorbol 12-myristate 13-acetate (PMA) also induced MAP kinase activity, but pretreatment of the cultured cells with PMA to down-regulate protein kinase C did not abolish the activation of MAP kinase by NE. In addition, although phosphorylation of Raf-1 kinase was stimulated by PMA, this phosphorylation was not induced by either NE or A23187. These results demonstrate that NE activates MAP kinase directly in GT1–1 cells, and that the effect of NE is mediated by increase in the cAMP level and by calcium influx, but not by PMA-sensitive protein kinase C or Raf-1 kinase.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE INTERMITTENT secretion of GnRH from the hypothalamus is responsible for the regulation of gonadotropins during reproductive processes. In the past, it was difficult to study the regulatory mechanisms at the level of the GnRH-releasing neuron because of the small number and scattered localization of these neurons in the hypothalamus (1). For instance, it was unknown which signaling pathways coupled the actions of neurotransmitters, neuromodulators, and hormones to GnRH secretion (2). In vitro studies using mediobasal hypothalamus or median eminence tissue fragments demonstrated the calcium dependency of GnRH release induced by depolarization with K+ (3, 4, 5). It was also shown that GnRH was released in response to manipulation of ion channels (5), PGE2 (6, 7), leukotrienes C4 and D4 (8), cAMP (9, 10), and protein kinase C (11, 12). However, these preparations contain many local neuronal networks, making it difficult to determine whether activation of these signaling pathways alters GnRH neuronal activity directly or via transsynaptic effects.

Recently, many groups reported the regulatory mechanisms of GnRH using hypothalamic GnRH neurons (GT1 cell lines). GT1 cell lines (GT1–1, GT1–3, and GT1–7) were developed by genetically targeted tumorigenesis in a transgenic mouse. Cell-specific expression of the potent oncogene simian virus 40 T antigen is regulated in these cells by the promotor/enhancer elements of the GnRH gene (13). These cells have a characteristic neuronal phenotype, and the GnRH gene is expressed and processed at high levels (13, 14).

Norepinephrine (NE) (15), depolarization induced by high K+ (3, 4, 5), the entry of extracellular calcium (16), and protein kinase C (11, 12) are known to induce the secretion of GnRH from GT1 cells. The mechanism of these stimulatory effects is not clearly understood. Recently, it has been reported that membrane depolarization and calcium influx stimulate mitogen-activated protein kinase (MAP kinase) in the nervous system (17). MAP kinase is a serine/threonine kinase that has a diverse array of cellular targets, suggesting that it is a central regulator of many cellular responses (18, 19, 20, 21). Although the genes of MAP kinase (ERK1 and ERK2) are highly expressed in the nervous system (22), the role that MAP kinase activation may play in neurons is not yet clear. MAP kinase appears to be localized primarily in neuronal cell bodies and dendrites, consistent with a postsynaptic function in neuronal signaling (23). Taken together, these concepts led us to examine the effects of agents that are known to stimulate the secretion of GnRH on the MAP kinase activity and the tyrosine phosphorylation of MAP kinase in GT1–1 cells. We show here that MAP kinase is acutely activated and tyrosine phosphorylated by NE, which is mediated by an increase in cAMP and by calcium influx and is not mediated by phorbol ester-sensitive protein kinase C. In addition, we provide evidence that the phosphorylation of Raf-1 kinase does not seem to be involved in the NE-induced MAP kinase activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
NE, phorbol 12-myristate 13-acetate (PMA), and KCl were purchased from Sigma Chemical Co. (St. Louis, MO). A23187 was purchased from Wako Pure Chemical Industries (Tokyo, Japan), BayK 8644 was obtained from Research Biochemicals (Natick, MA), and H8 and KN-62 were obtained from Peptide Institute (Osaka, Japan). ECL Western blotting detection reagents were obtained from Amersham Corp. (Arlington Heights, IL). [{gamma}-32P]ATP (3000 Ci/mmol) was obtained from New England Nuclear (Bannockburn, IL). Antiphosphotyrosine (PY20) and mouse monoclonal anti-MAP kinase were obtained from Upstate Biotechnology (Lake Placid, NY). ERK1 rabbit polyclonal anti-MAP kinase antiserum was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Raf-1 antiserum was developed as described previously (24). Anti-MAP kinase kinase (anti-MEK) antiserum was the gift of Dr. Kunliang Guan (25).

Cell culture
Murine GT1–1 cells (a generous gift from Dr. Richard I. Weiner, University of California-San Francisco) were cultured in DMEM with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were plated on 60-mm culture dishes for tyrosine phosphorylation of MAP kinase assay or on 100-mm culture dishes for MAP kinase, MEK, and Raf-1 kinase assay and were maintained at 37 C in a water-saturated atmosphere of 95% O2 and 5% CO2 until they reached confluence. The medium was replaced by DMEM without serum 16 h before the experiments. Only cells between passages 8–24 were used for experiments.

Assay of MAP kinase activity
Cells were incubated in the absence of serum for 16 h and then treated with various materials. They were washed twice with PBS and lysed in ice-cold HNTG buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EDTA, 10 mM sodium pyrophosphate, 100 µM sodium orthovanadate, 100 mM NaF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonylfluoride] (26). The extracts were centrifuged to remove cellular debris, and the protein content of the supernatants was determined using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Richmond, CA). ERK1 rabbit polyclonal antibody was bound to protein A-Sepharose beads, and 300 µg protein from the lysate samples were immunoprecipitated at 4 C for 2 h. The immunoprecipitated products were washed once in HNTG buffer, twice in 0.5 M LiCl-0.1 M Tris, pH 8.0, and once in kinase assay buffer [25 mM HEPES (pH 7.2–7.4), 10 mM MgCl2, 10 mM MnCl2, and 1 mM dithiothreitol], and the samples were resuspended in 30 µl kinase assay buffer containing 10 µg myelin basic protein and 40 µM [{gamma}-32P]ATP (1 µCi) as described previously (21). The kinase reaction was allowed to proceed at room temperature for 5 min and was stopped by the addition of Laemmli SDS sample buffer (27). Reaction products were resolved by 15% SDS-PAGE.

Assay of MEK activity
MEK was assayed as previously described (21). Cells were serum deprived for 16 h. After treatments, cells were lysed in HNTG buffer, and lysates were immunoprecipitated with anti-MEK antiserum (1:200 dilution) for 2 h at 4 C. This antiserum precipitates both MEK1 and MEK2. Immunoprecipitates were mixed with protein A-Sepharose beads for 30 min, and the beads were washed twice with 1 ml HNTG buffer. The sample was then resuspended in 100 µl reaction buffer containing 20 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 1 mM MnCl2, and 1 mM EGTA. Reactions were initiated by the addition of 10 µCi [{gamma}-32P]ATP (50 µM) and 10 µg of a glutathione-S-transferase (GST) fusion protein containing p44MAPK with a lysine to alanine mutation at position 71 (MAPK/KA) (28). As this mutation eliminates the kinase activity of MAPK, kinase activity is attributable solely to the added MEK. After a 15-min incubation at 25 C, the reactions were stopped by the addition of 20 µl Laemmli sample buffer, and phospho-MAPK/KA was detected by SDS-PAGE followed by autoradiography.

Assay of Raf-1 kinase activity
Raf-1 kinase activity was assayed as previously described (24). Briefly, after hormonal treatment, cells were lysed in 1 ml HNTG buffer. Lysates were precleared with rabbit IgG-agarose and precipitated with anti-Raf-1 antiserum. Immunoprecipitates were resuspended in 20 µl reaction buffer, and 2 µl 20 µM [{gamma}-32P]ATP (10 µCi) were added. The reactions were stopped by the addition of Laemmli sample buffer, and equal amounts of protein were electrophoresed by 8% SDS-PAGE, followed by autoradiography.

Immunoblots
For analysis of tyrosine phosphorylation of MAP kinase, cells were grown in 60-mm dishes. After treatment, the cells were washed, and then 100 µl 1% SDS was added. Lysates were heated for 5 min at 100 C and diluted 1:10 with ice-cold HNTG buffer, followed by incubation with anti-MAP kinase antiserum. Immune complexes were precipitated with protein A-Sepharose, and the isolated proteins were analyzed by electrophoresis on 8% SDS-PAGE. Transfer to nitrocellulose, immunoblotting with antiphosphotyrosine antiserum, and washing was performed as described previously (29).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NE stimulation of MAP kinase activity
To evaluate whether MAP kinase is involved in GnRH secretion, we first examined the effect of NE, which is known to directly stimulate the release of GnRH from GT1–1 cells (15), on MAP kinase activity. GT1–1 cells were treated with 10 µM NE for the indicated times. Cell lysates were immunoprecipitated with anti-MAP kinase antibody and examined for MAP kinase by assaying the incorporation of 32P into MBP, followed by SDS-PAGE and autoradiography (Fig. 1AGo). NE produced an increase in this kinase activity within 2.5 min, with the maximum at 5 min and a decline thereafter. Forskolin induced MAP kinase activity (Fig. 1AGo, lane 5) comparable to that induced by NE.



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  		Figure 1. NE stimulation of MAP kinase activity (A) and tyrosine phosphorylation of MAP kinase (B). A, Cells were grown in 100-mm dishes and treated with 10 µM NE for the indicated times (lanes 2–4), with 50 µM forskolin for 5 min (lane 5), or with 75 mM KCl for 1 min (lane 6). Lysates of cells were subsequently immunoprecipitated with an anti-MAP kinase antiserum, and the immunoprecipitates were incubated with [{gamma}-32P]ATP in the presence of myelin basic protein (MBP), as described in Materials and Methods. After the reactions were stopped with the addition of Laemmli sample buffer, SDS-PAGE and autoradiography were performed. Experiments were repeated three times with essentially identical results. B, Cells were grown in 60-mm dishes and treated with 10 µM NE for the indicated times (1, lanes 2–4) or were treated with the indicated concentrations of NE for 5 min (2, lanes 2–5). The cells were then harvested and lysed in 100 µl 1% SDS. The cell lysates were diluted with HNTG buffer and centrifuged. The supernatant was precipitated with an anti-MAP kinase antiserum, and the immunoprecipitated MAP kinase was subjected to SDS-PAGE, followed by immunoblotting with antiphosphotyrosine antiserum. Experiments were repeated three times with essentially identical results.

 
Mitogenic stimuli activate MAP kinase by increasing tyrosine and serine or threonine phosphorylation of the protein due to the activity of the dual specificity of MEK (30). Therefore, hormone-dependent phosphorylation of the predominant form of MAP kinase was evaluated by anti-phosphotyrosine Western blotting of the anti-MAP kinase immunoprecipitates. The GT1–1 cells were treated with 10 µM NE for the indicated times, followed by lysis and evaluation of tyrosine phosphorylation of MAP kinase (Fig. 1BGo). NE produced an increase in tyrosine phosphorylation of MAP kinase within 2.5 min, with the maximum at 5 min and a decline thereafter, showing a similar time frame to that of NE-induced MAP kinase activity. The dose dependence of NE-induced tyrosine phosphorylation of MAP kinase was also evaluated (Fig. 1BGo). GT1 cells were treated with various concentrations of NE for 5 min. Tyrosine phosphorylation of MAP kinase was detected with 100 nM NE; it was maximal at 10 µM and declined slightly at higher concentrations. The dose of NE that stimulates MAP kinase tyrosine phosphorylation is similar to that which stimulates GnRH release (15).

NE stimulation of MEK activity
MAP kinase was phosphorylated and activated by an immediately upstream activating kinase, MEK. Cells were treated with 10 µM NE for the indicated times. Cell lysates were immunoprecipitated with anti-MEK antibody and assayed for MEK activity by examining the incorporation of 32P into GST-ERK fusion protein (Fig. 2Go). NE produced an increase in this activity within 2.5 min, with the maximum at 5 min and a decline thereafter, showing a similar time frame to the NE-induced MAP kinase activity. Forskolin also induced MEK activity (Fig. 2Go, lane 5) comparable to that induced by NE.



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  		Figure 2. NE stimulation of MEK activity. Cells were grown in 100-mm dishes and treated with 10 µM NE for the indicated times (lanes 2–4) or with 50 µM forskolin (lane 5) for 5 min. Lysates of cells were subsequently immunoprecipitated with an anti-MEK antiserum, and the immunoprecipitates were incubated with [{gamma}-32P]ATP in the presence of GST-ERK fusion protein, as described in Materials and Methods. After the reactions were stopped with Laemmli sample buffer addition, SDS-PAGE and autoradiography were performed. Experiments were repeated three times with essentially identical results.

 
Effect of membrane depolarization and calcium influx on tyrosine phosphorylation of MAP kinase
As it was reported that hypothalamic GnRH release also depends on membrane depolarization and calcium influx (3, 4, 5), we evaluated the effect of calcium influx on the NE-induced tyrosine phosphorylation of MAP kinase. Elimination of extracellular calcium by EGTA completely blocked the NE-induced tyrosine phosphorylation of MAP kinase (Fig. 3AGo), indicating that calcium influx is required for NE-induced MAP kinase phosphorylation. Moreover, we examined the effect of depolarization and calcium influx on the tyrosine phosphorylation of MAP kinase. Depolarization of GT1–1 cells with 75 mM KCl induced rapid activation of MAP kinase (Fig. 1AGo, lane 6) and tyrosine phosphorylation of MAP kinase (Fig. 3BGo, lane 6). The omission of calcium from the extracellular medium by EGTA completely abolished this MAP kinase tyrosine phosphorylation, indicating that activation of MAP kinase is due to calcium influx rather than membrane depolarization per se. To explore this possibility further, we examined the effect of BayK 8644, a calcium L channel opener, or of A23187, a calcium ionophore, on MAP kinase tyrosine phosphorylation: MAP kinase was tyrosine phosphorylated after incubation with BayK 8644 or A23187, and the omission of calcium from the extracellular medium by EGTA completely abolished the phosphorylation (Fig. 3BGo). Elevation of intracellular calcium in response to a variety of stimuli, therefore, causes tyrosine phosphorylation of MAP kinase in GT1–1 cells.



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  		Figure 3. Effect of membrane depolarization and calcium influx on tyrosine phosphorylation of MAP kinase. A, Cells were grown in 60-mm dishes and treated with 10 µM NE for 5 min in the absence (lane 2) or presence (lane 3) of 3 mM EGTA. B, Cells were treated with 10 µM BayK 8644 (lanes 2 and 3) for 5 min, with 1 µM A23187 (lanes 4 and 5) for 5 min, or with 75 mM KCl (lanes 6 and 7) for 1 min in the absence or presence of 3 mM EGTA as indicated. The cells were then harvested and lysed in 100 µl 1% SDS. The cell lysates were diluted with HNTG buffer and centrifuged. The supernatant was precipitated with an anti-MAP kinase antiserum, and the immunoprecipitated MAP kinase was subjected to SDS-PAGE, followed by immunoblotting with antiphosphotyrosine antiserum. Experiments were repeated three times with essentially identical results.

 
Effect of down-regulation of protein kinase C on NE-induced MAP kinase activity
Protein kinase C (11, 12) is also known to induce the secretion of GnRH from GT1 cells. To explore a possible contribution of protein kinase C to NE-induced MAP kinase activity, we used PMA for direct activation of protein kinase C. PMA stimulated MAP kinase activity (Fig. 4Go, lane 4), as did NE. However, the ability of PMA to induce the activation of MAP kinase does not necessarily mean that the protein kinase C pathway is involved in the signaling pathway leading to activation of MAP kinase in response to NE. To test this, we pretreated the cultured cells with PMA for 24 h before NE treatment. The effectiveness of the pretreatment for the down-regulation of protein kinase C was confirmed by the total loss of the acute effect of PMA (Fig. 4Go, lane 5). In contrast, the MAP kinase activity induced by NE was not attenuated by pretreatment with PMA (Fig. 4Go, lane 3), which excludes the possible involvement of a PMA-sensitive protein kinase C.



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  		Figure 4. Effects of down-regulation of protein kinase C on NE-induced MAP kinase activity. Cells were grown in 100-mm dishes and treated with (lanes 3 and 5) and without (lanes 1, 2, and 4) 1 µM PMA for 24 h and then with 1 µM PMA (lanes 4 and 5) or 10 µM NE (lanes 2 and 3) for 5 min. Lysates of cells were subsequently immunoprecipitated with an anti-MAP kinase antiserum, and the immunoprecipitates were incubated with [{gamma}-32P]ATP in the presence of MBP, as described in Materials and Methods. After the reactions were stopped with Laemmli sample buffer, SDS-PAGE and autoradiography were performed. Experiments were repeated three times with essentially identical results.

 
Effect of NE on the phosphorylation of Raf-1 kinase
It was reported that membrane depolarization and calcium influx stimulate MEK and MAP kinase via activation of ras in the nervous system (17). The serine/threonine kinase encoded by the raf protooncogene exists upstream of MAP kinase and is thought to function as the activator of MAP kinase kinase. Protein kinase C activates Raf-1 by direct phosphorylation (31). Protein kinase C does not seem to be involved in NE-induced tyrosine phosphorylation of MAP kinase (Fig. 4Go). Therefore, we investigated whether NE stimulates the phosphorylation of Raf-1 kinase. Cells were treated with NE, A23187, or PMA for 5 min, and lysates were immunoprecipitated with an anti-Raf-1 antiserum. The resulting immunoprecipitates were subjected to an in vitro kinase assay with [{gamma}-32P]ATP, followed by SDS-PAGE, and autoradiography (Fig. 5Go). Activation of phosphorylation of the 74-kDa Raf-1 protein was observed in response to PMA, but was not induced by either NE or A23187.



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  		Figure 5. Effect of NE on the phosphorylation of Raf-1 kinase. Cells were grown in 100-mm dishes and treated with 10 µM NE (lane 2), 1 µM PMA (lane 3), or 1 µM A23187 (lane 4) for 5 min. Lysates were immunoprecipitated with anti-Raf-1 antiserum. Immunoprecipitates were incubated with 2 µl 20 µM [{gamma}-32P]ATP (10 µCi) for 5 min in kinase buffer at 24 C. After the reactions were stopped with Laemmli sample buffer, SDS-PAGE and autoradiography were performed. Experiments were repeated three times with essentially identical results.

 
Effect of protein kinase inhibitor on NE-induced MAP kinase activity
NE is known to bind to adrenergic receptor, followed by stimulation of protein kinase A (15). Stimulation of protein kinase A is reported to increase intracellular calcium (32). To examine the role of A kinase and calcium in NE-induced MAP kinase more clearly, we treated GT-1 cells with a selective inhibitor of cAMP-dependent protein kinase, H8 (33), or a selective inhibitor of calcium/calmodulin-dependent kinase, KN-62 (34). Both the pretreatment with H8 (Fig. 6Go) and that with KN62 (Fig. 7Go) partially attenuated NE-induced MAP kinase activity and tyrosine phosphorylation.



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  		Figure 6. Effect of H8 on NE-induced MAP kinase activity (A) and tyrosine phosphorylation of MAP kinase (B). Cells, grown in 100-mm (A) or 60-mm (B) dishes, were pretreated with 10 µM H8 for 3 h (lane 3), followed by treatment with 10 µM NE for 5 min (lanes 2 and 3). A, Lysates of cells were subsequently immunoprecipitated with an anti-MAP kinase antiserum, and the immunoprecipitates were incubated with [{gamma}-32P]ATP in the presence of MBP, as described in Materials and Methods. After the reactions were stopped with Laemmli sample buffer, SDS-PAGE and autoradiography were performed. Experiments were repeated three times with essentially identical results. B, The cells were then harvested and lysed in 100 µl 1% SDS. The cell lysates were diluted with HNTG buffer and centrifuged. The supernatant was precipitated with an anti-MAP kinase antiserum, and the immunoprecipitated MAP kinase was subjected to SDS-PAGE, followed by immunoblotting with antiphosphotyrosine antiserum. Experiments were repeated three times with essentially identical results.

 


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  		Figure 7. Effect of KN-62 on NE-induced MAP kinase activity (A) and tyrosine phosphorylation of MAP kinase (B). Cells, grown in 100-mm (A) or 60-mm (B) dishes, were pretreated with 1 µM KN-62 for 30 min (lane 3), followed by treatment with 10 µM NE for 5 min (lanes 2 and 3). A, Lysates of cells were subsequently immunoprecipitated with an anti-MAP kinase antiserum, and the immunoprecipitates were incubated with [{gamma}-32P]ATP in the presence of MBP, as described in Materials and Methods. After the reactions were stopped with Laemmli sample buffer, SDS-PAGE and autoradiography were performed. Experiments were repeated three times with essentially identical results. B, The cells were then harvested and lysed in 100 µl 1% SDS. The cell lysates were diluted with HNTG buffer and centrifuged. The supernatant was precipitated with an anti-MAP kinase antiserum, and the immunoprecipitated MAP kinase was subjected to SDS-PAGE, followed by immunoblotting with antiphosphotyrosine antiserum. Experiments were repeated three times with essentially identical results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A variety of neuromediators appear to be involved in signaling in the release of GnRH from hypothalamic neurosecretory neurons (1, 2). One of the first neurotransmitters shown to be involved in the regulation of the ovulatory surge of LH in the rabbit (35, 36) and rat (37) was NE. Although a good deal is known concerning the neuroanatomy of the noradrenergic neurons (38, 39), it is still not clear whether the observed effects on the release of gonadotropins are mediated via noradrenergic synapses on GnRH neurons (40, 41, 42) or via multisynaptic pathways (43) involving {gamma}-aminobutyric acidergic (44) or other interneurons (45, 46). The pharmacological evidence and the messenger RNA analysis are consistent with NE acting through a ß1-adrenergic receptor positively coupled to adenylate cyclase in GT1 cells (15). However, the effect of cAMP on the MAP kinase cascade is dependent on the cell types; it antagonizes the growth factor-activated (47, 48, 49, 50, 51) or oxytocin-activated (19) MAP kinase in some cell types, whereas cAMP itself has a stimulative effect in other cells (52, 53); thus, the mechanisms involved are likely to be complex. In this study, forskolin, which is known to elevate intracellular cAMP, led to activation of MAP kinase in GT1–1 cells comparable with that induced by NE, and inhibitor of protein kinase A attenuated the NE-induced activation of MAP kinase, suggesting that the effect of MAP kinase is mediated by protein kinase A.

Stimulation of protein kinase A is reported to increase intracellular calcium (32). Calcium is a critical mediator of transsynaptic signaling in neurons and affects a wide variety of neuronal responses (54). The cytosolic calcium concentration in resting cells is low (<0.1 µM), but extracellular calcium (1 mM) can rapidly enter neurons in response to membrane depolarization through calcium channels. Presynaptic calcium influx is required for neurotransmitter release and neurite outgrowth (54). Postsynaptic calcium influx can enhance neuronal survival (55). Secretion of GnRH from GT1 cells is coupled to depolarization induced by high K+. Furthermore, superfused GT1 cells secrete GnRH in a calcium-dependent rhythmic pattern of discrete pulses with a frequency similar to that observed in vivo in castrated mice. In this study, we clarified that both membrane depolarization and calcium influx stimulated MAP kinase activation and that the inhibitor of calcium/calmodulin-dependent protein kinase attenuated the NE-induced MAP kinase activation, suggesting that MAP kinase may be a central mediator of calcium signaling in GT1–1 GnRH neuronal cells.

Activation of MAP kinase is induced by phosphorylation of both threonine and tyrosine residues of the enzyme as a result of successive stimulations of Ras, MAP kinase kinase kinase (which may be Raf-1, MEK kinase, or an alternative kinase), and MEK (56, 57). Protein kinase C{alpha} activates Raf-1 by direct phosphorylation (31). We, therefore, analyzed possible involvement of protein kinase C, which is involved in many types of receptor-mediated activation of MAP kinase cascade (56, 57). Direct stimulation of protein kinase C with PMA led to activation of MAP kinase in GT1–1 cells. However, this pathway is not responsible for the effect of NE, because NE still activated MAP kinase, even in the cells in which protein kinase C had been down-regulated. Similar data were reported in NE-induced MAP kinase activation in adipocytes (58). In addition, we examined whether NE induced the phosphorylation of Raf-1 kinase. Although PMA stimulated the phosphorylation of Raf-1 kinase, NE and A23187 had no effect on the phosphorylation of Raf-1 kinase. Thus, it can be concluded that stimulation by NE of MAP kinase in GT1–1 cells involves neither PMA-sensitive protein kinase C nor Raf-1 kinase. Another candidate MEK activator may be the Raf-1-independent MEK kinase (25) that was cloned by homology with yeast kinases (59). Alternatively, a novel MEK kinase may be involved (60, 61).

In conclusion, we have demonstrated that the sympathetic neurotransmitter NE stimulates a MAP kinase signaling pathway through an increase in cAMP, calcium influx, and the subsequent activation of the dual specificity kinase MEK, not mediated by a phorbol ester-sensitive protein kinase C. Determining the biological effects of MAP kinase activation in GT1 GnRH neuronal cells should further understanding of the molecular mechanisms involved in GnRH release and synthesis.

Received June 23, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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