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Endocrinology Vol. 141, No. 12 4496-4502
Copyright © 2000 by The Endocrine Society

ARTICLES

Adrenergic Regulation of Mitogen-Activated Protein Kinase in Rat Pinealocytes: Opposing Effects of Protein Kinase A and Protein Kinase G1

A. K. Ho and C. L. Chik

Departments of Physiology and Medicine (C.L.C.), Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

Address all correspondence and requests for reprints to: Dr. A. K. Ho, Department of Physiology, 7–26 Medical Sciences Building, Edmonton, Alberta, Canada T6G 2H7. E-mail: anho{at}ualberta.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The role of adrenergic stimulation in the regulation of mitogen-activated protein kinase (MAPK) in rat pinealocytes was investigated by measuring phosphorylated MAPK using Western blot analysis and a MAPK enzymatic assay. Stimulation with the endogenous neurotransmitter, norepinephrine (NE; a mixed {alpha}- and ß-adrenergic agonist), concentration dependently increased the phosphorylation of both p44 and p42 isoforms of MAPK. This effect of NE was blocked by PD98059 and UO126 (two inhibitors of MEK). Treatment with prazosin or propranolol significantly reduced the effect of NE on MAPK phosphorylation, suggesting the involvement of both {alpha}- and ß-adrenergic receptors. Investigation into the intracellular mechanisms of NE action revealed that the increase in MAPK phosphorylation was blocked by KT5823 (a protein kinase G inhibitor), but was enhanced by H89 (a protein kinase A inhibitor). Calphostin C (a protein kinase C inhibitor) and KN93 (a Ca2+/calmodulin-dependent protein kinase inhibitor) also attenuated NE-mediated MAPK activation, but to a lesser degree. Furthermore, inhibition of MAPK phosphorylation by (Bu)2cAMP was effective in reducing MAPK activation by (Bu)2cGMP, an active phorbol ester or ionomycin. These results indicate that the effect of NE on MAPK phosphorylation represents mainly the integration of two signaling mechanisms, protein kinase A and protein kinase G, each having an opposite effect on MAPK phosphorylation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN RAT PINEALOCYTES, the synthesis of cyclic nucleotides is stimulated by the release of norepinephrine (NE) from the sympathetic nerves at night (1). This stimulation involves activation of both {alpha}1- and ß-adrenergic receptors (1, 2). Stimulation of ß-adrenergic receptors, which activates both adenylyl and guanylyl cyclases, produces a 7- to 10-fold increase in cAMP and a 2- to 4-fold increase in cGMP accumulation (1). In contrast, stimulation of {alpha}1-adrenergic receptors, which activates protein kinase C (PKC) (3, 4) and elevates intracellular Ca2+ ([Ca2+]i) (5, 6), has no direct effect on cAMP or cGMP accumulation. However, simultaneous stimulation of {alpha}1- and ß-adrenergic receptors results in a 50-fold increase in cAMP and a 100-fold increase in cGMP accumulation (1, 2). Both PKC and elevation of [Ca2+]i are involved in the {alpha}1-adrenergic potentiation of ß-adrenergically stimulated cyclic nucleotide accumulation (3, 4, 5, 6). A role of nitric oxide in the NE-stimulated cGMP accumulation has also been confirmed (7). The primary function of cAMP in the rat pineal gland is induction of arylalkyl-N-acetyltransferase (AA-NAT), the rate-limiting enzyme in melatonin synthesis (1). Potentiation of ß-adrenergically stimulated AA-NAT activities by {alpha}1-adrenergic agonist has also been demonstrated (8). In contrast, the physiological consequence of the large increase in cGMP response after NE stimulation remains largely unknown.

The importance of mitogen-activated protein kinase (MAPK) in the regulation of cellular processes such as proliferation, differentiation, secretion, and metabolism has been well established (9, 10). Upon stimulation by growth factors, hormones, or neurotransmitters, MAPK is activated through a signaling cascade that includes Ras, Raf, and phosphorylation of MAPK kinase (MEK) (11, 12). Activated MEK then phosphorylates a threonine and a tyrosine residue on the activation loop of MAPK and activates this enzyme in the process (13, 14). Signaling pathways including those involving cAMP (15, 16, 17, 18, 19), cGMP (19, 20, 21), PKC (22, 23), and elevation of [Ca2+]i (24) have all been shown to play a role in modulating the state of MAPK phosphorylation. Therefore, in cells in which these signaling pathways can be activated simultaneously, MAPK may serve as a site of integration for these input signals in regulating cellular function.

In the rat pineal gland, the presence of p42 and p44 isoforms of MAPK and their activation by cGMP have been established (25, 26). As several signaling pathways are activated after NE stimulation (3, 4, 5, 6, 7), it is quite probable that in addition to cGMP, other signaling mechanisms, such as cAMP, PKC, and elevation of [Ca2+]i, may also modulate the state of MAPK phosphorylation. Therefore, the objectives of the present study were 1) to determine whether MAPK is under adrenergic regulation in rat pinealocytes, 2) to identify the signaling pathways involved, and 3) to determine how these signaling pathways interact in controlling MAPK activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
(Bu)2cGMP, (Bu)2cAMP, isoproterenol (ISO), NE, phenylephrine (PE), 4ß-phorbol 12-myristate 13-acetate (PMA), prazosin (Praz), and propranolol (Prop) were obtained from Sigma (St. Louis, MO). H89, H7, KT5823, KN93, calphostin C, UO124, UO126, PD98059, and ionomycin (ION) were obtained from Calbiochem (San Diego, CA). Polyclonal antibodies against MAPK and a monoclonal antibody against phosphorylated MAPK were purchased from Sigma. The MAPK enzymatic assay kit was obtained from Amersham Pharmacia Biotech (Piscataway, NJ). All other chemicals were of the purest grade available commercially.

Preparation of pinealocytes and drug treatment
Sprague Dawley rats (male; weighing 150 g) were obtained from the University of Alberta Animal Unit. Pinealocytes were prepared from freshly dissected rat pineal glands by trypsinization as described previously (27, 28). The cells were suspended in DMEM containing 10% FCS and were maintained at 37 C for 24 h in a gas mixture of 95% air and 5% CO2 before the experiments. For determination of MAPK activation, aliquots of pinealocytes (5 x 105 cells/0.5 ml) were treated with drugs that had been prepared in concentrated solutions in water or dimethylsulfoxide for 20 min (unless otherwise indicated). Treated cells were collected by centrifugation (2 min, 12,000 x g). Samples for Western blot analysis were solubilized in 1 x sample buffer by boiling for 5 min and were stored until electrophoresis. Samples for the determination of MAPK phosphorylation activities were immediately frozen in dry ice and stored at -75 C.

Western blot
SDS-PAGE was performed according to the procedure of Laemmli (29) using 10% acrylamide in the presence of 1 mg/ml SDS (Mini-Protein II gel system, Bio-Rad Laboratories, Inc., Hercules, CA). After electrophoresis, gels were equilibrated for 20 min in transfer buffer (25 mM Tris, 190 mM glycine, and 20% methanol). Proteins were transferred onto polyvinylidene difluoride membranes (1 h, 100 V), which were then incubated with a blocking solution [5% dried skim milk in 100 mM Tris (pH 7.5) with 140 mM NaCl and 0.01% Tween-20] for a minimum of 1 h. The blots were incubated overnight at 4 C with diluted specific antisera as indicated. After washing twice with the blocking solution, the blots were then incubated with diluted horseradish peroxidase-conjugated second antibodies (Bio-Rad Laboratories, Inc.) for 1 h at room temperature. They were then washed again extensively and developed using enhanced chemiluminescence (Amersham Pharmacia Biotech).

MAPK phosphorylation activities
Frozen cell pellets were homogenized in 10 mM Tris buffer (pH 7.4) containing 150 mM NaCl, 2 mM EGTA, 2 mM dithiothreitol, 1 mM orthovanadate, 1 mM phenylmethylsulfonylfluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. After centrifugation (15,000 x g, 10 min, 4 C), the supernatant was collected for assay using a commercial available kit that used [{gamma}-32P]ATP and an epidermal growth factor receptor-based peptide that is specific for MAPK phosphorylation.

Results and statistical analysis
For the Western blots, a typical blot from at least three similar experiments is shown. MAPK phosphorylation activities are presented as the mean ± SEM from three independent observations performed in duplicate. Statistical comparisons were analyzed by unpaired t test for the kinase measurements.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Activation of MAPK by NE
Using a monoclonal antibody that detected the diphosphorylated form of MAPK (30), NE was found to cause a concentration- and time-dependent increase in phosphorylation of the 42- and 44-kDa isoforms of MAPK (p-MAPK) in rat pinealocytes (Fig. 1Go). An increase in p-MAPK was observed with the addition of 1 µM NE (Fig. 1AGo). At 10 µM NE, an increase in p-MAPK was observed within 5 min, was sustained for at least 15 min, and returned toward the basal level after 60 min (Fig. 1BGo). The increase in p-MAPK after NE stimulation occurred in the absence of any change in MAPK immunoreactivity (Fig. 1Go, A and B). In parallel experiments, NE caused similar increases in MAPK activities using an in vitro MAPK enzymatic assay (Table 1Go).



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  		Figure 1. Effect of NE on MAPK activation. Pinealocytes (5 x 105 cells/0.5 ml) were cultured for 24 h and treated with NE (A; 0.1–10 µM) for 15 min or NE (B; 10 µM) for different time periods as indicated. The cells were then collected by centrifugation, dissolved in 1 x sample buffer, and analyzed by Western blotting using a monoclonal antibody against p-MAPK or polyclonal antibodies against MAPK as described in Materials and Methods. The blot presented is representative of three independent experiments.

 

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  		Table 1. Activation of MAPK activities in rat pinealocytes by NE

 
Role of MEK on NE-stimulated MAPK phosphorylation
PD98059 and UO126, two inhibitors of MEK activation (31, 32), were used to establish that the NE-stimulated MAPK activation was mediated by MEK. Treatment with PD98059 (0.1–10 µM) or UO126 (0.01–1 µM), concentration dependently reduced the NE (10 µM)-stimulated increase in p-MAPK levels (Fig. 2Go). In contrast, UO124 (1 µM), an inactive analog, had no effect on MAPK phosphorylation (Fig. 2BGo). PD98059 (10 µM) and UO126 (1 µM), but not UO124 (1 µM), also blocked the NE-stimulated increase in MAPK activities (Table 1GoB).



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  		Figure 2. Role of MEK in NE-mediated MAPK activation. Pinealocytes (5 x 105 cells/0.5 ml) were treated for 15 min with A) NE (10 µM) in the absence or presence of PD58059 (PD; 0.1–10 µM) and B) NE (10 µM) in the absence or presence of UO126 (10 nM to 1 µM) or UO124 (1 µM). The cells were analyzed by Western blotting using a monoclonal antibody against p-MAPK as described in Materials and Methods. The blot presented is representative of three independent experiments.

 
Receptor characterization of the adrenergic regulation of MAPK activation
Selective agonists and antagonists were used to determine the subtype of adrenergic receptors involved in the activation of MAPK. Selective activation of ß-adrenergic receptors by ISO (1 µM in the presence of 1 µM Praz) had no effect, whereas activation of {alpha}1-adrenerigc receptors by PE (1 µM in the presence of 1 µM Prop) caused a small increase in MAPK activation (Fig. 3AGo). However, combined treatment with ISO and PE caused a large increase in MAPK activation, an effect similar to that of NE (10 µM) (Fig. 3AGo). Furthermore, the presence of either Praz (1 µM), an {alpha}1-adrenergic blocker, or Prop (10 µM), a ß-adrenergic blocker, significantly reduced NE-mediated MAPK activation (Fig. 3BGo). Measurement of MAPK activities showed that combined treatment with ISO and PE produced an effect bigger than the summation of the individual effects of the two agonists (Table 2Go). These results suggest that interaction between the {alpha}1- and ß-adrenergic-mediated signaling pathways probably plays an important role in the regulation of MAPK activation.



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  		Figure 3. Receptor characterization of NE-mediated MAPK activation. Pinealocytes (5 x 105 cells/0.5 ml) were treated for 15 min with A) NE (1 µM), ISO (1 µM) in the presence or absence of PE (1 µM), or PE (1 µM) alone or B) NE (1 µM) in the absence or presence of Prop (1 µM) or Praz (1 µM). The cells were analyzed by Western blotting using a monoclonal antibody against p-MAPK as described in Materials and Methods. The blot presented is representative of three independent experiments.

 

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  		Table 2. Characterization of the receptors involved in NE-stimulated MAPK activities in rat pinealocytes

 
Effects of protein kinase inhibitors on NE-, ION-, and PMA-mediated MAPK activation
To establish the signaling pathways involved in NE-mediated MAPK activation, the effects of several protein kinase activators and inhibitors were tested. Treatments with (Bu)2cGMP (50 µM), PMA (0.1 µM), and ION (1 µM) were effective in increasing MAPK phosphorylation (Fig. 4Go). In contrast, (Bu)2cAMP (50 µM) reduced the phosphorylated level of MAPK (Fig. 4Go). Similar results were obtained when MAPK activities were determined (Table 3Go). When the effects of different protein kinase inhibitors were determined, it was found that H7 (100 µM; a nonselective serine and threonine kinase inhibitor) and KT5823 (1 µM; a PKG selective inhibitor with Ki <0.3 µM) significantly reduced NE-stimulated phosphorylation of MAPK. Both calphostin C (1 µM; a selective PKC inhibitor with Ki <0.05 µM) and KN93 (1 µM; a selective Ca2+/calmodulin-dependent protein kinase inhibitor) also attenuated NE-mediated MAPK activation, but to a lesser degree than KT5823 (Fig. 5AGo). In contrast, H89 (1 µM; a PKA-selective inhibitor with Ki <0.05 µM) caused a significant enhancement of the NE-stimulated response (Fig. 5AGo). Calphostin C and KN93, but not KT5823, were also found to reduce MAPK activation stimulated by PMA or ION, respectively (Fig. 5Go, B and C). None of the five inhibitors had an effect on the basal level of p-MAPK (Fig. 5DGo). The effects of the five inhibitors on NE-stimulated MAPK activities also paralleled those of p-MAPK (Table 4Go).



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  		Figure 4. Effects of protein kinase activators on MAPK activation. Pinealocytes (5 x 105 cells/0.5 ml) were treated for 15 min with NE (10 µM), (Bu)2cAMP (dbcAMP, 50 µM), (Bu)2cGMP (dbcGMP, 50 µM), PMA (0.1 µM), or ION (1 µM). The cells were analyzed by Western blotting using a monoclonal antibody against p-MAPK as described in Materials and Methods. The blot presented is representative of three independent experiments.

 

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  		Table 3. Stimulation of MAPK activities by different protein kinase activators in rat pinealocytes

 


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  		Figure 5. Effects of protein kinase inhibitors on NE-, PMA-, and ION-mediated MAPK activation. Pinealocytes (5 x 105 cells/0.5 ml) were treated for 15 min with NE (A; 10 µM), PMA (B; 0.1 µM), ION (C; 1 µM), or Control (D; Cont) in the absence or presence of H89 (1 µM), H7 (100 µM), KT5823 (1 µM), calphostin C (Cal-C; 1 µM), or KN93 (1 µM). The cells were analyzed by Western blotting using a monoclonal antibody against p-MAPK as described in Materials and Methods. The blot presented is representative of three independent experiments.

 

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  		Table 4. Effects of different protein kinase inhibitors on NE-mediated activation of MAPK activities in rat pinealocytes

 
Contributions of PKA and PKG in the adrenergically mediated MAPK activation
The contributions of PKA and PKG to the adrenergic regulation of MAPK activation were investigated by using specific inhibitors of each pathway. Whereas the PKA inhibitor H89 (1 µM) enhanced NE-mediated MAPK activation, the PKG inhibitor KT5823 (0.1 and 1 µM) reduced NE-activated MAPK activation (Fig. 6AGo). Enhancement by H89 (1 µM) and inhibition by KT5823 (0.1 and 1 µM) were observed when MAPK was activated by ISO (Fig. 6BGo). Thus, it appears that the degree of MAPK activation represents a summation of the opposing effects of cAMP and cGMP.



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  		Figure 6. Effects of KT5823 and H89 on NE- and ISO-mediated MAPK activation Pinealocytes (5 x 105 cells/0.5 ml) were treated for 15 min with NE (A; 10 µM) or ISO (B; 1 µM) in the presence or absence of KT5823 (0.1 and 1 µM) or H89 (0.1 and 1 µM). The cells were analyzed by Western blotting using a monoclonal antibody against p-MAPK as described in Materials and Methods. The blot presented is representative of three independent experiments.

 
Inhibitory effects of cAMP on MAPK activation by different agonists
The inhibitory effect of (Bu)2cAMP on MAPK activation was further investigated. It was found that (Bu)2cAMP (0.01–1 mM) concentration dependently reduced MAPK activation stimulated by (Bu)2cGMP (50 µM). (Bu)2cAMP also reduced PMA (0.1 µM)- and ION (1 µM)-mediated MAPK activation in a concentration-dependent manner (Fig. 7Go, B and C).



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  		Figure 7. Effect of (Bu)2cAMP on MAPK phosphorylation caused by different activating agents. Pinealocytes (5 x 105 cells/0.5 ml) were treated for 15 min with (Bu)2cGMP (A; dbcGMP, 1 mM), PMA (B; 0.1 µM), or ION (C; 1 µM) in the absence or presence of (Bu)2cAMP (A; dbcAMP, 0.01–1 mM). The cells were analyzed by Western blotting using a monoclonal antibody against p-MAPK as described in Materials and Methods. The blot presented is representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The presence of MAPK in the rat pineal gland was first reported by Kiyama et al. (26) using immunohistochemical techniques. Our recent study also established the presence of Raf1, MEK1, and two isoforms of MAPK (p42 and p44), indicating that the components of the MAPK signaling cascade are well established in this neuroendocrine tissue (25). In the present study, by monitoring the phosphorylation state of MAPK as well as measuring the subsequent changes in MAPK activities directly, we showed that NE treatment, although having no effect on the MAPK protein level, significantly increases the levels of p-MAPK and MAPK activities.

Our results on NE-mediated MAPK activation also indicate the involvement of both {alpha}1- and ß-adrenergic receptors. This is based on the observation that the NE-mediated MAPK activation is reduced by treatment with either an {alpha}1- or a ß-adrenergic receptor antagonist. Previously, it has been shown that whereas activation of ß-adrenergic receptors is an absolute requirement, activation of {alpha}1-adrenergic receptors potentiates the ß-adrenergically stimulated cAMP and cGMP accumulation as well as the AA-NAT response (1, 2, 8). Our results show that unlike the cyclic nucleotide or AA-NAT response, activation of ß-adrenergic receptors has no effect on MAPK activation, whereas activation of {alpha}1-adrenergic receptors causes only a small increase in MAPK phosphorylation. However, when both receptors are activated, similar to the cAMP and cGMP accumulation (1, 2, 8), their effects on MAPK activation are bigger than the summation of the individual effect. These observations suggest that even though interactions between the {alpha}1- and ß-adrenergically mediated signaling pathways are important in the regulation of MAPK activation, the postreceptor mechanisms involved are probably different from those that mediate the potentiation of the cyclic nucleotide synthesis or AA-NAT induction (see below).

Downstream from the adrenergic receptors, our study with different protein kinase inhibitors indicates that PKG is the most likely kinase responsible for MAPK activation in rat pinealocytes, because the PKG-selective inhibitor (KT5823) is most effective in blocking the effect of NE. This result is consistent with our earlier report that activation of PKG alone can activate MAPK in rat pinealocytes (25). The effect of KT5823 is specific for the NE-mediated MAPK activation, as a similar concentration of KT5823 has no effect on MAPK phosphorylation stimulated by PMA or ION. As the effect of cGMP can be blocked by PD98059 (25), an inhibitor of MEK (31), it is unlikely that PKG has a direct effect on MAPK phosphorylation. Therefore, our results support an effect of PKG acting upstream of MEK in modulating MAPK activation.

Apart from PKG, our results indicate that PKA is also involved in the NE-mediated MAPK activation. Depending on the cell type, PKA has been shown to inhibit (15, 18, 19), activate (16, 17), or have no effect on MAPK activities (33). In rat pinealocytes, PKA has an inhibitory effect on MAPK activation. This is based on the observation that the NE-mediated MAPK activation is enhanced by H89, a PKA inhibitor. Furthermore, an inhibitory effect of (Bu)2cAMP on MAPK activation is also in keeping with an inhibitory effect of PKA. Therefore, our results indicate that in rat pinealocytes, the effect of NE on MAPK phosphorylation represents the integration of two signaling mechanisms, PKA and PKG, each having an opposite effect on MAPK phosphorylation. Similar antagonism between cAMP and cGMP in MAPK activation has also been shown in vascular smooth muscle cells (19).

In addition to antagonizing the stimulatory effect of cGMP on MAPK phosphorylation, our results showed that PKA activation is effective in abolishing MAPK phosphorylation stimulated by an activator of PKC or a [Ca2+]i-elevating agent. This suggests that the cAMP/PKA signaling pathway functions as a general inhibitory signal to MAPK activation in the rat pineal gland. The specific mechanism through which PKA inhibits MAPK phosphorylation in rat pinealocytes remains unclear. However, depending on the cell type, cAMP/PKA could inhibit MAPK activation through a Ras-dependent (15, 34) or -independent mechanism (35).

The opposite effect of PKA and PKG on MAPK activation may provide a mechanistic explanation for the requirement of simultaneous activation of both {alpha}1- and ß-adrenergic receptors in inducing an increase in MAPK phosphorylation in rat pinealocytes. When ß-adrenergic receptors are activated alone, the increase in cyclic nucleotide contents, in particular the cGMP response, is relatively small (1, 2). Under this condition, the inhibitory effect of PKA on MAPK activation is adequate to counteract the stimulatory effect of PKG, hence resulting in little or no effect on MAPK activation. In contrast, when both {alpha}1- and ß-adrenergic receptors are activated, MAPK phosphorylation stimulated by the 100-fold increase in cGMP level (1, 2) is sufficient to overcome the inhibitory effect of PKA. In agreement with this is our observation that the ISO- or NE-mediated MAPK activation is enhanced by a PKA inhibitor, but is reduced by a PKG inhibitor.

In addition to PKA and PKG, other mechanisms activated by NE (through {alpha}1-adrenergic receptors), including elevation of [Ca2+]i and activation of PKC (2), may participate in MAPK activation in rat pinealocytes. Consistent with a modulating role of these pathways, MAPK phosphorylation is increased by activation of PKC (with PMA) or elevation of [Ca2+]i (with ION) in rat pinealocytes. However, neither calphostin C (a specific inhibitor of PKC) nor KN93 (a specific inhibitor of Ca2+/calmodulin-dependent protein kinase) has an effect on NE-mediated MAPK activation. Therefore, our results suggest that whereas activation of PKC and elevation of [Ca2+]i are effective activators of MAPK phosphorylation under pharmacological conditions, their contributions to the NE-mediated MAPK activation are relatively minor. This difference can be explained by the smaller NE-mediated increases in membrane-associated PKC and elevation of [Ca2+]i compared with those of PMA or ION (28, 36).

Although the function of MAPK in rat pinealocytes is unknown, the finding that (Bu)2AMP alone can activate NAT while inhibiting MAPK phosphorylation indicates that MAPK is unlikely to be required for NAT induction. However, we cannot exclude a modulatory role of MAPK on NAT induction, because MAPK has been reported to mediate cAMP response element-binding protein phosphorylation stimulated by PMA and Ca2+ in other cell types (37). Furthermore, MAPK may also be involved in cellular events regulated by PKG, including regulation of the cGMP-dependent phosphodiesterase activities (38), inhibition of the L-type Ca2+ channel (39), and changes in synaptic ribbons (40).

In summary, we have demonstrated the importance of activating both {alpha}1- and ß-adrenergic receptors in the adrenergic regulation of MAPK activities in rat pinealocytes. Intracellularly, we showed the participation of multiple signaling pathways in the activation of MAPK by NE, with PKG being the major activating mechanism and PKA being inhibitory. As the pineal gland is stimulated by the release of NE from the sympathetic neurons at night, our results suggest that there is probably a circadian rhythm for MAPK activation. Indeed, circadian activation of MAPK has recently been shown to play an important role in the chick pineal clock oscillation (41).


   Footnotes
 
1 This work was supported by grants from the Medical Research Council of Canada. Back

Received July 6, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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