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Departments of Physiology and Medicine, 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.
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Abstract |
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1- and ß-adrenergic receptors. Treatment with dibutyryl cGMP, 4ß-phorbol 12-myristate 13-acetate, or ionomycin mimicked norepinephrine-stimulated p90RSK phosphorylation, whereas dibutyryl cAMP caused a decrease in p90RSK phosphorylation. Inhibition of p42/44MAPK activation by UO126 was effective in reducing norepinephrine-stimulated p90RSK phosphorylation. Moreover, UO126 had an inhibitory effect on norepinephrine-stimulated arylalkyl-N-acetyltransferase activity. These results indicate that the adrenergically regulated nocturnal increase in p90RSK phosphorylation is mainly mediated through a cGMP
p42/44MAPK-dependent mechanism. |
Introduction |
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1- and ß-adrenergic receptors, causes a 100-fold increase in cAMP and cGMP accumulation (1). These large increases in cyclic nucleotide responses are the results of the potentiation of ß-adrenergically stimulated cyclic nucleotide synthesis by the 1-adrenergic receptor-mediated activation of protein kinase C (PKC) and elevation of intracellular Ca2+ (2, 3, 4, 5). The primary function of cAMP in the rat pineal gland is to initiate a series of cellular events that leads to the synthesis of arylalkyl-N-acetyltransferase (AA-NAT), the rate-limiting enzyme in the synthesis of the pineal hormone, melatonin (6). The physiological importance of NE-stimulated cGMP accumulation in the pineal gland is unclear. However, we have shown that cGMP is involved in regulation of the L-type Ca2+ channel (7) as well as NE activation of p42/44 mitogen-activated protein kinase (p42/44MAPK) in rat pinealocytes (8). MAPKs, a large family of serine/threonine protein kinases, function as mediators of signal transduction and are activated by diverse stimuli, such as cytokines, growth factors, neurotransmitters, hormones, and cellular stresses (9, 10). Three main groups of MAPKs have been identified: p42/44MAPK (11, 12), p38MAPK (13, 14), and c-Jun N-terminal kinase (15, 16). The best studied member of MAPKs is p42/44MAPK. Upon activation, p42/44MAPK can regulate gene expression either directly by phosphorylating transcription factors (9, 17, 18, 19) or indirectly through phosphorylation of protein kinases such as the 90-kDa ribosomal S6 kinase (p90RSK) (20). One of the cellular functions of p90RSK that has been identified is regulation of gene expression by phosphorylating transcription factors, including cAMP response element-binding protein (CREB) (21, 22, 23). Considering the importance of CREB phosphorylation in the circadian activation of AA-NAT (24), NE activation of p90RSK may represent an important signaling mechanism that modulates the induction of AA-NAT at night. Therefore, the objectives of the present study are 3-fold. First, we will determine whether there is a day/night variation in the activation state of p90RSK in the rat pineal gland. Second, if there is a diurnal difference in the activation of p90RSK, we will investigate whether this is mediated by NE and will characterize the receptor and signaling pathways involved in the regulation of p90RSK. Third, we will determine the effect of inhibition of p42/44MAPK/p90RSK on NE-stimulated AA-NAT activity.
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Materials and Methods |
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Animals
All procedures were reviewed and approved by the health sciences animal and welfare committee of the University of Alberta (Edmonton, Alberta). Sprague Dawley rats (male; weighing 150 g) were obtained from the University of Alberta animal unit. Animals were housed under a lighting regimen that provides 12 h of light every 24 h with lights on at 0600 h [zeitgeber time zero (ZT 0)]. For the preparation of pinealocyte cell culture, animals were killed 3 h after the onset of light by decapitation, and the pineal glands were rapidly removed and stored in ice-cold PBS until trypsinization. For the circadian study, groups of animals were killed at various time points as indicated. The pineal glands were collected, washed in ice-cold PBS, and frozen in dry ice immediately until preparation for Western blot analysis or AA-NAT determination. When the animals were killed during the dark period, only a dim red light was used, except in the experiment when the animals were deliberately exposed to light during darkness.
Preparation of cultured pinealocytes and drug treatment
Pinealocytes were prepared from freshly dissected rat pineal glands by trypsinization as described previously (25). The cells were suspended in DMEM containing 10% fetal calf serum and were maintained at 37 C for 24 h in a gas mixture of 95% air and 5% CO2 before the experiments. For the determination of the activation states of p42/44MAPK and p90RSK, aliquots of pinealocytes (5 x 105 cells/0.5 ml) were treated with drugs that were added to the medium at 200x concentrated solutions in water or dimethylsulfoxide for the duration indicated. Treated cells were collected by centrifugation (2 min, 12,000 x g). Samples for Western blot analysis were solubilized in 1x sample buffer by boiling for 5 min and stored until analysis. For the determination of AA-NAT activity, treated cells were collected by centrifugation (2 min, 12,000 x g), and cell pellets were immediately frozen in dry ice and stored at -75 C.
Preparation of pineal glands for Western blot analysis
Frozen pineal glands were homogenized in a buffer (20 mM Tris-HCl; 2 mM EDTA; 0.5 mM EGTA; 2 mM phenylmethylsulfonylfluoride; 1 µg/ml each of aprotinin, leupeptin, and pepstatin; 1 mM sodium vanadate; and 1 mM sodium fluoride, pH 7.5) by sonication. Sodium vanadate and sodium fluoride were included to inhibit phosphatase activity. An aliquot of the homogenate was used for protein determination (26). The sample buffer that contained an identical amount of protease inhibitors, EGTA and EDTA, was used as a blank. The sample, after adjusting for protein content, was mixed with 2x sample buffer, boiled for 5 min, and stored until electrophoresis.
Western blot
SDS-PAGE was performed according to the procedure described by Laemmli (27), using 10% acrylamide in the presence of 1 mg/ml sodium dodecyl sulfate (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 incubated with diluted horseradish peroxidase-conjugated second antibodies (Bio-Rad Laboratories, Inc.) for 1 h at room temperature. The blots were washed extensively and developed using enhanced chemiluminescence (Amersham Pharmacia Biotech, Arlington Heights, IL).
AA-NAT assay
AA-NAT activity was determined as described previously (28). Briefly, the collected pineal glands or cell pellets were homogenized in a reaction mixture of 0.1 M phosphate buffer (pH 6.8) containing 30 nmol [3H]acetyl coenzyme A (specific activity, 1 mCi/mmol) and 1 µmol tryptamine hydrochloride in a final volume of 60 µl. The reaction mixture was incubated at 37 C for 1 h. At the end of the incubation period, the reaction was stopped by the addition of 1 ml methylene chloride. After vortexing, the aqueous phase was removed, and the organic phase was washed three times with 0.1 M phosphate buffer (pH 6.8). The organic phase was transferred to a scintillation vial and evaporated to dryness. The radioactive acetylated product was determined, and NAT activity was expressed as picomoles per microgram of protein per hour or nanomoles per hour per 105 cells.
Result presentation and statistical analysis
For the Western blots, a typical blot from at least three similar experiments is shown. Selected results were quantified using densitometric measurements, analyzed by Sigmagel (Jandel Scientific, San Rafael, CA), normalized to the control level, and presented as the mean ± SEM from at least three separate experiments. AA-NAT activity was presented as the mean ± SEM of three independent observations performed in duplicate. Statistical comparisons were analyzed by either unpaired t test or one-way ANOVA, followed by Tukey’s post hoc test. Statistical significance was set at P < 0.05.
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Results |
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, pineal glands collected during the subjective night (ZT 14, 18, and 22; lights on at ZT 0; lights off at ZT 12) were found to have 3-fold higher levels of phosphorylated p90RSK than tissues collected during the subjective day (ZT 3 or 9; Fig. 1B). However, there was no significant difference in the level of total p90RSK (both phosphorylated and unphosphorylated) protein between any time point regardless of the light-dark cycle.
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). Exposure to light for 30 or 60 min at ZT 17 during the dark period caused a significant decline in the level of phosphorylated p90RSK (Fig. 3A). In comparison, acute light exposure abolished the increase in AA-NAT activity that occurred at night (Fig. 3B), as reported previously (29).
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). The time profile of NE-stimulated p42/44MAPK activation followed closely the time profile of NE activation of p90RSK (Fig. 4A). At 15 min, the effects of NE on phosphorylated p90RSK and p42/44MAPK levels were dependent on concentration (Fig. 4B).
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1- and ß-adrenergic receptors are involved in NE-stimulated p90RSK activation in rat pinealocytes). Activation of 1-adrenerigc receptors by phenylephrine (1 µM) in the presence of propranolol (1 µM) caused a small increase in phosphorylated p90RSK (Fig. 5). Combined treatment with isoproterenol (1 µM) and phenylephrine (1 µM) caused a 3-fold increase in the phosphorylated 90RSK level, an effect similar to that of NE (10 µM; Fig. 5). Furthermore, the presence of either prazosin (1 µM) or propranolol (1 µM) caused a reduction in NE-mediated p90RSK activation (Fig. 5). These results suggest that both 1- and ß-adrenergic receptors are involved in the NE activation of p90RSK. The requirement for both 1- and ß-adrenoceptors was similar to that seen in the regulation of p42/44MAPK by NE as reported previously (30) and shown in Fig. 5 for comparison.
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1- and ß-adrenergic receptors, causes activation of protein kinase A (PKA), protein kinase G (PKG), and PKC as well as elevation of intracellular Ca2+ (2, 3, 4, 5). To investigate the relative contributions of NE-activated signaling pathways in NE activation of p90RSK, pharmacological agents were used. Phosphorylated levels of p42/44MAPK were measured for comparison. As shown in Fig. 6, activation of PKG by dibutyryl cGMP (1 mM) and of PKC by PMA (0.1 µM) as well as elevation of intracellular Ca2+ by ionomycin (1 µM) were effective in increasing the phosphorylated levels of p90RSK. In comparison, treatment with dibutyryl cAMP (1 mM) caused a reduction in the phosphorylated p90RSK level. Thus, it appears that whereas pharmacological activation of PKG and PKC as well as elevation of intracellular Ca2+ enhance the phosphorylation of p90RSK, activation of PKA has the opposite effect. The pharmacological profiles of these agents in activating p90RSK were similar to their profiles in activating p42/44MAPK (Fig. 6A).
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). Whereas H89 (1 µM), a selective PKA-selective inhibitor with a Ki less than 0.05 µM, was effective in enhancing the NE activation of p90RSK, the effect of KT5823 (1 µM), a selective PKG inhibitor with a Ki less than 0.3 µM, was inhibitory (Fig. 6B). Calphostin C (1 µM), a selective PKC inhibitor with a Ki less than 0.05 µM, and KN93 (1 µM), a selective Ca2+/calmodulin-dependent protein kinase inhibitor, had little effect (Fig. 6B). The effects of these kinase inhibitors on NE-stimulated p90RSK activation were similar to their effects on NE-stimulated p42/44MAPK activation (Fig. 6B).
p42/44MAPK mediates the effect of NE on p90RSK on rat pinealocytes
To confirm that p42/44MAPK mediates the effect of NE on p90RSK, inhibitors against MAPK kinase (MEK1/2) and p38MAPK were used. Pretreatment of pinealocytes with a MEK1/2 inhibitor, UO126 (1 µM), caused a significant reduction in NE activation of p90RSK and p42/44MAPK (Fig. 7 and Table 1). In comparison, UO124 (1 µM), the inactive analog, had no effect. Inhibition of p38MAPK by SB203580 (10 µM), a treatment that we have previously shown to enhance NE-stimulated p42/44MAPK activation (31), caused a further increase in NE-stimulated p90RSK activation. The inactive analog, SB202474 (10 µM), had no effect on the NE-mediated increase in phosphorylated p90RSK (Fig. 7). Together these results suggest that NE increases phosphorylated p90RSK through activation of the p42/44MAPK pathway.
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). The presence of UO126 (1 µM) caused a 40% reduction in NE-stimulated AA-NAT activity (Table 1). In contrast, UO124 (1 µM) had no effect on NE-stimulated AA-NAT activity (Table 1). |
Discussion |
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In support of nocturnal activation of p90RSK, subjecting the animals to continuous light from the onset of darkness is effective in blocking the nocturnal increase in p90RSK phosphorylation. Moreover, treatment with propranolol reduces the nocturnal increase in p90RSK phosphorylation, an observation indicating that activation of p90RSK is under adrenergic regulation. Considering that the pineal gland is stimulated by the release of NE from the sympathetic nerve at night (1, 32), these results suggest that adrenergically stimulated p90RSK phosphorylation in the pineal gland may be driven by the endogenous circadian clock in the suprachiasmatic nucleus (33, 34), similar to other cellular processes that have a circadian rhythm.
Our result also showed that the nocturnal increase in p90RSK phosphorylation is inhibited by acute light exposure. Within 30 min of light exposure, there is a decline in the increase in phosphorylated p90RSK during the dark phase. Although the decline in phosphorylated p90RSK is fairly rapid after light exposure, this appears slower than the decline in AA-NAT activity after similar light exposure. These results indicate that similar to the regulation of AA-NAT, a sustained stimulation is required for the nighttime activation of p90RSK. However, the mechanism involved in the inactivation of p90RSK may not be identical to that of AA-NAT.
In cultured pinealocytes we also found that both 1- and ß-adrenergic receptors are involved in NE-stimulated p90RSK activation. Furthermore, potentiation at the postreceptor level also constitutes part of the intracellular mechanism through which NE regulates p90RSK activation. This is based on observations that NE-mediated p90RSK phosphorylation can be reduced by treatment with either an 1- or a ß-adrenergic receptor antagonist. Moreover, treatment with an 1-adrenergic receptor agonist causes a small increase in phosphorylated p90RSK, whereas treatment with a ß-adrenergic receptor has no effect. However, combined treatment with both an 1- and a ß-adrenergic receptor agonist results in a large increase in the level of phosphorylated p90RSK.
Downstream from the adrenergic receptors, it has previously been shown that NE causes elevation of cAMP and cGMP, elevation of intracellular Ca2+, as well as activation of PKC (2). When these signaling pathways are activated pharmacologically, treatment that either activates PKG or PKC or causes elevation of intracellular Ca2+ is effective in activating p90RSK. In comparison, activation of PKA reduces the level of phosphorylated p90RSK. Our results also indicate that, similar to p42/44MAPK (8, 30), PKG appears to be the main pathway involved in NE activation of p90RSK phosphorylation. This is based on observations that whereas selective inhibition of PKG by KT5823 is effective in blocking the NE activation of p90RSK, inhibition of either PKC or the Ca2+/calmodulin pathway has only a minimal effect on NE-stimulated p90RSK phosphorylation. Although treatment with either PMA or ionomycin causes a large increase in p90RSK phosphorylation, activation of 1-adrenergic receptors alone only has a modest effect on p90RSK phosphorylation. 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 ionomycin (35, 36).
The opposite effect of PKA and PKG on the activation state of p90RSK also provides an explanation for the requirement for activation of 1- and ß-adrenergic receptors for enhancement of p90RSK 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 (2). Under these conditions, the inhibitory effect of PKA on p90RSK phosphorylation is adequate to counteract the stimulatory effect of PKG, hence resulting in little or no effect on p90RSK phosphorylation. In contrast, when both 1- and ß-adrenergic receptors are activated, p90RSK phosphorylation stimulated by the large increase in cGMP is sufficient to overcome the inhibitory effect of PKA. In agreement with this is the observation that the isoproterenol- or NE-mediated p90RSK activation is enhanced by a PKA inhibitor, but is reduced by a PKG inhibitor.
The similarities between the adrenergic receptors involved in the activation of p90RSK and p42/44MAPK (30) as well as the predominant involvement of the PKG pathway (8, 30) suggest that NE-mediated activation of p90RSK may occur as a consequence of p42/44MAPK activation. Other supportive evidence includes the observation that reducing the NE-mediated p42/44MAPK phosphorylation by inhibition of MEK1/2 reduces the NE-mediated activation of p90RSK. Moreover, increasing NE-mediated p42/44MAPK phosphorylation by an inhibitor of p38MAPK (31) increases NE-mediated activation of p90RSK.
Previously, p42/44MAPK has been shown to play an important role in pineal clock oscillation in chicks (37). In this study we found that UO126, an inhibitor of MEK1/2, has an inhibitory effect on NE-stimulated AA-NAT, hence supporting a modulating role of p42/44MAPK/p90RSK in the adrenergic regulation of melatonin synthesis. The specific mechanism through which p42/44MAPK modulates AA-NAT activity remains unclear. However, previous studies have shown the presence of cAMP response element in the promoter of the AA-NAT gene (38) and regulation of this gene by the binding activity of activating protein-1 (39). Therefore, p42/44MAPK/p90RSK could modulate AA-NAT activity through phosphorylation of CREB and activation of activating protein-1 as shown in other cell types (22, 40). Moreover, p90RSK may be the downstream element through which PKG regulates cellular targets such as the L-type Ca2+ channel (7) and synaptic ribbons in the rat pineal gland (41).
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Footnotes |
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Abbreviations: AA-NAT, Arylalkylamine N-acetyltransferase; CREB, cAMP response element-binding protein; MEK, MAPK kinase; NE, norepinephrine; p-, phosphorylated; PKA, protein kinase A; PKC, protein kinase C; PKG, protein kinase G; PMA, 4ß-phorbol 12-myristate 13-acetate; p90RSK, 90-kDa ribosomal S6 kinase; ZT, zeitgeber time.
Received February 18, 2003.
Accepted for publication April 14, 2003.
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1-Adrenoceptor activation elevates cytosolic calcium in rat pinealocytes by increasing net influx. J Biol Chem 262:741–745This article has been cited by other articles:
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A. K. Ho, D. L. Terriff, D. M. Price, M. T. Wloka, and C. L. Chik The Role of Inducible Repressor Proteins in the Adrenergic Induction of Arylalkylamine-N-Acetyltransferase and Mitogen-Activated Protein Kinase Phosphatase-1 in Rat Pinealocytes Endocrinology, February 1, 2007; 148(2): 743 - 751. [Abstract] [Full Text] [PDF]
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D. M. Price, C. L. Chik, and A. K. Ho Norepinephrine Induction of Mitogen-Activated Protein Kinase Phosphatase-1 Expression in Rat Pinealocytes: Distinct Roles of {alpha}- and {beta}-Adrenergic Receptors Endocrinology, December 1, 2004; 145(12): 5723 - 5733. [Abstract] [Full Text] [PDF]
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C. L. Chik, M. Mackova, D. Price, and A. K. Ho Adrenergic Regulation and Diurnal Rhythm of p38 Mitogen-Activated Protein Kinase Phosphorylation in the Rat Pineal Gland Endocrinology, November 1, 2004; 145(11): 5194 - 5201. [Abstract] [Full Text] [PDF]
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J. R. Man, S. Rustaeus, D. M. Price, C. L. Chik, and A. K. Ho Inhibition of p38 Mitogen-Activated Protein Kinase Enhances Adrenergic-Stimulated Arylalkylamine N-Acetyltransferase Activity in Rat Pinealocytes Endocrinology, March 1, 2004; 145(3): 1167 - 1174. [Abstract] [Full Text] [PDF]
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, Animals remaining in the dark; 
, animals subjected to light from ZT 17. The glands were homogenized, mixed with 2x sample buffer, and analyzed by Western blotting using a polyclonal antibody against p-p90RSK or were assayed for AA-NAT activity. See Materials and Methods for details. A, Densitometric measurements of p-p90RSK from four experiments presented as the fold increase over the value at ZT 12; B, AA-NAT activity from three independent experiments. AA-NAT activity from glands obtained at ZT 12 was not detectable. C, A representative blot of p-p90RSK from four separate experiments. *, P < 0.05, significantly different from ZT 17.
