Norepinephrine (NE) is one of the major neurotransmitters that determine melatonin production in the pineal gland. Although a substantial amount of Ca2+ influx is triggered by NE, the Ca2+ entry pathway and its physiological relevance have not been elucidated adequately. Herein we report that the Ca2+ influx triggered by NE significantly regulates the protein level of serotonin N-acetyltransferase, or arylalkylamine N-acetyltransferase (AANAT), a critical enzyme in melatonin production, and is responsible for maintaining the Ca2+ response after repetitive stimulation. Ca2+ entry evoked by NE was dependent on PLC activation. NE evoked a substantial amount of Ca2+ entry even after cells were treated with 1-oleoyl-2-acetyl-sn-glycerol (OAG), an analog of diacylglycerol. To the contrary, further OAG treatment after cells had been exposed to OAG did not evoke additional Ca2+ entry. Moreover, NE failed to induce further Ca2+ entry after the development of Ca2+ entry induced by thapsigargin (Tg), suggesting that the pathway of Ca2+ entry induced by NE might be identical to that of Tg. Interestingly, Ca2+ entry evoked by NE or Tg induced membrane hyperpolarization that was reversed by iberiotoxin (IBTX), a specific inhibitor of large-conductance Ca2+-activated K+ (BK) channels. Moreover, IBTX-sensitive BK current was observed during application of NE, suggesting that activation of the BK channels was responsible for the hyperpolarization. Furthermore, the activation of BK channels triggered by NE contributed to regulation of the protein level of AANAT. Collectively, these results suggest that NE triggers Ca2+ entry coupled to BK channels and that NE-induced Ca2+ entry is important in the regulation of AANAT.
- serotonin N-acetyltransferase
- pineal gland
the pineal gland is a neuroendocrine organ in which melatonin (5-methoxy-N-acetyltryptamine) is synthesized and released with daily and seasonal rhythms. It has important roles in the regulation of circadian rhythm, seasonal reproduction, and retinal function (11). Pinealocytes in the mammalian pineal gland express the genes encoding the melatonin-synthesizing enzymes. Serotonin N-acetyltransferase, or arylalkylamine N-acetyltransferase (AANAT; EC 18.104.22.168), is considered the melatonin rhythm-generating enzyme because it catalyzes the conversion of serotonin to N-acetylserotonin, which is the rate-limiting step in the biosynthetic pathway of melatonin (28). In the rat pineal gland, norepinephrine (NE), one of the major neurotransmitters, regulates melatonin production by stimulating the synthesis and enzymatic activity of AANAT (28, 30). NE acts through α1- and β1-adrenergic receptors (ARs) to activate PLC and adenylyl cyclase, respectively (32). We recently reported (6) that the regulation of stability and activity of AANAT by PKC contributes to the level as well as the de novo synthesis of AANAT through the adenylyl cyclase pathway (13, 31). Moreover, it was reported that stimulation of α1-AR results in the activation of PKC, which was shown to be dependent on increased intracellular Ca2+ concentration ([Ca2+]i) rather than on diacylglycerol (DAG) (14). Therefore, it is plausible that the [Ca2+]i increase triggered by NE plays a role in the modulation of AANAT. NE-induced elevation of [Ca2+]i consists of a rapid increase (primary phase) followed by a decrease to an elevated plateau (secondary phase) well above the basal level (25, 32). The [Ca2+]i response is considered to be due to both release of Ca2+ from intracellular stores and subsequent Ca2+ influx across the plasma membrane (23). Although a substantial amount of Ca2+ influx is triggered by NE (23, 25, 27), the Ca2+ entry pathway and its physiological relevance have not been elucidated adequately.
Stimulation of G protein-coupled receptors, tyrosine kinase receptors, and nonreceptor tyrosine kinases activates PLC and catalyzes the breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) into the second-messenger molecules inositol 1,4,5-trisphosphate (IP3) and DAG. IP3 evokes rapid Ca2+ release by activating IP3 receptors in the intracellular stores, whereas DAG activates PKC (2). After this initial Ca2+ release, external Ca2+ enters through plasma membrane channels, providing a secondary, more prolonged Ca2+ signal (20). The channels through which Ca2+ enters can be defined loosely within two major categories: store-operated Ca2+ channels (SOCs) and receptor-operated Ca2+ channels (ROCs) (2, 20). SOC is a store-operated Ca2+ entry (SOCE) channel activated as a direct result of Ca2+ store depletion that can also be activated by the endoplasmic reticulum Ca2+ pump blocker thapsigargin (Tg), which depletes stores independently of receptor activation (19). To the contrary, ROC refers to a receptor-operated Ca2+ entry channel activated after an agonist binds to a PLC-coupled receptor, but it does not necessarily depend on store depletion (20). Both types of channels are implicated in the modulation of cytosolic Ca2+ entry, which regulates several cellular processes such as neurotransmitter release (18, 19), cell proliferation (12), differentiation (8), and apoptosis (15, 19). In the present study, we have provided evidence that the Ca2+ entry evoked by NE is attributable mainly to the activation of SOCs. In addition, we show that inhibitors of SOCs suppressed the increase in the protein level of AANAT induced by NE. Furthermore, the SOCs were coupled to large-conductance Ca2+-activated K+ (BK) channels, thereby inducing membrane hyperpolarization. Taken together, these data suggest that Ca2+ entry triggered by NE plays an important role in pineal gland function (i.e., in modulation of NE signaling and regulation of AANAT).
MATERIALS AND METHODS
Chemicals were purchased from Sigma (St. Louis, MO) unless otherwise stated.
Preparation of cultured pinealocytes.
Sprague-Dawley rats were maintained in a controlled environment (12:12-h light-dark cycle). Pineal glands from male and female rats (aged 7–9 wk) were prepared using methods described in a previous study (9). Briefly, pineal glands removed from rat brains were washed in ice-cold HEPES buffer and transferred to DMEM (GIBCO-BRL, Grand Island, NY) containing papain (20 U/ml; Worthington Biochemical, Lakewood, NJ). After 1-h incubation, the tissue was washed in DMEM, mechanically dispersed in clean medium, and plated onto poly-d-lysine-coated coverslips. The cells were suspended in DMEM containing 10% FBS and maintained at 37°C in a 5% CO2-containing atmosphere until use (1–3 days). Animal experiments were performed after being approved by the University Ethics Committee.
Fluorescence measurements of [Ca2+]i.
Cells were loaded with 3 μM fura-2 AM (Molecular Probes, Eugene, OR) at room temperature (20–23°C) for 50 min and subsequently washed in fura-2-free solution for a minimum of 10 min. Single-cell Ca2+ measurements were performed as described previously by Lee and Lee (17). Briefly, cells loaded with fura-2 were mounted on an experimental chamber and illuminated with UV light (75-W xenon lamp) applied via an epifluorescence microscope (Nikon, Tokyo, Japan). A filter wheel in front of the UV light was rotated continuously at 50 Hz, and excitation filters of 340 and 380 nm were used alternately (Cairn Research, Kent, UK).
Measurement of membrane potential.
Pinealocytes in the experimental chamber were superfused continuously with Tyrode solution containing (in mM) 140 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose (pH 7.4 with NaOH) at ∼37°C. To measure membrane potential, we used conventional microelectrodes pulled from filamented thin-wall glass [1.5-mm outer diameter (OD), TW150F-6; World Precision Instruments, Sarasota, FL]. They were filled with 300 mM KCl and had resistances between 40 and 55 MΩ. The electrode resistance and capacitance were compensated to ∼85% of their initial values. Microelectrode potential was measured with an AxoClamp 2A amplifier (0.1-gain headstage; Axon Instruments, Foster City, CA).
Measurement of BK current.
To record BK current, cells were superfused with HEPES solution containing (in mM) 140 NaCl, 3 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 3 × 10−4 TTX, 1 4-aminopyridine (4-AP), 5 × 10−3 glibenclamide, and 10 glucose (pH adjusted to 7.4 with NaOH). 4-AP (1 mM) was used to reduce voltage-gated K+ currents and unmask the Ca2+ dependence of K+ currents. The concentration of 4-AP was chosen according to the concentration used in a previous study (33) to minimize any possible nonspecific blockade of other K+ currents. Recording pipettes were made of filamented borosilicate capillary glass (1.5-mm OD, TW150F-6; World Precision Instruments), and resistances ranged from 2 to 5 MΩ when the pipettes were filled with the solutions listed below. Patch-pipette solutions for K+ current recording contained (in mM) 110 K+-gluconate, 10 KCl, 5 NaCl, 2 MgCl2, 10 HEPES, 0.5 EGTA, 1 ATP, and 0.2 GTP (pH adjusted to 7.3 with KOH). Membrane currents were recorded using an Axopatch 200A amplifier (Axon Instruments). Signals were obtained at sampling rates of 5 kHz. WinWCP software (John Dempster, Strathclyde University, Strathclyde, UK) was used to control the generation of stimuli and to collect data. Capacitance subtraction was performed for all recordings. The series resistances were within 10 MΩ. Experiments were conducted at room temperature (20–23°C).
Western blot analysis.
Proteins were extracted with lysis buffer containing (in mM) 100 Tris·HCl (pH 7.0), 1 EGTA, 1 MgCl2, 1 PMSF, 0.1 DTT, 1 Na3VO4, and 1% Triton X-100. Proteins were separated on a 12.5% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The nitrocellulose membrane was blocked with 5% nonfat dry milk in TBST solution containing 20 mM Tris·HCl (pH 7.5), 140 mM NaCl, and 0.05% Tween 20. Detection of AANAT protein was performed as described previously (6). The immunosignal was detected using the SUPEX detection system (Neuronex, Pohang, Korea).
Data analysis and statistics.
Each experiment was repeated a minimum of three times, and the results are expressed as means ± SE when appropriate. Numerical data were analyzed using SigmaPlot 2001 for Windows (SPSS, Richmond, CA) and Origin 6.1 software (OriginLab, Northampton, MA). Statistical differences were determined using Student's t-test. Differences were considered statistically significant at P < 0.05.
Ca2+ influx triggered by NE regulates the protein level of AANAT.
NE stimulates transcription of AANAT through activation of adenylyl cyclase-cAMP pathway (11). Because NE activates the Ca2+ pathway as well as the cAMP pathway, we investigated the possible role of Ca2+ influx triggered by NE in the modulation of AANAT. For this purpose, we examined whether low (0.2 mM) extracellular Ca2+ concentration ([Ca2+]o) would affect the protein level of AANAT elevated by NE. As shown in Fig. 1, when Ca2+ entry was limited by lowering [Ca2+]o, the protein level of AANAT was significantly reduced. In addition, Gd3+ and SKF-96365, both of which are general inhibitors of Ca2+ entry channels, also caused a reduction in the level of AANAT upregulated by NE.
We next examined the Ca2+ response to repetitive NE stimulation, focusing on Ca2+ entry. To this end, we first compared the Ca2+ response in the absence and presence of 2 mM [Ca2+]o. As shown in Fig. 2A, when NE was applied five times, the amplitude of the NE-evoked Ca2+ peak gradually decreased and the amplitude of the fifth peak was 64 ± 4% (n = 7) of the first one. However, the biphasic Ca2+ response trace maintained its shape. On the other hand, removal of extracellular Ca2+ dramatically decreased the fifth NE-evoked Ca2+ peak to 3 ± 1% (n = 4) of the first one, and the secondary sustained phase was not detected. Similarly, in the presence of Gd3+ or La3+, inhibitors of Ca2+ entry, the amplitude of the fifth NE-evoked Ca2+ peak was reduced 6 ± 1% (n = 4) and the secondary sustained phase was not detectable. These results suggest that Ca2+ entry evoked by NE is necessary to maintain the Ca2+ response by repetitive stimulation and seems to contribute to potentiation of the NE-induced increase in the AANAT protein level.
Ca2+ influx evoked by NE is SOCE.
The secondary plateau phase in the NE-induced Ca2+ response reflected Ca2+ entry after receptor activation, because it was completely inhibited by removal of extracellular Ca2+ as well as by Gd3+ (Fig. 3A). To test whether the activation of voltage-gated Ca2+ channels (VGCCs) was involved in the secondary phase of the Ca2+ response, we examined the effect of nifedipine, a specific inhibitor of L-type Ca2+ channels, which is the major type of VGCC expressed in rat pinealocytes (4). As shown in Fig. 3A, nifedipine did not affect the secondary phase of [Ca2+]i elevation. To the contrary, the secondary phase of the Ca2+ response was abolished by 2-aminoethoxydiphenylborane, a known inhibitor of IP3 receptors and SOCs (7). Next, we examined the involvement of PLC. In the absence of [Ca2+]o, application of NE increased [Ca2+]i because of Ca2+ release from intracellular Ca2+ stores. When 2 mM [Ca2+]o was added to the medium, we observed a substantial amount of Ca2+ entry in response to NE (Fig. 3B, left). In the presence of U-73122, a selective inhibitor of PLC, NE did not evoke Ca2+ release and subsequent Ca2+ entry (Fig. 3B, middle), whereas its inactive analog U-73343 had no effect (Fig. 3B, right), suggesting that NE-induced Ca2+ entry is dependent on PLC signaling.
PLC hydrolyzes PIP2 into DAG and IP3, both of which could induce increase in [Ca2+]i (20, 34). To investigate whether DAG was involved in the NE-induced Ca2+ entry, we used a membrane-permeable DAG analog, 1-oleoyl-2-acetyl-sn-glycerol (OAG). Figure 3C shows that OAG alone increased a small amount of [Ca2+]i. PMA, an agonist of PKC that can be activated by DAG, failed to induce an increase in [Ca2+]i (Fig. 3C, middle). Moreover, GF-109203X, an inhibitor of PKC, did not affect the OAG-induced [Ca2+]i increase (data not shown). These results suggest that OAG is able to induce a small amount of [Ca2+]i increase independently of PKC activation. As shown in Fig. 3C, right, NE evoked further Ca2+ influx even after cells were treated with 100 μM OAG, although a higher concentration of OAG (200 μM) did not evoke further Ca2+ influx after treatment with 100 μM OAG (Fig. 3C, left). These results suggest that NE evokes Ca2+ entry, probably through a pathway other than OAG.
Next, we applied Tg, which is widely used to induce depletion of Ca2+ stores and to activate subsequent SOCE. As shown in Fig. 3D, OAG evoked an additional [Ca2+]i increase even after Tg had induced SOCE, whereas NE failed to induce further Ca2+ influx. Therefore, we concluded that the NE-induced Ca2+ entry would be included in SOCE induced by Tg.
SOCE hyperpolarizes membrane potential.
In excitable cells, activation of SOC current not only may be the path for Ca2+ to enter the cell but also may provide a depolarizing trigger (21). To elucidate whether activation of SOC induces changes in membrane potential, we recorded single-cell [Ca2+]i level and membrane potential simultaneously. When the pinealocytes were penetrated using conventional microelectrodes, the mean membrane potential was −65 ± 5 mV (n = 45), which is in agreement with the findings reported previously (10). Unexpectedly, the addition of 2 mM [Ca2+]o to the superfusion solution containing NE resulted in the hyperpolarization of the membrane potential and removal of extracellular Ca2+ depolarized the membrane potential (Fig. 4A), suggesting that Ca2+ influx has an important role in the regulation of membrane potential. BK channels require both depolarization and elevated [Ca2+]i to become activated, which results in hyperpolarization of membrane potential coupled with increased [Ca2+]i (16). To test the possible involvement of BK channels in the hyperpolarization induced by Ca2+ influx, we examined the effect of iberiotoxin (IBTX), a specific inhibitor of BK channels. Ca2+ entry evoked by NE and Tg induced −17 ± 8-mV (n = 7) and −18 ± 8-mV (n = 5) membrane hyperpolarization, respectively, which was completely reversed by application of IBTX (Fig. 4, B and C). To test whether the membrane hyperpolarization accompanied by Ca2+ entry induced by NE and Tg could be mimicked by similar amounts of Ca2+ influx, we treated the cells with Ach, an activator of nicotinic Ach receptors. Ach treatment, however, resulted in a 29 ± 4-mV (n = 10) increase in membrane potential (i.e., depolarization) (Fig. 4D), which was reduced by nifedipine (48 ± 7%; n = 5). Nifedipine significantly inhibited the Ca2+ influx induced by Ach (49 ± 5%; n = 5), which presents a striking contrast to its effect on Ca2+ influx evoked by NE (Fig. 3A), excluding the involvement of VGCCs in NE-induced Ca2+ influx. Taken together, these results suggest that SOCE induces membrane hyperpolarization because of the coupling of BK channels.
IBTX-sensitive BK current is developed by NE.
To confirm that the BK channels were activated in response to NE stimulation, IBTX-sensitive BK current was measured before and during application of NE. Figure 5A shows the macroscopic outward currents from a pinealocyte to which NE was not applied. The depolarization-induced BK current is defined as the outward current sensitive to blockage by IBTX. In the absence of NE, outward currents were not sensitive to IBTX (Fig. 5A). However, IBTX reduced outward current by 40 ± 7% at +30 mV (n = 7) during application of NE (Fig. 5B), suggesting that BK current is developed upon stimulation by NE. When Gd3+ was applied to inhibit SOCE, NE did not induce IBTX-sensitive BK current (Fig. 5E). When Tg was used as an alternative activator of SOCE, IBTX-sensitive BK current was also detected (data not shown).
Inhibition of BK channels reduces the protein level of AANAT.
NE-dependent melatonin synthesis is negatively regulated by parasympathetic stimulation, such as that induced by nicotinic Ach receptors. Ach depolarizes membrane potential, which leads to the secretion of l-glutamate, thereby inhibiting the synthesis of AANAT (28). Because activation of BK channels is responsible for the membrane hyperpolarization induced by NE (Fig. 4), it is possible that BK channels might contribute to the regulation of the level of AANAT, the synthesis of which is influenced by the status of membrane potential. To test this possibility, we investigated whether the protein level of AANAT elevated by NE would be affected by the inhibition of BK channels. As shown in Fig. 6, 100 μM Ach partially decreased the protein level of AANAT upregulated by NE, as expected. Importantly, IBTX alone caused a reduction in AANAT protein level in the presence of NE. Furthermore, application of IBTX resulted in a further reduction in the AANAT protein level elevated by NE in the presence of Ach compared with the level inhibited by Ach. Similar results were obtained when lower concentrations of Ach (10 and 50 μM) were applied (data not shown). These results suggest that activation of BK channels triggered by NE participates in the regulation of AANAT.
SOCE evoked by NE occurs through activation of α1-AR.
Because NE stimulates both α1- and β1-AR, we examined the effect of phenylephrine (PE) and isoproterenol (Iso), specific agonists of α1- and β1-AR, respectively, to determine which receptor is responsible for Ca2+ influx. Stimulation of α1-AR with PE in the presence of propranolol (Prop), an antagonist of β1-AR, caused a biphasic Ca2+ increase and maintained the Ca2+ response after repetitive stimulation as observed in NE stimulation (Fig. 7, top). However, stimulation of β1-AR with Iso in the presence of prazosin, an antagonist of α1-AR, failed to evoke a detectable Ca2+ increase (Fig. 7, middle). Taken together, these results suggest that Ca2+ entry stimulated by NE occurs through activation of α1-AR.
In the present study, we have provided evidence that NE triggers Ca2+ entry that is sensitive to inhibitors of SOCE (Figs. 2 and 3) in rat pinealocytes. We suggest that this Ca2+ entry allows for continual Ca2+ transients in response to repetitive stimulation of NE (Fig. 2) and have shown that NE-induced Ca2+ entry significantly contributes to the upregulation of AANAT (Fig. 1). Interestingly, NE-evoked Ca2+ entry was associated with membrane hyperpolarization due to the activation of BK channels (Figs. 4 and 5).
Upon noradrenergic stimulation of the rat pineal gland, intracellular cAMP contents increase ∼100-fold compared with the basal level (30), leading to the induction of AANAT gene expression and elevation of AANAT activity. This action of NE is initiated by the β1-AR coupled to Gs protein and adenylyl cyclase (35). However, activation of the β1-AR leads to only a 10-fold increase in the cAMP contents. The maximal increase in cAMP is actually reached when the α1-AR is activated simultaneously with the β1-AR (36). Activation of the α1-AR alone has no effect on cAMP, but it potentiates the β1-AR-induced increase (35). Therefore, the role of Ca2+ increased by stimulation of α1-AR has been suggested to potentiate the β1-AR-induced elevation of cAMP contents and the subsequent AANAT level (38). Herein we report that α1-AR is responsible for the Ca2+ transients observed in response to repetitive NE application (Fig. 7). Together with the results suggesting that SOCE might be responsible for continual Ca2+ responses to repetitive stimulation (Fig. 2), the assumed role of SOCE in NE signaling is to refill the Ca2+ stores to enable α1-AR-mediated [Ca2+]i increase, which contributes to the potentiation role of α1-AR.
Outward K+ current plays an important role in determining the membrane potential. In rat pinealocytes, three types of K+ currents (BK current, transient A current, and delayed-rectifier current) have been identified (1, 5). Although activation of BK channels has been reported, its physiological relevance and relationship to Ca2+ entry channels have not yet been elucidated. We have shown that in the absence of NE, BK current is scarcely observed (Fig. 5), a finding that is in agreement with previously published results (1, 3). NE activates Ca2+ and cAMP, called a biochemical “AND” gate to stimulate melatonin production that has been suggested to activate BK current (3). In neurons and neuroendocrine cells, BK channels have been suggested to play an important role in controlling hormone secretion by altering the duration and frequency of action potentials (16, 24, 29). BK channels require both depolarization and elevated [Ca2+]i to become activated, and their electrical activity is tightly coupled to changes in [Ca2+]i level. Previous in vivo and in vitro electrophysiological studies showed that pinealocytes exhibit action potentials (22, 26). The firing is suggested to be modulated by NE, which is the primary neurotransmitter regulating melatonin synthesis. Herein we suggest that upon application of NE, although a substantial amount of Ca2+ entered through SOCs, hyperpolarization in membrane potential was detected as a result of the activation of the BK channel (Fig. 4). This hypothesis is in line with a previous finding that NE decreased the firing rate in an ex vivo pineal gland (26). Thus it is plausible to suggest that the BK channel coupled to NE-induced Ca2+ entry would reduce excitability by hyperpolarizing the membrane potential. In the rat pineal gland, parasympathetic activation during daylight hours induces depolarization followed by activation of L-type Ca2+ channels, which results in the secretion of l-glutamate and thus eventually decreases melatonin synthesis (37). Because the longer depolarization could induce the secretion of l-glutamate, coupling of BK channels to NE-induced SOCE might contribute to maintenance of the state of melatonin synthesis by preventing depolarization.
This work was supported by the Brain Neurobiology Research Program Grant M10412000088-04N1200-08810, System Bio-Dynamics National Research Center of the Ministry of Science and Technology Grant R15-2004-033-03001-0, the Brain Korea 21 Program of the Ministry of Education, and Korea Research Foundation Grant KRF-2003-015-C00513.
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