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Originally published In Press as doi:10.1074/jbc.M508130200 on September 15, 2005

J. Biol. Chem., Vol. 280, Issue 46, 38228-38234, November 18, 2005
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Rhythmic Expression of Adenylyl Cyclase VI Contributes to the Differential Regulation of Serotonin N-Acetyltransferase by Bradykinin in Rat Pineal Glands*

Sung Han{ddagger}§, Tae-Don Kim{ddagger}, Dae-Cheong Ha{ddagger}, and Kyong-Tai Kim{ddagger}1

From the {ddagger}System Bio-Dynamics NCRC, Division of Molecular and Life Science, Pohang University of Science and Technology, San 31, Hyoja Dong, Pohang 790-784 and the §Drug Discovery Group, LG Life Sciences, Ltd., R&D Park, Daejeon, 305-380, Republic of Korea

Received for publication, July 25, 2005 , and in revised form, August 31, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The rhythmic nocturnal production of melatonin in pineal glands is controlled by the periodic release of norepinephrine from the superior cervical ganglion. Norepinephrine binds to the {beta}-adrenergic receptor and stimulates an increase in intracellular cAMP levels, leading to the transcriptional activation of serotonin N-acetyltransferase, which in turn promotes melatonin production. In the present study, we report that bradykinin inhibits basal- and forskolin-stimulated adenylyl cyclase activity, norepinephrine-induced cAMP generation, and N-acetyltransferase expression in a calcium-dependent manner. These effects were blocked by pretreatment with U73122 [GenBank] (a selective phospholipase C inhibitor), and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (a Ca2+ chelator), but not pertussis toxin. The calcium ionophore, ionomycin, inhibited isoproterenol-mediated cAMP generation, similar to bradykinin. Interestingly, the inhibitory effect of bradykinin was evident only during the daytime. At midday, bradykinin inhibited the cAMP level by ~50% but markedly stimulated cAMP production (by ~50%) at night. Northern blotting and immunoblotting data disclosed circadian expression of calcium-inhibitable adenylyl cyclase type 6. Expression of adenylyl cyclase type 6 was maximal at Zeitgeber Time (ZT) 01 and very low at ZT 13. Our results suggest that bradykinin-induced calcium inhibits melatonin synthesis through the mediation of adenylyl cyclase type 6 expression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The mammalian pineal gland is a neuroendocrine transducer that rhythmically synthesizes and secretes melatonin, a regulator of circadian rhythm, sleep, mood, reproduction, and aging (1). Melatonin synthesis is modulated by the hypothalamic circadian clock in the suprachiasmatic nucleus as well as light signals through a multisynaptic neuronal pathway projecting from the retina to the suprachiasmatic nucleus of the anterior hypothalamus via the retinohypothalamic tract (2). A dark signal perceived by the retina triggers norepinephrine release from postganglionic neurons originating in the superior cervical ganglion regulated by suprachiasmatic nucleus activation (3, 4). Norepinephrine interacts with the {beta}-adrenergic receptor on pinealocytes, inducing an increase in pineal cAMP generation. This elevation of cAMP, in turn, stimulates the conversion of tryptophan to melatonin through triggering transcription and activity of serotonin N-acetyltransferase (AANAT,2 acetyl coenzyme A:arylalkylamine N-acetyltransferase; EC 2.3.1.87 [EC] ), the key enzyme catalyzing the important step of melatonin synthesis (5, 6). This {beta}-adrenergic receptor-mediated cAMP signaling is further potentiated by stimulation of {alpha}1-adrenergic receptors through increment of cytosolic Ca2+ and activation of protein kinase C (PKC) (7). Protein kinase C additionally stabilizes the AANAT protein via phosphorylation (8).

Previous studies have suggested that multiple receptors for amino acids, neuropeptides, and biogenic amines other than norepinephrine present on pinealocytes participate in the modulation of melatonin synthesis (9). However, to date, no studies have established the presence of BK receptors on pinealocytes.

BK, a nonapeptide hormone, is generated from the high molecular weight precursor, kininogen, by the proteolytic action of kallikrein and functions as a potent mediator of inflammation, pain, asthma, and hypertension (10, 11). The kallikrein-kinin system has been identified in the mammalian nervous system (12, 13). Interestingly, kallikrein mRNA and enzyme activity are higher in the rat pineal gland than other brain regions (14, 15). Consistent with this finding, immunoelectron microscopy data by Kudo et al. (16) disclosed that kallikrein is localized in perivascular spaces in the rat pineal gland. However, the functional role of the kallikrein-kinin system in rat pineal glands is currently unclear.

A number of earlier investigations reported that BK inhibits {beta}-adrenergic receptor-mediated cAMP accumulation in various cell types (17–19). Moreover, Ca2+-inhibitable adenylyl cyclase (AC) is involved in the BK-induced inhibition mechanism, because stimulation of the BK receptor leads to increased [Ca2+]i. To date, at least nine isoforms of AC have been cloned and characterized in mammals (20). Among these, types V and VI constitute a subfamily defined as Ca2+-inhibitable.

Here, we report that the treatment of rat pinealocytes with BK results in [Ca2+]i increase and diurnal inhibition of pineal AC activity. Inhibition of {beta}-adrenergic receptor-mediated cAMP generation and AANAT expression by BK is mediated by direct suppression of Ca2+-inhibitable AC type VI (AC6). Additionally, the expression patterns of AC6 mRNA and protein follow a diurnal rhythm.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Fura-2 pentaacetoxymethyl ester (Fura-2/AM) and BAPTA/AM were obtained from Molecular Probes (Eugene, OR). [3H]IP3 and [3H]cAMP were purchased from PerkinElmer Life Sciences. U73122 [GenBank] was acquired from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). BK, Me2SO, (±)-sulfinpyrazone, Triton X-100, EGTA, ethidium bromide, pertussis toxin (PTX), charcoal, acetyl coenzyme A, tryptamine, isoproterenol, and IP3 were purchased from Sigma. DArg[Hyp3,Thi5,dTic7,Oic8]BK (HOE140) was obtained from Research Biochemicals, Inc. (Natick, MA). [des-Arg10]Kallidin (DAK), and [des-Arg10]HOE140 (dHOE140) were from Peninsula laboratories, Inc. (Belmont, CA). [3H]Acetyl coenzyme A and Econofluor were obtained from PerkinElmer Life Sciences and an enhanced chemiluminescence kit (ECL, SUPEX) was purchased from Neuronex (Pohang, Korea). Peroxidase-conjugated anti-mouse immunoglobulin G (IgG) and anti-sheep IgG were obtained from Kirkegaard and Perry Laboratories Inc. (Gaithersburg, MD). X-ray film was from AGFA (Agfa-gevaert N. V. Belgium). Anti-rat AANATp22–37 sera 2500 and 3352, anti-rat AANAT25–200 serum 3314, and purified rat AANAT protein were kindly supplied by Dr. David C. Klein (National Institutes of Health, Bethesda, MD).

Animals—Adult male Sprague-Dawley rats (150–200 g) were obtained from Hyochang Science (Seoul, Korea). Rats were maintained under a 12-h light/12-h dark cycle (LD12:12) with lights off at 7:00 pm for at least 1 week before the day of the experiment.

Rat Pinealocyte Culture—Pinealocytes were isolated from the pineal glands of male Sprague-Dawley rats (150 g, Hyochang Science) at postnatal week 6. Male Sprague-Dawley rats were decapitated, and pineal glands were washed with ice-cold Locke's solution (154 mM NaCl, 5.6 mM KCl, 10 mM glucose, 2.2 mM CaCl2, 1.2 mM MgCl2 and 5 mM HEPES, adjusted to pH 7.4). Glands were dissected into small sections and treated with 0.1% collagenase solution (2 mg/ml collagenase type V in Locke's solution, Invitrogen) at 37 °C for 30 min with gentle shaking. This was followed by treatment with 0.025% trypsin solution (Invitrogen) at 37 °C for 15 min, and centrifugation at 180 x g for 5 min. Dispersed pinealocytes were washed three times with Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum and 1% (v/v) antibiotics comprising 5000 units/ml penicillin G (sodium) and 5000 µg/ml streptomycin sulfate in 0.85% saline (Invitrogen), pH 7.4. Pinealocytes were placed in coverslips or culture dishes coated with poly-L-lysine, and maintained in the above medium in a humidified atmosphere of 5% CO2 at 37 °C.

Single-cell Ca2+ Imaging—Intracellular Ca2+ levels were measured by fluorescence ratio imaging of the Ca2+ indicator dye, Fura-2. Briefly, pinealocyte-attached coverslips were transferred to a 35-mm culture dish containing prewarmed medium. After 10 min, the medium was replaced with 1.5 ml of the same culture medium containing 3 µM Fura-2/AM dissolved in Me2SO, and incubated for 1 h. Coverslips were rinsed twice with 1 ml of medium, placed in a perfusion chamber, and perfused with imaging buffer (154 mM NaCl, 5.6 mM KCl, 5.0 mM HEPES, 1.2 mM MgCl2, 2.2 mM CaCl2, 10 mM glucose, 1 mg/ml bovine serum albumin, pH 7.4). Rat pinealocytes were imaged using the ImageMaster system (Photon Technology International). The average Ca2+ level in individual rat pinealocytes was determined from the ratio of fluorescence emissions obtained using two different excitation wavelengths (340 and 380 nm).

Measurement of IP3—The IP3 concentration in cells was determined using a competition assay with [3H]IP3, as described previously (21), with minor modifications. In brief, pinealocytes were stimulated as indicated, and the reaction was terminated by aspirating medium off the cells, followed by the addition of 5% (w/v) ice-cold trichloroacetic acid containing 4 mM EGTA. Samples were left on ice for 30 min to extract water-soluble inositol phosphates. Samples were transferred to Eppendorf tubes, and trichloroacetic acid was removed by extraction with diethyl ether four times. The final preparation was neutralized with 200 mM Trizma base, and the pH was adjusted to ~7.4. Assay buffer (50 mM Tris/HCl, pH 8.0, 4 mM EGTA, 4 mg/ml bovine serum albumin), [3H]IP3 (0.1 Ci/ml), and IP3-binding protein were added to cell extracts. The mixture was incubated on ice for 15 min and centrifuged at 10,000 x g for 5 min. Scintillation mixture was added to the pellet to measure radioactivity. The IP3 concentration was determined using a standard curve. IP3-binding protein was prepared from bovine adrenal cortex according to the method of Challiss et al. (22).

Quantitation of cAMP—The cAMP concentration in rat pineal glands was determined using the [3H]cAMP competition assay for evaluating interactions with cAMP-binding protein (23). Rats were decapitated at midday (12:00), and glands were placed directly into ice-cold DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin. After the removal of extraneous tissue, glands were placed on nylon mesh that rested on DMEM medium, and incubated at 37 °C in a humidified atmosphere of 5% CO2 for 1 h before the experiment. Glands were stimulated with agonists for 3 min at 37 °C and transferred to a 1.5-ml Eppendorf tube containing 200 µl of absolute ethanol for termination of the reaction. This was followed by incubation for 2 h at –20 °C to extract cAMP. Glands in ethanol were sonicated for 10 s and centrifuged with 2,500 x g for 10 min at 4 °C. The supernatant was evaporated to dryness in a SpeedVac (Savant instruments, Farmingdale, NY). Residues were dissolved in 0.2 ml of Tris-HCl (pH 7.5) and 4 mM EDTA. Sample solution (50 µl) was employed in the cAMP assay. The assay is based on competition between [3H]cAMP and unlabeled cAMP in the sample for crude cAMP-binding protein prepared from bovine adrenal cortex according to the method of Brown et al. (24). Free [3H]cAMP was adsorbed using charcoal and removed by centrifugation, and bound [3H]cAMP in the supernatant was measured by liquid scintillation. Each unknown sample was incubated with 50 µlof[3H]cAMP (5 µCi/ml) and 100 µl of binding protein for 2 h at 4°C. Separation of protein-bound cAMP from unbound cAMP was achieved by adsorption of free cAMP onto charcoal (100 µl), followed by centrifugation (12,000 x g)at4°C. Supernatant fractions (200 µl) were placed in Eppendorf tubes containing 1.2 ml of scintillation mixture to measure radioactivity. The cAMP concentrations in samples were determined using a standard curve and expressed as picomoles/gland.

AANAT Activity Assay—AANAT activity was measured as described previously (25). Briefly, rats were decapitated at midday (12:00), and pineal glands were placed directly into ice-cold DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin. After the removal of extraneous tissue, pineal glands were placed on nylon mesh that rested on DMEM medium, and incubated at 37 °C in a humidified atmosphere of 5% CO2 for 1 h before the experiment. Following stimulation, rat pineal glands were individually disrupted by ultrasound in 100 µl of ice-cold phosphate buffer (50 mM, pH 6.8). Debris was removed by centrifugation (15,000 x g, 5 min at 4 °C), and the supernatant was transferred to a new tube and stored at –80 °C until use. An amount (8 µl or indicated volumes) of the supernatant was incubated in the presence of 5 µl of tryptamine-HCl (10 mM), 1 µl of acetyl CoA (0.5 mM), and 1 µlof[3H]acetyl CoA (3.6 Ci/mmol, 250 µCi/ml). Phosphate buffer (50 mM, pH 6.8) was added to obtain a final volume of 20 µl. Incubation was performed at 37 °C for 30 min, and the was reaction terminated by dilution with an additional 180 µl of ice-cold phosphate buffer (50 mM, pH 6.8). Econofluor was rapidly added to the whole diluted reaction mixture, and after incubation for 15 min, the amount of radiolabeled acetyltryptamine was determined in a liquid scintillation counter.

Reverse Transcription-PCR Analysis—Total RNA was extracted from rat pineal glands with the TRI reagent (Molecular Research Center, Inc., Cincinnati, OH). First-strand cDNA was synthesized for 90 min at 42 °C in the presence of 1 mM of each of the four deoxynucleotide triphosphates (Invitrogen) and Superscript II (Invitrogen). cDNA was amplified with 10 pmol of primers (Bioneer, Korea) specific for each AC isoform, using ExTaq polymerase (Takara, Japan). The primers of AC isoforms were selected from published cDNA sequences (26, 27), including AC type I, 5'-AGCACTTCCTAATGTCCAACCCT-3' and 5'-AGCACTTCCTAATGTCCAACCCT-3'; AC type II, 5'-CGTGTCACTCTCCATATTC-3' and 5'-CCTTGTTCACATCTGACTC-3'; AC type III, 5'-CATCGAGTGTCTACGCTTC-3' and 5'-GGATGACCTGTGTCTCTTCT-3'; AC type IV, 5'-TTCTTCACACTCCTCGTCC-3' and 5'-CGTCCTTGTTGTGTGTCCTG-3'; AC type V, 5'-ATCGAGCTCATCTACGTGC-3' and 5'-AGCATGCAGATACAGAGCC-3'; AC type VI, 5'-CTGCTTGTGTTCATCTCTG-3' and 5'-GACGCTAAGCACTAGATCA-3'; AC type VII, 5'-CCAGTTATTTAGAGAGAGACCTG-3' and 5'-CTTGCTCATCAGGGCCATGCTAA-3'; AC type VIII, 5'-GGACAGCAGCTGGAGTACACAGC-3' and 5'-CCTGATCCTTCAGGATGAGATAG-3'; AC type IX, 5'-AGCTTATCCTCACCTTCTTCTTCCTC-3' and 5'-AGGACACGGTAGCACTCCTTGCC-3'. The reaction was performed for 30 cycles of denaturation at 95 °C for 1 min, annealing at 48 °C for 1 min, and extension at 72 °C for 1 min with a final 10-min extension at 72 °C. The specificities of amplified cDNA fragments were confirmed by sequence analysis.

Northern Blotting—Total RNA (15 µg) was resolved on 1% (w/v) agarose gels containing 0.66 M formaldehyde and transferred to nylon membranes (ICN, East Hills, NY). Blots were hybridized to rat AC type VI probes labeled with [{alpha}-32P]dCTP by the random primer extension method. Hybridization was performed at 65 °C in solution containing 10% polyethylene glycol, 7% SDS, 10 mM EGTA, 250 mM NaCl, 85 mM NaH2PO4 (pH 7.2), denatured salmon sperm DNA (100 µg/ml), and radiolabeled probe (5 x 105 cpm/ml). After hybridization, blots were briefly washed twice with 1x saline sodium citrate (SSC: 300 mM NaCl and 30 mM sodium citrate) containing 0.1% SDS at room temperature, three times with 0.2x SSC containing 0.1% SDS at 65 °C, and twice with 0.1x SSC at room temperature. Blots were reprobed with rat glyceraldehyde-3-phosphate dehydrogenase cDNA as a control.

Immunoblotting—Proteins from pineal glands prepared at indicated times were separated by SDS-PAGE and blotted onto nitrocellulose membrane (0.45 mm, Bio-Rad), as described previously (28). Membranes were incubated with the AC5/6 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or AANAT antibody (serum 2500, 1:30,000 dilution, a kind gift from Dr. D. C. Klein, National Institutes of Health, Bethesda, MD). Signals were detected with the ECL detection system.

Statistical Analysis—All quantitative data are presented as means ± S.E. of a minimum of three experiments. Comparisons between two groups were analyzed via t test, and values of p < 0.05 were considered to be significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Following perfusion of pinealocytes with 100 nM BK, [Ca2+]i increased transiently in 90% of the cell population. Serial perfusion of 100 nM BK, 1 µM 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), 1 µM phenylephrine, and 70 mM KCl (High K+), which are known [Ca2+]i-mobilizing agents in rat pinealocytes (29, 30), led to transient elevation of [Ca2+]i (Fig. 1A). These responses correlate with the typical characteristics of rat pinealocytes. BK triggered an increase in [Ca2+]i in a concentration-dependent manner (Fig. 1B). The concentration dependence curve discloses maximal and half-maximal effective concentration (EC50) values for BK of 1 µM and 50 nM, respectively. The B1 BK receptor-specific agonist, [des-Arg10]kallidin (DAK), had little effect on [Ca2+]i mobilization (Fig. 2A). To further define the subtype of BK receptor involved, cells were perfused with specific BK antagonists. Fig. 2B depicts the responses of BK-induced [Ca2+]i increase in the presence of [des-Arg10]HOE140 (dHOE140) and HOE140, B1 and B2 BK receptor specific antagonists, respectively. Pretreatment with HOE140 completely eliminated the BK-induced [Ca2+]i increase. In contrast, dHOE140 had little inhibitory effect on BK-induced [Ca2+]i mobilization. The antagonists themselves had no effects on [Ca2+]i increase.



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  		FIGURE 1.
BK increases the [Ca2+]i in rat pinealocytes. A, trace of Fura-2/AM fluorescence ratio (alternate excitation at 340 and 380 nm) depicting the [Ca2+]i transients from a single rat pinealocyte. The following compounds were added for the indicated times represented by bars: 100 nM BK, 1 µM DMPP, 1 µM phenylephrine, and 70 mM KCl (high K+). B, concentration dependences for the [Ca2+]i transients stimulated by BK (•). Fura-2/AM-loaded pinealocytes were treated with various concentrations of BK, and peak fluorescence ratios were measured. Each BK concentration was independently tested four times, and data are presented as mean ± S.E. values.

 
In view of the finding that IP3 induces Ca2+ mobilization from intracellular stores through specific receptors, we measured the IP3 generated as a result of BK treatment. Fig. 2C shows that BK treatment stimulated IP3 generation, whereas DAK, a B1 BK receptor-specific agonist, had little effect. Antagonists of BK receptors had similar effects on IP3 generation, as evident from [Ca2+]i mobilization data. HOE140, a B2 BK receptor-specific antagonist, blocked BK-induced IP3 generation. In contrast, dHOE140, a B1 BK receptor-specific antagonist, did not affect BK-induced IP3 generation. The results collectively indicate that phospholipase C (PLC)-linked BK receptors exist in rat pinealocytes, and the subtype of BK receptors expressed in rat pinealocytes is B2.

To elucidate the physiological roles of BK, we focused on its effect on {beta}-adrenergic receptor signaling, the major pathway of melatonin synthesis. Previous studies have shown that [Ca2+]i increase potentiates the effects of {beta}-adrenergic stimulation in pineal glands (31, 32). However, BK-mediated modulation of {beta}-adrenergic receptor signaling was distinct from the well established concept of Ca2+ signaling on melatonin synthesis. BK differentially modulates {beta}-adrenergic signaling according to the circadian time. At ZT05, isoproterenol-induced cAMP generation was inhibited by about 30% by BK (Fig. 3A). On the other hand, at ZT17, isoproterenol-induced cAMP generation was increased 1.8-fold in the presence of BK (Fig. 3B). Furthermore, forskolin-induced AC activity was differentially regulated by BK, dependent on the time of day. At ZT05, co-treatment with BK resulted in about 50% inhibition of forskolin-stimulated AC activity (Fig. 3A), whereas at ZT17, BK enhanced forskolin-stimulated AC activity by about 1.5-fold (Fig. 3B). Additionally, differential regulation of basal pineal AC activity by BK was observed. At ZT05, BK inhibited basal AC activity about 80%, whereas at ZT17, enzyme activity was stimulated ~20-fold (Fig. 3C). These results indicate that BK diurnally inhibits {beta}-adrenergic receptor signaling through suppression of AC activity.



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  		FIGURE 2.
Effects of BK and BK analogs on [Ca2+]i and IP3 production in rat pinealocytes. A, traces of the Fura-2/AM fluorescence ratio signifying the [Ca2+]i transients evoked by 100 nM BK and 100 nM DAK for the indicated times, represented by bars. B, inhibition of BK-induced [Ca2+]i transients by BK receptor antagonists from a single rat pinealocyte. Prior to the addition of 100 nM BK, HOE140 and dHOE140 (100 nM each, BK receptor antagonists) were preincubated with Fura-2/AM-loaded rat pinealocytes for the indicated times represented by bars. C, the left panel displays IP3 production, following treatment with BK and DAK (100 nM each) for 30 s. The right panel shows that BK-induced IP3 production was inhibited by pretreatment with the BK receptor antagonists, HOE140 and dHOE140. Rat pinealocytes were treated with the indicated BK receptor antagonists (100 nM each) for 10 min, and cells were stimulated with 100 nM BK for 1 min. The IP3 concentration was determined from cells. Data are presented as means ± S.E. (bars) values from three individual experiments.

 
Because Ca2+-mediated potentiation of {beta}-adrenergic stimulation in pineal glands is a well known concept, we focused on the opposing inhibitory effect of BK on {beta}-adrenergic signaling during the daytime. Co-treatment with BK resulted in concentration-dependent inhibition of isoproterenol-induced cAMP generation (Fig. 4A) and forskolin-stimulated AC activation (Fig. 4B) in rat pineal glands. Furthermore, concentration-dependent blockage of isoproterenol-induced AANAT activation (Fig. 4C) was also observed in rat pineal glands. All the above experiments were conducted with rat pineal glands excised at ZT05. The results indicate that BK acts as a negative regulator of melatonin synthesis at daytime through direct inhibition of AC.



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  		FIGURE 3.
Zeitgeber Time-dependent differential regulation of {beta}-adrenergic signaling and AC activity by BK. Rat pineal glands were stimulated with 10 µM isoproterenol (Iso)or10 µM forskolin (Fsk) in the presence (dashed bar) or absence (filled bar)of1 µM BK for 3 min at ZT05(A) and ZT17 (B). Glands were prepared at the indicated times and stimulated with various drugs after 30 min of stabilization. Next, glands were homogenized, and cAMP content assayed, as described under "Experimental Procedures." C, basal AC activity was additionally measured by measuring cAMP levels in rat pineal glands in the presence (dashed bar) or absence (filled bar)of1 µM BK. Data are presented as means ± S.E. of three independent experiments (bars). *, p < 0.05.

 
We further investigated the mechanism of BK signaling that inhibits {beta}-adrenergic receptor-mediated cAMP generation (TABLE ONE). To confirm the hypothesis that BK-evoked inhibition of {beta}-adrenergic signaling is mediated through a guanine nucleotide-binding regulatory protein (G protein), such as Gi/o, rat pineal glands were preincubated with 300 ng/ml PTX, which uncouples the Gi/o protein from its receptor by catalyzing ADP-ribosylation of the {alpha}i/o subunit, for 6 h. PTX had no effect on BK-specific inhibition of isoproterenol-mediated cAMP generation. Because the B2 BK receptor generally links to PLC through Gq protein (33), we additionally evaluated whether PLC-mediated [Ca2+]i rise contributes to the inhibitory effect of BK. U73122 [GenBank] , a PLC inhibitor, completely reversed BK-induced suppression of isoproterenol-mediated cAMP generation. Next, we examined the possibility that BK-specific inhibition of cAMP production in rat pineal glands was due to the downstream event of PLC activation. Intact rat pineal glands were preincubated with 20 µM BAPTA/AM, a cell-permeable Ca2+ chelator, to block the [Ca2+]i increase. As shown in TABLE ONE, inhibition of cAMP generation by BK was completely reversed following pretreatment with BAPTA/AM. In addition, treatment of rat pineal glands with ionomycin (200 nM) led to blockage of isoproterenol-mediated cAMP generation. These results indicate that the inhibitory effect of BK on isoproterenol-induced cAMP generation in rat pineal glands is due to the direct effect of [Ca2+]i increase on pineal AC.


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  		TABLE ONE
Involvement of the [Ca2+]i in BK-specific inhibition of {beta}-adrenergic signaling in rat pineal glands

Rat pineal glands were pretreated with the following compounds for the indicated time: 300 ng/ml PTX for 12 h, 10 µM U73122 [GenBank] for 10 min, 20 µM BAPTA/AM for 10 min, and 200 nM ionomycin for 10 min, respectively. Glands were stimulated with 10 µM isoproterenol in the presence or absence (Control) of 1 µM BK for 3 min. Following stimulation, glands were homogenized, and cAMP generation was assayed, as described under "Experimental Procedures." Results were expressed as relative values ± S.E. (three independent experiments). Isoproterenol (10 µM)-induced cAMP generation in the absence of BK was taken as 100%.

 



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  		FIGURE 4.
BK-mediated inhibition of{beta}-adrenergic signaling and AC activity at ZT05. A, BK inhibits isoproterenol-induced cAMP generation in a concentration-dependent manner. Glands were co-treated with 10 µM isoproterenol and various concentrations of BK for 3 min, and cAMP generation was measured using a competition assay, as described under "Experimental Procedures." Data are presented as means ± S.E. values from three independent experiments. B, BK additionally inhibits forskolin-induced cAMP generation in a concentration-dependent manner. Glands were co-treated with 10 µM forskolin and various concentrations of BK for 3 min, and cAMP generation was measured using a competition assay, as described under "Experimental Procedures." Data are presented as mean ± S.E. values from three independent experiments. C, BK inhibits isoproterenol-induced AANAT activation in a concentration-dependent manner. Glands (three each) were co-incubated with 10 µM isoproterenol and various concentrations of BK for 7 h. Following incubation, glands were collected, and AANAT activity was measured with the liquid biphasic diffusion assay, as described under "Experimental Procedures." Results were expressed as relative values ± S.E. (three independent experiments). Isoproterenol (10 µM)-induced AANAT activity in the absence of BK was taken as 100%.

 
To explain the differential modulation of {beta}-adrenergic signaling by BK, which are dependent on the time of day, we hypothesize that "Ca2+-inhibitable" AC proteins are present in the pineal gland with expression patterns following circadian rhythms. To verify this theory, the expression of AC isoforms in rat pineal glands was examined by reverse transcription-PCR analysis. PCR primer sets specific for known rat AC genes were selected from the reported cDNA sequences (26, 27). As shown in Fig. 5, amplified products of the expected sizes for all AC isoforms (except type V) were detected in rat pineal glands. These products were identified in at least three sets of experiments using different rat pineal glands. Moreover, nucleotide sequences of the amplified DNA products were identical to those of rat AC isoforms. Among the AC isoforms expressed in rat pineal glands, the Ca2+-inhibitable AC is type VI (AC6). Accordingly, AC6 expression was investigated during the day-night cycle in rat pineal glands by Northern blot and immunoblot analyses. AC6 mRNA and protein expression displayed significant circadian patterns of expression (Fig. 6). Specifically, levels of AC6 mRNA and protein started to increase at midnight (ZT17), and peaked at midday (ZT05). This expression pattern is complementary to that of AANAT mRNA and protein. In this case, the levels of AANAT mRNA and protein started to decrease at midnight (ZT17), and reached a minimum at midday (ZT05). Our results strongly support the theory that the differential modulation of BK on {beta}-adrenergic signaling and pineal AC activity during the day and night is mainly due to diurnal expression of AC6.



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  		FIGURE 5.
Analysis of AC isoform expression in rat pineal glands. Reverse transcription-PCR data on transcripts for AC isoforms reveal that all known AC isoforms, except type V, are expressed in rat pineal glands. Primer pairs specific for AC isoforms I, II, III, IV, V, VI, VII, VIII, and IX were employed, as described under "Experimental Procedures." The template for PCR analysis was analyzed in the presence (+) and absence (–) of reverse transcriptase.

 



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  		FIGURE 6.
Circadian variation of AC6 expression in the rat pineal gland. Rats were housed in a controlled lighting environment (LD12:12), with lights on at circadian time (CT) 07. The filled bar represents the times when lights were off. A, Northern blot and immunoblot analysis display the circadian expression of rat AC type 6 (rAC6). AANAT mRNA and protein levels are shown as a comparing control. B, time course of relative AC6 protein (open circle) and AANAT mRNA expression (filled circle). Band intensities of AANAT transcripts and rAC6 protein were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA and protein to correct for variations in loading. AANAT/glyceraldehyde-3-phosphate dehydrogenase ratio at ZT17 and rAC6/glyceraldehyde-3-phosphate dehydrogenase ratio at ZT01 were adjusted to 100%.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The present study reveals that the B2 BK receptors are present and differentially modulate {beta}-adrenergic signaling, the major pathway of melatonin synthesis, by direct suppression of Ca2+-inhibitable AC type VI (AC6) in rat pinealocytes, and that the differential modulation of {beta}-adrenergic signaling by BK-evoked [Ca2+]i increase is due to the diurnal expression of pineal AC6 mRNA and protein.

Recently, the presence of BK receptors in mammalian neuronal and neuroendocrine systems was demonstrated by immunohistochemical (34, 35) and radioligand binding experiments (36). Although the physiological roles of BK are currently unclear, the presence of BK receptors indicates a role in the brain or neuroendocrine system. BK increases the permeability of the blood-brain barrier by activation of B2 receptors on brain endothelial cells (37). Moreover, BK stimulates the release of hormones and neurotransmitters, such as gonadotropin-releasing hormone and norepinephrine, through B2 receptor activation (38, 39). Activation of B2 receptors triggers norepinephrine release from rat sympathetic neurons (40). Our present data show that stimulation of BK results in concentration-dependent inhibition of {beta}-adrenergic receptor-mediated cAMP generation and AANAT activation in rat pineal glands during the daytime. Our results also indicate that inhibition of {beta}-adrenergic receptor-induced cAMP generation by BK is mediated by the increase in [Ca2+]i, which is inconsistent with the well established concept of intracellular free Ca2+ on melatonin synthesis. Previous studies have shown that [Ca2+]i increase potentiates the effects of {beta}-adrenergic stimulation (30–32). However, the effects of intracellular Ca2+ mobilization in pineal glands at various circadian time points remain to be determined. Tzavara et al. (41) demonstrated inhibition of AC activity in rat pineal glands by calcium, both in the presence and absence of calmodulin at midday but marked activation by the calcium-calmodulin complex at night. In agreement with this finding, we showed that BK-evoked [Ca2+]i diurnally suppresses {beta}-adrenergic signaling through inhibition of pineal AC activity in rat pineal glands. To elucidate the differential modulation mechanism of BK, we focused on the Ca2+-inhibitableAC proteins. To date, nine isoforms of AC have been cloned (20). Among these, types V and VI constitute a subfamily, which has the remarkable property of being inhibited by submicromolar Ca2+ concentrations in addition to G{alpha}i-mediated processes (42). Several previous studies showed the involvement of Ca2+-inhibitable AC in the inhibitory mechanism of BK in various cell types. Stimulation of BK receptors markedly reduced cAMP production in C6–2B rat glioma cells through the PTX-insensitive elevation of intracellular Ca2+ concentration (17). In NCB-20 cells, BK stimulates Ca2+ mobilization, leading to direct suppression of AC (18). These reports suggest the possibility of direct inhibition of {beta}-adrenergic receptor-linked AC by BK-mediated [Ca2+]i in rat pineal glands. Initially, we identified the AC isoforms expressed in rat pineal glands. Reverse transcription-PCR analyses revealed the expression of types I, II, III, IV, VI, VII, VIII, and IX AC proteins, of which type VI (AC6) is the Ca2+-inhibitable AC. We examined the time-dependent expression patterns of AC6 mRNA and protein. Northern blot and immunoblot analyses revealed circadian expression of AC6 mRNA and protein, with maximal levels at ZT01 and minimal levels at ZT13. Moreover, the expression profiles of AC6 and AANAT were complementary. Specifically, when the AANAT mRNA level started to decrease, AC6 transcript expression increased. These findings support the possible involvement of AC6 in diurnal inhibition of {beta}-adrenergic receptor-mediated cAMP generation and AANAT activity by B2 bradykinin receptor signaling in rat pineal glands.

Previous studies have reported the diurnal expression of AC1 mRNA in rat pineal glands (41, 43) and circadian patterns of AC1 mRNA and protein activity. AC1 mRNA levels are maximal during the daytime and minimal at night. These results imply that expression of AC1 and AC6 mRNA follow the same circadian pattern. Interestingly, unlike AC6, AC1 mRNA expression and protein activity are not in phase, but rather they are inversely related. Whereas the AC1 transcript level peaks during the day, activity is maximal at night. Chan et al. (44) explained the uncoupling of AC1 mRNA expression and protein activity by showing the presence of the cAMP-inhibitable element in the AC1 promoter region. Based on our results, in association with earlier data, we have formed an interesting hypothesis. AC1 and AC6 are part of the Ca2+-regulated AC subclass. One is Ca2+-stimulatable (AC1), whereas the other is Ca2+-inhibitable (AC6). The activities of these two ACs follow the circadian rhythm with inverse patterns in rat pineal glands. During the daytime, AC6 activity reaches a peak. Conversely, AC1 activity peaks at night. In view of these findings, we propose that [Ca2+]i increase acts as a Janus-faced modulator of AC signaling in pineal glands. At night, the [Ca2+]i increase potentiates {beta}-adrenergic receptor-mediated cAMP signaling through activation of AC1 (41) and protein kinase C (45). During the daytime, {beta}-adrenergic receptor-mediated cAMP signaling is suppressed via direct inhibition of AC6 by [Ca2+]i. In other words, differential circadian modulation of the pineal cAMP level by [Ca2+]i is influenced by circadian changes in the AC1/AC6 ratio in rat pineal glands. Indeed, this hypothesis is based on speculation rather than fact, and more extensive studies are necessary for confirmation.

Until now, several mechanisms have been proposed to explain the termination of nocturnal melatonin synthesis. There is no doubt that termination of melatonin synthesis is mainly initiated by the termination of norepinephrine release (46). Other mechanisms have been proposed, such as rapid proteasomal proteolysis of AANAT (47) and inhibition of {beta}-adrenergic signaling by metabotropic glutamate receptors through the inhibitory guanine nucleotide-binding protein (Gi) (48). In addition, we have shown that rhythmic degradation of AANAT mRNA is also essential to achieve circadian oscillation of melatonin synthesis (49). Here, we propose an additional termination mechanism of nocturnal melatonin synthesis by the inhibition of {beta}-adrenergic signaling through blocking Ca2+-inhibitable AC6 with [Ca2+]i-mobilizing agents, such as BK. We additionally provide evidence that [Ca2+]i differentially modulates the pineal cAMP level according to circadian time, due to periodic changes in AC1/AC6 activity in rat pineal glands.


   FOOTNOTES
 
* This work was supported by the Brain Neurobiology Research Program (Grant M10412000088-04N1200-08810), the System Bio-Dynamics National Core Research Center of the Ministry of Science and Technology, the Brain Korea 21 Program of the Ministry of Education, and the Korea Research Foundation Grant for Young Scientist (Grant KRF-2005-213-C00036). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 82-54-279-2297; Fax: 82-54-279-2199; E-mail: ktk{at}postech.ac.kr.

2 The abbreviations used are: AANAT, arylalkylamine N-acetyltransferase; BK, bradykinin;BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; -AM, -acetoxymetyl ester; AC1, adenylyl cyclase type I; AC6, adenylyl cyclase type VI; HOE140,DArg[Hyp3,Thi5,dTic7,Oic8]BK; DAK, [des-Arg10]kallidin; dHOE140, [des-Arg10]HOE-140; DMEM, Dulbecco's modified Eagle's medium; DMPP, 1,1-dimethyl-4-phenylpiperazinium iodide; ZT, Zeitgeber Time; PTX, pertussis toxin; PLC, phospholipase C; Fura-2/AM, Fura-2 pentaacetoxymethyl ester; IP3, inositol 1,4,5-trisphosphate; Back


   ACKNOWLEDGMENTS
 
We thank Dr. Seungwon Lee for critical discussions and ideas.



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