The pineal glands regulate circadian rhythm through the synthesis and secretion of melatonin. The stimulation of nicotinic acetylcholine receptor due to parasympathetic nerve activity causes an increase in intracellular Ca2+ concentration and eventually downregulates melatonin production. Our previous report shows that rat pinealocytes have spontaneous and nicotine-induced Ca2+ oscillations that are evoked by membrane depolarization followed by Ca2+ influx through voltage-dependent Ca2+ channels (VDCCs). These Ca2+ oscillations are supposed to contribute to the inhibitory mechanism of melatonin secretion. Here we examined the involvement of large-conductance Ca2+-activated K+ (BKCa) channel conductance on the regulation of Ca2+ oscillation and melatonin production in rat pinealocytes. Spontaneous Ca2+ oscillations were markedly enhanced by BKCa channel blockers (1 μM paxilline or 100 nM iberiotoxin). Nicotine (100 μM)-induced Ca2+ oscillations were also augmented by paxilline. In contrast, spontaneous Ca2+ oscillations were abolished by BKCa channel opener [3 μM 12,14-dichlorodehydroabietic acid (diCl-DHAA)]. Under whole cell voltage-clamp configurations, depolarization-elicited outward currents were significantly activated by diCl-DHAA and blocked by paxilline. Expression analyses revealed that the α and β3 subunits of BKCa channel were highly expressed in rat pinealocytes. Importantly, the activity of BKCa channels modulated melatonin secretion from whole pineal gland of the rat. Taken together, BKCa channel activation attenuates these Ca2+ oscillations due to depolarization-synchronized Ca2+ influx through VDCCs and results in a recovery of reduced melatonin secretion during parasympathetic nerve activity. BKCa channels may play a physiological role for melatonin production via a negative-feedback mechanism.
- calcium oscillation
- calcium-activated potassium channel
- pineal gland
- parasympathetic nerve
the pineal glands can play a pivotal role in the generation of circadian rhythm through the synthesis and secretion of melatonin. Melatonin production begins with adrenergic β1 receptor stimulation by norepinephrine (NE), which is released from nerve endings of sympathetic axons in the superior cervical ganglia (33). The stimulation increases cAMP production and activates a melatonin-synthesizing enzyme, arylalkylamine-N-acetyltransferase (AANAT). This signal is thought to be enhanced synergistically by inositol 1,4,5-trisphosphate-mediated Ca2+ release via adrenergic α1 activation by NE. In addition to this adrenergic pathway, parasympathetic neurons, which originate from the pterygopalatine ganglia, are involved in the regulation of melatonin production (28). The stimulation of nicotinic acetylcholine receptors (nAChRs) elicits membrane depolarization, and thus induces Ca2+ influx mediated by voltage-dependent Ca2+ channels (VDCCs). The increase in intracellular Ca2+ concentration ([Ca2+]i) enhances the release of glutamic acid from pinealocytes (37). The released glutamic acid is likely to activate metabotropic glutamate receptor type 3 (mGluR3), which couples to Gi protein and reduces adenylate cyclase activity (38). The decrease in cAMP synthesis reduces AANAT activity and melatonin synthesis. Therefore, the parasympathetic innervation is considered to regulate negatively the NE-dependent melatonin synthesis in pineal glands.
Recently, we have found both spontaneous and nAChR-mediated Ca2+ oscillations in tissue slice preparations from pineal glands and also in single pinealocytes from the rat (27). These Ca2+ oscillations are mediated by Ca2+ influx through VDCCs and involved in the inhibitory mechanism of melatonin secretion, which is known to be regulated by the parasympathetic activity in mammalian pineal glands. In addition to VDCCs (1, 9, 22, 40), molecular and functional expressions of voltage-dependent K+ channels (1, 4, 7, 8), nonselective cation channels (12, 29), store-operated Ca2+ channels (21), and cyclic nucleotide-gated channels (32) have been suggested in mammalian pineal glands. Furthermore, membrane currents presumably due to the large-conductance Ca2+-activated K+ (BKCa; also known as KCa1.1) channel activation have been recorded in mammalian pinealocytes (7, 8, 21, 22). However, the molecular basis of pineal BKCa channel and its physiological significances remain to be fully elucidated.
The present study was undertaken to elucidate the regulation of Ca2+ oscillation and melatonin secretion by the activity of BKCa channels during parasympathetic activation in mammalian pineal glands, using Ca2+ imaging techniques, electrophysiological recordings, expression analyses, and melatonin assay. Here we report that spontaneous and nicotine-evoked Ca2+ oscillations in rat pinealocytes are modulated by the BKCa channel conductance. Moreover, melatonin secretion in rat pineal glands is also affected by the BKCa channel activity.
MATERIALS AND METHODS
All experiments were approved by the Ethics Committee of Nagoya City University, Japan, and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the Japanese Pharmacological Society.
The pineal glands were removed from male Wistar/ST rats (6–9 wk) and then incubated in PBS containing 0.1% collagenase (Wako Pure Chemical Industries, Osaka, Japan) and 0.02% trypsin (Type I; Sigma-Aldrich, St. Louis, MO) for 30 min at 37°C (27). The dispersed pinealocytes were cultured on coverslips coated with 5 μg/ml poly-l-lysine (Sigma-Aldrich) in DMEM supplemented with 10% heat-inactivated FBS (Invitrogen/Gibco, Carlsbad, CA), 20 U/ml penicillin, and 20 μg/ml streptomycin (Wako Pure Chemical Industries). Experiments were performed at 24–96 h after cell culture.
[Ca2+]i measurements were performed using an Argus/HiSCA imaging system (Hamamatsu Photonics, Hamamatsu, Japan). Single pinealocytes were loaded with 10 μM fura-2 acetoxymethyl ester (Invitrogen/Molecular Probes, Eugene, OR) for 40 min at room temperature (23 ± 2°C). The HEPES-buffered solution had an ionic composition of 137 mM NaCl, 5.9 mM KCl, 2.2 mM CaCl2, 1.2 mM MgCl2, 14 mM glucose, and 10 mM HEPES. The pH was adjusted to 7.4 with 10 N NaOH. Ca2+ images were scanned approximately every 1.3–2.8 s. The fura-2 signal was converted to [Ca2+]i by the in vitro calibration method (14) as follows: [Ca2+]i = Kd(Rmin + R)/(R + Rmax), where Kd is the dissociation constant of fura-2 (224 nM), R is the fluorescence ratio (F340/F380), and Rmin and Rmax are the fluorescence ratios in the absence of and with saturation of Ca2+, respectively.
Electrophysiological studies were performed using a whole cell voltage-clamp technique with a CEZ-2400 amplifier (Nihon Kohden, Tokyo, Japan), an analog-digital converter (Digidata 1440A), and pCLAMP software (version 10; Molecular Devices/Axon Instruments, Foster City, CA) (15, 39). The HEPES-buffered solution was used as an extracellular recording solution. The pipette solution for whole cell currents had the following ionic composition: 140 mM KCl, 4 mM MgCl2, 5 mM ATPNa2, 10 mM HEPES, and 0.05 mM EGTA. The pH was adjusted to 7.2 with 1 N KOH. When BKCa currents were recorded using the pipette solution (pCa 6.0), 50 μM CdCl2 was added to the extracellular solution. The pipette solution (pCa 6.0) contained 140 mM KCl, 2.8 mM MgCl2, 2 mM ATPNa2, 10 mM HEPES, 4.2 mM CaCl2, and 5 mM EGTA. The pH was adjusted to 7.2 with 1 N KOH.
Quantitative real-time PCR.
The total RNA extraction from homogenates of rat pineal glands, the RT method, and quantitative real-time PCR analysis were performed as reported previously (27). Specific primers for rat BKCa genes were designed as follows: α (GenBank Accession number NM_031828), (+) CCC AAT AGA ATC CTG CCA GA, (−) GCA ATA AAC CGC AAG CCA AA; β1 (NM_019273), (+) AGA AGA CAC TCG GGA TCA AA, (−) GAA ATT GGC TCT GAC CTT CTT CAC; β2 (NM_176861), (+) AAG AGC GTC ATC CTG ACC AAA, (−) GTT TCA CCA TAG CAA CGA TTG C; β3 (NM_001104560), (+) CTT AGC CAC TCG GGA CAG AAA G, (−) ATC CCT GTC TCC GTG ACA CTT G; β4 (NM_023960), (+) ATC GGT TCC CAG CCA TTC A, (−) GAA GCA GTG CAG GAG AGC AAT; β-actin (NM_031144), (+) AGG CCA ACC GTG AAA AGA TG, (−) ACC AGA GGC ATA CAG GGA CA.
Immunocytochemical staining was performed as reported previously (27). In brief, pinealocytes were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. These pinealocytes were treated with primary antibody [1:100 dilution; anti-BKα (APC-107), anti-BKβ1 (APC-036), anti-BKβ2 (APC-034), and anti-BKβ4 (APC-061), Alomone Lab, Jerusalem, Israel; and anti-BKβ3 (H00027094-A01), Abnova, Taipei, Taiwan] for 12 h at 4°C, and then covered with Alexa Fluor 488-labeled secondary antibody solution (1:1,000 dilution; Invitrogen/Molecular Probes) for 1 h at room temperature. Confocal images were obtained using a laser scanning confocal fluorescent microscope (A1R; Nikon, Tokyo, Japan).
Melatonin secretion assay.
Freshly dissected pineal glands were incubated for 1 h at 37°C and then exposed to 1 μM NE or vehicle (control) for 2 h. Test compounds were added at the beginning of incubation prior to NE addition. The amount of melatonin secreted from the whole pineal gland was quantitatively determined using a melatonin ELISA kit (IBL International, Hamburg, Germany) and then normalized by that of NE-induced melatonin release (100%).
Pharmacological reagents were obtained from Sigma-Aldrich except for 4-aminopyridine, CdCl2 (Wako Pure Chemical Industries), 12,14-dichlorodehydroabietic acid (diCl-DHAA; Helix Biotech, New Westminster, Canada), EGTA, HEPES (Dojin, Kumamoto, Japan), iberiotoxin (IbTx; Peptide Institute, Osaka, Japan), nicotine, and tetraethylammonium (TEA) chloride (Tokyo Chemical Industry, Tokyo, Japan). All hydrophobic compounds were dissolved in DMSO at a concentration of 1 or 3 mM as a stock solution.
All pooled data are shown as means ± SE with the numbers of cells (n) or glands (N) examined. Statistical significance between two groups was determined by Student's t-test. Statistical significance among groups was determined by Scheffé's test after one-way ANOVA or Wilcoxon's rank sum test (U-test) after the nonparametric Kruskal-Wallis test. Significant differences are expressed as P < 0.05 or P < 0.01.
Enhanced Ca2+ oscillations by BKCa channel blockers in rat pinealocytes.
When resting [Ca2+]i in normal bath solution was measured in primary cultured rat pinealocytes, a subset of cells (15–20%) showed spontaneous and oscillatory [Ca2+]i changes (Figs. 1 and 2), as reported previously (27). The amplitude (100–500 nM) and frequency (0.3–1.0 min−1) of spontaneous Ca2+ oscillations varied somewhat among cells. Spontaneous Ca2+ events usually continued for >30 min during [Ca2+]i measurements.
The involvement of BKCa channel activity in the regulation of spontaneous Ca2+ oscillations was examined in rat pinealocytes. The application of 1 μM paxilline, a selective BKCa channel blocker (13, 18), significantly increased the amplitude of spontaneous Ca2+ oscillations from 167 ± 35 to 464 ± 67 nM (n = 15, P < 0.01; Fig. 1, A and B). Application of another BKCa channel blocker, 100 nM IbTx (13, 18), showed the same effect (211 ± 35 to 576 ± 52 nM, n = 5, P < 0.01; Fig. 1, D and E). The frequency of spontaneous Ca2+ oscillations was slightly but not significantly increased in the presence of paxilline (0.36 ± 0.07 to 0.47 ± 0.06 min−1, n = 15, P > 0.05; Fig. 1, A and C) or IbTx (0.43 ± 0.04 to 0.53 ± 0.08 min−1, n = 5, P > 0.05; Fig. 1, D and F). These findings indicated that the mechanism underlying the generation of spontaneous Ca2+ oscillations was modulated by the activity of BKCa channels in rat pinealocytes.
Attenuated Ca2+ oscillations by BKCa channel opener in rat pinealocytes.
In addition to BKCa channel blockers, effects of a BKCa channel opener, diCl-DHAA (31), on spontaneous Ca2+ oscillations were also examined in rat pinealocytes. The addition of 3 μM diCl-DHAA markedly reduced the amplitude (461 ± 76 to 159 ± 63 nM, n = 5, P < 0.05; Fig. 2, A and B) and frequency (0.70 ± 0.06 to 0.36 ± 0.05 min−1, n = 5, P < 0.05; Fig. 2, A and C) of spontaneous Ca2+ oscillations. The attenuation by diCl-DHAA was partly removed by its withdrawal. The inhibitory effects by 0.1 μM diCl-DHAA on the amplitude of spontaneous Ca2+ oscillations (from 182 ± 26 to 80 ± 12 nM, n = 6, P < 0.05) were reversed by the addition of 1 μM paxilline (to 511 ± 96 nM, n = 6, P < 0.01 vs. diCl-DHAA alone; Fig. 2, D–F). These results suggested that the activation of BKCa channel conductance decreased VDCC-mediated Ca2+ influx, presumably due to membrane hyperpolarization in rat pinealocytes.
Effect of BKCa channel blocker on nicotine-induced Ca2+ oscillations in rat pinealocytes.
In some pinealocytes (20–25%), Ca2+ oscillations were observed following a short application of nicotine, which induced a transient Ca2+ rise, as reported previously (27). Therefore, effect of BKCa channel blockade on cytosolic Ca2+ mobilization during stimulation of nAChRs was examined in rat pinealocytes. The application of 100 μM nicotine caused a transient increase in [Ca2+]i in pinealocytes (by 2,575 ± 48 nM, n = 7; Fig. 3A). After withdrawal of nicotine, Ca2+ oscillations were observed in some pinealocytes. The amplitude of nicotine-induced Ca2+ oscillations was markedly enhanced by 1 μM paxilline (152 ± 43 to 742 ± 220 nM, n = 7, P < 0.05; Fig. 3, A and B). There was no significant change in frequency in the presence of paxilline (0.36 ± 0.12 to 0.46 ± 0.17 min−1, n = 7, P > 0.05; Fig. 3, A and C). These observations suggest that the pharmacological properties of nicotine-induced Ca2+ oscillations are similar to those of spontaneous Ca2+ oscillations.
BKCa channel currents in rat pinealocytes.
Next, the function of BKCa channels in rat pinealocytes was examined by the whole cell patch-clamp technique. When single pinealocytes were depolarized from a holding potential of −40 to +40 mV in 10-mV steps for 150 ms every 15 s, using normal pipette solution containing 0.05 mM EGTA, outward currents were elicited at test potentials positive to −10 mV (Fig. 4, A–D). Depolarization-evoked outward currents were significantly reduced by a nonspecific BKCa channel blocker, 1 mM TEA (11) (101 ± 7 to 42 ± 7 pA/pF at +40 mV, n = 5, P < 0.01; Fig. 4, A and B). The outward currents were also decreased by 1 μM paxilline (86 ± 13 to 47 ± 12 pA/pF at +40 mV, n = 4, P < 0.01; Fig. 4, C and D). The paxilline-insensitive component was reduced by further addition of nonselective blockers of voltage-dependent K+ channels, 10 mM TEA, or 3 mM 4-aminopyridine (18) (to ∼20% of control, n = 3–5). Thus, paxilline-sensitive BKCa current was apparently the major component of outward currents upon depolarization in rat pinealocytes.
To analyze BKCa channel currents in detail, the pCa in the pipette solution was set at 6.0 with Ca2+/EGTA, and VDCCs were blocked by 50 μM Cd2+ in the external solution. Single pinealocytes were depolarized from a holding potential of −40 to +40 mV in 10-mV steps for 150 ms every 15 s (Fig. 4, E and F). Depolarization-elicited outward currents were significantly enhanced by 3 μM diCl-DHAA (100 ± 13 to 161 ± 10 pA/pF at +40 mV, n = 4, P < 0.05). Further addition of 1 μM paxilline reduced the outward currents to ∼85% of the control (to 84 ± 4 pA/pF at +40 mV, n = 4, P < 0.05 vs. diCl-DHAA alone). These results strongly suggested that rat pinealocytes functionally expressed the BKCa channels that modulate spontaneous and nicotine-induced Ca2+ oscillations.
Molecular basis of BKCa channels in rat pinealocytes.
BKCa channels are constituted from tetrameric sets of pore-forming α subunits and auxiliary β subunits in a reciprocal manner (3, 11, 35). Therefore, the molecular components of BKCa channels in rat pinealocytes were identified by quantitative real-time PCR and immunocytochemical analyses. Quantitative real-time PCR analysis showed the substantial expression of the α transcript (0.050 ± 0.005 ratio to β-actin, N = 5) in rat pineal glands (Fig. 5A). Among accessory subunits, mRNA expression of the β3 subunit (0.021 ± 0.002, N = 4) was clearly detected, but the β1, β2, and β4 subunits were not (<0.002, N = 3–4) in rat pineal glands.
The expression of BKCa channel proteins in rat pinealocytes was confirmed by an immunocytochemical approach using subunit-specific polyclonal antibodies. Immunoreactivities to the α and β3 proteins were clearly observed at the plasma membrane of all pinealocytes examined (5 and 8 cells, respectively), but those of other subunits were not (β1, 1 of 10 cells; β2, all negative of 5 cells; β4, all negative of 6 cells; Fig. 5B). These findings strongly suggested that the BKCa channels in rat pinealocytes potentially consisted of the α and β3 subunits.
Modulation of melatonin secretion by BKCa channel activity.
Melatonin secretion from rat pineal glands by neurotransmitters and its modulation by the activity of BKCa channels were quantitatively analyzed using a melatonin ELISA kit. Melatonin secretion from whole pineal glands was observed by treatment with 1 μM NE for 1 h at 37°C (26.9 ± 1.5 ng/ml, N = 28, P < 0.01 vs. vehicle of 9.1 ± 1.6 ng/ml, N = 14), as reported previously (27). The amount of released melatonin was normalized by that of NE-induced melatonin release (100%; Fig. 6). Melatonin secretion evoked by 1 μM NE was reduced by ∼50% in the copresence of 100 μM ACh (to 50.5 ± 5.3%, N = 13, P < 0.01 vs. NE). The suppression of NE-induced melatonin release by ACh was not significantly affected by 1 μM paxilline (53.0 ± 5.3%, N = 11, P > 0.05 vs. NE + ACh). Interestingly, ACh-induced inhibition of NE-induced melatonin secretion was significantly recovered by 3 μM diCl-DHAA (84.9 ± 9.8%, N = 12, P < 0.01 vs. NE + ACh). The recovery effect of diCl-DHAA was partly antagonized by the further addition of 1 μM paxilline (60.2 ± 5.1%, N = 12, P > 0.05 vs. NE + ACh + diCl-DHAA by Wilcoxon's rank sum test but P = 0.036 by Student's t-test). These data suggested that this inhibitory effect of ACh on NE-evoked melatonin secretion in rat pineal glands was modulated by the activity of BKCa channels.
Our previous report showed that rat pinealocytes have spontaneous Ca2+ oscillations evoked by VDCC-mediated Ca2+ spikes following membrane depolarization (27). Spontaneous Ca2+ oscillations are supposed to contribute to the inhibitory mechanism of melatonin secretion due to parasympathetic nerve activity. The present data show that the α and β3 subunits of BKCa channels are highly expressed in rat pinealocytes and that the BKCa channel activity strongly modulates spontaneous Ca2+ oscillations and thereby, more importantly, regulates melatonin production.
The regulation of melatonin production in pinealocytes depends on a balance of activity due to sympathetic and parasympathetic neuronal activities (28, 33). The inhibitory mechanism of melatonin production driven by nAChR stimulation via parasympathetic activity is considered to be closely related to the rise of [Ca2+]i in mammalian pinealocytes (37, 38). However, it is not well understood how ion channels contribute to cellular Ca2+ mobilization for circadian regulation in mammalian pineal glands (19). Our previous work demonstrated spontaneous and nAChR-mediated Ca2+ oscillations in tissue slice preparations and single pinealocytes of the rat (27). These Ca2+ oscillations are triggered by synchronized periodic membrane depolarizations, and thus induced Ca2+ influx mediated by VDCCs (mainly α1F subunit). In addition to nifedipine-sensitive VDCCs in pinealocytes, the involvement of BKCa channels in the regulation of Ca2+ oscillations and the subsequent inhibition of melatonin production were clarified in this study. The parameters of spontaneous and nicotine-induced Ca2+ oscillations mediated by Ca2+ influx through VDCCs (27) were markedly changed by BKCa channel openers and blockers. Furthermore, the activity of BKCa channels completely modified the ACh-induced inhibition of melatonin synthesis by NE, which is known to be regulated by parasympathetic activities in mammalian pineal glands (28). In other types of mammalian glands, the specific role of BKCa channels on the regulation of exocrine functions has been reported, for example, in salivary glands (6), pituitary glands (34), parathyroid glands (5), lacrimal glands (24), airway submucosal glands (30), and adrenal medulla (10). Based on our present results, pineal BKCa channels play an obligatory role in the regulation of melatonin secretion.
BKCa channels are highly expressed in excitable cells including neurons, secretory cells, and smooth muscle cells (3, 11). BKCa channels contribute to the regulation of membrane excitability including the suppression of action potential firing and the formation of the action potential repolarizing phase (3). The activity of BKCa channels is triggered by both membrane depolarization and elevated cytosolic Ca2+. Membrane hyperpolarization following BKCa channel activation protects cells from excessive excitability and Ca2+ overload by closing VDCCs (11). Therefore, the BKCa channel is considered a key molecule in the negative-feedback mechanism in [Ca2+]i regulation (16, 17). Similarly, in pineal glands, activation of the BKCa channel is likely to cause a membrane hyperpolarization and, following a decrease in Ca2+ influx, to result in the reduction of Ca2+ oscillation parameters and, finally, the recovery of melatonin synthesis. Therefore, pineal BKCa channels may form a negative-feedback mechanism in cytosolic Ca2+ mobilization during parasympathetic innervation.
The BKCa channel is a tetrameric assembly of pore-forming α subunits with four β subunits (3, 11, 35). The expression pattern of the α subunit is ubiquitous in excitable cells, such as neurons, secretory glands, and smooth muscles, except in the heart. The auxiliary β subunit (β1, β2, β3, and β4) binds with the α subunit in a one-to-one manner and modulates Ca2+ sensitivity, voltage dependence, and pharmacological characteristics. It has been revealed that genetic deficiency of the α subunits in mice significantly disrupts the regulation of circadian behavioral rhythms (26). The β subunit shows tissue-specific distribution and is responsible for the tissue-specific variation of BKCa channel characteristics (2). Expression analyses at the mRNA and protein levels in the present study revealed that the BKCa channel was predominantly composed of a combination of α and β3 subunits. It is known that the β3 subunits mainly distribute in the spleen, placenta, pancreas, testis, and prostate, but that the β4 subunits are predominant in neuronal tissues (2, 11). Some reports, however, indicate that β subunits other than β4 are also localized in the brain and may contribute to these neuronal functions (2, 20, 23, 36). One of the characteristic modulations of BKCaα channel currents by β4 subunits is a marked reduction of sensitivity to IbTx and charybdotoxin (25). In the present study, spontaneous Ca2+ oscillations were highly susceptible to IbTx. In addition, electrophysiological results suggested that BKCa channel currents in rat pinealocytes are completely blocked by IbTx and charybdotoxin (7, 8, 21, 22). Therefore, β3 rather than β4 is the predominant β subunit of BKCa channel functionally expressed in rat pineal glands.
One of the most important findings in this study is that melatonin secretion from whole pineal gland following nAChR stimulation is strongly modulated by the activity of BKCa channels. The parasympathetic signals mediated by nAChR is considered to regulate negatively the NE-dependent melatonin synthesis due to sympathetic activity in pineal glands (28). The negative-feedback model for melatonin secretion is summarized in Fig. 7. Spontaneous and nAChR (α3β4)-mediated Ca2+ oscillations in pinealocytes are triggered by synchronized periodic membrane depolarizations, and thus induce Ca2+ influx though VDCCs (mainly α1F subunits). The VDCC-mediated [Ca2+]i rise is supposed to elicit the release of glutamic acid from pinealocytes. Consequently, the released glutamate stimulates mGluR3, by both autocrine and paracrine mechanisms, and thus suppresses adenylate cyclase via Gi protein activation (37, 38). Finally, the activation of this pathway leads to the reduction of melatonin secretion. Because the BKCa channel opener abolished the suppression of NE-induced melatonin secretion by ACh, it is strongly suggested that membrane hyperpolarization via the activation of BKCa channels (α and β3 subunits) is essential for this inhibitory regulation of melatonin secretion following nAChR stimulation. The finding that nAChR-induced Ca2+ oscillation was strongly suppressed by the BKCa channel opener supports the hypothesis that Ca2+ oscillations are involved in glutamate release.
In conclusion, we found that spontaneous and nAChR-mediated Ca2+ oscillations were completely modified by the activity of the BKCa channel that was composed of α and β3 subunits. Changes in membrane potential modulated by BKCa channel activity are involved in the inhibitory mechanism of melatonin secretion, which is known to be regulated by parasympathetic activities in mammalian pineal glands.
This investigation was supported by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (23136512 and 25136717; to Y. Imaizumi), and by Grants-in-Aid for Scientific Research (B) (26293021; to Y. Imaizumi) and Scientific Research (C) (25460104; to H. Yamamura) from the Japan Society for the Promotion of Science. This work was also supported by a Grant-in-Aid from the Smoking Research Foundation (to Y. Imaizumi).
No conflicts of interest, financial or otherwise, are declared by the author(s).
Author contributions: H.M., H.Y., M.M., and Y.H. performed experiments; H.M., H.Y., M.M., Y.H., and Y.I. analyzed data; H.M., H.Y., M.M., Y.H., Y.S., and Y.I. interpreted results of experiments; H.M., H.Y., M.M., and Y.H. prepared figures; H.M., H.Y., M.M., Y.H., Y.S., and Y.I. approved final version of manuscript; H.Y. and Y.I. conception and design of research; H.Y. and Y.I. drafted manuscript; H.Y. and Y.I. edited and revised manuscript.
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