Modulation of Ca2+ oscillation and melatonin secretion by BKCa channel activity in rat pinealocytes

Abstract

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 Ca(2+) concentration and eventually downregulates melatonin production. Our previous report shows that rat pinealocytes have spontaneous and nicotine-induced Ca(2+) oscillations that are evoked by membrane depolarization followed by Ca(2+) influx through voltage-dependent Ca(2+) channels (VDCCs). These Ca(2+) oscillations are supposed to contribute to the inhibitory mechanism of melatonin secretion. Here we examined the involvement of large-conductance Ca(2+)-activated K(+) (BKCa) channel conductance on the regulation of Ca(2+) oscillation and melatonin production in rat pinealocytes. Spontaneous Ca(2+) oscillations were markedly enhanced by BKCa channel blockers (1 μM paxilline or 100 nM iberiotoxin). Nicotine (100 μM)-induced Ca(2+) oscillations were also augmented by paxilline. In contrast, spontaneous Ca(2+) 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 Ca(2+) oscillations due to depolarization-synchronized Ca(2+) 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.

Keywords: calcium oscillation; calcium-activated potassium channel; parasympathetic nerve; pineal gland.

Fig. 1.

Enhancement of spontaneous Ca²⁺ oscillations by large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel blockers in rat pinealocytes. Effects of BK_Ca channel blockers on spontaneous Ca²⁺ oscillations were examined in rat pinealocytes. A: representative recording of changes in Ca²⁺ oscillations before and after the application of 1 μM paxilline (Pax). B and C: amplitude (B) and frequency (C) of spontaneous Ca²⁺ oscillations in the presence of paxilline (n = 15). D: spontaneous Ca²⁺ oscillations in the absence and presence of 100 nM iberiotoxin (IbTx). E and F: amplitude (E) and frequency (F) of spontaneous Ca²⁺ oscillations after the application of IbTx (n = 5). **P < 0.01, statistical significance vs. control (Cont). [Ca²⁺]_i, intracellular Ca²⁺ concentration.

Fig. 2.

Attenuation of spontaneous Ca²⁺ oscillations by BK_Ca channel opener in rat pinealocytes. Effects of BK_Ca channel opener on spontaneous Ca²⁺ oscillations were examined in rat pinealocytes. A: application of 3 μM 12,14-dichlorodehydroabietic acid (diCl-DHAA) blocked spontaneous Ca²⁺ oscillations. B and C: amplitude (B) and frequency (C) of spontaneous Ca²⁺ oscillations after application of diCl-DHAA (n = 5). D: inhibitory effect of 0.1 μM diCl-DHAA on spontaneous Ca²⁺ oscillations was removed by addition of 1 μM paxilline. E and F: amplitude (E) and frequency (F) of spontaneous Ca²⁺ oscillations after coapplication of diCl-DHAA and paxilline (n = 6). *P < 0.05 or ^##P < 0.01, statistical significance vs. control or diCl-DHAA, respectively.

Fig. 3.

Effect of BK_Ca channel blocker on Ca²⁺ oscillations induced by nicotinic acetylcholine receptor (nAChR) stimulation in rat pinealocytes. Effect of BK_Ca channel blockade on cytosolic Ca²⁺ mobilization following nAChR stimulation was examined in rat pinealocytes. A: Ca²⁺ oscillations were often observed following the short application of 100 μM nicotine, which induced a transient [Ca²⁺]_i rise. Effects of 1 μM paxilline on nicotine-induced Ca²⁺ oscillations are shown in a representative recording. B and C: enhancing effects of paxilline on the amplitude (B) and frequency (C) of nicotine-induced Ca²⁺ oscillations (n = 7). *P < 0.05, statistical significance vs. control.

Fig. 4.

BK_Ca channel currents in rat pinealocytes. Electrophysiological and pharmacological characteristics of BK_Ca channel currents in rat pinealocytes were analyzed using the whole cell patch-clamp technique. Depolarizing pulses of 150 ms were applied from a holding potential of −40 to +40 mV in 10-mV increments every 15 s. A: current traces before and after the application of 1 mM tetraethylammonium (TEA). B: current-voltage relationships under control conditions and in the presence of TEA (n = 5). C: current recordings in the absence and presence of 1 μM paxilline. D: current-voltage relationships before and after the application of paxilline (n = 4). E: when the pCa of the recording pipette solution was fixed at 6.0 with Ca²⁺/EGTA, outward currents were activated by application of 3 μM diCl-DHAA and blocked by further addition of 1 μM paxilline. F: current-voltage relationships under control conditions, in presence of diCl-DHAA, and in presence of diCl-DHAA plus paxilline (n = 4). *P < 0.05 or **P < 0.01, statistical significance.

Fig. 5.

Molecular identification of BK_Ca channels in rat pinealocytes. Expression pattern for BK_Ca channels was obtained from rat pinealocytes using quantitative real-time PCR and immunocytochemical techniques. A: expression of mRNA encoding BK_Ca channel subunits in rat pineal glands was measured using the quantitative real-time PCR method. Expression levels of BK_Ca channel subunits were normalized by that of endogenous β-actin. Expression of α and β3 genes was clearly identified but β1, β2, and β4 subunits were not. Data were obtained from 3–5 independent experiments. B: immunoreactivity of BK_Ca channel subunit proteins in rat pinealocytes is shown using specific antibodies. Specific signals of the α and β3 subunits were detected on the plasma membrane in rat pinealocytes. Data were obtained from 5–10 cells.

Fig. 6.

Contribution of BK_Ca channels to the regulation of melatonin secretion by neurotransmitters. Effects of ACh and BK_Ca channel modulators on melatonin secretion elicited by 1 μM norepinephrine (NE) were examined in freshly dissected rat pineal glands. Measurement of melatonin secretion from 1 pineal gland was quantitatively determined using a melatonin ELISA kit and then normalized by that of NE-induced melatonin release (100%). The NE (1 μM)-induced melatonin secretion was reduced by 100 μM ACh. Opening of BK_Ca channels by 3 μM diCl-DHAA clearly reduced inhibition of NE-induced melatonin secretion by ACh. This effect of diCl-DHAA was partly compensated for by addition of 1 μM paxilline. Data were obtained from 7–28 pineal glands. Statistical significance: **P < 0.01 vs. vehicle, ^##P < 0.01 vs. NE alone, or ^$$P < 0.01 vs. NE + ACh.

Fig. 7.

Diagram of mechanism underlying melatonin production modulated by activities of voltage-dependent Ca²⁺ channel (VDCC) and BK_Ca channel (BK) in pinealocytes. In pinealocytes, ACh is released from nerve terminal of parasympathetic axons in the pterygopalatine ganglion (PPG) and activates nAChRs (α3β4 subunits). Stimulation of nAChR causes a membrane depolarization (Depo.) followed by Ca²⁺ influx (Ca²⁺ oscillation) through VDCCs (mainly α1F subunit). [Ca²⁺]_i increase is likely to elicit the release of glutamic acid (l-glutamate; Glu) from pinealocytes. The released glutamic acid is supposed to activate G_i protein-coupled metabotropic glutamate receptor type 3 (mGluR3), and results in inhibitory regulation of melatonin synthesis. Therefore, parasympathetic innervation is considered to regulate negatively the NE-dependent melatonin synthesis in pineal glands. Activation of BK_Ca channels (α and β3 subunits) controls negatively the activity of VDCCs and thus induces recovery of melatonin production.