Am J Physiol Cell Physiol 306: C726–C735, 2014.
First published February 19, 2014; doi:10.1152/ajpcell.00354.2013.
CALL FOR PAPERS
Cellular Circadian Rhythms
Modulation of nicotinic receptor channels by adrenergic stimulation
in rat pinealocytes
Jin-Young Yoon,1 Seung-Ryoung Jung,1 Bertil Hille,1 and Duk-Su Koh1,2
1
Department of Physiology and Biophysics, University of Washington, Seattle, Washington; and 2Department of Physics,
POSTECH, Pohang, Kyungbuk, Republic of Korea
Submitted 20 November 2013; accepted in final form 18 February 2014
acetylcholine receptors; ␤-adrenergic receptors; pineal gland; melatonin; cross talk
AT NIGHT, THE PINEAL GLAND
secretes melatonin, a neuroendocrine hormone that helps to regulate activity and the sleepwake cycle (21, 46). The pineal gland is composed of melatonin-secreting pinealocytes and small numbers of other cell
types including glial-cell-like interstitial cells (31). In mammals, melatonin production is tightly regulated by norepinephrine (NE) released from sympathetic nerve fibers with perikaryal origin in the superior cervical ganglia. NE release to
pinealocytes occurs exclusively at night. Synthesis of melatonin is powerfully upregulated by Gs-coupled ␤1-adrenergic
receptors and Gq-coupled ␣1B-adrenergic in pinealocytes (3,
49). Coincident activation of both receptors by NE increases
cAMP by ⬃100-fold (52). In turn, cAMP induces the expression and activation of arylalkylamine N-acetyltransferase (AANAT),
Address for reprint requests and other correspondence: D.-S. Koh, Dept. of
Physiology and Biophysics, Univ. of Washington School of Medicine, Box
357290, Seattle, WA 98195-7290 (e-mail: [email protected]).
C726
a rate-limiting enzyme in melatonin production (22). Morphological and immunohistochemical evidence supports parasympathetic innervation of mammalian pineal glands with perikaryal origin in the sphenopalatine and otic ganglia (25; for
reviews, see Refs. 32, 35). However, in contrast to the
well-characterized adrenergic sympathetic inputs, roles for
cholinergic innervation and when it might be active are not
established. Acetylcholine can act on both nicotinic and
muscarinic acetylcholine receptors (nAChRs and mAChRs)
in pinealocytes (34, 38, 47, 53). Nicotinic cholinergic signals from the parasympathetic or the central nervous system
have been suggested to have inhibitory effects on pineal
melatonin synthesis (56, 58). In many cell types including
central neurons, nAChRs are modulated via protein phosphorylation by second messenger-dependent protein kinases
(15, 51). Protein kinase A (PKA) and protein kinase C can
phosphorylate nAChRs, resulting in faster desensitization
(36, 41). Physiological modulation of pineal nicotinic receptors has not been reported so far.
Considering that sympathetic inputs counter parasympathetic actions in various organs and that pineal adrenergic
signals are coupled to PKA and protein kinase C (46), we
hypothesized that adrenergic input would inhibit nAChRs in
the rat pineal gland. Here we demonstrate that NE downregulates nAChRs via cAMP/PKA-dependent pathways and reduces the current, Ca2⫹ influx, and membrane depolarization
mediated by nAChRs.
MATERIALS AND METHODS
Preparation of rat pinealocytes. Pinealocytes were isolated from
pineal glands of male Sprague-Dawley rats (Harlan Laboratories,
Indianapolis, IN) at postnatal weeks 5– 6 as described previously (20).
Animals were on a 14:10 day-night cycle and killed in the midmorning during the light period. All procedures followed the animal
use guidelines of the University of Washington and the protocol was
approved by Institutional Animal Care and Use Committee. Rats were
euthanized using carbon dioxide and subsequently decapitated. After
removal of the brain, the pineal gland was washed with ice-cold
Eagle’s balanced salt solution (Invitrogen, Carlsbad, CA). The gland
was cut into a few pieces with scissors and treated with 4 mg/ml
collagenase (type I; Sigma-Aldrich, St. Louis, MO) in HBSS (Invitrogen) containing 5 mM D-glucose, 20 mM HEPES, and 1 mg/ml
bovine serum albumin for 35– 40 min at 37°C with gentle shaking.
Cell clusters were centrifuged at 180 g for 5 min and dispersed into
single cells by trituration in Ca2⫹-free HBSS containing 1 mM
EGTA, 20 mM HEPES, and 10 mg/ml bovine serum albumin. Isolated
cells were resuspended in DMEM (Invitrogen) supplemented with 10
mM glucose, 10% fetal bovine serum, 100 ␮g/ml streptomycin, and
0363-6143/14 Copyright © 2014 the American Physiological Society
http://www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.4 on June 16, 2017
Yoon JY, Jung SR, Hille B, Koh DS. Modulation of nicotinic receptor
channels by adrenergic stimulation in rat pinealocytes. Am J Physiol Cell
Physiol 306: C726–C735, 2014. First published February 19, 2014;
doi:10.1152/ajpcell.00354.2013.—Melatonin secretion from the pineal
gland is triggered by norepinephrine released from sympathetic terminals at night. In contrast, cholinergic and parasympathetic inputs,
by activating nicotinic cholinergic receptors (nAChR), have been
suggested to counterbalance the noradrenergic input. Here we investigated whether adrenergic signaling regulates nAChR channels in rat
pinealocytes. Acetylcholine or the selective nicotinic receptor agonist
1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) activated large
nAChR currents in whole cell patch-clamp experiments. Norepinephrine (NE) reduced the nAChR currents, an effect partially mimicked
by a ␤-adrenergic receptor agonist, isoproterenol, and blocked by a
␤-adrenergic receptor antagonist, propranolol. Increasing intracellular
cAMP levels using membrane-permeable 8-bromoadenosine (8-Br)cAMP or 5,6-dichlorobenzimidazole riboside-3=,5=-cyclic monophosphorothioate (cBIMPS) also reduced nAChR activity, mimicking the
effects of NE and isoproterenol. Further, removal of ATP from the
intracellular pipette solution blocked the reduction of nAChR currents, suggesting involvement of protein kinases. Indeed protein
kinase A inhibitors, H-89 and Rp-cAMPS, blocked the modulation of
nAChR by adrenergic stimulation. After the downmodulation by NE,
nAChR channels mediated a smaller Ca2⫹ influx and less membrane
depolarization from the resting potential. Together these results suggest that NE released from sympathetic terminals at night attenuates
nicotinic cholinergic signaling.
C727
ADRENERGIC-CHOLINERGIC CROSS TALK IN PINEALOCYTES
A
tion for 45 min at room temperature and further incubated without the
dye for ⬃30 min for deesterification of the dye by cellular esterase.
Single pinealocytes were imaged using an inverted microscope
(TE2000-U; Nikon) equipped with a Polychrome monochromator
(TILL Photonics, Graefelfing, Germany) and an Evolve CCD camera
(Photometrics, Tucson, AZ). The excitation wavelengths were 340
and 380 nm, and fluorescence images were recorded at 510 nm using
Metafluor software (Molecular Devices, Sunnyvale, CA) every 2 s.
Background fluorescence determined at cell-free areas was subtracted
from the signal in single cells. We calculated the ratio of fluorescence
at two excitation wavelengths, F340/F380, in regions of interest and
report Ca2⫹ transients as the change of this ratio, ⌬F340/F380, without
absolute calibration. For Ca2⫹ imaging experiments, solutions were
exchanged by a local perfusion system that allowed complete exchange of extracellular solutions within 0.5 s (23). These cells were
not patch clamped, and therefore intracellular calcium transients were
not attenuated by EGTA. All electrophysiological and Ca2⫹ measurements were made at room temperature (22–24°C).
Chemicals. 8-Bromoadenosine (8-Br)-cAMP and Rp-adenosine cyclic 3=,5=-phosphorothioate (Rp-cAMPS) were from Biolog Life Science Institute (Bremen, Germany), 5,6-dichlorobenzimidazole riboside-3=,5=-cyclic monophosphorothioate (cBIMPS) from Enzo Life
Sciences (Farmingdale, NY), and H-89 from Calbiochem (now EMD
Millipore, Billerica, MA), and all other chemicals were obtained from
Sigma-Aldrich.
Statistics. All data are expressed as means ⫾ SE, and n values refer
to the number of analyzed cells or measurements. The statistical
difference between two groups was evaluated by Student’s t-test.
Probabilities of P ⱕ 0.05 were considered significant.
RESULTS
Adrenergic signaling inhibits cholinergic receptor current
in pinealocytes. Application of ACh evoked an inward ion
current in every rat pinealocyte we tested with patch-clamp
recording. The mean current density was large (97 ⫾ 12 pA/pF
at ⫺60 mV; n ⫽ 22). Figure 1A shows a typical response to
rapid application of 50 ␮M ACh at a holding potential of ⫺60
mV. The induced inward current activated quickly, reaching a
peak within 30 ms, and then, in 21 of 22 cells, desensitized
gradually during 15-s ACh stimulation (n ⫽ 22). The speed of
onset and the agonist and antagonist selectivity given in the
next paragraph show that this response is from nAChRs. The
time course of desensitization was quite variable and partial
with an average time constant of desensitization of 2.5 ⫾ 1.3 s (n ⫽
22). There was also a slow and variable rundown of current
B
ACh
C
0
50
NE
NE
Inhibition (%)
I (pA)
Current
-100
-200
-300
Control
40
30
**
**
20
10
50 pA
2s
0
5
10
15
20
Time (min)
0
I Peak
I 15 s
Fig. 1. Reduction of acetylcholine receptor (AChR) currents by norepinephrine (NE) in rat pinealocytes. A: currents activated by rapid application of 50 ␮M ACh
for 15 s without (control) or with 2-min pretreatment of 1 ␮M NE. Membrane potential was held at ⫺60 mV and the dashed line indicates zero-current level.
B: representative time course of NE-mediated modulation of ACh-induced current. Peak current (IPeak; A, closed circles) and current at 15 s during ACh
application (I15s; A, open circles) were recorded every 2 min. The same experiment as in A. C: average inhibition of Ipeak and I15s of AChR currents by NE;
n ⫽ 4. **P ⬍0.01, compared with control currents.
AJP-Cell Physiol • doi:10.1152/ajpcell.00354.2013 • www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.4 on June 16, 2017
100 IU/ml penicillin. The cells were plated on glass coverslips coated
with 2 ␮g/ml of poly-D-lysine (Invitrogen) and cultured in supplemented DMEM for 1–3 days before experiments. The pinealocytes
were visually identified by tiny spots on the cell surface (20).
Patch-clamp recording and analysis. To measure nAChR currents
and the membrane potential, we used standard patch-clamp technique
in the whole cell configuration (12). For most voltage-clamp experiments, pipettes were filled with an internal solution containing the
following (in mM): 145 Cs-aspartate, 20 creatine phosphate, 10
EGTA, 5 HEPES, 3 Na2ATP, 0.1 Na3GTP, and 0.1 BAPTA pH 7.4.
For some current-clamp experiments the internal solution was K⫹based and contained the following (in mM): 137 K aspartate, 10
EGTA, 3 MgATP, 5 MgCl2, 5 HEPES, 0.1 Na3GTP, and 0.1 BAPTA.
Both of these internal solutions contained enough EGTA to eliminate
most of the intracellular calcium signaling during patch-clamp experiments. When filled with internal solution, patch pipettes had a
resistance of 3–7 M⍀. The liquid junction between these pipette
internal aspartate solutions and the bath external chloride solution
gives a measured junction potential of ⬃10 mV, pipette negative. The
records were not corrected for this value, but the effect is noted in text
in a couple of places. The mean membrane capacitance of pinealocytes and series resistance were 13 ⫾ 1 pF and 11 ⫾ 1 M⍀ (n ⫽ 22),
respectively. Control bath saline contained the following (in mM):
137.5 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 8 glucose, and 10 HEPES,
pH 7.4. Currents were recorded using an EPC-9 patch-clamp
amplifier (HEKA Instruments; Lambrecht/Pfalz, Germany), lowpass filtered at 100 Hz, and digitized at 200 Hz using Pulse
software (HEKA Instruments). Data were analyzed using Igor Pro
software (WaveMetrics). As reported previously, some properties
of neuronal nAChR change during the first few minutes of patchclamp recordings, probably due to dialysis of the cytoplasm (17,
27). Therefore, we waited 3–5 min after formation of whole cell
configuration before making records.
Fast agonist application. To avoid fast desensitization of nAChRs,
agonists had to be applied quickly to measure peak current amplitudes
(19). A double-barreled pipette was fabricated from theta-glass tubing
(cat. no. BT-150-10; Sutter Instruments, Novata, CA) and manipulated by a piezo-electric device (Burleigh LSS-3200; Thorlabs, Newton, NJ) under computer control. The pipette was positioned close
(⬃100 ␮m) to the patched pinealocyte. Solution exchange time
(20 – 80%) was estimated as ⬃3 ms when measured as the junctionpotential change with an open patch pipette during solution changes
between 10 and 100% saline solutions.
Ca2⫹-imaging. Intracellular Ca2⫹ concentration ([Ca2⫹]i) was
measured using the Ca2⫹-sensitive fluorescent dye fura-2. The cells
were preincubated with 2 ␮M membrane-permeable fura-2 AM (Molecular Probes, Life Technologies, Grand Island, NY) in saline solu-
C728
ADRENERGIC-CHOLINERGIC CROSS TALK IN PINEALOCYTES
A
specific antagonist mecamylamine (Meca) confirmed that
DMPP activates nAChRs selectively (33). When cells were
preincubated with NE, both peak and steady-state (I2s) nAChR
currents were reduced at all negative membrane potentials.
There was no statistically significant difference in NE-mediated nAChR inhibition measured at ⫺80 and ⫺40 mV (Fig.
2C). If there is any voltage dependence of this modulation, it is
weak. Likewise, desensitization time constants measured by
fitting a single exponential were not significantly modified by
NE at ⫺80, ⫺60, ⫺40, and ⫺20 mV (n ⫽ 5).
␤-Adrenergic signaling inhibits nAChRs via cAMP-dependent phosphorylation. Pinealocytes express two classes of
adrenergic receptor (AR), ␣1-AR and ␤-AR (3). ␣-ARs couple
to Gq and phospholipase C, elevate intracellular Ca2⫹, and
activate protein kinase C and K(Ca) channels, whereas the
␤-ARs elevate cAMP and cGMP (26, 46, 49, 59). We identified the adrenergic signal pathway to nAChRs using selective
agonists and antagonists.
The ␤-AR agonist isoproterenol partially mimicked the NE
effect. A 2-min application of 1 ␮M isoproterenol reduced the
DMPP-evoked nAChR current (Ipeak by 19 ⫾ 5% and I2s by
31 ⫾ 6%; Fig. 3, A and B). By comparison, 1 ␮M NE was
somewhat more effective, reducing the currents by 38 ⫾ 5 and
46 ⫾ 5%, respectively (Fig. 3B). We tested the involvement of
cAMP signaling using membrane-permeable 8-Br-cAMP and
the phosphodiesterase-resistant cAMP analog cBiMPS to activate PKA. Each analog applied alone decreased the nAChR
current (Fig. 3, Ab, Ac, and B). Thus a 2-min application of
DMPP
DMPP
DMPP
+60 mV
-20 mV
Current
+Meca
+NE
500 pA
Control
500 ms
-80 mV
40
-40
-0.2
-0.4
-0.6
-0.8
-1.0
C
-80
Vhold (mV)
-0.2
-0.4
-0.6
-0.8
-1.0
N.S.
N.S.
60
40
-40
40
20
0
-80
-40
-40
-80
IPeak
AJP-Cell Physiol • doi:10.1152/ajpcell.00354.2013 • www.ajpcell.org
I2 s
Vhold (mV)
Vhold (mV)
Normalized I2 s
-80
Normalized IPeak
B
Inhibition by NE (%)
Fig. 2. Voltage-independent modulation of nAChR
currents by NE. A: currents activated by 50 ␮M
dimethylphenylpiperazinium (DMPP) without (control),
with 1 ␮M NE, or with 10 ␮M mecamylamine
(Meca) at membrane potentials between ⫺80 and
⫹60 mV in 20-mV steps. All records were from the
same cell. B: current-voltage (I–V) relationships of
IPeak (left) and I2s (right) in control (circles), with
NE (squares), or with Meca (triangles). Currents
were normalized to the control peak current at ⫺80
mV. Each point is the average of 4 – 6 cells. C:
average inhibition of nAChR currents at ⫺80 and
⫺40 mV. N.S.: not significantly different between
recordings at ⫺80 mV and ⫺40 mV (n ⫽ 5; P ⬎
0.05).
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.4 on June 16, 2017
during the experiment, perhaps due to dialysis into the patch
pipette of components critical for maintenance of the channel
activity.
We tested whether this cholinergic response could be modulated by adrenergic signaling in pinealocytes. Activation of
adrenergic receptors with the physiological agonist NE gradually and substantially reduced both the peak and steady-state
cholinergic currents (Fig. 1, A and B). After 2 min in NE (1
␮M), the peak amplitude and the current measured at the end
of 15 s ACh (I15s) had decreased by 29 ⫾ 6 and 36 ⫾ 6%,
respectively (Fig. 1C). Currents did not recover from downregulation within 5 min.
NE-induced modulation of nAChRs has little voltage dependence.
Next, we studied whether the modulation occurs at all membrane potentials. Since rat pinealocytes express both nicotinic
and muscarinic acetylcholine receptors (46), for this and the
following experiments we switched from ACh to a selective
agonist dimethylphenylpiperazinium (DMPP) to activate only
nAChRs. In the experiments shown in Fig. 2A, we applied 50
␮M DMPP at different holding potentials. Currents reversed
sign near ⫹5 mV after junction potential correction, suggesting
little selectivity between Cs⫹ and Na⫹ (PNa/PCs ⫽ 1.2), a
nonselective cation channel. The channel conducted large inward currents at negative membrane potentials and almost no
outward current at corresponding positive potentials (Fig. 2B).
Such strong inward rectification, typical of neuronal nAChRs,
is likely due to voltage-dependent block by cytoplasmic polyamines (11). Block of the currents by a neuronal-nAChR-
ADRENERGIC-CHOLINERGIC CROSS TALK IN PINEALOCYTES
C729
A
Iso
1.0
IPeak
b
I2 s
1.0
Relative currents
Relative currents
a
0.8
0.6
0.4
0
2
4
6
8-Br-cAMP
0.8
0.6
0.4
8
0
2
Relative currents
1.0
0.8
0.6
0.4
2
4
6
1.0
0.8
AMPPCP
0.6
0.4
8
2
0
**
**
40
**
0
6
60
40
**
**
** **
8
20
NE
I so
r-c
A
cB M P
8-B iMPS
rRp cAM
- cA P
M
AM PS
PPC
P
H89
Pr
op
8-B
NE
I so
r -c
AM
cB P
8-B iMP
S
rRp cAM
- cA P
AM M P
P- S
PC
P
H89
Pr
op
0
8 -B
20
**
4
Time (min)
Inhibition of I2 s (%)
60
8
Iso
Time (min)
B
Inhibition of IPeak (%)
d
cBiMPS
0
6
+ Iso
+ Iso
8-Br-cAMP reduced Ipeak by 26 ⫾ 2% and I2s by 30 ⫾ 6%, and
a pretreatment with 8-Br-cAMP occluded further inhibition of
nAChRs by isoproterenol (Fig. 3B). In addition, the isoproterenol-induced nAChR inhibition could be prevented by incubating cells with PKA-inhibitors Rp-cAMPS and H-89 (Fig.
3B). Similarly, internal perfusion of cells with an ATP-free
pipette solution containing nonhydrolyzable AMP-PCP prevented nAChR modulation (Fig. 3Ad). Overall, these results
suggest that adrenergic inhibition of nAChRs largely uses the
␤-AR pathway, cAMP, and PKA.
An earlier study (52) found that high concentrations of
isoproterenol may act on ␣1-AR as well as on ␤-AR in
pinealocytes. Therefore, it was important for us to guard
against this possible artifact as follows. We always used a
lower isoproterenol concentration (1 ␮M) that was reported not
to affect ␣1-ARs (52). Control experiments showed that in the
presence of the ␤-AR antagonist propranolol, isoproterenol
was prevented from inhibiting nACh current (Fig. 3B), whereas
the ␣1-AR antagonist doxazosin (10 nM) had no affect on the
inhibition. Tests on intact pinealocytes (no pipette) revealed no
Ca2⫹ elevations during application of isoproterenol, and all
whole cell experiments included 10 mM EGTA in the pipette
solution to depress any Ca2⫹ signaling. Thus we attribute
actions of isoproterenol seen here only to its agonist actions at
␤-ARs.
Downregulation of nAChRs reduces Ca2⫹ influx into pinealocytes.
It was previously reported that ACh and NE increase the
intracellular Ca2⫹ concentration in pinealocytes (28, 44).
The adrenergic response was attributed to ␣1-AR stimulation of Ca2⫹ release from internal stores followed by additional store-operated calcium entry (26, 49). Using Ca2⫹
imaging with AM-loaded fura-2, we confirmed the Ca2⫹
rises. In these experiments there was no whole cell pipette or
dialysis with EGTA. The transient increments of fluorescence ratio F340/F380 with 50 ␮M ACh and with 1 ␮M NE
were 3.7 ⫾ 0.8 and 0.8 ⫾ 0.2, respectively (Fig. 4). Here we
focus on the cholinergic response and ask whether Ca2⫹
entry is mediated by nAChR channels and whether it is
modulated by ␤-adrenergic signals.
In the first set of Ca2⫹ experiments, we activated nAChRs
specifically with DMPP (Fig. 5A, top and left trace). Repeated
applications of the agonist evoked repeated Ca2⫹ transients.
AJP-Cell Physiol • doi:10.1152/ajpcell.00354.2013 • www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.4 on June 16, 2017
Relative currents
c
4
Time (min)
Time (min)
Fig. 3. Signal transduction pathways for the modulation of nAChRs. A: currents activated by DMPP (50
␮M) are depressed by isoproterenol (Iso; 1 ␮M; a),
8-bromoadenosine (8-Br-cAMP; 1 mM; b), or 5,6dichlorobenzimidazole riboside-3=,5=-cyclic monophosphorothioate (cBIMPS; 0.1 mM; c). Isoproterenol
elicited little inhibition when intracellular pipette solution did not include ATP but 3 mM ␤,␥-methyleneadenosine 5=-triphosphate (AMP-PCP; d). Currents were
activated by DMPP for 2 s every 30 s at a membrane
potential of ⫺60 mV. Current amplitude was normalized to the initial value in each cell. Solid and dashed
lines are for Ipeak and I2s, respectively. B: summary of
inhibition of Ipeak (left) or I2s (right) current by application of different modulators including NE (n ⫽ 13),
isoproterenol (n ⫽ 10), and 8-Br-cAMP (n ⫽ 4).
Treatments of cells with 8-Br-cAMP (1 mM; n ⫽ 7),
Rp-cAMPS (0.1 mM; n ⫽ 3), AMP-PCB (n ⫽ 5),
H-89 (1 ␮M; n ⫽ 6), and propranolol (Prop; 1 ␮M;
n ⫽ 2) for 2 minutes occluded the inhibitory effect of
isoproterenol. Since nAChR currents showed a slow
spontaneous run down, we compared their values just
before and after 2 min of drug treatments. **P ⬍ 0.01,
significantly different from control currents activated
by DMPP. Other values are not significantly different
from controls.
C730
ADRENERGIC-CHOLINERGIC CROSS TALK IN PINEALOCYTES
A
B
5
4
ΔF340 /F380
Fig. 4. Ca2⫹-transients evoked by activation of
acetylcholine receptors and adrenergic receptors in
rat pinealocytes. A: representative Ca2⫹ rises upon
15 s application of 50 ␮M ACh with 2 mM extracellular concentration Ca2⫹ ([Ca2⫹]o) or of 1 ␮M
NE with 0 mM [Ca2⫹]o, plotted as dimensionless
fura-2 fluorescence ratios (F340/F380). B: average
change of intracellular Ca2⫹ signals (⌬F340/F380)
evoked by 50 ␮M ACh or 1 ␮M NE. Measurements
from 11 and 14 cells, respectively, as in A.
Calcium (F340 /F380)
ACh
3
2
NE
1
0
2
ACh
NE
cantly different from that without isoproterenol treatment (P ⬍
0.05) due to incomplete recovery.
Open nAChR channels might initiate Ca2⫹ entry in two
ways. They should pass a small Ca2⫹ influx and would also
depolarize the membrane potential, potentially activating voltage-gated Ca2⫹ channels (28, 57). ␤-AR stimulation may also
have multiple effects on Ca2⫹ signaling. In addition to downregulating nAChR channels, it can modulate several types of
voltage-gated Ca2⫹ channels (7, 8), mAChR-mediated Ca2⫹
A
DMPP
Calcium (F340/ F380)
The ⌬F340/F380 of the first Ca2⫹ peak was 3.1 ⫾ 0.3 (n ⫽ 11).
The peak amplitude was slightly reduced in successive applications (by 9 ⫾ 2 and 14 ⫾ 5% for the 2nd and 3rd DMPP
application; Fig. 5B). Application of isoproterenol to activate
␤-ARs significantly attenuated the subsequent DMPP-induced
Ca2⫹ increase (Fig. 5A, bottom trace, and Fig. 5B for summary). The inhibitory effect was slightly but significantly
reversed by 5 min of washing (P ⬍ 0.01), but after the
isoproterenol treatment the third response remained signifi-
Iso
C
0.8
200 s
Iso
200 s
Relative ΔF340/ F380
200 s
D
0.8
0.4
F
1.0
1.0
0.8
0.8
KCl
Iso
200 s
0.8
B
E
Nife+Atr/DMPP
200 s
0.8
200 s
0.8
1.4
1.2
***
0.6
*
1.0
***
0.6
**
Iso
0.2
0.4
Iso
0.2
0.2
0
0
1st
2nd
DMPP application
3rd
**
0.6
0.4
0.4
0.8
0
1st
2nd
3rd
DMPP application
1st
2nd
3rd
KCl application
Fig. 5. Downregulation of Ca2⫹ transients by nAChR modulation. A, top trace: Ca2⫹-transients evoked by 3 applications of 10 ␮M DMPP (15 s every 5 min).
A, bottom trace: the cell was incubated with 1 ␮M isoproterenol (Iso) for 2 min before the second DMPP application. B: summary of experiments like those
in A. The increase of F340/F380 ratio from the basal level was normalized to the first response (n ⫽ 5–13 for each point). Open and closed symbols represent
Ca2⫹ signals without and with isoproterenol before the second DMPP stimulation. C: same measurement as A but with 5 ␮M nifedipine (Nif) and 1 ␮M atropine
(Atr) to isolate Ca2⫹ transients mediated by nAChRs. D: summary of the experiments like those in C analyzed as in B. E: same measurement as A. Ca2⫹ transients
were evoked by 15 s applications of a saline solution containing 20 mM KCl. F: summary of the experiments like those in E analyzed as in B. The second and
third Ca2⫹ responses of the cells treated with isopterenol once before the second DMPP or KCl stimulations were compared with the values of the control cells
(*P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 0.001). The Ca2⫹ concentration in the bath was 2 mM.
AJP-Cell Physiol • doi:10.1152/ajpcell.00354.2013 • www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.4 on June 16, 2017
15 s
C731
ADRENERGIC-CHOLINERGIC CROSS TALK IN PINEALOCYTES
A
NE
DMPP
Control
ΔVm (mV)
Vm (mV)
40
B
NE
DMPP
-10
during subsequent stimulation. The second Ca2⫹ signal was
97 ⫾ 7% of the first. Pretreatment with 1 ␮M isoproterenol
decreased the Ca2⫹ rise significantly (49 ⫾ 5% compared with
the first Ca2⫹ rise and significantly different from the second
KCl of control cells; P ⬍ 0.01; n ⫽ 19). Thus VGCC also can
be negatively modulated by cAMP in pinealocytes.
Adrenergic modulation of nAChRs reduces membrane voltage responses of pinealocytes. Activating the nAChR nonselective cation channels depolarizes pinealocytes (28). We
tested whether NE attenuates the nicotinic membrane depolarization. The resting membrane potential (Vm) measured with
the current-clamp configuration of the patch-clamp technique
and a K⫹-based pipette solution (Fig. 6A) was ⫺49 ⫾ 3 mV
(n ⫽ 15). This value becomes about ⫺59 mV after correction
for the electrode potential, more negative than in previous
reports (1, 6, 20, 28, 59) for two reasons. First, most authors
did not correct for junction potentials, and second, unlike the
others, our internal solution contained no chloride and thus
could induce membrane hyperpolarization if pinealocytes have
a resting chloride conductance. When cells were treated with
10 ␮M DMPP, Vm depolarized as expected (by 20 ⫾ 3 mV;
n ⫽ 3). Addition of NE did not change Vm by itself, but it did
attenuate the DMPP-induced depolarization. This attenuation
-20
-30
-40
30
*
20
+ NE
10
-50
0
50
100
150
200
0
250
5
Time (s)
C
10
50
[DMPP] (µM)
D
Control or DMPP
0 pA
40
Control
20
-50
IInject (pA)
50
100
150
DMPP
-60
Control
-80
Vm (mV)
Voltage
DMPP
-100
20 mV
0.5 s
Fig. 6. Membrane potential responses to activation of nAChRs. A: depolarization induced by selective activation of nAChRs (10 ␮M DMPP), NE (1 ␮M) alone,
and nAChRs during modulation by NE. Reagents were applied to the cell by a rapid perfusion system. B: dose response of DMPP-induced depolarization before
and after treatment with NE (1 ␮M). Each point is the mean of 3–5 measurements. *P ⬍ 0.05, significantly different between control (open symbols) and the
modulated state (filled symbols). C: changes of membrane potential induced by applied currents without (control) and with activation of nAChRs (10 ␮M DMPP).
Currents from ⫺40 to 140 pA in 20 pA steps were injected using current-clamp configuration. All traces are from the same cell. Dashed lines indicate 0 mV.
D: voltage-current relationships of steady-state membrane potential (Vm) evoked by current injection (Iinject). Experiments indicated with symbols were done with
our standard K⫹-based pipette solution as in the rest of this figure (open circles for control and filled circles with 10 ␮M DMPP). Experiments indicated with
lines used a pipette solution in which Cs⫹ replaced K⫹ in the pipette solution to eliminate K⫹ currents (dash line for control and line with 10 ␮M DMPP). To
maintain the resting potential at ⫺50 mV with Cs⫹ in the pipettes, we injected a small negative holding current between 0 and ⫺20 pA. Averages from 5 to
15 cells.
AJP-Cell Physiol • doi:10.1152/ajpcell.00354.2013 • www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.4 on June 16, 2017
release (29), and store-operated calcium entry (26). To study
the modulatory effect of ␤-AR on the nAChR-mediated Ca2⫹influx in isolation, we repeated the same experiments with a
cocktail of 10 ␮M nifedipine (to block of L-type voltage-gated
Ca2⫹ channels) and 1 ␮M atropine (to block muscarinic
receptors that might evoke Ca2⫹ release from the stores and
activate store-operated calcium entry; Fig. 5, C and D). As
expected, in a control experiment, nifedipine completely
blocked the Ca2⫹ transient evoked by direct membrane depolarization with 50 mM extracellular K⫹ (n ⫽ 4, data not
shown). Ca2⫹ transients mediated by DMPP were also reduced
with nifedipine and atropine (⌬F340/F380 ⫽ 1.1 ⫾ 0.1; n ⫽ 9;
Fig. 5C), but pretreatment with isoproterenol still reduced the
Ca2⫹ transients further (by 63%, n ⫽ 9 compared with control,
n ⫽ 28; Fig. 5C, bottom trace, and Fig. 5D). Thus, like the
inward cation current, direct Ca2⫹ influx through nAChR
channels is significantly reduced by ␤-AR activation in rat
pinealocytes.
Similarly we tested whether voltage-gated Ca2⫹ channels
(VGCC) are modulated by isoproterenol (Fig. 5, E and F). We
measured Ca2⫹ transients evoked directly by KCl-depolarization. The Ca2⫹ rise induced by 20 mM KCl was 1.9 ⫾ 0.3
(F340/F380, n ⫽ 12) and did not show significant run-down
C732
ADRENERGIC-CHOLINERGIC CROSS TALK IN PINEALOCYTES
6). This ceiling would restrict firing of action potentials in
pinealocytes. In agreement, we observed no action potentials in
our current-clamp experiments even with strong current injection. There are repeated older reports of action potentials seen
with extracellular recording in pineal glands of anesthetized
animals (39, 40, 48). We favor the hypothesis that these
originate from nerve fibers terminating in the pineal rather than
from the pinealocytes themselves. Indeed, some of the single
units recorded were reported to be driven directly by shocks to
the habenula (40). Nevertheless, the opposite view is common,
and more clarification may still be needed.
We implicate an adrenergic pathway involving cAMP and
PKA in downregulation of nAChRs. nAChRs can be phosphorylated by PKA, protein kinase C, and protein tyrosine kinases
(16, 54). Such phosphorylation is likely to underlie the modulation we report here. In published experiments on neuronal
nAChRs, indirect evidence suggests a speeding of receptor
desensitization after stimulation of PKC (16) and more direct
evidence suggests a slowing of desensitization with chronic
increase of cAMP (9). In our experiments, cAMP and PKA
signals evoked by NE decrease the evoked current in nAChRs.
They also may accelerate receptor desensitization slightly during several-second DMPP treatment since the inhibition of
current is more prominent on I2s than Ipeak (Figs. 2 and 3). This
effect is small. The direction of change may be affected by
other accessory proteins, such as 14 –3-3 proteins, which are
know to interact with some nAChR ␣ subunits in a PKAdependent manner and to control membrane targeting of the
Sympathetic
(active at night)
NE
Cholinergic
(?)
α1-AR
β-AR
DISCUSSION
mAChR
[Ca2+]i
+
We have described functional properties of the nAChRs of
rat pinealocytes and their downregulation by adrenergic signaling. The nAChRs of pinealocytes have functional properties
typical of neuronal nAChR subtypes including strong inward
rectification, desensitization (5, 37), block by Meca, a blocker
of ganglionic and brain nAChRs (33), and block by D-tubocurarine but not ␣-bungarotoxin (13, 28). In agreement, published immunoprecipitation, RNAase protection assays, and
gene-chip analysis have identified the pineal nAChRs as the
neuronal ␣3␤4-subunit subtype (3, 13). The gene chips show
that these subunits are expressed ⬎16-fold higher in pineal
than in several other brain regions (3). Accordingly, the current
density we see from nAChRs in rat pinealocytes is remarkably
high, especially considering the high resting resistance of these
cells. Therefore, ACh released from cholinergic nerve terminals could evoke enough inward current through nAChRs to
depolarize the membrane and activate voltage-gated Ca2⫹
channels. Since nAChRs themselves are also appreciably Ca2⫹
permeable (28, 56, and Figs. 4 and 5), this cholinergic system
could initiate robust Ca2⫹ signal generation in pinealocytes.
However, depolarizing membrane excitation by nAChRs would be
capped at membrane potentials approaching ⫺20 mV (or ⫺30
mV) by the activation of counteracting K⫹ conductances (Fig.
ACh
nAChR
[cAMP]
mGluR
Depolarization
[Ca2+]i
[Ca2+]i
AANAT
VGCC
MEL
Glutamate
KV
MEL
Fig. 7. A partial model for the control of melatonin (MEL) secretion in
pinealocytes. NE released from sympathetic varicosities activates ␤-adrenergic
receptors to increase cellular cAMP which, in turn, increases melatonin
synthesis by the enzyme arylalkylamine N-acetyltransferase (AANAT). The
nature and function of cholinergic inputs still remain to be clarified. Their
suggested roles are indicated in this diagram (18, 43, 56, 58). ACh from
cholinergic terminals increases intracellular Ca2⫹ through nAChR and voltagegated Ca2⫹ channel (VGCC). It is suggested that the Ca2⫹ increase evokes
glutamate release from pinealocytes and triggers autocrine glutamatergic
signal inhibitory for melatonin production. We postulate a novel cross talk
where sympathetic NE suppresses cholinergic input via downregulation of
nAChR and VGCC using the canonical cAMP pathway in pinealocytes.
Voltage-gated potassium channels (Kv) limit membrane depolarization beyond
⫺20 mV and may limit the generation of action potential firing and therefore
cell electrical excitability. Straight and dashed lines are positive and negative
controls, respectively. Circles in metabotropic and ionotropic receptors denote
the binding sites of neurotransmitters. The diagram omits potential signals
from mAChR.
AJP-Cell Physiol • doi:10.1152/ajpcell.00354.2013 • www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.4 on June 16, 2017
was large and statistically significant for low concentrations of
DMPP (5 ␮M) but less clear for high concentrations (50 ␮M;
Fig. 6B).
To understand the voltage responses to DMPP and the effect
of NE better, we had to explore the electrical properties of
pinealocytes further by injecting small hyperpolarizing and
depolarizing test currents (Fig. 6C). Injecting hyperpolarizing
current gave large negative changes of Vm in accordance with
the high resting membrane resistance (⬃1.8 G⍀ in Fig. 6C;
1–2 G⍀ in Refs. 1, 20) and reflecting the absence of many open
ion channels at rest. Injecting small depolarizing currents
depolarized Vm, but larger currents failed to depolarize Vm
beyond ⫺20 mV (Fig. 6D, symbols; after correction for electrode junction potential, this would become closer to ⫺30 mV).
It is apparent that other ion channels become active that oppose
further membrane depolarization. As shown before with voltage-clamp experiments, depolarization to ⫺20 mV activates
several voltage-gated K⫹ channels that are highly expressed in
pinealocytes (1, 6, 20). Their large outward current would
counteract the applied depolarizing current, establishing a
ceiling on the evoked depolarization. Quite likely, these potassium channels limit the normal physiological operating voltage
range of pinealocytes to values negative of ⫺20 mV (59). The
voltage-current relations of Fig. 6D (symbols) also show that
by opening nAChR channels, DMPP decreases the resting
resistance (slope of the I–Vm curve), and again the depolarization it induces is limited by potassium channels at ⫺20 mV.
Thus, as we saw in Fig. 6B, at high DMPP concentrations,
where many nAChR channels are opened and the membrane
potential is near its “ceiling,” a fractional reduction of functional nAChR channels by adrenergic modulation would have
only a small impact on the DMPP-induced depolarization. This
ceiling effect was absent when K currents were precluded by
replacing all K⫹ in the patch pipette with Cs⫹ (dashed and
solid lines).
ADRENERGIC-CHOLINERGIC CROSS TALK IN PINEALOCYTES
on muscarinic receptors also inhibits melatonin synthesis both
through presynaptic inhibition of NE release from sympathetic
terminals and by inhibiting the enzyme arylalkylamine Nacetyltransferase in rat pinealocytes (10, 34). Thus ACh is a
candidate “day signal” or a signal to terminate the “night”
actions of NE, sharpening the night-to-dawn transition. NE,
acting as a night signal, not only stimulates melatonin synthesis
but also antagonizes cholinergic actions, perhaps thus sharpening the transition from dusk-to-night.
Convergence and antagonism of adrenergic and cholinergic
inputs to several areas of the central and peripheral nervous
systems have been postulated based on anatomical and functional assays (2, 14, 30). In this study, we clearly demonstrate
potential cross talk between sympathetic and cholinergic inputs
to rat pinealocytes by studying modulation of nAChR by
␤-adrenergic receptors.
ACKNOWLEDGMENTS
We thank Dr. Morten Møller for initial advice and Drs. Eamonn Dickson,
Horacio de la Iglesio, Andrea Meredith, Møller, and Haijie Yu for commenting
on the manuscript and Lea M. Miller for technical help.
Present address of J.-Y. Yoon: Division of Cardiovascular Medicine, Internal
Medicine, University of Iowa Carver College of Medicine, Iowa City, IA.
GRANTS
This research was supported by National Institute of General Medical
Sciences Grant GM-83913, National Institute of Neurological Disorders and
Stroke Grant NS-08174, and National Institute of Diabetes and Digestive and
Kidney Diseases Grant DK-080840.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: J.-Y.Y., B.H., and D.-S.K. conception and design of
research; J.-Y.Y. and S.-R.J. performed experiments; J.-Y.Y. and S.-R.J.
analyzed data; J.-Y.Y., S.-R.J., B.H., and D.-S.K. interpreted results of experiments; J.-Y.Y., S.-R.J., and D.-S.K. prepared figures; J.-Y.Y. and D.-S.K.
drafted manuscript; J.-Y.Y., S.-R.J., B.H., and D.-S.K. approved final version
of manuscript; S.-R.J., B.H., and D.-S.K. edited and revised manuscript.
REFERENCES
1. Aguayo LG, Weight FF. Characterization of membrane currents in
dissociated adult rat pineal cells. J Physiol 405: 397–419, 1988.
2. Archakova LI, Bulygin IA, Netukova NI. The ultrastructural organization of sympathetic ganglia of the cat. J Auton Nerv Syst 6: 83–93, 1982.
3. Bailey MJ, Coon SL, Carter DA, Humphries A, Kim JS, Shi Q,
Gaildrat P, Morin F, Ganguly S, Hogenesch JB, Weller JL, Rath MF,
Møller M, Baler R, Sugden D, Rangel ZG, Munson PJ, Klein DC.
Night/day changes in pineal expression of ⬎600 genes: central role of
adrenergic/cAMP signaling. J Biol Chem 284: 7606 –7622, 2009.
4. Boehm S, Kubista H. Fine tuning of sympathetic transmitter release via
ionotropic and metabotropic presynaptic receptors. Pharmacol Rev 54:
43–99, 2002.
5. Bohler S, Gay S, Bertrand S, Corringer PJ, Edelstein SJ, Changeux
JP, Bertrand D. Desensitization of neuronal nicotinic acetylcholine
receptors conferred by N-terminal segments of the ␤2 subunit. Biochemistry 40: 2066 –2074, 2001.
6. Castellano A, López-Barneo J, Armstrong CM. Potassium currents in
dissociated cells of the rat pineal gland. Pflügers Arch 413: 644 –650,
1989.
7. Catterall WA. Voltage-gated calcium channels. Cold Spring Harb Perspect Biol 3: a003947, 2011.
8. Chik CL, Liu QY, Li B, Klein DC, Zylka M, Kim DS, Chin H,
Karpinski E, Ho AK. ␣1D L-type Ca2⫹-channel currents: inhibition by a
␤-adrenergic agonist and pituitary adenylate cyclase-activating polypeptide (PACAP) in rat pinealocytes. J Neurochem 68: 1078 –1087, 1997.
AJP-Cell Physiol • doi:10.1152/ajpcell.00354.2013 • www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.4 on June 16, 2017
subunit proteins. Tonic activation of cAMP-PKA signaling
may change the stoichiometry of ␣- and ␤-subunit expression
and channel properties (24). This may explain why Di Angelantonio et al. (9) saw slowing of receptor desensitization with
chronic treatments whereas we saw slight speeding with an
acute treatment.
The key biochemical and physiological roles of sympathetic
input to the pineal are well understood (Fig. 7). NE released as
a night signal from varicosities and endings of superior cervical
ganglion sympathetic neurons initiates new gene expression
and synthesis and release of melatonin (46). Thus nocturnal NE
governs the nocturnal endocrine secretion of the pineal. We
have considered possible antagonism between adrenergic and
cholinergic signaling in pinealocytes. Here we showed that NE
reduces the depolarization and calcium elevation responses to
nicotinic agonists in the rat pineal. Previous work also showed
that ACh reduces the release of NE from the nerve terminals of
sympathetic ganglion neurons (10, 23; reviewed by Ref. 4) and
the synthesis of melatonin (see below). Such reciprocal interactions between sympathetic and cholinergic inputs could
fine tune nocturnal melatonin synthesis. In our experiments,
the pinealocytes were initially in what we call a “neutral”
condition that is probably most related to the daylight
portion of the diurnal cycle since they had been incubated
without NE for more than 24 h (3). The very brief exposure
to NE could be like the very first minutes of a night signal.
These few minutes would be quite insufficient to accomplish
the 1–2 h program of new gene expression that marks a full
transition to night state (46).
The findings here would be of greatest physiological interest
if we knew more about cholinergic input into the pineal gland
and its possible circadian activity profiles. Previous work
shows that the pineal glands of several mammals contain
acetylcholine (by HPLC), choline acetyl transferase (by immunoreactivity), vesicular acetylcholine transporter (by immunoreactivity) (35, 42, 55) with some reported exceptions (e.g.,
Ref. 45), and nerve fibers staining for cholinergic markers (31,
35, 50). Further, as already discussed, pinealocytes express
functional nAChRs abundantly and mAChRs. The amounts of
ACh and of vesicular ACh transporter show 10-fold diurnal
cycling in rat pineal (55). Cholinergic nerve fibers in the pineal
seem to originate both peripherally (parasympathetic) and
centrally (e.g., habenula). Many peptides that are candidate
cotransmitters in parasympathetic fibers are present in the
pineal, and the nerve terminals and varicosities remaining after
lesioning the superior cervical ganglia show a vesicular morphology that is parasympathetic like (31, 35). However, we
cannot yet point to specific fiber pathways and describe when
they might be active.
Figure 7 summarizes some acute signaling pathways in the
pineal gland. Cholinergic signals from the parasympathetic or
central nervous systems (31) may have inhibitory effects on
pineal melatonin synthesis. An inhibitory action of ACh
through nAChRs has been described (18, 56, 58). In rat
pinealocytes, acetylcholine-activated nAChRs depolarize the
membrane, open L-type voltage-gated Ca2⫹ channels, and
ultimately trigger glutamate release. The paracrine secreted
glutamate is postulated to decrease cAMP via inhibitory Gicoupled type-3 metabotropic glutamate receptors on nearby
pinealocytes and to suppress NE-induced melatonin biosynthesis (for review, see Ref. 43). In addition, acetylcholine acting
C733
C734
ADRENERGIC-CHOLINERGIC CROSS TALK IN PINEALOCYTES
34. Phansuwan-Pujito P, Govitrapong P, Ebadi M. Inhibitory actions of
muscarinic cholinergic receptor agonists on serotonin N-acetyltransferase
in bovine pineal explants in culture. Neurochem Res 16: 885–889, 1991.
35. Phansuwan-Pujito P, Møller M, Govitrapong P. Cholinergic innervation and function in the mammalian pineal gland. Microsc Res Tech 46:
281–295, 1999.
36. Pitchford S, Day JW, Gordon A, Mochly-Rosen D. Nicotinic acetylcholine receptor desensitization is regulated by activation-induced extracellular adenosine accumulation. J Neurosci 12: 4540 –4544, 1992.
37. Quick MW, Lester RA. Desensitization of neuronal nicotinic receptors.
J Neurobiol 53: 457–478, 2002.
38. Reuss S, Schroder B, Schroder H, Maelicke A. Nicotinic cholinoceptors
in the rat pineal gland as analyzed by western blot, light- and electron
microscopy. Brain Res 573: 114 –118, 1992.
39. Reuss S, Vollrath L. Electrophysiological properties of rat pinealocytes:
evidence for circadian and ultradian rhythms. Exp Brain Res 55: 455–461,
1984.
40. Rønnekleiv OK, Kelly MJ, Wuttke W. Single unit recordings in the rat
pineal gland: evidence for habenulo-pineal neural connections. Exp Brain
Res 39: 187–192, 1980.
41. Safran A, Sagi-Eisenberg R, Neumann D, Fuchs S. Phosphorylation of
the acetylcholine receptor by protein kinase C and identification of the
phosphorylation site within the receptor delta subunit. J Biol Chem 262:
10506 –10510, 1987.
42. Schäfer MK, Eiden LE, Weihe E. Cholinergic neurons and terminal
fields revealed by immunohistochemistry for the vesicular acetylcholine
transporter. I. Central nervous system. Neuroscience 84: 331–359, 1998.
43. Schomerus C, Korf HW. Cholinergic signal transduction cascades in rat
pinealocytes: functional and ontogenetic aspects. Reprod Nutr Dev 39:
305–314, 1999.
44. Schomerus C, Laedtke E, Korf HW. Calcium responses of isolated,
immunocytochemically identified rat pinealocytes to noradrenergic, cholinergic and vasopressinergic stimulations. Neurochem Int 27: 163–175,
1995.
45. Schrier BK, Klein DC. Absence of choline acetyltransferase in rat and
rabbit pineal gland. Brain Res 79: 347–351, 1974.
46. Simonneaux V, Ribelayga C. Generation of the melatonin endocrine
message in mammals: a review of the complex regulation of melatonin
synthesis by norepinephrine, peptides, and other pineal transmitters. Pharmacol Rev 55: 325–395, 2003.
47. Stankov B, Cimino M, Marini P, Lucini V, Fraschini F, Clementi F.
Identification and functional significance of nicotinic cholinergic receptors
in the rat pineal gland. Neurosci Lett 156: 131–134, 1993.
48. Stehle J, Reuss S, Vollrath L. Electrophysiological characterization of
the pineal gland of golden hamsters. Exp Brain Res 67: 27–32, 1987.
49. Sugden LA, Sugden D, Klein DC. Alpha 1-adrenoceptor activation
elevates cytosolic calcium in rat pinealocytes by increasing net influx. J
Biol Chem 262: 741–745, 1987.
50. Swiatkiewicz G. The presence of acetylcholinesterase-positive nerve
fibres in the deep pineal gland and the pineal stalk but not in the superficial
part of adult albino rats. Folia Biol (Krakow) 48: 97–103, 2000.
51. Swope SL, Moss SJ, Raymond LA, Huganir RL. Regulation of ligandgated ion channels by protein phosphorylation. Adv Second Messenger
Phosphoprotein Res 33: 49 –78, 1999.
52. Vanecek J, Sugden D, Weller J, Klein DC. Atypical synergistic ␣1- and
␤-adrenergic regulation of adenosine 3=,5=-monophosphate and guanosine
3=,5=-monophosphate in rat pinealocytes. Endocrinology 116: 2167–2173,
1985.
53. Wada E, Wada K, Boulter J, Deneris E, Heinemann S, Patrick J,
Swanson LW. Distribution of ␣2, ␣3, ␣4, and ␤2 neuronal nicotinic
receptor subunit mRNAs in the central nervous system: a hybridization
histochemical study in the rat. J Comp Neurol 284: 314 –335, 1989.
54. Wang K, Hackett JT, Cox ME, Van Hoek M, Lindstrom JM, Parsons
SJ. Regulation of the neuronal nicotinic acetylcholine receptor by SRC
family tyrosine kinases. J Biol Chem 279: 8779 –8786, 2004.
55. Wessler I, Reinheimer T, Bittinger F, Kirkpatrick CJ, Schenda J,
Vollrath L. Day-night rhythm of acetylcholine in the rat pineal gland.
Neurosci Lett 224: 173–176, 1997.
56. Yamada H, Ogura A, Koizumi S, Yamaguchi A, Moriyama Y. Acetylcholine triggers L-glutamate exocytosis via nicotinic receptors and
inhibits melatonin synthesis in rat pinealocytes. J Neurosci 18: 4946 –
4952, 1998.
AJP-Cell Physiol • doi:10.1152/ajpcell.00354.2013 • www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.4 on June 16, 2017
9. Di Angelantonio S, Piccioni A, Moriconi C, Trettel F, Cristalli G,
Grassi F, Limatola C. Adenosine A2A receptor induces protein kinase
A-dependent functional modulation of human ␣3␤4 nicotinic receptor. J
Physiol 589: 2755–2766, 2011.
10. Drijfhout WJ, Grol CJ, Westerink BH. Parasympathetic inhibition of
pineal indole metabolism by prejunctional modulation of noradrenaline
release. Eur J Pharmacol 308: 117–124, 1996.
11. Haghighi AP, Cooper E. Neuronal nicotinic acetylcholine receptors are
blocked by intracellular spermine in a voltage-dependent manner. J
Neurosci 18: 4050 –4062, 1998.
12. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved
patch-clamp techniques for high-resolution current recording from cells
and cell-free membrane patches. Pflügers Arch 391: 85–100, 1981.
13. Hernandez SC, Vicini S, Xiao Y, Dávila-García MI, Yasuda RP,
Wolfe BB, Kellar KJ. The nicotinic receptor in the rat pineal gland is an
␣3␤4 subtype. Mol Pharmacol 66: 978 –987, 2004.
14. Hösli L, Hösli E, Käser H. Colocalization of cholinergic, adrenergic and
peptidergic receptors on astrocytes. Neuroreport 4: 679 –682, 1993.
15. Huganir RL, Greengard P. cAMP-dependent protein kinase phosphorylates the nicotinic acetylcholine receptor. Proc Natl Acad Sci USA 80:
1130 –1134, 1983.
16. Huganir RL, Greengard P. Regulation of neurotransmitter receptor
desensitization by protein phosphorylation. Neuron 5: 555–567, 1990.
17. Ifune CK, Steinbach JH. Modulation of acetylcholine-elicited currents in
clonal rat phaeochromocytoma (PC12) cells by internal polyphosphates. J
Physiol 463: 431–447, 1993.
18. Ishio S, Yamada H, Hayashi M, Yatsushiro S, Noumi T, Yamaguchi
A, Moriyama Y. D-aspartate modulates melatonin synthesis in rat pinealocytes. Neurosci Lett 249: 143–146, 1998.
19. Jonas P. Fast application of agonists to isolated membrane patches. In:
Single-channel Recording (2nd ed.), edited by Sakmann B, Neher E. New
York: Plenum, 1995, p. 231–243.
20. Kim MH, Uehara S, Muroyama A, Hille B, Moriyama Y, Koh DS.
Glutamate transporter-mediated glutamate secretion in the mammalian
pineal gland. J Neurosci 28: 10852–10863, 2008.
21. Klein DC, Coon SL, Roseboom PH, Weller JL, Bernard M, Gastel JA,
Zatz M, Iuvone PM, Rodriguez IR, Bégay V, Falcón J, Cahill GM,
Cassone VM, Baler R. The melatonin rhythm-generating enzyme: molecular regulation of serotonin N-acetyltransferase in the pineal gland.
Recent Prog Horm Res 52: 307–357, 1997.
22. Klein DC. Arylalkylamine N-acetyltransferase: “the Timezyme”. J Biol
Chem 282: 4233–4237, 2007.
23. Koh DS, Hille B. Modulation by neurotransmitters of catecholamine
secretion from sympathetic ganglion neurons detected by amperometry.
Proc Natl Acad Sci USA 94: 1506 –1511, 1997.
24. Krashia P, Moroni M, Broadbent S, Hofmann G, Kracun S, Beato M,
Groot-Kormelink PJ, Sivilotti LG. Human ␣3␤4 neuronal nicotinic
receptors show different stoichiometry if they are expressed in Xenopus
oocytes or mammalian HEK293 cells. PLoS One 5: e13611, 2010.
25. Laitinen JT, Laitinen KS, Kokkola T. Cholinergic signaling in the rat
pineal gland. Cell Mol Neurobiol 15: 177–192, 1995.
26. Lee SY, Choi BH, Hur EM, Lee JH, Lee SJ, Lee CO, Kim KT.
Norepinephrine activates store-operated Ca2⫹ entry coupled to largeconductance Ca2⫹-activated K⫹ channels in rat pinealocytes. Am J Physiol
Cell Physiol 290: C1060 –C1066, 2006.
27. Lester RA, Dani JA. Time-dependent changes in central nicotinic acetylcholine channel kinetics in excised patches. Neuropharmacology 33:
27–34, 1994.
28. Letz B, Schomerus C, Maronde E, Korf HW, Korbmacher C. Stimulation of a nicotinic ACh receptor causes depolarization and activation of
L-type Ca2⫹ channels in rat pinealocytes. J Physiol 499: 329 –340, 1997.
29. Marin A, Urena J, Tabares L. Intracellular calcium release mediated by
noradrenaline and acetylcholine in mammalian pineal cells. J Pineal Res
21: 15–28, 1996.
30. McCormick DA. Cellular mechanisms underlying cholinergic and noradrenergic modulation of neuronal firing mode in the cat and guinea pig
dorsal lateral geniculate nucleus. J Neurosci 12: 278 –289, 1992.
31. Møller M, Baeres FM. The anatomy and innervation of the mammalian
pineal gland. Cell Tissue Res 309: 139 –150, 2002.
32. Møller M. Fine structure of the pinealopetal innervation of the mammalian pineal gland. Microsc Res Tech 21: 188 –204, 1992.
33. Papke RL, Sanberg PR, Shytle RD. Analysis of mecamylamine stereoisomers on human nicotinic receptor subtypes. J Pharmacol Exp Ther 297:
646 –656, 2001.
ADRENERGIC-CHOLINERGIC CROSS TALK IN PINEALOCYTES
57. Yamada H, Yamamoto A, Takahashi M, Michibata H, Kumon H,
Moriyama Y. The L-type Ca2⫹ channel is involved in microvesiclemediated glutamate exocytosis from rat pinealocytes. J Pineal Res 21:
165–174, 1996.
58. Yamada H, Yatsushiro S, Ishio S, Hayashi M, Nishi T, Yamamoto A,
Futai M, Yamaguchi A, Moriyama Y. Metabotropic glutamate receptors
C735
negatively regulate melatonin synthesis in rat pinealocytes. J Neurosci 18:
2056 –2062, 1998.
59. Zemkova H, Stojilkovic SS, Klein DC. Norepinephrine causes a biphasic
change in mammalian pinealocye membrane potential: role of alpha1Badrenoreceptors, phospholipase C, and Ca2⫹. Endocrinology 152: 3842–
3851, 2011.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.4 on June 16, 2017
AJP-Cell Physiol • doi:10.1152/ajpcell.00354.2013 • www.ajpcell.org