Biophysical properties of voltage-gated Na+ channels in frog parathyroid cells and their modulation by cannabinoids
Yukio Okada1,*,
Kotapola G. Imendra4,
Toshihiro Miyazaki2,
Hitoshi Hotokezaka3,
Rie Fujiyama1,
Jorge L. Zeredo1,
Takenori Miyamoto5 and
Kazuo Toda1
1 Integrative Sensory Physiology, Graduate School of Biomedical Sciences,
Nagasaki University, Nagasaki, Nagasaki 852-8588, Japan
2 Oral Cytology and Cell Biology, Graduate School of Biomedical Sciences,
Nagasaki University, Nagasaki, Nagasaki 852-8588, Japan
3 Orthodontics and Biomedical Engineering, Graduate School of Biomedical
Sciences, Nagasaki University, Nagasaki, Nagasaki 852-8588, Japan
4 Department of Physiology, Faculty of Medicine, University of Rhuna, Galle,
Sri Lanka
5 Department of Chemical and Biological Sciences, Faculty of Science, Japan
Women's University, Bunkyo-ku, Tokyo 112-8681, Japan
*
Author for correspondence (e-mail:
okada{at}net.nagasaki-u.ac.jp)
Accepted 2 November 2005
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Summary
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The membrane properties of isolated frog parathyroid cells were studied
using perforated and conventional whole-cell patch-clamp techniques. Frog
parathyroid cells displayed transient inward currents in response to
depolarizing pulses from a holding potential of 84 mV. We analyzed the
biophysical properties of the inward currents. The inward currents disappeared
by the replacement of external Na+ with NMDG+ and were
reversibly inhibited by 3 µmol l1 TTX, indicating that
the currents occur through the TTX-sensitive voltage-gated Na+
channels. Current density elicited by a voltage step from 84 mV to
24 mV was 80 pA pF1 in perforated mode and
55 pA pF1 in conventional mode. Current density was
decreased to 12 pA pF1 by internal GTP
S (0.5
mmol l1), but not affected by internal GDPßS (1 mmol
l-1). The voltage of half-maximum (V1/2)
activation was 46 mV in both perforated and conventional modes.
V1/2 of inactivation was 80 mV in perforated mode
and 86 mV in conventional mode. Internal GTP
S (0.5 mmol
l1) shifted the V1/2 for activation to
36 mV and for inactivation to 98 mV. A putative endocannabinoid,
2-arachidonoylglycerol ether (2-AG ether, 50 µmol l1) and
a cannabinomimetic aminoalkylindole, WIN 55,212-2 (10 µmol
l1) also greatly reduced the Na+ current and
shifted the V1/2 for activation and inactivation. The
results suggest that the Na+ currents in frog parathyroid cells can
be modulated by cannabinoids via a G protein-dependent mechanism.
Key words: parathyroid, voltage-gated Na+ channel, G protein, activation, inactivation, cannabinoid, frog
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Introduction
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Parathyroid hormone (PTH) regulates extracellular free Ca2+
concentration ([Ca2+]o) in cooperation with
1,25-dihydroxycholecalciferol (1,25(OH)2D3) and
calcitonin (CT). On the other hand, [Ca2+]o regulates
the secretion of PTH from parathyroid cells through extracellular
Ca2+-sensing receptor (CaR;
Brown et al., 1993
;
Hofer and Brown, 2003
). High
[Ca2+]o inhibits and low [Ca2+]o
enhances PTH secretion. It is believed that extracellular Ca2+
inhibits the secretion of PTH via the intracellular free
Ca2+ concentration ([Ca2+]i). However, the
molecular mechanism by which [Ca2+]i regulates PTH
secretion is not well elucidated.
Several electrophysiological studies have been performed in mammalian
parathyroid cells. Those using classical intracellular microelectrodes
indicated that rodent parathyroid cells display a deep resting potential
(about 70 mV), which is depolarized by increasing
[Ca2+]o (Bruce and
Anderson, 1979
;
López-Barneo and Armstrong,
1983
). Later, the patch-clamp technique was applied on bovine,
human and rodent parathyroid cells
(Castellano et al., 1987
;
Jia et al., 1988
;
Komwatana et al., 1994
;
Kanazirska et al., 1995
;
McHenry et al., 1998
;
Välimäki et al.,
2003
). These studies showed that parathyroid cells possess some
types of K+ channels. Other studies suggested the presence of
voltage-gated Ca2+ channels in bovine and goat parathyroid cells
(Sand et al., 1981
;
Chang et al., 2001
). However,
voltage-gated Na+ channels could not be found in any of the
aforementioned studies.
Ion channels are regulated by neurotransmitters and hormones via G
protein-coupled receptors (GPCRs; Wickman
and Clapham, 1995
; Dascal,
2001
). GPCRs dissociate heterotrimeric G proteins
(G
ß
) to G
-GTP and Gß
. Both subunits can
regulate a variety of ion channels directly (via physical
interactions between G protein subunits and the channel protein) or indirectly
(via second messengers and protein kinases).
In the present study, we report that frog parathyroid cells possess
voltage-gated Na+ channels and that their activity may be modulated
by cannabinoids.
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Materials and methods
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Cell preparation
Adult bullfrogs Rana catesbeiana Shaw weighing 250550 g
were used for the experiment over the course of a year. Experiments were
performed in accordance with the Guidelines for Animal Experimentation of
Nagasaki University. Parathyroid cells were isolated from the parathyroid
glands of decapitated and pithed animals. Two pairs of the oval parathyroid
glands in both sides were quickly dissected from the pre-cardial region, where
they lie near the ventral branchial bodies and attached to the carotid
arteries (Sasayama and Oguro,
1974
). The glands were cut into small pieces in
Ca2+-free saline containing 2 mmol l1 EDTA and
incubated for 1215 min in 2 ml of the same saline containing 10 mmol
l1 L-cysteine and 10 units ml1
papain (Sigma, St Louis, MO, USA). The glands were then rinsed with normal
saline. The individual cells were dissociated by gentle trituration in normal
saline. Isolated parathyroid cells displayed an oval-shape, with a diameter of
about 10 µm (Fig. 1).

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Fig. 1. Freshly isolated frog parathyroid cells are white and oval-shaped in this
phase-contrast image. Two large cells in the center of the image are the
erythrocytes. Bar, 50 µm.
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Electrophysiological recording
Voltage-clamp recording was performed in whole-cell configuration
(Hamill et al., 1981
) using a
CEZ 2300 patch-clamp amplifier (Nihon Kohden, Tokyo, Japan). The patch
pipettes were pulled from Pyrex glass capillaries containing a fine filament
(Summit Medical, Tokyo, Japan), using a two-stage puller (Narishige PD-5,
Tokyo, Japan). The tips of the electrodes were heat-polished with a microforge
(Narishige MF-80). The resistance of the resulting patch electrode was
510 M
when filled with internal solution. The formation
520 G
seals between the patch pipette and the cell surface was
facilitated by applying weak suction to the interior of the pipette. The patch
membrane was broken by applying strong suction, resulting in a sudden increase
in capacitance. Amphotericin B (133160 µg ml1,
Sigma) was added to the pipette solution when using the perforated method
(Rae et al., 1991
). The
perforated whole-cell condition was obtained within 5 min of the establishment
of a G
seal. Recordings were made from parathyroid cells that had been
allowed to settle on the bottom of a chamber placed on the stage of an
inverted microscope (Olympus IMT-2, Tokyo, Japan). The recording pipette was
positioned with a hydraulic micromanipulator (Narishige WR-88). The current
signal was low-pass-filled at 5 kHz, digitized at 125 kHz using a TL-1
interface (Axon Instruments, Union City, CA, USA), acquired at a sampling rate
of 210 kHz using a computer running the pCLAMP 5.5 software (Axon
Instruments), and stored on hard disk. The pCLAMP was also used to control the
digitalanalogue converter for the generation of the clamp protocol. The
indifferent electrode was a chlorided silver wire. The voltages were corrected
for the liquid junction potential (4 mV) between normal saline solution
and the standard K+ internal solution. Capacitance and series
resistance were compensated for, as appropriate. The series resistances after
compensation in perforated mode were in the range 815 M
. The
whole-cell currentvoltage (IV) relationship
was obtained from the current generated by the 70 ms voltage step pulses
between 74 and +56 mV in 10 mV increments from a holding potential of
84 mV. The voltage-dependence of steady-state inactivation was studied
in a conventional manner by applying stepwise 200 ms conditioning pulses over
the range from 124 mV to 34 mV and a single depolarization to
34 mV near the peak of the IV curve. The time course of
recovery from inactivation was measured by double-pulse protocols. Input
resistance was calculated from the slope conductance generated by the voltage
ramp from 104 to 54 mV.
Data analysis
Data were analyzed with pCLAMP and Origin 7.5 (Origin Lab, Northampton, MA,
USA). Unless stated otherwise, the data are presented as means ±
S.E.M., significance was tested by Student's
t-test and a difference was considered significant if
P<0.05. The steady-state inactivation curves were fitted with a
Boltzmann equation as follows:
 | (1) |
where I/Imax is the current magnitude normalized
to its maximum value, V designates the conditioning voltage,
V1/2 is the voltage at which the magnitude is
half-maximum, and k is a Boltzmann slope factor, which reflects the
voltage sensitivity of steady-state inactivation of the current. The
conductance for activation was estimated according to the following equation:
 | (2) |
where G is the peak conductance, I is the current magnitude
at the test voltage (V), and Vrev is the apparent
reversal potential for the Na+ current. The activation curve was
also fitted with the Boltzmann equation as follows:
 | (3) |
where G/Gmax is the conductance magnitude
normalized to its maximum value, V designates the test voltage, and
other parameters are the same as above. Recovery from inactivation was fitted
using:
 | (4) |
where t is time, a is maximum magnitude, and
is time
constant for the recovery.
Solution and drugs
Normal saline solution consisted of (in mmol l1): NaCl
115, KCl 2.5, CaCl2 1.8, Hepes 10; glucose 20, pH 7.2. The pH of
normal saline and other solutions was adjusted by Tris base. The extracellular
Na+-free solution was prepared by the replacement of Na+
with NMDG+. The solution exchange was done by gravity flow. For
stock solution, tetrodotoxin (3 mmol l1; Sigma), spermine
tetrahydrochloride (100 mmol l1; Sigma) and adrenaline
bitartrate (10 mmol l1; Sigma) were dissolved in deionized
water. WIN 55,212-2 mesylate (10 mmol l1; Tocris, Bristol,
UK), phorbol 12,13-dibutyrate (PDBu, 10 mmol l1; Sigma),
forskolin (10 mmol l1; Sigma) and chelerythrine chloride (10
mmol l1; Sigma) were dissolved in dimethylsulphoxide (DMSO).
2-arachidonoylglycerol ether (2-AG ether, 13.7 mmol l1)
dissolved in ethanol was purchased from Tocris. Samples of the stock solutions
were added to normal saline solution to give the desired final
concentrations.
The standard K+ internal solution contained (in mmol
l1): KCl 100, CaCl2 0.1, MgCl2 2, EGTA
1, Hepes 10, pH 7.2. GTP
S (0.5 mmol l1, Sigma) and
GDPßS (1 mmol l1, Sigma) were dissolved in the internal
solution on every experimental day.
All experiments were carried out at room temperature
(2025°C).
 |
Results
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Basal properties of isolated frog parathyroid cells
Under conventional whole-cell mode in a standard K+ internal
solution, frog parathyroid cells displayed resting potential of 10 to
70 mV (30±2 mV, N=44). The input resistance
ranged from 4.7 to 25.0 G
(13.7±0.9 G
, N=44) and
the membrane capacitance was 5.811.0 pF (7.6±0.2 pF,
N=44). On the other hand, under perforated whole-cell mode using
amphotericin B, the cell displayed resting potentials of 10 to
87 mV (26±3 mV, N=26). In perforated mode, the
input resistance ranged from 4.5 to 50.0 G
(11.1±1.8 G
,
N=26), and the membrane capacitance was 6.013.0 pF
(7.9±0.4 pF, N=26). Differences in basal membrane properties
were not significant between conventional and perforated conditions.
Activation
After attaining the perforated whole-cell configuration in normal saline
solution, almost all frog parathyroid cells displayed transient inward
currents in response to depolarizing voltage steps from a holding potential of
84 mV (Fig. 2A).
Although the pipette contained a K+ internal solution, leak
currents, but not the voltage-gated outward current could be found. The
threshold for the inward current activation ranged from 64 to 54
mV (Fig. 2B). The inward
currents were activated more rapidly with successive depolarizing steps. The
activation time reaching a peak at 24 mV, for instance, was
0.69±0.04 ms (N=27). Removal of external Na+
completely abolished the inward currents (3 cells;
Fig. 3A), indicating that the
currents were caused predominately by Na+. Application of 3 µmol
l1 TTX (a voltage-gated Na+ channel blocker)
totally eliminated the inward currents (8 cells;
Fig. 3B) after about 4 min. The
currents recovered to 92.2±12.2% (N=8) of the controls after
about 10 min of washing with normal saline solution.

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Fig. 2. A representative example of perforated whole-cell current of a frog
parathyroid cell in normal saline solution. (A) Transient inward currents were
elicited in response to 15 ms voltage steps between 74 to +56 mV in 10
mV increments from a holding potential of 84 mV. The leak currents were
not subtracted from the current traces. (B) Pooled currentvoltage
(IV) relationships for the inward currents (N=17)
elicited by the voltage steps. Values are means ±
S.E.M.
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Fig. 3. Effects of the elimination of external Na+ (A) and the addition
of 3 µmol l1 TTX to external normal saline solution (B)
on the transient inward currents. The currents were elicited by 15 ms step
pulses from a holding potential of 84 mV to a test potential of
24 mV.
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Membrane-delimited G protein-regulation of voltage-gated Na+
channels has been suggested. We also investigated the effect of GTP
S on
the Na+ channels in frog parathyroid cells. Activation parameters
were compared with each other in the presence and absence of GTP
S. Peak
inward current elicited by a voltage step from 84 to 24 mV was
663±74 pA (N=27) in perforated mode, whereas the
current decreased to 419±33 pA (N=30,
P<0.05) in conventional mode. GDPßS (1 mmol
l1) added to the intracellular solution did not affect the
current amplitude (413±88 pA, N=7) as compared with
conventional mode, but internal 0.5 mmol l1 GTP
S
significantly decreased the current to 93±10 pA (N=8,
P<0.05). Similar results were observed for current density
(expressed as the ratio of current amplitude to cell capacitance). The
magnitudes of the current density were 79.5±6.6 pA
pF1 (N=27) in perforated mode,
55.2±4.4 pA pF1 (N=30) in
conventional mode, 49.8±9.2 pA pF1
(N=7) in the condition containing 1 mmol l1
GDPßS and 11.6±1.3 pA pF1 (N=8)
in the condition containing 0.5 mmol l1 GTP
S,
respectively. The voltage of half-maximum activation V1/2
in perforated mode was estimated from the activation curve
(V1/2=45.7±0.8 mV, N=17;
Fig. 4A). The
V1/2 in conventional mode (46.1±1.1 mV,
N=18) was almost the same. Addition of 1 mmol l1
GDPßS into the pipette solution did not change the
V1/2 (45.1±1.7 mV, N=6) as compared
with conventional mode, but internal 0.5 mmol l1 GTP
S
shifted the V1/2 to 35.5±3.3 mV
(N=8, P<0.05; Fig.
4B).

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Fig. 4. Voltage dependence of activation of the Na+ currents, determined
by IV relationships for the Na+ currents elicited
by the voltage steps. Smooth curves are simple Boltzmann functions. (A)
V1/2=45.7 mV, k=6.4 mV for
perforated recording (filled circles), V1/2=46.1
mV, k=5.2 mV for conventional recording (open circles). (B)
V1/2=35.5 mV, k=7.6 mV for 0.5 mmol
l1 GTP S (filled triangles),
V1/2=45.1 mV, k=4.4 mV for 1 mmol
l1 GDPßS (open triangles). For comparison, the broken
line for conventional recording is plotted in B. The values are means ±
S.E.M. obtained from 618 cells.
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Inactivation
We measured inactivation time constant (
) for inactivation onset
during a test pulse to 24 mV. The inactivation time constant in
perforated mode (0.87±0.08 ms, N=27) was shorter than the
constant in conventional mode (1.13±0.06 ms, N=30,
P<0.05). Internal 1 mmol l1 GDPßS and 0.5
mmol l1 GTP
S did not significantly change the
constant as compared with conventional mode (data not shown).
The voltage-dependence of steady-state inactivation was studied in a
conventional manner. As the level of the conditioning pulses was shifted to
the depolarizing direction, the magnitude of the inward currents decreased
(Fig. 5A). The point of the
half-maximum inactivation (V1/2) was
79.8±2.1 mV (N=14) in perforated mode
(Fig. 5B). The
V1/2 in conventional mode shifted to hyperpolarizing
direction (86.3±2.0 mV, N=12, P<0.05).
Internal 1 mmol l1 GDPßS did not affect the
V1/2 (86.1±3.0 mV, N=7) as compared
with conventional mode, but internal 0.5 mmol l1 GTP
S
significantly shifted the V1/2 to 98.4±2.1
mV (N=12, P<0.05; Fig.
5C).

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Fig. 5. Voltage dependence of steady-state inactivation of the Na+
currents. (A) The dependence was determined by measuring peak current elicited
by a single depolarization to 34 mV from a range of 200 ms conditioning
voltages. (B) V1/2=79.8 mV, k=8.2 mV for
perforated recording (filled circles),
V1/2=86.3 mV, k=9.4 mV
for conventional recording (open circles). (C)
V1//2=98.4 mV, k=9.6 mV for 0.5 mmol
l-1 GTP S (filled triangles),
V1/2=86.1 mV, k=10.0 mV for 1 mmol
l1 GDPßS (open triangles). For comparison, the broken
line for conventional recording is plotted in B. The values are means ±
S.E.M. obtained from 714 cells.
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The time course of recovery of the inward currents from inactivation was
investigated using double-pulse protocol with varying pulse intervals. The
two-step pulses (control and test pulse) were identical and consisted of a
depolarization to 24 mV, lasting 20 ms
(Fig. 6A). The time course of
recovery was single exponential (Fig.
6B). The time constant was 9.6±1.3 ms (N=5) in
perforated mode and 13.1±0.7 ms (N=5) in conventional mode,
respectively. Addition of 0.5 mmol l1 GTP
S to the
internal solution further prolonged the time constant to 16.5±2.4 ms
(N=5). Recovery was significantly more rapid in perforated mode than
in conventional mode and the condition containing 0.5 mmol
l1 GTP
S. However, there was no significant difference
in the time constant between the conventional mode and after addition of
GTP
S.

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Fig. 6. Time course of recovery from inactivation. (A) Inward currents recorded
using a double pulse protocol in which a 20 ms control pulse from 84 to
24 mV was followed by a second identical voltage pulse. (B) The plots
show the recovery time course of the Na+ currents. The curves
represent the fits of a single exponential function, giving values of time
constants of 9.6 ±1.2 ms (N=5) for perforated mode (filled
circles), 13.1±0.7 ms (N=5) in conventional mode (open
circles) and 16.5±2.4 ms (N=5) for the condition containing
0.5 mmol l1 GTP S in the internal solution (open
triangles).
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Effects of cannabinoids on Na+ current
In the present study, dialysis of GTP
S into internal solution
modulated activation and inactivation of Na+ currents, suggesting
regulation by a G protein-coupled mechanism. We therefore searched for the
ligand that can modulate the Na+ current activity. Parathyroid
cells express the CaR and the PTH release from the cells is decreased by a CaR
agonist, spermine (Quinn et al.,
1997
). Spermine (1 mmol l-1), however, did not affect
the Na+ current activity (Fig.
7C). By contrast, beta-adrenergic agonists stimulate cAMP
production and PTH release in parathyroid cells
(Brown et al., 1977
).
Adrenaline (10 µmol l1) also did not significantly
inhibit the Na+ current (Fig.
7C).

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Fig. 7. Effects of WIN 55,212-2 and 2-AG ether on the Na+ currents. (A)
Reversible inhibition of the Na+ current by 10 µmol
l1 WIN 55,212-2. The currents were elicited by a 20 ms pulse
from a holding potential of 84 mV to a test potential of 24 mV.
The current traces labelled ac were obtained at the times indicated by
the same letter on the time course. (B) Representative example of time course
of the current signal. (C) Na+ current magnitudes before and after
superfusion with each drug. Values are means ±
S.E.M. Numerals within parentheses are number
of the cells sampled.
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Although the cannabinoid receptors were not found on the cell membrane of
parathyroid cells, frog parathyroid cells under perforated whole-cell mode
were exposed to a putative endocannabinoid, 2-AG ether (50 µmol
l1). The drug did not change the basal properties of the
cells, but reduced the Na+ current. The peak current at 24
mV decreased to 36±8% (P<0.05) of the controls in 5 of 5
cells (Fig. 7C). Even when the
external solution was returned to normal saline solution, the Na+
current did not recover to the initial level. A cannabinomimetic
aminoalkylindole, WIN 55,212-2 (10 µmol l1) reversibly
inhibited the Na+ current (Fig.
7A,B). The peak current at 24 mV decreased to 33±7%
(N=5, P<0.05) of the controls. WIN 55,212-2 shifted the
V1/2 of activation by 11.7±1.6 mV (N=4,
P<0.05; Fig. 8A,C)
and the V1/2 of inactivation by 17.5±1.9 mV
(N=4, P<0.05; Fig.
8B,D). 2-AG ether also induced similar shift in the
V1/2 of activation and inactivation
(Fig. 8C,D).

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Fig. 8. Effects of WIN 55,212-2 and 2-AG ether on activation and inactivation of
the Na+ currents. (A) Voltage dependence of activation before and
after superfusion with 10 µmol l1 WIN 55,212-2.
V1/2=48.6 mV, k=5.1 mV for control
(open squares), V1/2=37.0 mV, k=5.0
mV for WIN 55,212-2 (filled circles). (B) Voltage dependence of inactivation
before and after superfusion with 10 µmol l1 WIN 55,212-2
(filled circles). V1/2=76.9 mV, k=8.1 mV
for control (open squares), V1/2=94.4 mV,
k=9.0 mV for WIN 55,212-2. (C) V1/2 of activation
before (white bars) and after superfusion with each drug (hatched bars). (D)
V1/2 of inactivation before (white bars) and after
superfusion with each drug (hatched bars). Values are means ±
S.E.M. Numerals within parentheses are number
of the cells sampled.
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Effects of protein kinases on the Na+ current
The cytoplasmic loop in the Na+ channels possesses several
protein kinase A (PKA) and protein kinase C (PKC) phosphorylation sites
(Cantrell et al., 2003). To determine the role of PKC on Na+
current, we tested the effect of a PKC activator, PDBu on the properties of
Na+ current. PDBu (10 µmol l1) decreased the
Na+ current at 24 mV to 37±8% of the controls
(Fig. 9C). A wash-out with
normal saline solution was not observed
(Fig. 9A). PDBu did not
significantly shift the V1/2 of activation
(Fig. 10A), but induced the
hyperpolarizing shift of 11.9±2.6 mV (N=5, P<0.05)
in the V1/2 of inactivation
(Fig. 10B). An adenylyl
cyclase activator, forskolin (10 µmol l1) did not
significantly inhibit the Na+ current
(Fig. 9C).

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Fig. 9. Effects of PDBu and forskolin on the Na+ currents. (A)
Representative example of time course of the current signal after superfusion
with 10 µmol l1 PDBu. (B) Representative example of time
course of the current signal after superfusion with 10 µmol
l1 forskolin. (C) Na+ current magnitude before
(white bars) and after superfusion with each drug (hatched bars). The currents
were elicited by 20 ms pulse from a holding potential of 84 mV to a
test potential of 24 mV. Values are means ±
S.E.M. Numerals within parentheses are number
of the cells sampled.
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Fig. 10. Effect of 10 µmol l1 PDBu on activation and
inactivation of the Na+ currents. (A) Voltage dependence of
activation. V1/2=47.7 mV, k=5.3 mV
for control (open squares), V1/2=44.3 mV,
k=6.0 mV for PDBu (filled circles). (B) Voltage dependence of
inactivation. V1/2=81.8 mV, k=8.1 for
control (open squares), V1/2=93.7 mV,
k=9.0 mV for PDBu (filled circles). Values are mean ±
S.E.M. obtained from 58 cells.
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We further evaluated the effect of a PKC inhibitor (chelerythrine) on the
modulation of Na+ current elicited by cannabinoids. Chelerythrine
(10 µmol l1) itself did not affect the Na+
current (Fig. 11A). Even when
frog parathyroid cells were pre-incubated for 10 min in a solution containing
10 µmol l1 chelerythrine, subsequent application of 10
µmol l1 WIN 55,212-2 still inhibited the Na+
currents (Fig. 11A,B).
Furthermore, in the presence of PKC inhibitor, WIN 55,212-2 shifted the
V1/2 of activation by 11.3±2.6 mV (N=3,
P<0.05; Fig. 11C)
and the V1/2 of inactivation by 18.3±5.0 mV
(N=3, P<0.05; Fig.
11D). These results indicate that WIN 55,212-2 acts on the
Na+ currents, probably through a mechanism independent of PKC.

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Fig. 11. Effect of WIN 55,212-2 on the Na+ currents in the presence of an
inhibitor of PKC. (A) Representative example of time course of the current
signal. The currents were elicited by a pulse from a holding potential of
84 mV to a test potential of 24 mV. (B) Mean values of
Na+ current magnitudes before (white bar) and after superfusion
with 10 µmol l1 chelerythrine and 10 µmol
l1 WIN 55,212-2 (hatched bar). Numerals within parentheses
are number of the cells sampled. (C) Voltage dependence of activation.
V1/2=42.0 mV, k=6.1 mV for control
(open squares), V1/2=30.7 mV, k=8.0
mV for chelerythrine plus WIN 55,212-2 (filled circles). (D) Voltage
dependence of inactivation. V1/2=83.5 mV,
k=8.4 mV for control (open squares),
V1/2=103.8 mV, k=7.6 mV for chelerythrine
plus WIN 55,212-2 (filled circles). The values are mean ±
S.E.M. obtained from three cells.
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Discussion
|
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The present study shows that frog parathyroid cells express TTX-sensitive
voltage-gated Na+ channels in the cell membrane. However, most
Na+ channels were silent at resting potential (30 mV). The
silent Na+ channels in the parathyroid cells could only be restored
if a hyperpolarizing voltage was applied. Similarly, rat gonadotropes in the
anterior lobe of the pituitary glands fire action potentials at the
termination of the hyperpolarization elicited by gonadotropin-releasing
hormone (GnRH; Tse and Hille,
1993
); in these cells, fast depolarizations in action potentials
open voltage-gated Ca2+ channels that the allow entry of
extracellular Ca2+. Nevertheless, in the present experiments, the
V1/2 for inactivation was about 80 mV in perforated
mode, and few cells that possess such a deep resting potential are
excitable.
By comparing the activation and inactivation processes in the presence and
absence of GTP
S, we observed evidence that the activation and
inactivation of Na+ currents are regulated by guanine nucleotides,
perhaps as a G protein-coupled mechanism. In the absence (conventional) of
GTP
S, the mean V1/2 for Na+ current
activation was about 46 mV. A similar mean value was observed in the
presence of GDPßS in the pipette solution. Since internal GTP
S
shifted the V1/2 to a more positive level (36 mV),
the presence of GTP
S results in the activation of a G protein-dependent
mechanism, which may be functionally important in regulating Na+
current activation. A similar effect of GTP
S was also observed in the
inactivation process. Our results suggest that a G protein-dependent
inhibitory mechanism may be involved in both activation and inactivation
processes in the voltage-gated Na+ channels of frog parathyroid
cells. Furthermore, in perforated mode, the V1/2 for
inactivation shifted to a more positive value than in conventional mode. This
also suggests that the Na+ channels may be regulated by another
enhancing mechanism. It has been reported that voltage-gated Na+
channels are under the regulation of G proteins. In rat cardiac myocytes,
internal GTP
S inhibits the Na+ channel by shifting the
steady-state inactivation to more negative potentials (Schubert et al.,
1989
and
1990
). In contrast,
GTP
S enhances Na+ current in frog olfactory receptor neurons
(Pun et al., 1994
).
Two types (CB1 and CB2) of cannabinoid receptors have
been cloned from many vertebrates including mammals, birds, amphibians and
fish (Lutz, 2002
).
CB1 is expressed predominantly in the central and peripheral
nervous system, while CB2 is present exclusively in immune cells.
CB1 is a typical Gi/o-coupled receptor and can initiate
various signaling events (Piomelli,
2003
). These include closure and opening of ion channels,
inhibition of adenylyl cyclase activity and stimulation of protein kinases. In
the present study we demonstrate that external application of a putative
endocannabinoid, 2-AG ether, and cannabinomimetic aminoalkylindole WIN
55,212-2, produced potent inhibition of the voltage-gated Na+
current in frog parathyroid cells, although their receptors are not found on
the cells. External cannabinoids as well as internal GTP
S shifted the
V1/2 for activation and inactivation, suggesting that
cannabinoids could inhibit the Na+ current via a G
protein-dependent mechanism. Although a PKC activator, PDBu, also inhibited
the Na+ current and shifted the V1/2 for
inactivation, the modulating effect of cannabinoids on the Na+
current cannot be explained by PKC activity. Further studies will be required
to elucidate the mechanism for modulation induced by cannabinoids.
In conventional whole-cell configuration, human parathyroid cells hardly
displayed macroscopic K+ current in low intracellular
Ca2+ concentration (<10 nmol l1;
Välimäki et al.,
2003
). We also could not record voltage-gated outward currents in
response to the depolarizing voltage steps from a holding potential of
84 mV. The free Ca2+ concentration of the present internal
solution is calculated to be about 16 nmol l1 using the
Webmaxc software. As expected from those results, both human and frog
parathyroid cells displayed low resting potential in the condition of low
intracellular Ca2+. In human parathyroid cells, the K+
current increases and the membrane potentials are hyperpolarized when
extracellular Ca2+ concentration is elevated
(Välimäki et al.,
2003
). On the other hand, high extracellular Ca2+
elicits Ca2+-activated Cl conductance in frog
parathyroid cells (Okada et al.,
2001
). The activation of Cl conductance may
hyperpolarize the cells if intracellular Cl concentration is
low. Further experiments should clarify more fully the relationships between
changes in extracellular Ca2+ concentration and the regulation of
Ca2+-activated Cl channel in frog parathyroid
cells.
In conclusion, frog parathyroid cells possess voltage-gated Na+
channels whose activity may be modulated by cannabinoids.
 |
List of symbols and abbreviations
|
---|
- 2-AG
- 2-arachidonoylglycerol
- [Ca2+]o
- extracellular free Ca2+ concentration
- 1,25(OH)2D3
- 1,25-dihydroxycholecalciferol
- a
- maximum magnitude
- CaR
- Ca2+-sensing receptor
- CT
- calcitonin
- DMSO
- dimethyl sulphoxide
- G
- peak conductance
- GPCR
- G protein-coupled receptor
- I
- current
- k
- Boltzmann slope factor
- PKA
- protein kinase A
- PKC
- protein kinase C
- PTH
- parathyroid hormone
- t
- time
- V
- voltage
- Vrev
- apparent reversal potential

- time constant
 |
Acknowledgments
|
---|
This work was supported by Grants-in-Aid (14540630) from Japan Society for
the Promotion of Science to Y.O.
 |
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