From the Departments of Stimulation-regulated fusion of vesicles to the
plasma membrane is an essential step for hormone secretion but may also
serve for the recruitment of functional proteins to the plasma
membrane. While studying the distribution of G protein-gated
K+ (KG) channels in the anterior
pituitary lobe, we found KG channel subunits Kir3.1 and
Kir3.4 localized on the membranes of intracellular dense core vesicles
that contained thyrotropin. Stimulation of these thyrotroph cells with
thyrotropin-releasing hormone provoked fusion of vesicles to the plasma
membrane, increased expression of Kir3.1 and Kir3.4 subunits in the
plasma membrane, and markedly enhanced KG currents
stimulated by dopamine and somatostatin. These data indicate a novel
mechanism for the rapid insertion of functional ion channels into the
plasma membrane, which could form a new type of negative feedback
control loop for hormone secretion in the endocrine system.
The G protein-gated K+
(KG)1 channel, a
member of the inwardly rectifying K+ (Kir) channel family,
is directly activated by pertussis toxin-sensitive G proteins. This
system was first discovered in the muscarinic deceleration of the
heartbeat (1). More recently, it is considered to play an essential
role in the hormone-mediated inhibitory regulation of neural
excitability (1, 2). Electrophysiological studies have revealed that a
variety of inhibitory receptors, including M2-muscarinic,
A1-purinergic, D2-dopamine, The main subunit of KG channels has been cloned from the
heart and designated GIRK1/Kir3.1 (8, 9). In the brain and heart,
Kir3.1 forms functional KG channels by assembling with other Kir3.0 subunits such as GIRK2/Kir3.2, GIRK3/Kir3.3 (10, 11), and
GIRK4/CIR/Kir3.4 (12). Because Kir3.1 mRNA was detected in the
anterior pituitary lobe (13) and pancreatic islet (14), this subunit
may also contribute to the formation of KG channels in
endocrine cells. The anterior pituitary lobe contains several kinds of
endocrine cells: lactotrophs, somatotrophs, corticotrophs, and
thyrotrophs. Although electrophysiological experiments have shown that
somatostatin or dopamine activate KG currents in
lactotrophs (4) and also in several cell lines derived from the
anterior pituitary lobe, such as GH3 (5) and AtT20 (6), the cellular and subcellular localizations of KG channels in in
vivo pituitary endocrine cells have not been examined.
In this study, using a polyclonal antibody specific to Kir3.1, we found
that this subunit was expressed only in thyrotroph cells and,
surprisingly, was localized predominantly on intracellular secretory
vesicles. Kir3.4 was co-localized on the vesicles with Kir3.1.
Thyrotropin-releasing hormone (TRH) stimulation of thyrotrophs caused
fusion of the vesicles to the cell membrane, increase of cell
capacitance, and enhancement of dopamine- or somatostatin-induced KG current. These data indicate a novel mechanism for the
rapid insertion of functional ion channels into the plasma membrane, which could form a new type of negative feedback control loop for
thyrotrophs by tuning up their inhibitory regulatory signaling system
in response to the stimulatory signal.
Preparation of Polyclonal Antibodies--
The polyclonal
antibody for Kir3.1 (aG1C-1) was raised in rabbit against a synthetic
peptide corresponding to amino acid residues 488-501 (LPAKLRKMNSDRFT)
of Kir3.1/GIRK1 (15, 16). We have also developed the antibody to Kir3.4
(aG4N-10) in rabbit using the antigenic peptide, DSRNAMNQDMEIGV, which
corresponds to amino acids 4-17 of Kir3.4 (17). These antibodies have
been successfully used for immunoprecipitation and Western blotting
analyses of Kir3.1 protein (15, 16) and Kir3.4
protein2 in the brain and
heart. Furthermore, in the rat heart, the immunoreactivities were
detected with the antibodies only in the atrium but not in the
ventricle (data not shown). This finding coincides with the expression
of the cardiac KG channel, which is composed of the subunits Kir3.1 and Kir3.4 in the heart atrium (13).
Immunohistochemical Study--
Immunohistochemistry was
performed according to a method described elsewhere (15). Rats were
anesthetized with sodium pentobarbital and perfused transcardially with
4% (w/v) paraformaldehyde, 0.5% (w/v) glutaraldehyde, and 0.2% (w/v)
picric acid, 0.1 M phosphate buffer (pH 7.4). The anterior
pituitary lobe was removed, postfixed in a fixative containing 4%
paraformaldehyde and 0.2% picric acid for 48 h at 4 °C, and
transferred to 0.1 M phosphate buffer containing 15% (w/v)
sucrose and 0.1% (w/v) sodium azide at 4 °C. Sections were cut with
a cryostat at 20 µm of thickness and stored at 4 °C in the same
solution until used. After rinsing, the sections were placed in
free-floating states into aG1C-1 that had been diluted (1:10,000) in
phosphate-buffered saline containing 0.3% (v/v) Triton X-100 and 1%
(w/v) bovine serum albumin and incubated for 3 days at 4 °C. After
incubation with biotinylated goat anti-rabbit IgG, staining was
accomplished using the avidin-biotin complex method (Vectastain Elite
kit, Vector Laboratories, Burlingame, CA), with nickel-diaminobenzidine
as the chromogen. Control procedures consisted of preabsorbing aG1C-1
(1:10,000 dilution) by incubating with a saturating concentration of
the antigenic peptide.
For double immunofluorescent staining, sections were first incubated
with aG1C-1 and a monoclonal antibody for each pituitary hormone,
i.e. anti-prolactin monoclonal antibody (QED Bioscience Inc., San Diego, CA), anti-growth hormone monoclonal antibody (Quartett, Berlin, Germany), anti-luteinizing hormone monoclonal antibody (Quartett), anti-adrenocorticotropic hormone (ACTH) monoclonal antibody (YLEM, Rome, Italy), or anti-thyrotropin (TSH) monoclonal antibody (Quartett). The sections were washed thoroughly and incubated with fluorescein isothiocyanate-labeled anti-rabbit IgG (E. Y. Laboratories, San Mateo, CA) and Texas Red-labeled anti-mouse IgG
(Protos ImmunoResearch, San Francisco, CA). These sections were
examined with a confocal microscope (MRC-1024, Bio-Rad Laboratories, Hertfordshire, U. K.).
Electron Microscopic Study--
For electron microscopy,
sections were processed according to the method described elsewhere
(18). TRH stimulation involved the injection of 10 µg of TRH into the
femoral vein 10 min before the perfusion, which was enough to stimulate
the thyrotroph in vivo (19). The vibratome sections were
dehydrated in a graded series of ethanol and incubated in a mixture of
ethanol and LR Gold resin (London Resin, Berkshire, U. K.) followed by
flat embedment in fresh LR Gold containing 0.1% (v/v) benzyl on
silicon-coated glass slides. The embedded sections were polymerized for
6 h in an ultraviolet cryochamber (Pelco, Ted Pella Inc., Redding,
CA) at Whole Cell Patch-Clamp Recordings--
Anterior pituitary glands
were dissected from adult female Wistar rats. Cells were dispersed
enzymatically using a non-trypsin dissociation protocol modified from
that used by Einhorn and Oxford (4). In brief, the glands were removed
and minced in sterile Hanks' balanced salt solution that was free of
calcium and magnesium (Hanks' CMF). After washing, the fragments were
incubated in a shaking water bath for 1 h in 37 °C in Hanks'
CMF containing trypsin inhibitor (Sigma, 0.1 mg/100 ml), collagenase
(Worthington, 0.3%), and DNase I (Sigma, 1.0 mg/100 ml). Fragments
were then mechanically dispersed by trituration with a siliconized
Pasteur pipette, washed with Dulbecco's modified Eagle's medium,
filtered through nylon mesh (20 µm), and harvested via
centrifugation. Cells were incubated in Dulbecco's modified Eagle's
medium containing 10% fetal bovine serum. Whole cell recordings were
made utilizing the gigaohm seal patch-clamp technique (20). Recordings
were made using a patch-clamp amplifier (EPC-7, List Electronics,
Darmstadt, Germany) and recorded on videocassette tapes with a PCM
converter system (RP-880, NF Electronic Circuit Design, Yokohama,
Japan), reproduced and low pass-filtered at 1 kHz ( Single-cell RT-PCR Analysis--
Cytoplasmic RNAs of a
dissociated single pituitary cell, which was aspirated and transferred
into a microcentrifuge tube with a glass tip used for patch-clamp
experiments, were extracted, and cDNAs were synthesized as
described elsewhere (21). To eliminate the contamination of genomic
DNAs, we treated RNA samples with DNase I (Takara) followed by cDNA
synthesis with reverse transcriptase (Life Technologies, Inc.). To
ascertain that cytoplasmic RNAs were correctly aspirated, samples were
conducted to RT-PCR of Cell Capacitance Measurement--
Simultaneous measurements of
changes in membrane capacitance (Cm), conductance
(Gm), and membrane current (Im)
were carried out using a patch-clamp amplifier (EPC-7, List
Electronics) and a two-phase lock-in amplifier (NF5610B, NF Electronic
Circuit Design) as described previously (24). The capacitance
transients originating from the capacitance of the plasma membranes
were minimized by adjusting the capacitance and the time constant of
the capacitance cancellation circuit in the patch-clamp amplifier while
applying voltage pulses with an amplitude of 10 mV. This adjustment
gave whole cell capacitance values of 9.0-13.4 picofarads and series
resistance values of 5-9 megaohms. After the cancellation, a 600-Hz,
3.4-mV peak to peak sine-wave voltage was superimposed on the holding
potential of KG channels are heteromultimeric proteins
composed of the subunits Kir3.1 + Kir3.4 in the heart (17) and Kir3.1 + Kir3.2 in the brain (25, 26). The Kir3.1 subunit may therefore be common to the KG channels of various tissues (8, 9). We examined the distribution of Kir3.1 immunoreactivity in sections of rat
pituitary using a specific polyclonal antibody (aG1C-1) (15, 16) (Fig.
1). The Kir3.1-positive cells were
scattered in the anterior pituitary lobe and made up ~3% of the
anterior pituitary cells (Fig. 1A). Under the light
microscope Kir3.1 immunoreactivity was detected diffusely in the
cytosol of each positive cell (Fig. 1, A and B).
The Kir3.1-positive cells had a polygonal or stellate shape between 20 and 30 µm in size. To identify which types of pituitary cells express
Kir3.1, sections were double-stained for Kir3.1 and various pituitary
hormones. Fig. 1, B and C, shows that Kir3.1
immunoreactivity (green) was detected only in the cells that
were stained with anti-TSH antibody (red). In 176 TSH-positive cells (i.e. thyrotrophs), 149 cells (~85%)
were also Kir3.1-positive. No Kir3.1 immunoreactivity was detected in
cells stained with anti-prolactin, anti-growth hormone,
anti-luteinizing hormone, or anti-ACTH antibodies. Prominent
orange-yellow signals generated by the double staining of
green (Kir3.1) and red (TSH) suggest that they
may be localized in close proximity in thyrotrophs (Fig. 1D).
The subcellular localization of Kir3.1 was examined using
immunoelectron microscopy (Fig. 1, E, F, and H).
The immunoreactive signals (gold particles) were detected predominantly
on secretory vesicles and rarely on the plasma membrane of
Kir3.1-positive pituitary cells (Fig. 1E). The cells were
~30 µm in diameter and polygonal in shape. The reactive
intracellular secretory vesicles were ~100 nm in diameter. These
morphological features of Kir3.1-positive cells are those of the
thyrotroph (27). Furthermore, with double immunolabeling under the
electron microscope, we found that Kir3.1 (detected by 15-nm gold
particles) and TSH (10-nm gold particles) were localized in the same
secretory vesicles (Fig. 1E, inset). In contrast,
the adjacent cell, which contained large 200-300-nm-diameter electron
dense vesicles, did not show any immunoreactivity to aG1C-1.
Fig. 1, F and H, depicts examples of Kir3.1
immunoreactivity in the thyrotrophs obtained from TRH-administered
rats. TRH stimulation caused an increase in the detection of gold
particles on the plasma membrane (Fig. 1F,
arrows). The number of gold particles detected on the plasma
membrane in the control was 0.25 ± 0.25% (mean ± S.E.,
n = 15 cells) of the total number of particles that
existed within 1.5 µm of the plasma membrane. This number increased
to 7.56 ± 1.80% (n = 15 cells) after TRH
stimulation (Fig. 1G). Furthermore, as indicated by the
arrowheads in Fig. 1H, after TRH stimulation various stages of fusion of the gold particle-positive vesicles with
the plasma membrane were detected. These results suggest that the
KG channels on the secretory vesicles of thyrotrophs could
be translocated to the plasma membrane during TRH-induced exocytosis.
It is known that dopamine-D2 and somatostatin receptors can
couple to KG channels via pertussis toxin-sensitive G
proteins in pituitary cells (2). Therefore, TRH-induced recruitment of
the KG channels to the plasma membrane of thyrotrophs may
in turn facilitate the effect of inhibitory transmitters such as dopamine and somatostatin. To examine this possibility,
electrophysiological techniques were applied to dissociated pituitary
cells (Fig. 2). From 82 cells examined,
we observed three distinct types of cells in terms of their responses
to bromocriptine, somatostatin, and TRH (Types I-III). Fig.
2A depicts a representative cell current record of the
response of Type I cells (n = 6). The dopamine receptor agonist bromocriptine induced a small inwardly rectifying
K+ (Kir) current, which was markedly enhanced upon the
addition of TRH to the bath. The enhanced bromocriptine-induced Kir
current was inhibited by sulpiride, an antagonist for the dopamine
receptor. After ~10 min of wash-out of bromocriptine and sulpiride,
somatostatin induced a small Kir current similar to that induced by
bromocriptine under control conditions. The somatostatin-induced Kir
current was also enhanced by the addition of TRH. In the six Type I
cells, TRH increased the bromocriptine-induced KG current
by 6.0 ± 0.5-fold (mean ± S.E.) and the
somatostatin-induced KG current by 7.0 ± 0.8-fold,
measured at the command pulse to Pharmacology II and
§ Obstetrics and Gynecology,
Department of Physiology,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
2-adrenergic,
serotonin,
-aminobutyric acid type B, opioid, and somatostatin
receptors in the brain, are coupled to KG channels (3). In
endocrine organs such as the anterior pituitary lobe and pancreatic
islet, it was also reported that some neurotransmitters including
dopamine and somatostatin hyperpolarize the membrane by activating
KG channels, which results in the inhibition of hormone
secretion (4-7).
EXPERIMENTAL PROCEDURES
20 °C. Small blocks containing anterior pituitary lobe were cut out and glued to black resin. Ultrathin sections were cut using an
ultramicrotome (Ultracut UCT, Leika, Vienna, Austria) and mounted on
collodion and carbon-coated nickel grids (thin bar grid, Nishin EM Co.
Ltd., Tokyo, Japan). The nickel grids with ultrathin sections were
incubated with 3% (v/v) normal goat serum in 0.1 M
phosphate-buffered saline containing 0.2% bovine serum albumin, 0.2%
(w/v) saponin, and 0.05% (w/v) NH4Cl for 30 min at room
temperature. The grids were then incubated with aG1C-1 (1:100) or
aG4N-10 (1:50) in the same buffer for 2 h and then with a 15-nm
immunogold conjugated goat anti-rabbit IgG (British BioCell
International Inc., Cardiff, U. K., diluted 1:40) for 1.5 h at
room temperature. In the case of double immunolabeling, the grids were
incubated with aG1C-1 (1:100) or aG4N-10 (1:50), and anti-TSH
monoclonal antibody (1:10) followed by incubation with
immunogold-conjugated anti-rabbit IgG (15-nm gold particles) and
immunogold-conjugated anti-mouse IgG (10 nm gold particles). The grids
were washed with 0.1 M phosphate-buffered saline and then
with distilled water before staining with uranyl acetate and Reynold's
lead citrate. These sections were examined with an electron microscope
(H7100TE, Hitachi, Hitachinaka, Japan).
3 db) by a filter
with Bessel characteristics (48 db/octave slope attenuation) for an
analysis and illustration. Experiments were performed at 22-24 °C
in a bath solution composed (in mM) of the
following: NaCl (91), KCl (50), MgCl2 (1),
glucose (5.5), and Hepes buffer (5), pH 7.2. The composition of the pipette filling solution was as follows (in mM): KCl (140),
MgCl2 (8), EGTA (0.1), CaCl2 (0.0415), GTP
(0.1), and Hepes (5), pH 7.2.
-actin by the
-actin PCR kit
(CLONTECH). We could detect the
-actin
transcripts in all of the Type I cells, 16 Type II cells, 45 Type III
cells, and 45 cells after capacitance measurement. Using the
-actin
detectable samples, nested PCR was done for Kir3.1, Kir3.2, and Kir3.4.
Primers for Kir3.1 PCR corresponded to nucleotides
10 to 10 and
760-781 (first PCR) and
10 to 10 and 736-757 (second PCR) of rat
Kir3.1 cDNA (8). Primers for Kir3.2 PCR corresponded to nucleotides
171-190 and 893-912 (first PCR) and 285-305 and 879-897 (second
PCR) of rat Kir3.2 cDNA (22). Primers for Kir3.4 PCR corresponded
to nucleotides 904-923 and 1276-1294 (first PCR) and 989-1006 and
1246-1265 (second PCR) of rat Kir3.4 cDNA (17). For TSH
PCR,
primers corresponded to nucleotides 101-121 and 378-398 of rat TSH
cDNA (23). PCR amplification was performed for 30 cycles at
94 °C for 45 s, at 58 °C (Kir3.1), 60 °C (Kir3.2),
55 °C (Kir3.4), or 56 °C (TSH
) for 1 min, and at 72 °C for
1 min and then at 72 °C for 8 min. Because the products from the
single pituitary cell were insufficient for visualization, second
cycles of reactions using the second PCR primers and the same
amplification conditions were performed with a part of the first PCR
products. The nucleotide sequence of the PCR products was confirmed
with the dye primer method and DNA sequencer (A-381, Perkin-Elmer)
after TA cloning (Invitrogen).
100 mV. The resulting current output was fed into the
lock-in amplifier. The phase offset of the lock-in amplifier was
adjusted so that when the capacitance of the cancellation circuit was
modified to calibrate Cm, there was no change in the
output for Gm (closed circles at the
Cm trace in Fig. 3A).
RESULTS AND DISCUSSION
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Fig. 1.
Kir3.1 channel immunoreactivity in the rat
anterior pituitary lobe. A, Kir3.1-positive cells were
scattered in the anterior pituitary lobe. PL, posterior
pituitary lobe; AL, anterior pituitary lobe. Scale
bar = 1 mm. B-D, immunofluorescent staining of
Kir3.1 (green, B) and TSH (red, C). Kir3.1
immunoreactivity was detected in the cytoplasm of the polygonal or
stellate cells. Both immunoreactivities were represented by
orange-yellow staining when the images were superimposed
(D). Scale bar = 70 µm. E-G,
ultrastructural localization of Kir3.1 immunoreactivity on the
secretory vesicles in the thyrotroph. E, Kir3.1
immunoreactivity was localized on the secretory vesicles of the
thyrotroph. On the plasma membrane, no signals were detected. Kir3.1
signals (15-nm gold particles) and TSH (10-nm gold particles) were
detected on the same vesicles (inset). Scale
bar = 0.5 µm (inset, 0.15 µm), which is common
in E and F. F, after TRH
administration, some sets of Kir3.1-positive secretory vesicles align
near the plasma membrane, and gold signals were detected on the plasma
membrane (arrows). G, the number of
Kir3.1-immunoreactive gold particles was counted on the thyrotrophs.
The number of gold particles on the plasma membrane, in the area within
0.3 µm adjacent to the plasma membrane, and in the area 0.3-1.5 µm
from the plasmalemma was counted under the electron microscope on 15 thyrotrophs of control and after TRH administration. The ratios of the
number of particles in each area divided by the total number of
particles in these three areas were represented in each column
(means ± S.E.). Closed bars, control; open
bars, TSH administered. **, p < 0.01. H, Kir3.1-positive secretory vesicles fuse with the plasma
membrane (arrowheads). Scale bar = 0.2 µm.
100 mV. TRH alone, in the absence of
bromocriptine or somatostatin, did not induce any appreciable Kir
current (data not shown). Both bromocriptine- and somatostatin-induced
currents reversed at approximately
30 mV close to the equilibrium
potential for K+ with 50 mM extracellular
K+ and exhibited clear inward rectification (Fig.
2A, lower right panel). The currents activated
slowly during hyperpolarizing command voltage pulses, which is a
feature of KG channels containing Kir3.1 subunits (Fig.
2A, lower left panels) (17, 25). In RT-PCR analysis of cytoplasmic RNA aspirated from the cell presented in Fig.
2A, we detected transcripts of Kir3.1 and TSH
as shown in
lane 1 of Fig. 2D. The same results were obtained
in the remaining five Type I cells (including the result shown in
lane 4 of Fig. 2D). Thus, Type I cells are
thyrotrophs that may possess KG channels containing the
Kir3.1 subunit.
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Fig. 2.
Three types of cell current responses of
dissociated pituitary cells to bromocriptine, somatostatin, and
TRH. The external solution contained 50 mM
K+. Command pulses of 500 ms in duration to 100 and +40
mV were applied from the holding potential of
30 mV. A,
perfusion of bromocriptine (10 nM) induced an inward
current without any effect on the outward current. Addition of TRH
(final concentration in the bath, 3 µM) drastically
augmented the inward current, which was almost completely inhibited by
the application of sulpiride (100 nM). After more than 10 min (double slashes) of wash-out, perfusion of somatostatin
(0.1 µM) evoked a small inward current, and TRH
application further enhanced this current. Lower left panel,
superimposed whole cell current traces evoked by bromocriptine (traces
2-1 and 3-1) or
somatostatin (5-4 and 6-4)
before (2-1 and 5-4) and
after TRH stimulation (3-1 and
6-4). Currents were recorded with voltage steps
from
120 to +40 mV in 20-mV steps. Arrowheads indicate a
current level of 0 in A-C. Bars in
upper and lower left panels are 1 nA for 1 min
and 100 pA for 500 ms, respectively. These scales are applicable also
to B and C. Lower right panel,
current-voltage relationships of bromocriptine- and
somatostatin-induced currents after TRH administration. Open
circles, bromocriptine-induced currents (trace
3-1). Closed circles,
somatostatin-induced currents (trace 6-4).
B, in a Type II cell, perfusion of bromocriptine induced a
small inward current, which was not affected by TRH. Lower
panels of B and C, superimposed whole cell
currents evoked by bromocriptine before (trace
2-1) and after TRH stimulation (trace
3-1) with the same voltage steps as in
A. C, in most of the dissociated pituitary cells
(Type III cells), dopamine and TRH did not affect the membrane current.
D, representative results of single-cell RT-PCR. The
cytosols were sucked through the patch pipettes after current
recording, and then RT-PCR was done. All of the Type I cells
(n = 6) expressed the Kir3.1 and TSH
transcripts
(lanes 1 and 4). Lane 1 exhibits the
transcripts obtained from the cell whose current record is shown in
A. Type II (lanes 2 and 5) and Type
III (lanes 3 and 6) cells did not express the
Kir3.1 transcripts, although a few cells expressed the TSH
mRNAs
(lane 2). Lanes 5 and 3 illustrate
transcripts obtained from the cells whose current recordings are shown
in B and C, respectively. Lane P, PCR
product of whole pituitary cDNA; lane C, PCR product of
each cDNA. E, Kir3.4 immunoreactivity (detected by 15-nm
gold particles) was also located on the TSH (10-nm
particles)-containing vesicles. Scale bar = 0.25 µm.
Because it is known that homomeric KG channels composed of Kir3.1 are not functional (17) but that heteromers of Kir3.1 and another Kir3.0 subunit form functional KG channels, we have tried to identify the Kir3.0 subunit that may assemble with Kir3.1 in the vesicular KG channels. Kir3.3 was not detected by RT-PCR in the mRNA from whole anterior pituitary lobe (data not shown). Therefore, the expression of Kir3.2 and Kir3.4 subunits was examined by single-cell RT-PCR. All six Type I cells expressed a Kir3.4 transcript, and one of them also expressed Kir3.2. Furthermore, we detected Kir3.4 immunoreactivity (15-nm gold particles) on the vesicles containing TSH (10-nm particles) with electron microscopic immunocytochemistry (Fig. 2E). Kir3.2 immunoreactivity was not detected on the vesicles (data not shown). These results suggest that Kir3.4 may be the subunit that makes up the vesicular KG channel with Kir3.1.
In Type II cells (n = 21), bromocriptine or
somatostatin induced a very small Kir current, which was not affected
by TRH (Fig. 2B). Lanes 2 and 5 in
Fig. 2D depict examples of RT-PCR analysis of Type II cell
mRNA; lane 2 is from the cell shown in Fig.
2B. In the cytoplasmic RNAs of 10 cells of this type, we
detected transcripts of Kir3.2 and/or Kir3.4 but not of Kir3.1. TSH
mRNA was detected only in the cell shown in lane 2 but
not in the remaining nine Type II cells. In Type III response cells
(n = 55), bromocriptine or somatostatin did not induce
any Kir current, and TRH showed no appreciable effect (Fig.
2C). We could not detect any transcripts of Kir3.1, Kir3.2,
or TSH
in 10 Type III cells examined (lane 3 in Fig.
2D is from the cell shown in Fig. 2C). Two of the
10 Type III cells expressed Kir3.4 mRNA. In conclusion, TRH
enhances bromocriptine or somatostatin activation of KG
current specifically in thyrotrophs, which express Kir3.1 mRNA.
To examine whether the TRH-induced augmentation of KG
channel activity in Type I thyrotrophs is derived from the exocytosis of secretory vesicles, as was suggested by the electron microscopic examination (Fig. 1, E-H), we simultaneously measured
Cm, Gm, and Im
in Type I cells (Fig. 3A). The
cells were bathed in 5 mM K+ bathing solution
and held at 100 mV. A 600-Hz sine wave with a peak to peak amplitude
of 3.4 mV was applied to the cell under voltage clamp. The application
of bromocriptine caused some increase of Gm and of
the small inward K+ current without any significant effect
on Cm. When TRH was added in the continued presence
of bromocriptine, the inward K+ current was markedly
enhanced in parallel with the simultaneous increase of both
Cm and Gm. The increase of Cm may reflect the fusion of secretory vesicles to
the plasma membrane induced by TRH (28). In the continued presence of
TRH, Cm, Gm, and
Im fluctuated in parallel and slowly returned toward
their basal levels. The decrease of Cm may represent
endocytosis of membrane containing KG channels. This
TRH-induced response was observed in 5 of 45 cells examined. In these 5 cells, expression of mRNAs of Kir3.1, Kir3.4, and TSH
was
confirmed by retrospective single-cell RT-PCR (data not shown). The
remaining 40 cells did not express any Kir3.1transcript. The
simultaneous measurement of Cm,
Gm, and Im indicates that
TRH-induced augmentation of KG channel activity in Type I
cells is associated with the exocytosis of secretory vesicles.
|
TRH-induced TSH secretion may be composed of voltage-dependent and voltage-independent components, as is the case for prolactin secretion by TRH in the lactotroph (29). The increase in Cm measured in Fig. 3A must represent a voltage-independent component because these experiments were conducted under voltage clamp. The TRH-induced voltage-independent fusion of secretory vesicles causes an increase in the number of KG channels on the plasma membrane. The increased KG channels should then facilitate the hyperpolarization induced by dopamine and somatostatin, which may suppress the depolarization-induced secretion. TRH-induced recruitment of vesicular KG channels to the plasma membrane may act as a feedback control mechanism to avoid excessive stimulation of the cell (Fig. 3B).
At the crustacean neuromuscular junction, repetitive stimulation of
motor nerves resulted in an increase of the number of active zones
containing a voltage-dependent Ca2+ channel at
the axonal terminus, which was implicated in the long term facilitation
of neurotransmitter release (30). The increase of active zones is
thought to be the result of the translocation to the plasma membrane of
a voltage-dependent Ca2+ channel localized on
secretory vesicles (31). Similarly, it was shown that the number of
postsynaptic -aminobutyric acid type A receptors was rapidly
increased by insulin (32) or in an experimental model of temporal lobe
epilepsy (33). The stimulation-induced increase of
-aminobutyric
acid type A receptors may underlie the long term modification of
synaptic transmission in hippocampal inhibitory synapses (32, 33).
These previous studies require a mechanism that enables stimulation to
translocate ion channels from an intracellular pool to the plasma
membrane. This study provides, for the first time, clear evidence to
indicate that the localization of ion channels on secretory vesicles
actually plays a role in the physiological regulation of membrane
excitability. Further studies are needed to elucidate the molecular
mechanisms localizing ion channels on secretory vesicles. This line of
study may provide novel insights into the stimulation control of
cellular excitability, including long term modification of synaptic transmission.
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ACKNOWLEDGEMENTS |
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We thank Dr. Ian Findlay (University of Tours, Tours, France) for critical reading of this manuscript. We also thank Kiyomi Okuto for technical assistance and Keiko Tsuji for secretarial support.
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FOOTNOTES |
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* This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan (to Y. K.), the "Research for the Future" Program of The Japan Society for the Promotion of Science, and the Human Frontier Science Program.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed. Tel.: 81-6-6879-3512; Fax: 81-6-6879-3519; E-mail: ykurachi{at}pharma2.med.osaka-u.ac.jp.
2 A. Inanobe and Y. Kurachi, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: KG, G protein-gated potassium channel; Kir, inwardly rectifying potassium channel; TSH, thyroid-stimulating hormone (thyrotropin); TRH, thyrotropin-releasing hormone; ACTH, adrenocorticotropic hormone; RT-PCR, reverse transcriptase-polymerase chain reaction; Cm, membrane capacitance; Gm, membrane conductance; Im, membrane current.
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REFERENCES |
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