1Department of Physiology, Cornell University, New York, New York 10021; 2Department of Neurology, University of California Los Angeles School of Medicine, Los Angeles, California 90095; 3Department of Pharmacology, The George Washington University, Washington, DC 20037; and 4Department of Psychiatry, University of California Los Angeles School of Medicine, Los Angeles, California 90095
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ABSTRACT |
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Piros, Elemer T.,
Rew C. Charles,
Lei Song,
Chris
J. Evans, and
Tim G. Hales.
Cloned -Opioid Receptors in GH3 Cells Inhibit
Spontaneous Ca2+ Oscillations and Prolactin Release Through
KIR Channel Activation.
J. Neurophysiol. 83: 2691-2698, 2000.
Opioid
receptors can couple to K+ and Ca2+ channels,
adenylyl cyclase, and phosphatidyl inositol turnover. Any of these
actions may be important in the regulation of neurotransmitter and
hormone release from excitable cells. GH3 cells exhibit
spontaneous oscillations of intracellular Ca2+
concentration ([Ca2+]i) and prolactin
release. Activation of cloned
-opioid receptors stably expressed in
GH3 cells inhibits both spontaneous Ca2+
signaling and basal prolactin release. The objective of this study was
to examine a possible role for K+ channels in these
processes using the patch-clamp technique, fluorescence imaging, and a
sensitive ELISA for prolactin. The selective
receptor agonist
[D-Pen2,
D-Pen2]enkephalin (DPDPE) inhibited
[Ca2+]i oscillations in GH3 cells
expressing both µ and
receptors (GH3MORDOR cells) but
had no effect on control GH3 cells or cells expressing µ receptors alone (GH3MOR cells). The inhibition of [Ca2+]i oscillations by DPDPE was unaffected
by thapsigargin pretreatment, suggesting that this effect is
independent of inositol 1,4,5-triphosphate-sensitive Ca2+ stores. DPDPE caused a concentration-dependent
inhibition of prolactin release from GH3MORDOR cells with
an IC50 of 4 nM. DPDPE increased inward K+
current recorded from GH3MORDOR cells but had no
significant effect on K+ currents recorded from control
GH3 cells or GH3MOR cells. The µ receptor
agonist morphine also had no effect on currents recorded from control
cells but activated inward K+ currents recorded from
GH3MOR and GH3MORDOR cells. Somatostatin activated inward currents recorded from all three cell lines. The
DPDPE-sensitive K+ current was inwardly rectifying and was
inhibited by Ba2+ but not TEA. DPDPE had no effect on
delayed rectifier-, Ca2+-, and voltage-activated or A-type
K+ currents, recorded from GH3MORDOR cells.
Ba2+ attenuated the inhibition of
[Ca2+]i and prolactin release by DPDPE,
whereas TEA had no effect, consistent with an involvement of
KIR channels in these actions of the opioid.
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INTRODUCTION |
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Opioids activate µ, , and
receptors that
couple through pertussis-toxin-sensitive G proteins to adenylyl cyclase
and ion channels (for review, see Piros et al. 1996a
).
Opioid receptors can also increase phosphatidyl inositol turnover
elevating inositol 1,4,5-triphosphate (IP3) levels and
subsequently releasing intracellular Ca2+
(Harrison et al. 1998
). In central neurons, opioid
receptor activation inhibits N- and P/Q-type Ca2+
channels and adenylyl cyclase, and activates inwardly rectifying K+ (KIR)
channels (Childers et al. 1992
; North and
Williams 1985
; Rhim and Miller 1994
;
Williams et al. 1988
; Wimpey and Chavkin 1991
). Any one of these actions can reduce neuronal
excitability and neurotransmitter release (see Boehm and Huck
1997
; Miller 1998
). Opioid receptors also couple
to multiple effectors in peripheral neurons and chromaffin cells
(Kleppisch et al. 1992
; Moises et al.
1994
; Schroeder et al. 1991
; Twitchell
and Rane 1994
). The activation of µ opioid receptors in
chromaffin cells inhibits Ca2+ channel activity and
increases the activity of Ca2+ and voltage-activated
K+ channels (KCa,V) (Twitchell and Rane
1994
). This raises the question: why are so many transduction
pathways required for opioid receptors to inhibit neurotransmitter and
hormone release? One possibility is that the combined regulation of all
of the effectors is required to effectively attenuate release. Several
reports suggest that this is not the case, instead the regulation of
neurotransmitter release by G-protein-coupled receptors can be
explained by the involvement of specific effectors. The identity of the
relevant effector varies according to the receptor and/or the
preparation being studied (Boehm and Huck 1997
;
Capogna et al. 1993
; Cherubini and North
1985
; Koyama et al. 1999
). An
alternative possibility is that specific effectors must be regulated to
inhibit release from cells receiving different stimuli. For example,
the activation of K+ channels may be sufficient to inhibit
spontaneous release, whereas the inhibition of adenylyl cyclase and
Ca2+ channels might be necessary to prevent release evoked
by specific stimuli.
In this study, we used a combination of electrophysiological,
fluorescence imaging ,and biochemical techniques to examine the
contribution of K+ channels to the opioid-induced
inhibition of spontaneous changes in intracellular Ca2+
concentration ([Ca2+]i) and
release. We required a homogeneous population of cells that both
expressed opioid receptors and released measurable quantities of a
neurotransmitter or hormone. Although there are opioid
receptor-expressing cell lines that have been studied extensively using
electrophysiological and Ca2+ imaging techniques
(Connor and Henderson 1996; Seward et al. 1991
) none of these is suitable for the study of spontaneous
release. We circumvented this problem by expressing cloned opioid
receptors in the prolactin-secreting rat pituitary GH3 cell
line (Piros et al. 1995
, 1996b
) and developed a
sensitive enzyme-linked immunosorbent assay (ELISA) for measuring
secreted prolactin levels (Charles et al. 1999
;
Piros et al. 1996a
). GH3 cells have been
extensively studied for several years; they have well characterized
K+ and Ca2+ channels and exhibit spontaneous
oscillations of intracellular Ca2+ concentration and
hormone release (Barros et al. 1992
; Bauer et al.
1990
, 1994
; Charles et al. 1999
; Dubinsky
and Oxford 1985
; Matteson and Armstrong 1986
;
Oxford and Wagoner 1989
). The inhibition of
KIR current stimulates the frequency of
[Ca2+]i oscillations and leads to an increase
in prolactin release (Charles et al. 1999
). Conversely,
inhibition of L-type Ca2+ channel activity inhibits
[Ca2+]i oscillations and prolactin secretion.
We stably transfected GH3 cells with µ receptor cDNA to
establish GH3 cells stably expressing rat µ receptors
(GH3MOR) (Piros et al. 1995
). This cell
clone was subsequently stably transfected with
receptor cDNA to
establish GH3 cells expressing both rat µ and mouse
receptors (GH3MORDOR) (Piros et al. 1996b
).
The activation of µ receptors expressed in either clone or
receptors expressed by GH3MORDOR cells, leads to the
inhibition of adenylyl cyclase and L-type Ca2+ channel
activity. Either or both of these actions may be involved in the
opioid-induced reduction in prolactin release from
GH3MORDOR cells.
The goal of this study was to use the whole cell recording technique to
examine whether opioid receptors, stably expressed in
GH3MOR and GH3MORDOR cells, couple to
K+ channels. We also sought to examine the contribution of
KIR channels in the receptor-mediated
inhibition of spontaneous [Ca2+]i
oscillations and prolactin release.
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METHODS |
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Cell culture
GH3 cells, obtained from the American Type
Culture Collection, Rockville, MD (CCL 82.1), were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% (vol/vol)
calf serum, penicillin (100 IU/ml), and streptomycin (100 µg/ml) and
incubated in a humid atmosphere of 5% CO2-95%
air, at 37°C. GH3MOR or
GH3MORDOR cells were grown under positive
selection using geneticin (G418) alone or G418 plus hygromycin B, as
described previously (Piros et al. 1995, 1996b
). Cells
were harvested once a week by treatment with a phosphate-buffered
saline containing EDTA (3 mM) and reseeded at 20% of their original
density, either into six-well plates for prolactin release assays,
35-mm-diameter culture dishes for electrophysiological studies, or
poly-D-lysine-coated coverslips for
Ca2+ imaging studies. The incubation medium was
changed every 2-3 days.
Electrophysiological recordings
Single GH3, GH3MOR,
or GH3MORDOR cells were voltage-clamped and
voltage-activated K+ channel activity was
recorded using a List EPC-7 patch-clamp amplifier (Cambell, CA). Cells
were superfused with a solution containing (in mM) 140 NaCl, 2.8 KCl, 2 MgCl2, 1 CaCl2, 10 HEPES, 6 glucose, and 5 × 104 TTX (pH 7.2 with
NaOH) when recording outward K+ currents. The
recording electrode contained a solution composed of (in mM) 120 KCl, 1 EGTA, 1 MgCl2, 3 Mg-ATP, and 10 HEPES (pH 7.2 with KOH). Currents were activated by step-depolarizations of membrane
potential from a holding potential of
80 mV for 100 ms every 10 s. Capacitance compensations were achieved using the patch-clamp
amplifier. Residual artifacts and leakage currents were nulled using a
P/4 subtraction.
Whole cell KIR current recordings were
performed at room temperature (20-22°C) using an extracellular
solution containing (in mM): KCl 140, MgCl2 4, CaCl2 1, HEPES 10, glucose 7, TTX 5 × 104 (pH 7.2 with KOH). Recording electrodes
contained a solution comprised of (in mM): KCl 140, EGTA 10, MgCl2 2, HEPES 10, Mg-ATP 3 (pH 7.2 with KOH).
Two protocols were used: Currents were evoked by a step
hyperpolarization from the
40 mV holding potential to
90 mV
(duration 1.5 s, frequency 0.03 Hz), no leak subtraction was
employed. Alternatively, [D-Pen2,
D-Pen2]enkephalin (DPDPE), morphine,
or somatostatin was bath applied to GH3,
GH3MOR, and GH3MORDOR cells
clamped at
60 mV. In some cells there was a gradual increase in the
amplitude of inward current even in the absence of agonist. To
compensate for this effect, a linear extrapolation was used to compare
control current amplitude to the current amplitude recorded in the
presence of an agonist. The current-voltage relationship of the DPDPE
activated KIR current was examined by
ramping the potential from
100 to 50 mV (1-s duration). Leak
subtraction was achieved by subtracting the control current recorded in
response to the ramp depolarization in the absence of DPDPE.
Patch electrodes were manufactured from thin-walled borosilicate glass pipettes (World Precision Instruments, New Haven, CT) using a Flaming/Brown P-87 micropipette puller (Sutter, Novato, CA). Whole cell currents recorded using the EPC-7 amplifier were low-pass filtered with an 8-pole filter at 1 kHz and digitized (Labmaster DMA, Axon Instruments, Burlingame, CA) at a frequency of 5 kHz onto the hard drive of a personal computer. Data were analyzed using pClamp software (Axon Instruments).
Measurement of [Ca2+]i
[Ca2+]i was
measured using a fluorescence imaging system that has previously been
described in detail (Charles et al. 1991). Briefly,
cells grown on poly-D-lysine-coated glass coverslips were
loaded with fura2 by incubation in 5 µM fura2-AM for 40 min. Cells
then were washed and maintained in normal medium for 30 min before
experimentation. Coverslips were excited with a mercury lamp through
340- and 380-nm band-pass filters, and fluorescence at 510 nm was
recorded through a ×10 or ×20 objective with a SIT camera to an
optical memory disk recorder. Images then were digitized and subjected
to background subtraction and shading correction, after which
[Ca2+]i was calculated on
a pixel-by-pixel basis, as previously described, by a frame grabber and
image analysis board (Data Translation). Dr. Michael Sanderson wrote
data-acquisition and -analysis software. Tracings in all figures are
based on fluorescence of a 4 × 4 pixel area located within each
cell body.
Experiments were carried out in Hanks' balanced salt solution with 10 mM HEPES buffer, pH 7.4 (HBSS/HEPES) at 20-22°C. Agents were applied in HBSS/HEPES by perfusion of the recording chamber the base of which was a coverslip supporting the cells.
ELISA for measuring prolactin
The competitive ELISA makes use of an antibody raised in rabbit
against rat prolactin. Both antisera (PRL-S-9) and standards (PRL-RP-3)
were provided by the National Institute of Diabetes and Digestive and
Kidney Diseases (NIDDK). GH3 cells (0.5-0.7 million cells per well) were seeded into six-well tissue-culture plates
48 h before experimentation. Release experiments were conducted at
37°C in a humidified atmosphere of 5% CO2-95%
air. Before each experiment, cells were washed gently with media (DMEM
with 20 mM HEPES and 0.1% BSA, pH 7.4 with NaOH). After washing,
aliquots of media (1 ml) were added to each well for 0.5 h at a
time so that release could be monitored before, during, and after
exposure to drugs. Prolactin (ng/ml/106 cells)
released in 0.5 h in the presence of drugs was expressed as a
percentage of release from the same cells during 0.5 h under control conditions. After incubation with the cells, each media aliquot
was centrifuged at 1000 g at 4°C to pellet cells the
supernatant was stored at 20°C or assayed directly to determine the
prolactin concentration. Each well of a 96-well Nunc-Immuno Maxisorp
Plate (Life Sciences, Denver, CO) was coated with prolactin by
incubation of 100 µl of 0.1 M NaHCO3, pH 9.5, containing 1 ng prolactin for 20-24 h at 4°C. Before the assay,
prolactin-coated plates were washed with assay buffer (AB) containing
0.5 M NaCl, 20 mM NaH2PO4, 0.05% Tween20, 0.5% BSA, pH to 7.4, and then incubated with AB for
0.5 h at room temperature to remove prolactin bound weakly to the
plate. After further washing with AB, undiluted samples (100 µl) or
prolactin standards (0.02-40 ng) dissolved in 100 µl media were
added to the wells, followed by the addition of 50 µl prolactin
antibody at a dilution of 1:40,000. After incubation for 2 h at
room temperature, bound antibody was detected using peroxidase-conjugated anti-rabbit antibody (Vector, Burlingame, CA)
with tetramethylbenzidine (GIBCO/BRL, Gaithersburg, MD) as substrate.
H2SO4 (1 N) terminated the
peroxidase reaction and a microplate reader (Molecular Devices)
measured absorbance at 450 nm. All samples were assayed in
quadruplicate from three separate determinations.
Data analysis
Data are expressed as means ± SE. Statistical significance
was established using Student's t-test. Comparisons between
multiple data sets were made using ANOVA followed by the post hoc
application of Student's t-test. Data points in the graph
of DPDPE concentration versus prolactin release were fitted using the
logistics equation
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Drugs and reagents
Pertussis toxin (Sigma, MO) was added to the culture media at 200 ng/ml and cells were incubated for 24 h before performing the prolactin assay. Parallel control experiments were performed on the same batch of cells. All tissue culture reagents, including geneticin (G418) and hygromycin, were obtained from GIBCO/BRL. Nimodipine (a gift from Miles Pharmaceuticals) was diluted fresh each day from a 10 mM stock solution in ethanol. K+ channel inhibitors 4-AP, apamin, charybdotoxin, and iberiotoxin from RBI (Natick, MA) were put into solution on the day of the experiment. [D-Pen2, D-Pen2]enkephalin (DPDPE), [D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAMGO), and somatostatin were obtained from Peninsula Laboratories (Belmont, CA). Morphine sulfate was a gift from NIDA Drug Supply Program. All other reagents were obtained from Sigma Chemical (St. Louis, MO). Agonists were diluted from frozen stocks on the day of experimentation.
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RESULTS |
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Opioid receptor activation inhibits spontaneous prolactin release
Previously we have determined that prolactin release from
GH3MOR and GH3MORDOR cells
but not GH3 cells can be modulated by opioid
ligands (Piros et al. 1996a). In this study, we examined the cellular mechanisms by which
-opioid receptors inhibit prolactin release from GH3MORDOR cells. The
receptor
selective agonist DPDPE (0.1 nM to 1 µM), inhibited prolactin release
from GH3MORDOR cells with an
IC50 of 3.8 nM (Fig.
1). The inhibition of prolactin release
by DPDPE (100 nM) was attenuated by pertussis toxin pretreatment (200 ng/ml, for 24 h), demonstrating the involvement of inhibitory (Gi
and/or Go) G proteins (Fig. 1).
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We previously have observed that the activation of µ or receptors inhibits Ca2+ channel and adenylyl
cyclase activity recorded from GH3MORDOR cells.
Both of these pertussis-toxin-sensitive effects could be involved in
the opioid-induced inhibition of prolactin release (Piros et al.
1996b
). K+ channel activation is
thought to be important for the inhibitory actions of opioids on
neurotransmitter release from peripheral neurons (Cherubini and
North 1985
). However, µ-opioid-induced presynaptic inhibition
of GABA release from hippocampal neurons appears to be independent of
K+ channel activity (Capogna et al.
1993
). We previously demonstrated that in
GH3 cells blockade of
KIR channels, and
K(Ca,V) channels increased the
frequency and amplitude of
[Ca2+]i oscillations,
respectively. Only the former caused enhanced prolactin release
(Charles et al. 1999
).
receptor activation inhibits spontaneous
[Ca2+]i oscillations
Bath application of DPDPE (10 nM) reversibly abolished spontaneous Ca2+ oscillations and decreased baseline [Ca2+]i in the majority of GH3MORDOR cells (122/189 cells in 6 experiments, Fig. 2). In some cells, it reduced the frequency of Ca2+ oscillations without abolishing them altogether (42/189 cells), whereas in a smaller percentage of cells (25/189), it had no effect. DPDPE (10-100 nM) had no effect on spontaneous Ca2+ oscillations in untransfected GH3 cells (n = 3 coverslips) or GH3MOR cells expressing µ receptors alone (n = 3 coverslips). The inhibitory effect of DPDPE on GH3MORDOR cells was blocked by pretreatment of cells with pertussis toxin (200 ng/ml, for 24 h, n = 100 cells in 3 experiments, data not shown) as well as by the opioid receptor antagonist naloxone (1 µM, n = 90 cells in 3 experiments, data not shown). The ability of DPDPE to inhibit [Ca2+]i oscillations was unaffected by pretreatment with 10 µM thapsigargin (n = 60 cells in 3 experiments, Fig. 2B), a compound that dumps Ca2+ from IP3-sensitive intracellular stores, indicating that the effects of DPDPE are not mediated through modulation of Ca2+release.
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[Ca2+]i oscillations in
GH3 cells involve the activity of
K+ and L-type Ca2+ channels
(Charles et al. 1999). Regulation of either
K+ or L-type Ca2+ channels
by
receptors therefore could lead to the observed changes in
[Ca2+]i and subsequent
reduction in prolactin release. We have shown previously that
activation of
receptors inhibits Ca2+ channel
activity recorded from GH3MORDOR cells
(Piros et al. 1996b
). Here we examined whether the
activation of
receptors in GH3MORDOR cells
also modulates K+ channel activity and, if so,
whether this effect is involved in the opioid regulation of
[Ca2+]i and prolactin release.
Regulation of K+ channel activity in GH3MOR and GH3MORDOR cells by opioids
Depolarization of GH3MORDOR cells from 80
to 20 mV activates outward voltage-activated currents (Fig.
3). Such currents were mediated by the
outward flow of K+, and they were abolished when
CsCl replaced KCl in the electrode solution and were inhibited by
K+ channel antagonists (e.g., Fig.
3A). Under similar recording conditions, several studies
have provided evidence for the presence of K+
channels that mediate delayed rectifier-, Ca2+-,
and voltage-activated- and A current in GH3 cells
(Dubinsky and Oxford 1985
; Oxford and Wagoner
1989
; Ritchie 1987
; Simasko 1991
). We have demonstrated previously that
Ba2+ and TEA inhibit K+
currents activated in GH3 cells in response to
depolarizing pulses from
80 to 20 mV. Bath application of TEA
preferentially inhibited a sustained K+ current
component that is likely to be mediated by delayed rectifying K+ channels (Charles et al. 1999
).
By contrast, 5 mM 4-aminopyridine (4-AP) reduced a fast activating
current leaving a slowly rising component (Fig. 3A)
reminiscent of current through the delayed rectifier (Rudy
1988
). By subtracting the current in the presence of 4-AP from
the control recording, the amplitude of the A current component
recorded from GH3 cells was observed. The
4-AP-sensitive current constituted 56 ± 4% (n = 4) of the peak K+ current activated by
depolarizing from
80 to 20 mV (Fig. 3B). The inhibition of
transient K+ current induced by 4-AP was measured
by comparing the mean current amplitudes averaged between 5 and 10 ms
after depolarizing to 20 mV in the presence and absence of the drug.
Apamin (100 nM) and iberiotoxin (100 nM), inhibitors of small
(Blatz and Magelby 1986
) and large (Giangiacomo
et al. 1992
) conductance
Ca2+-activated K+ channels,
caused significant parallel inhibitions of the outward K+ current amplitude of 2.7 ± 0.6 and
21 ± 6% (n = 4). The effects of these agents
were averaged over 10 ms at the end of each depolarizing step and were
determined to be significant using the paired t-test when
compared with the current in the absence of drug application (P < 0.05). The effects of the
K+ channel inhibitors reversed during washout.
Taken together with our previous observations (Charles et al.
1999
), these data demonstrate that outward currents recorded
from GH3 cells are mediated by several classes of
K+ channels. DPDPE (1 µM) had no discernible
effect on outward K+ currents (n = 10), suggesting that activation of
receptors does not effect
K+ channels mediating delayed rectifier,
K(Ca,V), or A current recorded from
GH3MORDOR cells (Fig. 3B).
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An inwardly rectifying K+ current component
can also be recorded from GH3 cells under
appropriate conditions (Bauer et al. 1990;
Charles et al. 1999
). Hyperpolarizing
GH3 cells from
40 to
90 mV with equal
K+ concentrations on either side of the cell
membrane reveals inward currents. Such currents are inhibited by
Cs+ (1 mM) and Ba2+ (1 mM)
but are insensitive to 1 mM TEA (Charles et al. 1999
). The bath application of DPDPE (1 µM) increased the amplitude of currents (by 31 ± 4%, n = 6) observed by
hyperpolarizing GH3MORDOR cells from
40 to
90
mV, suggesting that opioid receptors couple to
KIR channels (Fig.
4A). This suggestion is
supported by the demonstration that currents recorded in the presence
of DPDPE are inhibited by bath application of 1 mM
Ba2+ (Fig. 4B). To test whether the
DPDPE-induced current was truly inwardly rectifying, we examined the
relationship between current amplitude and holding potential using
voltage ramps from
100 to 50 mV (Fig. 4C). Currents
generated by the voltage ramp under control conditions were subtracted
from those observed in the presence of DPDPE. In an attempt to restrict
the actions of the opioid to K+ channel
activation, we performed experiments in the presence of the
dihydropyridine nimodipine (100 µM) at a sufficient concentration to
abolish Ca2+ channel activity in
GH3 cells (Piros et al. 1995
).
Subtraction of ramp currents recorded in the presence of nimodipine
alone from those recorded with both nimodipine and DPDPE (100 nM)
revealed inwardly rectifying currents (n = 5) that
reversed in sign at the K+ equilibrium potential
of 0 mV (Fig. 4C).
|
To confirm that DPDPE was selectively activating receptors in
GH3MORDOR cells, we tested the sensitivity of
control GH3 cells and
GH3MOR cells expressing µ receptors alone.
Cells were held at
60 mV while DPDPE, somatostatin (an agonist of
somatostatin receptors native to GH3 cells), or
morphine (a µ receptor agonist) were bath applied (Fig.
5). Clearly discernible inward currents (87 ± 11 pA, n = 9) recorded from control
GH3 cells developed in the presence of
somatostatin (1 µM). No increase in the amplitudes of inward currents
were observed on application of morphine (1 µM) or DPDPE (100 nM) to
control GH3 cells (n = 5 and 8, respectively). By contrast, DPDPE (100 nM) activated robust currents
(168 ± 23 pA, n = 16) recorded from
GH3MORDOR cells (Fig. 5) but had no significant
effect on currents recorded from GH3MOR cells
(n = 11, Fig. 5). Both somatostatin (1 µM) and
morphine (1 µM) activated currents recorded from
GH3MOR cells (169 ± 25 pA,
n = 13 and 197 ± 46 pA, n = 12, respectively). The more selective µ receptor agonist DAMGO (1 µM)
also activated inward currents recorded from GH3MOR and GH3MORDOR cells
(data not shown).
|
DPDPE inhibits [Ca2+]i oscillations and prolactin release by activating KIR channels
We exploited the selective K+ channel
blocking actions of extracellular Ba2+ and TEA to
examine whether DPDPE induces inhibition of
[Ca2+]i oscillations and
prolactin release through activation of
KIR current. The inhibition of
[Ca2+]i oscillations by
DPDPE (10-100 nM) was attenuated by the preapplication of 1 mM
Ba2+ (n = 60 cells in 3 experiments, Fig. 6A) but was
unaffected by 1 mM TEA (n = 75 cells in 3 experiments,
Fig. 6B). Likewise, the DPDPE (1 µM)-evoked inhibition of
prolactin release was inhibited by coapplication with
Ba2+ (1 mM) or Cs+ (1 mM),
another ion capable of inhibiting KIR channel
activity (Fig. 6C). The increase in prolactin release in the
presence of Ba2+ and DPDPE was not significantly
different from that observed when Ba2+ was
applied alone to GH3 cells (Charles et al.
1999), confirming that DPDPE has no discernible effect on
prolactin release when KIR channels
are inhibited (Fig. 6C). There was also no significant difference between the change in prolactin release when
Cs+ (1 mM) was applied alone to
GH3 cells (Charles et al. 1999
) or coapplied with DPDPE (1 µM; Fig. 6C). TEA had no
significant effect on the inhibition of prolactin release by DPDPE.
DPDPE (1 µM) inhibited prolactin levels by 29 ± 7%
(n = 4) and 39 ± 4% (n = 11) in
the presence and absence of TEA (1 mM). TEA (1 mM) applied alone to
GH3 cells had no effect on prolactin release
(Charles et al. 1999
).
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Taken together, these data suggest that activation of KIR channels by DPDPE reduces spontaneous [Ca2+]i oscillations and prolactin release.
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DISCUSSION |
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We previously established excitable, prolactin-secreting anterior
pituitary-derived GH3 cell lines stably
expressing either µ receptors alone (GH3MOR
cells) or µ and receptors (GH3MORDOR cells)
together (Piros et al. 1995
, 1996b
). Here we demonstrate that opioid receptors in GH3MORDOR cells activate
inwardly rectifying K+ channels leading to a
reduction in spontaneous
[Ca2+]i oscillations and
prolactin release.
GH3 cells were chosen as the system for
expression of cloned opioid receptors as they contain a variety of
well-characterized G proteins and several effectors regulated by native
somatostatin and muscarinic receptors (Hescheler and Schultz
1993). Unlike unexcitable cell lines more commonly used to
study cloned opioid receptors, GH3 cells also
express a range of voltage-activated ion channels as well as adenylyl
cyclase. Furthermore these cells exhibit spontaneous oscillations of
[Ca2+]i and prolactin
release. Therefore opioid receptor properties, from ligand binding to
regulation of hormone release, can be studied using this single clonal
cell system (Piros et al. 1996a
).
The activation of either µ or receptors expressed in
GH3 cells leads to the inhibition of the
activities of adenylyl cyclase and dihydropyridine-sensitive
Ca2+ channels (Piros et al. 1995
,
1996b
). Reductions in the intracellular concentration of cAMP
and Ca2+ entry may contribute to the
opioid-induced reduction in prolactin release. Indeed the inhibition of
Ca2+ channel activity by nimodipine caused both a
reduction in spontaneous [Ca2+]i oscillations and
prolactin release, suggesting that the entry of
Ca2+ through L-type channels plays an important
part in these processes in GH3 cells
(Charles et al. 1999
).
In the present study DPDPE caused a marked reduction in spontaneous
[Ca2+]i oscillations in
GH3MORDOR but not GH3MOR or
control GH3 cells, demonstrating that this effect
required receptor activation. We examined the possibility that
receptors could be regulating [Ca2+]i through
modulation of IP3-sensitive intracellular
Ca2+ stores, a phenomenon that has been observed
in NG108-15 cells (Jin et al. 1994
). This pathway does
not appear to be important in the actions of
receptors in
GH3MORDOR cells because the DPDPE-induced reduction in [Ca2+]i
oscillations persisted after application of thapsigargin. The application of thapsigargin had little effect on spontaneous
Ca2+ oscillations, suggesting that
IP3-sensitive intracellular
Ca2+ stores play only a minor role in the control
of this process in GH3 cells (Charles et
al. 1999
).
The activation of receptors increased the amplitude of a
Ba2+-sensitive inward rectifying
K+ current recorded from
GH3MORDOR cells. Consistent with the idea that
this effect is mediated specifically by
receptors, DPDPE did not
activate currents in GH3 or
GH3MOR cells. GH3 cells
express endogenous somatostatin receptors that are known to couple to KIR channels. Somatostatin activated
inward currents in all three cell lines, whereas the µ receptor
agonists morphine and DAMGO activated resolvable currents only in
GH3MOR and GH3MORDOR cells. This action of the opioids appears to be specific to
KIR channels as DPDPE did not regulate
outward K+ currents mediated by TEA-, 4-AP-,
apamin-, charybdotoxin-, or iberiotoxin-sensitive channels. TEA,
Ba2+, and Cs+ have been
used previously to examine the roles of
K(Ca,V) and KIR channels in spontaneous
[Ca2+]i oscillations and
prolactin release from GH3 cells (Charles et al. 1999
). TEA (1 mM) selectively inhibits outward
K+ current, Cs+ (1 mM)
selectively inhibits inward currents, whereas
Ba2+ (1 mM) inhibits both
K+ current components. The application of
Ba2+ but not TEA increased the frequency of
Ca2+ oscillations, an action that
is accompanied by an increase in prolactin release (Charles et
al. 1999
). In the present study, we demonstrated that,
consistent with an effect primarily involving KIR, the DPDPE-mediated inhibitions of
Ca2+ signaling and prolactin release were
attenuated by Ba2+ but not by TEA.
Opioid receptors are known to couple to
KIR channels in a variety of neurons
(Grudt and Williams 1993; North and Williams 1985
; Wimpey and Chavkin 1991
), an effect that
can be mimicked by expressing opioid receptors with G-protein-coupled
inwardly rectifying K+ channels (GIRK) in
Xenopus oocytes (Chen and Yu 1994
;
Henry et al. 1995
; Ikeda et al. 1995
;
Ma et al. 1995
). This action appears to be mediated by
activation of inhibitory G proteins liberating
subunits that
bind directly to an amino acid motif found on GIRK channels and several
other effectors regulated by G-protein-coupled receptors (Ford
et al. 1998
). The molecular identity of the G-protein-regulated KIR channel in
GH3 cells remains unknown (Falk et al.
1995
). Early reports implicate G-protein
subunits in the
activation of KIR channels in
GH3 cells (Codina et al. 1987
;
Yatani et al. 1988
). Whether G-protein
subunits are
involved in the regulation of KIR
channels by recombinant opioid receptors expressed in
GH3 cells remains to be examined.
In summary, taken together the results of this study indicate that on
activation cloned opioid receptors stimulate
KIR channels native to
GH3 cells preventing depolarization and the
influx of Ca2+ through voltage-activated
Ca2+ channels thus reducing spontaneous
Ca2+ oscillations and prolactin release. Our
findings with this model system may be more widely applicable to other
neuronal and endocrine cells at times of tonic neurotransmitter or
hormone release when opioid-receptor-mediated activation of
KIR channels can prevent spontaneous
depolarization. We speculate that the other actions mediated by opioid
receptors in GH3MOR and
GH3MORDOR cells, inhibition of L-type
Ca2+ channel activity and adenylyl cyclase
(Piros et al. 1995, 1996b
), may play a role when hormone
release is stimulated by exogenous signals causing depolarization and
increased intracellular cAMP levels, respectively. Multiple effector
mechanisms may enable opioid receptor agonists to inhibit either basal
or evoked Ca2+ signaling and hormone release.
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ACKNOWLEDGMENTS |
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This work was supported by National Institutes of Health Grants NS-32283 and NS-02808 (A. C. Charles), DA-05010 (T. G. Hales and C. J. Evans), and DA-05627 (E. T. Piros).
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FOOTNOTES |
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Address for reprint requests: T. G. Hales, Dept. of Pharmacology, The George Washington University, 2300 Eye St. NW, Washington, DC 20037.
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.
Received 3 December 1999; accepted in final form 7 February 2000.
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REFERENCES |
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