Expression of dopamine D2 receptor in PC-12
cells and regulation of membrane conductances by
dopamine
Wylie H.
Zhu,
Laura
Conforti, and
David E.
Millhorn
Department of Molecular and Cellular Physiology, College of
Medicine, University of Cincinnati, Cincinnati, Ohio 45267-0576
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ABSTRACT |
PC-12 cells depolarize during hypoxia and release dopamine. The
hypoxia-induced depolarization is due to inhibition of an O2-sensitive K+ current. The role of dopamine
released during hypoxia is uncertain, but it could act as an autocrine
to modulate membrane conductance during hypoxia. The current study was
undertaken to investigate this possibility. Reverse
transcription-polymerase chain reaction and sequence analysis revealed
that the D2 isoform of the dopamine receptor is expressed
in rat PC-12 cells. Exogenously applied dopamine and the D2
agonist quinpirole elicited inhibition of a voltage-dependent
K+ current (IK) that was prevented by
sulpiride, a D2 receptor antagonist. Dopamine and
quinpirole applied during hypoxia potentiated the inhibitory effect of
hypoxia on IK. We also found that quinpirole caused
reversible inhibition of a voltage-dependent Ca2+ current
(ICa) and attenuation of the increase in
intracellular free Ca2+ during hypoxia. Our results
indicate that dopamine released from PC-12 cells during hypoxia acts
via a D2 receptor to "autoregulate" IK and ICa.
reverse transcription-polymerase chain reaction; fura 2; potassium
current; calcium current; hypoxia
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INTRODUCTION |
THE RAT PHEOCHROMOCYTOMA (PC-12) clonal cell
line has been used by our laboratory as a model system to
study the cellular and molecular mechanisms that mediate O2
chemosensitivity. We reported previously that reduced O2
tension (i.e., hypoxia) caused inhibition of an
O2-sensitive K+ current
[IK(O2)], membrane
depolarization, and an increase in cytosolic Ca2+ in PC-12
cells (39). In addition, our laboratory reported that the gene for
tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of
dopamine, is stimulated in PC-12 cells during hypoxia (10). Although
the synthesis and release of dopamine from PC-12 cells is tightly
coupled to O2 tension, the actual role of the released
dopamine remains unknown. However, it was reported recently that
dopamine released from the O2-chemosensitive (type I) cells
of the mammalian carotid body, which are morphologically and
functionally similar to PC-12 cells, acts to autoregulate a
voltage-dependent Ca2+ channel (2). The present study was
undertaken to determine whether dopamine receptors are expressed in
PC-12 cells and whether activation of these receptors modulates
membrane currents during hypoxia. We report here that the
D2 receptor is expressed in rat PC-12 cells and
that stimulation of this receptor leads to inhibition of a
voltage-dependent K+ current (IK) and a
voltage-dependent Ca2+ current (ICa).
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MATERIALS AND METHODS |
Cell culture.
PC-12 cells were obtained from the American Type Culture
Collection (Rockville, MD) and were prepared as described previously (39). Cells were maintained with Dulbecco's modified Eagle's medium/Ham's F-12 (DMEM/F-12) containing 15 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid
(HEPES), L-glutamine, 10% fetal bovine serum, and
penicillin-streptomycin (100 U/ml and 100 µg/ml, respectively) and
were grown at 37°C with 95% air-5% CO2. They were
passed once each week and plated at low density on sterilized 12-mm
round glass coverslips. The electrophysiological experiments were
performed 1-3 days after plating.
Reverse transcription-polymerase chain reaction.
Total cellular RNA was isolated from PC-12 cells and from rat whole
brain using TRI-REAGENT (Molecular Research Center, Cincinnati, OH).
Reverse transcription-polymerase chain reaction (RT-PCR) was performed
using a GeneAmp RNA PCR kit that was purchased from Perkin-Elmer Cetus
(Norwalk, CT). In these experiments, 3 µg of total RNA were reverse
transcribed with 2.5 µM oligo(dT) (16-mer) primer, 1 mM dNTPs, 1 U/µl ribonuclease inhibitor, and 2.5 U/µl murine leukemia virus
reverse transcriptase for 15 min at 42°C. PCR amplification of the
D2 message was performed as follows. The primers were
designed to span a portion of the 3' coding region as well as part of
the noncoding region of the D2 receptor sequence (GenBank
accession number M36831; Ref. 6). The forward primer was 5'(1308) ACC
CCA TCA TCT ACA CCA CCT T 3'; the backward primer was 5' TGG CGT GTT
CCC TGC TTT 3'(1823). PCR was carried out for 35 cycles for 1 min at
95°C, for 1 min 30 s at 57°C, and for 1 min 30 s at 72°C. The
amplification product was a 533-base pair fragment. This primer set did
not discriminate between the long and short forms of the D2
receptor sequence (14, 20). The PCR product was sequenced using an
automated DNA sequencer.
Electrophysiology.
Detailed protocols were described previously (39). Briefly, whole cell
voltage-clamp recordings were performed in conventional patch-clamp
mode. Patch pipettes had resistances of 3-5 M
(when filled with
the internal solution) and were made with a Mecanex three-stage puller
using borosilicate glass (World Precision Instruments, Sarasota, FL).
Recordings were performed using an Axopatch 200A amplifier (Axon
Instruments, Foster City, CA). Whole cell IK were elicited with step depolarization pulses to +50 mV from
70 mV holding potential (HP). The current signals were filtered with a
four-pole Bessel filter at 1 kHz and sampled at 5 kHz. To study ICa, Ba2+ was used as the charge
carrier and currents were recorded during steps to +20 mV for 160 ms
from a HP of
80 mV. The voltage pulses were given at 0.1 Hz. All
traces were leak subtracted using currents elicited by small
hyperpolarizing pulses, filtered at 1 kHz, and sampled at 10 kHz. The
digitized signals were analyzed on a personal computer using the pCLAMP
5.5.1 analysis programs. All experiments were carried out at room
temperature (25°C). Statistical analyses were performed using
Student's t-test. A value of P
0.05 was considered
statistically significant.
Measurement of intracellular free Ca2+.
Intracellular free Ca2+ was measured using the fluorescence
indicator fura 2 and a Ca2+-imaging system (Intracellular
Imaging, Cincinnati, OH). The cells were plated on a 12-mm round cover
glass and loaded with fura 2 (5 µM) for 30 min. The postload
incubation was 15 min before each experiment. Cells were perfused with
serum-free culture medium (DMEM/F-12) in a homemade chamber that was
mounted on the stage of a Nikon TMS microscope. Light from a 300-W
xenon arc illuminator passed through a computer-controlled filter
changer and shutter unit that contained 340- and 380-nm filters. Light
from selected cells was collected by an integrating charge-coupled
device video camera. The ratio of light intensity at the two
wavelengths was calculated on-line and stored on the computer. All
experiments were performed at 37°C.
Experimental solutions and drugs.
The composition of the standard external solution was as follows (in
mM): 140 NaCl, 2.8 KCl, 2 CaCl2, 2 MgCl2, 10 HEPES, and 10 glucose. The pH was adjusted to 7.4 with NaOH. The
pipette solution consisted of (in mM) 140 potassium gluconate, 1 CaCl2, 11 ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), 2 MgCl2 , 0.2 NaGTP, 3 Na2 ATP,
and 10 HEPES. The pH of the pipette medium was adjusted to 7.2 with
KOH. For recording of ICa, the
external solution contained (in mM) 113 N-methyl-D-glucamine glutamate, 20 BaCl2, 2 MgCl2, 2.8 CsCl, 10 glucose, and 10 HEPES, pH adjusted to 7.4 with tris(hydroxymethyl)aminomethane base. The pipette solution contained (in mM) 140 cesium gluconate, 2 MgCl2, 1 CaCl2, 10 EGTA, 10 HEPES, 3 Na2 ATP, and 0.2 Na GTP. The pH was adjusted to 7.2 with
CsOH. Dopamine was dissolved in external recording medium and was made
fresh for every experiment. Quinpirole and sulpiride were dissolved in
distilled water as stock solutions (10 mM), and aliquots were stored at
80°C until use. Experiments involving dopaminergic agents were
performed in a dark environment to minimize light-induced degradation
of these drugs.
Hypoxia was achieved by equilibrating the perfusion medium with 10%
O2 (balanced with N2). In some experiments, a
PO2 of 0 mmHg was achieved with 1 mM sodium
hydrosulfite in addition to saturating the medium with 100%
N2. The PO2 in the medium was measured with an O2 electrode connected to a polarographic
amplifier as described previously (39).
Dopamine, protein kinase A inhibitor fragment 6-22 amide (PKI),
staurosporine, and tetraethylamonium chloride (TEA-Cl) were obtained from Sigma (St. Louis, MO). (
)-Quinpirole,
(
)-sulpiride, and pertussis toxin (PTX) were purchased from Research
Biochemicals International (Natick, MA).
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RESULTS |
Dopamine D2 receptor modulation of IK in PC-12
cells.
RT-PCR was used to determine whether PC-12 cells derived from rat
pheochromocytomas express the D2 dopamine receptor. RNA extracted from rat brain was used as a positive control. Total RNA
extracted from PC-12 cells and used for PCR without RT served as a
negative control. PCR primers were designed to amplify the 3' region of
the cDNA that is distal to the alternative splicing site involved in
the short and long forms of the D2 receptor (14, 20). The
PCR amplification yielded a single band of 533 bases (Fig.
1). Sequence analysis of this
PCR product showed an ~100% homology with the rat D2
receptor (6, 20). We did not attempt to study the D2L or
D2S forms of the D2 receptor, since it is suggested that they tend to coexpress and have similar pharmacological functions (14, 20, 21, 24, 37). We also performed RT-PCR studies to
examine the expression of D3 and D4 dopamine
receptors in PC-12 cells. Our study was successful in obtaining a
positive signal from a D3 clone (27), but we were unable to
detect any expression from PC-12 cells. This suggests that
D3 expression may be very low or not present. In addition,
we did not detect D4 receptor mRNA.

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Fig. 1.
Dopamine D2 receptor expression in PC-12 cells. Reverse
transcription-polymerase chain reaction (RT-PCR) study of
D2 receptor gene expression in PC-12 cells and rat brain.
Rat whole brain RNA was used as a positive control (+). For negative
control ( ), PC-12 cell total RNA was used to undergo PCR without RT.
Both brain and PC-12 cells expressed D2 receptor gene
[533-base pair (bp) fragment]. M, DNA size marker.
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High-performance liquid chromatography (HPLC) studies in our laboratory
suggested that prolonged exposure to hypoxia (5% O2, 1-3 h) increased dopamine secretion from PC-12 cells (measured dopamine concentration in the culture media during normoxia,
2.4 ± 0.1 µg/ml; dopamine concentration during hypoxia,
4.7 ± 0.5 µg/ml, n = 5). To determine whether dopamine
mediates a cellular response through the D2 receptor, we
studied the effect of exogenously applied dopamine on ionic
conductances. Figure 2A shows the
effect of dopamine on IK. Depolarizing voltage
pulses (to +50 mV from
70 mV holding potential, delivered every 10 s) elicited a fast-activating, slow-inactivating outward
IK that was shown previously to be inhibited by
reduced O2 tension (39). Dopamine (100 µM) applied
extracellularly led to inhibition of IK within
30-60 s of its application [the concentration of dopamine used in
our study was determined based on the reported effective range of
dopamine concentration (7), as well as on our HPLC results]. A similar
time course for recovery of the IK inhibition was
measured during the washout of dopamine. Steady-state current
amplitudes were measured during the last 100 ms of the depolarizing
voltage pulses, and the average percentage of current inhibition by
dopamine in these experiments was 18 ± 2% (means ± SE,
n = 10). In a separate set of experiments, we found that
sulpiride (20 µM), a D2 antagonist, blocked the
inhibitory effect of dopamine on IK (Fig.
2B). Quinpirole (10 µM), a D2 agonist, caused
inhibition of IK in a manner similar to that
measured with dopamine (Fig. 2C). The dose effect of
quinpirole on IK is shown in Fig.
3. To avoid possible complications
involving receptor desensitization, individual cells were exposed to
only a single dose of quinpirole. The 10 µM dose of quinpirole
elicited the maximal inhibitory effect on IK (Fig.
3A). Therefore, this dose was used in our subsequent
experiments. To confirm that the effect of this concentration of
quinpirole was due solely to the stimulation of D2
receptors, experiments were performed in the presence of the
D2 antagonist sulpiride (Fig. 3B, n = 4). We
found that sulpiride (10 µM) alone had no effect on
IK but blocked the inhibitory effect of quinpirole
on IK. Taken together, these data show that
dopamine causes inhibition of IK in PC-12 cells and
that this response is mediated by the D2 receptor.

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Fig. 2.
Dopamine inhibits voltage-dependent K+ current
(IK) through D2 mechanism. Whole cell
voltage-clamp recordings of IK were performed at a
depolarizing potential of +50 mV from a holding potential of 70 mV.
Depolarizing voltage pulses lasted 800 ms and were delivered at 0.1 Hz.
A: dopamine (DA, 100 µM) was applied extracellularly through
perfusion at a rate of 2 ml/min. Dopamine induced inhibition of
IK in PC-12 cells. Control, baseline
IK. Current during dopamine exposure was recorded
after 1 min of drug application, when steady-state response was
reached. B: in presence of D2-specific antagonist sulpiride (Sulp, 10 µM), the inhibitory effect of dopamine on IK was prevented completely (n = 4).
C: under similar experimental paradigm, exogenous application
of D2-specific agonist quinpirole (Quin, 10 µM) elicited
similar IK inhibition in PC-12 cells
(n = 4).
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Fig. 3.
D2-specific IK modulation. A:
different concentrations of D2-specific agonist quinpirole
were used to study their effects on IK modulation
as follows: 1 µM (n = 5), 10 µM (n = 5), and 50 µM (n = 4). Average current amplitudes were measured during last 100 ms of depolarizing voltage pulses (at +50 mV). Data from different experiments were then averaged and are presented as means ± SE of %IK inhibition over log[dose]. B:
sulpiride specifically blocked quinpirole effect on
IK. Average current amplitudes were measured as in
A, and data from a representative experiment are presented as
time course of current amplitude. Application of sulpiride
(D2-specific antagonist) did not affect
IK amplitude. However, the presence of sulpiride
prevented quinpirole from inhibiting IK. Removal of
sulpiride allowed quinpirole to exert its inhibitory effect
(n = 4).
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Mechanism of D2-mediated IK inhibition.
The PTX-sensitive Go/Gi protein is
involved in a variety of D2-mediated signal transduction
pathways (17, 30). To examine the possible role of
Go/Gi in the D2 receptor
regulation of IK, PC-12 cells were pretreated with
PTX (200 ng/ml, overnight), a Go/Gi
inhibitor, and the effects of dopamine and quinpirole on IK were measured. PTX treatment had no effect on
the characteristics of IK recorded from PC-12
cells. However, our results show that PTX treatment prevented both the
dopamine (n = 4) and quinpirole (n = 5) induced
inhibition of IK (Fig.
4). It is important to note that these
cells were still responsive to extracellular application of TEA (5 mM,
n = 3), which indicated that the cells were viable and
responsive to other pharmacological agents (Fig. 4C).

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Fig. 4.
Dopamine/D2 modulation of IK is
pertussis toxin (PTX) sensitive. PC-12 cells were pretreated with PTX
(200 ng/ml overnight). IK recordings were performed
under whole cell voltage clamp. Normal pipette medium was used.
A: application of dopamine (100 µM) induced IK inhibition in control cells. However, in
PTX-treated cells (B), inhibitory effect of dopamine was not
observed (n = 4). C: effect of PTX pretreatment (200 ng/ml overnight) on quinpirole-mediated IK
inhibition was studied using similar voltage-clamp paradigm as in
A. Data from a representative experiment are presented as time
course of current amplitude change. Arrows indicate points of
application of quinpirole, tetraethylammonium (TEA), and wash. In
PTX-treated PC-12 cells, application of quinpirole (10 µM) failed to
cause any IK modulation. However, on application of TEA (5 mM), there was marked current inhibition that is completely reversible (inset: current traces of that experiment).
Superimposed current traces of baseline control (a) and after
applications of quinpirole (b) and TEA (c) are shown.
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It is well established that the D2 receptor mediates
inhibition of adenylate cyclase, which, in turn, leads to decreases in both cytosolic adenosine 3',5'-cyclic monophosphate (cAMP) and protein
kinase A (PKA) activity (for review see Ref. 17). To determine whether
PKA is involved in the D2-mediated inhibition of
IK, a specific inhibitor of PKA, PKI (100 µM),
was added to the standard pipette medium. PKI alone had no effect on
IK in PC-12 cells. We found that PKI failed to
prevent the quinpirole-induced inhibition of IK
(Fig. 5, hatched bar). This finding
indicates that D2 receptor modulation of
IK is not mediated by PKA. Other investigators have
reported that a much smaller dose of PKI (10-25 µM) inhibits
PKA-mediated current modulation (1, 29). Thus the effect of
D2 on IK appears not to be mediated by
PKA.

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Fig. 5.
D2 modulation of IK is independent of
protein kinase A (PKA) or protein kinase C (PKC). Steady-state
IK was measured during the last 100 ms of
depolarizing pulses. Data are means ± SE of %IK
inhibition. PKA was inhibited using a PKA-specific inhibitory peptide
(PKI), whereas PKC was blocked by staurosporine (ST). In both PKI (100 µM, hatched bar, n = 5) treated and ST (1 µM, solid bar,
n = 4) treated cells, quinpirole (10 µM) was able to induce
IK inhibition as in control (open bar,
n = 7).
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There have been several recent reports that the D2 receptor
might also mediate cellular responses via protein kinase C
(PKC)-related pathways (13, 31). Experiments were performed to
determine whether staurosporine, a potent PKC inhibitor (19, 32, 35), blocks the effect of quinpirole on IK.
Staurosporine treatment did not affect the characteristics of whole
cell IK recorded from PC-12 cells. In four cells
pretreated (30 min) with staurosporine (1 µM), the inhibitory effect
of quinpirole was retained (Fig. 5, solid bar). The dose of
staurosporine used in our experiments has been shown to be sufficient
for inhibition of the PKC enzyme activity (5, 9, 35).
D2 receptor modulation of cellular excitability during
hypoxia.
Earlier studies from our laboratory showed that hypoxia reversibly
inhibits IK(O2), which is a
component of whole cell IK (39). We wondered,
therefore, whether activation of the dopamine D2 receptor
modulates the effect of hypoxia on IK. To
investigate this possibility, the same voltage-pulse paradigm described
above was used, with the exception that the cells were exposed first to
hypoxia (10% O2). After a stable recording level was
achieved, the response to either dopamine or quinpirole was measured in the presence of hypoxia. The average current amplitudes were measured during the last 100 ms of the 800-ms depolarizing voltage pulses (+50
mV given at 0.1 Hz). The effect of hypoxia alone on
IK is shown in Fig. 6
(open bar; n = 10). The addition of dopamine (100 µM, Fig.
6, hatched bar, n = 5) or quinpirole (10 µM, Fig. 6, solid
bar, n = 5) during hypoxia caused additional
inhibition of IK. These results suggest that
dopamine and quinpirole enhance the hypoxic inhibition of
IK in PC-12 cells.

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Fig. 6.
Effects of dopamine/D2 receptor stimulation on hypoxic
IK inhibition. Presence of dopamine and/or
quinpirole potentiated hypoxia-induced IK
inhibition. PC-12 cells were exposed sequentially to 1-2 min of
either 1) hypoxia (H; 10% O2) and then addition of
dopamine during hypoxia or 2) hypoxia and then addition of
quinpirole during hypoxia. IK was recorded at 0.1 Hz, and average steady-state IK amplitudes were
measured at last 100 ms of 800-ms depolarizing pulses. Data from
different experiments are presented as means ± SE of percent change
of IK compared with control
(%IK inhibition). Effects of hypoxia on
IK from 2 sets of experiments were grouped, and
averaged data are presented. Hypoxia (10% O2) caused 16 ± 2% (n = 10) IK inhibition (open
bar). Addition of dopamine (100 µM) during hypoxia caused 27 ± 3%
IK inhibition (n = 5, hatched bar). With
addition of quinpirole (10 µM), current inhibition was 32 ± 2%
(solid bar, n = 5). * P 0.05 vs. H (open
bar).
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It has been shown that dopamine caused inhibition of a
voltage-dependent ICa in carotid body type I cells
(2) and bovine adrenal chromaffin cells (34). These authors speculated
that this response was mediated by D2. To test this
possibility, we examined the effect of quinpirole on voltage-dependent
ICa in PC-12 cells (Fig.
7, n = 4). The divalent charge
carrier BaCl2 (20 mM) was used to measure conductance
through the voltage-dependent Ca2+ channel.
Voltage-dependent currents were recorded at +20 mV (every 10 s) from a
HP of
80 mV. Peak inward current was measured and presented as a
percentage of initial current amplitude (set as 100%) vs. time. After
a steady baseline current was recorded, quinpirole (10 µM) was
applied extracellularly. This led to a reduction of the inward current,
which was readily reversible on return to the control recording media.
This finding shows that activation of D2 inhibits
ICa in PC-12 cells.

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Fig. 7.
D2 modulation of voltage-dependent Ca2+
current. Cells were depolarized to +20 mV for 160 ms from a holding
potential of 80 mV. Voltage pulses were given at 0.1 Hz. Data were
shown as time course of current changes. Beginning of line indicates
point of application of quinpirole and the end indicates washout.
Application of 10 µM quinpirole caused reversible inhibition of
voltage-dependent Ca2+ current. All currents
were leak subtracted, filtered at 1 kHz, and sampled at 10 kHz.
Ca2+ current was measured using Ba2+ as charge
carrier (20 mM BaCl2) and in Na+- and
K+-free external and pipette solutions. Peak inward
currents were measured and presented as percent control current
amplitude (baseline inward current). Inset: superimposed
current traces recorded before (a) and after (b)
quinpirole application.
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We next performed experiments to determine whether activation of the
D2 receptor would inhibit the hypoxia-induced increase in
cytosolic free Ca2+. PC-12 cells were loaded with fura 2, and the cytosolic levels of free Ca2+ were determined using
the fluorescence ratio between 340 and 380 nm. Hypoxia caused an
increase in intracellular free Ca2+ that was significantly
reduced by addition of quinpirole (10 µM) to the media
(P < 0.05, n = 11; Fig.
8A, first peak). We also found that
the addition of D2 receptor antagonist, sulpiride (10 µM), prevented the quinpirole-induced inhibition of cytosolic free
Ca2+ during hypoxia (P < 0.05,
n = 11; Fig. 8A, third peak).

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Fig. 8.
D2 modulation of the O2 sensitivity. A:
intracellular Ca2+ level was shown as fluorescence ratio at
340/380 nm. Hypoxia (0 mmHg) with addition of 10 µM quinpirole
produced a small increase in intracellular free Ca2+ (first
peak) compared with hypoxia alone (second peak). Addition of 10 µM
sulpiride antagonized the effect of quinpirole (third peak;
n = 11). B: membrane potential recording was
performed using current clamp with normal pipette and bath media.
D2 receptor activation with 10 µM quinpirole did not
alter the magnitude of hypoxia-induced membrane depolarization (first
depolarization) compared with hypoxia alone (second depolarization;
n = 5).
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Our results show that activation of the D2 receptor caused
inhibition of both IK and ICa
and modulated these currents during hypoxia. It is important to
determine whether modulation of these currents by the D2
receptor alters membrane depolarization in PC-12 cells during hypoxia.
Membrane potential was measured using current clamp. After a stable
resting membrane potential was recorded for 2 min, the cells were
exposed to 10 µM quinpirole plus hypoxia (0 mmHg). Hypoxia led to
membrane depolarization in quinpirole-treated cells (18 ± 2 mV,
Fig. 8B, first depolarization, n = 5). The cells were
allowed to repolarize in normoxia and then were exposed to hypoxia
alone (without quinpirole). The average membrane depolarization measured was 16 ± 1 mV (Fig. 8B, second depolarization,
n = 5). These results indicate that, although the
D2 receptor causes inhibition of both
IK and ICa, it does not affect
the ability of hypoxia to elicit membrane depolarization in PC-12
cells.
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DISCUSSION |
The O2-sensitive type I cells in the mammalian carotid body
depolarize and release dopamine during hypoxia (2, 18). We reported
recently that PC-12 cells respond in a similar manner to hypoxia (39).
HPLC measurement showed that hypoxia increases dopamine release in
PC-12 cells. Therefore, PC-12 cells provide a useful model for studying
the molecular events associated with cellular responses to hypoxia. The
current study was undertaken to investigate the potential role of
D2 dopamine receptor in regulation of ionic conductances in
PC-12 cells. A number of different dopamine receptors
(D1-D5) have been cloned (6, 23, 33, 36,
38) and subdivided into D1-like (D1 and
D5) and D2-like
(D2-D4) dopamine receptors (for reviews,
see Refs. 17 and 30). We found that PC-12 cells express the
D2 isoform of the dopamine receptor and that activation of
these receptors with exogenously applied dopamine or a D2
agonist led to inhibition of conductance through both K+
and Ca2+ channels. We also found that activation of
D2 receptors attenuated the increase in the intracellular
free Ca2+ during hypoxia. Although the D3
receptor also responds to quinpirole and sulpiride, our RT-PCR studies
argue against that possibility.
To our knowledge, this is the first direct evidence that the
D2 receptor is expressed in rat PC-12 cells and that
activation of these receptors leads to modulation of
IK and ICa. It has been shown
previously that the D2 receptor is expressed in human
pheochromocytoma cells (25) and in the dopaminergic type I cells of the
rat carotid body (11). There is also evidence that D2
receptors are expressed in adrenal chromaffin cells (3, 34). However,
the physiological role of these receptors remains unknown.
There is evidence that the dopamine D2 receptor is involved
in modulation of IK in a number of different cell
types. Among the earliest observations is a report by Sasaki and Sato
(28), which showed that dopamine increased IK in
Aplysia ganglion cells. Although these investigations did not
show direct evidence of D2, they did show that the effect
of dopamine was blocked by pretreatment with PTX and enhanced by
guanosine 5'-O-(3-thiotriphosphate). Studies in rat lactotrophs
and melanotrophs show similar effects of dopamine on
IK (8, 16). In lactotrophs, the D2
receptor increases voltage-dependent and Ca2+-activated
K+ currents. The effect of D2 receptor
activation on the regulation of IK in chromaffin
cells appears to differ from the responses in other cells. Sontag et
al. (34) showed that D2 activation caused inhibition of a
voltage-dependent IK in bovine adrenal chromaffin
cells. Stimulation of D2S dopamine receptors that were transfected into glioma cells caused inhibition of
IK through a PTX-insensitive receptor mechanism
that is secondary to the mobilization of intracellular Ca2+
(7). Our current study shows that the D2 receptor inhibits IK through a PTX-sensitive
Go/Gi protein. Therefore, effects of the
D2 receptor on IK seem to be cell type
specific.
We found that D2 stimulation also caused reversible
inhibition of a voltage-dependent ICa in PC-12
cells. This finding confirms earlier reports that dopamine inhibited
voltage-dependent ICa (17). Benot and Lopez-Barneo
(2) showed that dopamine caused inhibition of a voltage-dependent
ICa in type I cells without affecting
Na+ or K+ currents. Our current study shows
that dopamine inhibits both IK and
ICa in PC-12 cells. The lack of effect of dopamine
on IK in carotid body type I cells was probably due
to the concentration of dopamine (nanomolar compared with micromolar in
our study). There is also evidence that D2 activation has
an inhibitory effect on voltage-dependent ICa in
bovine chromaffin cells (3, 33).
A surprising finding was that the effect of the D2 receptor
on IK is independent of PKA or PKC. Dopamine
receptor functions are mediated through two G protein-linked
mechanisms. D1-like receptors are coupled to
Gs, whereas D2-like receptors are linked to
Go/Gi (17). It has been reported that the
D2 receptor modulates the activities of the intracellular
cAMP/PKA system through its inhibitory effect on adenylate cyclase (24;
for review see Ref. 17). In PC-12 cells, dopamine-mediated facilitation
of an ATP-activated inward current was shown to be independent of cAMP
or any intracellular protein kinases (22). In the present study, PKI, a
small heat-stable inhibitory protein of PKA, was used to examine the
role of PKA in mediating the effect of the D2 receptor on
IK. The high-affinity binding of PKI to the free
catalytic subunit of PKA causes the enzyme to remain in an inactive
state (15). We found that inhibition of PKA had no effect on the
D2-mediated inhibition of IK in PC-12 cells. It has also been reported that the D2 receptor
modulates signal transduction through the PKC pathway (13, 31). PC-12 cells express conventional PKC isoforms (
,
I,
II,
) and
unconventional isoforms (
,
,
), as well as the atypical
isoform
(4, 12). Staurosporine, an alkaloid isolated from
streptomycetes, is a potent nonselective inhibitor of PKC (19, 32, 35).
We tested the effect of staurosporine on the modulation of
IK by quinpirole. Our study showed that
staurosporine was ineffective in blocking the D2-mediated
inhibition of IK. It is possible that the
D2 receptor modulates IK through direct
coupling with PTX-sensitive Go/Gi protein. The
mechanism by which activation of D2 inhibits
IK is unknown and is a topic of current
investigation.
We showed previously that hypoxia depolarizes PC-12 cells by inhibition
of an IK(O2). The depolarization
during hypoxia leads to an elevation of intracellular free
Ca2+ (39). The elevated intracellular free Ca2+
during hypoxia is potentially important for a number of cellular functions, including neurotransmitter release and activation of second
messenger pathways involved in O2-mediated gene regulation (26). In the present study, we found that D2 receptor
activation maintained cellular depolarization by inhibition of
IK but reduced the increase in cytosolic free
Ca2+ during hypoxia. This modulatory effect on
intracellular free Ca2+ may play an important role in
feedback regulation of the cellular response to hypoxia. We propose
that modulations of IK and ICa by the D2 receptor maintain membrane excitability during
hypoxia, while regulating the steady-state level of intracellular free Ca2+. This can probably be explained by a balancing effect
caused by D2-mediated inhibition of both
IK and ICa.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Heart, Lung, and Blood
Institute Grant R37-HL-33831 to D. E. Millhorn.
 |
FOOTNOTES |
Address reprint requests to D. E. Millhorn.
Received 17 December 1996; accepted in final form 9 May 1997.
 |
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