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

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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).

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 MOmega (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(beta -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).

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.


View larger version (49K):
[in this window]
[in a new window]
 
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.

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.


View larger version (8K):
[in this window]
[in a new window]
 
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).


View larger version (10K):
[in this window]
[in a new window]
 
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).

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).


View larger version (17K):
[in this window]
[in a new window]
 
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.

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.


View larger version (15K):
[in this window]
[in a new window]
 
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).

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.


View larger version (16K):
[in this window]
[in a new window]
 
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).

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.


View larger version (10K):
[in this window]
[in a new window]
 
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.

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).


View larger version (19K):
[in this window]
[in a new window]
 
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).

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.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 (alpha , beta I, beta II, gamma ) and unconventional isoforms (delta , epsilon , eta ), as well as the atypical isoform zeta  (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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Artalejo, C. R., M. A. Ariano, R. L. Perlman, and A. P. Fox. Activation of facilitation calcium channels in chromaffin cells by D1 dopamine receptors through a cAMP/protein kinase A-dependent mechanism. Nature 348: 239-242, 1990[Medline].

2.   Benot, A. R., and J. Lopez-Barneo. Feedback inhibition of Ca2+ currents by dopamine in glomus cells of the carotid body. Eur. J. Neurosci. 2: 809-812, 1990[Medline].

3.   Bigornia, L., C. N. Allen, C. Jan, R. A. Lyon, M. Titeler, and A. S. Schneider. D2 dopamine receptors modulate calcium channel currents and catecholamine secretion in bovine adrenal chromaffin cells. J. Pharmacol. Exp. Ther. 252: 586-592, 1990[Abstract].

4.   Borgatti, P., M. Mazzoni, C. Carini, L. M. Neri, M. Marchisio, L. Bertolaso, M. Previati, G. Zauli, and S. Capitani. Changes of nuclear protein kinase C activity and isotype composition in PC12 cell proliferation and differentiation. Exp. Cell Res. 224: 72-78, 1996[Medline].

5.   Bruno, S., B. Ardelt, J. S. Skierski, F. Traganos, and Z. Darzynkiewiez. Different effects of staurosporine, an inhibitor of protein kinases, on the cell cycle and chromatin structure of normal and leukemic lymphocytes. Cancer Res. 51: 470-473, 1992.

6.   Bunzow, J. R., H. H. M. Van Tol, D. K. Grandy, P. Albert, J. Salon, M. Christie, C. A. Machida, K. A. Neve, and O. Civelli. Cloning and expression of a rat D2 dopamine receptor cDNA. Nature 336: 783-787, 1988[Medline].

7.   Castellano, M. A., L. Liu, F. J. Monsma, Jr., D. R. Sibley, G. Kapatos, and L. A. Chiodo. Transfected D2 short dopamine receptors inhibit voltage-dependent potassium current in neuroblastoma × glioma hybrid (NG108-15) cells. Mol. Pharmacol. 44: 649-656, 1993[Abstract].

8.   Castelleti, L., M. Memo, C. Missale, P. F. Spano, and A. Valerio. Potassium channels involved in the transduction mechanism of dopamine D2 receptors in rat lactotrophs. J. Physiol. (Lond.) 410: 251-265, 1989[Abstract].

9.   Conforti, L., K. Sumii, and N. Sperelakis. Dioctanoyl-glycerol inhibits L-type calcium current in embryonic chick cardiomyocytes independent of protein kinase C activation. J. Mol. Cell. Cardiol. 27: 1219-1224, 1995[Medline].

10.   Czyzyk-Krzeska, M. F., B. A. Furnari, E. E. Lawson, and D. E. Millhorn. Hypoxia increases rate of transcription and stability of tyrosine hydroxylase mRNA in pheochromocytoma (PC12) cells. J. Biol. Chem. 269: 760-764, 1994[Abstract/Free Full Text].

11.   Czyzyk-Krzeska, M. F., E. E. Lawson, and D. E. Millhorn. Expression of D2 dopamine receptor mRNA in the arterial chemoreceptor afferent pathway. J. Auton. Nerv. Syst. 41: 31-39, 1992[Medline].

12.   Gatti, A., X. Wang, and P. J. Robinson. Protein kinase C-alpha is multiply phosphorylated in response to phorbol ester stimulation of PC12 cells. Biochim. Biophys. Acta 1313: 111-118, 1996[Medline].

13.   Giambalvo, C. T., and R. L. Wagner. Activation of D1 and D2 dopamine receptors inhibits protein kinase C activity in striatal synaptoneurosomes. J. Neurochem. 63: 169-176, 1994[Medline].

14.   Giros, B., P. Sokoloff, M. P. Martres, J. F. Riou, L. J. Emorine, and J. C. Schwartz. Alternative splicing directs the expression of two D2 dopamine receptor isoforms. Nature 342: 923-926, 1989[Medline].

15.   Glass, D. B., L. J. Lundquist, B. M. Katz, and D. A. Walsh. Protein kinase inhibitor-(6-22)-amide peptide analogs with standard and nonstandard amino acid substitutions for phenylalanine 10---inhibition of cAMP-dependent protein kinase. J. Biol. Chem. 264: 14579-14584, 1989[Abstract/Free Full Text].

16.   Israel, J. M., C. Kirk, and J. D. Vincent. Electrophysiological responses to dopamine of rat hypophysial cells in lactotroph-enriched primary cultures. J. Physiol. (Lond.) 390: 1-22, 1987[Abstract].

17.   Jackson, D. M., and A. Westlind-Danielsson. Dopamine receptors: molecular biology, biochemistry and behavioural aspect. Pharmacol. Ther. 64: 291-369, 1994[Medline].

18.   Lopez-Lopez, J., C. Gonzalez, J. Urena, and J. Lopez-Barneo. Low pO2 selectively inhibits K channel activity in chemoreceptor cells of the mammalian carotid body. J. Gen. Physiol. 93: 1001-1015, 1989[Abstract].

19.   McGlynn, E., J. Liebetanz, S. Reutener, J. Wood, N. B. Lydon, H. Hofstetter, M. Vanek, T. Meyer, and D. Fabbro. Expression and partial characterization of rat protein kinase C-delta and protein kinase C-zeta in inset cells using recombinant baculovirus. J. Cell. Biochem. 49: 239-250, 1992[Medline].

20.   Monsma, F. J., Jr., L. D. McVittie, C. R. Gerfen, L. C. Mahan, and D. R. Sibley. Multiple D2 dopamine receptors produced by alternative RNA splicing. Nature 342: 926-929, 1989[Medline].

21.   Montmayeur, J.-P., J. Guiramand, and E. Borrelli. Preferential coupling between dopamine D2 receptors and G-proteins. Mol. Endocrinol. 7: 161-170, 1993[Abstract].

22.   Nakazawa, K., T. Watano, and K. Inoue. Mechanisms underlying facilitation by dopamine of ATP-activated currents in rat pheochromocytoma cells. Eur. J. Physiol. 422: 458-464, 1993. [Medline]

23.   O'Malley, K. L., S. Harmon, L. Tang, and R. D. Todd. The rat dopamine D4 receptor: sequence, gene structure, and demonstration of expression in the cardiovascular system. New Biol. 4: 137-146, 1992[Medline].

24.   Piomelli, D., and V. D. Marzo. Dopamine D2 receptor signaling via the arachidonic acid cascade: modulation by cAMP-dependent protein kinase A and prostaglandin E2. J. Lipid Med. 6: 433-443, 1993.

25.   Pupilli, C., R. Lanzillotti, G. Fiorelli, C. Selli, R. A. Gomez, R. M. Carey, M. Serio, and M. Mannelli. Dopamine D2 receptor gene expression and binding sites in adrenal medulla and pheochromocytoma. J. Clin. Endocrinol. Metab. 79: 56-61, 1994[Abstract].

26.   Raymond, R., and D. E. Millhorn. Regulation of tyrosine hydroxylase gene expression during hypoxia: role of Ca2+ and PKC. Kidney Int. 51: 536-541, 1997[Medline].

27.   Richtand, N. M., J. R. Kelsoe, D. S. Segal, and R. Kuczenski. Regional quantification of D1, D2, and D3 dopamine receptor mRNA in rat brain using a ribonuclease protection assay. Mol. Brain Res. 33: 97-103, 1995. [Medline]

28.   Sasaki, K., and M. Sato. A single GTP binding protein regulates K channel couples with dopamine, histamine and acetylcholine receptors. Nature 325: 259-262, 1987[Medline].

29.   Schackow, T. E., and R. E. Ten Eick. Enhancement of ATP-sensitive potassium current in cat ventricular myocytes by beta -adrenoreceptor stimulation. J. Physiol. (Lond.) 474: 131-145, 1994[Abstract].

30.   Seeman, P., and H. H. M. Von Tol. Dopamine receptor pharmacology. Trends Pharmacol. Sci. 15: 264-270, 1994[Medline].

31.   Senogles, S. E. The D2 dopamine receptor mediates inhibition of growth in GH4ZR7 cells: involvement of protein kinase-Cepsilon . Endocrinology 134: 783-789, 1994[Abstract].

32.   Seynaeve, C. M., M. G. Kazanietz, P. M. Blumberg, E. A. Sausville, and P. J. Worland. Differential inhibition of protein kinase C isozymes by UCN-01, a staurosporine analogue. Mol. Pharmacol. 45: 1207-1214, 1994[Abstract].

33.   Sokoloff, P., B. Giros, M. Martres, M. Bouthenet, and J. Schwartz. Molecular cloning and characterization of a novel dopamine receptor (D3) as a target for neuroleptics. Nature 347: 146-151, 1990[Medline].

34.   Sontag, J. M., P. Sanderson, M. Klepper, D. Aunis, K. Takeda, and M. F. Bader. Modulation of secretion by dopamine involves decreases in calcium and nicotinic currents in bovine chromaffin cells. J. Physiol. (Lond.) 427: 495-517, 1990[Abstract].

35.   Tamaoki, T., H. Nomoto, I. Takahashi, Y. Kato, M. Morimoto, and F. Tomita. Staurosporine, a potent inhibitor of phospholipid/Ca2+ dependent protein kinase. Biochem. Biophys. Res. Commun. 135: 397-402, 1986[Medline].

36.   Tiberi, M., K. R. Jarvie, C. Silvia, P. Falardeau, J. A. Gingrich, N. Godinot, L. Bertrand, T. L. Yang-Feng, R. T. Fremeau, Jr., and M. G. Caron. Cloning, molecular characterization, and chromosomal assignment of a gene encoding a second D1 dopamine receptor subtype: differential expression pattern in rat brain compared with the D1A receptor. Proc. Natl. Acad. Sci. USA 88: 7491-7495, 1991[Abstract].

37.   Watts, V. J., and K. A. Neve. Sensitization of endogenous and recombinant adenylate cyclase by activation of D2 dopamine receptors. Mol. Pharmacol. 50: 966-976, 1996[Abstract].

38.   Zhou, Q. Y., D. K. Grandy, L. Thambi, J. A. Kushner, H. H. M. Van Tol, R. Cone, D. Prbnow, J. Salon, J. R. Bunzow, and O. Civelli. Cloning and expression of human and rat D1 dopamine receptors. Nature 347: 76-79, 1990[Medline].

39.   Zhu, W. H., L. Conforti, M. F. Czyzyk-Krzeska, and D. E. Millhorn. Membrane depolarization in PC-12 cells during hypoxia is regulated by an O2-sensitive K+ current. Am. J. Physiol. 271 (Cell Physiol. 40): C658-C665, 1996[Abstract/Free Full Text].


AJP Cell Physiol 273(4):C1143-C1150
0363-6143/97 $5.00 Copyright © 1997 the American Physiological Society