• Abstract
  

• Introduction
  

• Materials and Methods
  

• Results
  

• Discussion
  

• Acknowledgments
  

• References
  

ABSTRACT

Two isoforms of the D₂ dopamine (DA) receptor have been identified. They are derived via alternative splicing of a single gene and have been termed D₂-short (D_2S) and D₂-long (D_2L) to indicate that they vary by only a 29 amino acid sequence within the third cytoplasmic loop. In the present study, the role of changes in intracellular calcium concentrations ([Ca²⁺]i) in the coupling of D_2S and D_2L receptors to a whole-cell potassium (K⁺) current was examined in stably transfected NG108-15 cells. It was noted that changes in intracellular concentration of the calcium chelator BAPTA (0.1-2.0 mM) attenuated or blocked the ability of D_2S (as previously reported), but not D_2L, receptor stimulation to reduce whole-cell K⁺ current. Bath application of thapsigargin (10-50 micromolar), which are known to increase(s) [Ca²⁺]i did not alter the magnitude of this current in D_2L transfected cells. The ability of the D₂ dopamine receptor agonists quinpirole (QUIN) to mobilize intracellular calcium was measured in both D_2L and D_2S transfected cells using the fluorescent probe fura-2. Bath application of QUIN (2-50 micromolar) dose-dependently increased [Ca²⁺]i in both transfected groups (D_2S and D_2L) but not untransfected cells with levels reaching a maximal concentration of approximately 100 nM (from resting levels of approximately 40 nM). This effect of QUIN was completely abolished in both D_2S and D_2L transfected NG108-15 cells by pretreatment with ryanodine (10 or 20 micromolar), demonstrating that receptor activation induced a mobilization of calcium from intracellular stores. Pretreatment with pertussis toxin completely blocked the mobilization of [Ca²⁺]i in D_2L transfected NG108-15 cells while having no effect on D_2S transfected cells. These results provide further evidence that the two isoforms of the D₂ receptor can couple to common effectors in a cell (whole-cell K⁺ current and intracellular calcium release) via two distinct signal transduction pathways.

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INTRODUCTION

Dopaminergic neurotransmission has been studied extensively because of hypothesized involvement of this neurotransmitter in a wide variety of psychiatric and neurological disorders. Two isoforms of the dopamine (DA) D₂ receptor termed D_2S and D_2L have been identified and are derived from the same gene via alternative mRNA splicing (Bunzow et al., 1988; Monsma et al., 1989; Sibley and Monsma, 1992). It is now clear that the activation of D₂ receptors modifies adenylyl cyclase activity, phosphotidylinositol hydrolysis, intracellular calcium concentrations, and several types of potassium and calcium currents (Kebabian and Calne, 1979; Valler and Meldosi, 1989; Spano et al., 1978; Niznik, 1987; Sibley and Monsma, 1992; Kanterman et al., 1991; Chiodo et al., 1995; Castellano et al., 1993; Liu et al. 1994, 1996). It has been demonstrated that when NG108-15 cells are transfected to stably express the individual D₂ receptor isoforms, these receptors couple to whole-cell K⁺ currents and stimulation by DA receptor agonists inhibits these currents (Liu et al., 1996). With respect to the D_2S-dependent modulation of K⁺ current, the coupling mechanism is pertussis toxin-insensitive and involves the mobilization of intracellular calcium ([Ca²⁺]_i) (Castellano et al., 1993).

The two D₂ receptor isoforms differ by a 29 amino acid sequence located in third cytoplasmic loop. It has been proposed by a number of investigators that this loop may be involved in receptor coupling to G proteins in this class of receptors, and thus it has been suggested that the two isoforms of the D₂ receptor might couple to different G protein and intracellular signal transduction pathways (Monsma et al.,1989; Dal Toso et al., 1989; Giros et al., 1989; Montmayeur and Borreli, 1991). For example, in transfected NG108-15 cells it has been demonstrated that D_2L receptors utilize a signal transduction pathway which utilizes G_O while D_2S receptors use G_s to couple to the same K⁺ current (Liu et al., 1996). To further address the role of intracellular calcium in the coupling of D_2S and D_2L DA receptors to K⁺ current in transfected NG108-15 cells, the mobilization of [Ca²⁺]_i was examined using the fluorescent probe fura-2 AM.

 

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MATERIALS AND METHODS

NG108-15 Cell Culture and Transfection: NG108-15 cells were routinely cultured in high-glucose Dulbecco's modified essential medium with L-glutamine (GIBCO), containing 1 mM sodium pyruvate, 10% fetal bovine serum, hypoxanthine-aminopterin-thymidine medium supplement (Sigma), and 30 microgram/ml gentamycin. The cDNAs encoding the D_2S and D_2L receptors were isolated from a rat striatal library and subcloned into eukaryotic expression vector pCD-SRa as previously detailed (Zhang et al., 1994). Cells seeded into 150 X 20-mm plates were transfected with 60 microgram of pSRa-D_2S or pSRa-D_2L plasmid DNA plus 6 microgram of pMam-neo (Invitrogen) by the CaPO4 precipitation method. Individual cell colonies were isolated after 1 week of selection and screened for DA receptor binding as described previously (Monsma, et al 1989). Receptor-expressing cell lines were subcloned by limiting dilution to generate clonal cell lines. The cell lines used in this study were found to express 80-120 fmol/mg of protein of specific D₂-like receptor binding activity.

Electrophysiological Methods: Coverslips with attached NG108-15 cells were placed into a flow through recording chamber where cells were continuously perfused at a rate of 2 ml/min in the normal extracellular medium which contained (in mM) NaCl 135; KCl 5.4; CaCl₂ 1.8; MgSO₄ 0.8 glucose, 20; HEPES, 5; plus 0.5 micromolar tetrodotoxin, adjusted to pH 7.3 with Tris base (315-325 mOsmoles). The chamber was placed on the stage of a inverted microscope (Zeiss ICM 405) and visualized using Hoffman differential interference contrast optics at X400. Cells were voltage-clamped in the whole-cell configuration using an EPC7 amplifier (Adams-List Associates., Westbury, NY). Patch electrodes were fabricated from 1.65 O.D. Micro-Hematocrit capillary tubes (VWR Scientific) pulled in a two-stage process on a vertical pipette puller (Narishigie PA-81). The holding potential and all test voltage command steps were controlled by PCLAMP software and hardware (Axon Instruments) using the CLAMPEX program. To activate steady-state outward currents, the cell was voltage clamped to a holding potential of -60 mV and the membrane was stepped to test membrane potentials between -80 and +70 mV for 300 msec (at a presentation frequency of 0.2 Hz). The patch pipette internal solution contained the following (in mM): KCl 140; MgCl₂ 2; CaCl₂ 1; HEPES 10; ATP 2; cAMP 0.25; and BAPTA 0.5 (pH adjusted to 7.3 with KOH, osmolarity 310-320 mOsmoles). The patch electrode resistance ranged form 2-5 megohms and after membrane rupture the series resistance ranged from 5.8-12 megohms and capacitance compensation ranged from 8-21 pF. All studies were conducted at room temperature (18-22 ^oC). To address the role of intracellular Ca²⁺, the concentration of the Ca²⁺-chelator BAPTA within the patch pipette was varied from 0.1-2 mM. In other experiments, 10-20 micromolar ryanodine was included in the external solution to inhibit Ca²⁺ release from internal stores (McPherson et al., 1991; Smith et al., 1988; Imagawa et al., 1987).

Fura-2 Loading: Coverslips with attached transfected or control NG108-15 cells were placed in a 35 mm petri dish containing 700 microliter of fura-2 acetoxymethyl ester (fura-2 AM) (Molecular Probes Inc.) at a final concentration of 2 micromolar, dissolved in anhydrous DMSO and 0.1% Pluronic F-127 (BASF). The cells were returned to the incubator (37 ^oC, 10% CO₂) for 25 minutes. After the incubation, cells were washed three times with basic external recording solution and finally left in this solution for 35-120 minutes to allow complete hydrolysis of the ester bond on the intracellularly incorporated fura-2 AM.

Fluorescence Microscopy and Image Analysis: Coverslips with attached, fura-2 loaded cells were placed in a flow chamber attached to the stage of a Nikon Diaphot 300 (Nikon, Japan) inverted microscope. A Nikon UV-F 40X (Nikon Fluor, NA 1.3) objective was used for the epifluorescence measurements together with a 75W xenon lamp. The images were obtained via an intensified CCD camera (Hamamatsu) linked to an image-processing system (Universal Imaging, Image-1/MetaFluor version 2.0). Images were obtained at two different excitation wavelengths (340 and 380 nm) via excitation filters mounted on a computer-controlled Lambda-10 optical filter changer (Sutter Instruments) and fluorescence emission was filtered with a 500 nm long pass filter (Chroma Filters). The frame capture period was 40 msec and only 4 frames were averaged (for each wavelength) once every 2-5 sec. the averaged images obtained at both 340 and 380 nm were ratioed on a pixel-by-pixel basis and [Ca²⁺]_i was calculated according to the equation presented by Grynkiewicz et al., (1985):

[Ca²⁺]_i = K_d * F_max/F_min*(R - R_min)/(R_max - R)

R_min is the ratio of fluorescence intensities at 340 and 380 nm obtained at zero [Ca²⁺]_i and R_max is the ratio at saturating [Ca²⁺]_i, K_d is the dissociation constant for fura-2 and F_min and F_max are the fluorescence intensities at 380 nm minus and plus calcium, respectively. The apparent dissociation constant for fura-2 used in all calibrations was 224 nM (Grynkiewicz et al., 1985). In situ calibration of fura-2 was carried out by bathing the cells in a calcium-free external solution with 10 micromolar ionomycin and 5 mM EGTA to determine R_min and 2 mM calcium in external solution with 10 micromolar ionomycin to determine R_max as suggested by Malgaroli et al., (1987).

In some experiments 10-20 micromolar ryanodine was included in the external solution to block the calcium release from internal stores (McPherson et al., 1991; Smith et al., 1988; Imagawa et al., 1987). Ryanodine stock solution was made in 95% ethanol and the experiments were carried out with the same final concentration of ethanol, to control for the effect of ethanol. There were no effect of this vehicle. Quinpirole (QUIN) was applied to the outside of the cell and the stock solution (1 mM) was prepared in normal external solution. All the solutions were filtered through 0.22 micron membrane filter. QUIN was the only DA receptor agonist used in these studies but it has been previously shown that these cells respond in a dose-dependent manner to DA, QUIN and apomorphine, and posses a D₂-like pharmacological profile (Castellano et al., 1993; Liu et al., 1996).

Pertussis Toxin Preincubation: Cultures were pretreated with pertussis toxin as follows: 500 ml of the medium was removed from the well of a 24 well plate and replaced with fresh medium with 1000 ng/ml of pertussis toxin giving a final concentration 500 ng/ml. The cultures were then returned to the incubator for about 3-5 hours. Prior to loading the cells with fura-2 AM , the coverslip was washed three times with external recording solution and image analysis were carried out as outlined above.

Data Analysis: Groups differences in [Ca²⁺]_i were determined using an analysis of variance with multiple post-hoc comparisons performed using the Bonferroni t-test. Analysis of current records was performed using the CLAMPFIT program.

 

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RESULTS

As mentioned above, the coupling of the D_2S receptor to the whole-cell K⁺ current in NG108-15 cells was suggested to be dependent upon the mobilization of intracellular calcium (Castellano et al., 1993). In the present study, the role of [Ca²⁺]_i in the coupling of both D_2S and D_2L receptors to the K⁺ current was further examined. As seen in Figure 1, increases in intracellular concentration of the calcium chelator BAPTA (0.1-2 mM) produced a concentration-dependent reduction in QUIN's ability to decrease the K⁺ current studied in D_2S- but not D_2L-transfected NG108-15 cells. In addition, the bath application of thapsigargin (50 micromolar) or ryanodine (10-20 micromolar), which are known to increase and decrease [Ca²⁺]_i respectively, did not alter the K⁺ current in NG108-15 cells expressing the D_2L receptor (Figure 2). It was shown earlier that thapsigargin mimicked while ryanodine blocked the QUIN-induced suppression of whole-cell K⁺ current in D_2S-transfected NG108-15 cells (Castellano et al., 1993).

Figure 1: The effect of different intracellular concentrations of the Ca2+ chelator BAPTA on the ability of D2S and D2L receptor activation to reduce the whole-cell K+ current present in NG108-15 cells. BAPTA was varied in the patch pipette at the concentrations shown and allowed to dialyze into the cell for at least 4 minutes after formation of the whole-cell patch configuration . Each data point represents the mean (+SEM) percent reduction in peak outward current observed at a test membrane potential of +70 mV (holding potential was -60 mV) and in the presence of the D2 receptor agonist QUIN (20 micromolar). It may be observed that the ability of D2S, but not D2L , receptor stimulation to decrease whole-cell K+ current was attenuated by increases in intracellular BAPTA concentration. *p<0.05, analysis of variance and Bonferroni t-test.

 

Figure 2: The effects on alterations in [Ca2+]i on the coupling of D2L receptors to whole-cell K+ current. It may be seen that bath application of ryanodine (RYAN 20 micromolar, left panel) or thapsigargin (THAPS 50 micromolar, right panel) had no effect on the K+ current observed in this cells. The inset whole-cell current and voltage traces are representative traces from a single cell in both treatment conditions. The cells were held at a membrane potential of -60 mV and stepped to a test potential of +70 mV for a duration of 300 msec. Data represents the mean +SEM for the individual cells studied (N=7 for both groups). Current trace calibration bar represents 1000 pA.

The ability of receptor stimulation to mobilize intracellular calcium was assessed directly using the ratiometric analysis of fura-2 fluorescence at 340 and 380 nM. Free [Ca²⁺]_i did not vary in cells incubated for 5 min in normal external recording medium (containing 1.8 mM Ca²⁺). The basal level of [Ca²⁺]_i in NG108-15 cells recorded ranged between 35-70 nM with a mean (+SEM) of 39 + 3.2 nM in wild type NG108-15 cells (n = 22), 45 + 1.9 nM in D_2L-transfected cells (n = 60), and 42 + 2.1 nM in D_2S-transfected cells (n = 67). The [Ca²⁺]_i in wild type NG108-15 cells did not respond to the bath application of the D₂ receptor agonist QUIN (Figure 3) and served as a control in our experiments. In contrast, both the D_2L- and D_2S-transfected cells demonstrated a dose-dependent increase in [Ca²⁺]_i in response to QUIN (10-50 mM, Figures 3 and 4). The [Ca²⁺]_i in D_2L-transfected NG108-15 cells increased transiently in response to 10 mM or 50 mM QUIN to 51 nM (mean, n = 15) and 57 nM (n = 37; p<0.05), respectively. Similarly, the [Ca²⁺]_i in D_2S-transfected NG108-15 cells increased in response to 10 mM, 20 mM or 50 mM QUIN to 54 nM (mean, n = 33; p<0.05), 62 nM (n = 17; p<0.05) and 65 nM (n = 18; p<0.05), respectively. The effect of QUIN was reversible upon washout (Figure 4).

Figure 3: Bar graph illustrating the effect of bath application of the D2 receptor agonists quinpirole (QUIN) on [Ca2+]i in transfected NG108-15 cells. It may be observed that in NG108-15 cells stably expressing either the D2L or the D2S receptor, QUIN application (50 micromolar) produced an increase in [Ca2+]i relative to control (C). In contrast, there was no effect of QUIN application on wild-type cells. Each bar represents the mean +SEM [Ca2+]i calculated for the group (number above each bar represents the number of cells sampled). *p<0.05, analysis of variance and Bonferroni t-test.

 

Figure 4: Photomicrographs demonstrating the mobilization of intracellular calcium upon stimulation of D2L receptors stably expressed in NG108-15 cells. The pictures represent pseudocolor images of the ratio of the fluorescence intensity measured at 340 and 380 nm. The top panel show the resting calcium levels in several NG108-15 cells. The bottom panel shows the same cells two minutes after bath application of the D2 receptor agonist quinpirole (20 micromolar). It may be observed that every cell in the field studied demonstrated an increase in [Ca2+]i as indicated by a shift in color from blue towards yellow-red (the relative concentration of calcium is indicated by the calibration bar, see text for details).

Additional experiments were undertaken to address the mechanism by which [Ca²⁺]_i was increased following receptor activation. The bath application of ryanodine, (20 micromolar), which prevents calcium release from internal stores (McPherson et al., 1991; Smith et al., 1988; Imagawa et al., 1987), completely abolished the QUIN-induced mobilization of [Ca²⁺]_i in D_2L- and D_2S-transfected NG108-15 cells (Figure 5). Furthermore, the increase in [Ca²⁺]_i was not affected when calcium was removed from the external medium or when voltage-dependent calcium channels were blocked by extracellular cadmium (1mM) (data not shown). Together these observations demonstrate that the increases in [Ca²⁺]_i observed herein result from the release of Ca²⁺ from internal stores not its entry into the cell.

Figure 5: Bar graph illustrating the ability of ryanodine to block the increase in [Ca2+]i which normally accompanies stimulation of D2 receptors. In both D2L and D2S expressing NG108-15 cells QUIN application (50 micromolar) produced a significant increase in [Ca2+]i relative to control levels (C). Ryanodine blocked the observed increase in both transfected groups (RYAN + QUIN). Each bar represents the mean +SEM [Ca2+]i calculated for the group (number above each bar represents the number of cells sampled). *p<0.05, analysis of variance and Bonferroni t-test.

The role of G proteins in the observed mobilization of [Ca²⁺]_i in D_2S- and D_2L-transfected NG108-15 cells was also determined. The pretreatment of cells with pertussis toxin abolished the QUIN-induced mobilization of [Ca²⁺]_i in D_2L-transfected cells but had no significant effect on cells transfected and expressing the D_2S receptor (Figure 6).

Figure 6: Bar graph illustrating the ability of pertussis toxin pretreatment to block the increase in [Ca2+]i which normally accompanies stimulation of D2S receptors. In both D2L and D2S expressing NG108-15 cells QUIN application (50 micromolar) produced a significant increase in [Ca2+]i relative to control levels (C). Pretreatment with pertussis toxin (PTX + QUIN) blocked the observed increase in D2S but not D2L transfected NG108-15 cells. Each bar represents the mean +SEM [Ca2+]i calculated for the group (number above each bar represents the number of cells sampled). *p<0.05, analysis of variance and Bonferroni t-test.

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DISCUSSION

It has been previously demonstrated that D_2S and D_2L receptors stably expressed in NG108-15 cells couple to whole-cell K⁺ currents using two different G proteins, G_s and G_O, respectively (Liu et al., 1996). Since an earlier study suggested that the D_2S receptor-mediated inhibition of K⁺ current was dependent on the alterations in [Ca²⁺]_i (Castellano et al., 1993), this was examined directly. The experiments herein have demonstrated that while the ability of D_2S and D_2L receptors to inhibit K⁺ current is differentially regulated by [Ca²⁺]_i the stimulation of either receptor by the direct-acting agonist QUIN results in a significant mobilization of [Ca²⁺]_i. It was observed that chelation of Ca²⁺ in the cytoplasm with BAPTA attenuated the D_2S receptor stimulation-induced reduction in K⁺ current while having no effect on the reduction in current amplitude normally produced by D_2L receptor stimulation. In addition, the mobilization of [Ca²⁺]_i by thapsigargin and the inhibition of Ca²⁺ release from intracellular stores by ryanodine had no effect on the D_2L receptor-mediated inhibition in K⁺ current. However, when the mobilization of intracellular Ca²⁺ in response to receptor stimulation was examined directly, it was shown that both D_2S and D_2L receptor stimulation produced a significant increase in [Ca²⁺]_i. The increase in [Ca²⁺]_i could arise from two sources, 1) the release of calcium from internal stores or 2) the influx of calcium through calcium channels. QUIN elicited a transient increase in [Ca²⁺]_i in these cells in a dose-dependent manner. Since these effects could be blocked by the application of ryanodine and were not altered when calcium was removed from the external medium, it was concluded that the QUIN-induced increase in [Ca²⁺]_i in these cells was the result of calcium release from internal stores.

In pertussis toxin treated D_2L transfected cells, there was no mobilization of intracellular calcium in response to quinpirole, which demonstrates that the D_2L receptor mobilizes intracellular calcium via pertussis toxin-sensitive G proteins. In contrast, pertussis toxin pretreatment of D_2S expressing cells did not result in a blockade of QUIN-induced mobilization of [Ca²⁺]_i. This suggests that the D_2S receptor mobilizes intracellular calcium via a different intracellular pathway which apparently does not involve pertussis toxin-sensitive G protein.

It has been previously reported that stimulation of D₂ receptors results in an increase in [Ca²⁺]_i in transfected Ltk^- fibroblasts but produces a decrease in intracellular calcium in transfected GH₄C₁ cells (Vallar et al. 1990). In contrast, stimulation of D4 receptors expressed in GH₄C₁ cells had no effect on [Ca²⁺]_i (Seabrook et al. 1994). Endogenous D₂ receptor stimulation has also been shown to produce a decrease in [Ca²⁺]_i in rat lactotrophs (Malgaroli et al. 1987). In the Ltk^- cells, D₂ receptor stimulation was shown to produce a rapid increase in inositol trisphosphate (IP₃) which is known to mobilize calcium from internal stores. We have observed that IP₃ mediated release of intracellular calcium is present in NG108-15 cells (unpublished observations) raising the possibility that the stimulation of both D_2S and D_2L dopamine receptors results in the generation of IP₃ and the subsequent mobilization of calcium. Further studies will be necessary to determine the signal transduction pathways involved in this effect for each of these receptors.

There are at least two different delayed rectifier channels present in NG108-15. The first is termed NGK1 (also termed RAK) and the second is termed NGK2 (Yokoyama et al., 1989). In addition, it has been shown that the NGK1 current may be inhibited by stimulation of the endogenous bradykinin receptors present in these cells via a mechanism which involves the activation of phospholipase C and an increase in [Ca²⁺]_i (Huang et al., 1993). This raises the possibility that the D_2S receptor isoform preferentially inhibits the NGK1 current via a similar mechanism since it is clear that mobilization of intracellular calcium plays a critical role in the D_2S receptor-mediated inhibition of the K⁺ current in these cells. It might therefore be suggested that the D_2L receptor isoform either inhibits this same current through a different, calcium-independent process or that this isoform is preferentially coupled to a different current, perhaps NGK2. Further indirect support that D_2S receptors may be coupling selectively to NGK1 channels in these cells comes from observations in the oocyte expression system. It has been shown that these channels couple to the M1 acetylcholine receptor (coexpressed in the oocyte) via a phospholipase C pathway which involves the mobilization of [Ca²⁺]_i (Huang et al., 1993). It was shown that intracellular injection of the calcium chelator EGTA, while having no effect by itself, did significantly attenuate the suppression of NGK1 current normally observed upon M1 receptor stimulation. Similar observations were made regarding the D_2S suppression of K⁺ current in NG108-15 cells (Castellano et al. 1994). Further studies will be necessary to determine whether or not D_2L and D_2S receptors are coupling to similar or different K⁺ current in NG108-15 cells.

 

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ACKNOWLEDGMENTS

This work was supported by MH-41557 (lac). We would like to thank Dr. Frederick J. Monsma, Jr. for his assistance in the preparation of the transfected NG108-15 cells.

 

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