Abstract
The role of drug efficacy in agonist-induced desensitization was studied in C-6 glioma cells transfected with the monkey dopamine D1A (mD1A) receptor. Dopamine pretreatment for 2 hr produced greater than 80% loss of responsiveness in the stimulation of cAMP accumulation that was blocked by the D1antagonist SCH23390. A series of full and partial D1agonists from structurally dissimilar classes were then examined. Three full agonists (dihydrexidine, SKF82958, A77636) desensitized the receptor to the same extent as dopamine, whereas two other full agonists (dinapsoline and A68930) and all the partial agonists tested (SKF38393, pergolide and d-lysergic acid diethylamide tartrate) produced only partial desensitization (i.e., 50% that of dopamine). Whereas partial agonists (i.e., SKF38393, pergolide and d-lysergic acid diethylamide tartrate) caused no alteration in ligand-accessible mD1A receptors, four of the full agonists (dopamine, dihydrexidine, dinapsoline, A68930) caused a 30 to 40% reduction in receptor number. One full agonist, A77636, caused nearly an 80% decrease in receptor number, despite the fact that the degree of functional desensitization was similar to the other full agonists. The desensitization of the D1 receptor was homologous, not affecting beta-2 adrenergic receptors endogenous to C-6 cells. Neither incubation with cAMP analogs, nor inhibition of protein kinase A, affected dopamine-induced desensitization, suggesting a cAMP-independent mechanism in this cell line. Together, these data suggest that functional desensitization of the mD1A receptor expressed in C-6 glioma cells is a cAMP-independent mechanism, cannot be predicted reliably from agonist efficacy for stimulating adenylate cyclase and can occur in the absence of changes in receptor number.
The D1 dopamine receptor is the product of one of five known dopamine receptor genes (for review, see Ogawa, 1995). The D1 and D5 dopamine receptors (referred to as D1A and D1B in rodents, respectively) are referred to as the “D1-like” receptors. As can be seen, this terminology for the D1-like receptors can lead to confusion. In this paper, when referring to drugs, the term “D1” will refer to compounds having affinity for both D1-like receptors (because no selective ligands are available presently). Conversely, when referring to receptors, we shall use the term for the specific receptor isoform (e.g., D1, D1A, D5 or D1B) or the general term D1-like, as appropriate.
The D1-like receptors often are coupled to stimulation of the enzyme adenylate cyclase. It also has been reported that D1-like receptors will stimulate phosphoinositide hydrolysis, although the pharmacology of this response is inconsistent with known characteristics of D1receptors (Undie and Friedman, 1990, 1992). In fact, a recent study has demonstrated that D1A agonist-stimulated phosphoinositide hydrolysis can be observed in transgenic mice lacking a functional D1A receptor gene, which suggests that this phenomenon is not mediated by D1Areceptors (Friedman et al., 1997).
At one time, studies of the function of D1-like receptors were hampered both by the lack of selective D1 antagonists, and by the fact that the only selective agonist, SKF38393, was of partial efficacy (Setler et al., 1978). The development of a selective D1 receptor antagonist (SCH23390; Iorio et al., 1983) and full D1 agonists like DHX (Lovenberg et al., 1989; Brewster et al., 1990;Mottola et al., 1992) have allowed the functional role of D1-like receptors in the central nervous system to be studied. Full D1 agonists apparently have a significant role in the therapy of Parkinson’s disease (Tayloret al., 1991; Kebabian et al., 1992), whereas partial D1 agonists are ineffective (Closeet al., 1985; Braun et al., 1987; Bedard and Boucher, 1989).
Receptor desensitization is defined as a loss of responsiveness after agonist exposure. One type of desensitization may be classified as heterologous, wherein exposure to a ligand causes a decreased responsiveness to activation of any receptor that uses the same downstream effector (e.g., cAMP). In contrast, in homologous desensitization, decreased responsiveness is limited to the receptor that induced the initial desensitization. The best characterized system for receptor desensitization is the beta-adrenergic system (for a review, see Benovic et al., 1988). Lefkowitz and colleagues have elegantly delineated many of the steps involved inbeta-2 adrenergic receptor desensitization, including the demonstration of both cAMP-dependent and -independent processes (seeHarden, 1983; Lefkowitz et al., 1983; Perkins, 1983). Although desensitization of the beta-2 adrenergic receptor has been well characterized, much less information is available for other G-protein coupled receptors. The role of D1-like receptors in the therapy of Parkinson’s disease provided a strong impetus to examine desensitization processes for this receptor subtype. The results of previous studies in this area suggest that D1 agonists can produce marked desensitization, although the relation between agonist efficacy and the degree of receptor desensitization depends on the D1 receptor expression system used. For example, in studies with the D1A receptors endogenous to NS20Y cells, pretreatment with both full and partial agonists resulted in similar decreases in D1 receptor-stimulated cyclic AMP accumulation (Barton and Sibley; 1990). Contrasting results were obtained by Balmforth et al. (1990), who studied the ability of agonists of varying efficacies to desensitize D1 receptors expressed endogenously in D384 cells. In this system, the efficacy to stimulate adenylate cyclase predicted the extent of desensitization. For example, incubation of D384 cells with dopamine or the full agonist 6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene produced significant desensitization. Conversely, the partial agonists 3,4-dihydroxynomifensine and fenoldopam produced moderate effects, and the partial agonist SKF38393 had virtually no effect on dopaminestimulated cAMP accumulation (Balmforth et al., 1990). Although the mechanism(s) responsible for D1 receptor desensitization remain to be elucidated, it has been shown that activation of D1 receptors expressed in Sf9 cells results in increased phosphorylation and palmitoylation of the D1 receptor, although the significance of these events is unknown (Ng et al., 1994, 1995).
The present studies took advantage of the availability of newly developed full D1 agonists from a variety of structural classes to expand previous comparisons of agonist efficacy and desensitization of the D1 receptor. We chose to examine desensitization in a simplified system, C-6 glioma cells expressing the rhesus macaque D1A(mD1A) receptor (Machida et al., 1992). Our choice of expression system was guided by our previous studies indicating that the expression level and pharmacological profile of D1 agonists in these C-6 mD1A-transfected glioma cells are consistent with results obtained in rat striatum, and thus represent a valid test system. In addition to our comparisons among D1full and partial agonists, we also examined the effects of direct manipulation of cAMP levels and of activation and inhibition of PKA on receptor desensitization. We now report that the desensitization mediated by the D1 dopamine receptor in this system is homologous, and that treatment with full, but not partial D1 agonists results in a decrease in [3H]SCH23390 binding density. We have found, however, that the extent of functional desensitization of adenylate cyclase cannot be predicted fully by agonist efficacy. This latter result implies a role for additional agonist-specific factors in desensitization. Finally, although the activation of the D1 receptor stimulates cyclic AMP accumulation, desensitization cannot be produced by simple direct elevation of cyclic AMP formation or PKA activation, which suggests that it occurs independently of these events.
Methods and Materials
Materials.
SCH23390, SKF38393, SKF82958, pergolide methanesulfonate, Rp-cAMPS, Sp-cAMPS, H-9 dihydrochloride and HA-1004 hydrochloride were purchased from Research Biochemicals, Inc. (Natick, MA). A68930 was a generous gift from Abbott Pharmaceuticals (Abbott Park, IL). LSD was obtained from the National Institute on Drug Abuse. The following drugs were synthesized according to previously described methods: (±)-DHX (Knoerzer et al., 1994); dinapsoline (Ghosh et al., 1996); and A77636 (DeNinno et al., 1991b). Dopamine, cAMP, dibutyryl cAMP, 8-Br-cAMP and IBMX were obtained from Sigma Chemical Co. (St. Louis, MO). Cyclic AMP primary antibody was obtained from Dr. Gary Brooker (George Washington University, Washington, DC), and secondary antibody (rabbit anti-goat IgG) covalently attached to magnetic beads was purchased from Advanced Magnetics, Inc. (Cambridge, MA). Finally, [3H]SCH23390 (specific activity, 85 Ci/mmol) was synthesized according to Wyrick et al. (1986).
Cell cultures.
The present studies were conducted with C-6 glioma cells transfected with the rhesus macaque D1A receptor (C-6-mD1A,Machida et al., 1992). Cells were grown in DMEM-H medium containing 4,500 mg/l glucose, l-glutamine, 5% fetal bovine serum and 600 ng/ml G418. In the present studies, the density of mD1A receptor binding sites in untreated cells was approximately 50 fmol/mg protein for C-6-mD1Acells. Cells were plated into 24-well plates and allowed to grow to confluence (usually 2–4 days), after which they were used for either dose-response or desensitization studies. For the binding studies, 75-cm2 flasks of confluent cells were treated as described below. All studies (functional and receptor binding) used cells from passages 2 to 20. Cells were maintained in a humidified incubator at 37°C with 95% O2 and 5% CO2.
Dose-response studies.
Agonist intrinsic activity was assessed by the ability of selected compounds to stimulate adenylate cyclase, as measured by cAMP accumulation in whole cells. Confluent plates of cells were incubated with drugs dissolved in DMEM-H supplemented with 20 mM HEPES, 0.1% ascorbic acid and 500 μM IBMX (pH 7.2; media A). The final volume for each well was 500 μl. In addition to the dose-response curves run for each drug, basal levels of cAMP and isoproterenol-stimulated cAMP accumulation were evaluated for each plate. Each condition was run in duplicate wells. After a 10-min incubation at 37°C, cells were rinsed briefly with media, and the reaction was stopped by the addition of 500 μl of 0.1 N HCl. Cells were then allowed to chill for 5 to 10 min at 4°C, the wells were scraped, and the contents placed into 1.7-ml centrifuge tubes. An additional 1 ml of 0.1 N HCl was added to each tube, for a final volume of 1.5 ml/tube. Tubes were vortexed briefly, and then spun in a BHG HermLe Z 230 M microcentrifuge for 5 min at 15,000 × gto eliminate large cellular particles. Cyclic AMP levels for each sample were determined as described under “Radioimmunoassay of cAMP.”
Desensitization studies.
Plates of confluent cells were incubated with test drugs dissolved in plain DMEM-H media supplemented with 20 mM HEPES and 0.1% ascorbic acid (pH 7.2; media B). Cells, in a final volume of 500 μl/well, remained in the incubator during the desensitization period. At the end of the desensitization period, cells were rinsed for 30 min at 37°C with 500 μl of media B. Cells were then challenged with 10 μM dopamine (dissolved in media A) for 10 min at 37°C, followed by a brief rinse with 500 μl of media A. The reaction was stopped with the addition of 500 μl of 0.1 N HCl, the plates were scraped and the contents placed into 1.7-ml centrifuge tubes. After vortexing briefly, these tubes were centrifuged and then cyclic AMP levels were evaluated by RIA. Basal activity (i.e., in the absence of drug) was measured before and after incubation with each concentration of test drug.
Radioimmunoassay of cAMP.
The concentration of cAMP in each sample was determined with an RIA of acetylated cAMP, modified from that described previously (Harper and Brooker, 1975). Iodination of cAMP was performed by a method described previously (Patel and Linden, 1988). Assay buffer was 50 mM sodium acetate buffer with 0.1% sodium azide (pH 4.75). Standard curves of cAMP were prepared in buffer at concentrations of 2 to 500 fmol/assay tube. To improve assay sensitivity, all samples and standards were acetylated with 10 μl of a 2:1 solution of triethylamine/acetic anhydride. Samples were assayed in duplicate. Each assay tube contained 10 μl of sample, 100 μl of buffer, 100 μl of primary antibody (sheep, anti-cAMP, 1:100,000 dilution with 1% BSA in buffer) and 100 μl of [125I]cAMP (50,000 dpm/100 μl of buffer); total assay volume was ∼300 μl. Tubes were vortexed and stored at 4°C overnight (approximately 18 hr). Antibody-bound radioactivity then was separated by the addition of 10 μl of BioMag rabbit, anti-goat IgG (Advanced Magnetics, Cambridge MA), followed by vortexing and further incubation at 4°C for 1 hr. To these samples 1 ml of 12% polyethylene glycol/50 mM sodium acetate buffer (pH 6.75) was added, and all tubes were centrifuged at 1700 × g for 10 min. Supernatants were aspirated and radioactivity in the resulting pellet was determined with an LKB Wallac gamma counter (Gaithersburg, MD).
Analysis of affinity for agonists at C-6-mD1A receptors.
Flasks of cells in the same passage were rinsed with 5 ml hypoosmotic buffer (1 mM HEPES, 2 mM EGTA, pH 7.4), and then incubated with 7 ml hypoosmotic buffer for 5 to 10 min at 4°C. Cells were then scraped off the bottom of the flask with a rubber policeman, collected into 50-ml tubes and centrifuged at 28,000 × g at 4°C for 20 min. The resulting pellet was resuspended in binding buffer (50 mM HEPES, pH 8.0), homogenized with a Brinkmann Polytron on a setting of 5 for 10 sec, and either used immediately or stored in 1-ml aliquots at −80°C until use in binding assays. Aliquots contained approximately 1 mg/ml of protein, as measured with the BCA protein assay reagent (Pierce, Rockford, IL).
Competition binding studies were done to evaluate the affinity of the different agonists for the mD1A receptor and were performed essentially following the protocol of Machida et al. (1992) with some minor modifications. Membranes were diluted in assay buffer A (50 mM HEPES, 0.9% NaCl, pH 8.0) and 100 μl of membranes (approximately 50 μg) was incubated with 0.3 nM [3H]SCH23390 and increasing concentrations of competing drug (0.01 nM–1 μM) in assay buffer B (50 mM HEPES, 0.9% NaCl, 0.001% BSA, pH 8.0). BSA was omitted from assay buffer A to determine protein levels in the samples accurately. (BSA was used as the standard in protein determinations.) Nonspecific binding was determined by 5 μM SCH23390, because there is no binding of SCH23390 in wild-type cells (Machida et al., 1992). Tubes were run in triplicate in a final volume of 500 μl. After incubation at 37°C for 15 min, tubes were filtered rapidly through Skatron glass fiber filter mats (11734) and rinsed with 5 ml of ice-cold wash buffer (10 mM Tris, 0.9% NaCl, pH 7.4) with a Skatron Micro Cell Harvester (Skatron Instruments Inc., Sterling, VA). Filters were allowed to dry, then punched into scintillation vials (Skatron Instruments Inc., Sterling, VA). OptiPhase ‘HiSafe’ II scintillation cocktail (1 ml) was added to each vial. After shaking for 30 min, radioactivity in each sample was determined on an LKB Wallac 1219 Rackbeta liquid scintillation counter.
Effect of agonist exposure on D1 receptor expression levels.
Flasks of cells in the same passage were exposed to 7 ml media B, or 7 ml media B supplemented with 10 μM concentrations of the various drugs for 2 hr. Cells were then rinsed with 7 ml media B (30 min), and then membranes were prepared as described above. Saturation binding studies were done to evaluate the level of expression of receptors in control and desensitized membranes and were the same as the competition studies with the following modifications. Membranes were diluted in assay buffer A and 100 μl of membranes (approximately 50 μg) was incubated with six concentrations of [3H]SCH23390 (0.09–1.1 nM), prepared in assay buffer B. Nonspecific binding was determined using 5 μM SCH23390.
Data analysis.
For dose-response studies, data were calculated for each sample and expressed initially as pmol cAMP per mg protein per min. Base-line values of cAMP were subtracted from the total amount of cAMP produced for each drug condition. To minimize interassay variation, data for each drug were expressed relative to the percentage of the stimulation produced by 100 μM dopamine in each assay. Normalized dose-response curves were analyzed by nonlinear regression with an algorithm for sigmoid curves in the curve-fitting program Prism (Graphpad Inc., San Diego, CA). In all cases, analysis of the residuals indicated an excellent fit with r values greater than 0.99. For each curve, the program provided point estimates of both the EC50 and the maximal stimulation. For desensitization studies, cAMP levels also were expressed initially as picomoles per minute, and then converted to percent dopamine-induced desensitization (dopamine = 100%) in each assay. These values then were averaged to obtain desensitization levels for all drugs studied. Desensitization data were analyzed by one-way analysis of variance, followed by Dunnett’s test. For competition binding studies, the raw data (expressed in dpm) were analyzed by nonlinear regression with a sigmoid dose-response model in Prism. The software generated estimates of both the IC50 and thenH. The IC50 was converted to an apparent K0.5 with the Cheng-Prusoff equation for bimolecular competitive interactions (Cheng and Prusoff, 1973). For saturation studies, the raw data (expressed in dpm) were analyzed by nonlinear regression with a one-site rectangular hyperbola model in Prism. The software generated estimates of both theKD and Bmax for each curve. Bmax estimates were transformed to fmol per milligram of protein, and then converted to percent of control Bmax. These values were analyzed by one-way analysis of variance, followed by Dunnett’s test.
Results
Dopamine-mediated desensitization of the D1receptor.
The initial studies characterized the time course of desensitization of mD1A receptors by pretreatment with dopamine. The desensitization of the mD1Areceptor-stimulated cAMP response was rapid, occurring in minutes, with a significant loss of responsiveness occurring by 5 min (fig.1). Pretreatment of C-6-mD1A cells with 10 μM dopamine produced, in 10 min, a 50% decrease in D1-stimulated cAMP accumulation (i.e., compared with vehicle-treated cells). After a 1-hr drug exposure, the amount of D1-stimulated cAMP accumulation was reduced to approximately 20% of control. It was found that desensitization of D1-stimulated cyclic AMP accumulation was maximal at 2 hr (fig. 1).
The next experiment examined the concentration dependence of dopamine-mediated desensitization of the D1receptor. Pretreatment with increasing concentrations of dopamine led to increasing levels of desensitization of D1-stimulated cAMP accumulation (fig.2). Whereas exposure of C-6-mD1A cells to 100 nM dopamine for 2 hr had little effect on subsequent D1-stimulated cAMP accumulation, pretreatment with 1 and 10 μM dopamine reduced D1-stimulated cAMP accumulation levels by 70 and 85%, respectively (fig. 2). Additional studies were designed to examine the ability of D1 antagonists to block desensitization. C-6-mD1A cells were pretreated with a D1 selective antagonist (1 μM SCH23390) alone, or with SCH23390 plus dopamine. After washing, the response to dopamine after the challenge phase was identical, which indicates that SCH23390 was able to be removed from the receptor and that it was able to prevent the desensitization caused by dopamine (fig. 2).
Affinity analysis of D1 agonists at mD1A receptors.
After characterization of dopamine-induced desensitization, we characterized the pharmacology and efficacy of a variety of D1 receptor agonists. The first set of experiments evaluated the mD1Areceptor affinity of the agonists in C-6 membranes. The rank order of affinity generally is consistent with published data for these compounds (table 1). The Hill slope for compounds that are purported to be full agonists differed markedly (ranging from 0.57 to 0.97). This was particularly evident with A77636, which had a Hill slope of 0.97. The remainder of the full agonists had Hill slopes that are in general agreement with those reported for other agonists.
Concentration-response analysis of D1agonists.
The second set of experiments characterized drugs as full or partial agonists by assessing their intrinsic activity (and potency) for D1-mediated cAMP accumulation in C-6-mD1 cells. The resulting EC50 for dopamine was 1,084 nM, with near-maximal stimulation occurring at 10 μM (table2). As can be seen, DHX, dinapsoline, SKF82958 and A68930 were full agonists compared with dopamine. A77636, a potent agonist at the D1 receptor, had high intrinsic activity but was not a full agonist (its intrinsic activity was 85% that of dopamine). Consistent with previous studies, SKF38393 was a partial agonist (Watts et al., 1995b). Pergolide was also a partial agonist in this preparation. LSD was not tested, because we had determined its partial efficacy in this expression system previously (Watts et al., 1995a). The most potent agonists were A68930 and A77636, members of the isochroman family (DeNinnoet al., 1991a, b). Dinapsoline (a napthisoquinoline), DHX (a hexahydrobenzo[a]phenanthridine) and SKF82958 (a 1-phenyl-tetrahydrobenzazepine) had similar potencies in this preparation, although these were slightly lower than have been observed in membranes from the same cell line (Watts et al., 1995b). SKF38393 and pergolide were the least potent of the agonists tested.
Desensitization of mD1A receptors by “full” and partial D1 agonists.
We examined the ability of full D1 agonists from several structural classes to desensitize D1dopamine receptors in C-6-mD1A cells. We found that 2 hr pretreatment with the high intrinsic activity agonists DHX, SKF82958 and A77636 resulted in marked desensitization of dopamine-stimulated cyclic AMP accumulation (fig.3 and table3). As with dopamine, the degree of desensitization for these agonists was dose dependent and evident after a 1-hr treatment (table 3). In contrast, pretreatment with A68930 and dinapsoline (also full agonists as assessed via stimulation of cAMP accumulation) produced smaller decreases in dopaminestimulated cyclic AMP accumulation and did not appear to be dose dependent. Specifically, pretreatment with A68930 or dinapsoline desensitized D1 receptors by only ∼50% compared with that produced by dopamine pretreatment, even at the highest concentration tested (10 μM) (table 3 and fig. 3).
We also examined the ability of partial agonists to desensitize dopamine-stimulated cyclic AMP accumulation. In these studies, pretreatment with SKF38393 (10 μM) produced a significant decrease in dopamine-stimulated cAMP accumulation, although the magnitude of this decrease was significantly lower than that induced by pretreatment with dopamine at either 1 or 2 hr (table 3 and fig. 3). Additionally, we examined the ability of pergolide and LSD to desensitize D1 receptors and found that both pergolide and LSD produced a pattern of desensitization similar to that of SKF38393, inducing approximately a 60% decrease in dopamine-stimulated cAMP accumulation (table 3). The effects of both pergolide and LSD were significantly different from both vehicle- and dopamine-treated cells.
Effect of agonist exposure on ligand-available receptors.
The relationship between agonist-induced desensitization and receptor down-regulation was examined by assessing the effects of agonist exposure on D1 dopamine receptor expression levels after agonist treatment. Similar to the desensitization studies, C-6-mD1A cells were treated with agonist for 2 hr and rinsed, and then the receptor expression level was evaluated in cell membranes. Saturation binding analysis revealed that pretreatment with the partial agonists SKF38393, pergolide or LSD resulted in no change in receptor expression level or receptor affinity (table4). Conversely, exposure of mD1A receptors to dopamine, DHX, A77636, SKF82958 or dinapsoline resulted in a significant reduction in [3H]SCH23390 binding sites, with each agonist producing an approximately 40% reduction in receptor number compared with vehicle-treated cells. The full agonist A68930 also appeared to reduce D1 dopamine receptors to a similar degree (30% decrease), although this decrease did not reach statistical significance. One unexpected finding was a more pronounced decrease in D1 binding sites after pretreatment with A77636 than after pretreatment with the other agonists. A77636 reduced D1 binding by nearly 80%, whereas pretreatment with the other agonists resulted in only a 30 to 40% reduction. Moreover, whereas pretreatment with most D1 agonists did not alter D1 receptor affinity for [3H]SCH23390, treatment with three of the compounds tested here (dinapsoline, A77636 and SKF82958) did result in small changes in affinity (table 4).
Homologous desensitization of D1 dopamine receptors.
In an effort to characterize further the desensitization of the D1 dopamine receptor in C-6 glioma cells we took advantage of the endogenously expressed BAR. To this end, we pretreated cells with dopamine or isoproterenol, and examined subsequent D1- or BAR-stimulated cAMP accumulation. We found that pretreatment with dopamine reduced subsequent D1-stimulated cAMP accumulation but did not reduce isoproterenol-stimulated cAMP accumulation significantly (fig. 4). In addition, pretreatment with isoproterenol failed to alter subsequent D1-stimulated cyclic AMP accumulation (data not shown). Thus, desensitization of the D1 receptor expressed in C-6 glioma cells is specific to the D1 receptor in that it does not alter the response to isoproterenol, which suggests that the desensitization observed in the C-6-mD1A cells is homologous.
Desensitization is not altered by the PKA pathway.
Stimulation of D1 receptors results in increased cAMP accumulation and an increase in PKA activity. Thus, we assessed the effects of activators of PKA on D1-stimulated cyclic AMP accumulation and D1 receptor desensitization. C-6-mD1A cells were pretreated with the cell-permeable cAMP analogs 8-Br-cAMP or dibutyryl-cAMP, or with the cell permeable activator of protein kinase A, Sp-cAMPS, after which dopamine-stimulated cAMP accumulation was measured. The results of these studies found that pretreatment with these analogs for 1 or 2 hr did not result in desensitization of D1-stimulated cAMP accumulation (table5), which suggests that activation of PKA does not result in significant desensitization. We also examined the ability of PKA activators to potentiate desensitization induced by the partial agonist, SKF38393. The results of these studies revealed that the PKA activators did not enhance the degree of desensitization after pretreatment with SKF38393 alone (table 5).
Although activation of the PKA pathway was not sufficient to result in significant desensitization, we tested the ability of inhibitors of PKA to alter D1-mediated desensitization. To this end, C-6-mD1A cells were pretreated with inhibitors of PKA in the absence or presence of dopamine. The results of these studies demonstrate that pretreatment with H9, HA-1004 or Rp-cAMPS for 1 or 2 hr does not alter dopamine-stimulated cAMP accumulation significantly (table 6). Further, pretreatment with these PKA inhibitors in combination with dopamine (10 μM) did not attenuate dopamine-induced desensitization. These observations suggest that desensitization of the mD1A receptor occurs independently of the cAMP and/or the PKA pathways.
Discussion
Receptor desensitization is characterized either by a loss of, or a reduction in, receptor responsiveness after agonist exposure. This process is important in understanding how chronic drug administration results in tolerance and dependence. Although desensitization has been well characterized for the BARs, the mechanisms appear to depend on the cell type, because both homologous and heterologous desensitization have been observed in different cell systems. Such findings suggest the need for caution in generalizing results to other G-protein-coupled receptors. The present study was designed to characterize more fully D1 agonist-mediated desensitization of C-6 cells transfected with the rhesus macaque D1A receptor (C-6-mD1A cells). Pretreatment of C-6-mD1A cells with dopamine resulted in significant desensitization of D1 receptor responsiveness. This desensitization occurred rapidly and was maximal after a 2-hr drug exposure. In addition, the desensitization was found to be concentration-dependent and specific to the D1 receptor because it was antagonized by the D1 antagonist SCH 23390. These results are similar to those reported in other cell lines with native or transfected D1 receptors, where desensitization occurs rapidly (in minutes to hours: Barton and Sibley, 1990; Balmforthet al., 1990; Ng et al., 1994). Thus, C-6-mD1A cells appear to represent an attractive model system for assessing the effects of intrinsic activity of D1 agonists on D1desensitization because they exhibit the same rank order of affinity and potency of D1 receptor agonists. Moreover, use of this simplified system facilitates exploration of the mechanisms by which desensitization occurs.
To identify potential mechanisms for D1 receptor desensitization, we first sought to characterize more fully desensitization in C-6-mD1A cells. To this end, we found that D1 dopamine receptor-mediated desensitization was homologous, consistent with reports describing D1 receptor desensitization in NS20Y cells and D384 cells (Barton and Sibley, 1990; Balmforth et al., 1990). We then sought to examine the role of cAMP and the PKA pathway involved in D1 receptor desensitization. Whereas activation of D1 receptors stimulated cAMP accumulation and 2 hr pretreatment with D1agonists resulted in desensitization to subsequent stimulation, simple increases in cAMP concentrations (e.g., produced by cell-permeable cAMP analogs) neither caused desensitization of the D1 receptor when applied alone, nor potentiated the partial desensitization observed when applied in combination with partial agonists. Additionally, pretreatment with a direct activator of PKA alone failed to result in desensitization. These results suggest that elevations of cAMP or activation of PKA are not responsible for desensitization. This hypothesis is consistent with earlier reports describing the lack of effect of elevations of cAMP on desensitization of the D1 dopamine receptor (Balmforth et al., 1990; Bates et al., 1991; 1993).
Although direct elevation of cAMP levels or stimulation of PKA alone does not mimic receptor activation for desensitization, blocking the downstream effectors during agonist occupation conceivably may influence desensitization. Thus, we examined the ability of several inhibitors of PKA to alter dopamine-mediated desensitization. None of the compounds tested, H9, HA-1004 and Rp-cAMPS, had any effect on desensitization. The results support the hypothesis that desensitization of the D1 receptor occurs independently of increases in cAMP and subsequent activation of PKA (Balmforth et al., 1990; Bates et al., 1991,1993). The present results differ, however, from those of Blacket al. (1994), who found a critical role for cAMP in prolonged D1 receptor desensitization. Although the precise explanation for these discrepant results is unknown, they likely are caused by different complements of G-proteins, expressed forms of adenylate cyclase and/or additional signal transduction mechanisms within these different cell types.
It is common practice with the D1 receptor to define its intrinsic efficacy based on its ability to stimulate cAMP synthesis. Although cAMP per se may not be involved in the desensitization (see above), it may be that intrinsic activity assessed at this biochemical locus nonetheless predicts the ability to desensitize. In this regard, there seems to be an excellent correlation between agonist efficacy and level of desensitization in many expression systems (Balmforth et al., 1990); although in some expression systems, all agonists produced similar levels of desensitization (Barton and Sibley, 1990). To address this issue in C-6-mD1A cells, we first evaluated the intrinsic activity, as measured by cAMP accumulation in whole cells, of the agonists to be tested for desensitization. A significant advantage of the design of the present study is the availability of “full” agonists from four different chemical classes, as well as several partial agonists.
Our data indicate that the partial agonists SKF38393, pergolide and LSD produced functional desensitization of the C-6-mD1A receptor, although not as efficaciously as dopamine (i.e., only 50% desensitization relative to dopamine). The novel full agonist, DHX, as well as the high intrinsic activity agonists SKF82958 and A77636, functionally desensitized the C-6-mD1A receptor to the same extent as dopamine. At first glance, these data suggest a relation between intrinsic activity and the ability to cause functional desensitization. Yet, pretreatment of C-6-mD1A cells with two other purported full agonists A68930 and dinapsoline resulted in significantly lower desensitization compared with dopamine. These data are summarized graphically in a correlation matrix in figure5. As can be seen clearly, the intrinsic activity (at least as defined by the D1-mediated stimulation of cAMP synthesis) does not predict desensitization.
The lack of relation between agonist efficacy and desensitization is based primarily on the failure of the full agonists A68930 and dinapsoline to fully desensitize the D1 receptor. It is thus important to rule out possible technical artifacts that may be involved in the results with these two compounds. One possibility is that A68930 and dinapsoline remained bound to the receptor and continued to activate adenylate cyclase, thus resulting in elevated cAMP levels. Support for this hypothesis comes from the observations that A68930 has a long biological half-life (DeNinno et al., 1991a). Additionally, A77636, a closely related compound that showed marked receptor reduction (present results), has been shown to increase basal levels of cAMP in SN-K-MC cells after pretreatment, presumably because of its slow off-rate (Lin et al., 1996). The hypothesis of residual bound agonist, however, is not likely for several reasons. First, there was no increase in our basal levels after agonist exposure (data not shown). Further evidence against this hypothesis comes from the observation that pretreatment with A77636, a compound shown previously to have a slow off-rate, resulted in desensitization that was similar to that caused by dopamine pretreatment. Finally, the methods used were able to remove the antagonist SCH23390, even though this ligand has been shown to have a long residence time on the receptor (Schulz et al., 1985).
Although receptor desensitization and down-regulation appear to be separate events (see Sibley et al., 1985), we nonetheless compared agonist-induced receptor alterations and desensitization of D1 dopamine receptors in our system. We found that full agonists from several structural classes (dopamine, DHX, A68930, SKF82958 and dinapsoline) induced significant changes in receptor number (30–40%), and one compound, A77636, reduced receptors by nearly 80%. In contrast, we found that pretreatment of C-6-mD1A cells with the partial agonists SKF38393, pergolide or LSD did not alter receptor levels. This latter finding differs from that of Gupta and Mishra (1993), who showed that extensive pretreatment of SK-N-MC cells with SKF38393 resulted in a 40% decrease in D1 receptors. Thus, changes in receptor number may require an extended pretreatment with partial agonists to alter the number of available receptors. The observation that pretreatment with partial agonists results in desensitization of the D1 receptor and does not cause receptor alterations is consistent with other studies which suggest a distinction between receptor down-regulation and functional desensitization, and support the hypothesis that these two events are separate phenomena (Bates et al., 1993; Ng et al., 1995).
In the present study we have characterized and examined potential mechanisms for D1 receptor desensitization, although the intricacies of the desensitization of the D1A receptor remain largely unknown. We have shown that the desensitization of the D1receptors in C-6 glioma cells occurs rapidly, is homologous in nature and occurs independently of cAMP elevations. The present study also found that structurally dissimilar full D1agonists cause differential effects after occupation of the D1 receptor. Although all full agonists were able to induce functional desensitization, desensitization by DHX, SKF82958 and A77636 were equal to dopamine, whereas A68930 and dinapsoline caused only partial desensitization. Thus, whereas intrinsic activity may be suggestive as to whether an agonist will cause desensitization, it does not account fully for differences observed in the degree of desensitization among agonists, at least in this cell line. Similarly, down-regulation also was not affected consistently, with all the full agonists causing similar changes except for the isochroman, A77636, that induced a significantly greater decrease. Although the mechanisms involved in these agonist-induced changes are not well understood, the present study provides clear evidence that intrinsic activity, functional desensitization and changes in receptor number are not correlated in structurally diverse drugs, and therefore most likely involve different mechanisms. These data also suggest that the consequences of long-term receptor occupation in vivo could differ dramatically among drugs.
The results reported here may have implications for understanding tolerance and dependence induced by drugs of abuse. Whereas many abused substances are thought to mediate their effects indirectly through dopaminergic systems (e.g., amphetamine, methamphetamine, cocaine), others have been shown to have direct effects on D1 or D2 dopamine receptors (Pieri et al., 1978; Watts et al., 1995a). Moreover, recent evidence suggests that drug-induced alterations in intracellular messengers play an important role in opiate and cocaine tolerance and dependence (for a review, see Nestler, 1993, and references therein). For example, chronic opiate or cocaine treatment results in an up-regulation of the cAMP pathway. Recent studies also have shown that activation of D2 dopamine receptors results in heterologous sensitization of the adenylate cyclase pathway (Watts and Neve, 1996). Thus, understanding the mechanisms for biochemical changes after drug exposure is likely to provide important clues for understanding drug dependence. Last, the potential utility of full D1 agonists in the treatment of Parkinson’s disease (Taylor et al., 1991;Kebabian et al., 1992) suggests that studies examining the effects of persistent D1 receptor activation also may have important therapeutic implications.
Acknowledgments
We thank Dr. Kim Neve of Oregon Health Sciences University for the gift of the C-6-mD1A cells, Penny Ferry-Leeper and Stan Southerland for their excellent technical assistance and Dr. Caryn Striplin for her helpful comments concerning the manuscript.
Footnotes
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Send reprint requests to: Dr. Richard B. Mailman, CB 7250, UNC Neuroscience Center (US Mail), 7011 NC Neurosciences Hospital (Express Mail), University of North Carolina School of Medicine, Chapel Hill, NC 27599-7250.
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↵1 This work was supported, in part, by Public Health Service research grants MH40537 and MH42705 from the National Institute of Mental Health, center grants MH33127 and HD03310 and training grants DA07244 and ES07126. Some of these data were presented at the 20th annual meeting of the Society for Neuroscience, Miami Beach, FL [(1994) Soc. Neurosci. Abstr. 20:520].
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↵2 Current address: Veterans Affairs Medical Center, Oregon Health Sciences University, Portland, OR 97201.
- Abbreviations:
- A68930
- 1-aminomethyl-5,6-dihydroxy-3-phenylisochroman
- A77636
- 1-aminomethyl-5,6-dihydroxy-3-adamantylisochroman
- BAR
- beta adrenergic receptor
- 8-Br-cAMP
- 8-bromoadenosine 3′:5′-cyclic monophosphate
- BSA
- bovine serum albumin
- DHX
- dihydrexidine [(±)-trans-10,11-dihydroxy-5,6,6a,7,8,12b-hexahydrobenzo[a]phenanthridine]
- dibutyryl cAMP
- N6,2′-o-dibutyryl adenosine 3′:5′-cyclic monophosphate
- DMEM-H
- Dulbecco’s minimum essential medium supplemented with HEPES
- EGTA
- ethyleneglycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
- H-9 dihydrochloride
- N-(2-aminoethyl)-5-isoquinolinesulfonamide dihydrochloride
- HA-1004 hydrochloride
- N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride
- HEPES
- N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid
- IBMX
- isobutylmethyl xanthine
- LSD
- d-lysergic acid diethylamide tartrate
- mD1A
- macaque D1A receptor
- PKA
- protein kinase A
- Rp-cAMPS
- Rp-cyclic 3′,5′-hydrogen phosphorothioate adenosine triethylamine
- RIA
- radioimmunoassay
- SCH23390
- R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine
- SKF38393
- (±)-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzapine
- SKF82958
- R(+)-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine
- Sp-cAMPS
- Sp-cyclic 3′,5′-hydrogen phosphorothioate adenosine triethylamine
- Received September 3, 1997.
- Accepted March 4, 1998.
- The American Society for Pharmacology and Experimental Therapeutics