Introduction

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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 D_1A and D_1B 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, D_1A, D₅ or D_1B) 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 D₁ receptors (Undie and Friedman, 1990, 1992). In fact, a recent study has demonstrated that D_1A agonist-stimulated phosphoinositide hydrolysis can be observed in transgenic mice lacking a functional D_1A receptor gene, which suggests that this phenomenon is not mediated by D_1A receptors (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 (Taylor et al., 1991; Kebabian et al., 1992), whereas partial D₁ agonists are ineffective (Close et 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 in beta-2 adrenergic receptor desensitization, including the demonstration of both cAMP-dependent and -independent processes (see Harden, 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 D_1A 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 D_1A (mD_1A) 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 mD_1A-transfected glioma cells are consistent with results obtained in rat striatum, and thus represent a valid test system. In addition to our comparisons among D₁ full 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

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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 D_1A receptor (C-6-mD_1A, 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 mD_1A receptor binding sites in untreated cells was approximately 50 fmol/mg protein for C-6-mD_1A cells. 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 × g to 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-mD_1A 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).

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 the n_H. The IC₅₀ was converted to an apparent K_0.5 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 the K_D and B_max for each curve. B_max estimates were transformed to fmol per milligram of protein, and then converted to percent of control B_max. These values were analyzed by one-way analysis of variance, followed by Dunnett's test.

	Results

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Dopamine-mediated desensitization of the D₁ receptor. The initial studies characterized the time course of desensitization of mD_1A receptors by pretreatment with dopamine. The desensitization of the mD_1A receptor-stimulated cAMP response was rapid, occurring in minutes, with a significant loss of responsiveness occurring by 5 min (fig. 1). Pretreatment of C-6-mD_1A 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).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Rapid desensitization of the mD1A receptor. C-6-mD1A cells were pretreated with dopamine (10 µM) for 2 min to 2 hr and washed with assay buffer. Cyclic AMP accumulation then was stimulated with dopamine (10 µM) for 10 min. Data shown are expressed as a percent of dopamine-stimulated cAMP accumulation in vehicle-treated cells and are the mean and standard error of the mean for three independent experiments conducted in duplicate.

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Dose dependence for desensitization of the C-6-mD1A receptor. C-6-mD1A cells were treated with increasing concentrations of dopamine, 1 µM SCH23390 or 1 µM SCH23390 + 10 µM dopamine for 2 hr and washed with assay buffer. Cyclic AMP accumulation then was stimulated with dopamine (10 µM) for 10 min. Data shown are expressed as a percent of dopamine-stimulated cAMP accumulation in vehicle-treated cells and are the mean and standard error of the mean for three independent experiments conducted in duplicate. *Significantly different compared with vehicle-treated cells (Dunnett's post hoc analysis of variance, P < .05).

Affinity analysis of D₁ agonists at mD_1A 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 mD_1A receptor 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 D₁ agonists. 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 (table 2). 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 (DeNinno et 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 mD_1A receptors by "full" and partial D₁ agonists. We examined the ability of full D₁ agonists from several structural classes to desensitize D₁ dopamine receptors in C-6-mD_1A 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 table 3). 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).

View larger version (39K): [in this window] [in a new window]   Fig. 3.   Desensitization of C-6-mD1A receptors by D1 dopamine receptor agonists. C-6-mD1A cells were exposed to drug (10 µM) for 2 hr and rinsed; then cyclic AMP accumulation was stimulated with dopamine (10 µM) for 10 min. Data are expressed as the percent desensitization that resulted after dopamine (10 µM) pretreatment. Data are the mean and standard error of three experiments conducted in duplicate. *Significant difference compared with cells pretreated with dopamine (Dunnett's post hoc analysis of variance, P < .01).

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-mD_1A 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 (table 4). Conversely, exposure of mD_1A 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-mD_1A cells is homologous.

View larger version (44K): [in this window] [in a new window]   Fig. 4.   Homologous desensitization of the mD1A in C-6 glioma cells. C-6-mD1A cells were pretreated with dopamine (10 µM) for 2 hr and rinsed, and cAMP accumulation was stimulated with dopamine (10 µM) or isoproterenol (1 µM). Data are expressed as the percent cyclic accumulation in vehicle-treated cells. Data are the mean and standard error of three experiments conducted in duplicate. *Significant difference compared with vehicle-treated cells (Dunnett's post hoc analysis of variance, P < .05).

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-mD_1A 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 (table 5), 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).

	Discussion

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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 D_1A receptor (C-6-mD_1A cells). Pretreatment of C-6-mD_1A 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; Balmforth et al., 1990; Ng et al., 1994). Thus, C-6-mD_1A cells appear to represent an attractive model system for assessing the effects of intrinsic activity of D₁ agonists on D₁ desensitization 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-mD_1A 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 D₁ agonists 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 Black et 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-mD_1A 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-mD_1A 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-mD_1A 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-mD_1A 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 figure 5. As can be seen clearly, the intrinsic activity (at least as defined by the D₁-mediated stimulation of cAMP synthesis) does not predict desensitization.

View larger version (11K): [in this window] [in a new window]   Fig. 5.   Correlation of intrinsic activity and ability to desensitize. This graph uses the data from tables 2 and 3 for DA, DHX, dinapsoline, SKF38393, SKF82958, A68930, A77536 and LSD. As shown by the nonsignificant linear correlation coefficient, full intrinsic activity, regarding cAMP accumulation, does not predict the ability to cause 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-mD_1A 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 D_1A receptor remain largely unknown. We have shown that the desensitization of the D₁ receptors 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 D₁ agonists 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.