Abstract
Dihydrexidine (DHX), the first high-affinity D1 dopamine receptor full agonist, is only 10-fold selective for D1versus D2 receptors, having D2 affinity similar to the prototypical agonist quinpirole. The D2 functional properties of DHX and its more D2 selective analogN-n-propyl-dihydrexidine (PrDHX) were explored in rat brain and pituitary. DHX and PrDHX had binding characteristics to D2 receptors in rat striatum typical of D2 agonists, binding to both high- and low-affinity sites and being sensitive to guanine-nucleotides. Consistent with these binding data, both DHX and PrDHX inhibited forskolin-stimulated cAMP synthesis in striatum with a potency and intrinsic activity equivalent to that of quinpirole. Unexpectedly, however, DHX and PrDHX had little functional effect at D2 receptors expressed on dopaminergic neurons that mediate inhibition of cell firing, dopamine release, or dopamine synthesis. Quantitative receptor competition autoradiography demonstrated that DHX bound to D2 receptors in striatum (predominantly postsynaptic receptor sites) with equal affinity as D2 sites in the substantia nigra (autoreceptor sites). The data from these experiments, coupled with what is known about the location of specific dopamine receptor isoforms, lead to the hypothesis that DHX, after binding to D2L and D2Sreceptors, causes agonist-typical functional changes only at some of these receptors. This phenomenon (herein termed “functional selectivity”) suggests that drugs may be targeted not only at specific receptor isoforms but also at separate functions mediated by a single isoform, yielding novel approaches to drug discovery.
The original D1 and D2pharmacological subclasses of dopamine receptors (Kebabian and Calne, 1979) is now known in mammals to consist of two (Garau et al., 1978) D1-like receptors (D1A/D1 and D1B/D5) and products of three D2-like genes that result in four receptors: D2long (D2L) and D2short (D2S), D3, and D4. D1-like receptors preferentially recognize 1-phenyl-tetrahydrobenzazepines (e.g., SCH23390) over benzamides (e.g., sulpiride), whereas the D2-like receptors have the opposite pharmacological specificity. D1- and D2-like receptors have been defined traditionally by their opposing effects on the enzyme adenylate cyclase, with D1 receptors positively coupled to this enzyme, whereas D2 receptors are either negatively coupled or uncoupled to this effector. More recently, the actions of dopamine D1- and D2-like receptors on signaling systems other than adenylate cyclase have been confirmed in a variety of systems, including coupling to G protein inwardly rectifying potassium channels, phosphatidylinositol hydrolysis, and voltage-activated calcium channels (Jaber et al., 1996).
Dopamine D2-like receptors have been a primary target of drug development efforts for treating disorders of dopamine neurotransmission such as Parkinson's disease and schizophrenia. The functional effects of dopamine D2 receptor ligands in brain arise from their interactions with dopamine D2 autoreceptors expressed on dopamine neurons and with D2 heteroreceptors on target cells. D2 autoreceptors regulate the firing of dopamine neurons, as well as the synthesis and release of dopamine. In the striatum, the major dopamine terminal field in mammalian brain, D2 heteroreceptors include those present on giant cholinergic interneurons that regulate the release of acetylcholine, as well as D2 receptors expressed on medium spiny γ-aminobutyric acidergic afferent neurons (Le Moine and Bloch, 1991;Le Moine et al., 1991; Weiner et al., 1991). In anterior pituitary, D2 heteroreceptors regulate the release of prolactin (Caron et al., 1978). Although there has been controversy about how the various molecular isoforms of D2-like receptors contribute to the myriad of functional events mediated by these receptors in brain and neuroendocrine tissues, it is clear that functional diversity is not defined solely, or even primarily, by molecular diversity. For example, D2L, D2S, and D3 receptors are expressed as both autoreceptors and heteroreceptors (Bouthenet et al., 1991; Snyder et al., 1991). Although it has been suggested that D3 receptors have a primary role as autoreceptors (Meller et al., 1993; Aretha et al., 1995; Kreiss et al., 1995), this notion has not been supported by more recent studies in which D2 or D3 receptors have been ablated using transgenic methods (Koeltzow et al., 1998; L'hirondel et al., 1998).
Dihydrexidine (DHX) and related members of the hexahydrobenzo[a]phenanthridine class have been shown in previous publications to comprise a particularly interesting class of dopamine agonists (Lovenberg et al., 1989; Brewster et al., 1990, 1995;Mottola et al., 1992). In addition to its properties as a high-affinity, full D1 receptor agonist, DHX also has affinity for D2 receptors similar to the prototypical D2 receptor agonist quinpirole. Although there has been extensive characterization of the D1 properties of DHX (Watts et al., 1993b, 1995), there is only preliminary data about the functional properties of DHX at D2 receptors. These latter data suggest, however, that DHX has typical D2 agonist properties, binding to both high- and low-affinity [3H]spiperone-labeled sites, inhibiting prolactin release in vivo, and also inhibiting D1-stimulated cAMP efflux much like dopamine or other D2 agonists (Mottola et al., 1992).
Together, it appears that members of the benzo[a]phenanthridine series might have particular pharmacological utility by virtue of their being high-affinity full agonists for both D1- and D2-like receptors. Moreover, Brewster et al. (1990) have shown that the D1/D2 selectivity of this series of rigid dopamine analogs can be “tailored” (e.g.,N-n-propyl-DHX has twice the D2 affinity of DHX, yet has much lower D1 affinity). The present studies were designed to provide a full pharmacological characterization of two key members of this series, dihydrexidine andN-n-propyl-dihydrexidine (PrDHX) (Fig.1) with functions in native tissues that are generally accepted as being mediated by D2-like receptors. Contrary to the initial hypothesis, the current data lead to the surprising conclusion that DHX can activate D2 receptors on striatal target cells to produce maximal inhibition of adenylate cyclase, while having essentially no effects at D2 autoreceptors coupled to dopamine release, synthesis, or cell firing (despite binding to these receptors with equal affinity). Understanding the mechanisms underlying such “functional selectivity” may provide novel directions for drug design.
Experimental Procedures
Materials
(6a,12b)-trans-10,11-Dihydroxy-5,6,6a,7,8,12b-hexahydrobenzo- [a]phenanthridine (DHX), N-methyl-DHX, and PrDHX were synthesized as described previously (Brewster et al., 1990). [3H]Spiperone was purchased from Amersham Biosciences (Piscataway, NJ), and125I-epidepride was synthesized by Dr. Chester A. Mathis (University of Pittsburgh, Pittsburgh, PA), using a published protocol (Bishop et al., 1991). Apomorphine and U86170E were from Pharmacia & Upjohn, Inc. (Kalamazoo, MI).125Iodine and [3H]NPA were purchased from PerkinElmer Life Sciences (Boston, MA). SCH23390 was a gift from Schering Plough (Kenilworth, NJ) or was purchased from Sigma/RBI (Natick, MA), as was SKF38393, R-(+)- andS-(+)-NPA, R-(+)- and S-(−)-3-PPP, and haloperidol. Quinpirole (LY171555) was a gift from Lilly Research Laboratories (Indianapolis, IN). Spiperone, ketanserin, and domperidone were gifts from Janssen Pharmaceutica (Titusville, NJ). Remoxipride was a gift from Astra Arcus AB (Södertälje, Sweden). EDTA, isobutylmethylxanthine, GppNHp, dopamine, (−)-sulpiride, reserpine, and chlorpromazine were purchased from Sigma-Aldrich (St. Louis, MO). Tris-HCl and HEPES were purchased from Research Organics (Cleveland, OH). The complexing agent 1,4,7,10,13,16-hexaoxacyclooctadecane (18-crown-6) was purchased from Fluka Chemical (St. Louis, MO). Anti-cAMP primary antibody for cAMP assays was obtained from Dr. Gary Brooker (Georgetown University School of Medicine, Washington, DC).
Animals and Tissue Preparation
All experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the National Institutes of Health. Male Sprague-Dawley rats (Charles River Laboratories, Raleigh, NC) were housed under a 12-h light/dark cycle and given food and water ad libitum. In all experiments, rats weighing 250 to 400 g were sacrificed by decapitation. Some rats were treated with 5 mg/kg reserpine 20 h before euthanasia. Except where indicated, brains were immediately removed then rinsed and chilled in ice-cold 0.9% (w/v) sodium chloride solution. The striatum was dissected from two 1.2-mm coronal slices that were made with the aid of a cold dissecting block. The tissue was either used immediately as indicated, or was stored at −70°C for later use.
Radioligand Receptor Binding
Membrane Preparation.
Radioligand binding followed published methods (Schulz et al., 1985) with minor modifications. Frozen rat striata were homogenized in ice-cold 50 mM HEPES, 4 mM MgCl2 buffer, pH 7.4, at 25°C. This buffer was used throughout the binding experiments, except that for experiments with GppNHp or [3H]NPA, the buffer was modified to include 1 mM EDTA and 0.002% ascorbic acid. Homogenized tissue was centrifuged at 27,000g for 10 min. The pellet was again homogenized (five strokes by hand) with resuspension and centrifugation as described above. The final pellet was suspended at a concentration of approximately 2.0 mg wet weight/ml of binding buffer. In some cases, membranes were preincubated for 30 min at room temperature with 100 μM GppNHp before performing the radioligand binding experiments. The final tissue concentration per tube in all binding assays was 1 mg/ml. Protein levels were later estimated using the Folin reagent method (Lowry et al., 1951).
[3H]Spiperone-Labeled D2Receptors.
Ten to 15 concentrations of unlabeled competitor were assessed at D2 receptors labeled with 0.07 nM [3H]spiperone, as described previously (Mottola et al., 1992).
[3H]SCH23390-Labeled D1 Receptors.
Competition binding assays at D1 receptors were performed as described previously (Watts et al., 1995). D1 receptors were labeled with 0.3 nM [3H]SCH23390. Nonspecific binding was defined by 1 μM unlabeled SCH23390.
[3H]NPA-labeled D2 Receptors.
D2 receptors in their high-affinity state were labeled with 0.05 nM [3H]NPA. Previous saturation binding experiments conducted with [3H]NPA in this laboratory had determined that [3H]NPA binds to striatal D2 receptors with aKD of ca. 0.1 nM. TheBmax for [3H]NPA binding was found to be approximately 200 fmol/mg of protein, roughly one-half that of [3H]spiperone, suggesting that, under our experimental conditions, half of the striatal D2receptors were in the high-affinity state. Competition binding of [3H]NPA-labeled sites was determined as described for [3H]spiperone, with the exception that the incubation period was 30 min.
Receptor and Other Data Analyses
Data for the receptor competition binding experiments were analyzed by nonlinear regression (Prism; GraphPad Software, San Diego, CA) to provide estimates of receptor affinity (IC50) and indirect Hill coefficients (nH). Apparent affinity (K0.5) was determined from IC50 values by adjusting for ligand concentration using the Cheng-Prusoff equation for bimolecular interactions. Other selected data were analyzed using a one-way analysis of variance, followed by Tukey-Kramer multiple comparison post hoc tests using InStat version 3.05 (GraphPad Software).
DHX Accumulation in Brain
Sample Preparation.
Rats were dosed with 3.0 mg/kg DHX or PrDHX s.c. dissolved in 0.1% ascorbic acid. Rats administered DHX were euthanized by decapitation at 10, 20, 40, or 120 min later. Rats dosed with PrDHX were euthanized at a single time point, 20 min after drug administration. The brains were removed quickly, and the striata excised as described above. Whole cerebella were gently peeled off the back of the brain and brain stem. Wet weights were obtained for each brain region dissected, the brain tissue placed in plastic vials, and stored at −70°C. On the day of the assay, brain nuclei were sonified using a sonicating tip (Branson Ultrasonics, Danbury, CT) in 1 ml of mobile phase containing a fixed amount of the internal standardN-methyl-DHX. After centrifugation (15,000g for 15 min), 600 μl of the supernatant was added to tubes containing 400 μl of 1 mM Tris buffer, pH 8.6, and 30 mg of alumina (Bioanalytical Systems, West Lafayette, IN). After shaking for 5 min, the alumina was separated by brief centrifugation and the fluid aspirated. The alumina was then washed twice by adding 1.5 ml of double-distilled H2O and shaking for 2 min. Catecholamines were eluted from alumina by addition of 400 μl of perchloric acid (0.05 M) and shaking for 5 min. The eluate was prepared for HPLC by passing it through a 20-μm syringe-type filter (Gelman Instrument Co., Ann Arbor, MI). Separation by HPLC was performed as described below using 100-μl sample injections. Drug concentrations in cerebellum and striatal tissue were determined at each sacrifice time point. Comparisons were made of the relative drug concentrations found in the two brain regions to assess high-affinity accumulation of DHX and PrDHX. Data in each brain area were expressed as picograms per milligram of tissue.
Separation.
To determine the time course of DHX accumulation in brain after s.c. dosing, we used ion-pair chromatography followed by electrochemical detection similar to the analysis of catecholamines and metabolites in brain tissue (Kilts et al., 1981). The mobile phase used to separate DHX and endogenous catechols consisted of 50 mM Na2PO4, 30 mM citric acid, 4 mM 18-crown-6, 86 μM sodium octyl sulfate, 0.1 mM disodium EDTA, and 11.5% acetonitrile (v/v). Diethylamine (0.1% v/v) was added to minimize secondary interactions between DHX and unreacted silanol residues. The mobile phase was adjusted to pH 4.5 before the addition of acetonitrile. The ion-pair reagent 18-crown-6 was added to the mobile phase to retain selectively the primary monoamines dopamine, DOPAC, and norepinephrine, but not the late-eluting secondary amine DHX. Dihydroxybenzoic acid was used as the internal standard with this mobile phase. When quantification of only DHX was desired, the 18-crown-6 and octyl sodium sulfate were removed and the acetonitrile concentration increased to 15%. Isocratic mobile phase flow at 0.75 ml/min was accomplished with an HPLC pump (Anspec Analytical, Geneva, IL). An Ultramex C18 stainless steel column (150 × 4.6-mm i.d.) packed with 3-μm microparticulate silica (Phenomenex, Torrance, CA) was used for all separations. Electrochemical detection was accomplished with a detector (model 400; Princeton Applied Research, Princeton, NJ). The potential of the glassy-carbon working electrode was set at 0.6 V versus an Ag/AgCl reference electrode.
Prolactin Release Assay
To measure the inhibition of prolactin release after activation of D2 dopamine receptors, male rats (350–400 g) were given i.p. injections of the serotonin precursor 5-HTP (30 mg/kg) to stimulate an increase in serum prolactin concentration. The 5-HTP pretreatment group consisted of four rats for each drug condition, whereas the control group (saline vehicle without 5-HTP) consisted of two rats. Five minutes after the 5-HTP injection, the rats were injected i.p. with DHX (10 mg/kg), quinpirole (10 mg/kg), PrDHX (5 mg/kg), or 0.1% ascorbic acid vehicle. Rats were placed singly in plastic cages containing bedding for 25 min, after which they were sacrificed by decapitation. Trunk blood was collected in chilled plastic centrifuge tubes, and allowed to clot slowly at 4°C. Serum was collected by centrifugation at 1000g for 15 min at 4°C and stored at −20°C until the time of the assay. Serum prolactin was assayed by radioimmunoassay (RIA) with reagents and procedures supplied by the Hormone Distribution Program of the National Institute of Diabetes and Digestive and Kidney Diseases.
cAMP Efflux from Striatal Slices
cAMP efflux was measured using fresh striatal tissue obtained as described above and sliced further in two directions (90° apart) using a McIlwain tissue chopper (400-μm setting). As they were being collected, slices were transferred to a test tube containing chilled, oxygenated Krebs' buffer (121 mM NaCl, 4.84 mM KCl, 1.89 mM CaCl2, 1.16 mM MgSO4, 1.17 mM KH2PO4, 25 mM NaHCO3, 0.06 mM ascorbic acid, and 10 mMd-glucose, pH 7.35). Slices were subsequently washed with three 5-ml aliquots of Krebs' buffer and allowed to settle; slices (35 μl) were transferred to each well of the superfusion apparatus, and washed at 0.1 ml/min for 90 min. The perfusing Krebs' buffer (37°C), but not drug-containing solutions, was oxygenated with 95% O2, 5% CO2. Baseline aliquots of perfusate were collected for the first three 15-min intervals, after which perfusion aliquots were collected in four 10-min intervals. Differences in amounts of tissue per well were normalized by permitting each superfusion chamber to serve as its own control. The amount of drug-induced cAMP efflux was expressed as percentage of the basal efflux. Duplicate samples were analyzed by RIA (vide infra).
Adenylate Cyclase Assays in Rat Striatal Homogenates
Adenylate cyclase assays in rat striatal homogenates were performed as described by Watts et al. (1993b). A 20-μl aliquot of a 2.5-mg/ml homogenate solution was added to the reaction tubes. Baseline values of cAMP were subtracted from the total amount of cAMP produced for each drug condition. Data for each drug were expressed initially as picomoles of cAMP per milligram per minute, and converted to the percentage of stimulation produced by 5 μM forskolin.
Radioimmunoassay of cAMP
The concentration of cAMP in samples from superfusion or striatal homogenates was assayed by RIA of acetylated cAMP (Watts et al., 1993a). Normalized dose-response curves were analyzed by nonlinear regression using an algorithm for sigmoid curves using Prism (GraphPad Software). For each curve, the program provided point estimates of both the EC50 and the maximal effect, either inhibition or stimulation produced (i.e., top or bottom plateau of sigmoid curve).
Tyrosine Hydroxylase Activity in Striatal Slices
Activity of tyrosine hydroxylase, the rate-limiting step in dopamine biosynthesis, was assessed by measuring the formation of [14C]carbon dioxide (14CO2) evolved during the synthesis of dopamine from [1-14C]l-tyrosine. Aliquots (ca. 7.5 mg of tissue) of a 25-mg/ml suspension were added to tubes containing test ligands. Values of 14C cpm/30 min/assay tube, less a blank value (tissue omitted) were determined. Basal (control) tyrosine hydroxylase activity previously was found to be ca. 16.9 ± 1.2 pmol of14CO2/30 min/mg of protein. Data (as percentage of corresponding basal control values) were pooled and expressed as mean ± S.E.M.
In Vitro Voltammetry: Dopamine Release from Striatal Slices
Slices (350 μm in thickness) were prepared from the striatum of male Sprague-Dawley rats using a Lancer vibratome as described previously (Kelly and Wightman, 1987). The slices were submerged in a Scottish-type chamber, and perfused at 1 ml/min using a flow system equipped with a two-position, six-port valve. The perfusion medium, preheated to 32°C and saturated with 95% O2, 5% CO2, consisted of Krebs' buffer containing 126 mM NaCl, 2.5 mM KCl, 1.2 mM NaH2PO4, 2.4 mM CaCl2, 1.2 mM MgCl2, 25 mM NaHCO3, and 11 mM d-glucose, pH 7.4. Nafion-coated carbon-fiber microelectrodes were prepared as described previously (Kristensen et al., 1986). Fast-scan cyclic voltammetry used a commercially available potentiostat (EI-400; Ensman Instrumentation, Bloomington, IN). A sodium-saturated calomel electrode was used as a reference electrode, and all voltages are reported versus this standard. The electrode potential was linearly scanned from −400 to 900 mV and back to −400 mV at 300 V/s every 100 ms. This voltage range encompasses the oxidation of dopamine (at 500–700 mV) and the reduction of the quinone form of dopamine (at 0 to −200 mV). The current in the voltage range of the oxidation of DA is proportional to the dopamine concentration at the electrode. To obtain DA concentration versus time plots, the current at the oxidation potential for DA, obtained for each potential scan, is integrated and converted to concentration based on postcalibration of the electrode. Thus, the time plots have 100-ms resolution in the time axis. The time response of the electrode is determined in a flow stream and was always <200 ms to reach 50% of maximum response.
Electrical stimulation was accomplished with a twisted wire, bipolar electrode with 100-μm tips (Plastics One, Roanoke, VA) separated by approximately 500 μm. Biphasic stimulation pulses, 2 ms in width each phase, were generated by a locally constructed stimulator and optically isolated by Neuro Log System stimulus isolators (Medical Systems, Greenvale, NY). Ten 300-μA pulses at 20 Hz were used. The stimulating electrodes were mounted on a micromanipulator and positioned at the surface of the slice near its center. The working electrode, mounted on a separate micropositioner, was lowered to 75 μm below the surface of the slice at a position 100 to 200 μm from the center of the stimulating electrode pair. Electrode placements were made with the aid of a stereomicroscope (Bausch & Lomb, Rochester, NY). Evoked DA overflow was first determined by obtaining responses to electrical stimulation with the slices perfused with physiological buffer. Then, physiological buffer containing drug was applied for 20 min and the responses recorded again. Data were initially expressed as current obtained after release, subtracting background current obtained before release. Values for dopamine D2 agonists were then converted to a percentage of electrically stimulated release. Data are reported as mean ± S.E.M. of three to six slices, typically from different animals.
Cerebral Microdialysis in Freely Moving Animals
Guide cannulae (CMA/12; Bioanalytical Systems) were implanted with the aid of stereotaxic instruments (David Kopf Instruments, Tujunga, CA) at the dorsal surface of the body of the caudate putamen in rats anesthetized with pentobarbital (35 mg/kg i.p.) and ketamine (90 mg/kg i.m.). Relative to bregma, the coordinates were 1 mm anterior, 3 mm lateral, and 4 mm below skull surface. The cannula was cemented in place using cranioplastic cement and stainless steel mounting screws (Plastics One). The animals were housed individually for recovery. Rats with implanted guide cannulae were transferred to an isolated room 7 days later, placed in experimental cages (CMA/120 microdialysis system for awake animals; Bioanalytical Systems), and probes inserted (CMA/12, 4-mm fiber length, concentric design; Bioanalytical Systems). Probes were perfused at 2 μl/min (model 22 syringe pump; Harvard Apparatus, South Natick, MA) with a physiological Ringer's solution [120 mM NaCl, 1.2 mM CaCl2, 5 mM KCl, 1.2 mM MgCl2, and 0.15% (v/v) phosphate-buffered saline, pH 7.4]. Twelve to 18 h after probe insertion, samples were collected with the aid of a refrigerated fraction collector (CMA/170 refrigerated fraction collector; Bioanalytical Systems) at 20-min intervals. After collection of six to eight baseline samples, drugs were administered by s.c. injection or through inclusion in the dialyzing solution. Postdrug samples were collected for a 2- to 3-h period. Samples for dopamine overflow studies were collected in tubes containing a small volume of the HPLC mobile phase described below, and containing isoproterenol as an internal standard. Samples were frozen immediately and analyzed for dopamine, DOPAC, HVA, and 5-HIAA by using reverse phase HPLC-EC.
Quantitative determinations for dopamine and metabolites were made using the same stationary phase and detector as described above for the DHX accumulation studies. The electrode potential was set at +0.75 V versus a Ag/AgCl reference electrode. The mobile phase consisted of 0.05 M Na2HPO4, 0.03 M citric acid, 0.1 mM disodium EDTA, 2 mM sodium octyl sulfate, and 20% methanol, with a final pH of 3.4 and a flow rate of 0.80 ml/min. The column temperature was maintained at 39°C. Standard curves for the quantification of dopamine, DOPAC, and HVA were prepared by analyzing a series of solutions containing a fixed amount of isoproterenol and varying amounts of each compound. The HPLC data were collected and digitized by Analytic 900 series modules and Turbochrom software (PerkinElmer Instruments, Norwalk, CT) by measuring the peak height for each compound relative to the internal standard. Absolute values for each analyte were then normalized relative to the baseline values obtained before drug administration.
Electrophysiology: Firing of Substantia Nigra Dopamine Neurons
Electrophysiological studies were performed in accordance with the methods described by Piercey et al. (1997). Briefly, adult rats were anesthetized with chloral hydrate (400 mg/kg i.p.) and the femoral artery and vein were cannulated for drug administration and blood pressure monitoring, respectively. Drugs, measured as their salt, were dissolved in distilled water and injected intravenously in volumes of 0.15 ml or less. Stereotaxic coordinates were used for electrode placement in the substantia nigra pars compacta. Glass microelectrodes filled with pontamine blue in 2 M NaCl (4–10-MΩ impedance) were used for extracellular recordings. Dopaminergic neurons were identified by their long-duration action potentials (>2.5 ms), shape, and firing rates (Bunney et al., 1991). Changes in neuronal firing rates were monitored as described previously (Piercey et al., 1997). Dose-response studies were based on cumulative dosing schedules. Injections were timed 1 to 2 min apart to allow maximal response, as well as sufficient accumulation of drug to approximate concentrations expected with single bolus injections.
D2 Receptor Autoradiography
Tissue Processing.
D2 receptor autoradiography buffer contained 50 mM HEPES, 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, and 4 mM MgCl2, pH 7.4. D2 receptors were labeled with 0.03 nM125I-epidepride, with nonspecific binding defined by 1 μM domperidone. Twelve consecutive sections were incubated with one of 12 concentrations of dihydrexidine (0.01 nM to 100 μM). Ketanserin (400 nM) was included in all mailers to mask binding of the radioligand to 5-hydroxytryptamine2 sites. Slides were incubated at room temperature for 4 h. Dried sections were then apposed to Kodak X-OMAT RP film, along with brain paste standards containing known amounts of radioactivity (Amersham Biosciences). Autoradiograph cassettes were stored in the dark at room temperature then the films developed after appropriate exposure duration (usually ca. 2–3 days). Films of substantia nigra sections were exposed for substantially longer periods due to much lower levels of D2 binding compared with caudate nucleus.
Image Analysis.
Films were developed and MCID image processing system (Imaging Research, St. Catherine's, Ontario, Canada) was used to construct a standard curve relating radioactivity to film density, and to quantify the amount of radioactivity bound to tissue. Film images of nonspecific binding were not visible (<5% of total binding); thus, nonspecific binding was not subtracted from either total binding or competition points. Prism (GraphPad Software) was used to obtain curve-fitting parameters (K0.5 andnH) from nonlinear regression analyses of competition curves.
Results
Competition Binding in Rat Striatum.
DHX has affinity for D2 receptors labeled with [3H]spiperone (Fig.2; Table1), as well as for sites labeled with the D1 receptor antagonist [3H]SCH23390 (Table 1). Compared with DHX, the analog PrDHX has 100-fold lower affinity for [3H]SCH23390 sites, and somewhat higher affinity for [3H]spiperone sites. Thus, DHX displays approximately 10-fold D1/D2 receptor selectivity, whereas PrDHX displays the reverse selectivity. The affinities of both DHX and PrDHX for [3H]spiperone sites are similar to that of the prototypical D2 receptor agonist quinpirole. Also similar to quinpirole, the slopes of the competition curves of DHX and N-propyl-DHX for [3H]spiperone-labeled D2sites are shallow, whereas the competition curve for the antagonist chlorpromazine displays normal steepness. Preincubation of striatal membranes with 100 μM GppNHp produces 2- to 3-fold rightward shifts in the competition curves for DHX and PrDHX, although no curve steepening is detected (Fig. 2, A and B); the antagonist chlorpromazine is unaffected by GppNHp. The high-affinity interaction of DHX and PrDHX with D2 receptors is evidenced also by the low nanomolar K0.5 of both compounds for D2 receptor sites labeled with the agonist [3H]NPA (Table 1; Fig. 2C).
Demonstration of Bioavailability of DHX and PrDHX.
Darney et al. (1991) and Smith et al. (1997) reported that administration of DHX and PrDHX by s.c. injection produces acute effects on behaviors that are known to be modulated by dopamine receptors in the central nervous system. We developed a sensitive HPLC-EC detection method to allow direct demonstration of the time course and extent of accumulation of DHX and PrDHX in dopamine terminal areas. These data provide a basis for evaluating the effects of these compounds in whole animal D2 receptor functional assays that entail peripheral drug administration.
As shown in Fig. 3, DHX (3 mg/kg) rapidly accumulates in brain after acute s.c. administration. Furthermore, there appears to be a greater concentration of DHX in brain areas with high dopamine innervation (e.g., striatum versus cerebellum), consistent with the idea that some of the DHX being measured is that bound to dopamine receptors (Rollema et al., 1986). In both striatum and cerebellum, the concentration of DHX is maximal within the first 20 min and declines relatively rapidly thereafter. Little drug is detected in brain 2 h after s.c. dosing, and thet1/2 is clearly small (i.e., on the order of minutes). PrDHX also distributes rapidly to dopamine terminal areas after s.c. dosing, and the amount accumulated in striatum appears to be at least 2-fold greater than for DHX at 20 min (data not shown). The limited quantities of PrDHX that were available precluded full characterization of the time course of its distribution in brain.
Prolactin Release.
One well characterized functional effect of D2 receptor activation is the inhibition of prolactin release in vivo. Dopamine and D2 agonists inhibit prolactin release, whereas D2 receptor antagonists increase prolactin release by inhibiting the effects of endogenous dopamine. Prolactin release was stimulated by the serotonin precursor 5-HTP, and rats were subsequently treated with the test drugs. DHX (10 mg/kg) significantly inhibited the release of prolactin, as did quinpirole (10 mg/kg; Fig.4), consistent with our previous report (Mottola et al., 1992). At a dose of 5 mg/kg, PrDHX inhibited the release of prolactin nearly as well as DHX, although its response is somewhat less than that of quinpirole.
cAMP Efflux from Striatal Slices.
As shown in Fig.5, in striatal minces, DHX or SKF38393 stimulates cAMP efflux. The net effect for DHX and dopamine has been hypothesized to represent a balance between D1receptor-mediated stimulation and D2receptor-mediated inhibition. Consistent with this, when D2 receptors are blocked with the selective D2 antagonist domperidone (10 μM), the total amount of cAMP in the superfusate is increased. In contrast, the addition of domperidone does not affect cAMP efflux induced by the selective D1 agonist SKF38393. These data are consistent with the idea that DHX activates striatal D2 receptors that are negatively coupled to adenylate cyclase, and replicate a previous study with DHX (Mottola et al., 1992) in which the D2 antagonist sulpiride was used. Thus, the net effect of DHX or DA on cAMP efflux can be interpreted as being a balance between D1receptor stimulation and D2 receptor inhibition due to the mixed agonist properties, whereas SKF38393 acts only at D1 receptors in this preparation.
Inhibition of Forskolin-Stimulated cAMP Synthesis in Striatal Membranes.
The preceding cAMP efflux data with ex vivo superfused striatal slices indicate that DHX has agonist properties at D2 receptors, but this technique has practical limitations on the number of samples that can be studied in a single assay. For this reason, further studies evaluating D2 agonist inhibition of adenylate cyclase were performed in striatal membranes. The levels of cAMP were elevated artificially using 5 μM forskolin to permit easier evaluation of the effects of activation of D2 receptors. This concentration of forskolin produces a 10- to 15-fold increase in basal levels of cAMP under our assay conditions.
In striatal homogenates, as in superfused striatal slices, nonselective dopamine agonists exhibit both stimulatory and inhibitory effects at adenylate cyclase through their interaction with D1 and D2 receptors, respectively. Figure 6A depicts four concentration-response curves for dopamine: alone, in the presence of the D1 receptor antagonist SCH23390, in the presence of the D2 receptor antagonist sulpiride, or in the presence of both antagonists. Whereas dopamine alone produces a net inhibitory effect on forskolin-stimulated cAMP levels, inclusion of sulpiride (to block D2 receptor-mediated inhibition) results in a 20% increase over forskolin-stimulated levels, reflecting the remaining stimulatory action of dopamine mediated via D1 receptors. Conversely, inclusion of the D1 receptor antagonist SCH23390 eliminates this stimulatory effect of dopamine, leaving only the inhibitory effect mediated via D2 receptors.
This interpretation is consistent with the data seen with quinpirole (Fig. 6D) that demonstrate a similar extent of D2-mediated inhibition. Unlike DA or DHX, the inclusion of sulpiride with quinpirole does not increase the cAMP synthesis produced by forskolin consistent with the very high D2/D1 receptor selectivity of quinpirole. The concentration-response curves for DHX (top right) and PrDHX (bottom left) indicate that both compounds produce D2 receptor-mediated decreases in cAMP synthesis, with potency and intrinsic activity similar to that of dopamine and quinpirole. Like dopamine, DHX also displays D1agonism, as indicated by the stimulation of cAMP accumulation observed with the inclusion of sulpiride. On the other hand, this D1 receptor effect only begins to be seen at very high concentrations of PrDHX, consistent with the relative D2/D1 selectivity of this compound.
The intrinsic activity of DHX, PrDHX, and several prototypical D2 agonists at forskolin-stimulated cAMP accumulation are shown in Table 2. To eliminate artifacts due to D1 receptor-mediated stimulation, the data shown represent the maximal inhibition elicited by each compound in the presence of SCH23390. DHX and PrDHX elicit inhibition comparable with dopamine and a variety of high intrinsic efficacy agonists, including quinpirole and R-(−)-NPA. Compounds that are structurally related to DHX (dinapsoline, 4-Me-PrDHX, 4-Me-DHX, and 3-Me-PrDHX) exhibit profiles that are similar to that of DHX, with only minor changes in intrinsic activity and potency (data not shown). In contrast, two compounds generally accepted as being partial agonists, S-(+)-NPA andS-(+)-3-PPP, had low intrinsic activity in this experiment. Table 2 also indicates that the inhibitory effects of all test compounds were fully reversed by inclusion of the D2 receptor antagonist sulpiride.
Effects on Dopamine Synthesis.
Although cAMP efflux in striatum is largely mediated by D2-like receptors on target cells, there are D2-like receptors on processes of dopamine neurons that control dopamine biosynthesis via modulation of tyrosine hydroxylase (TH), the rate-limiting enzyme of the dopamine synthetic pathway. Dopamine and D2agonists activate these autoreceptors, resulting in a decrease in the activity of TH. As shown in Fig. 7, a typical D2 receptor agonist such as quinpirole inhibited TH activity in a manner that is reversed by spiperone, but not SCH23390. In contrast, although DHX and PrDHX (1 μM) both induced decreases in TH activity (Fig. 7), these effects were notreversed by spiperone. It thus appears that DHX and other members of the benzo[a]phenanthridine class inhibit the enzyme by a mechanism that is not receptor-mediated. Table3 illustrates data in support of the hypothesis that any catechol may suppress tyrosine hydroxylase activity by a non-D2 receptor-mediated mechanism (Nagatsu et al., 1964). Although both (R)-NPA and (S)-NPA inhibit TH, their effects are reversed by D2antagonists. Conversely, several catechol-containing drugs (the selective D1 partial agonist SKF38393, as well as DHX or PrDHX) cause TH inhibition that is not reversed by D2 antagonists. Moreover, the noncatechol partial D1 agonist CY 208,243 has no effect, despite having a backbone similar to DHX.
Additional experiments were performed using tissue from reserpine-treated rats to rule out any indirect actions of DHX on dopamine synthesis via effects on intracellular dopamine release or availability. Although unlikely (Mottola et al., 1992), such effects could obscure any receptor-mediated actions of DHX on dopamine synthesis. As shown in Table 4, the inhibition of DHX and PrDHX on tyrosine hydroxylase, and its lack of antagonism by spiperone, are similar in reserpine-treated and intact rats (cf. Table 3). The availability of intracellular dopamine is thus not determinative of TH inhibition by DHX and its analogs.
Effects on Dopamine Release.
To assess whether DHX (or theN-propyl analog) affects the release-modulating D2 autoreceptors, fast-scan cyclic voltammetry was used to measure dopamine released from striatal slices after electrical stimulation. This technique is well suited for assessing autoreceptor effects on dopamine release because of the superior time resolution of the sampling (Kennedy et al., 1992). As shown in Fig.8, neither DHX norN-propyl-DHX significantly altered dopamine overflow after stimulation of striatal slices. In contrast, quinpirole inhibited dopamine release by greater than 50%, an effect that was blocked by the D2 receptor antagonist sulpiride (data not shown). Thus, these benzo[a]phenanthridine analogs do not appear to activate D2 autoreceptors controlling dopamine release.
Additional experiments were conducted to examine the ability of DHX and PrDHX to affect dopamine release in whole animals. Dopamine overflow was measured in central striatum using cerebral microdialysis in freely moving rats. As expected, peripheral administration of the D2 receptor agonist quinpirole decreases basal dopamine overflow, whereas dopamine overflow is markedly increased by administration of the dopamine D2 receptor antagonist haloperidol (Fig. 9A). In contrast, administration of DHX in a range of doses known to produce robust behavioral activation (1–10 mg/kg) has very little effect on basal overflow, producing less than 20% inhibition (Fig. 9B). Comparison of the effects of equimolar concentrations of quinpirole and DHX infused directly through the microdialysis probe reveals that quinpirole produces a marked reduction compared with DHX (Fig. 9C).
Effects on Dopamine Neuron Firing.
Two separate experiments were performed to evaluate the ability of DHX to activate D2 autoreceptors coupled to dopamine cell firing. The first experiment (Fig. 10A) demonstrated that, as expected, the D2 agonists quinpirole and U-86170, as well as the mixed agonist apomorphine (Fig.10B), inhibit the firing of substantia nigra pars compacta dopamine neurons via activation of somatodendritic autoreceptors. Conversely, DHX has no such effect at i.v. doses up to 1 mg/kg. In the second study (Fig. 10C), the total cumulative dose of DHX was increased to 3 mg/kg. Again, no effects on firing were detected. In two of the four cells in which a full DHX dose-response curve was performed, apomorphine was given after the last dose of DHX. In the other two cells, quinpirole was given after the DHX. In all cases, these same cells then responded to the latter administration of these typical dopamine agonists (Fig.10B).
Competition Binding in Substantia Nigra.
One hypothesis to explain the lack of functional presynaptic or autoreceptor effects of DHX or PrDHX is that these ligands do not bind to those D2-like receptors that mediate these functions. In the striatum, the levels of D2-like autoreceptors are much lower than postsynaptic D2receptors (ca. 10% or less). Because there are no ligands selective for only one of these populations, it is impossible to assess a selective binding mode for DHX in this brain region. On the other hand, D2-like dopamine receptors in the substantia nigra are located exclusively on DA soma or dendrites (i.e., there are no postsynaptic D2 receptors in this region;Morelli et al., 1988). Thus, D2 competition assays using receptor autoradiography were performed to compare the binding of DHX to D2 receptors in the striatum with that in the substantia nigra. As shown in Fig.11, DHX competed similarly for125I-epidepride-labeled D2sites in the substantia nigra and striatum of the same animal. The affinity of DHX was essentially identical in both brain regions (K0.5 values: striatum = 470 nM, substantia nigra = 340 nM). In three replicate experiments, there also was no significant difference in the slope of the competition curves.
Discussion
Although the binding characteristics of DHX and PrDHX suggested they would be typical agonists (Mottola et al., 1992), the current work demonstrates unprecedented functional properties in native tissue (Table 5). DHX has clear agonist effects at some functions mediated by D2-like receptors (e.g., inhibition of adenylate cyclase or prolactin secretion), yet binds to other receptors of the same type where it apparently has antagonist, or no, functional actions (e.g., regulation of dopamine synthesis, cell firing, and release). A similar functional pattern was found with PrDHX, suggesting that at least some other hexahydrobenzo[a]phenanthridines share these novel functional properties. These data suggest that some drugs may, after binding to a single receptor isoform, cause grossly disparate functional effects, a phenomenon we have labeled functional selectivity.
Non-D2 Dopamine Receptor Mechanisms and Functional Selectivity.
There are a variety of mechanisms that could falsely lead to this notion of functional selectivity. A failure to observe effects of DHX might be due to pharmacokinetic mechanisms (e.g., failure of DHX to reach appropriate receptors). The current data, however, show that parenteral administration of DHX or PrDHX leads to accumulation of the drugs in dopamine terminal areas, and Darney et al. (1991) and Smith et al. (1997) show that both DHX and PrDHX cause behavioral effects. Similarly, the autoradiographic studies demonstrate that DHX can access and bind to D2-like receptors in brain slices even though it has little intrinsic activity neurochemically, even when given directly through a microdialysis probe. Thus, functional selectivity cannot be explained by differences in distribution or metabolism.
A second possible explanation is that functional selectivity of DHX and PrDHX is an artifact caused by pharmacological mechanisms other than direct interaction at D2-like receptors. Yet, DHX neither acts as an indirect dopamine agonist nor induces release of dopamine nor blocks the dopamine transporter (Mottola et al., 1992). Moreover, both receptor screening of DHX (Mottola et al., 1992) and the current pharmacological data (e.g., PrDHX, differing markedly in receptor profile from DHX, had the same atypical D2-like functional profile), make it unlikely that nondopamine neuroreceptors are involved. Concomitant D1 activation is also unlikely to play a role. For example, although DHX is a D1 full agonist, PrDHX is a low intrinsic activity partial D1agonist (Knoerzer et al., 1995). Therefore, one would expect very different functional patterns if D1 receptors were critical to the current phenomena (cf. Ruskin et al., 1998). In addition, D1 receptor activation opposes the inhibitory actions of agonists at D2 receptors coupled to cAMP synthesis in striatum; thus, the D1 agonist properties of DHX would obscure, rather than enhance, this measure of postsynaptic D2 receptor activation. Moreover, D1 receptors are not expressed on dopamine neurons (Fremeau et al., 1991), and D1 receptor agonists generally do not directly affect the D2autoreceptor regulatory processes that we studied (Imperato et al., 1988; Wachtel et al., 1989; See et al., 1991). In the rare cases where such D1 effects have been reported, they indirectly produce actions that mimic, rather than oppose, D2 receptor-mediated autoreceptor inhibition (Imperato and Di Chiara, 1988; Abraini et al., 1994). Together, this suggests that any D1 receptor actions of DHX would increase, rather than decrease, the likelihood of detecting autoreceptor actions of DHX. Finally, it is notable that mixed dopamine agonists such as apomorphine are able to demonstrate robust action at D2 autoreceptors, despite their partial agonist actions at D1 receptors (Strait and Kuczenski, 1986; Imperato et al., 1988; Martin et al., 1990).
Functional Selectivity and Dopamine D2-Like Receptors.
Another possibility is that the current data are a consequence of low DHX intrinsic efficacy at all D2-like receptors. Yet, the literature data suggest that there is higher efficiency of dopamine autoreceptor coupling due to greater autoreceptor receptor reserve when measured in vivo (Meller et al., 1987; Yokoo et al., 1988; Cox and Waszczak, 1989). Autoreceptor inhibition of DA cell firing usually is sensitive to low intrinsic activity partial agonists; DHX didnot cause such activation. Conversely, DHX was a full agonist at striatal D2 receptors coupled to adenylate cyclase where both DHX potency and intrinsic activity were indistinguishable from quinpirole. Agonists of known low intrinsic efficacy [e.g., S-(−)-3-PPP and S-(−)-NPA] are unable to elicit maximal inhibition of adenylate cyclase. Thus, differences in receptor reserve cannot explain these data.
Another possibility is that DHX and PrDHX cause differential functional effects because of high selectivity for one of the D2-like isoforms. In the brain regions that were studied, the D4 isoform is unlikely to be involved, yet the roles of the D2L, D2S, and D3 receptors must be considered. DHX binds with similar affinity to both D2L and D2S in molecular expression systems, and can fully inhibit adenylate cyclase at both receptors (Watts et al., 1993b). Moreover, DHX competes similarly for D2 receptor-labeled sites in striatum and substantia nigra, suggesting that DHX can bind to almost all D2-like sites, whether hetero- or autoreceptors. Because some have suggested that D3 receptors, although expressed at extremely low levels, serve as the primary dopamine autoreceptors (Aretha et al., 1995), it is possible that DHX fails to activate D3 autoreceptors either by failing to bind or having low intrinsic efficacy (Meller et al., 1993;Kreiss et al., 1995). Yet, although quinpirole has some selectivity for D3/D2 receptors, DHX is nonselective (Watts et al., 1993b). Apomorphine, which like DHX has no D3 selectivity (Sokoloff et al., 1990), is able to activate D2 autoreceptors fully. Although mRNA for both D2L and D2S is expressed by dopamine neurons, receptor localization using subtype-selective antibodies indicates that D2Smay participate preferentially in autoreceptor action (Khan et al., 1998). Recent findings using transgenic methodologies suggest that each of the D2-like autoreceptor functional effects can be observed in the absence of D3 receptors (Koeltzow et al., 1998; L'hirondel et al., 1998), indicating that D3 receptors do not provide exclusive, or even primary, support for autoreceptor regulation. Thus, DHX/PrDHX selectivity for the D3, D2L, or D2S receptor is unlikely to play a major role in the phenomenon of functional selectivity.
Because the DHX used in these studies was not enantiomerically pure (i.e., the pure isomers were not available in quantity), functional selectivity could be an artifact resulting from competing actions of two chemical species, e.g., as shown by precedents such as the D2 actions of 3-PPP enantiomers (Clark et al., 1985a,b; 1991). In the present case, however, the active (+)-6aR,12bS diastereomer of DHX carries both the D1 and D2 properties of the drug (Knoerzer et al., 1994), with (+)-DHX nearly 2 orders of magnitude higher affinity than (−)-DHX for both cloned and brain D1 and D2 receptors. Key experiments were performed with the active (+)-isomer with identical results. Thus, it is unlikely that functional selectivity is due to chemical artifacts.
Cellular and Molecular Mechanisms.
In addition to their expression on presynaptic terminals of nigrostriatal afferents, D2-like receptors in striatum are expressed on giant cholinergic interneurons, medium spiny striatal efferent neurons, and possibly on the terminals of corticostriatal glutamatergic afferents (Gerfen et al., 1990; Weiner et al., 1991). The major source of D2 inhibition of adenylate cyclase probably arises from D2 heteroreceptors on cell bodies within the striatum, because this inhibition is lost when striatal cell bodies are ablated with kainic acid (Memo et al., 1986). This form of inhibition also can be reproduced entirely in cultures of striatal neurons (Onali et al., 1985). If one assumed that inhibition of adenylate cyclase in striatum is due to activation of D2 receptors on target striatal cells then one explanation of the available data is that DHX discriminates functionally between D2-like autoreceptors and heteroreceptors. Yet, a recent electrophysiological study (Ruskin et al., 1998) demonstrated that DHX did not activate D2-like heteroreceptors on globus pallidus neurons that mediate firing rate increases. Thus, although the present data seem to suggest auto- versus heteroreceptor functional selectivity, DHX also may exhibit selectivity among other postsynaptic D2 receptor functions, such as those controlling acetylcholine release (Stoof and Kebabian, 1982).
In all the systems studied, DHX had full agonist activity at D2 receptors linked to inhibition of adenylate cyclase. From this perspective, the inability of DHX to activate autoreceptors controlling dopamine synthesis was unexpected because this process was hypothesized to occur via inhibition of adenylate cyclase (Strait and Kuczenski, 1986). Although it is possible that direct enzyme inhibition caused by the catechol moiety (Nagatsu et al., 1964) obfuscated these studies (although this usually requires higher drug concentrations), cell-specific variations in the complement of G protein subunits and/or adenylate cyclase isoforms also may be involved.
Although DHX and PrDHX may bind to D2L, D2S, and D3 receptors, we believe the most interesting (yet parsimonious) hypothesis is that either DHX or PrDHX binds, for example, to all D2L receptors, yet only causes activation of some receptor-mediated functions. Thus, DHX or PrDHX can be both an agonist and antagonist at the same receptor. Molecular pharmacology has provided several mechanisms that could mediate such agonist-selective activation of effector pathways. Gαi2 is reported as a preferred coupling partner for inhibition of adenylate cyclase (Montmayeur et al., 1993; Guiramand et al., 1995; O'Hara et al., 1996), whereas Gαo (Liu et al., 1994) and Gαi3 (Lledo et al., 1992) have been linked to D2 receptor coupling to potassium channels (Liu et al., 1996). Thus, some drugs (such as DHX and PrHX) after binding may cause conformational changes that lead to activation of only some of the G proteins with which the receptor interacts. This would make this phenomenon of functional selectivity dependent not only on the drug but also on the cellular locale of the receptor (i.e., the molecular players involved in signal transduction).
This interpretation is supported by recent studies in molecular expression systems, and has resulted in several models that have conceptualized the notion that drugs may have differential functional effects by selection among various active and inactive forms of receptors. This has been formalized in schemes such as “agonist trafficking” and the “three-state model” (Spengler et al., 1993;Chabre et al., 1994; Gettys et al., 1994; Gurwitz et al., 1994; Journot et al., 1994, 1995; Robb et al., 1994; Kenakin, 1996; Weiss et al., 1996; Leff et al., 1997). Recent data have been put forth that address these models (Berg et al., 1998; Brink et al., 2000), and in Kilts et al. (2002), we provide more mechanistic studies that may influence how one conceptualizes the involved mechanism(s). The importance of the current work is that it clearly demonstrates that ligands may have selective functional effects on single isoforms of GPCRs (e.g., D2L receptors) in native tissue, as well as in molecular expression systems. Although there have been many successes in identifying new compounds with selectivity for specific receptor isoforms, the idea of selectively targeting receptor function is usually serendipitous (Lawler et al., 1999), but may deserve more attention.
Footnotes
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↵1 Dr. Montford Piercey is deceased.
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This work was supported by National Institutes of Health Grants MH-53356, MH-40537, and MH-42705, by Training Grants DA-07244 and ES-07126, and by Center Grants HD-01130 and MH-03327. Portions of this work have been presented in the following abstracts: Mottola DM, Cook LL, Jones SR, Booth RG, Nichols DE, and Mailman RB (1991) Dihydrexidine, a selective dopamine receptor agonist that may discriminate postsynaptic D2 receptors. Soc Neurosci Abstr17:818; and Kilts JD, Nichols DE, Mailman RB, and Lawler CP (1997) The functionally selective agonist dihydrexidine inhibits adenylate cyclase in rat striatum via the D2 dopamine receptor. Soc Neurosci Abstr23:1777.
- Abbreviations:
- DHX
- dihydrexidine
- PrDHX
- N-n-propyldihydrexidine
- NPA
- N-propylnorapomorphine
- 3-PPP
- 3-(3-hydroxyphenyl)-N-n-propylpiperidine
- GppNHp
- guanylylimidodiphosphate
- HPLC
- high-performance liquid chromatography
- 5-HTP
- 5-hydroxytryptophan
- RIA
- radioimmunoassay
- DA
- dopamine
- DOPAC
- 3,4-dihydroxyphenylacetic acid
- HVA
- high-voltage activated
- 5-HIAA
- 5-hydroxyindoleacetic acid
- HPLC-EC
- high-performance liquid chromatography with electrochemical detection
- SCH23390
- R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine
- SKF38393
- 2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine
- LY171555
- quinpirole
- CY 208,243
- (−)-(6aR,12bR)4,6,6a,7,8,12a-hexahydro-7-methylindolo[4,3-a]phenanthridine
- U-86170
- (R)-5-(di[2,3–3H2]propylamino)-5,6-dihydro-4H-imidazo[4,5,1-ij]quinolin-2(1H)-one
- Received August 24, 2001.
- Accepted March 5, 2002.
- The American Society for Pharmacology and Experimental Therapeutics