Molecular cloning and characterization of crustacean type-one dopamine receptors: D1alphaPan and D1betaPan

Dopamine (DA) differentially modulates identified neurons in the crustacean stomatogastric nervous system (STNS). While the electrophysiological actions of DA have been well characterized, little is known about the dopaminergic transduction cascades operating in this system. As a first step toward illuminating the molecular underpinnings of dopaminergic signal transduction in the crustacean STNS, we have cloned and characterized two type-one DA receptors (DARs) from the spiny lobster (Panulirus interruptus): D(1alphaPan) and D(1betaPan). We found that the structure and function of these arthropod DARs are well conserved across species. Using a heterologous expression system, we determined that DA, but not serotonin, octopamine, tyramine or histamine activates these receptors. When stably expressed in HEK cells, the D(1alphaPan) receptor couples with Gs, and DA elicits an increase in [cAMP]. The D(1betaPan) receptor responds to DA with a net increase in [cAMP] that is mediated by Gs and Gz.     

Photoaffinity labeling of dopamine D1 receptors

A high-affinity radioiodinated D1 receptor photoaffinity probe, (+/-)-7-[125I]iodo-8-hydroxy-3-methyl-1-(4-azidophenyl)-2,3,4,5-tetra hyd ro- 1H-3-benzazepine ([125I]IMAB), has been synthesized and characterized. In the absence of light, [125I]IMAB bound in a saturable and reversible manner to sites in canine brain striatal membranes with high affinity (KD approximately equal to 220 pM). The binding of [125I]IMAB was stereoselectively and competitively inhibited by dopaminergic agonists and antagonists with an appropriate pharmacological specificity for D1 receptors. The ligand binding subunit of the dopamine D1 receptor was visualized by autoradiography following photoaffinity labeling with [125I]IMAB and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Upon photolysis, [125I]IMAB incorporated into a protein of apparent agents in a stereoselective manner with a potency order typical of dopamine D1 receptors. In addition, smaller subunits of apparent Mr 62,000 and 51,000 were also specifically labeled by [125I]IMAB in these species. Photoaffinity labeling in the absence or presence of multiple protease inhibitors did not alter the migration pattern of [125I]IMAB-labeled subunits upon denaturing electrophoresis in both the absence or presence of urea or thiol reducing/oxidizing reagents. [125I]IMAB should prove to be a useful tool for the subsequent molecular characterization of the D1 receptor from various sources and under differing pathophysiological states.     

Differential Effects of Cocaine-Induced Seizures and Lethality on M1-Like Muscarinic and Dopaminergic D1- and D2-Like Binding Receptors in Mice Brain

Summary This work was designed to study the changes produced by cocaine-induced seizures and lethality on dopaminergic D₁- and D₂-like receptors, muscarinic M₁-like binding sites, as well as acetylcholinesterase activity in mice prefrontal cortex (PFC) and striatum (ST). Binding assays were performed in brain homogenates from the PFC and ST and ligands used were [³H]-N-methylscopolamine, [³H]-NMS (in the presence of carbachol), [³H]-SCH 23390 and [³H]-spiroperidol (in presence of mianserin), for muscarinic (M₁-like), D₁- and D₂-like receptors, respectively. Brain acetylcholinesterase (AChE) activity was also determined in these brain areas. Cocaine-induced SE decreased [³H]-SCH 23390 binding in both ST and PFC areas. A decrease in [³H]-NMS binding and an increase in [³H]-spiroperidol binding in PFC was also observed. Cocaine-induced lethality increased [³H]-spiroperidol binding in both areas and decreased [³H]-NMS binding only in PFC, while no difference was seen in [³H]-SCH 23390 binding. Neither SE, nor lethality altered [³H]-NMS binding in ST. AChE activity increased after SE in ST while after death the increase occurred in both PFC and ST. In conclusion, cocaine-induced SE and lethality produces differential changes in brain cholinergic and dopaminergic receptors, depending on the brain area studied suggesting an extensive and complex involvement of these with cocaine toxicity in central nervous system.     

Comparative biochemical pharmacology of central nervous system dopamine D1 and D2 receptors

The biochemical properties of central nervous system (CNS) dopamine (DA) D1 and D2 receptors were examined using the specific antagonists [3H]SCH23390 and [3H]raclopride, respectively. There is a different participation of sulfhydryl (-SH) and disulfide (-SS-) groups in the binding site and/or coupling to second messenger systems of D1 and D2 receptors. The ionic studies with [3H]SCH23390 showed slight agonist and antagonist affinity shifts for the D1 receptor. On the other hand, the D2 receptor is very sensitive to cations; even if lithium and sodium influence specific [3H]raclopride binding in a similar manner, there appear to be quantitative differences between these two ions that cannot be explained by surface charge mechanisms. The distribution of D1 and D2 receptors was heterogenous in both species, with the greatest densities in the neostriatum, where the highest concentrations of DA and metabolites were measured. Regions with low endogenous DA content (cerebral cortex and hippocampus) had lower densities of DA receptors. Furthermore, these binding sites were differentially localized within the various regions, and there were substantially more D1 than D2 receptors. The functional significance and heterogeneities in the distribution of D1 and D2 receptors can be related to dopaminergic innervation and turnover.     

Effects of the stimulation of D1 and D2 dopaminergic receptors on the electrically induced release of gamma-(3H)-aminobutyric acid in the prefrontal cortex of the rat

The effects of D1 and D2 dopaminergic agonists and antagonists on the electrically-evoked release of gamma-[3H] aminobutyric acid (3H-GABA) have been studied on rat prefrontal cortex slices. The major part of the electrically-evoked release of 3H-GABA appeared to be Ca++ dependent since a 62% decrease was observed when calcium was removed from the superfusion medium. Two specific D2 dopaminergic agonists, RU 24926 (10(-7) M) and lisuride (10(-6) M), respectively induced a 32% and a 50% inhibition of the electrically-evoked release of 3H-GABA. The selective D2 dopaminergic antagonists sulpiride (10(-5) M) totally abolished the effect of RU 24926 and partially abolished the effect of lisuride. The selective D1 agonist SKF 38393 (10(-5) M) did not affect 3H-GABA release. These results suggest that in the rat prefrontal cortex in vitro, the dopaminergic modulation of 3H-GABA release is mediated through D2 but not D1 receptors. The activation of D2 dopaminergic receptors induces an inhibition of the electrically-evoked release of 3H-GABA.     

The alpha 1-adrenergic photoaffinity probe [125I]arylazidoprazosin binds to a specific peptide of P-glycoprotein in multidrug-resistant cells

Much evidence suggests that P-glycoprotein (P-gp) confers multidrug-resistance (MDR) in tumor cells by energy-dependent efflux of hydrophobic cytotoxic agents. In this study, we have used the alpha 1-adrenergic photoaffinity probe, [125I]arylazidoprazosin ([125I]AAP), and identified P-gp as a specific acceptor for prazosin. Drugs to which MDR cells are resistant, including vincristine, vinblastine, doxorubicin, actinomycin D and colchicine as well as agents reversing MDR, including verapamil, nicardipine, prenylamine, diltiazem, trifluoperazine, dibucaine, reserpine, monensin, and progesterone, differentially reduced [125I]AAP photolabeling of P-gp. We also analyzed the influence of alpha 2-adrenergic drugs and dopaminergic drugs on [125I]AAP photolabeling of P-gp. Limited proteolysis of [125I]AAP photolabeled P-gp with Staphylococcus aureus V8 protease revealed that prazosin binds to a single 8 kDa fragment of P-gp.     

Striatal CB1 and D2 receptors regulate expression of each other,CRIP1A and delta opioid systems

Although biochemical and physiological evidence suggests a strong interaction between striatal CB₁ cannabinoid (CB₁R) and D₂ dopamine (D₂R) receptors, the mechanisms are poorly understood. We targeted medium spiny neurons of the indirect pathway using shRNA to knockdown either CB₁R or D₂R. Chronic reduction in either receptor resulted in deficits in gene and protein expression for the alternative receptor and concomitantly increased expression of the cannabinoid receptor interacting protein 1a (CRIP1a), suggesting a novel role for CRIP1a in dopaminergic systems. Both CB₁R and D₂R knockdown reduced striatal dopaminergic‐stimulated [³⁵S]GTPγS binding, and D₂R knockdown reduced pallidal WIN55212‐2‐stimulated [³⁵S]GTPγS binding. Decreased D₂R and CB₁R activity was associated with decreased striatal phosphoERK. A decrease in mRNA for opioid peptide precursors pDYN and pENK accompanied knockdown of CB₁Rs or D₂Rs, and over‐expression of CRIP1a. Down‐regulation in opioid peptide mRNAs was followed in time by increased DOR1 but not MOR1 expression, leading to increased [D‐Pen2, D‐Pen5]‐enkephalin‐stimulated [³⁵S]GTPγS binding in the striatum. We conclude that mechanisms intrinsic to striatal medium spiny neurons or extrinsic via the indirect pathway adjust for changes in CB₁R or D₂R levels by modifying the expression and signaling capabilities of the alternative receptor as well as CRIP1a and the DELTA opioid system.     

Dopamine D1 receptors with enhanced agonist affinity and reduced antagonist affinity revealed by chemical modification

In order to investigate the possibility that there may be two conformationally distinct dopamine D1 binding sites, the effect of lysine-modifying agents on striatal dopamine D1 receptors was investigated. Treatment with the distilbene derivative, 4,4'-diisothiocyanostilbene-2,2'-disulfonate, (DIDS), resulted in an irreversible D1 receptor inactivation that was associated with a 70% loss of binding sites. The remaining DIDS-insensitive sites displayed both a decreased affinity (approximately 5 fold) for the D1 antagonist SCH-23390 and an enhanced affinity of dopaminergic agonists (approximately 10 fold) for the agonist high-affinity form of the receptor. Pretreatment with Gpp(NH)p, a non-hydrolysable guanine nucleotide, prevented the formation of the agonist high-affinity form, indicating that these sites are G-protein-linked. Prior occupancy of D1 receptors with dopaminergic agonists and antagonists afforded no protection against DIDS inactivation, suggesting that a site outside the ligand binding subunit of the D1 receptor was modified. Taken together, these data suggest that [3H]SCH-23390 labels two conformationally distinct populations of dopamine D1 receptors.     

6,7-Dihydroxy-2-dimethylaminotetralin (TL-99) stimulates postjunctional D-1 and D-2 dopamine receptors

6,7-Dihydroxy-2-dimethylaminotetralin (TL-99) mimicks the ability of dopamine either to enhance adenylate cyclase activity in homogenates of goldfish retina or to inhibit adenylate cyclase activity in homogenates of the intermediate lobe (IL) of the rat pituitary gland previously treated with cholera toxin. Both the dopamine stimulated adenylate cyclase activity in the fish retina and the dopamine inhibited adenylate cyclase activity in the rat IL are associated with cells postjunctional to the dopaminergic neurons innervating these tissues. Therefore, the present data do not support the contention that TL-99 is a selective presynaptic dopamine receptor agonist.     

Anomalously High Concentrations of Brain Extracellular Uric Acid Detected with Chronically Implanted Probes: Implications for In Vivo Sampling Techniques

The concentration of dopamine (DA) in the synaptic cleft in the mouse striatum in vivo was estimated from the competition between the synaptic DA and the 3H-labelled DA D2 receptor agonists N-n-propylnorapomorphine (NPA) or N,N-diethyl-N'-[(3 alpha, 4a alpha, 10 beta)-1,2,3,4,4a,5,10,10a-octahydro- 7-hydroxyl-1-propyl-3-benzo (g) quinolinyl]sulfamide (Sandoz 205-501) injected intravenously in tracer doses. Knowing the inhibitor constant for DA in inhibiting the binding of these receptor agonists in vitro, attempts were made to calculate the changes in the synaptic DA concentration from the changes in the in vivo binding of the receptor agonists evoked by various pharmacological agents. Inhibiting the firing of the dopaminergic neurons by gamma-butyrolactone (GBL) increased the binding of the receptor agonists corresponding to a decrease in the synaptic DA concentration of 55 +/- 2 nM in the experiments with [3H]Sandoz 205-501 and 48 +/- 3 nM in the experiments with tracer doses of [3H]NPA. These values may therefore approximate the normal DA concentration in the synaptic cleft in the mouse striatum. With this technique it was also possible to determine the synaptic concentration of NPA by its competition with [3H]Sandoz 205-501 for the DA D2 receptors in the striatum of GBL-treated mice in vivo. To compare the estimated synaptic concentration of DA with the affinity of DA to D1 and D2 receptors and to the DA transporter in the mouse striatum the kinetic parameters were determined at 37 degrees C in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional antagonism of D-1 and D-2 dopaminergic mechanisms affecting striatal acetylcholine release

Rat striatal slices prelabelled with [3H]choline were superfused with dopamine D-1 and D-2 agonists and antagonists, separately and in combination, during measurement of [3H]acetylcholine (ACh) release. SKF38393 (D-1 agonist), 10(-7)-10(-4) M, and SCH23390 (D-1 antagonist), 10(-7)-10(-5) M, produced a dose-dependent increase in [3H]ACh release when given separately. The increased [3H]ACh release induced by either drug could not be attenuated by sufficient L-sulpiride to block D-2 receptors. Yet both SKF38393, 10(-6)-10(-5) M, and SCH23390, 10(-6)-10(-5) M, were able to partially or fully overcome the [3H]ACh release-depressant effect of cosuperfused LY171555 (D-2 agonist), 10(-6) M. This suggests that a functional antagonism regarding striatal ACh release exists between D-1 and D-2 dopaminergic receptor-mediated mechanisms, but that D-1 modulation of local ACh release does not occur at the level of the recognition site of the striatal D-2 receptor. Finally, although attenuation of the increased release of striatal [3H]ACh induced by 10(-5) M SCH23390 by SKF38393 was seen, it is possible that such functional antagonism is not mediated by exclusively D-1 dopaminergic means.     

Facilitation of quantal release induced by a D1-like receptor on bovine chromaffin cells

Dopaminergic receptors are found on bovine adrenal chromaffin cells and have been implicated in the facilitation of an inward calcium current [Artalejo et al., (1990) Nature 348, 239-242] that could enhance release. However, previous studies using incubations of long duration (minutes) with dopaminergic receptor antagonists have found instead an inhibition of catecholamine release. In this work we used brief (subsecond) chemical depolarizing stimuli to reexamine the role of dopaminergic receptors on exocytosis from bovine adrenal chromaffin cells. Responses to consecutive depolarizing stimuli were compared using amperometry to monitor vesicular release events and intracellular fura-2 to examine Ca2+ dynamics within individual cells. Restoration of intracellular Ca2+ levels to their initial values following exposure to 60 mM K+ was found to be prolonged unless the exposure was brief (0.5 s) and the cells were maintained at 37 degrees C. However, with these optimum conditions, a second stimulation evoked more exocytotic events than the first. This effect was blocked by SCH-23390, a D1 antagonist, in a dose dependent fashion, but not by raclopride, a D2 antagonist. The D1 agonist, SKF-38393, enhanced the number of exocytotic events as did prior exposure of the cell to epinephrine. Taken together, the data indicate that released catecholamines can enhance their own release by interaction with a D1-like receptor on bovine adrenal chromaffin cells.     

Binding characteristics and sensitivity to endogenous dopamine of [11C]-(+)-PHNO, a new agonist radiotracer for imaging the high-affinity state of D2 receptors in vivo using positron emission tomography

[11C]-(+)-PHNO (4-propyl-9-hydroxynaphthoxazine) is a new agonist radioligand that provides a unique opportunity to measure the high-affinity states of the D2 receptors (D2-high) using positron emission tomography (PET). Here we report on the distribution, displaceablity, specificity and modeling of [11C]-(+)-PHNO and compare it with the well characterized antagonist D2 radioligand, [11C]raclopride, in cat. [11C]-(+)-PHNO displayed high uptake in striatum with a mean striatal binding potential (BP) of 3.95 +/- 0.85. Pre-treatment with specific D1 (SCH23390), D2 (raclopride, haloperidol) and D3 receptor (SB-277011) antagonists indicated that [11C]-(+)-PHNO binding in striatum is specific to D2 receptors. Within-subject comparisons showed that [11C]-(+)-PHNO BP in striatum was almost 2.5-fold higher than that measured with [11C]-(-)-NPA ([11C]-(-)-N-propyl-norapomorphine). Comparison of the dose-effect of amphetamine (0.1, 0.5 and 2 mg/kg; i.v.) showed that [11C]-(+)-PHNO was more sensitive to the dopamine releasing effect of amphetamine than [11C]raclopride. Amphetamine induced up to 83 +/- 4% inhibition of [11C]-(+)-PHNO BP and only up to 56 +/- 8% inhibition of [11C]raclopride BP. Scatchard analyses of [11C]-(+)-PHNO and [11C]raclopride bindings in two cats showed that the Bmax obtained with the agonist (29.6 and 32.9 pmol/mL) equalled that obtained with the antagonist (30.6 and 33.4 pmol/mL). The high penetration of [11C]-(+)-PHNO in brain, its high signal-to-noise ratio, its favorable in vivo kinetics and its high sensitivity to amphetamine shows that [11C]-(+)-PHNO has highly suitable characteristics for probing the D2-high with PET.     

Structural insights into the mechanisms of drug resistance in HIV-1 protease NL4-3

The development of resistance to anti-retroviral drugs targeted against HIV is an increasing clinical problem in the treatment of HIV-1-infected individuals. Many patients develop drug-resistant strains of the virus after treatment with inhibitor cocktails (HAART therapy), which include multiple protease inhibitors. Therefore, it is imperative that we understand the mechanisms by which the viral proteins, in particular HIV-1 protease, develop resistance. We have determined the three-dimensional structure of HIV-1 protease NL4-3 in complex with the potent protease inhibitor TL-3 at 2.0 A resolution. We have also obtained the crystal structures of three mutant forms of NL4-3 protease containing one (V82A), three (V82A, M46I, F53L) and six (V82A, M46I, F53L, V77I, L24I, L63P) point mutations in complex with TL-3. The three protease mutants arose sequentially under ex vivo selective pressure in the presence of TL-3, and exhibit fourfold, 11-fold, and 30-fold resistance to TL-3, respectively. This series of protease crystal structures offers insights into the biochemical and structural mechanisms by which the enzyme can overcome inhibition by TL-3 while recovering some of its native catalytic activity.     

Structural studies of FIV and HIV-1 proteases complexed with an efficient inhibitor of FIV protease

Three forms of feline immunodeficiency virus protease (FIV PR), the wild type (wt) and two single point mutants, V59I and Q99V, as well as human immunodeficiency virus type 1 protease (HIV-1 PR), were cocrystallized with the C2-symmetric inhibitor, TL-3. The mutants of FIV PR were designed to replace residues involved in enzyme-ligand interactions by the corresponding HIV-1 PR residues at the structurally equivalent position. TL-3 shows decreased (improved) inhibition constants with these FIV PR mutants relative to wt FIV PR. Despite similar modes of binding of the inhibitor to all PRs (from P3 to P3'), small differences are evident in the conformation of the Phe side chains of TL-3 at the P1 and P1' positions in the complexes with the mutated FIV PRs. The differences mimick the observed binding of TL-3 in HIV-1 PR and correlate with a significant improvement in the inhibition constants of TL-3 with the two mutant FIV PRs. Large differences between the HIV-1 and FIV PR complexes are evident in the binding modes of the carboxybenzyl groups of TL-3 at P4 and P4'. In HIV-1 PR:TL-3, these groups bind over the flap region, whereas in the FIV PR complexes, the rings are located along the major axis of the active site. A significant difference in the location of the flaps in this region of the HIV-1 and FIV PRs correlates with the observed conformational changes in the binding mode of the peptidomimetic inhibitor at the P4 and P4' positions. These findings provide a structural explanation of the observed Ki values for TL-3 with the different PRs and will further assist in the development of improved inhibitors.     

Preparation of an affinity chromatography matrix for the selective purification of the dopamine D2 receptor from bovine striatal membranes

A ligand affinity matrix has been developed and utilized to purify the dopamine D2 receptor approx. 2100 fold from bovine striatal membranes. 3-[2-Aminoethyl]-8-[3-(4-fluorobenzoyl)propyl]-4-oxo-1-phenyl-1,3,8- triazaspiro[4.5]decan-4-one (AES) was synthesized and used to prepare the affinity matrix by coupling to epoxy-activated Sepharose 6B (AES-Sepharose). AES (Ki approximately 1.7 nM) is similar in potency to the parent compound, spiperone (Ki approximately 0.8 nM), in competing for [3H]spiperone-binding activity. AES has no significant potency in competing for the dopamine D1 receptor as assessed by competition for [3H]SCH23390 binding (Ki greater than 1 microM). Covalent photoaffinity labeling of the dopamine D2 receptor in bovine striatal membranes with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS) was prevented by AES at nanomolar concentrations. The dopamine D2 receptor was solubilized from bovine striatal membranes using 0.25% cholate in the presence of high ionic strength, followed by precipitation and subsequent treatment with 0.5% digitonin. Nearly 100% of the [3H]spiperone-binding activity in the cholate-digitonin solubilized preparation was absorbed at a receptor-to-resin ratio of 2:1 (v/v). Dopamine D2 receptor was eluted from the affinity resin using a competing dopaminergic antagonist molecule, haloperidol. Recovery of dopamine D2 receptor activity from the affinity matrix was approx. 9% of the activity adsorbed to the resin. The [3H]spiperone-binding activity in AES-Sepharose affinity purified preparations is saturable and of high affinity (0.2 nM). Affinity-purified preparations maintain the ligand-binding characteristics of a dopamine D2 receptor as assessed by agonist and antagonist competition for [3H]spiperone binding.     

Enhanced Dopamine D1 and D2 Receptor Gene Expression in the Hippocampus of Hypoglycaemic and Diabetic Rats

Hypoglycaemic coma and brain injury are potential complications of insulin therapy. Hippocampal neurons are particularly vulnerable to hypoglycaemic stress leading to memory impairment. In the present article, we have investigated the dopamine (DA) content, homovanillic acid (HVA)/DA turnover ratio, DA D₁ and DA D₂ receptors in the hippocampus of insulin-induced hypoglycaemic (IIH) and streptozotocin induced diabetic rats where brain functions are impaired. The DA content decreased significantly in hippocampus of diabetic, diabetic +IIH and control +IIH rats compared to control. The HVA/DA turnover ratio also increased significantly in diabetic, diabetic +IIH and control +IIH rats compared to control. Scatchard analysis using [³H] DA in the hippocampus showed a significant increase in DA receptors of diabetic, diabetic +IIH and control +IIH rats with decreased affinity. Gene expression studies using Real-time PCR showed an increased expression of DA D₁ and DA D₂ receptors in the hippocampus of hypoglycaemic and diabetic rats. Our results indicate that the dopaminergic system is impaired in the hippocampus of hypoglycaemic and hyperglycaemic rats impairing DA related functions of hippocampus. We observed a prominent dopaminergic functional disturbance in the hypoglycaemic condition than in hyperglycaemia compared to control. This dopaminergic dysfunction in hippocampus during hypoglycaemia and hyperglycaemia is suggested to contribute to cognitive and memory deficits. This will have clinical significance in the treatment of diabetes.     

[3H]1-Methyl-4-Phenyl-2,3-Dihydropyridinium Ion Binding Sites in Mouse Brain: Pharmacological and Biological Characterization

Because 1-methyl-4-phenyl-2,3-dihydropyridinium ion (MPP+) appears to damage the dopaminergic neuron and cause neuronal death, we characterized [3H]MPP+ binding sites in mouse brain membranes. Among several compounds tested, debrisoquin [3,4-dihydro-2(1H)-isoquinolinecarboxamidine] and some analogues were able to antagonize [3H]MPP+ binding. Debrisoquin is able to block adrenergic transmission and inhibit the activity of monoamine oxidase A (MAO-A). We found a certain correlation between the ability of these agents to displace [3H]MPP+ from its binding sites and their capacity to inhibit MAO-A activity. These data and the finding of a higher number of [3H]MPP+ binding sites in human placenta compared to mouse brain suggest that these sites may correspond to MAO-A enzymes. Recently it has been demonstrated in human brain that neurons in regions rich in catecholamines are positive for MAO-A. Accordingly, we suggest MAO-A as a possible accumulation site of MPP+ within the dopaminergic neuron. We also indicate the chemical structural requirement associated with the best binding of debrisoquin analogues with [3H]MPP+ sites. It would be reasonable to test the effects of debrisoquin-like drugs able to pass the blood-brain barrier on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity.     

Synthesis and biological characterization of novel hybrid 7-[[2-(4-phenyl-piperazin-1-yl)-ethyl]-propyl-amino]-5,6,7,8-tetrahydro-naphthalen-2-ol and their heterocyclic bioisosteric analogues for dopamine D2 and D3 receptors

In a recent preliminary communication we described the development of a series of hybrid molecules for the dopamine D2 and D3 receptor subtypes. The design of these compounds was based on combining pharmacophoric elements of aminotetralin and piperazine molecular fragments derived from known dopamine receptor agonist and antagonist molecules. Molecules developed from this approach exhibited high affinity and selectivity for the D3 receptor as judged from preliminary [(3)H]spiperone binding data. In this report, we have expanded our previous finding by developing additional novel molecules and additionally evaluated functional activities of these novel molecules in the [(3)H]thymidine incorporation mitogenesis assay. The binding results indicated highest selectivity in the bioisosteric benzothiazole derivative N6-[2-(4-phenyl-piperazin-1-yl)-ethyl]-N6-propyl-4,5,6,7-tetrahydro-benzothiazole-2,6-diamine (14) for the D3 receptor whereas the racemic compound 7-([2-[4-(2,3-dichloro-phenyl)-piperazin-1-yl]-ethyl]-propyl-amino)-5,6,7,8-tetrahydro-naphthalen-2-ol (10c) showed the strongest potency. Mitogenesis studies to evaluate functional activity demonstrated potent agonist properties in these novel derivatives for both D2 and D3 receptors. In this regard, compound 7-[[4-(4-phenyl-piperazin-1-yl)-butyl]-prop-2-ynyl-amino]-5,6,7,8-tetrahydro-naphthalen-2-ol (7b) exhibited the most potent agonist activity at the D3 receptor, 10 times more potent than quinpirole and was also the most selective compound for the D3 receptor in this series. Racemic compound 10a was resolved; however, little separation of activity was found between the two enantiomers of 10a. The marginally more active enantiomer (-)-10a was examined in vivo using the 6-OH-DA induced unilaterally lesioned rat model to evaluate its activity in producing contralateral rotations. The results demonstrated that in comparison to the reference compound apomorphine, (-)-10a was quite potent in inducing contralateral rotations and exhibited longer duration of action.     

ACTH-(1-24) and alpha-MSH antagonize dopamine receptor-mediated inhibition of striatal dopamine and acetylcholine release

The effects of ACTH-(1-24), alpha-MSH and ACTH-(4-10) were studied on the electrically evoked release of 3H-dopamine and 14C-acetylcholine from striatal slices in the absence and presence of the dopamine receptor agonist TL-99. None of the peptides affected transmitter release when TL-99 was not present. ACTH-(1-24) and alpha-MSH concentration-dependently antagonized the inhibition of striatal transmitter release induced by dopamine receptor stimulation due to the presence of TL-99. ACTH-(1-24), 10(-7)M, reduced the TL-99-induced inhibition of the release of both dopamine and acetylcholine by approximately 50%, and 5 X 10(-6) M ACTH-(1-24) restored the release fully to control values. alpha-MSH was less effective by a factor 20-30 in counteracting the release-inhibiting effect of TL-99. ACTH-(4-10) had no effect at any of the concentrations tested. These results show that ACTH/MSH-like neuropeptides may act by modulating dopamine receptor functions in rat striatum.