D1 and D2 Dopamine Receptors in Caudate-Putamen of Nonhuman Primates (Macaca fascicularis)

D1 and D2 dopamine receptors were characterized in the caudate-putamen region of nonhuman primate brains (Macaca fascicularis). D1 dopamine receptors were identified with [3H]SCH 23390 and D2 receptors with [3H]-spiperone. Scatchard analysis of [3H]SCH 23390 saturation data using washed membranes revealed a single high-affinity binding site (KD, 0.352 +/- 0.027 nM) with a density (Bmax) of 35.7 +/- 2.68 pmol/g original wet tissue weight (n = 10). The affinity of [3H]spiperone for the D2 site was 0.039 +/- 0.007 nM and the density was 25.7 +/- 1.97 pmol/g original wet tissue weight (n = 10). D1 and D2 receptors in nonhuman primates may be differentiated on the basis of drug affinities and stereoselectivity. In competition experiments, RS-SKF 38393 was the most selective D1 agonist, whereas (+)-4-propyl-9-hydroxynaphthoxazine [(+)-PHNO] was the most selective D2 agonist. Apomorphine was essentially nonselective for D1 or D2 binding sites. Of the antagonists, R-SKF 83566 and SCH 23390 were the most selective for the D1 site, whereas YM-09151-2 was the most selective for the D2 site. cis-Flupentixol and (S)-butaclamol were the least selective dopamine antagonists. D1 receptors bound benzazepine antagonists (SCH 23390/SCH 23388, R-SKF 83692/RS-SKF 83692) stereoselectively whereas D2 receptors did not. Conversely D2 receptors bound (S)-sulpiride and (+)-PHNO more potently than their enantiomers whereas D1 receptors showed little stereoselectively for each of these isomeric pairs. These binding characteristics may be utilized for evaluation of individual receptor function in vivo.     

Dopamine D1 receptors characterized with [3H]SCH 23390. Solubilization of a guanine nucleotide-sensitive form of the receptor

Dopamine D1 receptors were solubilized from canine and bovine striatal membranes with the detergent digitonin. The receptors retained the pharmacological characteristics of membrane-bound D1 receptors, as assessed by the binding of the selective antagonist [3H]SCH 23390. The binding of [3H]SCH 23390 to solubilized receptor preparations was specific, saturable, and reversible, with a dissociation constant of 5 nM. Dopaminergic antagonists and agonists inhibited [3H]SCH 23390 binding in a stereoselective and concentration-dependent manner with an appropriate rank order of potency for D1 receptors. Moreover, agonist high affinity binding to D1 receptors and its sensitivity to guanine nucleotides was preserved following solubilization, with agonist dissociation constants virtually identical to those observed with membrane-bound receptors. To ascertain the molecular basis for the existence of an agonist-high affinity receptor complex, D1 receptors labeled with [3H] dopamine (agonist) or [3H]SCH 23390 (antagonist) prior to, or following, solubilization were subjected to high pressure liquid steric-exclusion chromatography. All agonist- and antagonist-labeled receptor species elute as the same apparent molecular size. Treatment of brain membranes with the guanine nucleotide guanyl-5'-yl imidodiphosphate prior to solubilization prevented the retention of [3H]dopamine but not [3H]SCH 23390-labeled soluble receptors. This suggests that the same guanine nucleotide-dopamine D1 receptor complex formed in membranes is stable to solubilization and confers agonist high affinity binding in soluble preparations. These results contrast with those reported on the digitonin-solubilized dopamine D2 receptor, and the molecular mechanism responsible for this difference remains to be elucidated.     

Autoradiographic Evidence of [3H]SCH 23390 Binding Site; in Human Prefrontal Cortex (Brodmann''s Area 9)

The distribution of dopamine D-1 receptors has been determined in human prefrontal cortex (Brodmann's area 9) by an in vitro light microscopic autoradiographic method. Dopamine D-1 receptors were localized by using [3H]SCH 23390 as a ligand. Our results indicated that [3H]SCH 23390 binding to slide-mounted tissue sections of human brain is specific, saturable, and of high affinity. Lamina Va contained the highest density of D-1 receptors, with a Bmax value of 11.2 +/- 1.3 fmol/mg tissue. The KD values for [3H]SCH 23390 in all laminae ranged from 2.6 to 3.2 nM. Competition studies performed with [3H]SCH 23390 indicated a pharmacologic profile consistent with labeling of the D-1 receptor.     

Binding of [3H]SCH23390 in Rat Brain: Regional Distribution and Effects of Assay Conditions and GTP Suggest Interactions at a D1-Like Dopamine Receptor

The compound [9-3H]SCH23390 [R-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7- ol] was synthesized, and the binding of this purportedly selective antagonist of D1 3,4-dihydroxyphenylethylamine (dopamine) receptors was characterized. The regional distribution of high-affinity, specific [3H]SCH23390 binding sites in the rat brain correlated well with levels of endogenous dopamine. Receptor densities were greatest in corpus striatum, nucleus accumbens, and olfactory tubercle; intermediate levels were found in several limbic and cortical areas, whereas few sites were detectable in cerebellum, brainstem, and ol-factory bulb. Specific binding in caudate-putamen was found to be both temperature- and pH-dependent, with optima at 25-30 degrees C and pH 7.8-8.0. Scatchard or Woolf analyses of binding in caudate-putamen suggest that most of the sites are either of a single class or of classes with similar characteristics (KD = 0.7 +/- 0.1 nM; Bmax = 347 +/- 35 fmol/mg of protein). Both dopamine and cis-flupenthixol altered the slope but not the intercept of lines generated by Scatchard analysis, suggesting a competitive mode of inhibition of [3H]SCH23390 binding. Competition for binding by dopamine or the D1 agonist SKF38393 was inhibited by guanine nucleotides, whereas GTP had little effect on the competition for binding by the antagonist cis-flupenthixol. The competition for [3H]SCH23390 binding sites by dopamine was much more sensitive to GTP than was competition for [3H]spiperone binding. These data support the hypotheses that [3H]SCH23390 binds to recognition sites that differ from those previously described using other radiolabeled dopamine antagonists and that these sites have the characteristics expected of dopamine receptors.     

In Vivo Partial Inactivation of Dopamine D1 Receptors Induces Hypersensitivity of Cortical Dopamine-Sensitive Adenylate Cyclase: Permissive Role of α1-Adrenergic Receptors

As shown by autoradiography, peripheral injections of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) induced a dose-dependent decrease of [3H]SCH 23390 and [3H]prazosin high-affinity binding sites in the rat prefrontal cortex. EEDQ showed similar efficacy in inactivating cortical and striatal dopamine (DA) D1 receptors, whereas prazosin-sensitive alpha 1-adrenergic receptors were more sensitive to the action of the alkylating agent, as for all doses of EEDQ tested (from 0.8 to 3 mg/kg, i.p.), the decrease in cortical [3H]SCH 23390 binding was less pronounced than that of [3H]prazosin. The effects of EEDQ on [3H]SCH 23390 binding and DA-sensitive adenylate cyclase activity were then simultaneously compared in individual rats. In the striatum, whatever the dose of EEDQ used, the decrease of DA-sensitive adenylate cyclase activity was always lower than that of D1 binding sites, suggesting the occurrence of a large proportion of spare D1 receptors. In the prefrontal cortex, a significant increase in DA-sensitive adenylate cyclase activity was observed in rats treated with a low dose of EEDQ (0.8 mg/kg), this effect being associated with a slight reduction in [3H]SCH 23390 binding sites (-20%). Parallel decreases in the enzyme activity and D1 binding sites were observed with higher doses. The EEDQ-induced supersensitivity of DA-sensitive adenylate cyclase did not occur in rats in which the decrease in [3H]prazosin binding sites was higher than 35%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Heavy Metal Cations and Other Sulfhydryl Reagents on Brain Dopamine D1 Receptors: Evidence for Involvement of a Thiol Group in the Conformation of the Active Site

To investigate aspects of the biochemical nature of membrane-bound dopamine D1 receptors, rat striatal homogenates were pretreated with heavy metal cations and some other chemical agents, and their effects on D1 receptors were subsequently determined using a standard [3H](R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1-N-3- benzazepine([3H]SCH 23390) binding assay. Incubation of striatal membranes with as little as 1 microM Hg2+, 10 microM Cu2+, and 10 microM Cd2+ completely prevented specific [3H]SCH 23390 binding. The effect of Cu2+, 1.5 microM, was noncompetitive in nature, whereas 3-5 microM Cu2+ afforded mixed-type inhibition. The inhibitory effect of Cu2+ was fully reversed by dithiothreitol (0.1-1 mM). Cu2+ (2 microM) did not affect the affinity of cis-flupenthixol or clozapine for remaining [3H]SCH 23390 sites. A second series of cations, Co2+ (30 microM), Ni2+ (30 microM), Mn2+ (1 mM), Ca2+ (25 mM), and Ba2+ (20 mM), inhibited specific [3H]SCH 23390 binding by 50% at the concentrations indicated. The thiol alkylating reagent N-ethylmaleimide (NEM) (0.2 mM) reduced specific binding by 70%. The effect of NEM was completely prevented by coincubation with a D1 receptor saturating concentration of SCH 23390 (20 nM) or dopamine (10 microM). The results indicated that the dopamine D1 receptor is a thiol protein and that a thiol group is essential for the ligand binding.     

Chronic estradiol treatment increases ovariectomized rat striatal D-1 dopamine receptors

Striatal D-1 dopamine (DA) receptors were investigated following chronic 17 beta-estradiol (10 micrograms, b.i.d., s.c., for two weeks) to ovariectomized (OVX) female rats. This treatment initiated the day after ovariectomy has revealed that the maximal density in homogenates of striatal D-1 DA receptors (Bmax) labelled with [3H] SCH 23390 was increased (44% without and 28% with 120 mM NaCl in the assay buffer). Estradiol treatments initiated 2 or 4 weeks after ovariectomy did not induce D-1 DA receptor binding modifications. The affinity (Kd) of the ligand for the receptor remains unchanged by the steroid treatment while NaCl increased both the density and the affinity of [3H] SCH 23390 binding to striatal D-1 DA receptors. By autoradiography, the increase of striatal [3H] SCH 23390 binding to D-1 DA receptors after chronic estradiol treatment was found to be homogenously distributed in this brain region. Thus, chronic treatment with estradiol of ovariectomized rats leads to an increased density of striatal D-1 DA receptors but, this hormonal modulation of D-1 DA receptors is lost when treatment is started 2 weeks after ovariectomy or later.     

[3H]SCH 39166, A New D1-Selective Radioligand: In Vitro and In Vivo Binding Analyses

SCH 39166 [(-)-trans-6,7,7a,8,9, 13b-hexahydro-3-chloro-2-hydroxy-N-methyl-5H-benzo-[d]naphtho[2, 1b]azepine] has recently been described as a selective D1 antagonist and has entered clinical trials for the treatment of schizophrenia. The tritiated analogue of this compound, [3H]SCH 39166, has now been synthesized and characterized for its in vitro and in vivo binding profiles. [3H]SCH 39166 binds to D1 receptors in a saturable, high-affinity fashion, with a KD of 0.79 nM. In competition studies, D1-selective antagonists like SCH 23390 displaced the binding of [3H]SCH 39166 with nanomolar affinities, whereas antagonists of other receptors exhibited poor affinity. In vivo, [3H]SCH 39166 bound to receptors in rat striatum in a fashion suggestive of D1 selectivity. Further, when the time course for the binding of [3H]SCH 39166 was compared with the behavioral time course of the unlabeled compound, the two durations of action were virtually indistinguishable. Similar studies were performed for SCH 23390 and its tritiated analogue, but the in vivo binding of this radioligand exhibited a duration of action far greater than the behavioral activity of the unlabeled drug. In concert, these data demonstrate that [3H]SCH 39166 selectively labels D1 receptors in vitro and in vivo, and that this drug is superior for in vivo imaging of the D1 receptor.     

Affinity chromatography of the D1 dopamine receptor from rat corpus striatum

The D1 dopamine receptor from rat corpus striatum has been purified 200-250-fold by using a newly developed biospecific affinity chromatography matrix based on a derivative of the D1 selective antagonist SCH 23390. This compound, (RS)-5-(4-aminophenyl)-8-chloro-2,3,4,5-tetrahydro-3-methyl-1H-3-benz azepin-7-o l (SCH 39111), possesses high affinity for the D1 receptor and, when immobilized on Sepharose 6B through an extended spacer arm, was able to adsorb digitonin-solubilized D1 receptors. The interaction between the solubilized receptor and the affinity matrix was biospecific. Adsorption of receptor activity could be blocked in a stereoselective fashion [SCH 23390 greater than SCH 23388; (+)-butaclamol greater than (-)-butaclamol]. The elution of [3H]SCH 23390 activity from the gel demonstrated similar stereoselectivity for antagonist ligands. Agonists eluted receptor activity with a rank order of potency consistent with that of a D1 receptor [apomorphine greater than dopamine greater than (-)-epinephrine much greater than LY 171555 greater than serotonin]. SCH 39111-Sepharose absorbed 75-85% of the soluble receptor activity, and after the gel was washed extensively, 35-55% of the absorbed receptor activity could be eluted with 100 microM (+)-butaclamol with specific activities ranging from 250 to 450 pmol/mg of protein. The affinity-purified receptor retains the ligand binding characteristics of a D1 dopamine receptor. This affinity chromatography procedure should prove valuable in the isolation and molecular characterization of the D1 dopamine receptor.     

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.     

Effect of Unilateral Perinatal Hypoxic-Ischemic Brain Injury in the Rat on Dopamine D1 and D2 Receptors and Uptake Sites: A Quantitative Autoradiographic Study

The effect of a unilateral perinatal hypoxic-ischemic brain injury on dopamine D1 and D2 receptors and uptake sites was investigated in rats by using in vitro quantitative binding autoradiography, 2-3 weeks after the insult. We observed significant decreases in the Bmax and KD for [3H]SCH 23390-labeled D1 and in the Bmax for [3H]spiperone-labeled D2 receptors in the lesioned caudate-putamen in rats with moderate brain injury (visible loss in hemispheric volume ipsilateral to the injury) compared with the nonlesioned contralateral caudate-putamen or with control rats. Changes in [3H]SCH 23390 and [3H]spiperone binding predominated in the dorsolateral part of the lesioned caudate-putamen. Pronounced reduction in [3H]SCH 23390 binding was also observed in the substantia nigra pars reticulata on the side of the lesion. In contrast, we did not observe any significant change in Bmax or KD for [3H]mazindol-labeled dopamine uptake sites. Similarly, no significant changes in the levels of dopamine or its metabolites were found on the side of the lesion. The observed reductions in striatal dopamine D1 and D2 receptors are a reflection of striatal cell loss induced by the hypoxic-ischemic injury. The absence of changes in [3H]mazindol binding or dopamine levels in the lesioned caudate-putamen indicates that the dopaminergic presynaptic structures are preserved.     

Evidence for Different States of the Dopamine Dl Receptor: Clozapine and Fluperlapine May Preferentially Label an Adenylate Cyclase-Coupled State of the Dl Receptor

Abstract: It has been shown previously that typical neuroleptics have higher affinities for 3,4-dihydroxyphenyl-ethylamine (dopamine) Dl receptors as labeled by(R)- (+)- 8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1 -N-3-benzazepine-7-ol ([³H]SCH 23390) than for inhibiting dopamine-stimulated adenylate cyclase. We now report that the atypical neuroleptics, clozapine and fluperlapine, exhibit characteristics opposite to typical neuroleptics, i.e., they have higher affinity for inhibiting dopamine-stimulated adenylate cyclase than [³H]SCH 23390 binding. A variety of compounds, i.e., clozapine, fluperlapine, and dopamine, were tested for their capacity to affect the rate constants of [³H]SCH 23390 binding; these experiments revealed no effect of any tested compound on on-rate or off-rate of [³H]SCH 23390 binding. Treatment of striatal membranes with phospholipase A₂ (PLA₂) caused a rapid decrease in the B_max value of the [³H]SCH 23390 binding with no effect on the K_d value. The adenylate cyclase, both the unstimulated, the dopamine-, fluoride-, and forskolin-stimulated activity, was far less sensitive than [³H]SCH 23390 binding to PLA₂. Treatment of striatal membranes with filipine and (NH₄SO₄ produced, as did PLA₂ treatment, a rapid decline in [³H]SCH 23390 binding. However, opposite to PLA₂ treatment, these agents stimulated the adenylate cyclase. In conclusion, a comparison of the pharmacological characteristics of [³H]SCH 23390 binding and dopamine-stimulated adenylate cyclase suggests the existence of two different Dl binding sites. The rate experiments exclude the possibility of allosterically coupled sites. Instead our results favor that the Dl receptor exists in different states/conformations, i.e., both adenylate cyclase-coupled and uncoupled, and further, that the atypical neuroleptics clozapine and fluperlapine may have adenylate cyclase-coupled dopamine Dl receptors as target.     

Stereospecific binding of a new benzazepine, [3H]SCH23390, in cortex and neostriatum

The binding of the D1 antagonist SCH23390 to membrane preparations from rat cerebral cortex was examined using enantiomers of dopamine agonists and antagonists to compete with the bound [3H]SCH23390 at its Kd value. The competition curves were compared with those obtained with preparations from the neostriatum. The results demonstrate that specific [3H]SCH23390 binding in the cerebral cortex has the same pharmacological profile as in the neostriatum, so that this radioligand can be used to label dopamine D1 receptors in brain regions with a sparse dopaminergic innervation.     

Effects of chronic antidepressant treatment on dopamine-related [3H]SCH 23390 and [3H]Spiperone binding in the rat striatum

1. The effects of chronic administration of antidepressants on dopamine-related [3H]SCH 23390 and [3H]spiperone binding to rat striatal membranes were assessed. 2. The monoamine oxidase inhibitors phenelzine (5 or 10 mg kg-1/day) and tranylcypromine (1 mg kg-1/day) and the tricyclic desipramine (10 mg kg-1/day) were administered for 28 days by constant subcutaneous infusion using Alzet (2ML4) osmotic minipumps. 3. These treatments did not alter Kd estimates for either [3H]SCH 23390 or [3H]spiperone binding sites. The monoamine oxidase inhibitors induced a decrease in the Bmax values for both [3H]SCH 23990 and [3H]spiperone binding sites. Desipramine induced a decrease in the Bmax value for [3H]SCH 23390 binding but had no effect on the Bmax value for [3H]spiperone binding.     

Comparison of 125I-SCH 23982 and [3H]SCH 23390 as Ligands for the D-1 Dopamine Receptor

125I-SCH 23982, an antagonist with high affinity and selectivity for the D-1 subtype of dopamine receptors, has recently been synthesized. Densities of D-1 receptors in rat brain obtained from autoradiographic studies using this iodinated ligand are 5- to 10-fold less than densities reported with tritiated analogues such as [3H]SCH 23390. A direct comparison of these two ligands using striatal homogenates confirmed this discrepancy. One explanation for this difference is that 125I-SCH 23982 labels a subset of the sites labeled by [3H]SCH 23390. However, the distributions of sites labeled by the ligands in autoradiograms of horizontal sections of rat brain were virtually identical. Furthermore, 127I-SCH 23982 displaced 100% of the specifically bound [3H]SCH 23390 in striatal homogenates with a Hill coefficient of approximately 1. These results are not consistent with the existence of a subset of receptors recognized by 125I-SCH 23982 and suggest that both ligands label the same population of receptors. An alternative explanation for the discrepancy in Bmax values is that an unlabeled inhibitor is present in commercial preparations of 125I-SCH 23982. When all of the solvent (including any volatile inhibitors) was removed from commercial preparations of 125I-SCH 23982 prior to use in radioligand binding experiments, the discrepancy in Bmax values was eliminated.     

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.     

Interactions of brain muscarinic acetylcholine receptors with plant lectins

We previously reported that muscarinic acetylcholine receptors (mAChRs) from porcine brains are glycoproteins. When porcine brain membranes were solubilized with digitonin or 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), approximately 20% of the receptors were solubilized, most (90% or more) of which bound to Sepharose 4B conjugated with wheat germ agglutinin (WGA). In contrast, when membranes were solubilized with Lubrol PX, a much larger fraction (approximately 60%) of the receptors were solubilized. However, about a third of this solubilized receptor population remained unbound to WGA-Sepharose even in the presence of an excess amount of the lectin-Sepharose. These results suggested a structural heterogeneity of the mAChR in terms of its carbohydrate moiety. The effects of lectins on the ligand binding properties of mAChRs were also studied. WGA or concanavalin A (ConA) was found to cause a 2- to 3-fold increase in the affinity of membrane-bound receptors to an antagonist [3H]quinuclidinyl benzylate [( 3H]QNB) without affecting the maximum number of sites, whereas the lectins had no significant effects on the binding of the agonist [3H]cis-methyldioxolane. When the membranes were dissolved with detergents, lectin did not increase the [3H]QNB affinity: These lectins caused an approximately 2 fold decrease in the affinity of digitonin-solubilized receptors for [3H]QNB. Thus the lectins exert differential effects on agonist and antagonist binding to the brain membrane mAChRs, most likely by modulating some intermolecular interactions.     

Differential effect of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) on [3H]SCH23390 and [3H]forskolin binding in rat striatum

The binding of [3H]forskolin to a homogeneous population of binding sites in rat striatum was enhanced by NaF, guanine nucleotides and MgCl2. These effects of NaF and guanylylimidodiphosphate (Gpp(NH)p) were synergistic with MgCl2, but NaF and Gpp(NH)p together elicited no greater enhancement of [3H]forskolin binding. These data suggest that [3H]forskolin may label a site which is modulated by the guanine nucleotide regulatory subunit which mediates the stimulation of adenylate cyclase (NS). The D1 dopamine receptor is known to stimulate adenylate cyclase via NS. In rat striatum, the Bmax of [3H]forskolin binding sites in the presence of MgCl2 and NaF was approximately two fold greater than the Bmax of [3H]SCH23390-labeled D1 dopamine receptors. Incubation of striatal homogenates with the protein modifying reagent EEDQ elicited a concentration-dependent decrease in the binding of both [3H]SCH23390 and [3H]forskolin, although EEDQ was approximately 14 fold more potent at inactivating the D1 dopamine receptor. Following in vivo administration of EEDQ there was no significant effect on [3H]forskolin binding sites using a dose of EEDQ that irreversibly inactivated greater than 90% of D1 dopamine receptors. These data suggest that EEDQ is a suitable tool for investigating changes in the stoichiometry of receptors and their second messenger systems.     

Changes in serotonin2A and GABA(A) receptors in schizophrenia: studies on the human dorsolateral prefrontal cortex

Having shown a decrease in serotonin2A receptors in the dorsolateral prefrontal cortex (DLPFC) from schizophrenic subjects, we have now determined if this change was reflective of widespread changes in neurochemical markers in DLPFC in schizophrenia. In Brodmann's area (BA) 9 from 19 schizophrenic and 19 control subjects, we confirmed a decrease in the density of [3H]ketanserin binding to serotonin2A receptors in tissue from the schizophrenic subjects [39 +/- 3.3 vs. 60 +/- 3.6 fmol/mg estimated tissue equivalents (ETE); p < 0.005]. In addition, the density of [3H]muscimol binding to GABA(A) receptors was increased in the schizophrenic subjects (526 +/- 19 vs. 444 +/- 28 fmol/mg ETE; p < 0.02). [3H]YM-09151-2, N-[1-(2-thienyl)cyclohexyl]-3,4-[3H]piperidine, [3H]SCH 23390, [3H]mazindol, and N(G)-nitro-L-[3H]arginine binding to BA 9 did not differ between groups, and there was no specific binding of [3H]raclopride or 7-hydroxy-2-(di-n-[3H]propylamino)tetralin to BA 9 from either cohort of subjects. This suggests the density of dopamine D1-like and NMDA receptors, the dopamine transporter, and nitric oxide synthase activity are not altered in BA 9 from schizophrenic subjects. The selective nature of the changes in serotonin2A and GABA(A) receptors in DLPFC could indicate that these changes are involved in the pathology of schizophrenia.     

Pharmacologic evidence for direct dopaminergic regulation of striatal acetylcholine release

This study was done to provide pharmacologic evidence for the location of those striatal dopamine D-1 and D-2 receptors that participate in the regulation of local acetylcholine (ACh) release. Striatal tissue slices from adult male Sprague-Dawley rats were preloaded with [3H]choline and superfused in separate experiments with buffer containing either: a D-2-specific agonist (LY141865 or LY171555), a D-2 specific antagonist (L-sulpiride), a D-1 specific agonist (SKF38393), or a D-1 antagonist (SCH23390), in the presence or absence of tetrodotoxin (TTX), used to block interneuronal activity. With either D-2 agonist there was a dose-dependent decrease in K+-stimulated [3H]ACh release, maximally at 5 X 10(-7)-10(-6) M [agonist] and to the same extent with each drug. Both SKF38393 and SCH23390 increased [3H]ACh release at tested concentrations of these agents. Results were unchanged when any of the drugs used was superfused in the presence of TTX, 5 X 10(-7) M. These data are consistent with the hypothesis that populations of striatal D-1 and D-2 receptors exist on local cholinergic neurons, where they regulate ACh release. Alternative interpretations are discussed.