A specific acylating agent for the [3H]phencyclidine receptors in rat brain

A derivative of phencyclidine (PCP, 1 in fig. 1) bearing an isothiocyanate moiety on the meta position of the aromatic ring (Metaphit, 3 in fig. 1) has been synthesized and identified as a rapid and specific site-directed acylating agent of the [3H]phencyclidine binding site in rat brain homogenates. The percentage of sites irreversibly inactivated by Metaphit was found to be the same in the hippocampus and striatum and the remaining sites were unaffected by Metaphit treatment under any conditions, suggesting that at least two distinct binding sites are present. An isomeric isothiocyanate derivative did not irreversibly inhibit [3H]phencyclidine receptors, indicating structural specificity for Metaphit in the inhibition of these receptors. The availability of Metaphit should greatly facilitate study of the structure and function of the phencyclidine receptors.     

Metaphit Irreversibly Inhibits [3H]threo- (±)-Methylphenidate Binding to Rat Striatal Tissue

Metaphit (1-[1-(3-isothiocyanatophenyl)cyclohexyl]-piperidine), a derivative of phencyclidine that contains an isothiocyanate group on the meta position of the aromatic ring, resembles its parent compound (phencyclidine) in its ability to inhibit the binding of the stimulant drug [3H]threo-(+/-)-methylphenidate to crude synaptosomal membranes from rat striatal tissue (IC50 = 1.4 and 6.2 microM for phencyclidine and Metaphit, respectively). Unlike phencyclidine, however, Metaphit appears to inhibit binding of the radiolabeled stimulant in an irreversible manner, as the degree of inhibition of binding of the stimulant does not diminish when the Metaphit-treated tissue is subjected to repeated washings before determination of the binding of [3H]threo-(+/-)-methylphenidate. This finding suggests that Metaphit may be a useful tool in the study of the molecular basis of stimulant action.     

Psychotomimetics moderately affect dopamine receptor binding in the rat brain

The hypothesis that psychotomimetics induce a rapid dopamine receptor regulation that could participate in the expression of the brain dopaminergic overactivation and in the early signs of psychotic-like behaviour, was checked by radioligand binding on rat brain cryosections. For this purpose, subchronic 7-day-d-amphetamine pretreatment was combined with acute amphetamine, phencyclidine or LSD challenge. Acute application of psychotomimetics affected only striatal and accumbens but not nigral and olfactory dopamine receptor binding after 40 min, while subchronic amphetamine expressed no effect, as revealed by two-way ANOVA. Post-hoc statistical analysis showed that only striatal and accumbens[3H]SCH 23390 binding decrease (10-12%) following phencyclidine and striatal [3H]spiperone binding increase (11%) after acute amphetamine were significant. It is assumed that such moderate dopamine receptor binding changes probably reflect the fast receptor regulation responses without important influence on a proposed drug-induced dopaminergic overactivity. The registered alterations of D1 receptor binding after phencyclidine are suggested to be capable to modify the activity of some other neural pathways in the basal ganglia and thus participate in a psychotic-like behaviour.     

Phencyclidine and related compounds evoked [3H]dopamine release from rat mesencephalic cell cultures by a mechanism independent of the phencyclidine receptor, sigma binding site, or dopamine uptake site

At concentrations greater than or equal to 100 microM, phencyclidine (PCP), N-(1-(2-thienyl)-cyclohexyl)piperidine (TCP), and MK-801 induced [3H]dopamine release from dissociated cell cultures of rat mesencephalon. This release was Ca2+ independent and tetrodotoxin insensitive. Tetrodotoxin (2 microM) itself had no effect on spontaneous release of [3H]dopamine. [3H]Dopamine release was induced by 1,3-di(2-tolyl)guanidine, a sigma ligand, and by 4-aminopyridine (1-3 mM), a K+ channel blocker. No stereoselectivity was observed for [3H]dopamine release evoked by the dioxadrol enantiomers, dexoxadrol, and levoxadrol, or by enantiomers of N-allylnormetazocine (SKF 10,047). The selective dopamine uptake inhibitor 1-(2-[bis(4-fluorophenyl)methoxy]ethyl)-4-(3-phenylpropyl)piperazine dihydrochloride (GBR 12909) did not affect spontaneous or TCP-evoked [3H]dopamine release. Together, these data suggest that the dopamine-releasing effects of PCP-like compounds on the mesencephalic cells were not mediated by actions at the PCP receptor or sigma binding site, Ca2+, or Na+ channels, or at the high affinity dopamine uptake site. It remains conceivable that blocking actions of PCP-like compounds at voltage-regulated K+ channels may at least partly explain the response. These results are discussed in comparison with findings in intact brain.     

Affinities of methylphenidate derivatives for dopamine,norepinephrine and serotonin transporters

We have synthesized several derivatives of dl-threo-methylphenidate (Ritalin) bearing substituents on the phenyl ring. IC₅₀ values for binding of these compounds to rat brain monoamine transporters were assessed using [³H]WIN 35,428 (striatal membranes, dopamine transporters, DAT), [³H]nisoxetine (frontal cortex membranes, norepinephrine transporters, NET) and [³H]paroxetine (brain stem membranes, 5HT transporters, 5HTT). Affinities (1/Ki) decreased in the order: DAT > NET ⪢ 5HTT. Substitution at the para position of dl-threo-methylphenidate generally led to retained or increased affinity for the dopamine transporter (bromo > iodo > methoxy > hydroxy). Substitution at the meta position also increased affinity for the DAT (m-bromo > methylphenidate; m-iodo-p-hydroxy > p-hydroxy). Substitution at the ortho position with bromine considerably decreased affinity. Similar IC₅₀ values for binding of o-bromomethylphenidate to the dopamine transporter were measured at 0, 22 and 37 degrees. N-Methylation of the piperidine ring of methylphenidate also considerably reduced affinity. The dl-erythro isomer of obromomethylphenidate did not bind to the DAT (IC₅₀ > 50,000 nM). Affinities at the dopamine and norepinephrine transporters for substituted methylphenidate derivatives were well correlated (r² = 0.90). Abilities of several methylphenidate derivatives to inhibit [³H]dopamine uptake in striatal synaptosomes corresponded well with inhibition of [³H]WIN 35, 428 binding. None of the compounds examined exhibited significant affinity to dopamine D1 or D2 receptors (IC₅₀ > 500 or 5,000 nM, respectively), as assessed by inhibition of binding of [³H]SCH 23390 or [¹²³I]epidepride, respectively, to striatal membranes.     

Chronic neuroleptic treatment in rats produces persisting changes in GABAA and dopamine D-2, but not dopamine D-1 receptors

The effects of continuous treatment with haloperidol (HAL) or fluphenazine (FLU) for 10 months on dopamine and GABA receptors in the rat brain was examined using in vitro autoradiography. Rats treated with HAL, but not FLU, showed an increase in D-2 receptor binding in the caudate-putamen as revealed by [3H]spiperone. Labeling of D-1 receptors by [3H]SCH23390 revealed no changes in either drug-treated group. Both drug-treated groups, however, exhibited a significant increase in [3H]muscimol binding in substantia nigra, pars reticulata (SNR). These dopaminergic-GABAergic receptor alterations may be related to previously reported changes in oral movement activity seen in these neuroleptic-treated animals.     

Interaction of non-steroidal antiestrogens with dopamine receptor binding

The ability of various estrogen antagonists and agonists to compete with [3H]spiroperidol, [3H]domperidone, [3H]dihydroalprenolol, [3H]dihydroergocryptine, [3H]dopamine or [3H]5-hydroxytryptamine for binding to membrane preparations from rat brain tissue was tested. The non-steroidal triphenylethylene-type antiestrogens with an amine side chain--enclomiphene, nitromifene, tamoxifen and zuclomiphene--were found to be competitive inhibitors of [3H]spiroperidol (Kd = 0.12 nM; Bmax = 101 fmol/mg protein) and [3H]domperidone (Kd = 0.62 nM; Bmax = 86 fmol/mg protein) binding to striatal membranes. The Ki values ranged from 4-12 microM. Estradiol-17 beta (Ki = 480 microM) or diethylstilbestrol (Ki = 63 microM) were much less effective inhibitors exhibiting noncompetitive interaction with the in vitro binding of [3H]spiroperidol. The pharmacological relevance of the antiestrogen interactions with dopamine receptor binding is discussed with respect to adverse effects of the in vivo administered compounds such as nausea and vomiting.     

Peripheral dopamine receptor identification: Properties of a specific dopamine receptor in the rat adrenal zona glomerulosa

Specific dopaminergic receptors were found in the rat adrenal zona glomerulosa. Specific binding as defined by the difference in [³H]-spiroperidol binding in the presence or absence of excess dopamine was saturable and of high affinity. Stereospecificity of binding to the dopaminergic receptor was demonstrated by the fact that (+)-butaclamol was 300-fold more active at displacing [³H]-spiroperidol from the binding site than (?)-butaclamol. A Scatchard analysis of the data revealed a K_D = 6.9 nM and a B_max = 173 pmol/gm for the binding of [³H]-spiroperidol to adrenal capsular homogenate binding site. Characteristics of this receptor place it in the recently defined D2 dopamine receptor subclass.     

Multiple dopamine binding sites: subcellular localization and biochemical characterization.

Abstract— The biochemical and pharmacological characteristics of dopamine agonist and antagonist binding to rat striatal subcellular fractions were studied and compared to the localization of dopamine–sensitive adenylate cyclase activity. The highest specific activity of adenylate cyclase sensitive to dopamine was associated almost exclusively with the crude synaptic membrane fraction (P₂). Using [³H]-haloperidol, [³H]apomorphine and [³H]spiroperidol as markers for the dopamine receptor, high affinity and stereoselective specific binding was observed for the crude synaptic fraction and the microsomal fraction (P₃). Analysis of the binding of [³H]haloperidol to the striatal microsomal preparation revealed a homogeneous receptor site with a K_d value of 3.0 nm . The data for [³H]haloperidol binding to the crude synaptosomal fraction showed two saturable binding sites with K_d values of 2.5 nm and 12.5 nm . A similar heterogeneous binding profile was observed in the P₂ fraction using [³H]apomorphine. The K_d values for [³H]apomorphine in this fraction were determined to be 1.2 nm and 7.2 nm . The effects of various biochemical parameters including ionic strength, salt concentration and pH on the binding of [³H]haloperidol to the P₂ fraction were also studied. Overall, these data show that the subcellular localization of multiple binding sites in the crude synaptosomal fraction and the identification of specific binding to purified synaptosomes correlate with the subcellular distribution of striatal dopamine-sensitive adenylate cyclase activity.     

Evidence for essential thiol groups and disulfide bonds in agonist and antagonist binding to the dopamine receptor

Dithiothreitol (DTT), a disulfide reducing agent, diminished the specific binding of [³H] dopamine to partially purified calf striatal membranes (P₂) but did not have an effect on [³H] spiroperidol binding. The thiol reagents, p-chloromercuribenzoate (PCMB), N-ethylmaleimide (NEM) and iodoacetamide (IA), were also tested for inhibitory effects on agonist and antagonist binding to the dopamine receptor. PCMB inhibited both [³H] dopamine and [³H] spiroperidol binding by changing the affinity (K_d) and the number of binding sites (B_max) for both of these ligands. This effect of PCMB was reversed by the addition of DTT. NEM inhibited binding to the dopamine agonist site but not to the antagonist site, while IA was ineffective on either site. These results indicate that a DTT-reducible disulfide bond may be an essential component for agonist binding to the dopamine receptor. Furthermore, the experiments with PCMB, NEM and IA suggest that the exposure of thiol groups in the dopamine receptor may play an important role in agonist and antagonist binding.     

Pharmacological characterization of mammalian D1 and D2 dopamine receptors expressed in Drosophila Schneider-2 cells

Mammalian D1 and D2 dopamine receptors were stably expressed in Drosophila Schneider-2 (S2) cells and screened for their pharmacological properties. Saturable, dose-dependent, high affinity binding of the D1-selective antagonist [3H]SCH-23390 was detected only in membranes from S2 cells induced to express rat dopamine D1 receptors, while saturable, dose-dependent, high affinity binding of the D2-selective antagonist [3H]methylspiperone was detected only in membranes from S2 cells induced to express rat dopamine D2 receptors. No specific binding of either radioligand could be detected in membranes isolated from uninduced or untransfected S2 cells. Both dopamine D1 and D2 receptor subtypes displayed the appropriate stereoselective binding of enantiomers of the nonselective antagonist butaclamol. Each receptor subtype also displayed the appropriate agonist stereoselectivities. The dopamine D1 receptor bound the (+)-enantiomer of the D1-selective agonist SKF38393 with higher affinity than the (-)-enantiomer, while the dopamine D2 receptor bound the (-)-enantiomer of the D2-selective agonist norpropylapomorphine with higher affinity than the (+)-enantiomer. At both receptor subtypes, dopamine binding was best characterized as occurring to a single low affinity site. In addition, the low affinity dopamine binding was also found to be insensitive to GTPgammaS and magnesium ions. Overall, the pharmacological profiles of mammalian dopamine D1 and D2 receptors expressed in Drosophila S2 cells is comparable to those observed for these same receptors when they are expressed in mammalian cell lines. A notable distinction is that there is no evidence for the coupling of insect G proteins to mammalian dopamine receptors. These results suggest that the S2 cell insect G system may provide a convenient source of pharmacologically active mammalian D1 and D2 dopamine receptors free of promiscuous G protein contaminants.     

Modulation of [3H]quinpirole binding at striatal D2 dopamine receptors by a monoamine oxidaseA-like site: evidence from radioligand binding studies and D2 receptor- and MAO(A)-deficient mice.

[3H]Quinpirole is a dopamine agonist with high affinity for the D2 and D3 dopamine receptors. A variety of monoamine oxidase inhibitors (MAOIs) inhibit equilibrium binding of [3H]quinpirole binding in rat striatal membranes suggesting that MAOIs interact with a novel binding site that is labeled by [3H]quinpirole or that allosterically modulates [3H]quinpirole binding. To determine whether the D2 receptor is essential for [3H]quinpirole binding and/or modulation of [3H]quinpirole binding by MAOIs, D2 receptor-deficient mice were studied. [3H]Quinpirole binding was decreased in D2 receptor-deficient mice to 3% of that observed in wild-type controls indicating that [3H]quinpirole binding is associated with the D2 dopamine receptors. Then, in an attempt to label the site mediating the modulation of [3H]quinpirole binding, binding of the MAOI [3H]Ro 41-1049 was characterized in rat striatal membranes. [3H]Ro-41-1049 labeled a single binding site with a pharmacological profile with respect to MAOIs that was similar to both [3H]quinpirole binding (Spearman r=0.976) and MAO(A) activity. To determine whether MAO(A) plays a role in the modulation of [3H]quinpirole binding by MAOIs, MAO(A)-deficient mice were examined. In these mice, [3H]Ro-41-1049 binding was decreased to 7% of wild-type control. [3H]Spiperone binding was unaltered. Spiperone-displaceable [3H]quinpirole binding was decreased to 43% of wild-type control; however, the remaining [3H]quinpirole binding in MAO(A)-deficient animals was inhibited by Ro 41-1049 similar to wild-type. [3H]Ro-41-1049 binding was not decreased in D2 receptor-deficient mice. These data suggest that [3H]Ro-41-1049 labels multiple sites and that MAOIs modulate [3H]quinpirole binding to the D2 receptor via interactions at a novel, non-MAO binding site with MAO(A)-like pharmacology.     

Modulation of dopamine receptors by thyrotropin-releasing hormone in the rat brain

Nanomolar concentration of thyrotropin-releasing hormone (TRH) in vitro caused a significant reduction of [3H]apomorphine binding sites (70% of the control) in the rat striatum and the limbic forebrain. [3H]Spiperone binding was not affected by TRH. On the other hand, dopamine and apomorphine displaced [3H]TRH binding partially, suggesting the presence of a TRH receptor subpopulation that has a high affinity for dopamine agonist. Most of the neuroleptics displaced [3H]TRH binding dose-dependently in the micromolar range. (-)-Sulpiride had no affinity to TRH receptors. These findings suggest that one of the important roles of TRH as a neuromodulator is to modulate receptors for classical neurotransmitters, and this receptor-receptor interaction may be of importance in explaining the well known stimulating effects of TRH on the dopaminergic system.     

Endogenous modulator of dopamine receptor(s)

An endogenous modulator(s) of the dopamine receptor(s) in bovine and rat brain striatum was detected by demonstrating that water extracts of the striatum inhibited [³H]apomorphine binding. This modulator(s) was partially purified by methanol extraction and then successive ion exchange chromatographies on SP-Sephadex C-25 and QAE-Sephadex A-25, and gel chromatography on Sephadex G-25. The partially purified (about 1,500-fold) modulator was a fluorescamine-positive substance, Mr = 500 1000, which was heat-stable (95°C, 10 min), and was destroyed by acid- and alkali-treatment, but not by treatments with various peptidases. The modulator inhibited binding of the dopamine agonist, [³H]apomorphine non-competitively, but did not inhibit binding of the dopamine antagonist, [³H]spiroperidol. Direct injection of the modulator into rat brain striatum depressed apomorphine-induced locomotor activity. Moreover the modulator inhibited dopamine-sensitive adenylate cyclase activity. These findings indicate that the modulator acts at a site(s) other than the ligand binding site of the dopamine receptor(s) and modulates the activities of dopamine agonists.     

[3H]Threo-(±)-Methylphenidate Binding to 3,4-Dihydroxyphenylethylamine Uptake Sites in Corpus Striatum: Correlation with the Stimulant Properties of Ritalinic Acid Esters

Saturable and stereoselective binding sites for [3H]threo-(+/-)-methylphenidate were characterized in rat brain membranes. The highest density of [3H]threo-(+/-)-methylphenidate binding sites was found in the synaptosomal fraction of corpus striatum. Scatchard analysis revealed a single class of noninteracting binding sites with an apparent dissociation constant (KD) of 235 nM and a maximum number of binding sites (Bmax) of 13.4 pmol/mg protein. Saturable, high-affinity binding of [3H]threo-(+/-)-methylphenidate to striatal synaptosomal membranes was dependent on the presence of sodium ions. A good correlation (r = 0.88; p less than 0.001) was observed between the potencies of various psychotropic drugs in displacing [3H]threo-(+/-)-methylphenidate from these sites and their potencies as inhibitors of [3H]3,4-dihydroxyphenylethylamine ( [3H]dopamine) uptake into striatal synaptosomes. A good correlation (r = 0.85; p less than 0.001) was also observed between the potencies of a series of ritalinic acid esters in inhibiting [3H]threo-(+/-)-methylphenidate binding to striatal synaptosomal membranes and their potencies as motor stimulants in mice. These observations suggest that the binding sites for [3H]threo-(+/-)-methylphenidate described here are associated with a dopamine uptake or transport complex, and that these sites may mediate the motor stimulant properties of ritalinic acid esters such as methylphenidate.     

The bicuculline-like properties of dopamine sulfate in rat brain

To determine whether the convulsive action of intraventricularly injected dopamine sulfate, a dopamine metabolite present in rat brain and human cerebrospinal fluid, could be due to its interaction with GABAergic pathway, we compared the convulsive effect of dopamine sulfate with that of bicuculline in the conscious rat and determined the interaction of dopamine sulfate with [3H] GABA binding and uptake in rat brain tissues. The results showed that the convulsive effects of dopamine sulfate and of bicuculline could be abolished by GABA agonists diazepam and muscimol, but not by DA antagonists haloperidol and metoclopramide. In addition they were additive. Both dopamine 3-O-sulfate and dopamine-4-O-sulfate, like bicuculline, could displace sodium-independent [3H] GABA binding to rat brain synaptic membranes (IC50 = 400 microM) but had no action on GABA uptake. DA sulfate had no effect on [3H] strychnine binding to rat brain homogenates. This evidence together with the structural resemblance between dopamine sulfate and GABA suggested that the convulsive activity of dopamine sulfate may result from its interaction with central GABA receptors.     

Is Metaphit a phencyclidine antagonist? Studies with ketamine, phencyclidine and N-methylaspartate

The dissociative anaesthetics, phencyclidine and ketamine, block excitation of central neurones by N-methylaspartate. Using the technique of microelectrophoresis on rat spinal neurones in vivo Metaphit, a phencyclidine receptor acylating agent, was tested to see whether it would antagonise this effect of dissociative anaesthetics. The predominant effect of Metaphit was, however, to reduce N-methylaspartate induced excitation. It is concluded that Metaphit has mixed agonist/antagonist effects at the phencyclidine receptor.     

Metaphit antagonizes phencyclidine-induced hypothermia in the rat

The acute administration of phencyclidine (PCP) causes hypothermia in the rat. Metaphit (1-[1-(3-isothiocyanatophenyl)cyclohexyl]-piperidine) is a derivative of PCP that has been shown to irreversibly acylate PCP receptors in vitro and in vivo and can antagonize the behavioral and electrophysiological effects of PCP in the rat. The purpose of the present study was to determine whether pretreatment with metaphit can block the hypothermic effects of PCP in the rat. Metaphit or PCP (1.0 mumol/rat) were injected into the lateral ventricles of rats, and 24 hr later the subjects were challenged with PCP (20.0 mg/kg s.c.). Pretreatment with metaphit blocked PCP-induced hypothermia; however, pretreatment with PCP did not affect the subsequent hypothermic response to PCP. These results indicate that the antagonism of PCP-induced hypothermia by metaphit was a specific effect and not due to PCP receptor desensitization.     

Binding of a novel dopaminergic agonist radioligand [3H]-fenoldopam (SKF 82526) to D-1 receptors in rat striatum

Fenoldopam (SKF 82526), a dopamine agonist which exhibits D-1 receptor subtype selectivity, was evaluated as a radioligand for this receptor subtype. In saturation studies in rat striatal membrane preparations, [3H]-fenoldopam appeared to label a single binding site with a KD of 2.3 +/- 0.1 nM and a Bmax of 590 +/- 40 fmoles/mg protein. In competition binding experiments, binding was shown to be stereoselective, and rank ordering of affinities of dopaminergic and non-dopaminergic compounds closely correlated with potencies of these compounds in stimulating or inhibiting dopamine-sensitive adenylate cyclase (D-1) and in binding to D-1 sites labelled with the antagonist [3H]-cis-flupenthixol. The most potent competitors were the recently identified D-1 selective antagonists, SCH 23390 and SKF R-83566. [3H]-Fenoldopam was also used to assess agonist/D-1 receptor interactions. The results suggest that [3H]-fenoldopam is a useful and selective agonist radioligand for the D-1 receptor.     

Chemical modification reveals involvement of tyrosine in ligand binding to dopamine D1 and D2 receptors

The effect of tyrosine-alkylating agents on the ligand-binding properties of bovine striatal dopamine D1 and D2 receptors was investigated. The tyrosine-alkylating agents, p-nitrobenzenesulphonylfluoride (pNBSF) and tetranitromethane (TNM) caused a time-and dose-dependent loss of the binding of [3H]SCH-23390 and [3H]spiroperidol, ligands specific for dopamine D1 and D2 receptors, respectively. The two dopamine receptors, however, showed a differential sensitivity to inactivation by these agents. The mechanism of inhibition of the two receptors appears to be complex as treatment of membranes with pNBSF and TNM resulted in a decrease of both the Kd and the Bmax of ligand binding. Spiroperidol almost completely protected the TNM-induced inhibition of [3H]spiroperidol binding to dopamine D2 receptors whereas SCH-23390 afforded only partial protection of the [3H]SCH-23390 binding by TNM suggesting that more than one tyrosine groups may be involved in the D1 receptor binding activity.