In vivo binding of 3H-N-methylspiperone to dopamine and serotonin receptors

3H-N-methylspiperone (3H-NMSP) was used to label dopamine-2 and serotonin-2 in vivo in the mouse. The striatum/cerebellum binding ratio reached a maximum of 80 eight hours after intravenous administration of 3H-NMSP. The frontal cortex/cerebellum ratio was 5 one hour after injection. The binding of 3H-NMSP was saturable in the frontal cortex and cerebellum between doses of 10 and 1,000 micrograms/kg. Between 0.01 and 10 micrograms/kg the ratio total/nonspecific binding increased from 14 to 21. Inhibition of 3H-NMSP binding in the frontal cortex and striatum by ketanserin, a selective serotonin-2 antagonist, demonstrated that 20% of the total binding in the striatum was to serotonin-2 receptors and 91% of the total binding in the frontal cortex was to serotonin-2 receptors. Compared to 3H-spiperone, 3H-NMSP results in a much higher specific/nonspecific binding ratio in the striatum and frontal cortex and displays more than a two-fold higher brain uptake.     

Evaluation of a Purification Procedure for the Muscarinic Receptor for the Purpose of Quantitative Receptor Assays of Anticholinergics

Abstract

The presented purification procedure for the muscarinic receptor from calf striatum includes the extraction of lipids with hexane in the first step and the removal of 39% of non-receptor proteins with 2 M NaCl in the second step. The simplicity of such an approach to the purification of the receptor warrants its use in the routine practice for quantitative purposes. The high affinity binding of tertiary ³H-dexetimide (³H-DEX) and quaternary ³H-N-methylscopolamine (³H-NMS) is preserved after the removal of irrelevant lipids and proteins from the P2-pellet.

The overall yield of receptors - 80%, when labelled with ³H-NMS, was satisfactory. Moreover, the final product, the NaCl-pellet, exerts a higher density of ³H-NMS binding sites per mg proteins by a factor of about 1.7. The overall yield of receptors and purification factor were lower, when measured with ³H-DEX. The total yield of ³H-DEX binding sites amounted to about 40% and the receptor density per mg protein decreased by a factor of 0.85.

We did not succeed in the improvement of the ratio specific/non-specific binding, neither for ³H-DEX nor for ³H-NMS for the purified receptor preparations. The use of ³H-NMS is preferable to ³H-DEX in plasma sample assays because of a negligible effect of plasma on ligand binding when compared with ³H-DEX.     

Alterations in central neurotransmitter receptor binding sites following estradiol implantation in female rats

The subcutaneous implantation of an estradiol pellet (10 mg) into female rats induced a hypophyseal hyperplasia with hyperprolactinaemia. Examination of neurotransmitter receptors in the hippocampus, striatum and cerebral cortex one month after the implantation revealed that estrogenization was associated with: an increased density of ³H-domperidone binding sites (D₂ receptors) in the striatum and reduced numbers of ³H-serotonin high affinity sites (5-HT₁ receptors) in the hippocampus and of ³H-muscimol binding sites (GABA receptors) in the hippocampus, striatum and cerebral cortex. In contrast, the characteristics of ³H-spiperone binding to 5-HT₂ receptors (in the cerebral cortex) and those of ³H-flunitrazepam binding to benzodiazepine sites (in the three brain regions examined) were not significantly different in estrogenized and in control female rats. However, the enhancing effect of GABA on ³H-flunitrazepam binding was markedly reduced in brain membranes from estrogenized animals. The respective roles of estradiol and prolactin in mediating these changes in neurotransmitter receptors are discussed notably with regard to the regional heterogeneity of estradiol binding capacity in the rat brain.     

Allosteric effects of four stereoisomers of a fused indole ring system with 3H-N-methylscopolamine and acetylcholine at M1-M4 muscarinic receptors

We previously demonstrated that brucine and some analogues allosterically enhance the affinity of ACh at muscarinic receptor subtypes M1, M3 or M4. Here we describe allosteric effects at human M1-M4 receptors of four stereoisomers of a pentacyclic structure containing features of the ring structure of brucine. All compounds inhibited 3H-NMS dissociation almost completely at all subtypes with slopes of 1, with similar affinity values at the 3H-NMS-occupied receptor to those estimated from equilibrium assays, consistent with the ternary complex allosteric model. Compound 1a showed positive cooperativity with H-NMS and small negative or neutral cooperativity with ACh at all subtypes. Its stereoisomer, 1b, showed strong negative cooperativity with both 3H-NMS and ACh across the subtypes. Compound 2a was positive with 3H-NMS at M2 and M4 receptors, neutral at M3 and negative at M1 receptors; it was negatively cooperative with ACh at all subtypes. Its stereoisomer, 2b, was neutral with 3H-NMS at M1 receptors and positive at the other subtypes; 2b was negatively cooperative with ACh at M1, M3 and M4 receptors but showed 3-fold positive cooperativity with ACh at M2 receptors. This latter result was confirmed with further 3H-NMS and 3H-ACh radioligand binding assays and with functional assays of ACh-stimulated 35S-GTPgammaS binding. These results provide the first well characterised instance of a positive enhancer of ACh at M2 receptors, and illustrate the difficulty of predicting such an effect.     

Effect of long-term L-dopa administration on the dopaminergic and cholinergic (muscarinic) receptors of striatum in 6-hydroxydopamine lesioned rats

L-Dihydroxyphenylalanine (L-Dopa) (200 mg/kg/day) was administered for 30 days to the rats whose nigrostriatal dopamine pathway was lesioned unilaterally with 6-hydroxydopamine and the receptor binding of ³H-spiperone and ³H-quinuclidinyl benzilate (³HQNB) was measured in the dopaminergic and muscarinic cholinergic receptors of the striatum. ³H-spiperone binding increased by 73% and ³HQNB binding decreased by 14% in the lesioned side when compared to the control side of L-Dopa-non-treated rats. ³H-spiperone binding was measured in the lesioned sides of L-Dopa-treated and L-Dopa-non-treated rats and was found to have decreased by 21% in the former. In the control side of the L-Dopa-treated lesioned rats, however, ³H-spiperone binding increased by 27% when compared to the opposite striatum of the same rats. ³HQNB binding in the lesioned side of L-Dopa-treated rats was not significantly different from that of the control side statistically. These results suggest that changes in functional equilibrium between the dopaminergic and cholinergic mechanisms influence the muscarinic cholinergic receptors and that supersensitivity of dopamine receptors after lesion of the nigrostriatal pathway also remains after long-term L-Dopa treatment.     

Dopamine receptor elevation by cholecystokinin

High concentrations of cholecystokinin (CCK) in the striatum and limbic areas of the brain suggest that this peptide may influence dopaminergic transmission. Thus, the effect of CCK on dopamine D₂ receptors in the striatum and nucleus accumbens of the rat brain both in vitro and in vivo (central and peripheral administration) was studied by determining the binding of ³H-spiperone. The density (B_max) of D₂ receptors was elevated (a) by 20% in the accumbens upon in vitro co-incubation with 10⁻⁶ M CCK. (A non-significant drop of 10% occurred in the striatum); (b) by about 40% in the accumbens and 25% in the striatum after continuous intraventricular infusion of CCK for 24 hr. The increase in receptor density in the accumbens was maintained for 14 days and in both tissues was specific to CCK (neurotensin infusion did not alter ³H-spiperone binding); (c) by 20% in the accumbens and 15% in the striatum 3 hr after a single IP injection of 50 μg/kg CCK or caerulein, and maintained up to 14 days later. These results suggest that CCK elevates dopamine D₂ receptors in the accumbens and striatum and may be a physiological modulator of the dopaminergic system.     

Neurotensin interacts with dopaminergic neurons in rat brain

Neurotensin (NT) injected intracerebroventricularly in rat increases dopamine (DA) turnover in the corpus striatum and nucleus accumbens. Significant increases in 3,4-dihydroxyphenylacetic acid (DOPAC) levels occurred within 15 minutes after injection with peak levels at 60 minutes. The effect on NT on DOPAC and homovanillic acid (HVA) accumulation was dose-dependent at 3–100 μg. NT, like haloperidol, stimulated 3,4-dihydroxyphenylalanine (DOPA) accumulation in striatal neurons, in the presence of DOPA decarboxylase inhibitor, after injection of gamma-butyrolactone (GBL). NT had a similar stimulatory effect on DOPA levels in the accumbens while haloperidol (0.25 mg·kg^?1) had no significant effect in this brain region. NT did not block the inhibitory effect of apomorphine on DOPA accumulation in both the striatum and accumbens, while haloperidol inhibited apomorphine effect in both regions. NT also failed to displace ³H-spiperone from DA receptors and the presence of NT in the binding assay did not alter the ability of DA to displace ³H-spiperone in either brain region. These experiments demonstrate that NT increases DA turnover in both the nigrostriatal and mesolimbic pathways.     

Taurine Modulation of the Benzodiazepine-γ-Aminobutyric Acid Receptor Complex in Brain Membranes

In unwashed brain membranes taurine produced an inhibition of [3H]flunitrazepam [( 3H]FNZ) binding with IC50 ranging between 31.5 and 11.9 microM; the IC20 varied between 18 and 26 nM. This inhibitory effect was of a mixed type, with a reduction in Bmax and an increase in KD. Various precursors and metabolites of taurine have a less inhibitory effect. Taurine also has little inhibitory effect (IC50 above 500 microM) on the binding of [3H]ethyl-beta-carboline-3-carboxylate. In extensively washed membranes, 10(-5) M taurine produces a 16-21% increase in the binding of [3H]FNZ while 10(-5) M gamma-aminobutyric acid (GABA) increases it between 31 and 42%. However, if 10(-5) M GABA plus 10(-5) M taurine is included in the assay there is a dramatic inhibitory effect. Taurine causes an inhibition of the GABAergic enhancement of [3H]FNZ binding with an IC50 between 7.3 and 7.8 microM. Binding experiments with [3H]taurine done under different conditions failed to detect a Na+-independent and specific [3H]taurine receptor. These results suggest that endogenous taurine, the second most abundant free amino acid in brain, may play an important modulatory role in the GABA-benzodiazepine receptor complex.     

In vivo studies of dopamine receptor ontogeny

Studies of the ontogeny of dopamine and neuroleptic receptors in the central nervous system of the rat were carried out $\text{in vivo}$ using ³H-spiperone as ligand. It was demonstrated that intraperitoneal injections can be successfully used to label these receptors in rat pups up to at least 30 days of age. The time course and characteristics of ³H-spiperone binding in the brain of 5, 15 and 30 day old rat pups were determined and found to include appropriate regional distribution, saturability and appropriate pharmacology. The developmental pattern of ³H-spiperone binding paralleled what has been seen using $\text{in vitro}$ techniques. In addition preliminary autoradiographic studies describe the neuroanatomical pattern of dopamine receptor ontogeny in the striatum.     

Effect of carbidine on the dopamine and serotonin receptors of the central nervous system

It has been shown in experiments on mice and rats that unlike haloperidol and sulpiride, carbidine does not influence the intensity and even increases the duration of apomorphine stereotypy. However, similarly to haloperidol carbidine decreases head twitches in mice, induced by administration of 5-hydroxytryptophan. In radioligand-binding experiments in vitro and in vivo carbidine displaced 3H-spiperone but in the brain cortex without having any effect on the binding in the striatum. Carbidine did not affect the dopamine-sensitive adenylate cyclase in rat striatum. Based on these data it is suggested that in contrast to haloperidol and sulpiride, carbidine selectively inhibits serotonin receptors of the brain.     

Chronic neuroleptic treatment specifically alters the number of dopamine receptors in rat brain

Trifluoperazine dihydrochloride (2.8–4.0 mg/kg/day) was administered continuously to rats in drinking water for six months. Animals killed at this time exhibited an increase in the number of dopamine receptors in the striatum and mesolimbic area, with a corresponding decrease in affinity (increase in the dissociation constant) for ³H-spiperone binding. In frontal cortex, ³H-spiperone binding to 5-HT receptors indicated no apparent change in numbers of receptors, but a slight increase in the dissociation constant. There was no obvious alteration in ³H-apomorphine binding in the striatum and mesolimbic area, but the individual results were very variable. The number and binding affinity of muscarinic receptors in striatum, mesolimbic area and cerebral cortex as identified by ³H-dexetemide were unchanged. Nor was there any alternation in the number or binding affinity of H-1 receptors identified by ³H-mepyramine, or of α-noradrenergic receptors identified by ³H-WB 4101, in cerebral cortex. The number and binding affinity of GABA receptors in the cerebellum identified by ³H-muscimol also was not altered.Chronic neuroleptic administration to rats appears to alter specifically the number of cerebral dopamine receptors.     

Comparison of Neuroleptic Binding Characteristics in Rat Striatum and Renal Cortex

Abstract

The binding characteristics of the dopaminergic ligand, ³H- spiperone, were compared in renal cortical and striatal membrane homogenates of the rat. This ligand labelled a single class of high affinity binding sites in striatum with an apparent dissociation constant (Kd) of 0.13 nM and a maximal number of binding sites (Bmax) of 890 fmol/mg protein representing D-2 receptors. In the renal cortex, ³H-spiperone identified a population of binding sites with a Bmax and a Kd of 310 fmol/mg protein and 5.1 nM, respectively. The antagonist displacing profile suggests the dopaminergic nature of the renal binding site. The affinities of dopamine antagonists for the peripheral ³H-spiperone binding site were in general in the micromolar range while the affinities of D-2 or D-2/D-1 dopamine antagonists in striatum were in the nanomolar range. Moreover, these sites showed differential stereoselectivity for (+)- and (-)-isomers of sulpiride. In conclusion, the presence of a D-2/DA-2 dopamine receptor population in renal cortex could not be confirmed. The pharmacological properties of the peripheral ³H-spiperone binding site are also different from the DA-1 receptor but seem to resemble those previously reported for dopamine receptors in sympathetic ganglia and adrenal medulla.     

125I-Spiperone: a novel ligand for D2 dopamine receptors

125I-Spiperone binds with high affinity (KD 0.3 nM) to a single specific site (Bmax 34 pmol/g wet weight) in homogenates of rat corpus striatum. Specific binding is about 40-60 percent of total binding and is displaced stereo-specifically by butaclamol and clopenthixol. Neuroleptic drugs of various classes are potent inhibitors of 125I-spiperone binding (Ki's 1-10 nM). Selective dopamine antagonists such as sulpiride (Ki 50 nM) and dopamine agonists such as apomorphine (Ki 200 nM) are also potent inhibitors. The drug specificity of 125I-spiperone binding correlates well with that of 3H-spiperone binding, providing good evidence that 125I-spiperone labels D2 dopamine receptors in striatal membranes. 125I-Spiperone, with its high specific activity (2200 Ci/mmol) may prove to be a useful ligand in studies examining D2 dopamine receptors in soluble preparations and by autoradiography. Furthermore iodinated spiperone may be useful in radioreceptor assays of neuroleptic drug levels and, in a 123I-labeled form, for imaging of dopamine receptors, in vivo, using single photon tomography.     

Characterization of specific in vivo binding of neuroleptic drugs in rat brain

$\text{In vivo}$ binding of ³H-spiperone is saturable in the striatum, the limbic system and the frontal cortex but not in the cerebellum. A specific binding is different in all the brain regions thus the amount of labelling in the cerebellum may not be considered as a blank value.³H-spiperone binding revealed a specific subcellular distribution only when a very low dose was injected into rats.$\text{Ex vivo}$ experiments allow the assessment of biochemical profiles of neuroleptic drugs according to their relative affinity for dopamine or serotonin receptors.     

77Br-p-bromospiperone: a ligand for in vivo labelling of dopamine receptors

A high striatum: cerebellum ratio of 77Br-p-bromospiperone (77Br-BrSp) was observed in rat brain following tail vein injection of the drug. Striatal 77Br-BrSp was stereospecifically displaced by the isomers of flupenthixol. After chronic haloperidol administration striatal dopamine receptor supersensitivity was demonstrated both by increased 3H-spiperone binding to striatal membranes in vitro and by increased striatal 77Br-BrSp content. These results confirm and extend previous findings and enhance interest in the use of 77Br-BrSp for the in vivo assessment of central dopamine receptors in man.     

Changes in the sensitivity of brain dopamine and serotonin receptors during the prolonged administration of carbidine

Male Wistar rats were treated chronically with either carbidine (10 mg/kg/day) or haloperidol (1 mg/kg/day) for 23 consecutive days. On days 4-5 after the treatment discontinuation the animals were challenged with apomorphine HCl (0.5 mg/kg) or 5-OTP (150 mg/kg i. p) in combination with pargiline (40 mg/kg i. p) and stereotype responses were scored. In carbidine-treated rats the intensity of stereotype sniffings was increased after apomorphine treatment. In contrast, animals treated with haloperidol exhibited more intensive gnawing after apomorphine in comparison to vehicle-treated rats. 5-OTP-induced head twitches were increased only in carbidine-treated rats. Prolonged carbidine treatment in contrast to haloperidol induced a decrease in 5H-spiperone and 3H-LSD binding in the frontal cortex, with the density of D-2 receptors in the striatum practically unchanged. It is concluded that neuroleptic carbidine in contrast to classical neuroleptics has a more selective effect in serotonin (S-2) receptors and antidepressive properties of this compound may be due to the down-regulation of S-2 receptors in the brain.     

Selective inhibition of the binding of 3H-(3-MeHis2) thyrotropin releasing hormone to rat amygdala membranes by some naturally occurring cannabinoids

The effect of naturally occurring cannabinoids, delta 9-tetrahydrocannabinol (THC), cannabinol (CBN) and cannabidiol (CBD), on the brain receptors for thyrotropin releasing hormone (TRH) was investigated. TRH receptors were labeled with 3H-(3-MeHis2)TRH (3H-MeTRH). 3H-MeTRH bound specifically to rat brain membranes at a single high affinity site with a Bmax value of 49.2 +/- 0.96 fmol per mg protein and a Kd value of 3.83 +/- 0.12 nM. The binding of 3H-MeTRH to whole brain membranes was inhibited when rats were injected intraperitoneally with 3 to 30 mg/kg of THC. The extent of inhibition in the binding at 10 and 30 mg/kg was similar. THC (10 mg/kg) significantly inhibited the binding of 3H-MeTRH to amygdala membranes but did not affect the binding to membranes prepared from hippocampus, septum, cortex, striatum and the rest of the brain. THC, CBN and CBD in doses of 3 to 30 mg/kg did not affect the binding of 3H-MeTRH to hypothalamic membranes. All the three cannabinoids at 30 mg/kg inhibited the binding of 3H-MeTRH to amygdala membranes. The inhibition in the binding of 3H-MeTRH by the cannabinoids was due to changes in the Kd values but the Bmax values remained unchanged. It is concluded that both psychotomimetic and nonpsychotomimetic cannabinoids inhibit the binding of 3H-MeTR to amygdala membranes selectively, which is accomplished by decreases in the affinity of the ligand to receptors, and the amygdala may be an important brain area in some of the actions of cannabinoids.     

Brain neurotransmitter receptor-binding characteristics in rats after oral administration of haloperidol, risperidone and olanzapine

The present study was conducted to characterize the binding of neurotransmitter receptors (dopamine D(2), serotonin 5-HT(2), histamine H(1), adrenaline alpha(1) and muscarine M(l) receptors) in the rat's brain after the oral administration of haloperidol, risperidone, and olanzapine. Haloperidol at 1 and 3 mg/kg displayed significant activity to bind the D(2) receptor (increase in the Kd value for [(3)H]raclopride binding) in the corpus striatum with little change in the activity toward the 5-HT(2) receptor (binding parameters for [(3)H]ketanserin). In contrast, risperidone (0.1-3 mg/kg) showed roughly 30 times more affinity for the 5-HT(2) receptor than D(2) receptor. Also, olanzapine (1-10 mg/kg) was most active toward the H(1) receptor in the cerebral cortex, corpus striatum, and hippocampus, was less active in binding 5-HT(2) and D(2) receptors, and showed the least affinity for alpha(1) and M(1) receptors. In conclusion, haloperidol and risperidone administered orally selectively bind D(2) and 5-HT(2) receptors, respectively, in the rat brain, while olanzapine binds H(1), 5-HT(2), and D(2) receptors more than alpha(1) and M(1) receptors.     

Effect of Unilateral Perinatal Hypoxic-Ischemic Brain Injury in the Rat on Striatal Muscarinic Cholinergic Receptors and High-Affinity Choline Uptake Sites: A Quantitative Autoradiographic Study

The binding characteristics and distribution of M1 and M2 muscarinic cholinergic receptors and high-affinity choline uptake sites were studied in the striatum of the rat at 3-4 and 9-12 weeks of age after exposure to unilateral perinatal hypoxic-ischemic brain injury. High-affinity choline uptake sites were labeled with [3H]hemicholinium-3, M1 receptors with [3H]pirenzepine, and M2 receptors with [3H]AF-DX 116. Saturation experiments revealed a significant decrease in the maximal binding capacity (Bmax) for [3H]pirenzepine-labeled M1 receptors in the lesioned caudate/putamen complex in immature rats with moderate brain injury, in comparison with controls. In contrast, the Bmax value for [3H]hemicholinium-3-labeled high-affinity choline uptake sites was significantly increased. No changes in dissociation constants (KD) were observed. These changes were most pronounced in the dorsolateral region of striatum. Striatal regional distribution of [3H]AF-DX 116 was not affected. In mature rats, binding of [3H]pirenzepine returned to control values, whereas [3H]hemicholinium binding showed a persistent increase (23%). The increase in [3H]hemicholinium-3 binding, as a specific marker of cholinergic nerve terminals, is consistent with our prior morphologic studies demonstrating relative preservation of cholinergic neurons and neuropil, and supports the concept that striatal cholinergic systems are resistant to hypoxic-ischemic injury.     

Different kinetic properties of neuroleptic receptor binding in the rat striatum and frontal cortex.

³H-spiperone binding in the striatum is 80% stereospecific and 6 % non-stereospecific. In the frontal cortex stereospecific sites account for 60 % of the total binding and non-stereospecific sites for 25 %. Stereospecific sites are of different nature in both brain regions : dopaminergic in the striatum and serotonergic in the frontal cortex. The non-stereospecific sites are saturable and can only be detected by the use of appropriate blanks.Release experiments demonstrate the occurrence of positive coöperative effects in the dissociation of spiperone from striatal receptors while this is not detectable in the frontal cortex. In both brain regions a rapidly and a slowly dissociating component have been observed. In the frontal cortex the rapid component is ascribed to the dissociation of spiperone from the stereospecific sites and the slow component to the dissociation from non-stereospecific sites. The nature of these sites is yet to be identified.