Effects of a series of substituted benzamides on rat prolactin secretion and 3H-spiperone binding to bovine anterior pituitary membranes

The potency of seven substituted benzamine drugs (AHR-5531B, AHR-5645B, AHR-6092, AHR-8764, bromopride, sultopride and tiapride) to stimulate rat prolactin (PRL) secretion $\text{in vivo}$ was found to be three orders of magnitude greater than that of non-benzamide neuroleptic drugs relative to their respective abilities to inhibit ³H-spiperone binding to bovine anterior pituitary membranes. Nevertheless, the IC₅₀ values for the inhibition of ³H-spiperone binding by the seven substituted benzamide drugs was significantly correlated with their high potency to stimulate rat PRL secretion $\text{in vivo}$. Further, the slope of the regression line for these substituted benzamides paralleled that of a series of butyrophenone, phenothiazine, morphanthridine and dibenzodiazepine neuroleptic drugs. Two benzamide (sulpiride and metoclopramide) and three non-benzamide neuroleptic drugs gave intermediate results. This data suggests that blockade of different subgroups of dopamine receptors in the anterior pituitary gland labeled by ³H-spiperone may be responsible for the $\text{in vivo}$ stimulation of PRL secretion by the benzamide and non-benzamide neuroleptioc drugs.     

In vivo receptor binding: attempts to improve specific/non-specific ratios.

$\text{In vivo}$ receptor binding was examined using ³H-spiperone and ³H-pimozide for dopamine receptors and ³H-LSD for serotonin receptors. Two strategies for improving total: nonspecific binding ratios were tested. The first was to deplete endogenous ligands by various pharmacological treatments prior to ³H-ligand administration in an attempt to increase specific receptor binding; the second was to perfuse the brain with ice-cold saline after ³H-ligand administration in an attempt to reduce nonspecific binding. Alteration of dopamine and serotonin by administering d-amphetamine, reserpine, alpha-methyl-paratyrosine or parachlorophenylalanine did not significantly elevate striatal: cerebellar or cortical: cerebellar (measures of total: nonspecific) bonding ratios. However, perfusion with ice-cold saline significantly improved the ratios for both dopamine and serotonin receptors. Thus, cold saline perfusion may be of value in reducing blank values in autoradiographic and other studies requiring $\text{in}$$\text{vivo}$ labelling of receptors.     

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.     

Identification of opiate receptor binding in intact animals.

After intravenous administration of ³H-naloxone to rats, particulate bound radioactivity accumulated in the brain is selectively associated with opiate receptor binding sites, providing a means of labeling the opiate receptor $\text{in vivo}$. The regional distribution of ³H-naloxone bound $\text{in vivo}$ closely parallels regional differences in opiate receptor binding $\text{in vitro}$ with highest levels in the corpus striatum, negligible receptor-associated binding in the cerebellum and intermediate levels in other regions. ³H-Naloxone binding $\text{in vivo}$ is saturable with the same total number of binding sites determined $\text{in vivo}$ as by $\text{in vitro}$ procedures. Nalorphine is markedly more potent than morphine in inhibiting ³H-naloxone binding $\text{in vivo}$ and non-opiates are ineffective. The half-life for dissociation of ³H-naloxone bound to particles $\text{in vivo}$ is the same as its dissociation rate after binding occurs $\text{in vitro}$, and sodium stabilizes ³H-naloxone bound $\text{in vivo}$ from initial rapid dissociation as predicted from the known properties of the opiate receptor $\text{in vitro}$.     

Effects of denzimol on benzodiazepine receptors in the CNS: Relationship between the enhancement of diazepam activity and benzodiazepine binding sites induced by denzimol in the rat

Denzimol, a new anticonvulsant drug with a pharmacological profile similar to that of phenytoin, enhances the ataxic and antimetrazol activity of diazepam in rats without affecting its activity against picrotoxin-induced seizures. $\text{In vivo}$ and $\text{ex vivo}$ denzimol enhances the binding of ³H-flunitrazepam in cortex and in hippocampus but not in cerebellum.The possibility of this increase in the number of benzodiazepine binding sites contributing in some way to enhancement of the depressive and anticonvulsant activity of diazepam is discussed.     

Specific in vivo binding of 77Br-p-bromospiroperidol in rat brain: A potential tool for gamma ray imaging

The binding of the gamma labeled neuroleptic, ⁷⁷Br-$\text{p}$-bromosprioperidol, in the rat brain was examined $\text{in vivo}$. This binding parallels the binding of ³H-spiroperidol, in that binding is especially high in dopaminergically innervated areas, is saturable, and is displaced by high doses of unlabeled spiroperidol (1–5). Thus, ⁷⁷Br-$\text{p}$-bromospiroperidol is a suitable ligand for use in gamma ray imaging techniques for $\text{in vivo}$ monitoring of receptor binding.     

Benzodiazepine binding in human brain: characterization using [3H]flunitrazepam.

[³H]Flunitrazepam was used to characterize benzodiazepine binding sites in human brain. The benzodiazepine binding sites exhibited high affinity, pharmacological specificity and saturability in their binding of [³H]flunitrazepam. The dissociation constant (K_D) of [³H]flunitrazepam binding was determined by three different methods and found to be in the range of 2–3 nM. The potency of several benzodiazepine analogs to inhibit specific [³H]-flunitrazepam binding $\text{in}$$\text{vitro}$ correlated well with their potency in several $\text{in}$$\text{vivo}$ human and animal tests. The density of [³H]-flunitrazepam binding sites was highest in the cerebrocortical and rhinencephalic areas, intermediate in the cerebellum, and lowest in the brain stem and commissural tracts.     

Specific invivo binding of 77Br-brombenperidol in rat brain

The $\text{in vivo}$ binding of the radiobrominated neuroleptic brombenperidol in rat brain was studied. The accumulation of the radiolabeled neuroleptic was high in the striatum and relatively low in the cerebellum, cortex, and blood. Striatal binding of brombenperidol was saturable and displaced by subsequent administration of benperidol. The rationale for the development of ⁷⁵Br-brombenperidol as a radiopharmaceutical for the non-invasive imaging of cerebral dopamine receptor areas is presented.     

Demonstration of [3H] diazepam binding to benzodiazepine receptors in vivo.

Benzodiazepine receptors were labeled with [³H] diazepam following intravenous injection in rats. Binding of [³H] diazepam $\text{in}$$\text{vivo}$ to rat forebrain membranes was displaceable by co-injection of clonazepam or the pharmacologically active enantiomers of two benzodiazepines, B9 and B10, but was not displaced by equal doses of the pharmacologically in-active enantiomers. Binding of [³H] diazepam $\text{in}$$\text{vivo}$ was bserved in kidney, liver, and abdominal muscle, but was not stereospecifically diplaced in any peripheral tissue studied. The regional distribution of benzodiazepine receptors in brain was uneven, with specific [³H] diazepam binding being highest in the cerebral cortex and lowest in the ponsmedulla. Preliminary studies of the subcellular distribution of [³H] diazepam binding demonstrated highest specific binding to synaptosomal membranes. These data demonstrate the feasibility of labeling benzodiazepine receptors in rat brain $\text{in}$$\text{vivo}$.     

Identification of diazepam binding in intack animals.

An $\text{in vivo}$ method for labeling specific benzodiazepine (BDZ) binding sites in brain was developed using intravenously injected [³H]diazepam. Labeling of these sites is blocked by pretreatment of animals with high doses of pharmacologically active BDZs (but not by an inactive BDZ). Using this $\text{in vivo}$ binding technique, specific BDZ binding is enhanced by pretreatment of rats with the GAB?A agonist muscimol or with amino-oxyacetic acid, which increases GABA levels in brain.     

Effect of phencyclidine on [3H]QNB binding

Intraperitoneal injection of phencyclidine before intravenous injection of [³H] Quinuclidinyl benzilate (QNE, 1.6 μg/kg) significantly increased the amount of radioactivity found in the brains of female C57BL/6J mice one hour after the ³H-QNB administration. This effect was found in hypothalamus, cortex, hippocampus and striatum and was decreased by pretreatment of the animal with atropine. The magnitude of the enhancement varied as a function of dose but did not change across the time span studied. These data are in contrast to our findings and those of others of inhibition of the specific binding of ³H-QNB to muscarinic cholinergic receptors by PCP $\text{in vitro}$. When atropine or PCP was administered $\text{in vivo}$ and the tissue later analyzed $\text{in vitro}$, no effects of the drugs were observed on ³H-QNB binding. The reasons for the differences remain a matter of speculation.     

Partial purification and properties of a non-ligandin (3H) 3-methylcholanthrene binding protein from liver cytosol

(³H) 3-Methylcholanthrene binds $\text{in vivo}$ to a macromolecule in addition to the previously reported binding to ligandin in liver cytosol. The properties of this second molecule are identical to those of the glucocorticosteroid receptor (Binder II) through 400 fold purification over the cytosol proteins (elution position from DEAE-Sephadex A-50 columns, molecular weight by gel filtration and pI value by isoelectrofocusing). The carcinogen, probably a metabolite, binds very strongly or covalently to the macromolecule $\text{in vivo}$, but non-covalently $\text{in vitro}$ in the absence of microsomes. Large amounts of unlabeled carcinogen administered $\text{in vivo}$ do not compete significantly with subsequent (³H) dexamethasone binding to the hormone receptor fraction $\text{in vitro}$. Methylcholanthrene and dexamethasone do not compete for binding sites $\text{in vitro}$ on isolated unlabeled Binder II leading to the conclusion that the glucocorticosteroid receptor and the methylcholanthrene binding protein are distinct entities.     

Interactions of chlorinated hydrocarbon pesticides with the 8S estrogen-binding protein in rat testes

The effect of certain DDT analogs on the binding of ³H-estradiol to the 8–9S estrogen binding protein of rat testicular cytosol was studied by sucrose sedimentation analysis. The binding of ³H-estradiol to testicular cytosol was inhibited by o,p'DDT, a DDT analog which is estrogenic in the intact female, but not by p,p'DDE which is a nonestrogen in the female. The pesticide methoxychlor, which is estrogenic $\text{in vivo}$ in the female, failed to inhibit ³H-estradiol binding, presumably requiring metabolic activation for binding to the testicular cytosol. In fact, its di-demethylated metabolite 2,2-bis(p-hydroxyphenyl)-1, 1,1-trichloroethane (HPTE), also estrogenic $\text{in vivo}$, caused $\text{marked}$ suppression of ³H-estradiol binding.     

Binding of opiates and endogenous opioid peptides to neuroleptic receptor sites in the corpus striatum.

Some opiates with morphinan- and benzomorphan-structures possess affinities for neuroleptic receptors as revealed by their abilities to compete with ³H-spiroperidol for common binding sites in rat striatum $\text{in vitro}$ (IC₅₀ in the range between 10^?6 and 10^?5M). The binding of these opiates to neuroleptic receptors appears to be of pharmacological significance, since $\text{in vivo}$ studies in mice revealed a small but significant displacement of spiroperidol by high doses of the opiate antagonist levallorphan from specific binding sites in the striatum. In addition, there exists some correlation between the ability of opiates to bind to neuroleptic receptor sites $\text{in vitro}$ and their potency to evoke “bizarre behavior” in rats $\text{in vivo}$. In contrast, a wide variety of other opiates having morphine-, morphinone- or oripavine-structure showed no affinity for neuroleptic binding sites $\text{in vitro}$ (IC₅₀ greater than 10^?4 M). Of the opioid peptides (methionine-enkephalin, leucine-enkephalin and β-endorphin) none has an affinity for neuroleptic binding sites. A variety of other peptides were also investigated but did not interfere with spiroperidol binding. Only ACTH showed a moderate affinity for neuroleptic binding sites.     

Norharman inhibition of [3H]diazepam binding in mouse brain

Norharman competitively inhibits specific binding of [³H]-diazepam in mouse brain homogenates. $\text{In vivo}$ this β-carboline produces a striking rigid catatonic-like appearance which is abolished by diazepam. It also causes a rapid tremor but has little anticonvulsant effect. Measurement of $\text{in vivo}$ concentrations and receptor occupancy demonstrate that these biological effects occur at doses which occupy a large proportion of benzo-diazepine receptors. It may represent a ligand of the benzo-diazepine receptors whose effects are opposite those of diazepam.     

Kinetics of in vivo binding of antagonist to muscarinic cholinergic receptor in the human heart studied by positron emission tomography

Positron Emission Tomography (PET) was used to analyse $\text{in vivo}$ antagonist binding to human myocardial muscarinic cholinergic receptor. The methiodide salt of the muscarinic antagonist, quinuclidinyl benzilate (MQNB), was labeled with the positron emitter, Carbon-11, and injected intravenously to 8 normal subjects. ¹¹C-MONB concentration was determined $\text{in vivo}$ in the ventricular septum from 40 cross-sectional images acquired at the same transverse level over a period of 70 minutes. In 4 subjects, various amounts of unlabeled atropine were rapidly injected at 20 minutes to study whether atropine competitively inhibited MQNB.The kinetics of binding of ¹¹C-MQNB were not the same $\text{in vivo}$ and $\text{in vitro}$. The apparent dissociation rate of ¹¹C-MQNB $\text{in vivo}$ was much slower (by 1 to 2 orders of magnitude) than that observed $\text{in vitro}$ with ³H-QNB. After atropine injection, ¹¹C-MNQB dissociated from its binding sites at a rate that apparently depended on the amount of atropine present. ¹¹C-MQNB kinetics were analysed with a mathematical model which assumes the existence of a boundary layer containing free ligand in the vicinity of the binding sites. The dissociation rate of the radioligand depends on the probability of its rebinding to a free receptor site.     

Kinetics of repair of O6-methylguanine in DNA by O6-methylguanine-DNA methyltransferase in, vitro and in, vivo

The demethylation of O⁶-methylguanine in double stranded DNA catalyzed by rat liver O⁶-methylguanine-DNA transmethylase was found to proceed much more rapidly when the DNA substrate was methylated to a high extent. When the content of O⁶-methylguanine in DNA was equal to 1 in 2000 guanines, the reaction was 90% complete within 2 min, but when the content was 1 in 500,000 it required 27 min at 37°C. These results suggest that the repair protein either moves along the DNA substrate or else has little selectivity for binding specifically to the sites containing O⁶-methylguanine rather than to the normal DNA. The repair of O⁶-methylguanine in rat liver $\text{in}\text{,}\text{vivo}$ occurred at rates comparable to those seen $\text{in}\text{,}\text{vitro}$ with the substrates alkylated to low extents and was virtually complete within 3 hours. These results provide strong evidence that this protein is the factor responsible for O⁶-methylguanine removal $\text{in}\text{,}\text{vivo}$ and explain the wide variation in time courses reported in the literature since substrates methylated to greatly different extents have been used for such experiments.     

In vitro and in vivo inhibition by zopiclone of benzodiazepine binding to rodent brain receptors.

Up to now the only drugs known to be able to inhibit the binding of benzodiazepines to rodent brain receptors are members of this chemical family.Zopiclone (RP 27 267), a new drug with a pharmacological profile similar to that of chlordiazepoxide and nitrazepam but entirely different chemically from benzodiazepines, has been tested for its ability to inhibit benzodiazepine binding. $\text{In vitro}$ and $\text{in vivo}$ studies have shown that zopiclone is able to inhibit the binding of [³H] diazepam and [³H] flunitrazepam to brain receptors. The potency of zopiclone is quite comparable to that of diazepam and nitrazepam $\text{in vitro}$ and to that of chlordiazepoxide $\text{in vivo}$.These results confirm the pharmacological similarities existing between zopiclone and the benzodiazepines.     

In vivo binding of spiroperidol and bromosphiroperidol at low drug loadings

The binding of spiroperidol and bromospiroperidol, $\text{in vivo}$, was studied over a wide range of drug dosages. It was found that while spiroperidol and bromospiroperidol bind selectively $\text{in vivo}$ to tissues known to be high in dopamine receptor binding sites, this specificity of binding does not persist at very low doses. Such anomalous binding behavior can have implications for the non-invasive imaging of these drugs.     

Etomidate stereospecifically stimulates forebrain,but not cerebellar, 3H-diazepam binding

³H-Diazepam binding to a total particulate fraction of rat forebrain is enhanced by (+)-etomidate and GABA, but not by (?)-etomidate. The enhancement of ³H-diazepam binding by (+)-etomidate was due to a two-fold increase in binding affinity, the maximal number of sites remained unchanged. The degree of stimulation with (+)-etomidate was higher than that obtained with GABA. THIP did not stimulate ³H-diazepam binding to forebrain, and did not reverse the enhanced binding seen with (+)-etomidate or GABA. In a synaptosomal membrane preparation of rat cerebellum, unlike (+)-etomidate, GABA and muscimol produced a marked stimulation of ³H-diazepam binding. (+)-Etomidate did not inhibit ³-muscimol binding to GABA receptors, nor did it activate or inhibit other $\text{in vitro}$ receptor binding assays. The effects of (+)-etomidate on the benzodiazepine binding are different from those of gabamimetic drugs. It is proposed that like barbiturates, (+)-etomidate may affect benzodiazepine binding by interaction with the chloride ionophore which is coupled to the GABA-receptor.