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.     

The actions of naloxazone on the binding and analgesic properties of morphiceptin (NH2Tyr-Pro-Phe-Pro-CONH2), a selective mu-receptor ligand

Active in both binding and biological assays, morphiceptin (NH₂ Tyr-Pro-Phe-Pro-CONH₂), a potent opioid peptide derivative of β-casamorphine, binds specifically and selectively to mu or morphine-type receptors with little affinity for delta sites. Displacement studies of a variety of ³H-labeled opiates and enkephalins show biphasic curves. Naloxazone, which blocks irreversibly and selectively high affinity opiate and enkephalin binding, abolishes morphiceptin's inhibition of binding at low concentrations, suggesting that the high affinity binding of enkephalins and opiates represents a mu or morphine-type receptor. Unlike the reversible antagonist naloxone, naloxazone treatment $\text{in}$$\text{vivo}$ inhibits for over 24 hours the analgesic activity of morphiceptin like it inhibits morphine, β-endorphin and enkephalin analgesia. Together, these studies imply that opiates and enkephalins bind with highest affinity to a mu receptor which mediates their analgesic activity. The ³H-D-ala²-D-leu⁵-enkephalin binding remaining after naloxazone treatment, representing a lower affinity site (K_D 4 nM), is quite insensitive to morphiceptin inhibition and has the characteristics of a delta receptor. However, the ³H-dihydromorphine binding present after naloxazone treatment, which also represents a lower affinity site (K_D 6 nM), is far more sensitive to both morphine and morphiceptin and may represent a second morphine-like, or mu, receptor.     

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.     

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.     

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.     

Temperature-sensitive high affinity [3H]serotonin binding: Characterization and effects of antidepressant treatment

Characterization of temperature-sensitive [³H]serotonin (5-HT) binding sites (1 and 4 nM Kd sites) revealed complex inhibition by neuroleptics and serotonin antagonists. There was no simple correlation with affinities for S₁ and S₂ receptors. $\text{In vivo}$ pretreatment (48 h before) with mianserin did not alter Bmax or Kd for the 1 nM Kd [³H]5-HT site, although [³H]ketanserin (S₂) densities were decreased by 50%. This suggested that possible S₂ components of [³H]5-HT binding must be negligeable, even though ketanserin competed with high affinity (IC₅₀ = 3 nM) for a portion of the 1 nM Kd [³H]5-HT site. Low concentrations of mianserin inhibited the 1 nM Kd [³H]5-HT site in a non-competitive manner, as shown by a decrease in Bmax with no change in Kd after $\text{in vitro}$ incubation. The complex inhibition data may therefore represent indirect interactions through another site.     

Benzodiazepine receptor: Heterogeneity in rabbit brain

Binding characteristics of benzodiazepine receptors were studied with synaptosomal and microsomal membranes from rabbit brain $\text{in}$$\text{vitro}$ utilizing [methyl-³H]diazepam. In synaptosomal membranes, both high and low affinity binding sites were identified with the dissociation constants (K_d) of 4.92 nM and 83.8 nM, respectively. However, only the high affinity site was identified with K_d of 3.96 nM with microsomal membranes. Benzodiazepine binding sites appear to include at least two subpopulations of receptors, one with high affinity and another with low affinity binding site.     

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 $\text{in vitro}$.     

Relation of cation binding sites on synaptic vesicles to opiate action.

The protein-sensitized fluorenscence of Tb³+ has been used to characterize cation binding sites on synaptic vesicles. In the presence of 100 mM KC1, the vesicles display a single class of sites having affinities for Ca²⁺ and Mg²⁺ in the mM range. Of subcellular fractions synaptic vesicles have the greatest number of sites, which involve tyrosine residues. Morphine, which is known to deplete Ca from nerve terminals $\text{in vivo}$ by a mechanism possibly involving low affinity sites on synaptic vesicles, does not compete $\text{in vitro}$ for the sites monitored by Tb fluorescence. In addition, acute morphine treatment does not alter the binding of Tb to the synaptic vesicle fraction. It is suggested that the depletion of brain Ca induced by morphine is not mediated by a direct action of opiates on these membrane sites.     

Stereospecific interaction of opiate narcotics in binding of 3H-dihydromorphine to membranes of rat brain

Membranes from homogenates of corpus striatum bound ³H-dihydromorphine in a saturable fashion with a Km value of 1 × 10^?9M. The binding of ³H-dihydromorphine to the membranes was reduced to about 10% by 10^?7M levorphanol but not by 10^?7M dextrorphan. The binding of ³H-dihydromorphine became less sensitive to 10^?7M levorphanol when the concentration of ³H-dihydromorphine was greater than 2 × 10^?9M. Other opiate narcotics, e.g. morphine and $\text{l}$-methadone, were as effective as levorphanol in competition for the binding ³H-dihydromorphine with ED₅₀ values of 2–4 × 10^?9M. $\text{d}$-Methadone and dextrorphan were about 1/50 and 1/2000 as effective as their respective levo-isomers. The opiate antagonist, naloxone, also competed effectively for the binding sites with an ED₅₀ value of 3.3 × 10^?9M. Substances like acetylcholine, choline, serotonin, norepinephrine and dopamine were ineffective. Only ionophores specific for divalent cations stimulated the binding of ³H-dihydromorphine suggesting that some endogenous divalent cations may be inhibitory to the binding of the opiate narcotic. The receptors of ³H-dihydromorphine probably exist in the membranes of nerve endings and have a density of 6 × 10¹² sites per g in corpus striatum. We conclude that the described technique can successfully detect the opiate narcotic receptors in the central nervous system without the usual method of displacement.     

Catecholamine receptors and cyclic AMP formation in the central nervous system: effects of tetrahydroisoquinoline derivatives.

The ability of a series of tetrahydroisoquinoline (THIQ) alkaloids to inhibit the binding of radioligands to catecholamine receptors in the CNS has been examined. $\text{(+)}$ THP was the most potent inhibitor of [³H] dihydroalprenolol binding to β-adrenergic receptors and of [³H] haloperidol to dopaminergic receptors and was the least potent inhibitor of [³H] WB-4101 binding to α-adrenergic receptors. Other THIQ alkaloids examined such as salsoline, salsolinol, and reticuline were less potent than $\text{(+)}$ THP in inhibiting radioligand binding to β-adrenergic and dopaminergic receptors, and more potent than $\text{(+)}$ THP in inhibiting radioligand to α-adrenergic receptors. The marked potency of $\text{(+)}$ THP in inhibiting radioligand binding to β-adrenergic receptors (IC₅₀ ～ 10^?7 M) was confirmed by the potency of this compound in inhibiting (?) isoproternol elicited accumulations of cyclic AMP in brain slice preparations. These data indicate that, if formed $\text{in}$$\text{vivo}$ during alcohol consumption, THIQ derivatives such as THP may affect catecholamine neurons in the CNS.     

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.     

Regional high affinity synaptosomal transport of choline in mouse brain — Influence of oxotremorine treatment

Kinetic parameters for high affinity [HA] uptake $\text{in vitro}$ in synaptosomes from different mouse brain regions were investigated. V_max was highest in the striatum [200 pmol.· mg protein^?1 · 4 min^?1], followed by the cortex [111 pmol · mg protein^?1 · 4 min^?1], hippocampus [63 pmol · mg protein^?1 · 4 min^?1], midbrain [21 pmol · mg protein^?1 · 4 min^?1] and, lowest, medulla oblongata [5 pmol · mg protein^?1 · 4 min^?1]. K_m was about the same in all brain regions [0.9–1.4 μM]. No sign of HA uptake was detected in synaptosomes from the cerebellum. A clear relationship between V_max for synaptosomal HA uptake of Ch $\text{in vitro}$ and apparent turnover of ACh $\text{in vivo}$ was found between the brain regions. Administration of oxotremorine [1 mg·kg^?1 i.p.] decreased V_max for HA uptake of Ch by 60% in the cortex and hippocampus, by 50% in the striatum and by 20% in the midbrain. This effect is in accordance with the previously observed marked decrease in turnover of ACh in these brain regions following oxotremorine treatment.     

Regulation of high- and low-affinity epidermal growth factor receptors by glucocorticoids

It is reported that receptors for epidermal growth factor (EGF) in HeLa S₃ cells exist in two forms, which differ in both affinity and capacity. Both the number of receptors and their distribution into low- and high-affinity forms are modulated by glucocorticoids. Scatchard analysis of saturation binding assays performed at 0 °C indicates that there is a low-affinity class of receptors ($\text{K}{}_{\mathrm{d}}\text{? 1.5}\text{nm}$), which contains approximately 6 × 10⁴ binding sites per cell, and a second, high-affinity class of receptors ($\text{K}{}_{\mathrm{d}}\text{? 0.16}\text{nm}$) containing approximately 5 × 10³ binding sites per cell. Exposure of HeLa S₃ cells to 10^?7m dexamethasone for 24 h increased EGF binding to whole cells by increasing the numbers of low- and high-affinity receptors by 20 and 114%, respectively. The increase in EGF binding depends upon the dose of dexamethasone, being raised from 10^?11 to 10^?6m. EGF binding is half-maximal near 2–4 × 10^?9m, a concentration equal to the K_d of dexamethasone for the glucocorticoid receptor in these cells. The increase in EGF binding is specific for glucocorticoids, occurring when the HeLa S₃ cells are exposed to 10^?7m cortisol or dexamethasone for 24 h, but not when the cells are similarly treated with testosterone, 5α-dihydroxytestosterone, 17β-estradiol, or progesterone. The effect on EGF binding appears to be biphasic; the initial rapid increase occurs between 8 and 12 h, is blocked by both 10^?6m cyclohexamide and 0.1 μg/ml actinomycin D, and is followed by a more gradual increase thereafter. These data indicate that glucocorticoids are able to regulate both the number of EGF receptors and their distribution into high- and low-affinity components. Press, Inc.     

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.     

Stereospecific 3H-nicotine binding to intact and solubilized rat brain membranes and evidence for its noncholinergic nature

³H-nicotine binding was performed on intact and solubilized rat brain membranes as well as membranes from the electric organ of the $\text{Torpedo}$ fish. The K_d for binding to intact and solubilized rat brain membranes was 5.6 × 10^?9 M and 1.1 × 10^?8M respectively, and the binding capacity 2.0 × 10^?14 and 3.0 × 10^?13 moles /mg protein respectively. The K_d for Torpedo membranes was 3.1 × 10^?7M and the binding capacity 6.8 × 10^?13 moles/mg protein. The binding was stereospecific with the affinity of the (?)-nicotine being about 8 times greater than the (+)-nicotine with all three preparations. The relative affinity for the nicotine binding site of nicotinic cholinergic drugs was considerably less in rat brain than in $\text{Torpedo}$ membranes, where the sites are mainly cholinergic. A comparison was made of the ability of a variety of cholinergic drugs and nicotine derivatives to compete with ³H-nicotine binding and their relative pharmacologic potency to produce or inhibit a characteristic prostration syndrome caused by (?)-nicotine administered intraventricularly to rats. From such studies it was concluded that nicotine, in part, may be interacting at noncholinergic sites in rat brain.     

Oxytocin receptors in rat oviduct.

Particles from rat oviduct homogenates sedimenting between 1,000 × g for 10 min and 48,000 × g for 30 min bound [³H]oxytocin $\text{in vitro}$. The apparent K_d for oxytocin binding to high affinity sites in particles prepared from estrogen-treated rats was 1.8 × 10^?9 M. About 215 fmoles of oxytocin were bound per mg of particulate protein. Oviducal preparations from untreated rats had about 25% the affinity for oxytocin of preparations from estrogen-treated rats. Oxytocin analogues were bound to oviducal particles in the same rank order as their uterotonic potencies: (desamino)oxytocin > (4-threonine)oxytocin > oxytocin > (8-lysine)vasopressin ? desaminotocinol. No oxytocin binding could be shown with the particulate fractions from rat ovary. The binding of oxytocin to the oviduct and uterus are similar in affinity, number of binding sites, ligand specificity, and the increase in response to estrogen treatment.     

A simple and rapid radio-receptor assay for the estimation of acetylcholine

The high potency with which acetylcholine (ACh) inhibits the binding of the specific muscarinic agonist, [³H]$\text{cis}$ methyldioxolane ([³H]CD), has provided the basis for the development of a radio-receptor assay for estimation of ACh. A synaptosomal preparation of the rat cerebral cortex was used as a source of muscarinic receptors. When binding assays were run at 0°C, the IC₅₀ value of ACh was approximately 5 × 10^?9 M, which corresponds to 2.5 – 10 pmoles of ACh, depending upon the assay volume. The ACh content of the rat cerebral cortex and corpus striatum was measured following fast microwave irradiation. By measuring the displacement of [³H]CD binding caused by aliquots of the supernatant from tissue homogenates and comparing the displacement values with an ACh standard curve, the ACh content of the cerebral cortex and corpus striatum was calculated to be 19 and 55 nmoles/g wet tissue weight, respectively.     

Buspirone: A potential atypical neuroleptic

Buspirone produces a dose-dependent but short-lived elevation in striatal dopamine (DA) metabolites in the rat. $\text{In}$$\text{vitro}$, buspirone possesses an affinity similar to sulpiride for DA receptors (3H-spiperone). A moderate affinity for α₁ receptors was also observed while buspirone was inactive at α₂, β, muscarinic and serotonin₂ receptors. This pharmacological profile as well as previous behavioral data indicate that buspirone may be a potential “atypical” neuroleptic.