Pierre M. Laduron

Receptor binding profile of R 41 468, a novel antagonist at 5-HT2 receptors.

For a new antiserotonergic agent, R 41 468 and 13 reference compounds with alleged antiserotonergic activity, the receptor binding profile is reported, comprising Ki-values measured in ten different receptor binding models. R 41 468 appeared to be a particularly selective agent with respect to differentiation between two 5-hydroxytryptamine (5-HT) receptor models; it primarily displayed high binding affinity for 5-HT2 receptors and was inactive at 5-HT1 receptors. Besides showing a moderate binding affinity for histamine1 and α1 adrenergic receptors, the compound was very weakly active at dopamine receptors and inactive at the remaining receptors. Receptor binding profiles of the reference compounds differed widely. Apart from R 41 468 no other compound showed a similar selectivity towards 5-HT2 receptors. Reference compounds either poorly differentiated between 5-HT2 and 5-HT1 receptors, showed other primary effects, or were only moderately active. In the 5-HT2 and 5-HT1 receptor binding models the ‘D-receptor’ antagonist phenoxybenzamine was weakly active and the ‘M-receptor’ antagonist morphine was inactive. It is concluded that R 41 468 will be a particularly suitable tool to antagonize 5-HT action mediated by 5-HT2 receptors.

Spiperone: A ligand of choice for neuroleptic receptors: 1. Kinetics and characteristics of in vitro binding

Abstract A binding assay for neuroleptic receptors has been developed with spiperone as the labelled ligand. As compared to haloperidol, spiperone showed a 2-times higher ratio of specific versus aspecific binding, a 10-fold greater association constant and a slower dissociation of the receptor ligand complex. The receptor sites labelled by spiperone appeared to be for a great deal similar to those of haloperidol, however certain differences were apparent; the number of receptor sites per gram of tissue was found to be higher for the former; spiperone showed a biphasic receptor ligand dissociation curve which was not observed for haloperidol; also a slight difference in physical stability between spiperone and haloperidol binding sites was noted. Inhibition studies using antagonists and agonists in comparison with the pharmacological profile of the compounds showed that the receptor sites labelled by both ligands are mainly of dopaminergic nature, but also a serotonergic and to a minor extent a noradrenergic component should be involved. Within the striatum haloperidol binding sites seemed to be relatively more related to dopaminergic sites whilst the spiperone binding sites appeared to comprise a higher serotonergic component. It is concluded that spiperone is a more suitable ligand than haloperidol for studying the neuroleptic receptors. The use of different labelled ligands provided evidence for the heterogeneity of the neuroleptic binding sites in the striatum.

Domperidone, a specific in vitro dopamine antagonist, devoid of in vivo central dopaminergic activity.

When tested in different in vitro binding assays and in dopamine-sensitive adenylate cyclase, domperidone was found to be a very potent and specifie dopamine antagonist. Accordingly, [3H]ldomperidone binding sites were exclusively detected in homogenates of brain dopaminergic regions. Despite its powerful dopamine antagonism, domperidone did not act centrally in a similar way to neuroleptic drugs. Ex vivo and in vivo experiments indicated that domperidone is unable to reach dopamine receptors in rat striatum. The very low amount of labelling and atypical distribution found in rat brain after administration of [3H]ldomperidone is compatible with a lack of penetration into brain structures. Finally, domperidone did not elicit marked HVA increase as is found with classical neuroleptic drugs and metoclopramide. From the present experiments, one may conclude that domperidone, though a very potent and specific dopamine antagonist, cannot readily cross the blood brain barrier, which thus prevents the occurrence of central effects.

Spiperone: a ligand of choice for neuroleptic receptors. 2. Regional distribution and in vivo displacement of neuroleptic drugs.

Abstract Specific in vitro as well as in vivo binding of, neuroleptic drugs was demonstrated by studying the regional distribution in rat brain. [ 3 H]spiperone and [ 3 H]pimozide were found to be specifically taken up in the dopaminergic areas of the brain, a fact which correlates with the distribution of neuroleptic receptors when measured under in vitro conditions. Larger doses of unlabeled neuroleptics only displaced the labeled neuroleptic in vivo in the dopaminergic areas (striatum, nucleus accumbens, tuberculum olfactorium and frontal cortex) but not in the cerebellum. Dopamine agonists were found to partly displace the labeled spiperone in the striatum, thus providing further evidence about the dopaminergic nature of the neuroleptic receptor. Nevertheless, some in vivo experiments suggest that the neuroleptic receptor is not the same in all the dopaminergic areas. It is concluded that spiperone is a ligand of choice for in vivo studies of neuroleptic receptors.

Axonal transport of opiate receptors in capsaicin-sensitive neurones

Opiate receptors measured in vitro or in vivo with [3H]lofentanil in the rat vagus nerve were found to accumulate on both sides of a ligature, thus indicating a bidirectional axoplasmic transport of these receptors. When rats were treated with capsaicin, the accumulation of opiate receptors was tremendously reduced in the vagus whereas muscarinic receptors in ligated sciatic nerves were unaffected. Since capsaicin is known to affect sensory neurones, mostly those containing substance P, the present results support the idea that the opiate receptors in the vagus are associated with substance P neurones.

Evidence for a displaceable non-specific [3H]neurotensin binding site in rat brain

SummaryLevocabastine is a potent antihistamine drug, structurally unrelated to neurotensin. In rat and mouse brain but not in other animal species, it inhibited 60% of the [3H]neurotensin binding displaced by unlabelled neurotensin or neurotensin (8–13).The levocabastine-sensitive site or “site 1” displayed high affinity properties for levocabastine (IC50=25 nM) and was highly stereospecific (IC50-value higher than 10 μM for one of the isomers). Binding to the “site 1” in rat brain corresponded to the [3H]neurotensin binding displaceable by 1 μM levocabastine, whereas binding to the “site 2” corresponded to the binding displaced by 1 μM neurotensin when the “site 1” was occluded by 1 μM levocabastine.Both “site 1” and “site 2” appeared to be saturable. Scatchard plots obtained in rat bulbus olfactorius allowed to calculate a KD-values of 7.1 nM and a Bmax-values of 37.2 fmol/mg original tissue for “site 1”, while “site 2” displayed a KD-value of 0.7 nM and a Bmax-value of 16.3 fmol/mg original tissue. The regional distributions of both sites showed marked differences. The “site 1” was homogeneously distributed throughout all rat brain areas, whereas the amount of “site 2” binding was markedly different in separate brain areas: bulbus olfactorius and substantia nigra had the highest amounts (8.9 and 7.8 fmol/mg tissue) while cerebellum had the lowest (0.4 fmol/mg tissue).In spite of its high affinity and stereospecificity, “site 1” has to be considered as an acceptor or recognition site for [3H]neurotensin because of its species-link, low saturability and homogeneous distribution in all rat brain areas.On the other hand, “site 2” had the characteristics of a physiological receptor: high affinity, saturability in the low nanomolar range and marked regional distribution in rat brain. “Site 2” corresponds therefore most probably to the physiological neurotensin receptor. The foregoing experiments provide evidence for the presence of a drug displaceable, non-specific (=unrelated to a physiological receptor) neurotensin binding site in rat brain; levocabastine should be an important tool to occlude this site in order to reveal, by means of in vitro binding assays, the specific neurotensin binding site in rat brain.

Characterization and regional distribution of serotonin S2-receptors in human brain

[3H]Ketanserin binding was characterized in vitro in human brain homogenates and the regional distribution of the sites was determined. In human brain, [3H]ketanserin was found to bind on serotonin (5-HT) S2-receptors; only 5-HT antagonists competed with the labelled ligand at nanomolar concentrations; other drugs were much less active or inactive. Special attention was paid to the choice of a displacer, here methysergide, to determine the blank value (non-displaceable binding). [3H]Ketanserin binding in human brain displayed similar binding characteristics to the S2-receptor in the rat frontal cortex, high affinity (Kd 0.69 nM) and relatively slow dissociation rate. The regional distribution of serotonin S2-receptors labelled with [3H]ketanserin was studied in 30 different regions of human brain. The highest number of receptors was measured in the cortex. However, within the cortex the distribution was also inhomogeneous, a much lower number of sites being found in the pre- and post-central gyri. In the dopaminergic areas and the cerebellum the number of sites was quite low, and only few binding sites were detected in the corpus callosum, the medulla and the hypophysis. The large number of serotonin S2-receptors in the human cortex suggests that serotonin has an important role in this brain region.

Criteria for receptor sites in binding studies

The study of binding is not an easy task especially because of the difficulty of interpreting the results in terms of binding on specific receptor sites. The problem is not new; what is new is the increasing amount of fanciful interpretation that such a technique has generated. The tendency to interpret anomalous or intriguing results in terms of new receptor subtypes seems to have reached its peak. The need to apply severe criteria becomes imperative before concluding that a binding site may be called a receptor site. Some pitfalls in binding studies will be discussed.

In vivo binding of [3H]ketanserin on serotonin S2-receptors in rat brain

In vivo binding of [3H]ketanserin was studied in various brain regions in rats. After i.v. injection of [3H]ketanserin (5 micrograms . kg-1), the highest labelling was found in the frontal cortex. Brain disposition of labelled drug correlated with the distribution of serotonin S2-receptors detected in vitro. The binding was saturable in the serotonergic areas but not in the cerebellum. Various drugs were tested for their ability to displace or to prevent [3H]ketanserin binding: these results were then compared to those obtained with [3H]spiperone in the frontal cortex. Although [3H]spiperone can be used to differentiate the affinity of a drug for serotonin (frontal cortex) and dopamine (striatum) receptors, the great advantage of [3H]ketanserin is that it labels serotonin S2-receptors exclusively.

Differential distribution of opiate and neuroleptic receptors and dopamine-sensitive adenylate cyclase in rat brain.

Abstract In rat brain, the regional distribution of the neuroleptic receptor and of dopamine-sensitive adenylate cyclase was found to be very similar, but it differed markedly from the distribution of the opiate receptor. Neuroleptic receptor sites were detectable in the cortex and the hypophysis. After differential centrifugation of rat striatum homogenate, opiate and neuroleptic receptors were enriched in the microsomal fraction while dopamine-sensitive adenylate cyclase revealed a mitochondrial distribution pattern. This different subcellular localization of the neuroleptic receptor and the dopamine-sensitive adenylate cyclase suggests a different function for both receptors.