C. Malgouris

Labelling of “Peripheral‐Type” Benzodiazepine Binding Sites in the Rat Brain By Using [3H]PK 11195, an Isoquinoline Carboxamide Derivative: Kinetic Studies and Autoradiographic Localization

Abstract: PK 11195 [1‐(2‐chlorophenyl)‐N‐methyl‐N‐(1‐methylpropyl)‐3‐isoquinolinecarboxamide] is a new ligand for the “peripheral‐type” benzodiazepine binding sites, chemically unrelated to benzodiazepines. It displaces with a very high potency (IC50× 10−9M) [3H]‐RO5–4864 (a benzodiazepine which specifically labels the peripheral‐type sites) from its binding sites. [3H]PK 11195 binds to a membrane fraction from rat brain cortex and rat olfactory bulb in a saturable and reversible manner with a very high affinity (KD= 10−9M). The number of maximal binding sites was ten times greater in the olfactory bulb than in the brain cortex. The order of potency of several compounds as displacers at 25°C (PK 11195 > RO5–4864 > diazepam > dipyridamole > clonazepam) demonstrates that [3H]PK 11195 binds to the peripheral‐type benzodiazepine binding sites. The KD value for the [3H]PK 11195 binding is not affected by temperature changes, whereas RO5–4864 and diazepam affinities decrease with increasing temperatures. Autoradiographic images of [3H]PK 11195 binding to rat brain sections show that binding sites are mainly localized in the olfactory bulb, median eminence, choroid plexus, and ependyma. This ligand could be a useful tool to elucidate the physiological and pharmacological relevance of these binding sites.

Labelling of peripheral-type benzodiazepine binding sites in human brain with [3H]PK 11195: Anatomical and subcellular distribution

The peripheral-type benzodiazepine binding site, erstwhile characterized in the rodent and feline brain, has now been characterized in post-mortem human brain using [3H]PK 11195. The kinetics and pharmacological properties of the binding of this ligand are similar to peripheral-type benzodiazepine binding sites elsewhere. The potency of RO5-4864 for this site in human brain is close to that seen in ruminant and carnivore tissues but considerably lower than in rodent tissues. The regional distribution of these binding sites would suggest a neuronal rather than a glial localization. [3H]PK 11195 bound in a similar fashion to slide-mounted sections of human brain, thus allowing quantitative studies of the regional distribution of peripheral-type benzodiazepine binding sites to be made. The binding sites were distributed heterogeneously, but were restricted to the grey matter. Highest densities of binding sites were found in forebrain structures. The localization was not limited to any functional system, nor did it resemble any previously described transmitter system. The similarities between peripheral-type benzodiazepine binding sites in human and in feline brain in terms of their pharmacological characteristics and their regional and subcellular distribution suggest that the cat, rather than the rat, may be the better model for studying a possible role for this site in human cerebral function.

Retrograde axonal transport of neurotensin in the dopaminergic nigrostriatal pathway in the rat

Although the existence of receptor transport has been clearly demonstrated in peripheral nerves, there is no clear cut evidence in the brain of such a process for neuropeptide receptors. Because of the localization of neurotensin receptors on dopaminergic terminals, the dopaminergic nigrostriatal pathway appears to be the system of choice for studying the axonal transport of neuropeptide receptors in the brain. When labelled neurotensin was injected into the rat striatum, a delayed accumulation of radioactivity in the ipsilateral substantia nigra was observed about 2 h after injection. An essential requirement to clearly observe this phenomenon was the pretreatment of animals with kelatorphan in order to prevent the labelled neurotensin degradation. The appearance of this labelling was prevented by injection of an excess of unlabelled neurotensin or of neurotensin 8-13, an active neurotensin fragment, but not by neurotensin 1-8, which had no affinity for neurotensin receptors. This process was saturable, microtubule-dependent and occurred only in mesostriatal and nigrostriatal dopaminergic neurons as identified after 6-hydroxydopamine lesion and by autoradiography. These results demonstrate that neurotensin was retrogradely transported by a process involving neurotensin receptors. The retrograde transport of receptor-bound neuropeptide may represent an important dynamic process which conveys information molecules from the synapse towards the cell body.

Neuroprotective effects of riluzole on N-methyl-d-aspartate- or veratridine-induced neurotoxicity in rat hippocampal slices

The neuroprotective activity of riluzole has been studied on N-methyl-D-aspartate (NMDA)- or veratridine-induced toxicity in immature rat hippocampal slices. Neurodegeneration was assessed by the measurement of LDH release and histology. Veratridine-induced LDH release can be inhibited by 100 microM riluzole (-90% and by tetrodotoxin (1 microM). Riluzole markedly reduced (-59%) the NMDA-induced LDH release and this protective effect was confirmed by histology. Riluzole inhibited the NMDA-induced LDH release in the presence of tetrodotoxin. Moreover, a pretreatment with pertussis toxin (1 microgram/ml) abolished the effect of riluzole against NMDA-induced neurotoxicity. These results support the view that the neuroprotective properties of riluzole could be exerted via two distinct mechanisms of action.

Autoradiographic studies of RP 62203, a potent 5-HT2 receptor antagonist. Pharmacological characterization of [3H]RP 62203 binding in the rat brain

The binding properties and localization of [3H]RP 62203, a novel ligand for 5-HT2 receptors, were investigated on rat brain sections. The specific binding of this 5-HT2 receptor antagonist was reversible and could be displaced by ritanserin (1 microM). Saturation experiments revealed a single class of binding sites with a KD of 0.128 +/- 0.018 nM and a Bmax of 1.67 +/- 0.06 pmol/mg protein. Pharmacological specificity was demonstrated by the potency order of displacing agents: RP 62203 > ritanserin > spiperone > methysergide > mianserin > pipamperone > cinanserin > 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI). Quantitative autoradiography showed a heterogeneous distribution of [3H]RP 62203 binding sites, with the highest densities in the frontal, parietal and auditory cortices (layer IV), claustrum and olfactory bulb. Binding densities in the occipital cortex, caudate putamen and thalamic nuclei were moderate, whereas the hippocampus and substantia nigra displayed a very low density of binding sites. The cerebellar cortex appeared almost devoid of [3H]RP 62203 binding sites. The anatomical distribution of binding sites demonstrated that [3H]RP 62203 essentially bound only to rat brain regions known to contain 5-HT2 receptors. This ligand could thus be a useful tool to visualize 5-HT2 receptors.

Quantitative Autoradiography of [3H]Indalpine Binding Sites in the Rat Brain: II. Regional Distribution

Abstract: The localization of binding sites for [3H]indalpine to sections of rat brain was studied by a quantitative autoradiographic technique. Binding sites for this specific neuronal 5‐hydroxytryptamine (5‐HT) uptake inhibitor are concentrated in areas rich in 5‐HT neuronal cell bodies, fibers, and synaptic terminals. One of the most interesting features of this regional distribution is the very high density of these sites found in the dorsal raphe, substantia nigra, ventral tegmental area, and locus ceruleus. Components of the visual system also show pronounced labelling with [3H]indalpine. The finding that limbic structures are strongly labelled by this drug may be related to the antidepressant activity of indalpine. The anatomical distribution of binding sites demonstrated is Consistent with the specific labelling of 5‐HT neurons by [3H]indalpine and confirms previous studies carried out with another 5‐HT uptake inhibitor, [3H]imipramine.

Autoradiographic studies of RP 62203, a potent 5-HT2 receptor antagonist. In vitro and ex vivo selectivity profile

In this study, quantitative autoradiography was used to determine the selectivity of RP 62203, a novel naphtosultam derivative, for 5-HT2 receptors in vitro and ex vivo, using [125I]7-amino-8-iodo-ketanserin ([125I]AMIK) and [3H]mesulergine as radioligands. The density of [125I]AMIK or [3H]mesulergine binding sites was determined by quantitative image analysis. In in vitro experiments, RP 62203 displaced [125I]AMIK from 5-HT2 receptors with an IC50 of 0.21 nM in rat frontal cortex. Its affinity for 5-HT1C receptors was 100-fold lower (IC50 25 nM versus [3H]mesulergine in rat choroid plexus). RP 62203 showed moderate affinity for alpha 1-adrenoceptors in the rat thalamus (IC50 14 nM) and for histamine H1 receptors in the guinea-pig cerebellum (IC50 13 nM). The tetrabenazine sites were not affected by RP 62203 at a concentration of 30 nM. In ex vivo experiments, RP 62203 was about 4 times more potent than ritanserin in displacing [125I]AMIK from 5-HT2 receptors (ED50 0.58 mg/kg p.o.). A dose of 10 mg/kg of RP 62203 did not displace [3H]mesulergine from 5-HT1C receptors or [125I]AMIK from alpha 1-adrenoceptors and tetrabenazine sites in the rat brain and from histamine H1 receptors in the guinea-pig brain. These results demonstrate that RP 62203 specifically recognizes 5-HT2 receptors in rodent brain.

Effect of mequitazine a non sedative antihistamine on brain H1 receptors

Abstract Marked species variations occured in the relative activity of mequitazine (10-[3-quinuclidinylmethyl]-phenothiazine) on the binding of [ 3 H]mepyramine to brain cortical membranes. Mequitazine was as potent as promethazine in the mouse but about 6 times less effective than promethazine in the guinea pig and human. On the other hand in the guinea pig mequitazine was as potent as promethazine on [ 3 H]mepyramine binding in a peripheral organ (lung). Although mequitazine did not displace [ 3 H]mepyramine in vivo in the mouse and guinea pig, its brain concentration (measured by [ 3 H]mequitazine) was largely sufficient and corresponds to 90% of inhibition in vitro. Moreover in the mouse the brain regional distribution of [ 3 H]mequitazine was very different from that of [ 3 H]mepyramine, highest level was obtained in the cerebellum and hypothalamus was the poorest region with mequitazine whereas the reverse was true with mepyramine. All these results could suggest that mequitazine possesses a greater affinity for peripheral H 1 receptors which could explain the absence of sedative side-effects of this potent H 1 antagonist.

Differential effects of typical and atypical neuroleptics on alpha-noradrenergic and dopaminergic postsynaptic receptors

Abstract Mezilamine (2-methylamino-4-N-methylpiperazino-5-methylthio-6-chloropyrimidine), a new antidopaminergic agent, is more effective, in vitro and in vivo, in competing for the binding of [3H]-haloperidol in rat tuberculum olfactorium than in rat striatum. Such effects, similar to atypical neuroleptics (clozapine, sulpiride), are opposite to classical neuroleptics like chlorpromazine or UK 177 (2-benzilamino-4-N-methylpiperazino-5-methylthio-6-chloropyrimidine). Mezilamine displaces 10 times more [3H]-clonidine binding than [3H]-WB 4101 binding in rat cerebral cortex and such a preference for α-agonist rather than antagonist sites is also opposite to most neuroleptics, including UK 177. A combination of phenoxybenzamine and mezilamine causes catalepsy and produces a preferential acceleration of striatal dopamine turnover whereas mezilamine alone is more effective on the mesolimbic system. Such results suggest that α-agonist activity in combination with antidopaminergic properties may limit the extrapyramidal effects and induce a selective neuroleptic action in the mesolimbic system.

Biochemical evidence that 2-phenyl-4[2-(4-piperidinyl) ethyl]quinoline, a quinoline derivative with pure anticonflict properties, is a partial agonist of benzodiazepine receptors

The atypical profile of 2-phenyl-4[2-(4-piperidinyl) ethyl]quinoline (PK 8165), a quinoline derivative with pure anticonflict properties, seems to be due to the fact that this compound is a partial agonist of benzodiazepine receptors. The drug PK 8165 is a competitive inhibitor of benzodiazepine binding sites with a Hill coefficient near unity. Opposite to 3-methyl-6-(3-trifluoromethylphenyl)2,4-triazolo(4,5-b)pyridazine (CL 218,872) it was unable to discriminate between BZ1 and BZ2 receptors in sections of brain. However, modulation by gamma-aminobutyric acid (GABA) and the effect of photolabelling by flunitrazepam on the affinity of PK 8165 indicated that GABA or photolabelling shifts of PK 8165 were between full agonists and antagonists. By itself PK 8165 was unable to modify the levels of cGMP in the cerebellum, but potentiated the lowering of levels of cGMP by diazepam and did not present antagonistic properties of this effect.