Neuroprotective effects of RPR 104632, a novel antagonist at the glycine site of the NMDA receptor, in vitro.

The NMDA antagonist and neuroprotective effects of RPR 104632 (2H-1,2,4-benzothiadiazine-1-dioxide-3-carboxylic acid), a new benzothiadiazine derivative, with affinity for the glycine site of the NMDA receptor-channel complex are described. RPR 104632 antagonized the binding of [3H]5,7-dichlorokynurenic acid to the rat cerebral cortex, with a Ki of 4.9 nM. This effect was stereospecific, since the (-)-isomer was 500-fold more potent than the (+)-isomer. The potent affinity of RPR 104632 for the glycine site was confirmed by the observation that RPR 104632 inhibited [3H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([3H]TCP) binding in the presence of N-methyl-D-aspartate (NMDA) (IC50 = 55 nM), whereas it had no effect on the competitive NMDA site or on the dissociative anaesthetic site. RPR 104632 inhibited the NMDA-evoked increase in guanosine 3',5'-cyclic monophosphate (cGMP) levels of neonatal rat cerebellar slices (IC50 = 890 nM) in a non-competitive manner and markedly reduced NMDA-induced neurotoxicity in rat hippocampal slices and in cortical primary cell cultures. These results suggest that RPR 104632 is a high-affinity specific antagonist of the glycine site coupled to the NMDA receptor channel with potent neuroprotective properties in vitro.

The mechanism of action of zopiclone.

The mechanism of action of the cyclopyrrolone hypnotic drug zopiclone involves allosteric modulation of the GABAA receptor. Zopiclone displaces the binding of [(3)H]-flunitrazepam with an affinity of 28 nM, and enhances the binding of the channel blocker [(35)S]-TBPS. The binding of zopiclone, unlike that of hypnotic benzodiazepines, is not facilitated by GABA. Zopiclone does not distinguish between GABAA receptors containing different alpha-subunits (BZ(1) and BZ(2) phenotype). Studies with protein-modifying agents (eg diethylpyrocarbonate) and photoaffinity labelling suggest that cyclopyrrolones bind to a domain on the GABAA receptor different from the benzodiazepine binding domain. The consequence of this interaction with the GABAA receptor is to potentiate responses to GABA, as can be demonstrated by electrophysiological methods. Subchronic treatment of mice with high doses of zopiclone does not produce the changes in sensitivity of the GABAA receptor that are observed with hypnotic benzodiazepines.

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.

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.

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.

Riluzole, a novel antiglutamate, prevents memory loss and hippocampal neuronal damage in ischemic gerbils.

The neuroprotective effects of riluzole, a novel antiglutamate, has been demonstrated in a model of ischemia induced in female Mongolian gerbils by transient bilateral carotid occlusion. Riluzole was administered at a dose of 4 mg/kg, i.p., just before, 4 hr after, and for the 14 d following the transient bilateral carotid occlusion (10 min). The functional sequelae of ischemic damage were assessed using a memory test (passive avoidance) and the extent of neuronal damage by histological examination and quantitative autoradiography of muscarinic cholinergic receptors in the hippocampus. The performance of the ischemic gerbils in the memory test was about half that of control animals. This memory deficit was completely reversed in animals treated with riluzole. This protective effect of riluzole was confirmed by histological and autoradiographic studies. The neuronal degeneration of CA1 pyramidal cells in the hippocampus observed in the ischemic group was not seen in the riluzole-treated animals, which resembled the control group. This neuronal degeneration in the CA1 area was confirmed by a quantitative measurement of muscarinic receptors: The binding was decreased by a third in the lacunosum moleculare, the stratum oriens, and the stratum radiatum. By contrast in riluzole-treated gerbils, this decrease was reversed by 50%. Finally, a clear-cut correlation was found between the deficit in the memory test and the decrease in muscarinic receptor binding in the CA1 fields. These results are compatible with the idea that glutamic acid may be involved in the neuronal degeneration of the hippocampus following ischemia, and could be foreseeable.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding sites for a peripheral type benzodiazepine antagonist ([3H]PK 11195) in human iris.

Peripheral-type benzodiazepine binding sites have been characterized on sections of 8 normal human iris/ciliary-body preparations. Saturability was determined at 25 degrees C with [3H] PK 11195 (1 nM) a specific ligand of peripheral type sites. The studies revealed a single class of binding sites for PK 11195 with a nanomolar range affinity (KD = 1.45 nM) and a maximal capacity (Bmax) of 35.5 fmol/mg protein. The displacement potency order of the benzodiazepines tested suggest that these sites belong to the peripheral type: PK 11211 (IC50 = 12 nM) greater than Ro 5-4864 (IC50 = 770 nM) greater than clonazepam (IC50 = 20,000 nM). The present data demonstrate that high affinity binding sites for peripheral type benzodiazepines are present in human iris/ciliary-body. This tissue is therefore a suitable tool for evaluation of the putative functional role of these binding sites.

Quantitative autoradiographic determination of binding sites for a peripheral benzodiazepine ligand ([3H]PK 11195) in human iris.

Specific binding sites of peripheral-type benzodiazepines were investigated in human iris/ciliary body (8 eyes). Examination of color-coded prints and densitometric quantification of autoradiograms were performed on slides (20 micron) labelled with [3H]PK 11195 (1 nM) at 25 degrees C. Nonspecific binding was determined with PK 11211 (5 microM) or Ro 5-4864 (5 microM). Binding sites were present on all the slides, with equivalent density in the 3 regions of the preparation (ciliary body, iris, and pupil margin). The numbers of binding sites in ciliary body, iris, and pupil margin, respectively, were: 42.7 +/- 0.2, 30.1 +/- 0.5, and 37.4 +/- 0.4 femtomol/mg protein. Labelling on the pupil margin seemed to coincide with the iris sphincter muscle. The presence of peripheral benzodiazepine binding sites in iris muscular tissue, and particularly in the pupil margin, suggests that the iris preparation may be a valuable tool to detect putative physiological effects of peripheral benzodiazepines on muscular motility.

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.

[Biochemical characterization and study by quantitative autoradiography of the binding sites of indalpine, a 5-HT uptake inhibitor, in cat brain]. / Caractérisation biochimique et étude par autoradiographie quantitative des sites de fixation de l'indalpine, un inhibiteur de la recapture de 5-HT, dans le cerveau de chat.

The [3H]indalpine binding sites have been characterized in slide-mounted cat brain sections. This inhibitor of 5-HT reuptake binds with a very high affinity to sites which have the pharmacological properties of the serotonin carrier. These sites can, however, be differentiated from the [3H]imipramine binding sites by their Na+ dependency and competitive inhibition by serotonin. Quantitative autoradiographic studies demonstrate that indalpine binding sites are localized in structures rich in serotonergic neurons. The widespread distribution of indalpine binding sites in limbic and associative areas is consisted with its well characterized antidepressant activity in human.

Quantitative autoradiography of [3H]indalpine binding sites in the rat brain: I. Pharmacological characterization.

The binding of [3H]indalpine (4-[2-(3-indolyl)]ethyl piperidine) to slide-mounted sections of rat brain has been characterized. This 5-hydroxytryptamine (5-HT) uptake blocker binds to sections with high affinity (KD approximately 1 nM). The binding is saturable, and can be displaced by the addition of clomipramine (1 microM). Other drugs inhibiting the uptake of 5-HT also have the capacity to inhibit the binding of [3H]indalpine. A significant correlation (r = 0.86) was found between the capacity of these compounds to inhibit the uptake of 5-HT and their potencies as inhibitors of [3H]indalpine binding. Binding was Na+ - and Cl- -dependent and was inhibited competitively by 5-HT. Furthermore, electrolytic lesions of the dorsal raphe or medial forebrain bundle, which cause a degeneration of 5-HT cell bodies and fibers, respectively, resulted in a 30-40% reduction in the binding of [3H]indalpine. [3H]Indalpine binds to the 5-HT uptake recognition sites in a different manner from imipramine-like compounds.

Quantitative autoradiography of [3H]indalpine binding sites in the rat brain: II. Regional distribution.

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.

Biochemical evidence that 2-phenyl-4[(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.

Autoradiographic localization of peripheral benzodiazepine binding sites in the cat brain with [3H]PK 11195.

"Peripheral type" benzodiazepine binding sites were labelled in cat brain membranes by using [3H]PK 11195. This ligand binds to the "peripheral type" binding sites in a reversible, specific and saturable manner. Cat brain binding sites density (congruent to 6 pmol/mg prot.) was higher than in the rat. Pharmacological specificity was demonstrated by the potency order of displacing agents: PK 11195 greater than RO5-4864 greater than dipyridamole greater than diazepam greater than clonazepam. A similar characterization was performed in slide mounted brain sections where [3H]PK 11195 also labelled the "peripheral type" benzodiazepine binding sites. The high percentage of specific binding (80%) at 1 nM of [3H]PK 11195 made possible the autoradiographic studies on binding sites distribution. These sites were heterogeneously distributed in the grey matter and absent in white matter. Visual, auditory and other specific sensory relay stations were highly labelled. The blood pressure regulating nuclei, the vestibulo-cerebellar and the extrapyramidal motor system also presented a very high binding density. As previously described in the rat brain, choroid plexus was also strongly labelled by [3H]PK 11195 in the cat.

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.

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 congruent to 10(-9) M) [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(-9) M). 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 degrees C (PK 11195 greater than RO5-4864 greater than diazepam greater than dipyridamole greater than 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.

"Peripheral type" benzodiazepine binding sites in rat adrenals: binding studies with [3H]PK 11195 and autoradiographic localization.

PK 11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinec arboxamide] is a compound chemically unrelated to benzodiazepines with a high affinity for the "peripheral type" binding sites for benzodiazepines (Le Fur et al., 1983a). [3H]PK 11195 binds to the adrenal membranes with a high affinity (KD congruent to 3 nM) in a specific, reversible and GABA-independent manner. Binding is also characterized by very high Bmax (34 pmol/mg protein). These binding sites are the "peripheral type" benzodiazepine binding sites as demonstrated by the potency order of displacement of the [3H]PK 11195 bound: PK 11195 greater than R05-4864 greater than diazepam greater than dipyridamole greater than clonazepam. The biochemical characteristic of the binding to rat adrenal sections has also been studied. In these conditions the affinity for [3H]PK 11195 is ten times smaller, but the potency order of displacing agents was the same, demonstrating the identity of the section binding sites. Using tritium sensitive film these sites have been visualized in adrenal sections. [3H]PK 11195 binding sites are localized in the adrenal cortex with some spare labelling in the medulla.