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'Peripheral type' benzodiazepine binding sites in several rat tissues were labelled by intravenous injection of [3H]PK 11195 and [3H] RO5 -4864. Binding was saturable in all tissues studied and regional distribution paralleled the in vitro binding. A similar potency order of displacing compounds was found in vivo and in vitro PK 11195 greater than PK 11211 greater than RO5 -4864 greater than diazepam greater than dipyridamole greater than clonazepam. These results demonstrate the feasibility of using this technique to examine the effects of pharmacological manipulation on the binding sites in their native state. However some properties (broader maximum during time course, higher percentage of particulate binding in the brain and independence of temperature) make [3H]PK 11195 the most suitable ligand for this kind of studies.

"Peripheral type" benzodiazepine binding sites have been solubilized with digitonin. Binding site density for the solubilized material is increased 1.7 times compared to membranes. A decrease in the affinity for [3H]-PK 11195 (a new ligand for the peripheral type benzodiazepine binding sites) was also observed. Pharmacological specificity of displacing agents was conserved during solubilization. The apparent molecular weight determined by gel filtration was 215,000 +/- 20,000. The high Bmax value of the solubilized preparation (greater than 50 pmole/mg protein) makes it advantageous as the starting point for a purification procedure.

Peripheral type of benzodiazepine binding sites were labelled in the kidney, the heart and the brain with [3H] RO5-4864 following intravenous injection in mice. The regional distribution of this in vivo binding parallels the in vitro binding: heart and kidney were more labelled than brain. Benzodiazepine potencies in reducing [3H] RO5-4864 binding in vivo parallel relative affinities for [3H] RO5-4864 binding sites in isolated organs membranes: RO5-4864 greater than diazepam greater than clonazepam. PK 11195 a new compound, chemically unrelated to benzodiazepines, which is a potent inhibitor of [3H] RO5-4864 in vitro is also very effective (more than RO5-4864) after I.P. injection and oral administration. These results emphasize the feasibility of using this technique to examine the effects on various pharmacological and physiological manipulations of these binding sites in vivo. Moreover the fact that PK 11195 binds to these sites in vivo might indicate that this compound could help to elucidate the physiological relevance of the peripheral type of benzodiazepine binding sites.

The comparative properties of heparin and PK 10169, a low molecular weight fraction, were studied using an antithrombotic test in anaesthetized dogs. The antithrombotic properties of the two compounds were evaluated by measuring inhibition of thrombus formation following transluminar stimulation of coronary artery with anodal current and by measuring anticoagulant properties, anti Xa and anti IIa activities. The results show that PK 10169 displayed significant antithrombotic activities above 0.625 mg/kg and was equipotent at 2.5 mg/kg s.c. with heparin 10 mg/kg s.c. No correlation could be observed between antithrombotic/anti Xa ratio of both compounds. Moreover it was shown that, unlike heparin, PK 10169 s.c. was devoid of obvious anticoagulant properties and induced a negligible anti IIa activity contrasting with a high anti Xa level. A similar dissociation between anti Xa and anti IIa activities was observed following i.v. administration of 2.5 mg/kg of PK 10169 but not with heparin. This low molecular weight heparin fraction might thus be regarded as a potential arterial antithrombotic agent devoid of appreciable anticoagulant effect.

The influence of endocrine factors on monoamine oxidase activity (MAO) and on noradrenaline metabolism has been evaluated by measuring platelet MAO activity and plasma levels of 3,4-dihydroxyphenylethylene glycol (DOPEG), the major deaminated metabolite of noradrenaline, as well as serum levels of steroid hormones weekly in 9 young healthy women during one menstrual cycle. A decrease in platelet MAO activity (correlated with high serum estradiol levels) was observed during the ovulatory period. In contrast, plasma free or sulfoconjugated DOPEG remained unchanged throughout the menstrual cycle. These results indicate that the hormonal status should be taken into consideration in studies dealing with platelet MAO activity in depressed women.

Platelet MAO activity was measured in 75 hospitalized depressed patients and in 31 healthy subjects. Plasmas post dexamethasone cortisol levels were examined in 73 patients. Results indicate that higher platelet MAO activity does not occur in all, but only in male major depressed patients. No relationship between changes of MAO activity and specific clinical subtypes was found. Platelet MAO activity is not different between DST suppressors and DST non suppressors. The authors suggest that platelet MAO activity may be related to non specific factors such as sex, age, but not to diagnosis of depression.

[3H]spiroperidol binding to lymphocytes was measured in untreated paranoid or disorganized and treated paranoid schizophrenic patients. An increase in the Bmax was detected in untreated paranoid patients but a decrease was found in the disorganized patients. No difference was detected in the KD value. Neuroleptic treatment produced a decrease in the Bmax without affecting the KD value. Such results did not comply with the down regulation but might be explained by a change in membrane viscosity as [3H]spiroperidol binding sites on lymphocytes were coupled to phospholipid methylation.

FM 24 [1-(2-exo-bicyclo[3,3,1]hept-2-ylphenoxy)-3-[(1-methylethyl)amino] 2-propanol hydrochloride] and propranolol were compared in mice with respect to their ability to displace in vivo 125I-hydroxybenzylpindolol which is selectively associated with beta-adrenergic receptor binding sites. After a simultaneous i.v. injection of the beta-blockers and 125I-hydroxybenzylpindolol propranolol was more active than FM 24. At an equiblocking dose i.e. a dose which inhibits by 80% the binding of 125I-hydroxybenzylpindolol, FM 24 was still effective after 6 h (40% inhibition in the brain and the heart, 60% in the lung) contrary to propranolol. After oral administration (2 mg/kg), 40% inhibition by FM 24 still persisted at 24 h in the heart whereas no effect of propranolol was detectable at 18 h. As the kinetics of [3H]FM 24 and [3H]propranolol after oral and i.v. administration are not very different we confirmed that the prolonged beta-blocking action of FM 24 was related to a tight irreversible binding to beta-receptors rather than to pharmacokinetic properties of this drug.

Two models have been chosen to study the effect of 2-amino-6-trifluoromethoxy benzothiazole (PK 26124) on excitatory amino acid neurotransmission: the pool of cyclic guanosine monophosphate (cGMP) in the cerebellum and the release of acetylcholine in the striatum and olfactory tubercles. The release of acetylcholine induced by N-methyl-DL-aspartate in the striatum and olfactory tubercles was antagonized by PK 26124 which was less potent on the release of acetylcholine induced electrically. The increase in levels of cGMP in the cerebellum induced by excitatory amino acids such as glutamate and quisqualate was antagonized by PK 26124, but the drug was inactive against N-methyl-DL-aspartate, L-aspartate, kainate and cysteine sulphinate. In vivo it antagonized the increases of cGMP in the cerebellum elicited by all these excitatory compounds. All these results are compatible with a possible antagonism by PK 26124 of the excitatory amino acid neurotransmission and may explain its anticonvulsant properties.

In a partially depolarized guinea pig papillary muscle preparation, BAY K8644 stimulated voltage-operated calcium channels, promoting slow action potentials; this effect was dose-dependent over a concentration range of 3 X 10(-7) M to 3 X 10(-6) M. Isoproterenol and histamine also induced slow action potentials by stimulating beta or H2 receptors, respectively. PK 11195, the antagonist of peripheral type benzodiazepine receptors, inhibited the effect of BAY K8644, but not those of histamine or isoproterenol. Moreover, PK 11195 "dose-dependently" antagonized the ability of RO5-4864 to inhibit the slow action potentials elicited by barium chloride. Thus, in the heart, PK 11195, an antagonist of peripheral type benzodiazepine receptors, can modulate voltage-operated calcium channels when they are activated directly, but not when they are activated by stimulation of neurotransmitter receptors.

2-Amino-6-trifluoromethoxy benzothiazole (PK 26124) prevented convulsions induced in rodents by maximal electroshock, inhibitors of the synthesis of gamma-aminobutyric acid (GABA) and ouabain, but was inactive against seizures provoked by GABA antagonists, unlike diazepam, chlordiazepoxide, phenobarbital and valproic acid. 2-Amino-6-trifluoromethoxy benzothiazole prevented seizures induced by sound stimuli in DBA/2 mice (ED50 = 0.66; 2.1 and 4.1 mg/kg, i.p. according to the seizure component), postural seizures in El mice (ED50 = 7.5 mg, i.p.) and seizures induced by photic stimulation in the baboon, Papio papio, at 4 and 8 mg/kg (i.v.). This spectrum of anticonvulsant activity closely resembles that reported previously for dicarboxylic amino acid antagonists. Indeed, PK 26124 prevented seizures induced by L-glutamate (ED50 = 8.5 mg/kg, i.p.) or by kainate (ED50 = 9.25 mg/kg, i.p.) and tremors induced by harmaline (ED50 = 2.5 mg/kg, i.p.) In these tests diazepam was inactive (L-glutamate) or as potent as PK 26124 (kainate, harmaline), whereas it was 10-20 times more potent than PK 26124 against seizures induced by inhibitors of the synthesis of GABA. Together, these data suggest that PK 26124 possesses antagonistic properties of excitatory dicarboxylic amino acids, which may contribute to its anticonvulsant action.

Disturbed noradrenaline (NA) metabolism is thought to play a causal role in certain types of endogenic depression. This study was based on data from 88 patients with depression. The metabolism of NA was investigated by measuring urinary MHPG and plasma DOPEG concentrations and platelet nonoamine oxidase activity to determine if there were any differences in subgroups of depression defined by the DSM3. There was no difference in plasma DOPEG concentrations or of MAO activity in the different subgroups of depression, especially between episodes of major and non-major depression. On the other hand, depressed patients with an episode of major depression had significantly higher urinary MHPG concentrations than those with non-major depression.

1. Metabolism of 10-(3-quinuclidinylmethyl)phenothiazine and the distribution and excretion of the metabolites, especially the sulphoxides, were studied in rat and dog after oral and intravenous administration. 2. Urine and bile contained relatively little unchanged drug. The sulphoxide, sulphone, N-oxide and N-oxide sulphoxide derivatives were identified as well as glucuronide and sulphate conjugates, suggesting the formation of hydroxylated products. Faeces contained mainly unchanged drug but also some sulphoxide and N-oxide. In the lung, brain and cerebro spinal fluid only unchanged drug and traces of sulphoxide were found, whereas in liver sulphone and N-oxide were also present. 3. After administration of the 35S-labelled sulphoxide, the distribution of radio-activity was very different from that observed with LM 209. The biological half-life (t0-5) of SM 209 was 3 to 4 times higher than that of the sulphoxide. LM 209 is better absorbed and its diffusion in the organism is superior. 4. Metabolism of LM 209 by liver microsome preparation was more rapid than metabolism of the sulphoxide. 5. These findings indicate that the activity of LM 209 is due more to the molecule itself than to its major metabolite.

The in vivo binding of [3H]spiroperidol was measured in discrete areas of the brain in 7-, 9- and 16-week-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto (WKY) controls. An increase in the [3H]spiroperidol binding in the striatum, tuberculum olfactorium and frontal cortex but not in the cerebellum was detected at all ages in SHR. The increase was more pronounced in 7- than in 9- or 16-week-old SHR. In vitro data indicated an increase in Bmax but no variation in Kd in the striatum of 7-week-old SHR. Moreover no difference was detectable in the dopaminergic cell bodies (A9, A10). This increase was specific to [3H]spiroperidol binding sites since no difference was observed in the in vivo binding of [3H]QNB and [3H]LSD in the same brain regions. No variation in dopamine level or dopamine utilization, as estimated by measuring the disappearance of the amine induced by alpha-methyl-p-tyrosine, was observed. The DOPA accumulation after injection of the DOPA decarboxylase inhibitor NSD 1015 was greater in the tuberculum olfactorium from 7-week-old SHR. An increase in [3H]spiroperidol binding sites was also observed in the striatum and tuberculum olfactorium after 7 weeks of DOCA-salt treatment. These results suggest that dopaminergic neurons might be implicated in the onset of hypertension in the rat.

The various clinical effects of benzodiazepines have been attributed to the presence of saturable binding sites, stereospecific and of high affinity in the central nervous system. Good correlations have been described between the inhibition of the binding of [3H]diazepam and the anticonvulsant and anticonflictual properties of benzodiazepines. Such results would suggest that these binding sites are the pharmacological receptors responsible for the therapeutic properties of benzodiazepines. In addition, the neuromediator which has been associated with benzodiazepines in terms of its functions is GABA. Up to the present, the anticonvulsant and anticonflictual properties of substances acting on benzodiazepine receptors could not apparently be dissociated. However, b using quinoline derivatives we have been able to dissociate anticonflictual and anticonvulsant properties for substances acting upon benzodiazepine receptors. These substances (PK 8165 and PK 9084) displace diazepam from binding sites (Ki of 100 to 400 nM) in the brain but not peripherally. The fact that PK 9084 and PK 8165 are more active on benzodiazepine derivatives in the presence of anions suggests that they act upon receptors coupled with a chloride ionophore-like. Benzodiazepines, PK 8165 and PK 9084 have anticonflictual properties but cause neither ataxia nor sedation even at doses 5 to 20 times greater than anticonflictual doses. Furthermore, these compounds are not anticonvulsant. In contrast to benzodiazepines, PK 8165 and PK 9084 do not decrease the cGMP of the Purkinje cells of the cerebellum. This cGMP pool being the reflection of the activation of GABAergic receptors, it would seem that these substances must act on a sub-unit of benzodiazepine receptors not coupled with GABA but associated with the chloride ionophore. This sub-unit could be responsible for the anticonflictual properties of substances acting upon benzodiazepine receptors.

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.

A series of 4-amino-6-chloro-2-piperazinopyrimidines were synthesized and evaluated for their ability to interact with alpha 1- and alpha 2-adrenoceptors in vitro in binding assays using [3H]WB-4101, [3H]clonidine, and [3H]idazoxan as radioligands. Some compounds were also tested as inhibitors of [3H]spiroperidol binding. Several members of this series showed high and selective affinity for alpha 2-adrenoceptors. The nature of the 4-amino substituent seems to be the most critical factor in determining the potency at these receptors.

A series of 30 6-chloro-2,4-diaminopyrimidines was synthesized and tested in vitro as inhibitors of [3H]spiroperidol binding. The affinity for the dopamine receptor was shown to be related to the 6-chloro-4-(N-methyl-piperazine)pyrimidine structure bearing a NH2 or NHR1 group as a substituent in position 2, provided that R1 was not an alpha branched alkyl group. The nature of the substituent in position 5 is also of importance for the affinity; 2-(benzylamino)-6-chloro-4-(N-methylpiperazino)-5-(methylthio)pyrimidine (22) is the most active member of the series. Molecular structures of three compounds were analyzed by X-ray diffraction and PCILO computation.

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.

52770 RP, the N-(3-chlorophenyl)-3-(3-pyridinyl)-1H,3H-pyrrolo[1,2-c]thiazole -7-carboxamide, displaces in a potent, specific and competitive manner [3H]PAF from its binding sites on rabbit platelets. Since 52770 RP is not structurally related to PAF and has low liposolubility with respect to PAF, it was selected as a potential radioligand for PAF receptor sites. [3H]52770 RP displayed high-affinity, specificity, as well as saturable and displaceable binding to a single class of recognition sites in intact platelets and crude platelet membranes. In these preparations, the values of binding parameters were, respectively, 8.5 and 7.6 nM for Kd, 0.2 pmol/5 X 10(7) platelets and 3.66 pmol/mg protein for Bmax and 0.96 and 0.91 for nH. Inasmuch as the (+)-52770 RP was 300-fold more potent than the (-)-isomer at displacing [3H]52770 RP in intact platelets, the studied binding site manifested stereospecific discrimination. A variety of pharmacological agents including pro- and anti-aggregant compounds did not exhibit affinity for [3H]52770 RP binding sites. In contrast, PAF, some of its active analogues and several recognized PAF antagonists (BN 52021, brotizolam, L-652,731, triazolam), displaced the [3H]52770 RP binding. Studies carried out using [3H]PAF demonstrated that 52770 RP was approximately 4- and 200-fold more potent than L-652,731 and BN 52021 respectively, as a PAF-receptor antagonist. In washed rabbit platelets, the rank order of potency (Ki) for several analogues of 52770 RP, to displace [3H]PAF from its binding sites, was highly correlated (r = 0.96) to their ability to antagonize [3H]52770 RP binding. In functional studies, 52770 RP antagonized not only the PAF-induced aggregation in washed rabbit platelets but also the hypotension evoked by PAF in the anesthetized rat. In this respect, it was 26 and 2 times more potent than L-652,731, respectively. In conclusion, [3H]52770 RP might represent a novel interesting tool for furthering our understanding of the role of PAF binding sites in pathophysiological processes.