Stimulation by neurotensin of (3H)5-hydroxytryptamine (5HT) release from rat frontal cortex slices.

The effect of neurotensin (NT) on the K+-evoked (3H)5HT release from brain frontal cortex slices was studied in rats. NT(1-13) and NT(8-13) increased (3H)5HT release with EC50 values in the nanomolar range and Emax values in the range of 100% of control, whereas D-tyr11-NT was inactive. Concerning NT receptor antagonists, SR 48692 and SR 142948A antagonized with IC50 values of 4.8+/-1.8 nM and 4.5+/-1.8 nM respectively, the NT stimulated K+-evoked (3H)5HT release. SR 48527 also antagonized NT induced (3H)5HT release with an IC50 value of 0.95+/-0.06 nM whereas the inactive R(-) enantiomer SR 49711 only inhibited this effect with IC50 value close to 10(-6)M. The 5HT-releasing effect of NT was completely inhibited by tetrodotoxin suggesting that NT receptors involved in the control of 5-HT release are not located on 5-HT terminals. After a first NT (10(-7)M) application, the NT (10(-7)M, 10(-6)M) effect under K+ depolarization was drastically decreased, indicating that the NT receptor could be desensitized. No potentiating effect of NT on K+-evoked (3H)5HT release was observed in striatal and hippocampal slices. These results suggest that, in the rat frontal cortex, NT regulates 5HT release through a high affinity NT receptor not associated with 5HT terminals.

Involvement of potentially distinct neurotensin receptors in neurotensin-induced stimulation of striatal [3H]dopamine release evoked by KCl versus electrical depolarization.

We intended to determine whether the effect of neurotensin (NT) on K+ and electrically evoked [3H]dopamine (DA) release from rat and guinea-pig striatal slices involved different mechanisms and/or receptors. In the two species, NT and three NT agonists were found to exhibit different relative potencies to enhance K+- and electrically-evoked [3H]DA release. NT(1-13) increased [3H]DA release with EC50 values in the nanomolar range and Emax values in the range of 100% of control. NT(8-13) and Eisai hexapeptide were both as active as NT(1-13) under K+ depolarization, but did not exceed 40% of the NT(1-13) effect under electrical depolarization. In rats, when [3H]DA release was stimulated with two successive K+ depolarizations, in the presence of NT(1-13), the NT effect during the second exposure to K+ was drastically decreased, suggesting that the NT receptor was desensitized. The desensitization process was essentially observed on Emax values, EC50 values being weakly affected. Similar results were obtained in guinea pig. In contrast, with two electrical depolarizations or with two different depolarizations (K+ followed by electrical), the NT effect during the second depolarization was not significantly affected. Concerning NT antagonists, SR 48692 antagonized with IC50 values in the nanomolar range the NT(1-13) stimulated K+-evoked [3H]DA release but did not affect, up to 10(-6) M, the NT(1-13) enhancement of electrically stimulated [3H]DA release. On the contrary, SR 142948A antagonized the NT(1-13) effect on K+- and electrically-evoked [3H]DA release. In conclusion, these results suggest the possible existence of potentially distinct neurotensin receptors differentially involved in the control exerted by NT on DA release under KCl vs electrical depolarization.

Neurochemical and behavioural effects of neurotensin vs [D-Tyr11]neurotensin on mesolimbic dopaminergic function.

Microinjection of neurotensin(1-13) or neurotensin(8-13) into the ventral tegmental area (VTA) of anaesthetized rats produced dose-dependent (1-100 pg) dopamine release in the nucleus accumbens as measured by differential pulse amperometry (DPA). Higher doses (100 pg-10 ng) of [D-Tyr11]neurotensin were required to produce an identical effect. In addition, the 3 peptides enhanced the K(+)-evoked [3H]DA release from nucleus accumbens slices. The stimulatory actions produced by 10(-8) M neurotensin(1-13) and neurotensin(8-13) were respectively of 96% and 72% while the effect of [D-Tyr11]neurotensin was only of 79% at 10(-6) M. Unilateral application of the 3 peptides in the VTA of cannulated rats produced contralateral circling. [D-Tyr11]neurotensin was effective in a dose-dependent manner, between 40 and 320 ng. Similar effects were observed with 80 ng of neurotensin(1-13) and neurotensin(8-13) in presence of the protease inhibitor thiorphan. In view of the higher potency of neurotensin(1-13) and neurotensin(8-13) versus [D-Tyr11]neurotensin to stimulate DA release both in vivo and in vitro and the higher efficacy of [D-Tyr11]neurotensin to induce circling, this study further strengthens the concept of neurotensin receptor heterogeneity.

SR 48692 inhibits neurotensin-induced [3H]dopamine release in rat striatal slices and mesencephalic cultures.

In rat striatal slices, the increase (114 +/- 11%) in K(+)-evoked [3H]dopamine release induced by neurotensin (10 nM) was antagonized by 2-[(1-(7-chloro-4-quinolinyl)-5-(2,6-dimethoxyphenyl)pyrazol-3-yl) carboxylamino]tricyclo(3.3.1.1.3.7)decan-2-carboxylic acid (SR 48692, IC50 = 1.2 +/- 0.11 nM). SR 48692 (100 nM) also suppressed the neurotensin (10 nM)-induced increase (47%) in K(+)-evoked [3H]dopamine release in primary cultures of fetal rat mesencephalic cells. These results further characterize SR 48692 as a potent antagonist of neurotensin receptors in the rat.

Biochemical and pharmacological profile of a potent and selective nonpeptide antagonist of the neurotensin receptor.

We describe the characteristics of SR 48692, a selective, nonpeptide antagonist of the neurotensin receptor. In vitro, this compound competitively inhibits 125I-labeled neurotensin binding to the high-affinity binding site present in brain tissue from various species with IC50 values of 0.99 +/- 0.14 nM (guinea pig), 4.0 +/- 0.4 nM (rat mesencephalic cells), 7.6 +/- 0.6 nM (COS-7 cells transfected with the cloned high-affinity rat brain receptor), 13.7 +/- 0.3 nM (newborn mouse brain), 17.8 +/- 0.9 nM (newborn human brain), 8.7 +/- 0.7 nM (adult human brain), and 30.3 +/- 1.5 nM (HT-29 cells). It also displaces 125I-labeled neurotensin from the low-affinity levocabastine-sensitive binding sites but at higher concentrations (34.8 +/- 8.3 nM for adult mouse brain and 82.0 +/- 7.4 nM for adult rat brain). In guinea pig striatal slices, SR 48692 blocks K(+)-evoked release of [3H]dopamine stimulated by neurotensin with a potency (IC50 = 0.46 +/- 0.02 nM) that correlates with its binding affinity. In a cell line derived from a human colon carcinoma (HT-29), SR 48692 competitively antagonizes neurotensin-induced intracellular Ca2+ mobilization with a pA2 (-log Kapp) values of 8.13 +/- 0.03, which is consistent with results obtained in binding studies. Moreover, SR 48692 is devoid of any intrinsic agonist activity. This compound is also active in vivo, since it reverses at low dose (80 micrograms/kg) the turning behavior induced by intrastriatal injection of neurotensin in mice with similar potency whatever the route of administration (i.p. or orally) and with a long duration of action (6 hr). Thus, being a potent and selective neurotensin receptor antagonist, SR 48692 may be considered as a powerful tool for investigating the role of neurotensin in physiological and pathological processes.

The sensitivity of gamma-aminobutyric acid antagonists to thiocyanate is related to the absence of a functional anionic group in their structure.

Pyridazinyl derivatives of gamma-aminobutyric acid (GABA) have recently been shown to be selective, reversible and competitive GABAA antagonists. Unlike what is observed with all other GABAA antagonists, the affinity of these compounds for the GABAA receptor is not modified by thiocyanate. The chemical structure of these pyridazinyl-GABA derivatives differs from that of other GABAA antagonists by the presence of a free carboxylic group in their structure. We speculated that this could explain their lack of sensitivity to thiocyanate. Consequently, we synthesized three structural analogues of these pyridazinyl-GABA derivatives in which we replaced the free carboxyl group by a cyano group. These compounds displaced [3H]GABA from rat brain membranes and reversed the GABA-induced enhancement of [3H]diazepam binding. However their affinity for the GABAA receptor increased 10- to 20-fold in the presence of thiocyanate. Thus, sensitivity to thiocyanate appears to be related more to the absence of an anionic functional group than to the agonist or antagonist nature of the GABAA ligand.

Characterization of the binding of [3H]SR 95531, a GABAA antagonist, to rat brain membranes.

A synthetic derivative of gamma-aminobutyric acid (GABA), SR 95531 [2-(3'-carboxy-2'-propyl)-3-amino-6-p-methoxyphenylpyridazinium bromide], has recently been reported, on the basis of biochemical and in vivo microiontophoretic studies, to be a potent, selective, competitive, and reversible GABAA antagonist. In the present study, the binding of [3H]SR 95531 to washed, frozen, and thawed rat brain membranes was characterized. Specific binding was linear with tissue concentrations, had a pH optimum at neutrality, and was maximal at 4 degrees C after 30 min of incubation. Pretreatment of the membranes with Triton X-100 resulted in a 50% decrease of specific binding. Addition of iodide, thiocyanate, or nitrate to the incubation mixture decreased the affinity of [3H]SR 95531 for its binding site; Na+ had no effect. Subcellular fractionation showed that 74% of the P2 binding was in synaptosomes; 31% of the total homogenate binding was in P2 and 50% in P3. The binding of [3H]SR 95531 was saturable; Scatchard analysis of the saturation isotherm revealed two apparent populations of binding sites (KD of 6.34 nM and Bmax of 0.19 pmol/mg of protein; KD of 32 nM and Bmax of 0.81 pmol/mg of protein). The binding of [3H]SR 95531 was reversible, and association and dissociation kinetics confirmed the existence of two binding sites. Only GABAA ligands were effective displacers of [3H]SR 95531. GABAA antagonists were relatively more potent in displacing [3H]SR 95531 than [3H]GABA; the inverse was true for GABAA agonists. There were marked regional differences in the distribution of binding sites: hippocampus = cerebral cortex greater than thalamus = olfactory bulb = hypothalamus = amygdala = striatum greater than pons-medulla and cerebellum. The surprisingly low density of binding sites in the cerebellum was owing to a marked reduction of Bmax values at both the high- and the low-affinity binding sites. In conclusion, the present results demonstrate specific, high-affinity, saturable, and reversible binding of [3H]SR 95531 to rat brain membranes and strongly suggest that this radioligand labels the GABAA receptor site in its antagonist conformation.

Specific binding of a phenyl-pyridazinium derivative endowed with GABAA receptor antagonist activity to rat brain.

SR 95531 has been shown to be a potent, selective, reversible and competitive GABAA antagonist. In the present study we report that (3H)SR 95531 binds with high affinity and in a specific and saturable manner to rat brain membranes. Scatchard analysis revealed two binding sites (KD: 6 nM; Bmax: 0.24 pmol/mg protein and KD: 38 nM; Bmax: 0.66 pmol/mg protein). Only GABA ligands were effective displacers of (3H)SR 95531. The respective IC50 values obtained with these compounds suggests that (3H)SR 95531 labels the GABA receptor in its antagonist conformation.

Biochemical characterization of the interaction of three pyridazinyl-GABA derivatives with the GABAA receptor site.

An arylaminopyridazine derivative of gamma-aminobutyric acid (GABA), SR 95103, has been shown to be a selective antagonist of GABA at the GABAA receptor site. Subsequent structure-activity studies showed that suppressing the methyl in the 4-position of the pyridazine ring, and substituting the phenyl ring at the para position with a chlorine (SR 42641) or a methoxy group (SR 95531) led to compounds which exhibited the highest affinities for the GABA receptor site in this series. In the present study we examined the biochemical interaction of these compounds with the GABA receptor as well as their biochemical selectivity for this receptor. SR 95531 and SR 42641 displaced [3H]GABA from rat brain membranes with apparent Ki values of 0.15 microM and 0.28 microM respectively and Hill numbers near 1.0. The two compounds antagonized the GABA-elicited enhancement of [3H]diazepam-binding in a concentration-dependent manner without affecting [3H]diazepam-binding per se. Scatchard and Lineweaver-Burk analysis of the interaction of the two compounds with the GABAA receptor sites, revealed that the compounds were competitive at the high affinity site, but non-competitive at the low affinity site. Neither compound interacted with other GABAergic processes or with a variety of central receptor sites. When administered intravenously, SR 95531 and SR 42641 elicited tonic-clonic seizures in mice. Based on these results, it is postulated that SR 95531 and SR 42641 are specific, potent and competitive GABAA antagonists.

Absence of modifications in gamma-aminobutyric acid metabolism after repeated generalized seizures in amygdala-kindled rats.

Alterations in gamma-aminobutyric acid (GABA) metabolism have been investigated in the kindling model of epilepsy. Numerous generalized seizures were induced by amygdala-kindling stimulations in rats. One week after the last stimulation, there were no changes in GABA levels nor in the activity of enzymes responsible for the synthesis (glutamic acid decarboxylase) and catabolism (GABA transaminase and succinyl semialdehyde dehydrogenase). These results do not exclude other changes in GABA function as modifications of transport or receptors.

Anticonvulsant activity of 4,9-dioxo-5,10-diazatetradecane (CM 40142), a new GABA derivative, in mice and photosensitive baboons).

The authors describe the anticonvulsant activity of a new gamma-amino-butyric acid (GABA) derivative in several animal models of generalized epilepsy including photoepileptic baboons. In all the studies, 4,9-dioxo-5,10-diazatetradecane (CM 40 142) revealed potencies against chemically, electrically and photic-induced seizures very similar to those observed with sodium valproate. In chemically elicited seizures in mice, CM 40142 exhibited a higher potency than sodium valproate in antagonizing anti-GABAergic agents. Although CM 40142 was synthesized as a compound which would cross the blood-brain barrier and liberate GABA within the central nervous system, preliminary biochemical investigations in mice failed to demonstrate a rise in brain GABA levels after treatment with CM 40142. Furthermore, CM 40142 increased spontaneous motility in mice at anticonvulsant doses. The data suggest that CM 40142 could be a broad spectrum nonsedative antiepileptic agent.

Specific and non-specific stimulation of unprimed and primed lymphocytes induced by plane-tree allergenic extracts in rats.

Whole allergenic extracts (WAE) from plane-tree pollen induces in vitro blastogenesis in splenic lymphocytes from primed and normal rats. Allergen P, the major allergen of plane-tree pollen, induces an early stimulation in primed cells. WAE induced in the same way an early stimulation in primed cells, but a late incorporation of 3H-thymidine occurs at 8 days of culture when lymphocytes from primed or normal rats were used. This late response appears to be non-specific and is not abolished when a short-pulse of antigen is given.

Pollen-allergen IgE response in the inbred rat. Regulation of allergen P-IgE antibodies in high and low responders by suppressive factors.

Cyclophosphamide treatment (100 mg/kg) significantly increased the IgE response to plane-tree allergen P in low-responder strains of rats. A suppressive serum was obtained by injection of CFA in normal low-responder strains. The injection of this serum 2 days after cyclophosphamide treatment and 1 day prior to primary immunization, reversed the cyclophosphamide-enhanced IgE antibodies response without affecting the IgG2a levels. This effect is strain specific.

Drug allergy: in vitro cross-allergenicity between amoxicillin and benzyl penicillin.

The capacity of amoxicillin to elicit allergenic reactions in vitro was determined on benzyl-penicillin sensitive patients. RAST and histamine release were performed on blood of these patients using B pen linked to human serum albumin (BPO-HSA) as allergen. A relationship was noted between the results of the in vitro tests and the date of the allergic manifestations. When results of RAST and histamine release were significant, amoxicillin was tested for its capacity to elicit histamine release and to inhibit RAST performed with BPO-HSA. A cross-allergenicity was observed in six sera out of eight. The results suggested that amoxicillin may be capable to react with cell-bound or serum IgE to give hypersensitive manifestations in some penicillin sensitive patients.

Denaturization of allergen P: effect on allergenicity, antigenicity and immunogenicity.

When allergen P was denatured by 8M urea, the modified molecule still reacted with IgE specific for the native allergen but not with hemagglutinating antibodies. Heating at 100 degrees C abolished the reaction in both cases. The results suggested differences between allergenic and antigenic capacities which may be based on structural differences of the antigenic determinants.

[Are rat alveolar macrophages capable of phagocytizing pollen grains?]. / Les macrophages alvéolaires de rat peuvent-ils phagocyter les grains de pollen?

Plane-tree pollen grains were incubated in vitro with alveolar macrophages from inbred Rats, and the possibility of phagocytosis was investigated. No phagocytosis was observed even after 48 hrs incubation. But alveolar cells were bound to pollen grains generally at the apertures. This binding did not require the Fc receptor on the macrophage membrane.

[Propranolol and arterial hypertension: study with frequent daily checking of blood pressure and serum levels after 1 week of treatment]. / Propranolol et hypertension arterielle: etude pluriquotidienne de la pression arterielle, de la frequence et des taux seriques apres une semaine de traitement

In order to establish correlations between serum levels of Propranolol, blood pressure in hypertensive patients and cardiac rate, these parameters have been recorded at three different times in a day and after a week. This study points out that therapeutic response is variable. However the patients may be classified into 4 groups. This classification and the value of serum levels of propranolol are discussed.