Differential effects of phosphonic analogues of GABA on GABAB autoreceptors in rat neocortical slices

The effects of five phosphonic derivatives of GABA on the release of [³H]-GABA from rat neocortical slices, preloaded with [³H]-GABA, were investigated. Phaclofen and 4-aminobutylphosphonic acid (4-ABPA) increased the overflow of [³H] evoked by electrical stimulation (2Hz) in a concentration-dependent manner, with similar potencies (phaclofen EC₅₀=0.3mmol/l, 4-ABPA EC₅₀=0.4mmol/l). At 3mmol/l, phaclofen increased the release of [³H]-GABA by 82.6±8.6%, and 4-ABPA increased the release by 81.3±9.0%. 2-Amino-ethylphosphonic acid (2-AEPA) increased the overflow of [³H] by 46.8±10.9% at the highest concentration tested (3mmol/l). In contrast, the lower phosphonic homologue 3-aminopropylphosphonic acid (3-APPA), and 2-amino-2-(p-chlorophenyl)-ethylphosphonic acid (2-CPEPA), a baclofen analogue, did not modify the stimulated overflow. These results suggest that phaclofen, 4-ABPA and 2-AEPA are antagonists at GABA_B autoreceptors, the latter being the weakest antagonist, whilst neither 3-APPA nor 2-CPEPA are active at these receptors. Since phaclofen, 4-ABPA and 2-CPEPA are antagonists and 3-APPA a partial agonist/antagonist on GABA_B heteroreceptors, the lack of effect of 3-APPA and 2-CPEPA on [³H]-GABA release in this study suggests that GABA_B autoreceptors may be pharmacologically distinct from the heteroreceptors. Received: 11 June 1997 / Accepted: 6 January 1998     

A cumulative dose-response technique for the characterization of presynaptic receptors modulating [3H]noradrenaline release from rat brain slices

A cumulative dose-response technique was developed for the characterization of presynaptic receptors involved in the modulation of [³H]noradrenaline (NA) release from rat hippocampus slices, using continuous K⁺ (20 mM) depolarization. The results obtained with this technique were compared with those obtained using a repetitive K⁺ stimulation procedure. The release of [³H]NA induced by continuous K⁺ stimulation as well as that caused by repetitive K⁺ stimulation was strongly Ca²⁺-dependent and consisted for more than 90% of unmetabolized [³H]NA. Using continuous K⁺ stimulation it was demonstrated that the presynaptic inhibition of ³H-NA release by exogenous NA reached a maximum 10 min after addition of NA. The inhibitory effect of NA appeared to be independent of the time of addition, suggesting that the sensitivity of the presynaptic α-adrenoceptors remained unchanged during the experiment. Cumulative dose-response curves were recorded by the successive addition, at 10 min intervals, of increasing concentrations of NA. It was shown that continuous stimulation and repetitive K⁺ stimulation were basically similar with regard to the characteristics of the resulting [³H]NA release as well as its presynaptic α-adrenoceptor-mediated modulation by exogeneous NA. However, the cumulative dose-response technique, which can be carried out only using continuous K⁺ stimulation, makes it possible to determine more rapidly and also more accurately the apparent affinities and intrinsic activities of drugs towards receptors involved in the modulation of neurotransmitter release from brain slices.     

Differential control of Ca2+-dependent [3H]noradrenaline release from rat brain slices through presynaptic opiate receptors and alpha-adrenoceptors

The inhibitory actions of the Ca2+ antagonist Cd2+, morphine and noradrenaline (exogenously added + endogenously released) on electrically evoked release of [3H]noradrenaline from superfused rat neocortical slices were strongly reduced when release was enhanced by 4-aminopyridine. In the presence of 4-aminopyridine the release inhibiting effects of these drugs were restored by lowering the extracellular Ca2+ concentration. When release was enhanced by prolonging the pulse duration, only the release inhibiting effect of noradrenaline was reduced but the effects of Cd2+ and morphine were unchanged. Irrespective of the pulse duration, blockade of presynaptic alpha-adrenoceptors with phentolamine did not affect the release inhibiting effects of Cd2+ and morphine. The inhibitory effects of morphine and noradrenaline remained unchanged in Cl--free medium. Furthermore, these drugs strongly reduced the [3H]noradrenaline release induced by 20 mM K+ in the presence of tetrodotoxin. The results suggest that activation of presynaptic opiate-receptors inhibits Ca2+ entry through voltage-sensitive Ca2+ channels, whereas presynaptic alpha-adrenoceptors affect a step in the secretory process subsequent to Ca2+ influx. Moreover, the involvement of (direct) changes in Na+, K+ or Cl- permeability appears unlikely for both receptor systems.     

Effects of vinconate on neurotransmitter receptor systems in aged rat brain

We investigated the effects of age and (±)-methyl-3-ethyl-2,3,3a,4-tetrahydro-1 H-indolo[3,2,1-de][1,5]naphthyridine-6-carboxylate hydrochloride (vinconate), an indolonaphthyridine derivative, on neurotransmitter receptor systems in the rat brain using quantitative receptor autoradiography. [³H]Quinuclidinyl benzilate (QNB), [³H]hemicholinium-3 (HC) and [³H]muscimol were used to label acetylcholine receptors, high-affinity choline uptake sites and γ-aminobutyric acid_A (GABA_A) receptors, respectively. [³H]QNB, [³H]HC and [³H]muscimol binding decreased in any brain areas of 24-month-old (aged) rats in comparison with 6-month-old (adult) animals. Chronic treatment with vinconate (10 and 30 mg/kg, i.p., once a day for 4 weeks) partly ameliorated the reduction in [³H]QNB, [³H]HC and [³H]muscimol biding in aged rat brains. This effect was especially noted in [³H]muscimol binding. The results suggest that vinconate may have beneficial effects on age-related changes in neurotransmitter receptor systems.     

Assessment of nicotinic acetylcholine receptor-mediated release of [(3)H]-norepinephrine from rat brain slices using a new 96-well format assay

The study of the modulatory effects of nicotinic acetylcholine receptor (nAChR) agonists on neurotransmitter release from tissue slices has been hampered by laborious and limiting superfusion techniques. A new methodology was developed utilizing 96-well filter plates. This new method produced comparable results to previously published data, yet expanded throughput to permit more complete pharmacological characterization. Rat brain slices, preloaded with [³H]-norepinephrine ([³H]-NE), were distributed onto 96-well filter plates. Following a 5 min preincubation, the slices were incubated for 5 min with nicotinic agonists or antagonists. (−)-Nicotine (NIC) and 1,1-dimethyl-4-phenylpiperazine (DMPP) evoked release of [³H]-NE from a number of brain regions and spinal cord, with the highest response seen in the hippocampus. Concentration–response curves revealed a rank order of potency of (±)-epibatidine>>anatoxin-a>A-85380>DMPP=NIC=(−)-cytisine in the hippocampus, thalamus, and frontal cortex. EC₅₀ values were approximately 0.005, 0.2, 1, 5, 5 and 5 μM, respectively. Concentration–inhibition curves of nicotine evoked [³H]-NE release from hippocampal and thalamic slices resulted in a rank order of potency of mecamylamine>hexamethonium>d-tubocurare>DHβE. Schild analysis revealed apparent noncompetitive antagonism of [³H]-NE release from hippocampus by mecamylamine, hexamethonium, and DHβE. In contrast, DHβE antagonism of [³H]-dopamine release from striatal slices using a similar methodology was competitive.     

Cholecystokinin-8 increases K-evoked [H]γ-aminobutyric acid release in slices from various brain areas

[³H]γ-Aminobutyric acid (GABA) release was studied in rat brain slices in the absebce or presence of cholecystokinin-8 (CCK-8). [³H]GABA release under the conditions used was Ca²⁺-dependent and insensitive tot he presence of the glial uptake blocker β-alanine. While the basal release of [³H]GABA was not affected by CCK-8, the K⁺-stimulated release of [³H]GABA wasm significantly enhanced by 300 nM of CCK-8 in the caudate putamen, the substantia nigra, the hippocampal formation and the parietofrontal cortex. In the cerebral cortex the CCK-8 enhancement of [³H]GABA release was concentration-dependent and abolished by the CCK_B receptor antagonists PD135,158 (1.0 nM) and L-365,260 (100 nM). A significant counteraction of the CCK-8 action was also found with the CCK_A receptor antagonist L-364,718 (100 nM) but only in concentrations at which both CCK_A and CCK_B receptors are blocked. No CCK-8 effects on [³H]GABA release were observed when tetrodotoxin was superfused 5 min before the K⁺-induced [³H]GABA release. It is suggested that the enhancing actions of CCK-8 on K⁺-stimulated [³H]GABA release is mainly related to an activation of CCK_B receptors.     

Beware the builders: Construction noise changes [C]GABA release and uptake from amygdaloid and hippocampal slices in the rat

The effects of exposure to chronic noise and vibration, produced by construction work, on the release and uptake of [³H]5-hydroxytryptamine ([³H]5-HT) and [¹⁴C]γ-aminobutyric acid ([¹⁴C]GABA) from rat amygdaloid and hippocampal slices were investigated. Noise-exposure resulted in increased release, with no significant change in uptake, of [¹⁴C]GABA from amygdaloid slices. In hippocampal slices, [¹⁴C]GABA release was also increased, but the changes in release were dependent upon the marked decrease in uptake of [¹⁴C]GABA into these slices. There was an increase in peak K⁺-evoked release of [³H]5-HT from hippocampal slices, but no other changes in [³H]5-HT release or uptake in either region were observed following noise-exposure. These findings may have important practical implications for the research carried out in laboratories exposed to construction noise and vibrations.     

High affinity [H]zolpidem binding in the rat brain: an imidazopyridine with agonist properties at central benzodiazepine receptors

[³H]Zolpidem, a novel hypnotic drug posessing a chemical structure unrelated to that of benzodiazapine (BZD) was employed as a new ligand to determine its binding characteristics to membrane preparations of rat cerebral cortex and cerebellum. In both structures, the imidazopyridine [³H]zolpidem bound with high affinity to a single population of recognition sites. The cerebellum possessed a similar number of [³H]zolpidem and [³H]diazepam binding sites, while the cerebral cortex possessed a lower density of [³H]zolpidem than [³H]diazepam binding sites. In contrast to [³H]diazepam binding, [³H]zolpiem binding was not detectable in the spinal cord. In the cortex, BZDs had a similar potency to displace [³H]zolpidem and [³H]diazepam binding while non-BZDs were more potent to inhibit [³H]zolpidem binding than [³H]diazepam binding. The binding of [³H]zolpidem was enhanced by GABA to the same extent as [³H]diazepam binding. The increase in [³H]zolpidem binding caused by chloride ions was less pronounced than that in [³H]diazepam binding. It is concluded that [³H]zolpidem possesses selectivity for BZD receptors with the pharmacological characteristics and regional distribution of the BZD₁ receptor subtype. [³H]Zolpidem as a radioligand offers a useful additional tool to study the mechanism of action of hypnotics acting through BZD receptor subtypes.     

Lindane inhibition of [35S]TBPS binding to the GABAA receptor in rat brain.

The inhibition of [³⁵S]t-butylbicyclophosphorothionate ([³⁵S]TBPS) binding to the GABA_A receptor by the insecticide γ-hexachlorocyclohexane, lindane, was studied in several brain regions and using different membrane preparation methods, both in vitro and after dosing the animals with the chemical. In the latter studies, the amount of lindane remaining in the membrane suspensions used for binding assays was determined. In vitro data showed values of IC₅₀ from 150 to 1675 nM, varying in function of the membrane preparation method used. This may account for the discrepancies in IC₅₀ values found in the literature. IC₅₀ values within the range of 150–250 nM were determined using extensively washed membranes from several brain regions, so no evidence arose for brain regional differences in the affinity of lindane for the TBPS binding site. After different schedules of acute treatment with lindane, we found a manifest relationship between the extent of the observable inhibition of [³⁵S]TBPS binding and the lindane amount remaining in the membrane suspensions used for binding assays. This relationship was in good agreement with the in vitro data, so no support for an in vivo acute regulation of the binding site was obtained.     

Cyclothiazide and AMPA receptor desensitization: analyses from studies of AMPA-induced release of [3H]-noradrenaline from hippocampal slices

1. Responses in brain produced by the activation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype of ionotropic receptor for L-glutamate are often rapidly desensitizing. AMPA-induced desensitization and its characteristics, and the potentiating effect of cyclothiazide were investigated in vitro by analysing AMPA-induced release of [³H]-noradrenaline from prisms of rat hippocampus.
2. AMPA (1–1000 μM) stimulated the release of [³H]-noradrenaline in a concentration-dependent manner that was both calcium-dependent and tetrodotoxin-sensitive, and attenuated by the AMPA-selective antagonists, NBQX (1 and 10 μM), LY 293558 (1 and 10 μM) and GYKI 52466 (10 and 30 μM).
3. By use of an experimental procedure with consecutive applications of AMPA (100 μM, 28 min apart), the second response was reduced, indicative of receptor desensitization, and was reversed by cyclothiazide in a concentration-dependent manner (1–300 μM). The concentration-response curve for AMPA-induced release of [³H]-noradrenaline was shifted leftwards, but the reversal by cyclothiazide of the desensitized response was partial and failed to reach the maximal response of the first stimulus.
4. Observations made with various schedules of cyclothiazide application indicated that the initial AMPA-evoked response was already partially desensitized (150% potentiation by 100 μM cyclothiazide) and that the desensitization was not likely to be due to a time-dependent diminution and was long-lasting (second application of cyclothiazide was ineffective).
5. Co-application of a number of drugs with actions on second messenger systems, in association with the second AMPA stimulus, revealed significant potentiation of the AMPA-induced release of [³H]-noradrenaline: forskolin (10 μM, +78%), Rp-cAMPS (100 μM, +65%), Ro 31-8220 (10 μM, + 163%) and thapsigargin (100 μM, +161%).
6. The AMPA receptor-mediated response regulating the release of [³H]-noradrenaline from rat hippocampal slices was desensitized and cyclothiazide acted to reverse partially the desensitization in a concentration-dependent manner. Since the time-course of desensitization was longer lasting than that noted in previous electrophysiological studies, multiple events may be involved in the down-regulation of AMPA receptor activity including receptor phosphorylation and depletion of intracellular Ca²⁺ stores.

     

Strychnine-sensitive glycine receptors inducing [3H]-acetylcholine release in rat caudatoputamen: a new site of action of ethanol?

In the present study acute effects of ethanol on [³H]-acetylcholine ([³H]-ACh) release induced by activation of strychnine-sensitive glycine receptors in superfused slices of rat caudatoputamen were investigated. The glycine-evoked [³H]-ACh release (lg EC₅₀ = –4.10, CI₉₅ = [–4.14, –4.05]) was inhibited by strychnine in a competitive manner (pA₂ = 6.86, CI₉₅ = [6.61, 7.08]). Release of [³H]-ACh could also be induced by L-serine. L-serine was less potent than glycine (lg EC₅₀ = –2.61, CI₉₅ = [–2.69, –2.52]). Both glycine and L-serine showed similar maximum effects (E_max(glycine) = 1.34, CI₉₅ = [1.24, 1.45]; E_{max(L-serine)} = 1.19, CI₉₅ = [1.09, 1.32]). Ethanol at concentrations of 2‰ (= 34 mM) and 4‰ (= 68 mM) inhibited glycine-evoked [³H]-ACh release in a manner like the competitive antagonist strychnine, however with lower potency. The pA₂ of ethanol was 1.19, CI₉₅ = [0.85, 1.41], at 2‰ [v/v] and 1.51, CI₉₅ = [1.19, 1.78] at 4‰ ethanol. Similar to its action on glycine-evoked [³H]-ACh release, ethanol at 4‰ [v/v] also inhibited L-serine-evoked transmitter release in a competitive-like fashion (pA₂ = 0.83, CI₉₅ = [–0.15, 1.18]). We conclude, that strychnine-sensitive glycine receptors, mediating [³H]-ACh release in the rat caudatoputamen, might represent a new site of action of ethanol. Received: 15 May 1997 / Accepted: 4 September 1997     

Inhibitory effects of GABA,L-glutamic acid and nicotine on the potassium-evoked release of subsaance P in substantia nigra slices of the rat

Rat substantia nigra slices were superfused with a physiological medium containing a diluted substance P (SP) antiserum, bacitracin and serum albumin to measure SP released in superfusates. As shown by measuring the degradation of a SP-labelled derivative incubated with cerebellar slices, this medium prevented the enzymatic inactivation of SP. Potassium (K⁺, 50 mM) and veratridine (5 × 10^?5M) stimulated SP release and these effects were respectively prevented in absence of calcium and in presence of tetrodotoxin (5 × 10^?7 M). GABA (5 × 10^?5 M) nicotine (10^?6 M) and L-glutamic acid (5 × 10^?5 M) reduced the K⁺ (50 mM)-evoked release of SP. In contrast, glycine (5 × 10^?5 M), oxotremorine (5 × 10^?5 M), D-glutamic acid (5 × 10^?5 M) and serotonin (5 × 10^?5 M) were without effect. Pempidine (10^? M) prevented the inhibitory effect of nicotine (10^? M) on the K⁺-evoked release of SP. Glutamic acid diethyl ester (10^?4 M) completely abolished the L-glutamic acid-induced inhibition of the K⁺-evoked release of SP. Picrotoxin (5 × 10^?5 M) did not influence the L-glutamic acid inhibitory effect excluding the intervention of GABAergic mechanisms.     

Autoradiographic study on the pharmacological characteristics of [H]3-OH-PCP binding sites in rat brain

The pharmacological characteristics and the regional distribution of [³H]3-OH-PCP (1-[1(3-hydroxyphenyl)-cyclohexyl]piperidine) binding were investigated in rat brain by quantitative autoradiography. Kinetic analysis of [³H]3-OH-PCP binding revealed fast and slow components, in the association and dissociation studies. The regional distribution of binding closely corresponded to those of binding sites labeled by [³H]N-[1-(2-thienyl)-cyclohexyl]3,4-piperidine (TCP) and [³H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK 801). High densities of [³H]3-OH-PCP binding sites were found in the stratum radiatum and oriens of field CA1 in the hippocampus and in the outer layers of cerebral cortices. In contrast, low levels of binding were seen in the brain stem and the granular cell layer of the cerebellum. [³H]3-OH-PCP binding was strongly inhibited by MK 801 and 3-OH-PCP, while the potency of (+)-SKF 10047 in inhibiting [³H]3-OH-PCP binding was less in the cerebral cortex and hippocampus. The antagonists for the glutamate, glycine and polyamine recognition sites at the NMDA/PCP receptor complex displaced [³H]3-OH-PCP binding sites with a potency similar to that of [³H]MK 801. These findings suggest that the [³H]3-OH-PCP binding site is similar or identical to the PCP binding site labeled by [³H]TCP and [³H]MK 801.     

Modulation by pertussis toxin of salbutamol- and arecoline-induced effects in the isolated heart and aorta of the rat

The modulatory effects of pertussis toxin pretreatment on responses mediated via β-adrenoceptors and muscarinic acetylcholine receptors were investigated in isolated rat hearts and aortic rings 4 days after in vivo administration of pertussis toxin. In isolated hearts, pertussis toxin increased heart weight and baseline coronary flow values but did not effect baseline left ventricular pressure values. In unpaced hearts, pertussis toxin inhibited the arecoline-induced cardiac standstill, while in paced hearts, the β₂-adrenoceptor agonist salbutamol produced a dose-dependent vasodilation with similar characteristics in pertussis toxin and control preparations. Pertussis toxin had no effect on myocardial or aortic cyclic nucleotide levels and the myocardial β-adrenoceptor density (B_max) and dissociation constant (K_d). In precontracted aortic rings, pertussis toxin had no effect on the salbutamol or arecoline induced vasorelaxation. In summary, we demonstrated a reduced cholinergic responsiveness in isolated hearts but an intact β₂-adrenoceptor pathway in isolated hearts as well as in isolated aortic rings after pertussis toxin pretreatment. In aortic rings no change in muscarinic acetylcholine receptor responsiveness occurred.     

Effect of treatment with mercury chloride and lead acetate during the second stage of rapid postnatal brain growth on δ-aminolevulinic acid dehydratase (ALA-D) activity in brain, liver, kidney and blood of suckling rats

The sensitivity of developing rodents to toxic metals differs considerably from that of adults. In the present study, we investigated the in vivo and in vitro effects of inorganic mercury and lead on δ-aminolevulinic acid dehydratase (ALA-D) from brain, liver, kidney and blood of young rats. Eight day-old rats were injected with one or five doses of lead acetate (0, 3.5, or 7.0 mg/kg) or HgCl₂ (0, 2.5, or 5.0 mg/kg). In vitro, the IC₅₀ for mercury inhibition of cerebral, renal and hepatic ALA-D was in the 124 to 160 μM range, while values for lead acetate was in the 7 to 12 μM range. The IC₅₀ of blood enzyme for lead (0.8 μM) and mercury (6.5 μM) was significantly lower than that observed for the other tissues. A single dose of lead did not affect the enzyme activity, but a single dose of HgCl₂ (5 mg/kg) caused a significant inhibition of ALA-D from kidney (40%, P < 0.01) and liver (25%, P < 0.05). Five doses of lead acetate (3.5 or 7 mg/kg) caused an inhibition of about 25 and 40%, respectively (P < 0.01), of hepatic ALA-D, and an increase of 1.4-fold (P < 0.05) and 2.6-fold (P < 0.01) of blood enzyme, respectively. Treatment with five doses of HgCl₂ (5 mg/kg) caused an inhibition of about 25, 60, 50, and 80% of ALA-D from brain, blood, liver and kidney, respectively (all P < 0.05). Five doses of 2.5 mg/kg HgCl₂ caused an inhibition of ALA-D from liver (40%, P < 0.01) and kidney (45%, P < 0.01). These results demonstrate that ALA-D from young rat tissues show different sensitivities to mercury and lead. The enzyme was more affected by mercury than by lead in vivo, while in vitro lead was more potent that mercury as an ALA-D inhibitor.     

Role of glutamate receptors and nitric oxide on the effects of glufosinate ammonium,an organophosphate pesticide,on in vivo dopamine release in rat striatum

The purpose of the present work was to assess the possible role of glutamatergic receptors and nitric oxide (NO) production on effects of glufosinate ammonium (GLA), an organophosphate pesticide structurally related to glutamate, on in vivo striatal dopamine release in awake and freely moving rats. For this, we used antagonists of NMDA (MK-801 and AP5) or AMPA/kainate (CNQX) receptors, or nitric oxide synthase (NOS) inhibitors (l-NAME and 7-NI), to study the effects of GLA on release of dopamine from rat striatum. So, intrastriatal infusion of 10 mM GLA significantly increased dopamine levels (1035 ± 140%, compared with basal levels) and administration of GLA to MK-801 (250 μM) or AP5 (650 μM) pretreated animals, produced increases in dopamine overflow that were ∼40% and ∼90% smaller than those observed in animals not pretreated with MK-801 or AP5. Administration of GLA to CNQX (500 μM) pretreated animals produced an effect that was not significantly different from the one produced in animals not pretreated with CNQX. On the other hand, administration of GLA to l-NAME (100 μM) or 7-NI (100 μM) pretreated animals, produced increases in dopamine overflow that were ∼80% and ∼75% smaller than those observed in animals not pretreated with these inhibitors. In summary, GLA appears to act, at least in part, through an overstimulation of NMDA (and not AMPA/kainate) receptors with possible NO production to induce in vivo dopamine release. Administration of NMDA receptor antagonists and NOS inhibitors partially blocks the release of dopamine from rat striatum.     

Effect of pentobarbitone and diethyl ether on the synthesis of monoamines in rat brain

Summary Rats were injected i.p. with pentobarbitone sodium 40 mg/kg or were exposed to diethyl ether anaesthesia for 15 to 60 min. Monoamine synthesis in vivo was measured in various brain regions by determining the accumulation of dopa and 5-hydroxytryptophan (5-HTP) during 15 or 30 min after the i.p. injection of NSD 1015 (3-hydroxybenzylhydrazine HCl, 100 mg/kg), an inhibitor of the aromatic amino acid decarboxylase.Pentobarbitone retarded the formation of dopa and 5-HTP by about 25% in most brain regions but had no effect on striatal dopa formation.Ether accelerated the formation of dopa and 5-HTP in most brain regions, the action on striatal dopa being most pronounced (to 250% of control). The effect was generally somewhat less marked during the second than during the first half-hour of anaesthesia. A postanaesthetic inhibition of dopa formation was found in the striatum and of 5-HTP formation in whole brain.If hypothermia was allowed to develop, the stimulating action of ether on dopa and 5-HTP formation tended to be partly antagonized. A complex interaction between NSD 1015 and the hypothermic response to ether was observed.     

Biochemical evidence for the 5-HT agonist properties of PAT (8-hydroxy-2-(di-n-propylamino)tetralin) in the rat brain

In vitro investigations revealed that PAT (8-hydroxy-2-(n-dipropylamino)tetralin) interacted with postsynaptic 5-HT receptors in the rat brain: the drug stimulated 5-HT-sensitive adenylate cyclase in homogenates of colliculi from new-born rats (K_Aapp 8.6 μM) and inhibited the specific binding of [³H]5-HT to 5-HT₁ sites. The PAT-induced inhibition of [³H]5-HT binding showed marked regional differences compatible with a preferential interaction of PAT (IC₅₀ 2 nM) with the 5-HT_1A subclass. As previously seen with 5-HT agonists, the efficacy of PAT for displacing [³H]5-HT bound to hippocampal membranes was markedly increased by Mn²⁺ (1 nM) and reduced by GTP (0.1 nM). PAT also affected presynaptic 5-HT metabolism since it inhibited competitively (K_i 1.4 μM) [³H]5-HT uptake into cortical synaptosomes and reduced (in the presence of the 5-HT uptake inhibitor fluoxetine) the K⁺-evoked release of [³H]5-HT previously taken up or newly synthesized from [³H]tryptophan in cortical or striatal slices. This latter effect was prevented by 5-HT antagonists (methiothepin, metergoline) suggesting that it was mediated by the stimulation of presynaptic 5-HT autoreceptors by PAT. Like 5-HT, PAT counteracted the stimulatory effect of K⁺-induced depolarization on the synthesis of [³H]5-HT from [³H]tryptophan in cortical slices. It is concluded that PAT is a potent 5-HT agonist acting on both post- and presynaptic 5-HT receptors in the rat brain.     

Opioid receptor agonists enhance the phosphorylation state of Fas-associated death domain (FADD) protein in the rat brain: functional interactions with casein kinase Ialpha, Galpha(i) proteins, and ERK1/2 signaling

Opioid drugs have been proposed to promote anti-apoptotic signals in brain through inhibition of FADD protein [García-Fuster et al., 2007. Effects of opiate drugs on Fas-associated protein with death domain (FADD) and effector caspases in the rat brain: Regulation by the ERK1/2 MAP kinase pathway. Neuropsychopharmacology 32, 399–411]. FADD phosphorylation by casein kinase Iα (CKIα) appears to regulate its non-apoptotic activity. This study investigated the effects of opioids on p-FADD in rat brain, as well as various mechanisms that could link opioid receptors with p-FADD, including the modulation of CKIα, Gα_i proteins and ERK1/2 signaling. In rat, mouse and human brains, various anti-p-FADD antibodies immunodetected the monomeric and oligomeric forms of this protein, irrespective of the antibody origin and specific Ser191 or Ser194 phosphorylation site. Acute μ- and δ-agonists increased, through specific opioid receptor mechanisms, the content of oligomeric and monomeric p-FADD forms in rat cortical homogenates (25–61%) and subcellular compartments, with most relevant effects for sufentanil in membrane (239%) and nucleus (136%). p-FADD induction vanished with repeated (5 days) morphine but not SNC-80, and opioid withdrawal induced a new (morphine) or sustained (SNC-80) stimulatory effect (32–33%). The κ-agonist (−)-U-50488H failed to stimulate p-FADD. Sufentanil reduced CKI protein and kinase activity in the cytosol (30–37%). Morphine, but not SNC-80, augmented CKIα in cytosol, membrane and nucleus (36–104%). In contrast to FADD, the ability of SNC-80 to stimulate p-FADD was not sensitive to ERK1/2 blockade. Pertussis toxin did not prevent the opposite effects of SNC-80 on p-FADD and FADD because the toxin by itself markedly altered their basal contents, indicating that FADD could be a novel toxin target. The upregulation of p-FADD induced by μ/δ-agonists could play a relevant role in the anti-apoptotic and/or neuroplastic effects of opioids.     

Evidence that 2-phenylpyrazolo[4,3-c]-quinolin-3(5H)-one antagonises pharmacological, electrophysiological and biochemical effects of diazepam in rats

The effects of the acute administration of 2-phenylpyrazolo[4,3-c]quinolin-3(5H)-one on diazepam-induced behaviour and electrophysiological activity were studied in rat. The compound, in doses of 5-10 mg/kg (i.p.), which per se did not induce alterations in spontaneous locomotor activity, antagonised the sedative effect induced by 5-10 mg/kg (i.p.) of diazepam. The injection of diazepam in rats, induced a profound reduction in the first negative wave of the recording of the visual evoked potential used as a sensitive electrophysiological test, in vivo. 2-Phenylpyrazolo[4,3-c]quinolin-3(5H)-one (10 mg/kg, i.p.) caused a recovery of the amplitude of the first negative wave within a few minutes. This result was confirmed by the finding that 2-phenylpyrazolo[4,3-c]quinolin-3(5H)-one, injected acutely in rats, pretreated with diazepam exhibited the capacity to antagonise the binding of [3H]diazepam determined in vitro on synaptic membrane preparations from cortex. The comparison of the pattern of the visual-evoked potential, recorded after the injection of 2-phenylpyrazolo[4,3-c]quinolin-3(5H)-one (50 mg/kg) with the patterns recorded after the injection of ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazolo(1,5a) (1,4)benzodiazepine-3-carboxylate (50 mg/kg) and ethyl-beta-carboline-3-carboxylate and 1-methyl-beta-carboline demonstrated that 2-phenylpyrazolo[4,3-c]quinolin-3(5H)-one is devoid of intrinsic activity.