Enhancement of benzodiazepine binding by progabide (SL 76002) and SL 75102

The new GABA derivatives, progabide (SL 76002) and SL 75102 (the corresponding carboxylic acid), enhanced [³H]diazepam binding to rat cortical membranes. SL 75102 was as effective as GABA, and both were less active than muscimol. Similar to other GABA agonists, progabide and SL 75102 enhanced binding in vitro by an increase in affinity. The elevation of [³H]diazepam binding in the presence of SL 75102 was antagonized by bicuculline. In ex vivo experiments, brain membrane preparations of mice pretreated with progabide were found to bind a greater amount of [³H]diazepam. This increased binding was characterized by an elevation in B_max, while K_D was unaffected. [³H]Flunitrazepam binding to brain in vivo was also enhanced in mice pretreated with progabide. Other GABA agonists (muscimol, THIP, isoguvacine) and GABAergic agents (sodium valproate) elicited an increase in [³H]flunitrazepam binding in vivo. Binding of intravenous [³H]flunitrazepam in vivo may be useful for discovering enhancers of benzodiazepine binding.     

3H]Ro 15-1788 binding to benzodiazepine receptors in mouse brain in vivo: marked enhancement by GABA agonists and other CNS drugs

Administration of the benzodiazepine receptor antagonist, [3H]Ro 15-1788, to mice intravenously was found to label these receptors in brain. Binding of [3H]Ro 15-1788 in vivo was strongly blocked by pretreating mice with clonazepam or diazepam. Marked enhancement of [3H]Ro 15-1788 binding in vivo was induced by progabide or sodium valproate. This effect was greater than a similar enhancement of [3H]flunitrazepam binding. The increased membrane-bound [3H]Ro 15-1788 elicited by progabide was completely dissociated on subsequent incubation with Ro 15-1788, diazepam or clobazam, indicating that the enhanced binding occurred at benzodiazepine receptors. Compounds that exert diazepam-like actions and/or indirect GABAergic activity (cartazolate, pentobarbital, methaqualone, levonantradol, phenytoin) elicited enhancement of [3H]Ro 15-1788 in vivo. Other CNS agents (atypical neuroleptics, GABA antagonists, baclofen, some 5-HT1 agonists) also induced elevation of [3H]Ro 15-1788 binding in vivo, as did drugs exerting vasodilatatory effects (papaverine, nimodipine, verapamil, prazosin, N6-cyclohexyladenosine). Possible explanations for enhancement of [3H]Ro 15-1788 binding in vivo include increase in the number of benzodiazepine receptors induced by GABA or GABAergic drugs or effects of binding enhancers that elevate brain levels of [3H]Ro 15-1788, such as accelerating cerebral blood flow, competing for radioligand binding sites in plasma or increasing metabolic stability of the radioligand.     

Pyrazolopyridines: Effect of structural alterations on activity at adenosine- and GABAA-receptors

A series of 4-substituted 1H-pyrazolo[3,4-b]pyridine- 5-carboxylic acid derivatives related in structure to the putative anxiolytics cartazolate, tracazolate, and etazolate were assessed for affinity at A₁- and A_2A-adenosine receptors and at GABA-, benzodiazepine-, and picrotoxinin- binding sites of the GABA_A-receptor-channel. None of the derivatives had markedly greater affinity at A₁-receptors than cartazolate (K_i-0.5 μM), but many had markedly lower affinity than cartazolate (K_i-1.5 μM) at A_2A-receptors. At the benzodiazepine-binding site of GABA_A-receptors some of the derivatives enhanced [³H]diazepam binding, as did cartazolate and GABA, some had no effect and some inhibited binding. Most of the derivatives inhibited binding of the benzodiazepine-antagonist [³H]Ro 15-1788. At the GABA-binding site, only a few of the derivatives inhibited binding of the antagonist [³H]SR-95531, as did GABA. At the picrotoxinin-binding site, many inhibited binding of [³⁵S]TBPS, but none were as potent as cartazolate or GABA. Analysis of the interactions indicates that stimulation of [³H]diazepam binding is allosteric and results from binding of the pyrazolopyridine at the GABA site or a subdomain of that site, while inhibition of [³H]Ro 15-1788 binding is competitive and due to binding at the benzodiazepine site. Inhibition of [³⁵S]TBPS binding at the picrotoxinin-channel site appears to be allosteric through the GABA site and/or by direct competition at the channel site. Alterations in structure markedly alter the affinities of pyrazolopyridines at such sites on the GABA_A-receptor-channel. Drug Dev. Res. 42:41–56, 1997. © 1997 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.     

The effect of cyclopyrrolones on GABAA receptor function is different from that of benzodiazepines

The effects of the cyclopyrrolones zopiclone and suriclone on the function of the central -aminobutyric acid type A (GABA_AA) receptor complex in mouse brain were evaluated both in vitro and in vivo. Added in vitro to mouse cerebral cortical membranes, these compounds potently inhibited [³H]flumazenil binding with IC₅₀ (50% inhibitory concentration) values of 35.8 nM (zopiclone) and 1.1 nM (suriclone). Similar results were obtained with cerebellar membranes, indicating that these drugs do not discriminate between putative type I and type II benzodiazepine receptors. The interaction of cyclopyrrolones with recognition sites present at the level of the GABA receptor complex appears to be competitive, because zopiclone decreased the affinity of the receptors for [³H]flumazenil without affecting the maximal number of binding sites. Moreover, zopiclone and suriclone did not affect the rate of dissociation of [³H]flumazenil from benzodiazepine receptors. The in vitro efficacy of zopiclone appeared different from that of suriclone and the benzodiazepines diazepam and flunitrazepam. Thus, zopiclone failed to affect muscimol-stimulated ³⁶Cl^– uptake and only slightly inhibited t-[³⁵S]butylbicyclophosphorothionate ([³⁵S]TBPS) binding. In contrast, like diazepam and flunitrazepam, suriclone increased muscimol-stimulated ³⁶Cl^– uptake and markedly inhibited [³⁵S]TBPS binding. On the other hand, suriclone, like zopiclone, did not modify [³H]muscimol binding to mouse cerebral cortical membranes. Moreover, zopiclone antagonized the reduction in [³⁵S]TBPS binding elicited by the benzodiazepine receptor full agonist diazepam. Consistent with its low efficacy in vitro, oral administration of zopiclone (2.5 to 100 mg/kg, p.o.) in mice failed to modify [³⁵S]TBPS binding subsequently measured in cerebral cortical membranes ex vivo. In contrast, suriclone (10 to 20 mg/kg, p.o.), like diazepam, decreased [³⁵S]TBPS binding measured ex vivo. Moreover, both zopiclone (50 to 100 mg/kg, p.o.) and suriclone (1 to 10 mg/kg, p.o.) abolished the increase in [³⁵S]TBPS binding induced by isoniazid (200 mg/kg, s.c.). These results suggest that suriclone may enhance GABAergic transmission with an efficacy similar to that of diazepam. In contrast, the low efficacy of zopiclone both in vitro and in vivo suggests that this drug may act as a partial agonist at benzodiazepine receptors.Abbreviations GABA -aminobutyric acid - TBPS t-butylbicyclophosphorothionate - ANOVA analysis of variance Correspondence to: A. Concas at the above address     

Studies on the binding of [3H]flumazenil and [3H]sarmazenil in post-mortem human brain

Two benzodiazepine analogues, [³H]flumazenil and [³H]sarmazenil, were used to study the GABA_A/benzodiazepine receptor complex in human post-mortem brain using in vitro receptor assays on homogenates and whole hemisphere autoradiography. Both radioligands bound in a saturable manner to single binding sites in the tissue preparations from any brain region. The highest levels of binding were found in the cortical regions and in cortex cerebelli. Both [³H]flumazenil and [³H]sarmazenil were excellent radioligands for autoradiography with high binding in cerebral and cerebellar cortex with no or very low binding in areas with white matter. The addition of a high concentration of flumazenil or clonazepam did not inhibit the binding of [³H]sarmazenil to granule cells in the cerebellum while the binding of [³H]flumazenil was abolished completely in all regions. The results show that with the two different radioligands, one an antagonist and one a partial inverse agonist, the binding pattern to GABA_A/benzodiazepine receptor complex is approximately similar in most brain regions. The additional binding seen in the cerebellum with [³H]sarmazenil is suggested to be due to binding to an α₆-containing complex.     

In vivo labelling of alpha5 subunit-containing GABA(A) receptors using the selective radioligand [(3)H]L-655,708

L-655,708 is an imidazobenzodiazepine possessing 30-70-fold selectivity for the benzodiazepine binding site of GABA_A receptors containing an α5 rather than α1, α2 or α3 subunit. In the present study, [³H]L-655,708 was used to label mouse brain benzodiazepine binding sites in vivo. When compared to inhibition of in vivo binding of the non-selective ligand [³H]Ro 15-1788, the pharmacology of mouse in vivo [³H]L-655,708 binding was consistent with selective in vivo labelling of α5 subunit-containing GABA_A receptors. Thus, diazepam was equipotent at inhibiting in vivo [³H]L-655,708 and [³H]Ro 15-1788 binding; zolpidem, which has very low affinity for α5-containing GABA_A receptors, gave no inhibition of in vivo [³H]L-655,708 binding despite inhibiting in vivo [³H]Ro 15-1788 binding; and L-655,708 was more potent at inhibiting the in vivo binding of [³H]L-655,708 compared to [³H]Ro 15-1788. This pharmacological specificity of in vivo [³H]L-655,708 binding was confirmed autoradiographically. Hence, the anatomical distribution of in vivo [³H]L-655,708 binding was comparable to the distribution of α5-containing GABA_A receptors identified in vitro. Moreover, this distribution was distinct from that identified using [³H]Ro 15-1788. These data therefore suggest that [³H]L-655,708 can be used to identify α5-containing GABA_A receptors in vivo and that this ligand can be used to measure receptor occupancy of α5-selective ligands.     

Biochemical evidence for a new benzodiazepine receptor antagonist

1-(3-Chlorophenyl)-3-diethylcarbamoyl-1 H-1,2,4-triazole (CP-32,961) inhibited [³H]diazepam binding to rat cortical membrances and [³H]flunitrazepam binding to mouse brain in vivo. Its inhibition of the binding of [³H]Ro 15-1788 (benzodiazepine receptor antagonist) to these membranes was not facilitated by added GABA; CP-32,961 exhibited a GABA ratio of 0.84 compared to 2.44 for diazepam. Cerebellar cyclic GMP content in rats was raised by CP-32,961, which also further elevated the increased cyclic GMP levels induced by isoniazid. These neurochemical actions are similar to those shown by ethyl β-carboline-3-carboxylate (β-CCE) and suggest that CP-32,961 is a benzodiazepine receptor antagonist with inverse agonist activity.     

Enhancement of benzodiazepine and GABA binding by the novel anxiolytic, tracazolate

Tracazolate (ICI 136,753) 4-butylamine-1-ethyl-6-methyl-1H-pyrazolo[3,4]pyridine-5-carboxylic acid ethyl ester is a non-benzodiazepine with anxiolytic-like activity in animal models. In contrast to the benzodiazepines, it enhances [3H]flunitrazepam binding in rat synaptic membrane fragments. The enhancement is potential by chloride ion and is due to an increase in affinity of the receptor. The enhancement of benzodiazepine binding by gamma-aminobutyric acid (GABA) is additive with that of tracazolate; however, the GABA antagonist bicuculline blocks the enhancement by both compounds. Tracazolate enhances [3H]GABA binding to frozen and thawed Triton X-100-treated membrane fragments. The enhancement is due to an increase in the number of sites and potentiated by chloride. Benzodiazepines also enhanced GABA binding but the effect was due to an apparent change in affinity and not potentiated by chloride. The rank order to chlorodiazepoxide, diazepam and flunitrazepam for enhancement of GABA binding and displacement of [3H]flunitrazepam binding were the same. The enhancement of [3H]GABA binding by flunitrazepam and tracazolate were additive. Possible interactions between these various receptors are discussed.     

Effect of 3-PPP,a putative dopamine autoreceptor agonist,on [3H]ligand binding to dopamine receptors of calf and rat striatum

3-(3-Hydroxyphenyl)-N-n-propyliperidine (3-PPP) is most effective in inhibiting [³H]apomorphine binding in rat striatal membranes, with Ki values of 63 nM. 3-PPP was six to 27 times less effective when it competed with the binding of [³H]dopamine or [³H]spiperone in calf and rat striatal membranes. At concentrations up to 10 μM, 3-PPP failed to substitute for dopamine in the activation of adenylate cyclase in rat striatal membranes. 3-PPP at 4.8-5 μM caused 50% inhibition of catecholamine uptake in synaptosomes of corpus striatum and hypothalamus, therefore appearing to be a relatively weak uptake inhibitor. The higher affinity of 3-PPP for [³H]apomorphine binding sites is consistent with its binding to a subset of dopamine receptors which are characterized by a high affinity for both the agonist and antagonist of dopamine.     

大鼠血小板外周型苯二氮结合部位的某些性质

用[~3H]PK11195和[~3H]氟硝西沣比较了外周型苯二氮(艹卓)(PBR)结合部位在大鼠各组织中的相对密度。结果表明,血小板为研究外周型结合部位的较好模型。以完整血小板为模型,观察了PBR结合部位的性质及温度对结合性质的影响。结果证实,这种结合部位具有饱和性、专一性、可逆性以及较高的亲和力,温度对配体的亲和力有明显的影响。     

450088-S,a ring-opened prodrug of a 1,4-benzodiazepine,inhibited [3H]flunitrazepam labeling of rat cerebral cortex in vivo

We report here that a peptidoaminobenzophenone (450088-S), a ring-opened prodrug of a 1,4-benzodiazepine, effectively inhibited the labeling of receptors for 1,4-benzodiazepines by [³H]flunitrazepam in vivo despite the lack of receptor affinity for 450088-S in vitro. Labeling of cerebral cortex after an intravenous injection of [³H]flunitrazepam was inhibited by about 80% after an intraperitoneal injection of 450088-S or diazepam. 450088-S was found to be four times more potent than diazepam. The inhibition occurred within 5 and 10 min of administration of diazepam and 450088-S, respectively. These findings demonstrate the occupancy of 1,4-benzodiazepine receptors in brain upon administration of the prodrug 450088-S in rats.     

Flunitrazepam photoaffinity labeling of the GABAA receptor reduces inhibition of [3H]Ro15-4513 binding by GABA

The benzodiazepine drugs modulate γ-aminobutyric acid (GABA)-mediated synaptic transmission via a high-affinity binding site that is part of the GABA_A receptor complex, but which is distinct from the GABA binding site. Ro15-4513 is a benzodiazepine negative modulator of GABA action that displays unique anti-ethanol properties both in vivo and in vitro. Ro15-4513 has been reported to photoaffinity label nearly 100% of the benzodiazepine binding sites in rat brain homogenates. In contrast, the benzodiazepine positive modulator flunitrazepam photoaffinity labels only 25% of the sites. Here, we have examined the reversible binding of [³H]Ro15-4513, [³H]flumazenil (Ro15-1788), and [³H]flunitrazepam to embryonic chick brain membranes, and to membranes that have been photoaffinity labeled with nonradioactive flunitrazepam. Photoaffinity labeling with flunitrazepam decreased the subsequent reversible binding of [³H]flunitrazepam and [³H]flumazenil, but increased the binding of [³H]Ro15-4513. The increase in [³H]Ro15-4513 binding after flunitrazepam photoaffinity labeling was due to a decrease in the apparent K_d, with no change in B_max. Following photoaffinity labeling, negative modulation of [³H]Ro15-4513 binding by GABA was lost, whereas positive modulation of residual [³H]flunitrazepam binding was retained. We conclude that the site photoaffinity labeled by flunitrazepam is distinct from the site responsible for reversible binding of [³H]Ro15-4513.     

Hypersusceptibility to DMCM-induced seizures during diazepam withdrawal in mice: evidence for upregulation of NMDA receptors

The present study investigated the role of NMDA (N-methyl-D-aspartate) receptors in the hypersusceptibility to seizures induced by the benzodiazepine inverse agonist DMCM (methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate) during diazepam withdrawal The seizure threshold of DMCM was markedly decreased during diazepam withdrawal, reflecting withdrawal hyperexcitability in response to physical dependence. The decrease in the seizure threshold of DMCM in diazepam-withdrawn mice was inhibited by the non-competitive NMDA receptor antagonists MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cycloheptan-5,10-imine maleate; 50 μg/kg, s.c.) and ifenprodil (20 mg/kg, i.p.). The effective doses of these compounds were lower than those required to prevent DMCM-induced seizures in chronically vehicle-treated mice. Since MK-801 and ifenprodil do not only bind to NMDA receptors but also to σ receptors, the present study also investigated the effects of σ receptor ligands. The decrease in the seizure threshold of DMCM in diazepam-withdrawn mice was not modified by the σ receptor agonist, (+)-pentazocine (5 mg/kg, s.c.), or the σ receptor antagonist, NE-100 (N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-ethylamine monohydrochloride; 5 mg/kg, i.p.). Furthermore, the latency to the expression of wild running induced by intracerebroventricular administration of NMDA (60 ng/mouse) was also significantly lower in diazepam-withdrawn mice than in vehicle-treated control mice. On the other hand, there was no difference in the spermidine concentration between vehicle-treated control and diazepam-withdrawn mice. In a receptor binding experiment, the B_max value for [³H]-MK-801 binding was significantly increased in cerebrocortical tissues from diazepam-withdrawn mice, while the K_d value did not change in either group. However, the acute addition of a high concentration of diazepam (10 and 100 μM) in vitro did not alter [³H]-MK-801 binding in cerebrocortical membrane preparations. The behavioral experiments suggest that NMDA receptor antagonists may suppress benzodiazepine withdrawal responses, while the biochemical study reveals upregulation of the NMDA receptor, which may play an important role in the hypersusceptibility to DMCM-induced seizure in diazepam-withdrawn mice. Received: 23 July 1997 / Accepted: 27 November 1997     

Benzodiazepine receptor binding: the interactions of some non-benzodiazepine drugs with specific [3H] diazepam binding to rat brain synaptosomal membranes

Summary The interaction of several non-benzodia-zepine drugs with [³H] diazepam binding to benzodiazepine receptors in rat brain synaptosomal membranes was investigated. Baclofen, benzoctamine, hydroxyzine, chlorpromazin, haloperidol, imipramine, and amitriptyline displace specific [³H] diazepam binding, but the concentrations needed are too high to explain pharmacological effects of these drugs by an interaction with benzodiazepine receptors. The most potent non-benzodiazepine drug for inhibiting specific [³H] diazepam binding was methaqualone (IC₅₀ value of 150 M). It is suggested that interactions with benzodiazepine receptors may account for the anxiolytic and anticonvulsive side effects of this drug. The analeptic drug pentylenetetrazole interacts with benzodiazepine receptor binding with an IC₅₀ value of about 1 mM, which is possibly too high to explain its convulsive properties by an antagonism at the benzodiazepine receptor.     

Action of pyrazolopyridines as modulators of [3H]flunitrazepam binding to the gaba/benzodiazepine receptor complex of the cerebellum

The pyrazolopyridines etazolate (SQ 20009) and cartazolate (SQ 65396) have strong modulatory effects on the GABA/benzodiazepine receptor complex of rate cerebellum. Thus, etazolate and cartazolate directly stimulate [3H]flunitrazepam binding (with EC50 values of 1.2 microM and 0.3 microM respectively) by increasing the apparent affinity of [3H]flunitrazepam for its binding sites. Stimulation of [3H]flunitrazepam binding by pyrazolopyridines is dependent on the presence of certain anions like chloride, bromide, iodide, nitrite, nitrate but not fluoride, acetate, formate or sulfate. If is inhibited by bicuculline-methiodide, and by the "chloride channel drugs' picrotoxinin and IPTBO. isoTHAZ, a GABA analogue with GABA antagonist properties in vivo, fails to inhibit binding stimulated by etazolate but antagonizes [3H]flunitrazepam binding stimulated by GABA. The pyrazolopyridines have also indirect effects on benzodiazepine receptor binding since they enhance the apparent sensitivity of those GABA recognition sites which are coupled to benzodiazepine binding sites. Thus, in the presence of 10 microM etazolate, GABA and muscimol enhance [3H]flunitrazepam binding, with EC50 values of 109 nM and 12 nM respectively. This sensitization effect is partially dependent on the presence of chloride ions. The pyrazolopyridines facilitate also the stimulation of benzodiazepine receptor binding by beta-alanine and taurine and by the rigid and flattened GABA analogues THIP and piperidine-4-sulfonic acid. Taken together, these results suggest that the pyrazolopyridines modulate [3H]flunitrazepam binding by acting at a site closely related to GABA receptor-regulated chloride ion channels.     

Effects of novel catechol ether imidazolidinones on calcium-independent phosphodiesterase activity, [3H]rolipram binding,and reserpine-induced hypothermia in mice

A series of new catechol ether imidazolidinones incorporating structural features of rolipram and Ro 20-1724 were synthesized as inhibitors of the calcium-independent phosphodiesterase (IPDE) from rat cerebral cortex. Several compounds were found to be more potent than rolipram as IPDE inhibitors; all of the compounds studied were more potent than Ro 20-1724. The new imidazolidinones also showed affinity for the [³H]rolipram binding site in mouse brain membranes and reversed reserpine-induced hypothermia in mice at relatively low doses. In vitro, the potency of these compounds as IPDE inhibitors did not correlate with their affinity for the [³H]rolipram binding site. Reversal of reserpine hypothermia by the imidazolidinones (“antidepressant effect”) could not be linked definitively to either in vitro activity.     

Platelet 5-HT uptake sites in depression: three concurrent measures using [3H] imipramine and [3H] paroxetine

Platelet 5-HT uptake sites were measured in 40 depressed patients and 40 controls using [³H] imipramine binding, defined with desmethylimipramine (DMI) and Na⁺ dependence, and [³H] paroxetine binding. In control subjects the B_max of DMI defined [³H] imipramine binding was significantly higher than both Na⁺ dependent [³H] imipramine (by 30%) and [³H] paroxetine binding (by 22%). The B_max of Na⁺ dependent [³H] imipramine and [³H] paroxetine binding did not differ significantly. The K_d of Na⁺ dependent [³H] imipramine binding was significantly lower than the K_d of DMI defined [³H] imipramine binding. The binding of DMI defined and Na⁺ dependent [³H] imipramine and [³H] paroxetine did not differ significantly between depressed patients and controls in the total group, in those depressed patients who had never taken antidepressants or in those depressed patients who had been recently with-drawn from antidepressants. This study provides no support for the view that the number of platelet 5-HT uptake sites are reduced in depression.     

Ketamine interacts with the noradrenaline transporter at a site partly overlapping the desipramine binding site

Effects of the intravenous anaesthetic ketamine on the desipramine-sensitive noradrenaline transporter (NAT) were examined in cultured bovine adrenal medullary cells and in transfected Xenopus laevis oocytes expressing the bovine NAT (bNAT). Incubation (1–3 h) of adrenal medullary cells with ketamine (10–300 μM) caused an increase in appearance of catecholamines in culture medium. Ketamine (10–1000 μM) inhibited desipramine-sensitive uptake of [³H] _{noradrenaline} (NA) (IC₅₀=97 μM). Saturation analysis showed that ketamine reduced V _max of [³H]NA uptake without changing K _m, indicating a non-competitive inhibition. Other inhibitors of NAT, namely cocaine and desipramine, showed a competitive inhibition of [³H]NA uptake while a derivative of ketamine, phencyclidine, showed a mixed type of inhibition. Ketamine (10–1000 μM) also inhibited the specific binding of [³H]desipramine to plasma membranes isolated from bovine adrenal medulla. Scatchard analysis of [³H]desipramine binding revealed that ketamine increased K _d without altering B _max, indicating a competitive inhibition. In transfected Xenopus oocytes expressing the bNAT, ketamine attenuated [³H]NA uptake with a kinetic characteristic similar to that of cultured adrenal medullary cells. These findings are compatible with the idea that ketamine non-competitively inhibits the transport of NA by interacting with a site which partly overlaps the desipramine binding site on the NAT. Received: 18 December 1997 / Accepted: 17 June 1998     

An integrated approach to study the molecular aspects of regulatory peptides biological mechanism

An integrated methodological approach to study the molecular aspects of short regulatory neuropeptides biological mechanism is proposed. The complex research is based on radioligand‐receptor method of analysis and covers such points of peptides molecular activity as: specific binding of peptides to brain cells plasmatic membranes, formation of tissue specific synacton, influence of peptides (as allosteric modulators) on functionality of different neuroreceptors as well as delayed in time effects of peptides on receptor‐binding activity of well‐known neuroreceptor systems. Radiolabeled ligands in such complex study are the one of the best and precision instruments to uncover the molecular mechanism of multiple and multitarget biological effects of regulatory peptides. In this issue we used heptapeptide Semax as a model regulatory peptide, [³H]Ach and [³H]GABA as an effector molecules, and the rat model of stress‐induced memory and behavior impairment as a morbid state. We showed the ability of Semax to modulate in a dose‐dependent manner [³H]Ach and [³H]GABA specific binding to some of its corresponding receptors as well as to affect the number of [³H]GABA specific binding places on rat neurons plasmatic membranes after complex stress exposure.