RESUMO
Schistosomiasis is one of the most prevalent infectious diseases worldwide and classified as a neglected disease for which there is an urgent need for searching new drug candidates. According to TDR/WHO, existing leads with proven schistosomicidal activity, like meclonazepam, might be the objects of further exploration. Here, we decided to investigate if the benzodiazepine binding sites that we recently characterized in adult Schistosoma mansoni could represent the molecular target of meclonazepam for its effect on worm motility and morphological appearance. The EC(50) of meclonazepam for its contracturant effect is 10-20 times lower than its IC(50) for binding to the worm benzodiazepine binding sites. On the contrary, benzodiazepines like flunitrazepam and diazepam have affinities at least 50 times higher than meclonazepam for these binding sites but did not induce contraction of the worms. We also confirmed the existence of a great similarity between the appearance, kinetics, Emax and external calcium dependency of the contractile effect of praziquantel and meclonazepam. Based on computer-aided molecular modeling calculations, we verified that a certain structural similarity exists between the active enantiomers of both drugs. We further proposed the hypothesis of common pharmacophoric elements including amide and imine subunits and the asymmetric carbons of S-(+)-meclozepam and R-(-)-praziquantel. As a whole, the present data indicate that the contracturant effect of meclonazepam is not a result of its binding to the worm benzodiazepine binding sites but that it shares some basic transduction pathway with praziquantel, even if not through identical molecular targets or binding sites.
Assuntos
Benzodiazepinonas/farmacologia , Músculos/efeitos dos fármacos , Schistosoma mansoni/efeitos dos fármacos , Schistosoma mansoni/metabolismo , Animais , Benzodiazepinas/metabolismo , Benzodiazepinonas/química , Benzodiazepinonas/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Técnicas In Vitro , Isoquinolinas/metabolismo , Isoquinolinas/farmacologia , Masculino , Modelos Moleculares , Conformação Molecular , Movimento/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Músculos/metabolismo , Músculos/fisiologia , Praziquantel/química , Praziquantel/metabolismo , Praziquantel/farmacologia , Receptores de GABA-A/metabolismo , Schistosoma mansoni/fisiologia , Transdução de Sinais/efeitos dos fármacos , Coloração e RotulagemRESUMO
Os benzodiazepínicos estão entre as drogas mais freqüentemente prescritas em razão de suas propriedades ansiolíticas. O objetivo deste trabalho foi avaliar a influência do diazepam sobre a resposta inflamatória peritoneal aguda induzida por lipopolissacarídeo. Para tanto, camundongos Swiss foram tratados com diazepam (1 ou 10 mg/kg de peso), em dose única, por via subcutânea, uma hora antes do desafio intraperitoneal com lipopolissacarídeo bacteriano. Após 16 horas do desafio, os animais foram sacrificados, coletando-se os lavados peritoneais para determinação do número total de células e das subpopulações de mononucleares e polimorfonucleares, além da atividade de TNF-α e da porcentagem de macrófagos espraiados. Observou-se que o tratamento com diazepam, nas doses de 1 ou 10 mg/kg, reduziu significativamente a porcentagem de macrófagos estimulados por LPS e a liberação de TNF-α independente de estímulo. Houve também significativa redução da migração de leucócitos nos animais estimulados com LPS e tratados com 10 mg/kg de diazepam em relação aqueles não tratados. Concluímos que a administração do diazepam, em dose única, pode influenciar significativamente o influxo celular, a estimulação de macrófagos e a atividade de TNF-α na resposta inflamatória aguda induzida por LPS em camundongos, com possíveis implicações na eficiência da resposta anti-infecciosa.
Benzodiazepines are one of the most frequently prescribed drugs due to their anxiolytic properties. The aim of this study was to evaluate the effects of diazepam on lipopolysaccharide-induced peritoneal acute inflammatory responses. Swiss mice were treated with diazepam in a single dose of 1 or 10 mg/kg- subcutaneously 1 h before an intraperitoneal injection of lipopolysaccharide or sterile saline solution. The mice were killed 16 h after and the cells were washed from the peritoneal cavity to determine the total number of cells and the mononuclear and polimorfonuclear subpopulations, as well as the TNF-alpha activity and percentage of spread macrophages. Our results showed that the diazepam treatment (1 and 10 mg/kg) induced a significant reduction in the LPS-induced macrophage stimulation and TNF-α activity. Diazepam (10 mg/kg) also reduced the inflammatory cellular migration when compared to the control. It can be concluded that the diazepam treatment in a single dose is able to influence the inflammatory cellular influx, macrophage stimulation and TNF-α activity in the acute inflammatory response in mice, having possible implications on the anti-infectious response efficiency.
Assuntos
Animais , Feminino , Camundongos , Benzodiazepinonas/metabolismo , Bioensaio/métodos , Fator de Necrose Tumoral alfa/análise , Lipopolissacarídeos/farmacologiaRESUMO
As we have recently shown that GABA should be considered a putative neurotransmitter in Schistosoma mansoni, the present work aimed to search for GABAA receptors in adult worms using [3H]-flunitrazepam to label the allosteric benzodiazepine binding site which is classically present on GABAA receptor complexes. We detected a large population (Bmax=8.25+/-1.1 pmol x mg protein(-1)) of high affinity (Kd=33.6+/-1.5 nM) binding sites for flunitrazepam. These sites harboured a singular pharmacological modulation that does not fit well with a mammalian central benzodiazepine receptor, mainly due to a very high affinity for Ro5-4864 and a very low affinity for clonazepam. We also detected a second population of benzodiazepine binding sites labelled with high affinity (IC50=85 nM) by [3H]-PK11195, a selective ligand of the mammalian peripheral benzodiazepine receptor. In conclusion, this work describes the pharmacological properties of a large population of central-like benzodiazepine receptors supporting their study as putative new targets for the development of anti-parasitic agents. We also describe, for the first time, the presence of peripheral benzodiazepine receptors in this parasite.
Assuntos
Benzodiazepinas/metabolismo , Receptores de GABA-A/metabolismo , Schistosoma mansoni/metabolismo , Animais , Benzodiazepinonas/metabolismo , Sítios de Ligação , Clonazepam/metabolismo , Clonazepam/farmacologia , Diazepam/metabolismo , Diazepam/farmacologia , Flunitrazepam/análise , Flunitrazepam/metabolismo , Flunitrazepam/farmacologia , Agonistas GABAérgicos/farmacologia , Moduladores GABAérgicos/farmacologia , Concentração Inibidora 50 , Isoquinolinas/metabolismo , Ligantes , Masculino , Piridinas/metabolismo , Piridinas/farmacologia , Ratos , Ratos Wistar , Receptores de GABA-A/classificação , Receptores de GABA-A/efeitos dos fármacos , Schistosoma mansoni/efeitos dos fármacos , Sinaptossomos/metabolismo , Temperatura , Fatores de Tempo , Trítio/análise , ZolpidemRESUMO
A significant increase in the number of measurable [3H]Ro 5-4864 receptors was found in forebrain membranes of chicks submitted to 15 min of acute swim stress compared to non-stressed chicks. In addition, low subsolubilizing concentrations of Triton X-100 caused a significant increase in the measurable [3H]Ro 5-4864 receptor number in forebrain membranes from non-stressed chicks. However, this increase caused by Triton X-100 was not observed when tested in forebrain membranes from stressed chicks. In all cases the affinity remained unchanged. These results suggest that: (1) acute stress and Triton X-100 induce receptor increase by enhancing [3H]Ro 5-4864 accessibility to a pool of receptors not detected before stress or in the absence of detergent; (2) the pool of non-measured receptors represents about a third of the total in control chicks; (3) the increments are not additive and could involve receptors coming from the same non-measured pool; (4) the receptor increase during a short time of stress could be explained by recruitment of receptors but not by an increase in the receptor protein biosynthesis; (5) stress induces a maximal recruitment of measurable [-3H]Ro 5-4864 receptors.
Assuntos
Benzodiazepinonas/metabolismo , Octoxinol/farmacologia , Prosencéfalo/metabolismo , Receptores de GABA-A/metabolismo , Estresse Psicológico/metabolismo , Animais , Membrana Celular/metabolismo , Galinhas , Convulsivantes/metabolismo , Cinética , Ensaio Radioligante , Receptores de GABA-A/efeitos dos fármacos , Valores de Referência , Natação , TrítioRESUMO
The effects of Ca2+ ions on 3H-RO 5-4864 binding to the peripheral benzodiazepine receptor were examined. Preincubation of rat kidney membranes with Ca2+ at 37 degrees C produced a dose-dependent inhibition of 3H-RO 5-4864 binding. No inhibition was observed in membranes preincubated at 0 degrees C. The effect of Ca2+ was competitive in nature and was fully reversed by the addition of EGTA. At 1 mM, the maximal effect was achieved with CaCl2, whereas CoCl2 and CdCl2 had lesser effects. No other divalent cation salts examined decreased 3H-RO 5-4864 binding to rat kidney membranes. Collectively, these data demonstrate that the affinity of 3H-RO 5-4864 binding to rat kidney membranes is regulated by Ca2+ and suggest the presence of cation recognition binding sites coupled to the peripheral benzodiazepine receptor.
Assuntos
Cálcio/farmacologia , Rim/química , Receptores de GABA-A/efeitos dos fármacos , Animais , Benzodiazepinonas/metabolismo , Sítios de Ligação , Relação Dose-Resposta a Droga , Masculino , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/metabolismoRESUMO
The binding of 3H-RO 5-4864 to the peripheral-type benzodiazepine receptors (PBZDR) in rat testicular interstitial cells (TIC) was characterized. The binding was saturable, reversible and showed a single high-affinity (Kd = 5.02 +/- 0.86 nM) class of binding sites. The maximal binding capacity (Bmax) in crude mitochondrial fractions (77.6 +/- 9.1 pmol/mg protein) represents the highest density of PBZDR in tissues thus far studied. In comparison with the crude mitochondrial fraction the subcellular fractionation of TIC revealed a 2-fold enrichment of 3H-RO 5-4864 binding sites to the purified mitochondria (Bmax = 140 +/- 23 pmol/mg protein). The ability of various drugs to displace 3H-RO 5-4864 from TIC binding sites was examined and the inhibition constants (Ki) for RO 5-4864, PK 11195, diazepam and flunitrazepam were 3.5, 4.4, 159, and 353 nM, respectively, whereas clonazepam and RO 15-1788 were inefficient in displacing 3H-RO 5-4864 (Ki greater than 10 microM). This pharmacological profile is characteristic of PBZDR described in other tissues. In conclusion, rat TIC possess a very high concentration of PBZDR primarily associated with mitochondrial membranes.
Assuntos
Células Intersticiais do Testículo/ultraestrutura , Mitocôndrias/metabolismo , Receptores de GABA-A/metabolismo , Animais , Benzodiazepinonas/metabolismo , Ligação Competitiva , Diazepam/metabolismo , Flunitrazepam/metabolismo , Membranas Intracelulares/metabolismo , Isoquinolinas/metabolismo , Cinética , Masculino , Ratos , Ratos EndogâmicosRESUMO
The in vivo and in vitro modulation of central benzodiazepine binding sites (BDZ-R) by phosphatidylserine purified from bovine cerebral cortex (BC-PS) was studied. Five days i.p. administration of 15 mg/kg/day of BC-PS liposomes increased the maximal number of binding sites (Bmax) for [3H]flunitrazepam in cerebral cortical membranes. In contrast, the density of hippocampal benzodiazepine recognition binding sites decreased. In cerebellar membranes, BC-PS treatment did not alter the characteristics of [3H]flunitrazepam binding. Similar experiments using phosphatidylcholine extracted from bovine brain (BC-PC) resulted in no changes in the [3H]flunitrazepam binding in the 3 neural structures studied. Confirming previous results, rats submitted to an acute swimming stress showed a decrease in the density of cerebral cortex BDZ-R. Animals treated with BC-PS liposomes before stress showed cortical [3H]flunitrazepam binding significantly below treated, unstressed animals but not below controls. The effects of BC-PS liposomes appeared to be selective for the central type of BDZ-R since no changes were observed in [3H]RO 5-4864 binding, a radioligand specific for the peripheral type BDZ-R. Preincubation of cerebral cortical and cerebellar synaptosomal membranes with BC-PS liposomes (1-300 micrograms per assay) significantly increased in a concentration-dependent manner (up to 100 micrograms) the [3H]flunitrazepam binding. Scatchard analysis revealed changes in the apparent affinity without alterations in the Bmax. Very similar results were obtained using a purified PS from spinal cord. BC-PC, phosphatidylinositol, phosphatidic acid and the lyso derivatives of PS and PC (lysoPS and lysoPC) were found to be ineffective.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Encéfalo/metabolismo , Fosfatidilserinas/farmacologia , Receptores de GABA-A/metabolismo , Animais , Benzodiazepinonas/metabolismo , Bovinos , Membrana Celular/metabolismo , Cerebelo/metabolismo , Córtex Cerebral/metabolismo , Convulsivantes/metabolismo , Flunitrazepam/metabolismo , Hipocampo/metabolismo , Cinética , Lipossomos , Ratos , Ratos Endogâmicos , Receptores de GABA-A/efeitos dos fármacos , Valores de ReferênciaRESUMO
The presence of both MAO and benzodiazepine (BZ) receptor binding inhibitory activities in rat tissues has been reported previously. The two activities were similarly and unevenly distributed in the tissues. This dual inhibitory activity has been termed tribulin. We report here the effect of 1 1/2 hrs cold restraint stress on tribulin activity in rat tissues together with biochemical evidence to support the concept of a physiological role of tribulin. Stress induced a significant increment of both activities in heart and kidney while no significant changes were observed in the other tissues studied. Hearts and kidneys from stressed rats also showed a significant decrease of MAO activity, a significant increase of dopamine content and a significant decrease of the binding of 3H-Ro 5-4864 to peripheral BZ receptors. Scatchard analysis of the saturation curves carried out using 3H-Ro 5-4864 (0.4-10 nM) showed significant Bmax decreases in both organs. No significant change in either of these inhibitory activities was observed in the other tissues studied. These data provide support for a role of tribulin in the biochemical response to stress.