Relationship between levels of mindfulness and substance use pattern / Relação entre níveis de mindfulness e padrão do uso de substâncias

OBJECTIVE: Evaluate the association between levels of mindfulness and sociodemographic characteristics and pattern of drug use of individuals seeking treatment in a University Service Specialized in Substance Use Disorders. METHODS: This is a cross-sectional study with 164 individuals over 18 years of age seeking treatment for the use of psychoactive substances in the June 2018-December 2019 period, using a questionnaire for sociodemographic data, the Mindful Attention Awareness Scale (MAAS) self- -reporting instrument, and the Alcohol, Smoking, and Substance Involvement Screening Test. RESULTS: An association was found between low levels of mindfulness mainly with the individual risk of being a medium/high-risk user of sedative-hypnotic drugs (p = 0.020). A borderline association was also found between MAAS and the risk of the individual being a medium/high risk of alcohol (p = 0.053) and with a more severe pattern of substance use (p = 0.065). CONCLUSION: Individuals seeking treatment for substance use presented impairments in the attentional aspect of mindfulness and levels of mindfulness seem to protect against behaviors related to substance use, especially against the use of high/ moderate risk of sedative-hypnotics.

OBJETIVO: Avaliar a associação entre níveis de mindfulness e características sociodemográficas e padrão do uso de drogas de indivíduos que buscam tratamento em Serviço Universitário Especializado em Transtorno por Uso de Substâncias. MÉTODOS: Estudo de corte transversal de 164 indivíduos acima de 18 anos que buscavam tratamento para uso de substâncias psicoativas no período de junho de 2018 a dezembro de 2019, utilizando questionário para dados sociodemográficos, o instrumento de autorrelato Mindful Attention Awareness Scale (MAAS) e o Alcohol, Smoking and Substance Involvement Screening Test. RESULTADOS: Foi encontrada associação entre baixos níveis de mindfulness principalmente com o risco de o indivíduo ser usuário de médio/alto risco de sedativos-hipnóticos (p = 0,020). Também foi encontrada associação limítrofe entre MAAS com risco de o indivíduo ser usuário de médio/alto risco de álcool (p = 0,053) e com padrão mais grave de uso de substâncias (p = 0,065). CONCLUSÃO: Indivíduos que buscavam tratamento para uso de substâncias apresentaram prejuízos no aspecto atencional de mindfulness, e níveis de mindfulness parecem proteger contra comportamentos relacionados ao uso de substâncias, principalmente contra o uso de alto/moderado risco de sedativos-hipnóticos.

4'-Chlorodiazepam Protects Mitochondria in T98G Astrocyte Cell Line from Glucose Deprivation.

The translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor (PBR), is considered an important regulator of steroidogenesis and a potential therapeutic target in neurological disorders. Previous evidence suggests that TSPO ligands can protect cells during injury and prevent apoptosis in central nervous system (CNS) cells. However, its actions on astrocytic cells under metabolic injury are not well understood. In this study, we explored whether 4'-chlorodiazepam (Ro5-4864), a TSPO ligand, might protect astrocyte mitochondria under glucose deprivation. Our results showed that 4'-chlorodiazepam preserved cell viability and reduced nuclear fragmentation in glucose-deprived cells. These effects were accompanied by a reduced production of free radicals and maintenance of mitochondrial functions in cells treated with 4'-chlorodiazepam. Finally, our findings suggest that TSPO might be involved in reducing oxidative stress by preserving mitochondrial functions in astrocytic cells exposed to glucose withdrawal.

The perceptions of medical researchers on qualitative methodologies / Percepción de los investigadores médicos en metodologías cualitativas / Percepção de pesquisadores médicos sobre metodologias qualitativas

We aimed to verify doctor's perception of the qualitative research method, via a qualitative study of interviews with questions on the academic profile of doctors and on the methodology. We interviewed 42 professionals, of which 18 had experience with the qualitative method and 24 with the quantitative method. The results showed that knowledge on the qualitative method was virtually nil among "quantitative researchers", who did not value qualitative research, although some of those realized that it would be important to be more accepting in clinical practice. Others only considered the method as subsidiary to quantitative. The majority considered qualitative methods as lacking academic structure, taking too long to conduct empirical studies, and being difficult to publish. All of them criticized the misuse of the method, and the "quantitatives" pointed out the problem of being unable to reproduce. We concluded that widening the use of the qualitative method by doctors requires investment from the beginning of the academic career and participation in qualitative research projects.

El objetivo es verificar la percepción de médicos sobre el método de investigación cualitativa. Se trata de un estudio cualitativo por medio de entrevistas con preguntas sobre el perfil de los médicos y sobre el método. Entrevistamos a 42 profesionales, 18 con experiencia en el método cualitativo y 24 con el cuantitativo. Los resultados mostraron que el conocimiento sobre lo cualitativo es casi nulo entre los "cuantitativistas", que no valoran la investigación cualitativa, aunque algunos se dan cuenta de que sería importante tener un enfoque más amplio en la práctica clínica. Otros la ven como subsidiaria a lo cuantitativo. Sus dificultades para utilizar ese abordaje son: falta de formación, cantidad de tiempo que exigen y problemas de publicación. Todos han criticado el mal uso del método. Los "cuantitativistas" han destacado como fragilidad, la no reproductibilidad. Llegamos a la conclusión de que para ampliar el uso de los abordajes cualitativos entre los médicos es importante invertir en su formación desde el inicio del curso y la participación en proyectos de investigación cualitativa.

Objetivamos verificar a percepção de médicos sobre o método qualitativo de pesquisa. Estudo qualitativo por meio de entrevistas com questões sobre o perfil acadêmico do médico e perguntas abertas a respeito do método. Entrevistamos 42 profissionais, sendo 18 com experiência no método qualitativo e 24 com o quantitativo. Os resultados evidenciaram que o conhecimento sobre o qualitativo é quase nulo entre os pesquisadores "quantitativistas", os quais não valorizam a pesquisa qualitativa, embora alguns percebam que seria importante ter uma postura mais compreensiva na prática clínica. Outros só a veem como subsidiária ao quantitativo. As principais dificuldades da maioria são: falta de formação, tempo longo despendido nos estudos empíricos e dificuldade de publicação. Todos os entrevistados criticaram o mau uso do método, e os "quantitativistas" ressaltaram, como problema, sua não reprodutibilidade. Concluímos que ampliar o uso do método qualitativo por médicos exige investimento na formação desde o início da graduação e participação em projetos de pesquisa qualitativa.

Synthesis, biological evaluation, and structure-activity relationship of clonazepam, meclonazepam, and 1,4-benzodiazepine compounds with schistosomicidal activity.

The inherent morbidity and mortality caused by schistosomiasis is a serious public health problem in developing countries. Praziquantel is the only drug in therapeutic use, leading to a permanent risk of parasite resistance. In search for new schistosomicidal drugs, meclonazepam, the 3-methyl-derivative of clonazepam, is still considered an interesting lead-candidate because it has a proven schistosomicidal effect in humans but adverse effects on the central nervous system did not allow its clinical use. Herein, the synthesis, in vitro biological evaluation, and molecular modeling of clonazepam, meclonazepam, and analogues are reported to establish the first structure-activity relationship for schistosomicidal benzodiazepines. Our findings indicate that the amide moiety [N(1) H-C(2) (=O)] is the principal pharmacophoric unit of 1,4-benzodiazepine schistosomicidal compounds and that substitution on the amide nitrogen atom (N(1) position) is not tolerated.

Actions of translocator protein ligands on neutrophil adhesion and motility induced by G-protein coupled receptor signaling.

The 18 kDa translocator protein (TSPO) also known as the peripheral benzodiazepine receptor (PBR), mediates the transportation of cholesterol and anions from the outer to the inner mitochondrial membrane in different cells types. Although recent evidences indicate a potential role for TSPO in the development of inflammatory processes, the mechanisms involved have not been elucidated. The present study investigated the ability of the specific TSPO ligands, the isoquinoline carboxamide PK11195 and benzodiazepine Ro5-4864, on neutrophil recruitment promoted by the N-formylmethionyl-leucyl-phenylalanine peptide (fMLP), an agonist of G-protein coupled receptor (GPCR). Pre-treatment with Ro5-4864 abrograted fMLP-induced leukocyte-endothelial interactions in mesenteric postcapillary venules in vivo. Moreover, in vitro Ro5-4864 treatment prevented fMLP-induced: (i) L-selectin shedding and overexpression of PECAM-1 on the neutrophil cell surface; (ii) neutrophil chemotaxis and (iii) enhancement of intracellular calcium cations (iCa(+2)). Intriguingly, the two latter effects were augmented by cell treatment with PK11195. An allosteric agonist/antagonist relation may be suggested, as the effects of Ro5-4864 on fMLP-stimulated neutrophils were reverted by simultaneous treatment with PK11195. Taken together, these data highlight TSPO as a modulator of pathways of neutrophil adhesion and locomotion induced by GPCR, connecting TSPO actions and the onset of an innate inflammatory response.

The effects of 3-methylclonazepam on Schistosoma mansoni musculature are not mediated by benzodiazepine receptors.

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.

Steroidogenesis in BeWo cells: role of protein kinase A and benzodiazepines.

The peripheral benzodiazepine receptor and protein kinase A have been proposed to modulate placental steroidogenesis. Binding of the radioactive benzodiazepine PK 11195 has been observed in membranes isolated from whole human placenta, but the presence of the peripheral benzodiazepine receptors, now called translocator protein, does not seem to be indispensable. We hypothesized that cAMP analogs could induce the translocator protein expression in BeWo cells increasing steroidogenesis in the presence of benzodiazepines. The effect of two benzodiazepines and of 8-Br-cAMP on steroidogenesis in BeWo cells or in isolated human placental mitochondria was studied. Benzodiazepines did not modify progesterone synthesis in either system. Progesterone increased three times in BeWo cells incubated in the presence of 8-Br-cAMP. The translocator protein was not identified by western blot in mitochondria isolated from either the human placenta or BeWo cells but it was present in isolated rat testicular mitochondria. Neither was it observed in isolated mitochondria from BeWo cells incubated with 8-Br-cAMP. An inhibitor of protein kinase A activity, H89, at 25 microM inhibited 90% the steroidogenesis in BeWo cells, even in the presence of 8-Br-cAMP, but protein phosphorylation in mitochondria increased in the presence of H89, suggesting that protein kinase A modulates the phosphorylation cycle of mitochondrial proteins. The results suggest that placental steroidogenesis is regulated via activation of protein kinase A modulated by cAMP.

Effects of peripheral-type benzodiazepine receptor ligands on Ehrlich tumor cell proliferation.

Peripheral-type benzodiazepine receptors have been found throughout the body, and particularly, in high numbers, in neoplastic tissues such as the ovary, liver, colon, breast, prostate and brain cancer. Peripheral-type benzodiazepine receptor expression has been associated with tumor malignity, and its subcellular localization is important to define its function in tumor cells. We investigated the presence of peripheral-type benzodiazepine receptors in Ehrlich tumor cells, and the in vitro effects of peripheral-type benzodiazepine receptors ligands on tumor cell proliferation. Our results demonstrate the presence of peripheral-type benzodiazepine receptor in the nucleus of Ehrlich tumor cells (85.53+/-12.60%). They also show that diazepam and Ro5-4864 (peripheral-type benzodiazepine receptor agonists) but not clonazepam (a molecule with low affinity for the peripheral-type benzodiazepine receptor) decreased the percentage of tumor cells in G0-G1 phases and increased that of cells in S-G2-M phases. The effects of those agonists were prevented by PK11195 (a peripheral-type benzodiazepine receptor antagonist) that did not produce effects by itself. Altogether, these data suggest that the presence of peripheral-type benzodiazepine receptor within the nucleus of Ehrlich tumor cells is associated with tumor malignity and proliferation capacity.

Antinociceptive effects of peripheral benzodiazepine receptors.

Pretreatment of mice with Ro5-4864 or PK11195 inhibited the first- and second-phase responses in the formalin test and this effect was significantly reversed by aminoglutethimide, an inhibitor of pregnenolone synthesis, suggesting that the antinociceptive effect of the peripheral-type benzodiazepine receptor ligands is dependent on steroid formation. Doses of Ro5-4864 that did not produce an antinociceptive effect when injected by the intraperitoneal route presented an analgesic effect, if infected by the intracerebroventricular, intrathecal or intraplantar routes. PK11195 pretreatments with intrathecal, intracerebroventricular or intraplantar doses had no effect in the formalin test. These results suggest that the antinociceptive effect of Ro5-4864 reflects the influence of this ligand on steroid production not only in many nonneuronal peripheral tissues but also in the nervous system, while the antinociceptive action of PK11195 may be due to the stimulation of steroid synthesis only in nonnervous tissues.

Involvement of steroids in anti-inflammatory effects of peripheral benzodiazepine receptor ligands.

Mouse paw oedema induced by carrageenan is used to determine if glucocorticoids are involved in the anti-inflammatory effects of peripheral benzodiazepine receptor ligands. The anti-inflammatory responses elicited by i.p. treatment with 1-(2-chlorophenyl)-N-methyl-N (1-methyl-propyl)-3-isoquinoline carboxamide (PK11195) and 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2-H-1, 4-benzodiazepin-2 (Ro5-4864) were reversed by aminoglutethimide, an inhibitor of steroidal synthesis. Intraplantar injection into the ipsilateral paw of Ro5-4864, but not PK11195, inhibited the formation of paw oedema and this effect was reversed by aminoglutethimide. These results suggest that glucocorticoids are involved in the systemic and local anti-inflammatory effects of Ro5-4864 and only in the systemic response to PK11195.

Effects of acute and long-term diazepam administrations on neutrophil activity: a flow cytometric study.

This study analyzed the effects of acute and long-term diazepam treatments on rat peripheral blood neutrophil activity and cortisol serum levels. Rats were acutely and long-term (21 days, once daily) treated with diazepam (10 mg/kg) or its vehicle (1.0 ml/kg). Blood was collected 1 h after treatments for flow cytometric analysis of neutrophil oxidative burst and phagocytosis. Corticosterone and diazepam concentrations were also determined. Results showed that: (1) both diazepam treatments increased lipopolysaccharide (LPS) and phorbol myristate acetate (PMA)-induced neutrophil oxidative burst; (2) the increase in oxidative burst after Staphylococcus aureus induction in acutely treated animals was higher than that observed after long-term treatment; (3) phagocytosis is increased by acute diazepam treatment and decreased by a long-term regimen; (4) acute, but not long-term, diazepam treatment increased corticosterone levels; (5) diazepam plasmatic levels after acute and long-term treatments were not different. These results indicate the development of tolerance to diazepam effects on corticosterone serum levels but not on neutrophil activity.

Alteration of CCK-induced satiety in post-Nippostrongylus brasiliensis-infected rats.

In rats, the nematode Nippostrongylus brasiliensis induces an intestinal inflammation, but after the inflammation had resolved and the worm burden eliminated, morphological alterations of the intestinal wall, mainly consisting of mast cell hyperplasia and enteric nerve remodeling, persist for several weeks. Intestinal signals reaching the brain through the vagus nerve and involving neuropeptides such as CCK, play a role in the control of food intake. Our hypothesis was that neuroimmune alterations of the intestine may alter this control. This work was aimed to evaluate whether post-infection alterations of the intestinal wall may affect the satiety effects of CCK and further, the role of mast cells and their mediators, histamine and serotonin, in post-N. brasiliensis-infected rats. In basal conditions, food intake was not different in control and post-infected groups of rats. Post-infected rats were characterized by prolonged satiety effects of both CCK and histamine but not serotonin. The prolonged effect of CCK was reduced when mast cells were previously stabilized by ketotifen, which had no effect per se on food intake. No difference was observed in the increase of food intake induced by CCK-A and CCK-B receptor antagonists in both control and post-infected rats. Mast cell degranulation with compound 48/80 induced severe anorectic effects that lasted for less than 24h in post-infected rats and as long as 6 days in controls. Thus, in our experimental conditions, i.e., within 30-50 days post-N. brasiliensis infection, we observed an enhancement of the anorectic effect of exogenous CCK involving mast cell degranulation and histamine.

Reduction of acute inflammation in rats by diazepam: role of peripheral benzodiazepine receptors and corticosterone.

Carrageenin causes a reproducible inflammatory reaction and remains the standard irritant for examining acute inflammation and anti-inflammatory drugs. High doses of diazepam (10.0-20.0 mg/Kg) were shown to reduce the volume of acute inflammatory paw edema in rats as a response to carrageenin administration. The present experiment was undertaken to investigate the possible roles of peripheral-type benzodiazepine receptors (PBRs) and corticosterone on the anti-inflammatory effects of diazepam. Five experiments were conducted to assess the effects of a single dose (10.0 mg/Kg) of diazepam on carrageenin-induced paw edema (CIPE), pleurisy and increase in vascular permeability in rats. Results showed that: 1. diazepam or Ro5-4864 (a PBR agonist) treatment reduced CIPE values; 2. prior treatment with PK11195 (a non-benzodiazepine PBR antagonist) suppressed the effects of either diazepam or Ro5-4864 on CIPE; 3. diazepam reduced the volume of the pleural exudate in carrageenin-injected rats, as well as its leukocyte count; 4. diazepam treatment reduced the magnitude of the increase in vascular permeability caused by carrageenin; 5. adrenalectomy suppressed the effects of diazepam on CIPE; and 6. diazepam treatment increased the serum concentration of corticosterone. These results suggest a relevant role of PBR and corticosterone on diazepam-induced changes in inflammation. They are discussed in the light of a possible activation of mitochondrial PBRs within the adrenal gland cells by diazepam, thereby increasing the serum levels of corticosterone and thus reducing CIPE.

Anti-inflammatory effects of peripheral benzodiazepine receptor ligands in two mouse models of inflammation.

In vivo treatment of mice with peripheral benzodiazepine receptor ligands exerts an inhibitory effect on the inflammatory response in two models of acute inflammation. In the first model, pretreatment of the animals (24 h) with 1-(2-chlorophenyl)-N-methyl-N(1-methylpropyl)-3-isoquinoline carboxamide (PK11195) and 7-chloro-5-(4-Chlorophenyl)-1, 3-dihydro-1-methyl-2-H-1,4-benzodiazepin-2 (Ro5-4864), at different doses (0.00001-10 mg/kg, i.p.) dose dependently inhibited the formation of mouse paw oedema induced by carrageenan with mean ID(50s) of 0.009 (95% confidence limits=0.0076-0.013) and 0.04 (95% confidence limits=0.025-0.0086) mg/kg, respectively. Both ligands (0. 1 mg/kg, i.p.) inhibited in the same way the mouse paw oedema induced by carrageenan in animals with and without adrenal glands. PK11195 and Ro5-4864 (0.1 mg/kg, i.p.) inhibited the mouse paw oedema induced by several inflammatory mediators. In the second model, the pretreatment (24 h) with peripheral benzodiazepine receptor ligands (0.1 mg/kg, i.p.) exerted an inhibitory effect on neutrophil influx and produce a marked inhibition of carrageenan-produced interleukin-13 and interleukin-6 in pleural exudation. Our results extend previous findings that peripheral benzodiazepine receptor is involved in the inflammatory response, and suggest that this action may be linked to the action of different inflammatory mediators, probably mainly by the inhibition of the release of pro-inflammatory cytokines.

Opposite modulation of apomorphine- or amphetamine-induced stereotypy by antagonists of CCK receptors.

Stereotyped behavior is elicited by activation of dopaminergic systems with drugs such as apomorphine and amphetamine. In previous studies, we have reported that the sulfated cholecystokinin octapeptide (CCK-8) decreased apomorphine-induced stereotypy in animals with normal and supersensitive dopamine receptors. The aim of the present study was to evaluate the effects of CCK(1) and CCK(2) receptor antagonists on stereotyped behavior induced by apomorphine or amphetamine. Rats were pretreated with the CCK(1) (SR 27897B; 1-[[2-(4-(2-chlorophenyl) thiazol-2-yl) aminocarbonyl]indolyl]acetic acid; 500 microg/kg; i.p.) or CCK(2) (L-365,260; 3R-(+)-N-(2,3-dihydro-1-methyl-2-oxo-5 phenyl-1H-1, 4-benzodiazepine-3-yl)-N'-(3-methyl phenyl)-urea; 500 microg/kg; i.p. ) receptor antagonists or saline 15 min before apomorphine (0.6 mg/kg; s.c.) or amphetamine (9.0 mg/kg; i.p.) injection. Both CCK(1) and CCK(2) receptor antagonists significantly increased apomorphine-induced stereotypy. In contrast, only the blockade of CCK(2) receptors significantly decreased amphetamine-induced stereotypy. The results suggest a dual opposite mechanism for CCK-dopamine interactions. These data also suggest that both apomorphine- and amphetamine-induced stereotypy should be used whenever effects of drugs acting on dopaminergic systems are being assessed.

Potential role of peripheral benzodiazepine receptors in inflammatory responses.

This study investigates the anti-inflammatory effects of 1-(2-chlorophenyl)-N-methyl-N(1-methylpropyl)-3-isoquinoline carboxamide and 7-chloro-5-(4-chlorophenyl)-1, 3-dihydro-1-methyl-2-H-1,4-benzodiazepin-2-one in paw oedema induced by carrageenan in mice. Pretreatment (24 h) with both ligands inhibited oedema formation in at different doses (0.00001-10 mg/kg, i.p.) with range of inhibition of 25% to 70%, in animals with or without adrenal glands. These results demonstrate, for the first time, an in vivo anti-inflammatory property of peripheral benzodiazepine receptor ligands.

Differential role of cholecystokinin receptor subtypes in opioid modulation of ongoing maternal behavior.

Cholecystokinin (CCK) can have effects opposite those of opioids. The present study was undertaken to determine whether peripheral injections of antagonists of the CCK1 receptor (lorglumide) and the CCK2 receptor (L-365,260) can influence the effects of morphine on maternal behavior during lactation. A total of 110 female Wistar rats were tested on days 5 and 6 postpartum. Groups were randomly assigned to morphine vehicle (MV-SC) + saline (S-IP), MV + lorglumide (LOR: 1.0 or 10.0 mg/kg), MV + L-365,260 (10 mg/kg), morphine chlorhydrate (MC: 7.0 mg/kg) + S, MC + LOR (1.0 or 10.0 mg/kg), and MC + L-365,260 (1.0 or 10 mg/kg). Maternal behavior testing was started 30 min after the injections, at which time pups were placed in the home cage of their mother. Latencies for retrieval, grouping, and crouching responses were scored. The results show that both lorglumide and L-365,260 potentiated the MC-induced inhibition of maternal behavior. In addition L-365,260 treatment alone inhibited maternal behavior. Blockade of both the CCK1 and CCK2 receptors potentiated the morphine-induced disruption of maternal behavior, while CCK2 antagonism alone also inhibited this behavior. The results suggest that CCK antagonism of opioid-induced disruption of maternal behavior occurs due to the action of CCK on both CCK1 and CCK2 receptor subtypes.

Inhibitory effects of diazepam in rat vas deferens: role of calcium.

Diazepam and Ro5-4864 effects on noradrenaline-induced rat vas deferens contractions were studied. We investigated whether central or peripheral type benzodiazepine receptors were involved, by studying the effects of the selective central or peripheral benzodiazepine receptor antagonists, flumazenil (Ro 151788) or PK 11195 respectively. Diazepam interactions with GABA, adenosine, theophylline, and hypercalcic medium (3.5 mM) were studied. Also, we investigate diazepam effect on KCl depolarized vas deferens. Results showed that diazepam (10(-4) to 1.7 x 10 (-4) M) and Ro 5-4864 (10(-5) to 5.5 x 10(-5) M) inhibited NA-induced vas deferens contractions and that neither flumazenil nor PK 11195 antagonized diazepam or Ro 5-4864 inhibitory effects respectively. GABA, adenosine and theophylline did not modify neither NA vas deferens response nor diazepam inhibitory action. Diazepam effect was significantly reduced in and 3.5 mM calcium medium and KCl vas deferens response was inhibited by diazepam 1.3 x 10(-5) and 1.3 x 10(-4) M. It is concluded that in rat vas deferens diazepam effect seems to be related with calcium mobilization.