Glycosylation of ß1 subunit plays a pivotal role in the toxin sensitivity and activation of BK channels

The accessory ß1 subunits, regulating the pharmacological and biophysical properties of BK channels, always undergo post-translational modifications, especially glycosylation. To date, it remains elusive whether the glycosylation contributes to the regulation of BK channels by ß1 subunits. Methods: Herein, we combined the electrophysiological approach with molecular mutations and biochemical manipulation to investigate the function roles of N-glycosylation in ß1 subunits. Results: The results show that deglycosylation of ß1 subunits through double-site mutations (ß1 N80A/N142A or ß1 N80Q/N142Q) could significantly increase the inhibitory potency of iberiotoxin, a specific BK channel blocker. The deglycosylated channels also have a different sensitivity to martentoxin, another BK channel modulator with some remarkable effects as reported before. On the contrary to enhancing effects of martentoxin on glycosylated BK channels under the presence of cytoplasmic Ca2+, deglycosylated channels were not affected by the toxin. However, the deglycosylated channels were surprisingly inhibited by martentoxin under the absence of cytoplasmic Ca2+, while the glycosylated channels were not inhibited under this same condition. In addition, wild type BK (α+ß1) channels treated with PNGase F also showed the same trend of pharmacological results to the mutants. Similar to this modulation of glycosylation on BK channel pharmacology, the deglycosylated forms of the channels were activated at a faster speed than the glycosylated ones. However, the V1/2 and slope were not changed by the glycosylation. Conclusion: The present study reveals that glycosylation is an indispensable determinant of the modulation of ß1-subunit on BK channel pharmacology and its activation. The loss of glycosylation of ß1 subunits could lead to the dysfunction of BK channel, resulting in a pathological state.(AU)

Glycosylation of ß1 subunit plays a pivotal role in the toxin sensitivity and activation of BK channels

The accessory ß1 subunits, regulating the pharmacological and biophysical properties of BK channels, always undergo post-translational modifications, especially glycosylation. To date, it remains elusive whether the glycosylation contributes to the regulation of BK channels by ß1 subunits. Methods: Herein, we combined the electrophysiological approach with molecular mutations and biochemical manipulation to investigate the function roles of N-glycosylation in ß1 subunits. Results: The results show that deglycosylation of ß1 subunits through double-site mutations (ß1 N80A/N142A or ß1 N80Q/N142Q) could significantly increase the inhibitory potency of iberiotoxin, a specific BK channel blocker. The deglycosylated channels also have a different sensitivity to martentoxin, another BK channel modulator with some remarkable effects as reported before. On the contrary to enhancing effects of martentoxin on glycosylated BK channels under the presence of cytoplasmic Ca2+, deglycosylated channels were not affected by the toxin. However, the deglycosylated channels were surprisingly inhibited by martentoxin under the absence of cytoplasmic Ca2+, while the glycosylated channels were not inhibited under this same condition. In addition, wild type BK (α+ß1) channels treated with PNGase F also showed the same trend of pharmacological results to the mutants. Similar to this modulation of glycosylation on BK channel pharmacology, the deglycosylated forms of the channels were activated at a faster speed than the glycosylated ones. However, the V1/2 and slope were not changed by the glycosylation. Conclusion: The present study reveals that glycosylation is an indispensable determinant of the modulation of ß1-subunit on BK channel pharmacology and its activation. The loss of glycosylation of ß1 subunits could lead to the dysfunction of BK channel, resulting in a pathological state.(AU)

Armazenamento de sêmen suíno líquido a 5ºC e criopreservação alteram o mecanismo de influxo de cálcio espermático.

A inseminação artificial tem grande relevância na suinocultura uma vez que propicia vários benefícios relacionados ao incremento genético. No entanto, o fato das doses inseminantes serem conservadas a 15-18°C traz uma série de inconvenientes, como sua logística de distribuição. Sendo assim, o interesse no desenvolvimento de protocolos de criopreservação do sêmen suíno tem atraido maior atenção. O espermatozoide suíno, devido a sua constituição da membrana plasmática é muito sensível às variações de temperatura abaixo de 15°C, bem como ao congelamento. Apesar de não ser claro, o mecanismo da maior sensibilidade celular às baixas temperaturas pode estar associado a características de membrana plasmática sendo possível que o metabolismo do Ca2+, possa estar envolvido. A hipótese deste estudo assume que a criopreservação afeta o funcionamento correto dos canais de Ca2+, prejudicando dessa forma, os índices de fertilidade na espécie suína. O objetivo deste trabalho foi avaliar os efeitos do resfriamento a 15°C, 5°C e da criopreservação sobre o influxo de Ca2+ em populações de espermatozoides suínos. O sêmen foi coletado de cinco reprodutores, processado em heterospermia. Os grupos experimentais foram compostos por: R0) sêmen resfriado a 15-18°C, avaliado no dia da coleta; R5) sêmen resfriado a 5°C, avaliado 24 h após a coleta; R15) sêmen resfriado a 15-18°C, avaliado 24 h após a coleta e Crio) sêmen criopreservado. A avaliação funcional dos espermatozoides em cada tratamento foi realizada por citometria de fluxo, utilizando os seguintes ensaios: a) viabilidade celular (iodeto de propídio (PI)); b) potencial de membrana mitocondrial (Rodamina123); c) concentração intracelular de Ca2+ (Fluo- 4AM) e; d) atividade de canais CatSper, utilizando um antagonista de canais de Ca2+ tipo T (NNC-55-0396). Os espermatozoides de todos os grupos foram incubados a 37°C em meio capacitante por uma hora antes da preparação das amostras para citometria. Os experimentos foram repetidos cinco vezes, analisando 10.000 espermatozoides (eventos) por ensaio. Análise de motilidade e vigor espermático de todas as amostras foi realizada anteriormente a avaliação citométrica. As diferenças foram consideradas significativas a 5%. A motilidade do sêmen descongelado (Crio) foi de 55%, diferindo dos grupos R0 (86,7%), R15 (83,3%) e R5 (81,7%). O vigor espermático dos grupos R0 (3,8), R15 (3,7) e R5 (3,5) foi superior ao do grupo Crio (3,0). Células positivas para PI nos grupos R0 (8,2), R15 (9,4%) e R5 (5,3) tiveram mesmo desempenho estatístico, entretanto foram diferentes do grupo Crio, com 29,9% de células PI-positivas. A atividade mitocondrial nos grupos R0 (98,4%) e R15 (93,9%) diferiu do grupo R5 (71,5%), mas não foi diferente do grupo Crio (85,3%). Entretanto, o grupo Crio teve atividade mitocondrial igual ao grupo R5. O influxo de Ca2+ no grupo R5 (58,8%) foi menor quando comparado aos demais grupos (98% para R0, 96,2% para R15 e 83,6% para Crio). O uso de um inibidor de CatSper não teve efeito no influxo de Ca2+ nos grupos R0 (91,3%), R5 (53,1%) e R15 (89,7%), entretanto reduziu a entrada deste íon quando o sêmen que foi criopreservado e descongelado (60,0%). Os dados encontrados sugerem que, apesar de não ser claro como o congelamento altera o efeito do composto NNC sobre as células criopreservadas, o resfriamento a 5°C e criopreservação alteram o influxo de Ca2+ em espermatozoides suínos.

Artificial insemination is relevant in swine as a tool for genetic improvement. However, the fact that the inseminating doses are conserved at 15-18°C brings limitations such as the transport of the genetic material to long distances and long-term storage. Therefore, the development of protocols for cryopreservation of swine semen has drawn substantial attention. Porcine sperm is very vulnerable to temperature fluctuation, especially those below 15°C, including cryopreservation. Although the mechanism by which low temperatures cause cell damage is not clear, it may be associated with plasma membrane characteristics and it is possible that Ca2+ metabolism may be involved. It is possible that cryopreservation and storage at low temperatures affect the appropriate operation of Ca2+ channels, thus impairing swine fertility. The goal of this work was to evaluate the effects of cooling porcine sperm at 15°C, 5°C and cryopreservation on the influx of Ca2+. The semen was collected from five mature males, pooling the ejaculate of two or three boars (heterospermia) at a time. The experimental groups were composed of: R0) semen cooled to 15-18°C, evaluated on the day of collection; R5) semen cooled to 5°C, evaluated 24 h after collection; R15) semen cooled to 15-18°C, evaluated 24 h after collection and cryo) cryopreserved semen. The functional evaluation of spermatozoa in each treatment was performed by flow cytometry, using the following assays: a) cell viability (propidium iodide (PI)); b) mitochondrial membrane potential (Rodamine123); c) intracellular concentration of Ca2+ (Fluo-4AM) and; d) CatSper channel activity using a T-type Ca2+ channel antagonist (NNC-55-0396). Spermatozoa of all groups were incubated at 37°C in capacitating medium for one hour prior to preparation of the samples for cytometry. The experiments were repeated five times, analyzing 10,000 sperm (events) per assay. Analyses of motility and sperm vigor of all samples was performed prior to the cytometric evaluation. Differences were considered significant if p < 0.05. The motility of thawed semen (Cryo) was 55%, differing from the R0 (86.7%), R15 (83.3%) and R5 (81.7%) groups. Sperm vigor of groups R0 (3.8), R15 (3.7), and R5 (3.5) was higher than that of the Cryo group (3.0). PI-positive cells in R0 (8.2), R15 (9.4%), and R5 (5.3) groups had same statistical performance, but were different from the Crio group, with 29.9% dead cells. Mitochondrial activity in R0 (98.4%) and R15 (93.9%) groups differed from the R5 group (71.5%), which was not different from Crio group (85.3%). However, Cryo and R5 groups had same mitochondrial activity. The proportion of cells displaying high intracellular Ca2+ in the R5 group (58.8%) was decreased when compared to the other groups (98% for R0, 96.2% for R15 and 83.6% for Crio). The use of a CatSper inhibitor had no effect on the Ca2+ influx in the R0 (91.3%), R5 (53.1%) and R15 (89.7%) groups, however, it reduced the entry of this ion when the semen was cryopreserved and thawed (60.0%). While the mechanism by which freezing alters the effect of the NNC compound on cryopreserved cells is not completely clear, these data suggest that cooling at 5°C and cryopreservation alter the influx of Ca2+ in swine spermatozoa.

Vasoconstrictor effect of Africanized honeybee (Apis mellifera L. ) venom on rat aorta

Background : Apis mellifera stings are a problem for public health worldwide, particularly in Latin America due to the aggressiveness of its Africanized honeybees. Massive poisoning by A. mellifera venom (AmV) affects mainly the cardiovascular system, and several works have described its actions on heart muscle. Nevertheless, no work on the pharmacological action mechanisms of the AmV in isolated aorta has been reported. Thus, the present work aimed to investigate the actions of AmV and its main fractions, phospholipase A2 (PLA2) and melittin, on isolated aorta rings and a probable action mechanism. Results : AmV and the complex PLA2 + melittin (0.1-50 μg/mL) caused contraction in endothelium-containing aorta rings, but neither isolated PLA2 nor melittin were able to reproduce the effect. Endothelium removal did not change the maximum vasoconstrictor effect elicited by AmV. Ca2+-free medium, as well as treatment with phentolamine (5 μM), verapamil (10 μM), losartan (100 μM), and U-73122 (10 μM, a phospholipase C inhibitor), eliminated the AmV-induced contractile effects. Conclusions : In conclusion, AmV caused contractile effect in aorta rings probably through the involvement of voltage-operated calcium channels, AT1 and α-adrenergic receptors via the downstream activation of phospholipase C. The protein complex, PLA2 + melittin, was also able to induce vasoconstriction, whereas the isolated proteins were not.(AU)

The effects of a sodium and a calcium channel blocker on lethality of mice injected with the yellow scorpion (Leiurus quinquestriatus) venom

Scorpion venom toxins generally produce similar effects by mainly acting on sodium channels, and to a lesser extent, on potassium, calcium, and chloride channels. This leads to increased release of neurotransmitters and mediators, resulting in a cascade of pathological events, involving the central nervous system, the autonomic nervous system, the cardiovascular and the respiratory system, eventually leading to death. The objective of this paper was to discover whether a sodium channel blocker, lidocaine, or a calcium channel blocker, verapamil, would prolong the survival of mice injected with the venom from the common yellow scorpion Leiurus quinquestriatus quinquestriatus (LQQ). For this purpose, mice were divided into 2 groups, each injected with a different venom dose (250 or 300 µg.kg-1, s.c.). Subgroups (n=10) from each group were given venom alone; different doses of lidocaine (4, 10, 15, or 20 mg.kg-1); or several doses of verapamil (0.01, 0.03, 0.1, 0.3, or 1 mg.kg-1). All doses of lidocaine and verapamil were intravenously administered 3 minutes before, 1, 5, and 15 minutes after venom injection. Percent surviving after 24 hours was recorded in addition to the time of death. In general, lidocaine significantly prolonged survival at the dose of 10 mg.kg-1 (P 0.05 and P 0.01, versus low and high dose of venom, respectively) or 15 mg.kg-1 (P 0.01 and P 0.001, versus low and high dose of venom, respectively; Covariance Wilcoxon survival statistics), especially when injected before the venom or in the early stages of envenomation. On the other hand, in all doses administered, verapamil was either toxic or showed non-significant results. Lidocaine, the sodium channel blocker, appears to play an important role in the protection from lethality of mice injected with LQQ venom, and significantly prolonged the survival time of mice whether injected before or in the early stages of envenomation.

The effects of a sodium and a calcium channel blocker on lethality of mice injected with the yellow scorpion (Leiurus quinquestriatus) venom

Scorpion venom toxins generally produce similar effects by mainly acting on sodium channels, and to a lesser extent, on potassium, calcium, and chloride channels. This leads to increased release of neurotransmitters and mediators, resulting in a cascade of pathological events, involving the central nervous system, the autonomic nervous system, the cardiovascular and the respiratory system, eventually leading to death. The objective of this paper was to discover whether a sodium channel blocker, lidocaine, or a calcium channel blocker, verapamil, would prolong the survival of mice injected with the venom from the common yellow scorpion Leiurus quinquestriatus quinquestriatus (LQQ). For this purpose, mice were divided into 2 groups, each injected with a different venom dose (250 or 300 µg.kg-1, s.c.). Subgroups (n=10) from each group were given venom alone; different doses of lidocaine (4, 10, 15, or 20 mg.kg-1); or several doses of verapamil (0.01, 0.03, 0.1, 0.3, or 1 mg.kg-1). All doses of lidocaine and verapamil were intravenously administered 3 minutes before, 1, 5, and 15 minutes after venom injection. Percent surviving after 24 hours was recorded in addition to the time of death. In general, lidocaine significantly prolonged survival at the dose of 10 mg.kg-1 (P<0.05 and P<0.01, versus low and high dose of venom, respectively) or 15 mg.kg-1 (P<0.01 and P<0.001, versus low and high dose of venom, respectively; Covariance Wilcoxon survival statistics), especially when injected before the venom or in the early stages of envenomation. On the other hand, in all doses administered, verapamil was either toxic or showed non-significant results. Lidocaine, the sodium channel blocker, appears to play an important role in the protection from lethality of mice injected with LQQ venom, and significantly prolonged the survival time of mice whether injected before or in the early stages of envenomation.(AU)

Effect of butanolic fraction of Desmodium adscendens on the anococcygeus of the rat

The chemical composition of plants can vary according to factors such as soil and time of collection. Desmodium adscendens (Sw.) D.C. var. adscendens (Papillionaceae) is a plant employed in the treatment of asthma in Ghana, Africa. Studies have shown that butanolic extract inhibits contraction of the ileum and trachea in guinea pigs. In Mato Grosso, this plant is used only in the treatment of ovarian inflammation.The objective of this work was to verify if the plant found in Mato Grosso also relaxes smooth muscle and to understand better its action.The cumulative application of the butanolic fraction relaxed the contraction maintained in the isolated anococcygeus of a rat, induced by high potassium, but not that induced by phenylephrine. Relaxation was not altered by methylene blue. The butanolic fraction reduced in a concentration-dependent way the maximum response of concentration-response curve to calcium in the anococcygeus muscle. The results suggest that the butanolic fraction acts, at least partly, through the blockade of voltage-sensitive Ca+2 channels.

A composição química das plantas pode variar em função de fatores como solo e época da coleta. Desmodium adscendens (Sw.) D.C. variedade adscendens (Papillionaceae) é uma planta empregada no tratamento da asma em Gana, África. Estudos indicam que o extrato butanólico inibe as contrações do íleo e traquéia de cobaio. Em Mato Grosso, essa planta é utilizada somente no tratamento de inflamação ovariana. O objetivo do trabalho foi verificar se fração butanólica da planta encontrada em Mato Grosso relaxa o músculo liso e compreender melhor seu mecanismo de ação. A aplicação acumulativa da fração butanólica relaxou a contração mantida do anococcígeo de rato induzida por potássio alto, mas não a induzida pela fenilefrina. O relaxamento não foi alterado pelo azul de metileno. Além disso, reduziu a resposta máxima da curva concentração-resposta ao cálcio. Os resultados sugerem que a fração butanólica atua, pelo menos em parte, por intermédio do bloqueio de canais de Ca+2 voltagem dependente.

Effect of butanolic fraction of Desmodium adscendens on the anococcygeus of the rat

The chemical composition of plants can vary according to factors such as soil and time of collection. Desmodium adscendens (Sw.) D.C. var. adscendens (Papillionaceae) is a plant employed in the treatment of asthma in Ghana, Africa. Studies have shown that butanolic extract inhibits contraction of the ileum and trachea in guinea pigs. In Mato Grosso, this plant is used only in the treatment of ovarian inflammation.The objective of this work was to verify if the plant found in Mato Grosso also relaxes smooth muscle and to understand better its action.The cumulative application of the butanolic fraction relaxed the contraction maintained in the isolated anococcygeus of a rat, induced by high potassium, but not that induced by phenylephrine. Relaxation was not altered by methylene blue. The butanolic fraction reduced in a concentration-dependent way the maximum response of concentration-response curve to calcium in the anococcygeus muscle. The results suggest that the butanolic fraction acts, at least partly, through the blockade of voltage-sensitive Ca+2 channels.

A composição química das plantas pode variar em função de fatores como solo e época da coleta. Desmodium adscendens (Sw.) D.C. variedade adscendens (Papillionaceae) é uma planta empregada no tratamento da asma em Gana, África. Estudos indicam que o extrato butanólico inibe as contrações do íleo e traquéia de cobaio. Em Mato Grosso, essa planta é utilizada somente no tratamento de inflamação ovariana. O objetivo do trabalho foi verificar se fração butanólica da planta encontrada em Mato Grosso relaxa o músculo liso e compreender melhor seu mecanismo de ação. A aplicação acumulativa da fração butanólica relaxou a contração mantida do anococcígeo de rato induzida por potássio alto, mas não a induzida pela fenilefrina. O relaxamento não foi alterado pelo azul de metileno. Além disso, reduziu a resposta máxima da curva concentração-resposta ao cálcio. Os resultados sugerem que a fração butanólica atua, pelo menos em parte, por intermédio do bloqueio de canais de Ca+2 voltagem dependente.

Efeitos de benzodiazepínicos sobre a atividade de neutrófilos de ratos avaliados por citometria de fluxo / Effects of benzodiazepines on rat neutrophil activity measured by flow cytometry

Benzodiazepínicos (BDZ) são fármacos ansiolíticos que se caracterizam por atuar em receptores GABAa presentes no sistema nervoso central. Além dos receptores centrais os BDZ também possuem afinidade por sítios ligantes periféricos presentes em vários tecidos, dentre eles nas glândulas adrenais e em células polimorfonucleares. O presente experimento analisou os efeitos de BDZ sobre a atividade de neutrófilos e sobre os níveis séricos de corticosterona de ratos. Especificamente, o burst e a fagocitose de neutrófilos foram estudados após o tratamento ex vivo e in vitro com diazepam, RO 5-4864, clonazepam e/ou PK 11195. Os efeitos do diazepam sobre a atividade de neutrófilos também foram avaliados após o uso prolongado deste fármaco. Finalmente, os efeitos in vitro dos BDZ foram estudados após a incubação com um bloqueador do canal de cálcio (Ca2+) o L-verapamil. Os resultados mostraram que (1) o tratamento agudo ex vivo com diazepam (10 mg/kg) produziu um aumento do burst oxidativo e da fagocitose induzida por PMA, LPS e Staphylococcus aureus; (2) o tratamento prolongado (21 dias) com diazepam (10 mg/kg) produziu um aumento do burst oxidativo induzido pelos mesmos estímulos e reduziu a fagocitose dos neutrófilos (porcentagem e intensidade); (3) o tratamento agudo ex vivo com diazepam, mas não o prolongado, aumentou os níveis séricos de corticosterona; (4) os efeitos da adição in vitro de diazepam (100 nM) foram na mesma direção daqueles observados após o tratamento agudo; (5) o RO 5-4864 (100 nM) induziu in vitro efeitos similares aos obtidos ex vivo (10 mg/kg) e in vitro (100 nM) com diazepam; (6) o tratamento ex vivo com clonazepam (10 mg/kg) diminuiu o burst oxidativo de neutrófilos induzido por PMA, LPS e Staphylococcus aureus enquanto que o tratamento com este fármaco na dose de 1 mg/kg aumentou o burst oxidativo destas células frente a todos os estímulos; (7) o tratamento ex vivo (1 e 10 mg/kg) e in vitro (100 nM) com clonazepam não alterou a fagocitose de neutrófilos; (8) os efeitos da adição in vitro de clonazepam sobre a atividade de neutrófilos vão na mesma direção daqueles observados após o tratamento ex vivo na dose de 1 mg/kg; (9) o clonazepam (1 e 10 mg/kg) aumentou os níveis séricos de corticosterona; (10) a adição in vitro do PK 11195 (100 nM) per se ou associado com diazepam (100 nM) ou RO 5-4864 (100 nM) aumentou o burst oxidativo e diminuiu a fagocitose realizada pelos neutrófilos; (11) a adição in vitro do PK 11195 (100 nM) associado com o clonazepam (100 nM) diminuiu o burst oxidativo induzido por PMA e diminuiu a fagocitose realizada por estas células; finalmente, (12) a pré incubação por 2 minutos com L-verapamil (100 nM) bloqueou todos os efeitos do diazepam sobre a atividade de neutrófilos estimulada por PMA, LPS e Staphylococcus aureus e reverteu os efeitos do RO 5-4864 no burst oxidativo induzido por PMA. Esses resultados sugeriram que os efeitos dos BDZ não estão relacionados com a ativação de PBR nas células da glândula adrenal e também que eles não estão relacionados a um aumento dos níveis séricos de corticosterona. Indicaram que os BDZ tenham produzido mudanças na concentração do Ca2+ intracelular dos neutrófilos através da ativação de PBR seguida de um aumento da permeabilidade da membrana plasmática ao Ca2+ através da abertura de canais sensíveis ao L-verapamil, permitindo a entrada do Ca2+. Os diferentes efeitos dos BDZ sobre o burst oxidativo e a fagocitose de neutrófilos foram atribuídos a ações específicas deste fármaco em subtipos de PBR presentes nos neutrófilos e nas membranas mitocondriais. Finalmente, os resultados sugeriram o desenvolvimento de tolerância aos efeitos do diazepam sobre os níveis séricos de corticosterona mas não sobre a atividade de neutrófilos

Benzodiazepines (BDZ) exert their anxiolytic effects via specific binding sites coupled to neuronal GABAa receptors. They also affect a second, pharmacologically different type of receptor, which has been found in many sites such as blood polimorphonuclear cells and adrenals. The present experiment was designed to analyze the effects of BDZ on neutrophil activity and corticosterone serum levels in rats. Specifically, neutrophil oxidative burst and phagocytosis were studied after ex vivo and in vitro treatments with diazepam, Ro 5-4864, clonazepam and/or PK 11195. Diazepam effects on neutrophil activity were also taken after long-term treatment. Finally, in vitro effects of BDZ were studied after incubation with L-verapamil, a Ca2+ channel blocking agent. Results showed that (1) acute and ex vivo diazepam (10.0 mg/kg) treatment induced an increment on PMA, LPS and Staphylococcus aureus induced oxidative burst and phagocytosis (2) long-term (21 days, 10 mg/kg/day) diazepam treatment increased the oxidative burst induced by the same stimuli and reduced both intensity of phagocytosis and the percentage of neutrophil performing phagocytosis; (3) acute and ex vivo diazepam treatment, but not long-term diazepam treatment increased corticosterone serum levels; (4) the effects induced by in vitro addition of diazepam (100 nM) were in the same direction of those observed after acute treatment; (5) Ro 5-4864 (100 nM) induced similar effects to those of diazepam after ex vivo (10.0 mg/kg) and in vitro (100 nM) treatments; (6) ex vivo (10.0 mg/kg) clonazepam treatment decreased the neutrophil oxidative burst induced by PMA and S. aureus after a 1.0 mg/kg dose and decreased these parameters after a higher (10 mg/kg) dose; (7) ex vivo (1.0 and 10.0 mg/kg) and in vitro (100 nM) clonazepam treatments did not change neutrophil phagocytosis; (8) in vitro clonazepam (100 nM) induced effects on neutrophil activity that were in the same direction of those observed after the 1.0 mg/kg dose (9) clonazepam (1.0 and 10.0 mg/kg) increased corticosterone serum levels; (10) in vitro PK 11195 (100 nM) per se or associated with diazepam (100 nM) or Ro 5-4864 (100 nM) increased PMA, LPS and S. aureus induced neutrophil oxidative burst and decreased cell phagocytosis; (11) in vitro PK 11195 (100 nM) together with clonazepam (100 nM) decreased PMA-induced oxidative burst and decreased S. aureus phagocytosis; finally, (12) in vitro L-verapamil (100 nM) incubation for 2 minutes prevented all effects induced by diazepam on PMA, LPS and S. aureus -induced neutrophil activity and reverted the effects induced by Ro 5-4864 (100 nM) on PMA-induced oxidative burst. These results suggest that BDZ effects on neutrophil activity were not related to PBR activation within the adrenal gland cells, leading to increments on corticosterone serum levels. They suggest instead that BDZ induced [Ca2+]i changes in neutrophils through PBR activation followed by an increase in plasma membrane permeability due to L-verapamil sensitive Ca2+ channels and subsequent extracelular Ca2+ entry. The differences of BDZ effects on neutrophil oxidative burst and phagocytosis were attributed to specific actions on different PBR subtypes present on neutrophil and mitochondrial membranes. Finally, results suggest the development of tolerance to diazepam effects on corticosterone serum levels but not on neutrophil activity