RESUMO
Delirium is a very common but annoying clinical state that interferes with the treatment of background disease and delays recovery. Delirium is a troublesome condition that exhausts not only the patient but also his/her family and healthcare professionals. Since aging is a risk factor for delirium, how to control delirium is an extremely important issue in an aging society. Phenotype of delirium is so diverse that it is difficult to elucidate the mechanism of individual symptoms, but it is clinically well known that maintaining sleep quality is important in preventing and improving delirium. Drugs and factors that are known to disrupt the sleep-wake cycle also overlap with the risk factors for delirium, indicating the close connection between delirium and sleep. Although the sleep-wake cycle is tightly regulated by many neurotransmitters and hormones, the role of each substance in this cycle is being elucidated in detail. It is well known that acetylcholine is one of the most important neurotransmitters involved in wakefulness, and anticholinergic drugs reduce rapid eye movement sleep. Anticholinergic drugs are also the major drug causing drug-induced delirium. Several clinical studies have reported that melatonin receptor agonists reduce delirium. Some clinical studies have examined the relationship between delirium and environmental factors that interfere with sleep, such as noise and brightness. The purpose of this review is to organize the cause of poor sleep underlying delirium and propose strategies to prevent delirium, based on rich neurological and pharmacological findings of sleep. We consider that elimination of causes of sleep deprivation underlying delirium is one of the most effective prevention strategies for delirium.
Assuntos
Antagonistas Colinérgicos/farmacologia , Delírio/induzido quimicamente , Melatonina/farmacologia , Sono/efeitos dos fármacos , Adenosina/metabolismo , Animais , Delírio/metabolismo , Histamina/metabolismo , Humanos , Qualidade do Sono , Vigília/efeitos dos fármacosRESUMO
Wogonin, a flavone in the roots of Scutellaria baicalensis, reduced etoposide-induced apoptotic cell death in normal cells, such as bone marrow cells and thymocytes. On the other hand, wogonin potentiated the proapoptotic or cytotoxic action of etoposide in tumor cells, such as Jurkat, HL-60, A549, and NCI-H226. These contradictory actions of wogonin on apoptosis are distinguished by normal or cancer cell types. Wogonin had no effect on apoptosis induced by other anticancer agents in the tumor cells. Thus, the potentiation effect of wogonin was observed only in etoposide-induced apoptosis in tumor cells. In a functional assay for P-glycoprotein (P-gp), wogonin suppressed excretion of calcein, a substrate for P-gp, in these tumor cells. Moreover, wogonin decreased the excretion of radiolabeled etoposide and accordingly increased intracellular content of this agent in the cells. P-gp inhibitors showed a similar potentiation effect on etoposide-induced apoptosis in these tumor cells. Thus, wogonin is likely to potentiate the anticancer action of etoposide due to P-gp inhibition and accumulation of this agent. These findings suggest that wogonin may be a useful chemotherapeutic adjuvant to potentiate the pharmacological action of etoposide and ameliorate its adverse effects.
Assuntos
Antineoplásicos/farmacologia , Etoposídeo/farmacologia , Flavanonas/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Animais , Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Etoposídeo/toxicidade , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Ratos Sprague-Dawley , Scutellaria baicalensis , Timo/citologiaRESUMO
ARTCEREB irrigation and perfusion solution (Artcereb) is a preparation intended for the irrigation and perfusion of the cerebral ventricles, and it is therefore important to evaluate the effects of Artcereb on brain cells. In vitro assessment of the effects of Artcereb in cell cultures of human fetal astrocytes was conducted in comparison with normal saline and lactated Ringer's solution. The effects of exposure to Artcereb were evaluated based on microscopic images of the mitochondria stained with rhodamine 123. The effects of exposure to Artcereb on cell function were also evaluated by quantitative analysis of mitochondrial activity based on rhodamine 123 and (3)H-thymidine incorporation. Morphological changes in nuclear structure were also evaluated. The results of the present study showed that cell function in cell cultures of human astrocytes was relatively unaffected by exposure to Artcereb as compared with normal saline or lactated Ringer's solution, suggesting that Artcereb has less effect on brain cells than normal saline or lactated Ringer's solution when used for the irrigation or perfusion of the cerebral ventricles.
Assuntos
Astrócitos/efeitos dos fármacos , Procedimentos Neurocirúrgicos , Perfusão , Soluções Farmacêuticas/farmacologia , Irrigação Terapêutica , Astrócitos/metabolismo , Astrócitos/ultraestrutura , Células Cultivadas , Ventrículos Cerebrais , Humanos , Mitocôndrias , Soluções Farmacêuticas/efeitos adversos , Rodamina 123 , Timidina/metabolismoRESUMO
Etoposide induces apoptotic cell death in normal and cancer cells. This apoptosis plays a role not only in anticancer effects but also in adverse reactions, such as myelosuppression. Because we had previously found that wogonin, a flavone found in a plant, suppresses thymocyte apoptosis induced by etoposide, we examined the effect of this flavone in cancer cells. Wogonin significantly potentiated etoposide-induced apoptosis in HL-60 cells. This flavone impaired the function of P-glycoprotein and then increased cellular content of etoposide in the cells. Thus, this flavone is likely to act as an inhibitor of P-glycoprotein and potentiate the apoptotic action of etoposide. On the other hand, wogonin inhibited etoposide-induced apoptosis in thymocytes, one of the normal cells. The potentiation by wogonin is likely to be a specific action for cancer cells but not normal cells. Therefore, this flavone may be used to reduce the excretion of the anticancer agents via P-glycoprotein and increase the pharmacological action of it in cancer cells. These results suggest that wogonin may play a role in overcoming multidrug resistance.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Etoposídeo/farmacologia , Flavanonas/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Citometria de Fluxo , Células HL-60 , Humanos , Marcação In Situ das Extremidades Cortadas , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patologiaRESUMO
The effect of wogonin, a flavone highly purified from the roots of Scutellaria baicalensis, on apoptotic cell death was re-evaluated in rat thymocytes. This flavone inhibited glucocorticoid-induced apoptotic changes such as DNA fragmentation, phosphatidylserine translocation, and nuclear condensation in rat thymocytes. Similar inhibition was also observed in apoptosis induced by other inducers such as etoposide. No significant changes of these apoptotic features were observed in rat thymocytes treated with wogonin alone, suggesting that this flavone protects against glucocorticoid-mediated immunosuppression caused by thymocyte apoptosis. Wogonin was reported to possess anti-inflammatory action in some previous studies, but this flavone had no effect on carrageenan-induced paw edema in this study. The simultaneous treatment of wogonin and glucocorticoid neither enhanced nor reduced the anti-inflammatory effect of glucocorticoid. These results indicate that wogonin is likely to prevent the immunosuppression of glucocorticoid without diminishing its drug efficacy as an anti-inflammatory agent.
Assuntos
Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Flavanonas/farmacologia , Animais , Anti-Inflamatórios/administração & dosagem , Transporte Biológico , Fragmentação do DNA/efeitos dos fármacos , Dexametasona/farmacologia , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/administração & dosagem , Edema/induzido quimicamente , Edema/tratamento farmacológico , Etoposídeo/farmacologia , Flavanonas/administração & dosagem , Glucocorticoides/farmacologia , Terapia de Imunossupressão , Técnicas In Vitro , Inflamação/tratamento farmacológico , Masculino , Fosfatidilserinas/metabolismo , Ratos , Ratos Sprague-Dawley , Scutellaria baicalensis/química , Timo/citologiaRESUMO
Cationic surfactants, such as benzalkonium chloride and benzethonium chloride, possess quaternary ammonium salt. These surfactants have antimicrobial action and are used as a preservative and an antiseptic. The positively charged polar head of cationic surfactants seems to play a role in the antimicrobial action of these compounds. Recently, benzalkonium chloride in eye drops has been reported to induce apoptosis in conjunctival cells. Here, we examined whether various types of surfactants including anionic and amphoteric surfactants induce apoptosis or not in mammalian cells. Antimicrobial cationic surfactants induced apoptosis at lower concentration than its critical micelle concentration (CMC) in rat thymocytes. Other quaternary ammonium surfactants, such as cetyltrimethylammonium bromide, similarly increased biochemical and morphological features of apoptosis, whereas both anionic and amphoteric surfactants had no significant effect on these apoptotic features. These results suggest that the positive charge of quaternary ammonium surfactants is involved with onset of the apoptotic process. The treatment of benzethonium chloride also led to apoptotic cell death in Jurkat cells. These results indicate that cationic surfactants induce apoptosis in the normal and cancer cells.
Assuntos
Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Leucemia de Células T/patologia , Tensoativos/toxicidade , Subpopulações de Linfócitos T/efeitos dos fármacos , Animais , Apoptose/fisiologia , Benzetônio/toxicidade , Cátions/toxicidade , Células Cultivadas , Humanos , Células Jurkat , Leucemia de Células T/tratamento farmacológico , Masculino , Ratos , Ratos Sprague-Dawley , Subpopulações de Linfócitos T/citologia , Timo/citologiaRESUMO
Glucocorticoid, such as dexamethasone, has anti-inflammatory and immunosuppressive action as major pharmacological effects. The latter action caused by lymphocyte apoptosis is not only a therapeutic effect but also an adverse reaction. Wogonin, a plant flavone found in Scutellaria baicalensis Georgi, inhibited dexamethasone-induced apoptotic changes, such as DNA fragmentation, nuclear condensation, phosphatidylserine translocation, and caspase activation in rat thymocytes. Since wogonin inhibited dexamethasone-induced DNA fragmentation in a noncompetitive manner, a target of this flavone is unlikely to be an antagonist of glucocorticoid receptor. Wogonin did not only act as an inhibitor of caspases, but also protected apoptosis induced by other glucocorticoids. Since wogonin reduced one of the major pharmacological effects of dexamethasone, we examined whether this flavone diminishes the anti-inflammatory action, another pharmacological effect. The anti-inflammatory action of dexamethasone was evaluated by carrageenan-induced paw edema model. Although dexamethasone significantly suppressed paw edema induced by carrageenan, wogonin had no effect on the anti-inflammatory action of dexamethasone. These results suggest that wogonin may be a useful compound to reduce the immunosuppressive side effect of glucocorticoid.
Assuntos
Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Dexametasona/farmacologia , Flavanonas/farmacologia , Hidrocortisona/farmacologia , Imunossupressores/antagonistas & inibidores , Animais , Células Cultivadas , Dexametasona/efeitos adversos , Dexametasona/antagonistas & inibidores , Medicamentos de Ervas Chinesas/efeitos adversos , Medicamentos de Ervas Chinesas/farmacologia , Edema/patologia , Edema/prevenção & controle , Hidrocortisona/efeitos adversos , Hidrocortisona/antagonistas & inibidores , Imunossupressores/farmacologia , Linfócitos/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-Dawley , Timo/citologia , Timo/efeitos dos fármacosRESUMO
Etoposide, a podophylotoxin anticancer agent, induces apoptotic cell death in normal and cancer cells. Etoposide-induced apoptosis plays a role in not only anticancer effect but also adverse reaction, such as myelosuppression. Since we have found that wogonin, a flavone found in Scutellaria baicalensis Georgi, prevents thymocyte apoptosis induced by various compounds including etoposide, we examined the effect of this flavone on etoposide-induced apoptosis in cancer cells. Although 100 muM wogonin itself significantly increased DNA fragmentation in HL-60 cells, this change was not observed in Jurkat cells. On the other hand, this flavone significantly potentiated etoposide-induced apoptosis in Jurkat and HL-60 cells. Similarly, wogonin accelerated etoposide-induced cell death in lung cancer cells. Since wogonin had no effect on the action of other anticancer agents, such as 5-FU and cisplatin, this flavone seems to accelerate only etoposide-induced apoptotic cell death in cancer cells. These results suggest that the modification of etoposide-induced apoptosis by wogonin may be available to reduce the adverse reaction of this agent.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Etoposídeo/farmacologia , Flavanonas/farmacologia , Leucemia Promielocítica Aguda/patologia , Leucemia de Células T/patologia , Animais , Sinergismo Farmacológico , Células HL-60 , Humanos , Células Jurkat , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia de Células T/tratamento farmacológico , Ratos , ScutellariaRESUMO
Selenium (Se) deficiency reduces glutathione peroxidase (GPx) activity, resulting in increased oxidative stress. We examined how Se deficiency induces renal injury via oxidative stress over time during the Se-deficient period. Seventy-two male Wistar rats were divided into two groups and fed either a control or Se-deficient diet. Rats were sacrificed on weeks 1, 2, 4, 6, 9, and 12. Blood and urine samples were collected, and the kidneys were removed. Urinalysis was performed, and creatinine clearance (Ccr) was calculated. Expressions of cellular GPx (cGPx) and phospholipid hydroperoxidase GPx (PHGPx) mRNA and GPx activity were measured. Histology was evaluated by light microscopy with immunohistochemistry for 4-hydroxy-2-nonenal (HNE) and vimentin. The Se-deficient diet caused significant decreases in GPx activity and cGPx mRNA expression but no change in PHGPx mRNA, together with significant proteinuria and glucosuria and slight decline in Ccr. The Se-deficient diet induced calcification in the kidney and increased the distribution of HNE and vimentin immunostaining in proximal tubuli, particularly around the outer medulla stripe. However, the histological damage did not progress after 6 weeks of deficiency. Se deficiency induces proteinuria and glucosuria with renal calcification, which may be primarily induced by injury of proximal tubuli via oxidative stress.
Assuntos
Túbulos Renais/patologia , Túbulos Renais/fisiopatologia , Selênio/deficiência , Animais , Creatinina/urina , Epitélio/patologia , Epitélio/fisiopatologia , Glutationa Peroxidase/metabolismo , Glicosúria/etiologia , Glicosúria/metabolismo , Masculino , Estresse Oxidativo/fisiologia , Proteinúria/etiologia , Proteinúria/metabolismo , Ratos , Ratos Wistar , Selênio/administração & dosagem , Selênio/fisiologiaRESUMO
Cisplatin (CP), a commonly used antineoplastic drug, is nephrotoxic. CP-induced nephrotoxicity involves oxidative pathways. A deficiency of selenium (Se) reduces glutathione peroxidase (GPx) activity resulting in oxidative stress. We investigated how Se deficiency or oral Se administration influences CP-induced nephrotoxicity. Thirty male Wistar rats were fed a Se-deficient or control diet for 4 weeks. Then they were given intraperitoneal (i.p.) CP alone, i.p. saline alone, or Se by gavage 24 and 1 h prior to i.p. CP. Blood and urine samples were collected and the kidneys were removed 5 days after CP treatment. Urinalysis, renal function, GPx activity, and expression of cellular GPx mRNA were measured. Histology was evaluated by light microscopy with immunohistochemistry for 4-hydroxy-2-nonenal (HNE), vimentin, and heme oxygenase (HO)-1. CP induced renal tubular damage with increased expression of vimentin, HO-1 and HNE staining, which represents lipid peroxidation. Se deficiency exacerbated CP-induced nephrotoxicity as shown by deterioration of the above parameters and depressed GPx activity and expression of GPx mRNA. Se treatment ameliorated CP-induced nephrotoxicity, but did not significantly improve renal function. These findings suggest that Se deficiency increases oxidative stress and enhances CP-induced nephrotoxicity, whereas oral Se treatment partially protects against the nephrotoxicity in rats.
Assuntos
Antioxidantes/metabolismo , Antioxidantes/farmacologia , Cisplatino , Nefropatias/induzido quimicamente , Nefropatias/fisiopatologia , Selênio/deficiência , Selênio/farmacologia , Administração Oral , Aldeídos/metabolismo , Animais , Antioxidantes/administração & dosagem , Citoproteção , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Heme Oxigenase-1/metabolismo , Imuno-Histoquímica , Rim/enzimologia , Rim/patologia , Rim/fisiopatologia , Nefropatias/metabolismo , Nefropatias/patologia , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Selênio/administração & dosagem , Selênio/sangue , Vimentina/metabolismoRESUMO
We have previously reported that YO-2, a selective plasmin inhibitor, induces thymocyte apoptosis. To elucidate the mechanism of YO-2-induced apoptosis, other YO compounds with different plasmin inhibitory action were tested for the pro-apoptotic activity in this study. The treatment of rat thymocytes with the YO compounds which had the hydrophobic but not the hydrophilic moiety at the C-terminal increased DNA fragmentation, the number of condensed nuclei and caspase-3-like activity. All pro-apoptotic YO compounds not only were potent plasmin inhibitors but also had the hydrophobic C-terminal as the common structure. Therefore, the target molecule of the YO compounds may be located not on the cell surface but rather inside the cells.
Assuntos
Apoptose/efeitos dos fármacos , Dipeptídeos/farmacologia , Fibrinolisina/antagonistas & inibidores , Animais , Fragmentação do DNA/efeitos dos fármacos , Dipeptídeos/química , Masculino , Conformação Molecular , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
The treatment of rat thymocytes with 10 microM terfenadine resulted in a significant increase in DNA fragmentation. The DNA fragmentation induced by terfenadine was dependent on its concentration and incubation time. In terfenadine-treated cells, the translocation of phosphatidylserine from the inside of plasma membrane to the outside, an early event of the apoptotic process, and chromatin condensation, the morphological characterization of apoptotic cell death, were observed. Terfenadine stimulated caspase-8, -9 and -3-like activities in an incubation time-dependent manner in thymocytes. The active forms of caspase-3 and -9 were detected in the extract from terfenadine-treated cells by immunoblotting analysis using specific antibodies to caspases, but active caspase-8 was not found in this fraction. Decrease in mitochondrial membrane potential and the release of cytochrome c from mitochondria to cytosol were observed in terfenadine-treated thymocytes. These results suggest that terfenadine induces apoptosis in rat thymocytes via mitochondrial pathway.
Assuntos
Apoptose/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Terfenadina/farmacologia , Animais , Apoptose/fisiologia , Células Cultivadas , Mitocôndrias/fisiologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Linfócitos T/fisiologiaRESUMO
We have previously found using inhibitors of protein phosphatase that phosphorylation of histones may be involved in thymocyte apoptosis. In this study, we examined whether histone modification occurs in astrocyte apoptosis induced by a pathological condition in the absence of drug. Incubation of cultured human astrocytes with growth medium for 24 h after exposure to saline solution for 30 min induced an increase in terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and nuclear condensation, biochemical and morphological hallmarks of apoptotic cell death. Acetic acid-urea-Triton X-100 (AUT) gel electrophoresis of the nuclear histone fraction and N-terminal peptide analysis showed that the treatment with saline solution caused rapid changes in phosphorylation of H2A subfamilies, but not in histone acetylation. The phosphorylation of the two subtypes increased markedly, whereas the phosphorylation of one subtype decreased. In contrast, exposure to ACF-95, an artificial cerebrospinal fluid (CSF), was associated with little induction of apoptotic cell death and induced less changes in histone phosphorylation. These results support the previous idea that chemical modification of histones is involved in the DNA fragmentation in astrocytes undergoing apoptosis.
Assuntos
Apoptose , Astrócitos/metabolismo , Histonas/metabolismo , Cloreto de Sódio , Sequência de Aminoácidos , Astrócitos/citologia , Histonas/química , Humanos , Marcação In Situ das Extremidades Cortadas , Dados de Sequência Molecular , FosforilaçãoRESUMO
To elucidate the role of chromatin structure in DNA fragmentation during apoptosis, we have examined whether chromatin structural change is observed after treatment with proapoptotic compounds. Analysis of the circular dichroism (CD) spectrum of the soluble chromatin from dexamethasone-treated thymocytes revealed a decrease in alpha-helical content. Mifepristone, an antagonist of glucocorticoid receptor, prevented both the change in chromatin structure and DNA fragmentation induced by dexamethasone. The effect of YO-2 [trans-4-aminomethylcyclohexanecarbonyl-l-(O-picolyl)tyrosine-n-octylamide], which possesses proapoptotic action, on chromatin structure was also examined. Judging from the CD spectrum of the soluble chromatin from YO-2-treated thymocytes, the structure was changed by this compound as well as by dexamethasone. A decrease in alpha-helical content was also observed in cells treated with etoposide, which is used clinically as an anticancer agent. These results suggest that the change in chromatin structure is likely to be an important process in DNA fragmentation of cells undergoing apoptosis.
Assuntos
Dipeptídeos/farmacologia , Timo/efeitos dos fármacos , Animais , Dicroísmo Circular , Ratos , Timo/citologiaRESUMO
Astrocytic apoptosis may play a role in the central nervous system injury. We previously showed that reperfusion of cultured astrocytes with normal medium after exposure to hydrogen peroxide (H(2)O(2))-containing medium causes apoptosis. This study examines the involvement of the lysosomal enzymes cathepsins B and D in the astrocytic apoptosis. Reperfusion after exposure to H(2)O(2) caused a marked increase in caspase-3 and cathepsin D activities and a marked decrease in cathepsin B activity. Pepstatin A, an inhibitor of cathepsin D, and acetyl-L-aspartyl-L-methionyl-L-glutaminyl-L-aspart-1-aldehyde (Ac-DMQD-CHO), a specific inhibitor of caspase-3, blocked the H(2)O(2)-induced decrease in cell viability and DNA ladder formation in cultured rat astrocytes. The (L-3-trans-(propylcarbamoyl)oxirane-2-carbonyl)-L-isoleucyl-L-proline methyl ester (CA074 Me), a specific inhibitor of cathepsin B, did not affect the H(2)O(2)-induced cell injury. On the other hand, CA074 Me decreased cell viability with DNA ladder formation when cultured in the presence of Ac-DMQD-CHO. This caspase-independent apoptosis was attenuated by the addition of the cathepsin D inhibitor pepstatin A. Caspase-3 like activity was markedly inhibited by Ac-DMQD-CHO and partially by pepstatin A. Pepstatin A and CA074 Me inhibited cathepsin B and cathepsin D activities, respectively, in the presence and absence of Ac-DMQD-CHO. These results suggest that cathepsins B and D are involved in astrocytic apoptosis: cathepsin D acts as a death-inducing factor upstream of caspase-3 and the caspase-independent apoptosis is regulated antagonistically by cathepsins B and D.
Assuntos
Astrócitos/patologia , Catepsinas/fisiologia , Traumatismo por Reperfusão/patologia , Animais , Apoptose/fisiologia , Caspase 3 , Inibidores de Caspase , Caspases/metabolismo , Catepsina B/antagonistas & inibidores , Catepsina B/fisiologia , Catepsina D/antagonistas & inibidores , Catepsina D/fisiologia , Catepsinas/antagonistas & inibidores , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , DNA/biossíntese , DNA/genética , Inibidores Enzimáticos/farmacologia , Peróxido de Hidrogênio/toxicidade , Peptídeo Hidrolases/metabolismo , Ratos , Ratos WistarRESUMO
The inhibitors of protein phosphatase such as calyculin A and okadaic acid induced thymocyte apoptosis. DNA fragmentation was increased in the nuclei from thymocytes treated with calyculin A, which accelerated phosphorylation of histones. Judging from the circular dichroism analysis, the structure of soluble chromatin was changed by the treatment with calyculin A. These results suggest that the change in chromatin structure may be one of the molecular mechanisms of internucleosomal DNA fragmentation.
Assuntos
Apoptose/fisiologia , Cromatina/ultraestrutura , Inibidores Enzimáticos/farmacologia , Histonas/metabolismo , Oxazóis/farmacologia , Linfócitos T/ultraestrutura , Apoptose/efeitos dos fármacos , Fragmentação do DNA , Humanos , Toxinas Marinhas , Modelos Biológicos , Fosforilação , Linfócitos T/fisiologiaRESUMO
The inhibitors of protein phosphatase such as calyculin A and okadaic acid induce the apoptotic cell death in rat thymocytes. To clarify the molecular mechanism of these inhibitor-induced apoptosis, the effect of calyculin A on DNA fragmentation in the isolated nuclei were studied. A significant increase in DNA fragmentation was observed in the nuclei prepared from the cells treated with calyculin A that caused histone hyperphosphorylation. No changes of the activities of caspase-8 and -3 were observed in the extract from the cells treated with calyculin A. The circular dichroism analysis of soluble chromatin from calyculin A-treated thymocyte nuclei indicated that phosphorylation of histones decreased its alpha-helical content. Thus, the change in the chromatin structure may be due to the chemical modification of histones. Moreover, the structural change in chromatin preceded DNA fragmentation in the nuclei. Therefore, these results suggest that the change of chromatin structure allow easy accessibility of nuclear DNase to chromosomal DNA.
Assuntos
Apoptose , Cromatina/metabolismo , Desoxirribonucleases/metabolismo , Histonas/metabolismo , Timo/citologia , Animais , Inibidores de Caspase , Cromatina/química , Dicroísmo Circular , Inibidores de Cisteína Proteinase/farmacologia , Toxinas Marinhas , Oxazóis/farmacologia , Fosforilação , RatosRESUMO
Heat shock proteins (HSPs) have been shown to act as inhibitors of apoptosis, but this anti-apoptotic effect is not known in the central nervous system. Prior heat shock has been demonstrated to protect astrocytes from cell death in a model of reperfusion injury (Brain Res. 735 (1996) 265). The present study examines the mechanism underlying the protective effect of the heat shock. Preincubation of astrocytes at 40 degrees C for 10 min attenuated the hydrogen peroxide (H(2)O(2))-induced decrease in cell viability, DNA ladder formation and nuclear condensation, and these effects were blocked by the protein synthesis inhibitor cycloheximide. The thermal stress inhibited the H(2)O(2)-induced increase in caspase-3 like protease activity, but it did not affect the H(2)O(2)-induced loss of mitochondrial membrane potential. The cytosol prepared from preheated cells did not affect Ca(2+)-induced swelling of mitochondria, a marker of the permeable transition pore. The protective effect of the thermal stress on the H(2)O(2)-induced decrease in cell viability was not affected by the mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor 2'-amino-3'-methoxyflavone, the phosphatidylinositol-3 kinase inhibitor wortmannin and the NF-kappaB inhibitor pyrrolidinedithiocarbamate. These findings suggest that HSPs inhibit apoptosis via an inhibition of caspase-3 activation without effect on mitochondrial dysfunction.
Assuntos
Apoptose/efeitos dos fármacos , Astrócitos/efeitos dos fármacos , Proteínas de Choque Térmico/farmacologia , Peróxido de Hidrogênio/antagonistas & inibidores , Oxidantes/toxicidade , Animais , Benzimidazóis , Caspase 3 , Caspases/metabolismo , Sobrevivência Celular/fisiologia , Células Cultivadas , DNA/biossíntese , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes , Temperatura Alta/efeitos adversos , Peróxido de Hidrogênio/toxicidade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , NF-kappa B/antagonistas & inibidores , Inibidores de Fosfoinositídeo-3 Quinase , Ratos , Ratos WistarRESUMO
We examined the effect of 3-ethyl-3-(ethylaminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC12), a nitric oxide (NO) donor, on apoptosis in cultured astrocytes. Reperfusion after hydrogen peroxide (H2O2) exposure caused a decrease in cell viability, loss of mitochondrial membrane potential, caspase-3 activation, DNA ladder formation, and nuclear condensation. NOC12 at 10-100 microM significantly attenuated these apoptotic changes, while the NO donor at 1 mM caused cell injury and exacerbated the H202-induced cell injury. NOC12 increased intracellular cGMP levels in a dose dependent manner with the maximal effect at 100 microM. The protective effect of NOC12 was mimicked by the NO-independent guanylate cyclase activator 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole, and was attenuated by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and the cGMP-dependent protein kinase inhibitor KT5823. ODQ and KT5823 did not block but rather exacerbated the cytotoxic effect of NOC12 at 1 mM. These findings demonstrate that lower concentrations of NOC12 inhibit the H2O2-induced apoptosis of astrocytes in a cGMP-dependent way, but higher concentrations of NOC12 show a toxic effect on astrocytes in a cGMP-independent way.
Assuntos
Apoptose/efeitos dos fármacos , Astrócitos/efeitos dos fármacos , GMP Cíclico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Compostos Nitrosos/farmacologia , Animais , Astrócitos/citologia , Astrócitos/enzimologia , Caspase 3 , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Ativação Enzimática , Peróxido de Hidrogênio/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Óxido Nítrico/fisiologia , Ratos , Ratos WistarRESUMO
The treatment of rat thymocytes with YO-2, a novel inhibitor of plasmin, resulted in an increase in DNA fragmentation. DNA fragmentation was also induced by another YO compounds such as YO-0, -3, -4 and -5. These YO compounds are the inhibitor of plasmin activity. On the other hand, YO-1, -6 and -8 that hardly inhibit plasmin activity had no effect on DNA fragmentation. Analysis of fragmented DNA from thymocytes treated with YO-2 by agarose gel electrophoresis revealed that the compound caused internucleosomal DNA fragmentation. In addition, judging from a laser scanning microscopy, annexin V-positive and propidium iodide-negative cells were increased by the treatment of the cells with the compound. Moreover, chromatin condensation was observed in thymocytes treated with the compound. These results demonstrated that YO-2 induces thymocyte apoptosis. There seemed to be some correlation between the apoptosis induced by YO compounds and their plasmin inhibitory effect. However, because the other protease inhibitors including pepstatin A, leupeptin, AEBSF, DFP and E-64-d did not affect DNA fragmentation, YO compounds are likely to have unique mechanism on plasmin or to show the effect on the other plasmin-like proteases. The plasmin inhibitory activity may have an important role in YO-2-induced apoptosis. Furthermore, the stimulations of caspase-8, -9 and -3-like activities were observed in thymocytes treated with YO-2. These results suggest that YO-2 induces thymocyte apoptosis via activation of caspase cascade.