RESUMO
Active oxygen and free radicals are involved in metabolism in cells and tissues. Immunohistological studies of related enzymes are few, and the morphological dynamics of these enzymes in dental pulp and odontoblasts remain to be elucidated. Nitric oxide synthase (NOS) has 3 isoforms: nNOS, iNOS, and eNOS. The aim of this study was to investigate the profiles of NOS isoforms in the absence of nNOS in dental pulp and odontoblasts. Five-week-old male C57BL/6 and nNOS knockout (KO) mice were sacrificed and expression of nNOS, iNOS, and eNOS determined immunohistochemically. Expression of nNOS was positive, whereas that of iNOS was negative and eNOS weakly positive in the dental pulp and odontoblasts of the control mice. In nNOS KO mice, expression of iNOS was positive in dental pulp and strongly positive in odontoblasts, whereas that of eNOS was stronger in fibroblasts, endothelial cells in the vicinity of blood vessels in the dental pulp, and odontoblasts. Expression of nNOS was negative in the nNOS KO mice. This suggests that iNOS and eNOS compensate for nNOS deficiency in vascular endothelial cells and fibroblasts in the dental pulp and odontoblasts.
Assuntos
Polpa Dentária , Odontoblastos , Animais , Células Endoteliais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase , Isoformas de ProteínasRESUMO
A close relationship between cell death and pathological calcification has recently been reported, such as vascular calcification in atherosclerosis. However, the roles of cell death in calcification by osteoblast lineage have not been elucidated in detail. In this study, we investigated whether cell death is involved in the calcification on osteoblastic differentiation of human bone marrow mesenchymal stem cells (hMSC) under osteogenic culture in vitro. Apoptosis and necrosis occurred in an osteogenic culture of hMSC, and cell death preceded calcification. The generation of intracellular reactive oxygen species, chromatin condensation and fragmentation, and caspase-3 activation increased in this culture. A pan-caspase inhibitor (Z-VAD-FMK) and anti-oxidants (Tiron and n-acetylcysteine) inhibited osteogenic culture-induced cell death and calcification. Furthermore, calcification was significantly promoted by the addition of necrotic dead cells or its membrane fraction. Spontaneously dead cells by osteogenic culture and exogenously added necrotic cells were surrounded by calcium deposits. Induction of localized cell death by photodynamic treatment in the osteogenic culture resulted in co-localized calcification. These findings show that necrotic and apoptotic cell deaths were induced in an osteogenic culture of hMSC and indicated that both necrotic and apoptotic cells of osteoblast lineage served as nuclei for calcification on osteoblastic differentiation of hMSC in vitro.
Assuntos
Apoptose , Calcificação Fisiológica , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Sal Dissódico do Ácido 1,2-Di-Hidroxibenzeno-3,5 Dissulfônico/farmacologia , Acetilcisteína/farmacologia , Clorometilcetonas de Aminoácidos/farmacologia , Antioxidantes/farmacologia , Caspase 3/metabolismo , Inibidores de Caspase/farmacologia , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Montagem e Desmontagem da Cromatina , Ativação Enzimática , Humanos , Técnicas In Vitro , Células-Tronco Mesenquimais/efeitos dos fármacos , Necrose , Osteoblastos/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Mitochondrial membrane permeability transition (MPT) plays a crucial role in apoptotic tail shortening during anuran metamorphosis. L-carnitine is known to shuttle free fatty acids (FFAs) from the cytosol into mitochondria matrix for ß-oxidation and energy production, and in a previous study we found that treatment with L-carnitine suppresses 3, 3', 5-triiodothyronine (T3 ) and FFA-induced MPT by reducing the level of FFAs. In the present study we focus on acetyl-L-carnitine, which is also involved in fatty acid oxidation, to determine its effect on T3 -induced tail regression in Rana rugosa tadpoles and spontaneous tail regression in Xenopus laevis tadpoles. The ladder-like DNA profile and increases in caspase-3 and caspase-9 indicative of apoptosis in the tails of T3 -treated tadpoles were found to be suppressed by the addition of acetyl-L-carnitine. Likewise, acetyl-L-carnitine was found to inhibit thyroid hormone regulated spontaneous metamorphosis in X. laevis tadpoles, accompanied by decreases in caspase and phospholipase A2 activity, as well as non-ladder-like DNA profiles. These findings support our previous conclusion that elevated levels of FFAs initiate MPT and activate the signaling pathway controlling apoptotic cell death in tadpole tails during anuran metamorphosis.
Assuntos
Acetilcarnitina/farmacologia , Anuros/genética , Anuros/metabolismo , Cauda/efeitos dos fármacos , Hormônios Tireóideos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Caspase 3/metabolismo , Caspase 9/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Larva , Masculino , Metamorfose Biológica/efeitos dos fármacos , Fosfolipases A2/metabolismoRESUMO
Ever since protoporphyrin IX (PpIX) was discovered to accumulate preferentially in cancer cells after 5-aminolevulinic acid (ALA) treatment, photodynamic treatment or therapy (PDT) has been developed as an exciting new treatment option for cancer patients. However, the level of PpIX accumulation in oral cancer is fairly low and insufficient for PDT. Ferrochelatase (FECH) and ATP-binding cassette transporter G2 (ABCG2) are known to regulate PpIX accumulation. In addition, serum enhances PpIX export by ABCG2. We investigated here whether and how inhibitors of FECH and ABCG2 and their combination could improve PpIX accumulation and PDT efficacy in an oral cancer cell line in serum-containing medium. ABCG2 inhibitor and the combination of ABCG2 and FECH inhibitors increased PpIX in the presence of fetal bovine serum (FBS) in an oral cancer cell line. Analysis of ABCG2 gene silencing also revealed the involvement of ABCG2 in the regulation of PpIX accumulation. Inhibitors of FECH and ABCG2, and their combination increased the efficiency of ALA-PDT even in the presence of FBS. ALA-PDT-induced cell death was accompanied by apoptotic events and lipid peroxidation. These results suggest that accumulation of PpIX is determined by the activities of ABCG2 and FECH and that treatment with a combination of their inhibitors improves the efficacy of PDT for oral cancer, especially in the presence of serum.
Assuntos
Ácido Aminolevulínico/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias Bucais/tratamento farmacológico , Fotoquimioterapia/métodos , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Clorometilcetonas de Aminoácidos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Sanguíneas/farmacologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Inibidores de Caspase/farmacologia , Linhagem Celular Tumoral , Desferroxamina/farmacologia , Ferroquelatase/antagonistas & inibidores , Inativação Gênica , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Proteínas de Neoplasias/genética , Fármacos Fotossensibilizantes/farmacologia , Protoporfirinas/metabolismo , Sideróforos/farmacologiaRESUMO
Cardiovascular risks are frequently accompanied by high serum fatty acid levels. Although recent studies have shown that fatty acids affect mitochondrial function and induce cell apoptosis, L-carnitine is essential for the uptake of fatty acids by mitochondria, and may attenuate the mitochondrial dysfunction and apoptosis of cardiocytes. This study aimed to elucidate the activity of L-carnitine in the prevention on fatty acid-induced mitochondrial membrane permeability transition and cytochrome c release using isolated cardiac mitochondria from rats. Palmitoyl-CoA-induced mitochondrial respiration that was observed with L-carnitine was inhibited with oligomycin. The palmitoyl-CoA-induced mitochondrial membrane depolarization and swelling were greatly inhibited by the presence of L-carnitine. In ultrastructural observations, terminally swollen and ruptured mitochondria with little or no distinguishable cristae structures were induced by treatment with palmitoyl-CoA. However, the severe morphological damage in cardiac mitochondria was dramatically inhibited by pretreatment with L-carnitine. Treatment with L-carnitine also attenuated 4-hydroxy-L-phenylglycine- and rotenone-induced mitochondrial swelling even when the L-carnitine could not protect against the decrease in oxygen consumption associated with these inhibitors. Furthermore, L-carnitine completely inhibited palmitoyl-CoA-induced cytochrome c release. We concluded that L-carnitine is essential for cardiac mitochondria to attenuate the membrane permeability transition, and to maintain the ultrastructure and membrane stabilization, in the presence of high fatty acid ß-oxidation. Consequently, the cells may be protected against apoptosis by L-carnitine through inhibition of the fatty acid-induced cytochrome c release.
Assuntos
Carnitina/farmacologia , Ácidos Graxos/farmacologia , Dilatação Mitocondrial/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Complexo Vitamínico B/farmacologia , Animais , Mitocôndrias Cardíacas/ultraestrutura , Consumo de Oxigênio/efeitos dos fármacos , Palmitoil Coenzima A/farmacologia , Permeabilidade/efeitos dos fármacos , RatosRESUMO
Accumulation of protoporphyrin IX (PpIX) in cancer cells is a basis of 5-aminolevulinic acid (ALA)-induced photodymanic therapy. We studied factors that affect PpIX accumulation in human urothelial carcinoma cell line T24, with particular emphasis on ATP-binding cassette transporter G2 (ABCG2) and serum in the medium. When the medium had no fetal bovine serum (FBS), ALA induced PpIX accumulation in a time- and ALA concentration-dependent manner. Inhibition of heme-synthesizing enzyme, ferrochelatase, by nitric oxide donor (Noc18) or deferoxamine resulted in a substantial increase in the cellular PpIX accumulation, whereas ABCG2 inhibition by fumitremorgin C or verapamil induced a slight PpIX increase. When the medium was added with FBS, cellular accumulation of PpIX stopped at a lower level with an increase of PpIX in the medium, which suggested PpIX efflux. ABCG2 inhibitors restored the cellular PpIX level to that of FBS(-) samples, whereas ferrochelatase inhibitors had little effects. Bovine serum albumin showed similar effects to FBS. Fluorescence microscopic observation revealed that inhibitors of ABC transporter affected the intracellular distribution of PpIX. These results indicated that ABCG2-mediated PpIX efflux was a major factor that prevented PpIX accumulation in cancer cells in the presence of serum. Inhibition of ABCG2 transporter system could be a new target for the improvement of photodynamic therapy.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Neoplasias/metabolismo , Protoporfirinas/metabolismo , Soro/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/genética , Ácido Aminolevulínico/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Bovinos , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Ferroquelatase/antagonistas & inibidores , Ferroquelatase/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Heme/biossíntese , Humanos , Indóis/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Compostos Nitrosos/farmacologia , Protoporfirinas/biossíntese , Soroalbumina Bovina/metabolismoRESUMO
Mitochondrial beta-oxidation is an important system involved in the energy production of various cells. In this system, the function of L-carnitine is essential for the uptake of fatty acids to mitochondria. However, it is unclear whether or not endogenous respiration, ADP-induced O(2) consumption without substrates, is caused by L-carnitine treatment. In this study, we investigated whether L-carnitine is essential to the beta-oxidation of quarried fatty acids from the mitochondrial membrane by phospholipase A(2) (PLA(2)) using isolated mitochondria from the liver of rats. Intact mitochondria were incubated in a medium containing Pi, CoA and L-carnitine. The effect of L-carnitine treatment on ADP-induced mitochondrial respiration was observed without exogenous respiratory substrate. Increase in mitochondrial respiration was induced by treatment with L-carnitine in a concentration-dependent manner. Treatment with rotenone, a complex I blocker, completely inhibited ADP-induced oxygen consumption even in the presence of L-carnitine. Moreover, the L-carnitine dependent ADP-induced mitochondrial oxygen consumption did not increase when PLA(2) inhibitors were treated before ADP treatment. The L-carnitine-dependent ADP-induced oxygen consumption did contribute to ATP productions but not heat generation via an uncoupling system. These results suggest that L-carnitine might be essential to the beta-oxidation of quarried fatty acids from the mitochondrial membrane by PLA(2).
Assuntos
Carnitina/farmacologia , Ácidos Graxos/química , Mitocôndrias Hepáticas/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Fosfolipases A2/metabolismo , Complexo Vitamínico B/farmacologia , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Ácidos Graxos/metabolismo , Masculino , Mitocôndrias Hepáticas/metabolismo , Membranas Mitocondriais/metabolismo , Oligomicinas/farmacologia , Oxirredução , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Ratos Wistar , Desacopladores/farmacologiaRESUMO
Phosphatidylserine (PS) externalization is a key feature of apoptotic cell death and plays an important role in clearance of apoptotic cells by phagocytes. PS externalization during apoptosis is generally an irreversible event mediated by caspase activation and is accompanied by other apoptotic events. We report here that an apoptosis inducer alpha-tocopheryl succinate (TOS) can induce PS externalization that is independent of apoptosis and reversible in the absence of fetal bovine serum (FBS) in histiocytic lymphoma U937 cells. In the presence of FBS, TOS induced PS externalization via a caspase-dependent mechanism accompanied by mitochondrial depolarization, cell shrinkage, increase of caspase-3 activity, and chromatin condensation. In contrast, in the absence of FBS, TOS induced the rapid PS externalization which was not accompanied by other apoptotic events. The PS externalization was reversible by removing TOS and was not involved in Ca(2+)-dependent scramblase activation and thiol oxidation of aminophospholipid translocase. A similar PS externalization was also induced by cholesteryl hemisuccinate (CS), the other succinate ester. These results suggested that the mechanism of TOS- and CS-induced PS externalization in the absence of FBS was different from it occurring during typical apoptosis.
Assuntos
Fosfatidilserinas/metabolismo , alfa-Tocoferol/farmacologia , Apoptose , Transporte Biológico/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular Tumoral , Ésteres do Colesterol/farmacologia , Meios de Cultura Livres de Soro/farmacologia , HumanosRESUMO
PURPOSE: The purpose of this study was to clarify the regulatory mechanism of protoporphyrin IX (PpIX) synthesis mediated by 5-aminolevulinic acid (ALA) in human urothelial carcinoma (UC), leading to improved accuracy in photodynamic diagnosis and therapy using ALA. EXPERIMENTAL DESIGN: PpIX accumulation in cultured UC cells after incubation for 1-5 h with 0.5-5 mM ALA was analyzed by fluorescence analysis using fluorescence microscopy and flow cytometry technique. RESULTS: PpIX fluorescence mediated by ALA was increased, and the intensity of PpIX fluorescence was time-dependently increased in UC cells compared to noncancerous cells. The distribution of endogenous PpIX fluorescence primarily coincided with mitochondria, and then increased at a specific perinuclear region in the cells during the time of incubation. The ALA-mediated PpIX synthesis in UC cells was suppressed by beta-alanine, an inhibitor of beta-transporters of cell membrane, and carbonylcyanide p-trifluoromethoxyphenyl hydrazone, an uncoupler of mitochondrial oxidative phosphorylation. In contrast, the ALA-mediated PpIX accumulation was increased by deferoxamine, an iron chelator, manganese and nitric oxide, which is contributed to PpIX metabolism by inhibiting ferrochelatase activity, generated by a nitric oxide-generating reagent NOC-18. As observed above, ALA-mediated PpIX synthesis in human UC cells was regulated by the process of ALA uptake, ALA conversion to PpIX and metabolism of accumulated PpIX to heme. CONCLUSIONS: This shows that the suppression of ferrochelatase increased PpIX accumulation in UC cells using small amount of ALA, thus leading to an improved clinical practicability of photodynamic diagnosis and therapy.
Assuntos
Ácido Aminolevulínico/farmacologia , Carcinoma de Células de Transição/metabolismo , Fármacos Fotossensibilizantes/farmacologia , Protoporfirinas/biossíntese , Neoplasias da Bexiga Urinária/metabolismo , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Carcinoma de Células de Transição/tratamento farmacológico , Linhagem Celular Tumoral , Desferroxamina/farmacologia , Ferroquelatase/antagonistas & inibidores , Ferroquelatase/metabolismo , Citometria de Fluxo , Humanos , Microscopia de Fluorescência , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Compostos Nitrosos/farmacologia , Fosforilação Oxidativa/efeitos dos fármacos , Fatores de Tempo , Neoplasias da Bexiga Urinária/tratamento farmacológico , Urotélio/efeitos dos fármacos , Urotélio/metabolismo , beta-Alanina/farmacologiaRESUMO
Photodynamic therapy (PDT) for tumors is based on the tumor-selective accumulation of a photosensitizer, protoporphyrin IX (PpIX), followed by irradiation with visible light. However, the molecular mechanism of cell death caused by PDT has not been fully elucidated. The 5-aminolevulinic acid (ALA)-based photodynamic action (PDA) was dependent on the accumulation of PpIX, the level of which decreased rapidly by eliminating ALA from the incubation medium in human histiocytic lymphoma U937 cells. PDA induced apoptosis characterized by lipid peroxidation, increase in Bak and Bax/Bcl-xL, decrease in Bid, membrane depolarization, cytochrome c release, caspase-3 activation, phosphatidylserine (PS) externalization. PDT-induced cell death seemed to occur predominantly via apoptosis through distribution of PpIX in mitochondria. These cell death events were enhanced by ferrochelatase inhibitors. These results indicated that ALA-based-PDA induced apoptotic cell death through a mitochondrial pathway and that ferrochelatase inhibitors might enhanced the effect of PDT for tumors even at low concentrations of ALA.
Assuntos
Ácido Aminolevulínico/farmacologia , Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Ferroquelatase/antagonistas & inibidores , Linfoma Difuso de Grandes Células B/fisiopatologia , Fármacos Fotossensibilizantes/farmacologia , Ferroquelatase/metabolismo , Humanos , Luz , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/enzimologia , Fotoquimioterapia , Protoporfirinas/farmacologia , Células U937RESUMO
We previously reported that the generation of reactive oxygen species (ROS) is the initial event in cell death induced by 6-hydroxydopamine (6-OHDA), an experimental model of Parkinsonism. Since recent studies suggested the important role of antioxidant activity of alpha-lipoic acid (LA) in the suppression of apoptosis of various types, we studied the effect on 6-OHDA-induced apoptosis of PC12 cells. Biochemical analysis revealed that LA suppressed the 6-OHDA-induced ROS generation, increase of caspase-like activity and chromatin condensation. The suppression of 6-OHDA-induced apoptosis by LA required pre-incubation of PC12 cells with LA for 12-24 h. LA increased the intracellular levels of heme oxygenase-1 (HO-1) and glutathione (GSH) and stimulated the expression of GSH synthesis-related genes such as cystine/glutamate antiporter and gamma-glutamylcysteine synthetase (gamma-GCS). However, Sn-mesoporphyrin IX, an inhibitor of HO-1, did not attenuate the LA-induced suppression of apoptosis. In contrast, buthionine sulfoximine, an inhibitor of gamma-GCS, attenuated the LA-induced suppression of ROS generation and chromatin condensation. In addition, a transcription factor Nrf2, which regulates the expression of antioxidant enzymes such as gamma-GCS, translocated to the nucleus by LA. These results suggested that LA suppressed the 6-OHDA induced-apoptosis by the increase in cellular glutathione through stimulation of the GSH synthesis system but not by the expression of HO-1.
Assuntos
Apoptose/efeitos dos fármacos , Glutationa/metabolismo , Heme Oxigenase-1/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ácido Tióctico/fisiologia , Sistemas de Transporte de Aminoácidos/metabolismo , Animais , Antioxidantes/fisiologia , Antiporters/metabolismo , Apoptose/fisiologia , Glutamato-Cisteína Ligase/metabolismo , Oxidopamina , Células PC12 , RatosRESUMO
Membrane permeability transition (MPT) of mitochondria has an important role in apoptosis of various cells. The classic type of MPT is characterized by increased Ca(2+) transport, membrane depolarization, swelling, and sensitivity to cyclosporin A. In this study, we investigated whether L-carnitine suppresses oleic acid-induced MPT using isolated mitochondria from rat liver. Oleic acid-induced MPT in isolated mitochondria, inhibited endogenous respiration, caused membrane depolarization, and increased large amplitude swelling, and cytochrome c (Cyt. c) release from mitochondria. L-Carnitine was indispensable to beta-oxidation of oleic acid in the mitochondria, and this reaction required ATP and coenzyme A (CoA). In the presence of ATP and CoA, L-carnitine stimulated oleic acid oxidation and suppressed the oleic acid-induced depolarization, swelling, and Cyt. c release. L-Carnitine also contributed to maintaining mitochondrial function, which was decreased by the generation of free fatty acids with the passage of time after isolation. These results suggest that L-carnitine acts to maintain mitochondrial function and suppresses oleic acid-mediated MPT through acceleration of beta-oxidation.
Assuntos
Carnitina/farmacologia , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Mitocôndrias Hepáticas/efeitos dos fármacos , Ácido Oleico/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Cefaloridina/farmacologia , Coenzima A/farmacologia , Citocromos c/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Dilatação Mitocondrial/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Permeabilidade/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Ratos , Ratos Wistar , Fatores de TempoRESUMO
3-Nitropropionic acid (3NP) functions as an irreversible inhibitor of succinic acid dehydrogenase (complex II) and induces neuronal disorders in rats similar to those in patients with Huntington's disease. It is well known that L-carnitine (LC), a carrier of long chain fatty acid into the mitochondrial matrix, attenuates the neuronal degeneration in 3NP-treated rats. From these findings it has been suggested that 3NP induces certain neuronal cell death through mitochondrial dysfunction and that LC preserves the neurons against the dysfunction of mitochondria caused by 3NP. However, the detailed mechanism of cell death by 3NP and the protective actions of LC against the mitochondrial dysfunction have not been fully elucidated yet. Thus, we studied the molecular mechanism of the effects of 3NP and LC on isolated rat liver mitochondria. 3NP inhibited succinate respiration and the decreased respiratory control ratio of isolated mitochondria without affecting oxidative phosphorylation. 3NP induced a membrane permeability transition (MPT), which plays an important role in the mechanism of apoptotic cell death. 3NP stimulated Ca2+ release from mitochondria, decreased membrane potential, induced mitochondrial swelling, and stimulated cytochrome c release from mitochondria. 3NP-induced swelling was suppressed by bovine serum albumin, inhibitors of phospholipase A(2) and by an inhibitor of classic MPT, cyclosporin A. Furthermore, LC suppressed the changes brought about by 3NP in mitochondrial functions in the presence of ATP. These results suggest that MPT underlies the mechanism of 3NP-induced cell death, and that LC attenuates mitochondrial MPT by decreasing long chain fatty acids generated by phospholipase A(2).
Assuntos
Carnitina/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Nitrocompostos/farmacologia , Propionatos/farmacologia , Animais , Cálcio/metabolismo , Morte Celular , Ciclosporina/metabolismo , Citocromos c/metabolismo , Inibidores Enzimáticos/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Dilatação Mitocondrial/efeitos dos fármacos , Nitrocompostos/antagonistas & inibidores , Inibidores de Fosfolipase A2 , Fosfolipases A2/metabolismo , Fosforilação , Propionatos/antagonistas & inibidores , Ratos , Ácido Succínico/metabolismoRESUMO
The membrane permeability transition (MPT) of mitochondria plays an important role in the mechanism of apoptotic cell death in various cells. Classic type MPT is induced by Ca(2+) in the presence of inorganic phosphate and respiratory substrate, and is characterized by various events including generation of reactive oxygen species (ROS), membrane depolarization, swelling, release of Ca(2+) and high sensitivity to cyclosporine A. However, the sequence of these events and the effect of antioxidants on their events remain obscure. Flow cytometry is a convenient method to investigate the order of events among various functions occurring in MPT using a limited amount of mitochondria (200 microl of 0.02 mg protein/ml) without contamination by other organelles. Flow cytometric analysis revealed that Ca(2+) sequentially induced ROS generation, depolarization, swelling and Ca(2+) release in mitochondria by a cyclosporine A-inhibitable mechanism. These results were supported by the finding that Ca(2+)-induced MPT was inhibited by antioxidants, such as glutathione and N-acetylcysteine. It was also revealed that various inhibitors of Ca(2+)-induced phospholipase A(2) suppressed all of the events associated with Ca(2+)-induced MPT. These results suggested that ROS generation and phospholipase A(2) activation by Ca(2+) underlie the mechanism of the initiation of MPT.
RESUMO
The purpose of this study was to profile gene expression in periapical lesions during root canal treatment (RCT). Periapical lesions were induced experimentally by exposing the pulp in Sprague-Dawley rats. After 3 wk, the animals received root canal filling (RCF) and were sacrificed 1 or 4 wk later. From the periapical tissues, total RNA was extracted and processed for cDNA-microarray analysis. The lesions were histologically and radiographically confirmed to expand 4 wk after pulp exposure (inflammation phase) and to stabilize 4 wk after RCF (healing phase). In approximately 30,000 genes on the microarray, 203 genes were up-regulated to more than 5-fold (e.g., IL-1beta), and 864 genes were down-regulated to less than 20% of baseline level (e.g., caspase 8) in inflammation phase. Compared with inflammation phase, we found that 133 genes were up-regulated (e.g., IL-1alpha) and 50 genes were down-regulated (e.g., defensin alpha5) in healing phase. Corresponding to the gene expression profiles, accumulation of IL-1alpha and IL-1beta was observed in the periapical lesions by immunohistochemistry. These gene profiles might be useful in diagnosing the healing process of periapical lesions.
Assuntos
Perfilação da Expressão Gênica , Periodontite Periapical/genética , Tratamento do Canal Radicular , Cicatrização/genética , Animais , Modelos Animais de Doenças , Regulação para Baixo , Técnicas Imunoenzimáticas , Interleucina-1/biossíntese , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Periodontite Periapical/terapia , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
To clarify the development of follicular growth and atresia in the immature ovary, rats. ovaries and blood were removed at fixed points during the period from 0 to 35 days after birth (Day 0 to Day 35). The ovaries were immunohistochemically examined, and blood concentrations of serum follicle-stimulating hormone (FSH) and estrogen (E) were measured. We investigated how time-course changes in follicular cell proliferation, estrogen receptor beta (ERbeta), apoptosis, and FSH and E concentrations are connected with follicular growth and atresia. Apoptosis was found in the ova from Day 0 to Day 3. On Day 15, apoptosis occurred in some granulosa cell nuclei in some follicles, but BrdU uptake and the presence of cyclin D2 and ER beta could be observed in other granulosa cells. From Day 17, apoptosis increased in the follicular granulosa cells, and BrdU uptake and the presence of cyclin D2 and ERbeta were decreased. Follicular atresia continued, reaching a peak on Day 30. Serum FSH and E concentrations increased until Day 15, then markedly decreased after Day 17. The mechanism of apoptosis in the ova from Day 0 to 3 has not been clarified. However, the onset of follicular atresia was caused by apoptotic degeneration from Days 15 to 17. These results showed that the oocytes were selected by apoptosis at 2 points in the time-course of the maturation of the ovary.
Assuntos
Atresia Folicular/fisiologia , Folículo Ovariano/citologia , Folículo Ovariano/crescimento & desenvolvimento , Ovário/citologia , Ovário/crescimento & desenvolvimento , Animais , Animais Recém-Nascidos , Apoptose/fisiologia , Senescência Celular/fisiologia , Ciclina D2 , Ciclinas/metabolismo , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Estrogênios/sangue , Feminino , Hormônio Foliculoestimulante/sangue , Folículo Ovariano/metabolismo , Ovário/fisiologia , Gravidez , Ratos , Ratos Wistar , Fatores de TempoRESUMO
The aim of the present work is to clarify the mechanism(s) that regulates the accumulation of protoporphyrin IX (PpIX) in human histiocytic lymphoma cell line U937 incubated with 5-aminolevulinic acid (ALA). Biosynthesis and accumulation of PpIX in the cells was determined after incubation with 0.1-5 mM ALA using a flow cytometric technique. The synthesized endogenous PpIX was found to localize predominantly in the mitochondrial region of the cells. The ALA-enhanced PpIX synthesis was suppressed by the presence of either beta-alanine, a competitive inhibitor of beta-transporters on cell membranes, or carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, an uncoupler of mitochondrial oxidative phosphorylation. In contrast, cellular accumulation of PpIX was enhanced by the presence of either deferoxamine (an iron chelater), MnCl2 (a ferrochelatase inhibitor), or Sn-mesoporphyrin (heme oxygenase inhibitor). These results suggest that ALA-enhanced accumulation of PpIX in U937 cells was regulated by cellular uptake and conversion of ALA to PpIX and by degradation of Heme.
Assuntos
Ácido Aminolevulínico/metabolismo , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Protoporfirinas/metabolismo , Cloretos , Desferroxamina , Humanos , Compostos de Manganês , Mesoporfirinas , Células U937RESUMO
Although cAMP protects neuronal cells from various apoptotic stimulations, its mechanism is not fully elucidated. We report here the molecular mechanism of the 6-hydroxydopamine (6-OHDA)-induced apoptosis of pheochromocytoma PC12 cells and its suppression by 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (pCPT-cAMP), which is a membrane permeable cAMP analog. Treatment of PC12 cells with 6-OHDA resulted in the activation of caspases and apoptosis, as detected by chromatin condensation. 6-OHDA also induced superoxide generation, Bid cleavage and mitochondrial membrane depolarization. In addition, Akt phosphorylation that was favorable to cell survival was decreased and p38 MAPK phosphorylation was increased by 6-OHDA. PC12 cell apoptosis was inhibited by pCPT-cAMP, Z-VAD-fmk (a broad-range caspase inhibitor) and tiron (a superoxide scavenger), although PC12 cell apoptosis was not inhibited by cyclosporine A (an inhibitor of mitochondrial membrane permeability transition). Moreover, pCPT-cAMP promoted Akt phosphorylation, but it did not prevent superoxide generation and mitochondrial membrane depolarization. Conversely, LY294002, an inhibitor of Akt upstream molecule PI3-kinase, enhanced 6-OHDA-induced apoptosis. These results indicated that the 6-OHDA-induced apoptosis of PC12 cells was initiated by superoxide generation followed by caspase cascade activation, which was associated with the suppressed Akt phosphorylation and increased p38 phosphorylation. It is likely that pCPT-cAMP prevented the 6-OHDA-induced apoptosis via activation of the PI3-kinase/Akt pathway without any effect on superoxide generation or mitochondrial membrane depolarization.
Assuntos
Adrenérgicos/farmacologia , Apoptose/efeitos dos fármacos , AMP Cíclico/análogos & derivados , Inibidores Enzimáticos/farmacologia , Oxidopamina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tionucleotídeos/farmacologia , Animais , Apoptose/fisiologia , Western Blotting/métodos , Caspases/metabolismo , AMP Cíclico/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Ativação Enzimática/efeitos dos fármacos , Citometria de Fluxo/métodos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Células PC12 , Fosfatidilinositol 3-Quinases , Ratos , Espécies Reativas de Oxigênio/metabolismo , Fatores de TempoRESUMO
Mitochondria play an important role in apoptosis by generating reactive oxygen species (ROS) and inducing membrane permeability transition (MPT). Recent studies on alpha-lipoic acid (LA) and its reduced form, dihydrolipoic acid, suggest that these agents (LAs) inhibit apoptosis of cells by means of their antioxidant activity. On the other hand, LAs also stimulate Ca2+-dependent mitochondrial MPT and induce apoptosis of certain cells. Thus, the role of LAs in apoptotic cell death remains obscure. We investigated the mechanism of LA-induced MPT of mitochondria. Biochemical analysis revealed, in the presence of Ca2+, inorganic phosphate and succinate, LA induced uncoupling of oxidative phosphorylation, stimulated oxidation of pyridine nucleotides and enhanced Ca2+-induced MPT, as characterized by decrease in Ca2+ loading, ROS generation, oxidation of thiol groups of adenine nucleotide translocator, membrane depolarization, swelling, and cytochrome c release in an incubation time and concentration dependent manner. LA also stimulated hydroxyl radical-induced MPT in a alpha-tocopherol-inhibitable manner. Cyclosporine A, a potent inhibitor of mitochondrial MPT, inhibited all these events induced by LA. These results indicate that, under certain conditions, LA stimulates Ca2+-induced MPT through the decrease in loading capacity of Ca2+ and that MPT is involved in LA-induced apoptotic cell death. Since fairly high doses of LA have been used as a dietary supplement, the possible occurrence of such side effects, including mitochondrial dysfunction and induction of apoptosis in normal tissues, should be studied.
Assuntos
Cálcio/metabolismo , Permeabilidade da Membrana Celular/fisiologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ácido Tióctico/administração & dosagem , Animais , Antioxidantes/administração & dosagem , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Masculino , Oxirredução/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
Nitric oxide (NO) is a free radical which is produced from a wide variety of cells and tissues in the human body. NO is involved in the regulation of many physiological processes, such as vascular relaxation, neurotransmission, immune regulation, and cell death. NO is generated by nitric oxide synthase (NOS), which has three identified isoforms: neuronal type NOS (nNOS), endothelial type NOS (eNOS), and inducible type NOS (iNOS). Different isoforms are expressed depending on the organs, tissues, and cells, and investigation of the types and functions of enzymes expressed in various tissues is underway. The oral cavity is a space in which marked changes have been detected in NO levels, and each tissue is constantly influenced by NO. NO is a component of saliva and is produced by oral bacteria in the oral cavity and released by NOS expressed in oral mucosa. NOS isoforms expressed under normal conditions differ among the oral organs. In addition, the overexpression of NOS was involved in carcinogenesis and tumor growth progression. This review summarized the expression of NOS and functions of NO in oral cavity organs, and their roles in diseases and the influences of treatments.