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1.
Cytokine ; 36(5-6): 245-53, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17376698

RESUMO

We have already demonstrated that interferon alfa-2b (IFN-alpha2b) induces apoptosis in isolated hepatocytes from preneoplastic rat livers via the secretion of transforming growth factor beta(1) (TGF-beta(1)), and this process is accompanied by caspase-3 activation. The aim of this study was to further investigate the mechanism of this activation. Isolated hepatocytes from preneoplastic livers induced DNA fragmentation in response to IFN-alpha2b, which was completely blocked when anti-TGF-beta(1) was added to the culture media. IFN-alpha2b mediated radical oxygen species (ROS) production that preceded the loss of mitochondrial transmembrane potential (DeltaPsi), release of cytochrome c, and activation of caspase-3. Bax levels increased in a time-dependent fashion, and Bcl-x(L) was down-regulated in the early hours of IFN-alpha2b treatment. The delayed translocation of Bid into the mitochondria was in concordance with late caspase-8 activation. In conclusion, endogenous TGF-beta(1) secreted under IFN-alpha2b stimulus seems to induce cytochrome c release through a mechanism related to Bcl-2 family members and loss of mitochondrial DeltaPsi. Bax protein could be responsible of the release of cytochrome c during the initial hours of IFN-alpha2b-induced apoptosis via TGF-beta(1). Activated Bid by caspases could amplificate the mitochondrial events, enhancing the release of cytochrome c.


Assuntos
Apoptose , Hepatócitos/citologia , Interferon-alfa/farmacologia , Neoplasias Hepáticas/metabolismo , Fígado/citologia , Lesões Pré-Cancerosas/metabolismo , Animais , Anexina A5/metabolismo , Caspase 3/metabolismo , Caspase 8/metabolismo , Células Cultivadas , Citocromos c/metabolismo , Fragmentação do DNA , Hepatócitos/metabolismo , Interferon alfa-2 , Masculino , Potencial da Membrana Mitocondrial , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes , Fator de Crescimento Transformador beta1/metabolismo
2.
Drug Metab Dispos ; 33(7): 888-91, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15843489

RESUMO

Ethinylestradiol (EE) induces cholestasis by affecting bile salt-dependent and -independent fractions of the bile flow. The decrease in bile salt-independent flow is thought to be due, in part, to a reduction in the expression of the canalicular transporter Mrp2. The impact of modulation of Mrp2 function by sodium ursodeoxycholate (UDC) in EE cholestasis is unknown. We evaluated the protective effect of UDC on EE-induced impairment of Mrp2 activity in vivo and in isolated hepatocytes, by using the substrate dinitrophenyl S-glutathione (DNP-SG). EE was administered to male Wistar rats at a dose of 5 mg/kg s.c. for 5 days. UDC was coadministered with EE at a dose of 25 mg/kg b.wt. i.p. for the same period. EE alone reduced DNP-SG biliary excretion by 55% when compared with controls. Coadministration with UDC partially restored the alteration. Secretion rate of DNP-SG was decreased by 30% in isolated hepatocytes from EE-treated rats, but, contrary to in vivo results, UDC coadministration did not restore DNP-SG transport, likely as a consequence of bile salt washout resulting from the isolation procedure. As a confirmation, tauroursodeoxycholate hepatocyte preloading significantly increased Mrp2 activity. Western blotting analysis of Mrp2 indicated that EE administration significantly reduced its level in total and plasma membranes and that UDC coadministration failed to revert this alteration. In conclusion, UDC improvement in Mrp2 transport activity in vivo likely derived from a direct enhancement of Mrp2 function rather than from a restoration of its expression levels. This provides a novel mechanism explaining the beneficial effects of UDC in EE-induced cholestasis.


Assuntos
Colestase/induzido quimicamente , Etinilestradiol/efeitos adversos , Proteínas Mitocondriais/fisiologia , Proteínas Ribossômicas/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Ácido Ursodesoxicólico/farmacologia , Animais , Bile/metabolismo , Hepatócitos/metabolismo , Masculino , Proteínas Mitocondriais/metabolismo , Transporte Proteico , Ratos , Ratos Wistar , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
3.
Hepatology ; 40(2): 394-402, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15368444

RESUMO

In previous work we showed that interferon alfa-2b (IFN-alpha2b) increases apoptosis on rat hepatic preneoplastic foci. The aim of this study was to determine if transforming growth factor beta1 (TGF-beta1) was involved in the programmed cell death on the foci. Animals were divided into 6 groups: subjected to a 2-phase model (diethylnitrosamine plus 2-acetylaminofluorene) of preneoplasia development (group 1); treated with IFN-alpha2b during the 2 phases (group 2); treated with IFN-alpha2b during initiation with diethylnitrosamine (group 3); treated with IFN-alpha2b during 2-acetylaminofluorene administration (group 4); subjected only to an initiation stage (group 5); and treated with IFN-alpha2b during the initiation period (group 6). Serum TGF-beta1 levels were increased in IFN-alpha2b-treated rats. Immunohistochemical studies showed that IFN-alpha2b significantly increased the quantity of TGF-beta1-positive hepatocytes in groups 2 to 4. Phosphorylated-Smads-2/3 (p-Smads-2/3) proteins in liver nuclear extracts were significantly elevated. To determine the source of TGF-beta1, isolated hepatocytes, Kupffer cells, and peritoneal macrophages from animals in groups 1 and 5 were cultured with or without IFN-alpha2b. IFN-alpha2b stimulus induced several-fold increases of TGF-beta1 secretion from hepatocytes. Neither Kupffer cells nor peritoneal macrophages secreted detectable TGF-beta1 levels when they were treated with IFN-alpha2b. IFN-alpha2b-stimulated cultured hepatocytes from preneoplastic livers showed enhanced apoptosis, measured by fluorescence microscopy and caspase-3 activity. They presented higher nuclear accumulation of p-Smads-2/3, indicating increased TGF-beta1 signaling. When anti-TGF-beta1 was added to the culture media, TGF-beta1 activation and apoptosis induced by IFN-alpha2b were blocked. In conclusion, IFN-alpha2b-induced production of TGF-beta1 by hepatocytes from preneoplastic liver is involved in the apoptotic elimination of altered hepatic foci.


Assuntos
Apoptose , Interferon-alfa/farmacologia , Neoplasias Hepáticas Experimentais/fisiopatologia , Fígado/efeitos dos fármacos , Fígado/fisiopatologia , Lesões Pré-Cancerosas/fisiopatologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Caspase 3 , Caspases/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Meios de Cultura/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Interferon alfa-2 , Fígado/metabolismo , Masculino , Fosforilação , Ratos , Ratos Wistar , Proteínas Recombinantes , Proteína Smad2 , Proteína Smad3 , Transativadores/metabolismo , Fator de Crescimento Transformador beta/sangue , Fator de Crescimento Transformador beta1
4.
Hepatology ; 37(5): 1026-33, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12717383

RESUMO

Hepatocytes express the water channel aquaporin-8 (AQP8), which is mainly localized in intracellular vesicles, and its adenosine 3',5'-cyclic monophosphate (cAMP)-induced translocation to the plasma membrane facilitates osmotic water movement during canalicular bile secretion. Thus, defective expression of AQP8 may be associated with secretory dysfunction of hepatocytes caused by extrahepatic cholestasis. We studied the effect of 1, 3, and 7 days of bile duct ligation (BDL) on protein expression, subcellular localization, and messenger RNA (mRNA) levels of AQP8; this was determined in rat livers by immunoblotting in subcellular membranes, light immunohistochemistry, immunogold electron microscopy, and Northern blotting. One day of BDL did not affect expression or subcellular localization of AQP8. Three days of BDL reduced the amount of intracellular AQP8 (75%; P <.001) without affecting its plasma membrane expression. Seven days after BDL, AQP8 was markedly decreased in intracellular (67%; P <.05) and plasma (56%; P <.05) membranes. Dibutyryl cAMP failed to increase AQP8 in plasma membranes from liver slices, suggesting a defective translocation of AQP8 in 7-day BDL rats. Immunohistochemistry and immunoelectron microscopy in liver sections confirmed the BDL-induced decreased expression of hepatocyte AQP8 in intracellular vesicles and canalicular membranes. AQP8 mRNA expression was unaffected by 1-day BDL but was significantly increased by about 200% in 3- and 7-day BDL rats, indicating a posttranscriptional mechanism for protein level reduction. In conclusion, BDL-induced extrahepatic cholestasis caused posttranscriptional down-regulation of hepatocyte AQP8 protein expression. Defective expression of AQP8 water channels may contribute to bile secretory dysfunction of cholestatic hepatocytes.


Assuntos
Aquaporinas/metabolismo , Colestase Extra-Hepática/metabolismo , Hepatócitos/metabolismo , Canais Iônicos , Animais , Aquaporinas/genética , Ductos Biliares , Colestase Extra-Hepática/fisiopatologia , Regulação para Baixo , Expressão Gênica , Ligadura , Fígado/citologia , Fígado/metabolismo , Masculino , RNA Mensageiro/análise , Ratos , Ratos Wistar , Frações Subcelulares/metabolismo
5.
Ren Fail ; 24(2): 111-26, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12071586

RESUMO

The purpose of this study was to assess the effects of the cyclooxygenase inhibitor, indomethacin, and some vasoactive agents on the renal functional parameters during the early liver injury induced by four days bile duct ligation (BDL). Wistar rats with four days-BDL and control-sham operated were used. Renal function was measured in anesthetized rat treated with a single dose of indomethacin (control, 0.3, 1.0, 3.0 mg/kg b.w.; i.v.) one hour before clearance studies. Sulindac effects were also evaluated (5 mg/kg b.w., i.p). Isolated rat kidney preparations from control and BDL donor rats were used to study renal vascular response to noradrenaline, carbachol or sodium nitroprusside. The bile duct ligation promoted a diminished renal cortical plasma flow (RCPF) on the fourth day post surgery accompanied with a diminution in the glomerular filtration rate (GFR), increased filtration fraction and increased fractional excretion of water and sodium. Indomethacin 0.3 mg/kg induced an increase in GFR and RCPF, maintaining the high filtration fraction in BDL rats. The other doses did not alter these parameters as compared with bile duct ligated rats without treatment, but indomethacin 3 mg/kg caused a significant increase in filtration fraction. Indomethacin induced dose-dependent diminution in natriuresis in sham and BDL groups. Sulindac did not modify hemodynamic parameters, but induced antinatriuresis and antidiuresis in both experimental groups. Maximal vascular responses to noradrenaline measured in isolated rat kidneys were statistically diminished in BDL-rats as compared with controls (C, n=7; 35.0+/-2.3 mmHg ml(-1) min; BDL-rats, n=5; 23.8+/-0.7 mmHg ml(-1) min; p<0.02), without changes in EC15. Maximal relaxation induced by sodium nitroprusside in the phenylephrine (PHE)-pre-constricted renal vasculature in control preparations did not differ from that observed in BDL group (C: n=6; 49.5+/-2.3%). Values of EC50 were 1.26+/-0.07 microM (n=6) in control preparations and 0.34+/-0.03 microM (n=4) in kidneys from BDL-rats (p<0.001). Carbachol induced a biphasic relaxation of PHE-pre-constricted renal vasculature. No differences in maximal responses were found. EC50 value of the second phase in BDL group was significantly decreased compared to control preparations (C: n=6, 0.47+/-0.05 microM; BDL: n=6, 0.22+/-0.03 microM p<0.001). The present results show that the altered renal function after a short time post bile duct ligation is determined, at least in part, by increased release of arachidonic derivatives in vascular bed and tubular cells. At this stage of liver injury, the alteration in the renal vascular response to different vasoactive agents is remarkable.


Assuntos
Ductos Biliares/fisiopatologia , Ductos Biliares/cirurgia , Inibidores de Ciclo-Oxigenase/farmacologia , Indometacina/farmacologia , Rim/efeitos dos fármacos , Rim/fisiopatologia , Fígado/lesões , Fígado/fisiopatologia , Vasoconstritores/farmacologia , Animais , Ductos Biliares/efeitos dos fármacos , Modelos Animais de Doenças , Taxa de Filtração Glomerular/efeitos dos fármacos , Taxa de Filtração Glomerular/fisiologia , Ligadura/efeitos adversos , Fígado/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar , Insuficiência Renal/etiologia , Insuficiência Renal/fisiopatologia , Fluxo Plasmático Renal/efeitos dos fármacos , Fluxo Plasmático Renal/fisiologia , Fatores de Tempo
6.
Anal Biochem ; 302(1): 104-7, 2002 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-11846382

RESUMO

We previously found that hepatocytes are able to control their osmotic membrane water permeability (P(f)) by regulating the number of surface aquaporin water channels. Hepatocyte P(f) has been assessed by phase-contrast microscopy and cell image analysis, an established but relatively laborious procedure. We report here an alternative method to assess hepatocyte P(f) based on a single silicone layer filtering centrifugation system. Isolated rat hepatocytes were incubated in hypotonic or isotonic buffers containing (3)H(2)O as a tracer and, then, were filtered by rapid centrifugation through a silicone layer down to a lysis layer. Osmotically driven radioactivity (i.e., (3)H(2)O) within hepatocytes was calculated as the difference between the dpm in lysis media measured under hypotonic and isotonic conditions. The P(f) calculated from the initial slope of the radioactivity-versus-time curve was 18 microm/s at 4 degrees C. Hepatocytes treated with dibutyryl cyclic AMP, to increase P(f) through the plasma membrane insertion of aquaporins, showed an increased P(f) value of 37 microm/s. The aquaporin blocker dimethyl sulfoxide selectively prevented the agonist-induced hepatocyte P(f). These data are in good agreement with the corresponding values determined by quantitative phase-contrast microscopy; thus, the method developed allows the rapid and reliable measurement of hepatocyte P(f).


Assuntos
Permeabilidade da Membrana Celular , Membrana Celular/metabolismo , Hepatócitos/metabolismo , Água/metabolismo , Animais , Aquaporinas , Centrifugação/métodos , Óxido de Deutério , Filtração , Hepatócitos/ultraestrutura , Masculino , Ratos , Ratos Wistar , Silicones
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