RESUMO
Hepatocytes are epithelial cells whose apical poles constitute the bile canaliculi. The establishment and maintenance of canalicular poles is a finely regulated process that dictates the efficiency of primary bile secretion. Protein kinase A (PKA) modulates this process at different levels. AKAP350 is an A-kinase anchoring protein that scaffolds protein complexes involved in modulating the dynamic structures of the Golgi apparatus and microtubule cytoskeleton, facilitating microtubule nucleation at this organelle. In this study, we evaluated whether AKAP350 is involved in the development of bile canaliculi-like structures in hepatocyte derived HepG2 cells. We found that AKAP350 recruits PKA to the centrosomes and Golgi apparatus in HepG2 cells. De-localization of AKAP350 from these organelles led to reduced apical cell polarization. A decrease in AKAP350 expression inhibited the formation of canalicular structures and impaired F-actin organization at canalicular poles. Furthermore, loss of AKAP350 expression led to diminished polarized expression of the p-glycoprotein (MDR1/ABCB1) at the apical "canalicular" membrane. AKAP350 knock down effects on canalicular structures formation and actin organization could be mimicked by inhibition of Golgi microtubule nucleation by depletion of CLIP associated proteins (CLASPs). Our data reveal that AKAP350 participates in mechanisms which determine the development of canalicular structures as well as accurate canalicular expression of distinct proteins and actin organization, and provide evidence on the involvement of Golgi microtubule nucleation in hepatocyte apical polarization.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Canalículos Biliares/metabolismo , Canalículos Biliares/ultraestrutura , Polaridade Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Hepatócitos/metabolismo , Hepatócitos/ultraestrutura , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Centrossomo/metabolismo , Centrossomo/ultraestrutura , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Imunofluorescência , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Células Hep G2 , Humanos , Immunoblotting , Microscopia Confocal , Microscopia de FluorescênciaRESUMO
The effect of vasopressin (VP) on canalicular function and hepatocellular morphology, with particular regard to actin cytoskeletal organization and the concomitant plasma membrane bleb formation, was studied in isolated rat hepatocyte couplets. VP induced the concentration-dependent formation of multiple plasma membrane blebs as well as simultaneous impairment in both canalicular vacuolar accumulation (cVA) and retention (cVR) of the fluorescent bile acid, cholyl-lysyl-fluorescein (CLF), which evaluate couplet secretory function and tight-junction integrity, respectively. These effects were mimicked by the protein kinase C (PKC) activator, phorbol dibutyrate (PDB), but not by the protein kinase A (PKA) activator, dibutyryl-cAMP. VP-induced bleb formation and canalicular dysfunction were fully prevented by the protein kinase inhibitor, H-7, but not by the PKA inhibitor, KT5720, further suggesting a specific role of PKC. VP-induced alterations were also prevented by pretreatment with the Ca2+-buffering agent, BAPTA/AM, but not with the calmodulin-dependent protein kinase II antagonist, calmidazolium. Neither the Ca2+-activated neutral protease inhibitor, leupeptin, nor the antioxidants, alpha-tocopherol or deferoxamine, were able to prevent either VP-induced plasma membrane blebbing or canalicular dysfunction. The Ca2+-ionophore, A23187, mimicked the VP-induced alterations, but its harmful effects were completely prevented by H-7. Bleb formation induced by VP and PDB was accompanied by an extensive redistribution of filamentous actin from the pericanalicular area to the cell body, and this effect was fully prevented by H-7. These results suggest that VP-induced canalicular and cytoskeletal dysfunction is mediated by PKC and that classical (Ca2+-dependent) PKC appear to be involved because intracellular Ca2+ is required for VP to induce its harmful effects.
Assuntos
Actinas/metabolismo , Canalículos Biliares/fisiologia , Carbazóis , Citoesqueleto/ultraestrutura , Fígado/fisiologia , Proteína Quinase C/metabolismo , Junções Íntimas/fisiologia , Vasopressinas/farmacologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Antioxidantes/farmacologia , Canalículos Biliares/efeitos dos fármacos , Canalículos Biliares/ultraestrutura , Bucladesina/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Células Cultivadas , Quelantes/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citoesqueleto/efeitos dos fármacos , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Indóis/farmacologia , Leupeptinas/farmacologia , Fígado/efeitos dos fármacos , Fígado/ultraestrutura , Masculino , Microscopia Eletrônica de Varredura , Dibutirato de 12,13-Forbol/farmacologia , Pirróis/farmacologia , Ratos , Ratos Wistar , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/ultraestrutura , Vacúolos/efeitos dos fármacos , Vacúolos/fisiologia , Vacúolos/ultraestruturaRESUMO
Cholestasis is associated with a marked increase in the release of canalicular membrane enzymes into bile. This phenomenon has been related to an increased lability of these canalicular membrane integral proteins to the solubilizing effects of secreted bile salts. To further characterize the effects of oral ursodeoxycholic acid (UDCA) administration on ethynylestradiol (EE)-induced cholestasis, the influence of this bile acid on changes in biliary excretion of membrane-bound enzymes was investigated. Bile flow, basal bile salt and biliary lipid secretory rates, the maximum secretory rate of taurocholate (TC SRm), and the biliary excretion of the canalicular membrane-bound ectoenzymes alkaline phosphatase (ALP) and gamma-glutamyl transpeptidase (GGT) were measured in rats after EE and/or UDCA administration. The activities of ALP, GGT and Na+,K(+)-ATPase in purified isolated canalicular and sinusoidal membrane fractions and the ultrastructure of hepatic acinus, including histochemical studies of ALP distribution, were also examined. EE significantly reduced bile flow, bile salt and biliary lipid secretory rates, and TC SRm, and caused dilatation and loss of microvilli at the canalicular pole of hepatocytes. Biliary excretion of ALP increased 2-fold, whereas biliary excretion of GGT was unchanged. The relationship between biliary excretion of ALP or GGT and bile salt secretion (units of enzyme activity secreted per nanomole of bile salt) was greater in EE-treated rats compared with controls (2.1- and 1.5-fold greater for ALP and GGT, respectively), indicating that in EE-induced cholestasis more enzyme was released into bile per nanomole of bile salt. Na+,K(+)-ATPase activity in sinusoidal membrane fraction was reduced significantly, whereas ALP activity increased in both membrane fractions in EE-treated rats. The histochemical distribution of ALP in the acinus showed a strong reaction in acinar zone 3 and at both the canalicular and sinusoidal membranes. Oral administration of UDCA prevented EE-induced bile secretory failure by normalizing bile flow, bile salt and biliary phospholipid secretory rates, and TC SRm. UDCA also prevented the EE-induced changes in the biliary excretion of enzymes. On the contrary, UDCA did not modify either the enzyme activity in isolated membrane fractions or the morphological or ALP histochemical changes associated with EE administration. These data indicate that in EE-induced cholestasis changes occur at the canalicular membrane, enabling this portion of the plasma membrane to be more susceptible to the solubilizing effect of bile salt, and that oral administration of UDCA prevents bile secretory failure and changes in the biliary excretion of ALP and GGT in EE-treated rats.
Assuntos
Canalículos Biliares/enzimologia , Bile/enzimologia , Colestase/enzimologia , Fígado/enzimologia , Ácido Ursodesoxicólico/administração & dosagem , Fosfatase Alcalina/análise , Animais , Ácidos e Sais Biliares/análise , Canalículos Biliares/ultraestrutura , Fracionamento Celular , Membrana Celular/enzimologia , Colestase/induzido quimicamente , Etinilestradiol , Masculino , Ratos , Ratos Wistar , Taxa Secretória , gama-Glutamiltransferase/análiseRESUMO
BACKGROUND/AIMS: Bile salts (BS) are cytotoxic agents, but cell damage is not observed in the hepatobiliary system. We hypothesized that biliary lipid vesicles (unilamellae and multilamellae) could have a protective role against BS-induced cytotoxicity. METHODS: Biliary lipid lamellar secretion was induced by feeding rats with 0.5% diosgenin. Cytoprotection was assessed in bile duct-obstructed rats and by incubating human erythrocytes with sodium taurocholate. RESULTS: Biliary cholesterol concentration increased > 300% in diosgenin-fed rats; electron microscopic examination showed a great abundance of lipid lamellar vesicles in bile and within the canaliculi. After bile duct obstruction, serum hepatic enzyme activities were significantly lower in diosgenin-fed rats. Histologically severe and confluent hepatocellular necrosis was only observed in control rats. Biliary lamellar lipid material significantly reduced the BS-induced hemolytic effect in vitro in a concentration-dependent manner. This protective effect correlated to a progressive decrease in the intermicellar BS concentration. Phosphatidylcholine or cholesterol, alone or as lamellar structures, also showed cytoprotective effect in vitro but always less than native biliary lamellae. CONCLUSIONS: These results support the concept that native biliary cholesterol phospholipid lamellae represent an important cytoprotective factor for hepatocytes and biliary epithelial cells against BS-induced damage.