RESUMO
UNLABELLED: Estradiol 17ß-D-glucuronide (E(2)17G) is an endogenous, cholestatic metabolite that induces endocytic internalization of the canalicular transporters relevant to bile secretion: bile salt export pump (Bsep) and multidrug resistance-associated protein 2 (Mrp2). We assessed whether phosphoinositide 3-kinase (PI3K) is involved in E(2)17G-induced cholestasis. E(2)17G activated PI3K according to an assessment of the phosphorylation of the final PI3K effector, protein kinase B (Akt). When the PI3K inhibitor wortmannin (WM) was preadministered to isolated rat hepatocyte couplets (IRHCs), it partially prevented the reduction induced by E(2)17G in the proportion of IRHCs secreting fluorescent Bsep and Mrp2 substrates (cholyl lysyl fluorescein and glutathione methylfluorescein, respectively). 2-Morpholin-4-yl-8-phenylchromen-4-one, another PI3K inhibitor, and an Akt inhibitor (Calbiochem 124005) showed similar protective effects. IRHC immunostaining and confocal microscopy analysis revealed that endocytic internalization of Bsep and Mrp2 induced by E(2)17G was extensively prevented by WM; this effect was fully blocked by the microtubule-disrupting agent colchicine. The protection of WM was additive to that afforded by the classical protein kinase C (cPKC) inhibitor 5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propanenitrile (Gö6976); this suggested differential and complementary involvement of the PI3K and cPKC signaling pathways in E(2)17G-induced cholestasis. In isolated perfused rat liver, an intraportal injection of E(2)17G triggered endocytosis of Bsep and Mrp2, and this was accompanied by a sustained decrease in the bile flow and the biliary excretion of the Bsep and Mrp2 substrates [(3)H]taurocholate and glutathione until the end of the perfusion period. Unlike Gö6976, WM did not prevent the initial decay, but it greatly accelerated the recovery to normality of these parameters and the reinsertion of Bsep and Mrp2 into the canalicular membrane in a microtubule-dependent manner. CONCLUSION: The PI3K/Akt signaling pathway is involved in the biliary secretory failure induced by E(2)17G through sustained internalization of canalicular transporters endocytosed via cPKC.
Assuntos
1-Fosfatidilinositol 4-Quinase/fisiologia , Colestase/induzido quimicamente , Proteína Quinase C/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Androstadienos/farmacologia , Animais , Canalículos Biliares/efeitos dos fármacos , Canalículos Biliares/fisiologia , Sistema Biliar/metabolismo , Carbazóis/farmacologia , Colchicina/farmacologia , Endocitose/efeitos dos fármacos , Estradiol/análogos & derivados , Glutationa/metabolismo , Técnicas In Vitro , Masculino , Microtúbulos/efeitos dos fármacos , Microtúbulos/fisiologia , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Perfusão , Proteína Quinase C/antagonistas & inibidores , Ratos , Ratos Wistar , Transdução de Sinais , Ácido Taurocólico/metabolismo , WortmaninaRESUMO
Abstract Bile is primarily secreted in hepatocytes (i.e. the canalicular bile) and subsequently delivered to the intrahepatic bile ducts, where is modified by cholangiocytes (i.e. the ductal bile). Bile formation is the result of the coordinated interactions of membrane-transport systems that generate the vectorial movement of solutes and osmotically driven water molecules. Hepatocytes and cholangiocytes express aquaporins, specialized membrane channel proteins that facilitate the osmotic transport of water. In this review, we provide a summary of what is known on liver AQPs and their significance in canalicular and ductal bile formation under normal and pathological conditions.
Assuntos
Aquaporinas/fisiologia , Canalículos Biliares/citologia , Ductos Biliares Intra-Hepáticos/citologia , Bile/metabolismo , Hepatócitos/metabolismo , Animais , Aquaporinas/metabolismo , Canalículos Biliares/fisiologia , Ductos Biliares Intra-Hepáticos/fisiologia , Transporte Biológico Ativo , Água Corporal/metabolismo , Humanos , Hepatopatias/fisiopatologiaRESUMO
Hormonal control of the restoration of hepatocanalicular polarity in short-term cultured hepatocyte couplets was analyzed. One hour following isolation, couplets were unable to accumulate the fluorescent bile acid analogue, cholyl-lysyl-fluorescein (CLF), and showed a nonpolarized distribution of F-actin and mrp2 over the cell body. A progressive, time-dependent restoration of couplet-polarized function and morphology was reached after 4 hours of culture. Both dibutyryl cyclic adenosine monophosphate (DBcAMP) and the Ca(2+)-elevating compound, thapsigargin, accelerated restoration of normal couplet morphology and function. The DBcAMP-mediated stimulus was inhibited by the Ca(2+) chelator, 1, 2-bis-(o-aminophenoxy)-ethene-N,N,N',N'-tetra-acetate tetra-(acetomethyl)ester (BAPTA/AM), but not by the protein kinase A (PKA) inhibitors, KT5720 or H89, suggesting that Ca(2+) elevation rather than PKA activation is involved. N-(6-aminohexyl-5-chloro-1-napththalenesulfonamide (W-7), a calmodulin inhibitor, and the protein kinase C (PKC) activator, phorbol dibutyrate, inhibited both the basal and the DBcAMP-stimulated recovery of functional polarity, whereas staurosporine and Gö 6976, 2 PKC inhibitors, accelerated the basal recovery of polarized function. Disruption of the microtubule cytoskeleton by colchicine induced only minor changes under basal, but not under DBcAMP-stimulated, conditions. The Golgi complex disruptor, brefeldin A, significantly delayed, and the microfilament-disrupting agent, cytochalasin D, fully blocked, both processes. However, DBcAMP stimulated trafficking of vesicles containing CLF to the pericanalicular region under the last condition. Our results indicate that restoration of couplet polarity following isolation occurs via a Ca(2+)-calmodulin-mediated mechanism, which depends on microfilament, but not on microtubule integrity. A second pathway is activated by DBcAMP activation via Ca(2+)-calmodulin formation, whose requirements with respect to cytoskeletal components are opposite. PKC has a negative regulatory role in both pathways.
Assuntos
Canalículos Biliares/metabolismo , Proteínas de Transporte/metabolismo , Citoesqueleto/fisiologia , Hepatócitos/metabolismo , Transdução de Sinais/fisiologia , Actinas/fisiologia , Animais , Proteínas de Transporte de Ânions , Canalículos Biliares/fisiologia , Membrana Celular/metabolismo , Células Cultivadas , Senescência Celular/fisiologia , Ácidos Cólicos/farmacocinética , Citoplasma/metabolismo , Fluoresceínas/farmacocinética , Corantes Fluorescentes/farmacocinética , Hormônios/fisiologia , Masculino , Ratos , Ratos Wistar , Vacúolos/fisiologiaRESUMO
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
Biliary solute concentrations measured at the tip of the cannula suffer a delay with respect to bile flow due to the transit time through the biliary tree volume. This study proposes a simple method, which is valid under variable bile flow conditions, to correct the distortion introduced by the biliary tree volume on the kinetic curves of the biliary excretion rate. The biliary transit time (tt) was calculated as the time needed to excrete a bile volume equal to the biliary tree volume by means of the interpolation of biliary cumulative volume versus time curves. Such tt permits one to estimate the canalicular concentration at time t, interpolating the biliary concentration curves at time t-tt. The product between the estimated canalicular concentration and the bile flow allows the calculation of the corrected biliary excretion rate. This method was evaluated by a comparison between biliary excretion rate curves of [14C]taurocholate [( 14C]TC) injected as a bolus under basal and sodium dehydrocholate (DHC)-induced choleresis conditions. Since the canalicular excretion rate of [14C]TC is considered independent of bile flow, the significant differences observed in its excretion kinetics under both conditions were attributed to distortion due to the biliary tree volume. After the correction, both curves showed a significant overlapping. This result indicates that the method improves the time-course representation of canalicular events in biliary excretion kinetic studies.