RESUMO
Cholestasis is a cardinal complication of liver disease, but most treatments are merely supportive. Here we report that the sulfonylurea glybenclamide potently stimulates bile flow and bicarbonate excretion in the isolated perfused rat liver. Video-microscopic studies of isolated hepatocyte couplets and isolated bile duct segments show that this stimulatory effect occurs at the level of the bile duct epithelium, rather than through hepatocytes. Measurements of cAMP, cytosolic pH, and Ca2+ in isolated bile duct cells suggest that glybenclamide directly activates Na+-K+-2Cl- cotransport, rather than other transporters or conventional second-messenger systems that link to secretory pathways in these cells. Finally, studies in livers from rats with endotoxin- or estrogen-induced cholestasis show that glybenclamide retains its stimulatory effects on bile flow and bicarbonate excretion even under these conditions. These findings suggest that bile duct epithelia may represent an important new therapeutic target for treatment of cholestatic disorders.
Assuntos
Ductos Biliares/fisiopatologia , Bile/metabolismo , Colestase/fisiopatologia , Glibureto/farmacologia , Hipoglicemiantes/farmacologia , Animais , Ductos Biliares/efeitos dos fármacos , Ductos Biliares/metabolismo , Transporte Biológico/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fígado/metabolismo , Fígado/patologia , Fígado/fisiopatologia , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND & AIMS: Bile secretion is regulated in part by adenosine 3',5'-cyclic monophosphate (cAMP) and cytosolic Ca2+ (Ca2+i). Hormone receptors that link to these second messengers are not uniformly distributed across the hepatic lobule, but both cAMP and Ca2+i cross gap junctions, so we tested whether gap junctional communication plays a role in changes in bile flow induced by the activation of these receptors. METHODS: cAMP levels in isolated perfused rat livers were increased by using glucagon, because glucagon receptors are predominantly on pericentral hepatocytes, or by using dibutyryl cAMP, which acts on hepatocytes throughout the hepatic lobule. Ca2+i concentration was increased by using vasopressin, because V1a receptors are most heavily expressed on pericentral hepatocytes, or by using 2,5-di(tert-butyl)-1, 4-benzo-hydroquinone (t-BuBHQ), which increases the Ca2+i concentration in hepatocytes throughout the hepatic lobule. We used 18alpha-glycyrrhetinic acid (alphaGA) to block gap junction conductance, which was assessed by fluorescence recovery after photobleaching. RESULTS: alphaGA blocked fluorescence recovery after photobleaching without altering the basal rate of bile flow. Glucagon and dibutyryl cAMP increased bile flow; alphaGA blocked the glucagon-induced increase but not that induced by dibutyryl cAMP. Vasopressin and t-BuBHQ decreased bile flow; alphaGA exacerbated the decrease induced by vasopressin but not by t-BuBHQ. CONCLUSIONS: Glucagon and vasopressin modulate bile flow in a manner that depends in part on gap junctional communication, even though the two hormones activate second messengers with opposing effects on bile flow. The organization of second messenger signals across the hepatic lobule may be an important component of hormonal regulation of bile secretion.
Assuntos
Bile/metabolismo , Comunicação Celular/fisiologia , Junções Comunicantes/fisiologia , Fígado/fisiologia , Animais , Bucladesina/farmacologia , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Comunicação Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fluoresceínas , Junções Comunicantes/efeitos dos fármacos , Glucagon/farmacologia , Glucose/metabolismo , Ácido Glicirretínico/análogos & derivados , Ácido Glicirretínico/farmacologia , Hidroquinonas/farmacologia , Técnicas In Vitro , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Perfusão , Ratos , Ratos Sprague-Dawley , Vasopressinas/farmacologiaRESUMO
In contrast to organic anions, substrates for the canalicular mdr1a and b are usually organic cations and are often sequestered in high concentrations in intracellular acidic compartments. Because many of these compounds are therapeutic agents, we investigated if their sequestration could be regulated. We used isolated perfused rat liver (IPRL), isolated rat hepatocyte couplets (IRHC), and WIF-B cells to study the cellular localization and biliary excretion of the fluorescent cation, daunorubicin (DNR). Despite rapid (within 15 minutes) and efficient (>90%) cellular uptake in the IPRL, only approximately 10% of the dose administered (0.2-20 micromol) was excreted in bile after 85 minutes. Confocal microscopy revealed fluorescence predominantly in vesicles in the pericanalicular region in IPRL, IRHC, and WIF-B cells. Treatment of these cells with chloroquine and bafilomycin A, agents that disrupt the pH gradient across the vesicular membrane, resulted in a loss of vesicular fluorescence, reversible in the case of bafilomycin A. Taurocholate (TC) and dibutyryl cAMP (DBcAMP), stimulators of transcytotic vesicular transport, increased the biliary recovery of DNR significantly above controls, by 70% and 35%, respectively. The microtubule destabilizer, nocodazole, decreased biliary excretion of DNR. No effect on secretion was noted in TR- mutant rats deficient in mrp2. Coadministration of verapamil, an inhibitor of mdr1, also decreased DNR excretion. While TC and DBcAMP did not affect the fluorescent intensity or pattern of distribution in IRHC, nocodazole resulted in redistribution of DNR to peripheral punctuate structures. These findings suggest that the organic cation, DNR, is largely sequestered in cells such as hepatocytes, yet its excretion can still be modulated.
Assuntos
Canalículos Biliares/metabolismo , Daunorrubicina/metabolismo , Fígado/metabolismo , Macrolídeos , Proteínas Mitocondriais , Proteínas de Saccharomyces cerevisiae , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Antibacterianos/farmacologia , Antineoplásicos , Bile/metabolismo , Transporte Biológico , Bucladesina/farmacologia , Cátions , Cloroquina/farmacologia , Cinética , Masculino , Nocodazol/farmacologia , Ratos , Ratos Wistar , Proteínas Ribossômicas/fisiologia , Ácido Taurocólico/farmacologia , Verapamil/farmacologiaRESUMO
BACKGROUND/AIMS: Liver reperfusion following cold ischemia is frequently associated with diminished bile flow in patients undergoing liver transplantation. Glutathione is a major determinant of bile-acid independent bile flow, and the effects of cold ischemia on biliary glutathione excretion are unknown. METHODS: We examined the effects of cold ischemia (University of Wisconsin solution (4 degrees C), 24 h) with subsequent reperfusion (100 min) on biliary glutathione excretion in a recirculating system. Since glutathione might represent an important antioxidant within the biliary tract and oxidative stress in the biliary tract during reperfusion could contribute to the pathogenesis of bile duct injury after liver transplantation, we also assessed bile duct morphology in reperfused livers of mutant TR- -rats, in whom biliary excretion of glutathione is already impaired. RESULTS: Hepatic bile formation was diminished in reperfused Wistar rat livers after cold ischemia. Biliary glutathione concentrations and output were significantly decreased and correlated with postischemic changes in bile secretion. An increased biliary oxidized glutathione/glutathione ratio, indicating oxidative stress, was detected only immediately after the onset of reperfusion. Basal bile flow rates in TR- -rat livers which were already markedly reduced in control-perfused livers, decreased further during the early but not the later reperfusion period. Reperfusion of both Wistar and TR- -rat livers was not associated with electron microscopic evidence of bile duct damage. CONCLUSIONS: We conclude that impaired biliary excretion of glutathione contributes to decreased bile flow after cold ischemia. The absence of biliary glutathione does not appear to promote ultrastructural evidence of bile duct injury during reperfusion in the isolated perfused rat liver.
Assuntos
Ductos Biliares/ultraestrutura , Bile/metabolismo , Glutationa/fisiologia , Isquemia/fisiopatologia , Fígado/fisiologia , Soluções para Preservação de Órgãos , Reperfusão , Adenosina , Alopurinol , Animais , Ductos Biliares/fisiologia , Temperatura Baixa , Glutationa/metabolismo , Técnicas In Vitro , Insulina , Fígado/irrigação sanguínea , Masculino , Preservação de Órgãos , Rafinose , Ratos , Ratos Wistar , Análise de Regressão , Fatores de TempoRESUMO
Interleukin-6 (IL-6) is an important mediator of parathyroid hormone (PTH)-induced bone resorption. Serum levels of IL-6 and its soluble receptor (IL-6sR) are regulated in part by PTH. The PTH/PTH-related protein type 1 receptor is highly expressed in the liver, and in the current study we investigated whether the liver produces IL-6 or IL-6sR in response to PTH. Perfusion of the isolated rat liver with PTH-(1-84) stimulated rapid, dose-dependent production of bioactive IL-6 and the IL-6sR. These effects were observed at near physiological concentrations of the hormone such that 1 pM PTH induced hepatic IL-6 production at a rate of approximately 0.6 ng/min. In vitro, hepatocytes, hepatic endothelial cells, and Kupffer cells, but not hepatic stellate cells, were each found to produce both IL-6 and IL-6sR in response to higher (10 nM) concentrations of PTH. Our data suggest that hepatic-derived IL-6 and IL-6sR contribute to the increase in circulating levels of these cytokines induced by PTH in vivo and raise the possibility that PTH-induced, liver-derived IL-6 may exert endocrine effects on tissues such as bone.