Instituto de Fisiología Experimental (IFISE), Facultad de Ciencias Bioquímicas y Farmacéuticas (CONICET, U.N.R.), Suipacha 570, S2002LRL, Rosario, Argentina.
Dig Dis Sci. 2013 Jun;58(6):1602-14. doi: 10.1007/s10620-013-2558-4. Epub 2013 Jan 31.
Estradiol-17β-D-glucuronide (E17G) induces cholestasis in vivo, endocytic internalization of the canalicular transporters multidrug resistance-associated protein 2 (Abcc2) and bile salt export pump (Abcb11) being a key pathomechanism. Cyclic AMP (cAMP) prevents cholestasis by targeting these transporters back to the canalicular membrane. In hepatocyte couplets, glucagon and salbutamol, both of which increase cAMP, prevented E17G action by stimulating the trafficking of these transporters by different mechanisms, namely: glucagon activates a protein kinase A-dependent pathway, whereas salbutamol activates an exchange-protein activated by cAMP (Epac)-mediated, microtubule-dependent pathway.
The present study evaluated whether glucagon and salbutamol prevent E17G-induced cholestasis in a more physiological model, i.e., the perfused rat liver (PRL). Additionally, the preventive effect of in vivo alanine administration, which induces pancreatic glucagon secretion, was evaluated.
In PRLs, glucagon and salbutamol prevented E17G-induced decrease in both bile flow and the secretory activity of Abcc2 and Abcb11. Salbutamol prevention fully depended on microtubule integrity. On the other hand, glucagon prevention was microtubule-independent only at early time periods after E17G administration, but it was ultimately affected by the microtubule disrupter colchicine. Cholestasis was associated with endocytic internalization of Abcb11 and Abcc2, the intracellular carriers being partially colocalized with the endosomal marker Rab11a. This effect was completely prevented by salbutamol, whereas some transporter-containing vesicles remained colocalized with Rab11a after glucagon treatment. In vivo, alanine administration increased hepatic cAMP and accelerated the recovery of bile flow and Abcb11/Abcc2 transport function after E17G administration. The initial recovery afforded by alanine was microtubule-independent, but microtubule integrity was required to sustain this protective effect.
We conclude that modulation of cAMP levels either by direct administration of cAMP modulators or by physiological manipulations leadings to hormone-mediated increase of cAMP levels (alanine administration), prevents estrogen-induced cholestasis in models with preserved liver architecture, through mechanisms similar to those arisen from in vitro studies.
雌二醇-17β-D-葡糖苷酸（E17G）在体内诱导胆汁淤积，胆小管转运蛋白多药耐药相关蛋白 2（Abcc2）和胆汁盐输出泵（Abcb11）的内吞内化是关键的病理机制。环磷酸腺苷（cAMP）通过靶向这些转运蛋白回到胆小管膜来防止胆汁淤积。在肝细胞对中，通过不同的机制，胰高血糖素和沙丁胺醇这两种能增加 cAMP 的物质，通过刺激这些转运蛋白的运输来防止 E17G 的作用，即：胰高血糖素激活蛋白激酶 A 依赖性途径，而沙丁胺醇激活 cAMP 激活的交换蛋白（Epac）介导的、微管依赖性途径。
本研究评估了胰高血糖素和沙丁胺醇是否能在更生理的模型，即灌注大鼠肝脏（PRL）中预防 E17G 诱导的胆汁淤积。此外，还评估了体内丙氨酸给药（诱导胰高血糖素分泌）的预防作用。
在 PRLs 中，胰高血糖素和沙丁胺醇预防了 E17G 诱导的胆汁流量和 Abcc2 和 Abcb11 分泌活性的降低。沙丁胺醇的预防作用完全依赖于微管的完整性。另一方面，胰高血糖素的预防作用在 E17G 给药后早期阶段与微管无关，但最终受到微管破坏剂秋水仙碱的影响。胆汁淤积与 Abcb11 和 Abcc2 的内吞内化有关，细胞内载体与内体标记物 Rab11a 部分共定位。这种作用完全被沙丁胺醇所阻止，而在胰高血糖素处理后，一些含有转运蛋白的囊泡仍与 Rab11a 共定位。在体内，丙氨酸给药增加了肝 cAMP，并加速了 E17G 给药后胆汁流量和 Abcb11/Abcc2 转运功能的恢复。丙氨酸最初的恢复与微管无关，但微管的完整性是维持这种保护作用所必需的。
我们的结论是，通过直接给予 cAMP 调节剂或通过导致激素介导的 cAMP 水平升高的生理操作（丙氨酸给药）来调节 cAMP 水平，通过与体外研究中出现的机制相似的机制，防止了在保留肝结构的模型中雌激素诱导的胆汁淤积。
