Ursodeoxycholic acid stimulates cholangiocyte fluid secretion in mice via CFTR-dependent ATP secretion
Ursodeoxycholic acid stimulates cholangiocyte fluid secretion in mice via CFTR-dependent ATP secretion
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DOI：
10.1053/j.gastro.2007.08.071
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发表时间：
2007-11-01
期刊：
影响因子：
29.4
通讯作者：
Strazzabosco, Mario
中科院分区：
文献类型：
--
作者：
Fiorotto, Romina;Spirli, Carlo;Strazzabosco, Mario
背景与目的：胆管疾病的特征是胆管细胞分泌功能受损。熊去氧胆酸（UDCA）被广泛用于胆管疾病的治疗，但其对胆管细胞分泌功能的影响尚不清楚，这是本研究的主题。研究方法：从野生型（WT）和B6-129-Cftr（tm 1 Kth）和Cftr（tm 1Unc）小鼠中获得管状（分离的胆管单位[IBDU]）或单层构型的极化小鼠胆管细胞，这些小鼠CFTR（胆管细胞中表达的腺苷3 '，5'-环磷酸（cAMP）刺激的Cl-通道）缺陷。通过视频光学测面法评估液体分泌，通过微量荧光法（6-甲氧基-N-乙基喹啉氯化物、fura-2和fluo-4）评估Cl-和Ca 2+流出，通过荧光素酶测定评估三磷酸腺苷（ATP）分泌，通过蛋白质印迹法评估蛋白激酶C（PKC）。结果：UDCA刺激WT-IBDU的液体分泌和Cl-外排，但在CFTR-KO-IBDU或暴露于CFTR抑制剂的WT-IBDU中不刺激。UDCA不影响细胞内cAMP水平，但增加WT中的[Ca 2 +]i，而不是CFTR-KO胆管细胞中的[Ca 2 +]i。UDCA刺激WT胆管细胞顶端ATP分泌，但不刺激CFTR KO胆管细胞。嘌呤能2 Y受体抑制剂苏拉明可抑制UDCA刺激的[Ca 2 +]i升高。UDCA刺激PKC-α和PKC-β向质膜的易位。UDCA刺激的分泌被2-双（2-氨基苯氧基）-乙烷-N N，N '，N'-四乙酸和磷脂酶C和PKC抑制剂抑制。UDCA增加WT小鼠离体灌注肝脏中的ATP输出，但不增加CFTR-KO小鼠的ATP输出。结论：我们的数据表明，UDCA刺激胆管细胞中CFTR依赖性顶端ATP释放。分泌的ATP激活嘌呤能2 Y受体，并通过[Ca 2 +]i增加和PKC激活刺激Cl-流出和液体分泌。这些数据支持CFTR在调节分泌上皮细胞中嘌呤能信号传导中发挥作用的概念，并提出了一种解释UDCA利胆作用的新机制。
Background & Aims: Cholangiopathies are characterized by impaired cholangiocyte secretion. Ursodeoxycholic acid (UDCA) is widely used for cholangiopathy treatment, but its effects on cholangiocyte secretory functions remain unclear and are the subject of this study. Methods: Polarized mouse cholangiocytes in tubular (isolated bile-duct units [IBDU]) or monolayer configuration were obtained from wild-type (WT) and B6-129-Cftr(tm1Kth) and Cftr(tm1Unc) mice that are defective in CFTR, an adenosine 3 ',5 '-cyclic monophosphate (cAMP)-stimulated Cl- channel expressed in cholangiocytes. Fluid secretion was assessed by video-optical planimetry, Cl- and Ca2+ efflux by microfluorimetry (6-methoxy-N-ethylquinolinium chloride, fura-2, and fluo-4), adenosine triphosphate (ATP) secretion by luciferin-luciferase assay, and protein kinase C (PKC) by Western blot. Results: UDCA stimulated fluid secretion and Cl- efflux in WT-IBDU but not in CFTR-KO-IBDU or in WT-IBDU exposed to CFTR inhibitors. UDCA did not affect intracellular cAMP levels but increased [Ca2+]i in WT and not in CFTR-KO cholangiocytes. UDCA stimulated apical ATP secretion in WT but not in CFTR KO cholangiocytes. UDCA-stimulated [Ca2+]i increase was inhibited by suramin, a purinergic 2Y-receptor inhibitor. UDCA stimulated the translocation of PKC-alpha and PKC-epsilon to the plasma membrane. UDCA-stimulated secretion was inhibited by 2-bis(2-aminophenoxy)-ethane-N N,N',N'-tetraacetic acid and by phospholipase C and PKC inhibitors. UDCA increased ATP output in isolated perfused livers from WT but not from CFTR-KO mice. Conclusions: Our data indicate that UDCA stimulates a CFTR-dependent apical ATP release in cholangiocytes. Secreted ATP activates purinergic 2Y receptors, and, through [Ca2+]i increase and PKC activation stimulates Cl- efflux and fluid secretion. These data support the concept that CFTR plays a role in modulating purinergic signaling in secretory epithelia and suggest a novel mechanism explaining the choleretic effect of UDCA.