<br>鲁比前列酮是一种 cAMP 门控离子通道和氯离子通道蛋白 2 (Clc-2) 的非选择性激活剂，在肠上皮细胞的促分泌作用中发挥次要作用,Molecular Pharmacology

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鲁比前列酮是一种 cAMP 门控离子通道和氯离子通道蛋白 2 (Clc-2) 的非选择性激活剂，在肠上皮细胞的促分泌作用中发挥次要作用
Molecular Pharmacology ( IF 3.2 ) Pub Date : 2022-08-01 , DOI: 10.1124/molpharm.122.000542
Apurva A Oak ₁ , Tifany Chu ₁ , Pattareeya Yottasan ₁ , Parth D Chhetri ₁ , Jie Zhu ₂ , Justin Du Bois ₂ , Onur Cil ₃

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分泌性Cl ^-通道囊性纤维化跨膜电导调节因子（CFTR）活性的丧失被认为是囊性纤维化胃肠道疾病（包括便秘和胎粪性肠梗阻）的关键原因。氯通道蛋白 2 (Clc-2) 被认为是肠上皮细胞中的替代 Cl ^-通道，可以补偿 CFTR 功能的丧失。鲁比前列酮是一种经美国食品和药物管理局批准的药物，其假定的作用机制是激活 Clc-2。然而，由于缺乏选择性 Clc-2 抑制剂，Clc-2 在肠道 Cl ^-分泌中的相对贡献以及鲁比前列酮的作用机制仍存在争议。使用最近鉴定的选择性 Clc-2 抑制剂 AK-42，我们表征了 Clc-2 在人肠上皮 T84 细胞 Cl ^-分泌中的作用。 Clc-2 抑制剂 AK-42 对 cAMP 激动剂诱导的分泌性短路电流 (I _sc ) 具有最小 (15%) 的抑制作用，随后应用 CFTR 抑制剂 (CFTR _inh -172) 引起 2 至 3 倍的抑制作用。类似地，AK-42 抑制鲁比前列酮诱导的 I _sc分泌达 20%，而 CFTR _inh -172 则导致 2 至 3 倍的抑制。除了增加 CFTR 和 Clc-2 介导的顶端 Cl ^-电导之外，鲁比前列酮还增加基底外侧膜 K ⁺电导，而 cAMP 激活的 K ⁺通道抑制剂 BaCl ₂可完全逆转这一现象。鲁比前列酮诱导的分泌的所有成分（Clc-2、CFTR 和 K ⁺通道）均被刺激 cAMP 水解的细胞外 Ca ²⁺感应受体 (CaSR) 激活剂西那卡塞抑制约 65%。最后，E 型前列腺素受体 4 (EP4) 前列腺素受体抑制剂 GW627368 预处理可抑制鲁比前列酮诱导的分泌 40%，且对毛喉素反应没有任何影响。我们的研究结果表明，Clc-2 在 cAMP 诱导的肠道 Cl ^-分泌中起次要作用；鲁比前列酮不是选择性 Clc-2 激活剂，而是人肠上皮细胞中 cAMP 门控离子通道的通用激活剂。

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Lubiprostone is a Non-Selective Activator of cAMP-Gated Ion Channels and Chloride Channel Protein 2 (Clc-2) Has a Minor Role in its Prosecretory Effect in Intestinal Epithelial Cells

Loss of prosecretory Cl^- channel cystic fibrosis transmembrane conductance regulator (CFTR) activity is considered the key cause of gastrointestinal disorders in cystic fibrosis, including constipation and meconium ileus. Chloride channel protein 2 (Clc-2) is proposed as an alternative Cl^- channel in intestinal epithelia that can compensate for CFTR loss-of-function. Lubiprostone is a Food and Drug Administration-approved drug with Clc-2 activation as its presumed mechanism of action. However, relative contribution of Clc-2 in intestinal Cl^- secretion and the mechanism of action of lubiprostone remain controversial due to lack of selective Clc-2 inhibitors. Using recently identified selective Clc-2 inhibitor AK-42, we characterized the roles of Clc-2 in Cl^- secretion in human intestinal epithelial T84 cells. Clc-2 inhibitor AK-42 had minimal (15%) inhibitory effect on secretory short-circuit current (I_sc) induced by cAMP agonists, where subsequently applied CFTR inhibitor (CFTR_inh-172) caused 2- to 3-fold greater inhibition. Similarly, AK-42 inhibited lubiprostone-induced secretory I_sc by 20%, whereas CFTR_inh-172 caused 2- to 3-fold greater inhibition. In addition to increasing CFTR and Clc-2-mediated apical Cl^- conductance, lubiprostone increased basolateral membrane K⁺ conductance, which was completely reversed by cAMP-activated K⁺ channel inhibitor BaCl₂. All components of lubiprostone-induced secretion (Clc-2, CFTR, and K⁺ channels) were inhibited by ∼65% with the extracellular Ca²⁺-sensing receptor (CaSR) activator cinacalcet that stimulates cAMP hydrolysis. Lastly, E-type prostanoid receptor 4 (EP4) prostaglandin receptor inhibitor GW627368 pretreatment inhibited lubiprostone-induced secretion by 40% without any effect on forskolin response. Our findings suggest that Clc-2 has a minor role in cAMP-induced intestinal Cl^- secretion; and lubiprostone is not a selective Clc-2 activator, but a general activator of cAMP-gated ion channels in human intestinal epithelial cells.

更新日期：2022-07-19

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