Oak Apurva A, Chu Tifany, Yottasan Pattareeya, Chhetri Parth D, Zhu Jie, Du Bois Justin, Cil Onur
Pediatrics, UCSF, United States.
Stanford University, United States.
Mol Pharmacol. 2022 Jun 9;102(2):106-15. doi: 10.1124/molpharm.122.000542.
Loss of prosecretory Cl channel CFTR activity is considered as the key cause of gastrointestinal disorders in cystic fibrosis including constipation and meconium ileus. Clc-2 is proposed as an alternative Cl channel in intestinal epithelia that can compensate for CFTR loss-of-function. Lubiprostone is an FDA-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) induced by cAMP agonists, where subsequently applied CFTR inhibitor (CFTR-172) caused 2-3 fold greater inhibition. Similarly, AK-42 inhibited lubiprostone-induced secretory I by 20%, whereas CFTR-172 caused 2-3 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 BaCl All components of lubiprostone-induced secretion (Clc-2, CFTR and K channels) were inhibited by ~65% with the extracellular Ca-sensing receptor (CaSR) activator cinacalcet that stimulates cAMP hydrolysis. Lastly, 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 minor role in cAMP-induced intestinal Cl secretion; and lubiprostone is not a selective Clc-2 activator, but general activator of cAMP-gated ion channels in human intestinal epithelial cells. Cl- channel Clc-2 activation is the proposed mechanism of action of the FDA-approved constipation drug lubiprostone. Using first-in-class selective Clc-2 inhibitor AK-42, we showed that Clc-2 has minor contribution in intestinal Cl- secretion induced by lubiprostone and cAMP agonists. We also found that lubiprostone is a general activator of cAMP-gated ion channels in human intestinal epithelial cells (via EP4 receptors). Our findings clarify the roles of Clc-2 in intestinal Cl- secretion and elucidate the mechanism of action of approved-drug lubiprostone.
促分泌性氯离子通道CFTR活性丧失被认为是囊性纤维化患者胃肠道疾病（包括便秘和胎粪性肠梗阻）的关键病因。Clc-2被认为是肠上皮细胞中的一种替代性氯离子通道，可补偿CFTR功能丧失。鲁比前列酮是一种经美国食品药品监督管理局（FDA）批准的药物，其作用机制推测为激活Clc-2。然而，由于缺乏选择性Clc-2抑制剂，Clc-2在肠道氯离子分泌中的相对作用以及鲁比前列酮的作用机制仍存在争议。利用最近鉴定出的选择性Clc-2抑制剂AK-42，我们对Clc-2在人肠上皮T84细胞氯离子分泌中的作用进行了表征。Clc-2抑制剂AK-42对cAMP激动剂诱导的分泌性短路电流（I）的抑制作用最小（15%），而随后应用的CFTR抑制剂（CFTR-172）引起的抑制作用则大2至3倍。同样，AK-42对鲁比前列酮诱导的分泌性I的抑制作用为20%，而CFTR-172引起的抑制作用大2至3倍。除了增加CFTR和Clc-2介导的顶端氯离子电导外，鲁比前列酮还增加了基底外侧膜钾电导，这被cAMP激活的钾通道抑制剂BaCl完全逆转。鲁比前列酮诱导的分泌的所有成分（Clc-2、CFTR和钾通道）均被刺激cAMP水解的细胞外钙敏感受体（CaSR）激活剂西那卡塞抑制约65%。最后，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%，而对福斯克林反应无任何影响。我们的研究结果表明，Clc-2在cAMP诱导的肠道氯离子分泌中作用较小；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门控离子通道的一般激活剂。氯离子通道Clc-2激活是FDA批准的便秘药物鲁比前列酮的作用机制。使用一流的选择性Clc-2抑制剂AK-42，我们表明Clc-2在鲁比前列酮和cAMP激动剂诱导的肠道氯离子分泌中作用较小。我们还发现，鲁比前列酮是人肠上皮细胞中cAMP门控离子通道的一般激活剂（通过EP4受体）。我们的研究结果阐明了Clc-2在肠道氯离子分泌中的作用，并阐明了已批准药物鲁比前列酮的作用机制。
