TY - JOUR T1 - 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 JF - Molecular Pharmacology JO - Mol Pharmacol SP - 106 LP - 115 DO - 10.1124/molpharm.122.000542 VL - 102 IS - 2 AU - Apurva A. Oak AU - Tifany Chu AU - Pattareeya Yottasan AU - Parth D. Chhetri AU - Jie Zhu AU - J. Du Bois AU - Onur Cil Y1 - 2022/08/01 UR - http://molpharm.aspetjournals.org/content/102/2/106.abstract N2 - 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 (Isc) induced by cAMP agonists, where subsequently applied CFTR inhibitor (CFTRinh-172) caused 2- to 3-fold greater inhibition. Similarly, AK-42 inhibited lubiprostone-induced secretory Isc by 20%, whereas CFTRinh-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.SIGNIFICANCE STATEMENT Cl- channel Clc-2 activation is the proposed mechanism of action of the Food and Drug Administration-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. ER -