Lubiprostone is non-selective activator of cAMP-gated ion channels and Clc-2 has a minor role in its prosecretory effect in intestinal epithelial cells.

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.