Abstract

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.