(216) Approved drug lubiprostone is not a selective Clc-2 activator and its prosecretory effect primarily depends on CFTR in human intestinal epithelial cells

Background: Loss of prosecretory Cl^- channel CFTR activity is considered as the key cause of gastrointestinal disorders in CF including constipation and meconium ileus. Clc-2 is considered as an alternative Cl^- channel in intestinal and other epithelia that can compensate for loss of CFTR function. Lubiprostone is an FDA-approved constipation treatment with Clc-2 activation as its proposed mechanism of action. Although lubiprostone had efficacy in various forms of constipation in the general population, its utility in CF-associated constipation is not well documented. In addition, relative contribution of Clc-2 in intestinal Cl^- secretion and the mechanisms of action of lubiprostone remain controversial due to lack of selective Clc-2 inhibitors.

Methods: 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.

Results: Clc-2 inhibitor AK-42 had minimal (15%) inhibitory effect on secretory short-circuit current (I_sc) induced by cAMP agonists forskolin, cholera toxin and vasoactive intestinal peptide. Whereas subsequently applied CFTR inhibitor (CFTR_inh-172) had 2-3 fold greater inhibitory effects on cAMP agonists-induced secretory currents. Similarly, AK-42 inhibited lubiprostone-induced secretory I_sc only by 20%, where subsequent CFTR_inh-172 application had 2-3 fold greater inhibitory effect. In addition to increasing CFTR and Clc-2 mediated apical membrane Cl^- conductance, lubiprostone also increased basolateral membrane K⁺ conductance in T84 cells that was studied by apical membrane permeabilization and apical-to-basolateral K⁺ gradient application. Lubiprostone-induced basolateral membrane K⁺ currents were completely inhibited by cAMP-activated K⁺ channel blocker BaCl₂. All components of lubiprostone-induced secretory I_sc (Clc-2, CFTR and K⁺ channels) were inhibited by ~65% with the extracellular Ca²⁺-sensing receptor (CaSR) activator cinacalcet which is known to stimulate cAMP hydrolysis. Since lubiprostone is a prostaglandin analog that appears to exert its prosecretory effects via cAMP, we tested the roles of G_s-coupled intestinal prostaglandin receptors (DP1, EP2, EP4 and IP) in its secretory effect. Pretreatment of T84 cells with selective DP1, EP2 or IP inhibitors had no effect on lubiprostone-induced secretory currents. However, EP4 prostaglandin receptor inhibitor GW627368 pretreatment inhibited lubiprostone-induced secretory I_sc by 40% without any effect on forskolin response.

Conclusions: 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 via EP4 receptors. Since its prosecretory effect primarily depends on CFTR activity, lubiprostone is predicted to be ineffective in CF-associated constipation.

Acknowledgements: This work was supported by CFF and NIDDK. We thank Drs. Justin Du Bois and Jie Zhu (Stanford University) for providing the Clc-2 inhibitor.

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