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 (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.
Footnotes
- Received April 16, 2022.
- Accepted May 27, 2022.
This work was supported by grants from the National Institutes of Health [Grant R01-DK126070] and [Grant P30-DK072517] (to O.C.) and the Cystic Fibrosis Foundation.
The authors have declared that no conflict of interest exists.
- Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|