Protease-activated receptor regulation of Cl- secretion in Calu-3 cells requires prostaglandin release and CFTR activation

Departments of 1 Physiology and 2 Animal Science, University of Minnesota, St. Paul, Minnesota; and 3 Department of Veterinary Pharmacology, Seoul National University, Seoul, Republic of Korea Submitted 16 September 2005 ; accepted in final form 20 November 2005 Human lung epithelial (Calu-3) cells were used to investigate the effects of protease-activated receptor (PAR) stimulation on Cl – secretion. Quantitative RT-PCR (QRT-PCR) showed that Calu-3 cells express PAR-1, -2, and -3 receptor mRNAs, with PAR-2 mRNA in greatest abundance. Addition of either thrombin or the PAR-2 agonist peptide SLIGRL to the basolateral solution of monolayers mounted in Ussing chambers produced a rapid increase in short-circuit current ( I sc : thrombin, 21 ± 2 µA; SLIGRL, 83 ± 22 µA), which returned to baseline within 5 min after stimulation. Pretreatment of monolayers with the cell-permeant Ca 2+ -chelating agent BAPTA-AM (50 µM) abolished the increase in I sc produced by SLIGRL. When monolayers were treated with the cyclooxygenase inhibitor indomethacin (10 µM), nearly complete inhibition of both the thrombin- and SLIGRL-stimulated I sc was observed. In addition, basolateral treatment with the PGE 2 receptor antagonist AH-6809 (25 µM) significantly inhibited the effects of SLIGRL on I sc . QRT-PCR revealed that Calu-3 cells express mRNAs for CFTR, the Ca 2+ -activated KCNN4 K + channel, and the KCNQ1 K + channel subunit, which, in association with KCNE3, is known to be regulated by cAMP. Stimulation with SLIGRL produced an increase in apical Cl – conductance that was blocked in cells expressing short hairpin RNAs designed to target CFTR. These results support the conclusion that PAR stimulation of Cl – secretion occurs by an indirect mechanism involving the synthesis and release of prostaglandins. In addition, PAR-stimulated Cl – secretion requires activation of CFTR and at least two distinct K + channels located in the basolateral membrane. cystic fibrosis transmembrane conductance regulator; KCNQ1; calcium-activated potassium channels; KCNN4; cAMP Address for reprint requests and other correspondence: S. M. O'Grady, Depts. of Physiology and Animal Science, 495 Animal Science/Veterinary Medicine Bldg., 1988 Fitch Ave., Univ. of Minnesota, St. Paul, MN 55110 (e-mail: ograd001{at}umn.edu )