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

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 mu A; SLIGRL, 83 +/- 22 mu A), which returned to baseline within 5 min after stimulation. Pretreatment of monolayers with the cell-permeant Ca2+-chelating agent BAPTA-AM (50 mu M) abolished the increase in Isc produced by SLIGRL. When monolayers were treated with the cyclooxygenase inhibitor indomethacin ( 10 mu M), nearly complete inhibition of both the thrombin- and SLIGRL-stimulated Isc was observed. In addition, basolateral treatment with the PGE(2) receptor antagonist AH-6809 (25 mu M) significantly inhibited the effects of SLIGRL on I-sc. QRT-PCR revealed that Calu-3 cells express mRNAs for CFTR, the Ca2+-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.