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 μA; SLIGRL, 83 ± 22 μA), which returned to baseline within 5 min after stimulation. Pretreatment of monolayers with the cell-permeant Ca²⁺-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 PGE₂ 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 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.